EP4688840A1 - Agonistic split antibodies - Google Patents

Agonistic split antibodies

Info

Publication number
EP4688840A1
EP4688840A1 EP24714516.2A EP24714516A EP4688840A1 EP 4688840 A1 EP4688840 A1 EP 4688840A1 EP 24714516 A EP24714516 A EP 24714516A EP 4688840 A1 EP4688840 A1 EP 4688840A1
Authority
EP
European Patent Office
Prior art keywords
seq
polypeptide
amino acid
acid sequence
antigen binding
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24714516.2A
Other languages
German (de)
French (fr)
Inventor
Petra Elisabeth Marilena BALBI
Jack Anthony BATES
Samuele CALABRO
Laura CODARRI DEAK
Stephan Gasser
Guy Georges
Xavier GUERIPEL
Ralf Joerg HOSSE
Christian Klein
Stéphane Gérard Alain LECLAIR
Ekkehard Moessner
Christian Mueller
Diana Angela PIPPIG
Laurene POUSSE
Camilla Elizabeth TREVOR
Pablo UMAÑA FERNÁNDEZ
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
F Hoffmann La Roche AG
Original Assignee
F Hoffmann La Roche AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by F Hoffmann La Roche AG filed Critical F Hoffmann La Roche AG
Publication of EP4688840A1 publication Critical patent/EP4688840A1/en
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against enzymes
    • 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
    • C07K2317/35—Valency
    • 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
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/66—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a swap of domains, e.g. CH3-CH2, VH-CL or VL-CH1
    • 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
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • the application relates to pairs of antigen binding molecules, each molecule comprising a target-binding domain, a cytokine receptor-binding domain and an Fc domain, wherein the target-binding domains simultaneously bind the target antigen and the cytokine receptor-binding domains bind subunits of a cytokine receptor complex.
  • Biparatopic assembly of the cytokine receptor-binding domains in presence of the target antigen allows to selectively activate a cytokine receptor and effectively mimic cytokine activity in a targeted manner.
  • IFNy interferon gamma or IFN-y
  • IFNy is a cytokine that is mainly produced by activated lymphocytes, such as CD4 + and CD8 + T cells, and natural killer cells (NK cells) in response to immune or inflammatory stimuli.
  • IFNy is a homodimer and its receptors (IFNyRl and IFNyR2) are expressed across hematopoietic and non-hematopoietic cells. On the cell surface, IFNyRl is stably expressed whereas IFNyR2 is differentially expressed and regulates IFNy.
  • IFNy When IFNy binds to its receptors, Janus kinases JAK1 and JAK2 are recruited and activated, which phosphorylate and activate STAT1. A phosphorylated STAT1 translocates to the nucleus, binds promoters, and modulates gene transcription under the control of IFNy. In contrast to other cancer therapies currently on the market or in development, IFNy can act on both tumor cells and immune cells including T cells and dendritic cells.
  • IFNy can also show undesirable side effects.
  • problems with administration, bioavailabilty and short half-life may arise.
  • new molecules able to selectively activate the IFNy pathway at the tumor site may also show undesirable side effects.
  • Cytokines are powerful immune modulators that initiate signaling through receptor dimerization, but natural cytokines have some limitations as therapeutics. Low stability and difficulties in the production process are just some of them. It is known for some time that antibodies can induce signaling on cells, and therefore substitute a natural ligand.
  • bispecific anti-IL-2RPy antibodies showed varying levels of agonist activity (Harris, K. E., et al. Sci Rep 11(1): 10592 (2021)).
  • a similar approach as described by Teneobio was undertaken by scientists at Synthekine, which described the functional induction of signaling of the two interleukin receptors (IL-2RP and IL-2Ry) via single domain antibodies (sdAbs; WO 2022/032040 Al), and reviewed by Saxton and colleagues (Saxton, R. A., et al. Nat Rev Drug Discov. 22, 21-37 (2022)).
  • Those peptides, with an molecular weight of less than 5 kDa fused to an IgGi-Fc domain exhibits biological properties similar to those of IL-7 in vitro, and when administered to non-human primates (Dower, W., et al. Journal for Immuno Therapy of Cancer 8 (Suppl 3): A341-A342 (2020)).
  • the present invention relates to novel antigen binding molecules comprising cytokine receptor-binding domains, which, under the desired conditions of biparatopic assembly on a target antigen, serve as cytokine mimetics selectively activating the receptor pathway.
  • the present invention relates to a pair of antigen binding molecules that specifically bind to a target antigen, comprising a) a first antigen binding molecule comprising i) a first target-binding domain, ii) a first cytokine receptor-binding domain, and iii) a Fc domain; and b) a second antigen binding molecule comprising i) a second target-binding domain, ii) a second cytokine receptor-binding domain, and iii) a Fc domain; wherein the first target-binding domain is capable of binding a first epitope on the target antigen and the second target-binding domain is capable of binding a second epitope on the target antigen, wherein the first and second target-binding domains do not compete for binding on the target antigen; and wherein the first cytokine receptor-binding domain is capable of binding a first cytokine receptor subunit and the second cytokine receptor-binding domain is capable of binding a second
  • the first and second target-binding domains are antibody fragments, such as Fv, Fab, scFv, scFab molecules or single domain antibodies.
  • the first and second target-binding domains are Fab molecules.
  • the first target-binding domain comprises a heavy chain variable domain (VHi), a light chain variable domain (VLi), a heavy chain constant domain (CHli) and a light chain constant domain (CLi)
  • the second target-binding domain comprises a heavy chain variable domain (VH2), a light chain variable domain (VL2), a heavy chain constant domain (CHI2) and a light chain constant domain (CL2).
  • the first and/or second target-binding domain is a cross-Fab molecule.
  • the first and second target-binding domains specifically bind to a tumor-associated antigen or a T-cell antigen. In one aspect, the first and second target-binding domains specifically bind to FAP, PD-1, Her2, Her3, LAG-3 or EGFR.
  • the first target-binding domain comprises a VHi of SEQ ID NO: 20 and a VLi of SEQ ID NO: 21 and the second target-binding domain comprises a VH2 of SEQ ID NO: 22 and a VL2 of SEQ ID NO: 23, or b) the first target-binding domain comprises a VHi of SEQ ID NO: 22 and a VLi of SEQ ID NO: 23 and the second target-binding domain comprises a VH2 of SEQ ID NO: 20 and a VL2 of SEQ ID NO: 21, or c) the first target-binding domain comprises a VHi of SEQ ID NO: 76 and a VLi of SEQ ID NO: 77 and the second target-binding domain comprises a VH2 of SEQ ID NO: 78 and a VL2 of SEQ ID NO: 79, or d) the first target-binding domain comprises a VHi of SEQ ID NO: 78 and a VLi of SEQ ID NO:
  • both the first and second cytokine receptor subunits are subunits of the IFNy receptor complex or, IL-2 receptor complex or IL-7 receptor complex.
  • a) the first cytokine receptor-binding domain is capable of binding IFNyRl and the second cytokine receptor-binding domain is capable of binding IFNyR2, or b) the first cytokine receptor-binding domain is capable of binding IFNyR2 and the second cytokine receptor-binding domain is capable of binding IFNyRl ; or c) the first cytokine receptor-binding domain is capable of binding IL-2RP and the second cytokine receptor-binding domain is capable of binding IL-2Ry, or d) the first cytokine receptor-binding domain is capable of binding IL-2Ry and the second cytokine receptor-binding domain is capable of binding IL- 2RP; or e) the first cytokine receptor-binding domain is capable of binding IL-2Ry and the second
  • the first and second cytokine receptor-binding domains are antibody fragments, such as Fv, Fab, scFv, scFab or single domain antibodies. In one aspect, the first and second cytokine receptorbinding domains are VHH domains.
  • the first cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 5 and the second cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, or b) the first cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10 and the second cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 5; c) the first cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58 SEQ ID NO: 60, and the second cytokine
  • the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 3 and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 7, or b) the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 7 and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 3;or c) the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 60 and the second cytokine receptorbinding domain comprises the sequence of SEQ ID NO: 62, or d) the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 62 and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 60; or e) the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 186, and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 62, or f) the first cytokine receptor-binding domain
  • the Fc domains of the first and second antigen binding molecules comprise a first Fc domain subunit and a second Fc domain subunit.
  • the Fc domains of the first and second antigen binding molecules are IgG, particularly an IgGi, Fc domains.
  • the Fc domains of the first and second antigen binding molecules are human Fc domains.
  • the Fc domains of the first and second antigen binding molecules comprise a modification promoting the association of the first and the second subunit of the Fc domains.
  • the Fc domains of the first and second antigen binding molecules comprise one or more amino acid substitution that reduces binding to an Fc receptor and/or effector function.
  • the cytokine receptor-binding domains are fused via peptide linkers to their respective fusion points.
  • the peptide linkers comprise an amino acid sequence selected from SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 279, SEQ ID NO: 280 or SEQ ID NO: 281.
  • the first cytokine receptor-binding domain is fused at its C-terminus to the N-terminus of VHi or VLi of the first target-binding domain and the second cytokine receptor-binding domain is fused at its C-terminus to the N-terminus of VH2 or VL2 of the second target-binding domain.
  • the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus the first cytokine receptorbinding domain, VHi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VLi and CLi; and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus the second cytokine receptor-binding domain, VH2, CHI 2 and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C- terminus VL2 and CL2; or b) the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N
  • the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus the first cytokine receptor-binding domain, VHi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VLi and CLi
  • the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus the second cytokine receptor-binding domain, VH2, CHI 2 and a second Fc domain subunit, and a third polypeptide comprising in order from the N- terminus to C-terminus VL2 and CL2; or b) the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the
  • the first and the second target-binding domains specifically bind to FAP, and the first and second cytokine receptor subunits are subunits of the IFNy receptor complex.
  • the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 27 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 30 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 38 and a third polypeptide comprising an amino acid sequence of S
  • first and the second target-binding domains specifically bind to EGFR, and the first and second cytokine receptor subunits are subunits of the IFNy receptor complex.
  • the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 260 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124
  • the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 259 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 127;
  • the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 119 and a third poly
  • the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 119 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 257, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 112 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 254; or e) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 260 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises
  • the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 261 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 112 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 254; or h) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 119 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 257, and the second antigen binding molecule comprises
  • the first and the second target-binding domains specifically bind to PD-1, and the first and second cytokine receptor subunits are subunits of the IL-2 receptor complex.
  • the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 86, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 44 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 83
  • the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 88 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 87; or
  • the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an
  • first and the second target-binding domains specifically bind to LAG-3, and the first and second cytokine receptor subunits are subunits of the IL-2 receptor complex.
  • a) first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 155 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 154
  • the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 165 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 163.
  • the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 161 and athird poly
  • first and the second target-binding domains specifically bind to EGFR
  • first and second cytokine receptor subunits are subunits of the IL-2 receptor complex.
  • a) first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 155 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 154
  • the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 165 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 163;
  • the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 161 and athird poly
  • the first and the second target-binding domains specifically bind to FAP, and the first and second cytokine receptor subunits are subunits of the IL-2 receptor complex.
  • the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 110 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 113 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an
  • first and the second target-binding domains specifically bind to Her2, and the first and second cytokine receptor subunits are subunits of the IL-2 receptor complex.
  • a) first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 136 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 134
  • the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 138 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 137;
  • the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 135 and a third polypeptide
  • the first and the second target-binding domains specifically bind to Her3, and the first and second cytokine receptor subunits are subunits of the IL-2 receptor complex.
  • the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 146 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 144
  • the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 148 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 147; or
  • the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 145 and a third polypeptid
  • the first and the second target-binding domains specifically bind to PD-1, and the first and second cytokine receptor subunits are subunits of the IL-7 receptor complex.
  • the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 194 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 193, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 195 and a third polypeptide compris
  • the first target-binding domain is fused at its C-terminus of CHI i to the N-terminus of the first Fc domain subunit and the first cytokine receptor-binding domain is fused at its C-terminus to the N-terminus of the second Fc domain subunit and the second target-binding domain is fused at its C- terminus of CHE to the N-terminus of the first Fc domain subunit and the second cytokine receptorbinding domain is fused at its C-terminus to the N-terminus of the second Fc domain subunit.
  • the first antigen binding molecule comprises a first polypeptide comprising in order from the N-terminus to C-terminus VHi, CHli and a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VLi and CLi, and a third polypeptide comprising in order from the N-terminus to C-terminus the first cytokine receptor-binding domain and a second Fc domain subunit
  • the second antigen binding molecule comprises a first polypeptide comprising in order from the N-terminus to C-terminus VH2, CH 12 and a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VL2 and CL2, and a third polypeptide comprising in order from the N-terminus to C-terminus the second cytokine receptor-binding domain and a second Fc domain subunit; or b) the first antigen binding molecule comprises a first polypeptide comprising in order from
  • the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 32, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 25 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 39
  • the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 34, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 29 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 40
  • the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 34, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 29 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 39
  • the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 32, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 25
  • the first target-binding domain is fused at its C-terminus of CHli to the N-terminus of one of the Fc domain subunits and the first cytokine receptor-binding domain is fused at its N-terminus to the C-terminus of the same Fc domain subunit and the second target-binding domain is fused at its C- terminus of CHE to the N-terminus of one of the Fc domain subunits and the second cytokine receptorbinding domain is fused at its N-terminus to the C-terminus of the same Fc domain subunit.
  • the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a VHi, a CHli, a second Fc domain subunit and a the first cytokine receptor-binding domain, and a third polypeptide comprising in order from the N-terminus to C-terminus a VLi and a CLi ; and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a VH2, a CHE, a second Fc domain subunit and a the second cytokine receptor-binding domain, and a third polypeptide comprising in order from the N- terminus to C-terminus VL2 and CL2; or b) the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a
  • the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 42 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 41
  • the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 45, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 44 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 43
  • the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 46 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 41
  • the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 47, a second polypeptide comprising an amino acid sequence
  • the first and second cytokine receptor-binding domains of the pair of antigen binding molecules are Fab molecules.
  • a) the first cytokine receptor-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 282 and a VL domain comprising the amino acid sequence of SEQ ID NO: 283 or VH domain comprising the amino acid sequence of SEQ ID NO: 284 and a VL domain comprising the amino acid sequence of SEQ ID NO: 285, and the second cytokine receptor domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 286 and a VL domain comprising the amino acid sequence of SEQ ID NO: 287 or VH domain comprising the amino acid sequence of SEQ ID NO: 288 and a VL domain comprising the amino acid sequence of SEQ ID NO: 289; or b) the first cytokine receptor-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 286 and a VL
  • the first and second cytokine receptor-binding domains of the pair of antigen binding molecules are scFv molecules.
  • a) the first cytokine receptor-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 290 and a VL domain comprising the amino acid sequence of SEQ ID NO: 291
  • the second cytokine receptor domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 292 and a VL domain comprising the amino acid sequence of SEQ ID NO: 293
  • the first cytokine receptor-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 292 and a VL domain comprising the amino acid sequence of SEQ ID NO: 293
  • the second cytokine receptor domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 290 and a VL domain comprising the amino acid sequence of SEQ ID NO: 291.
  • the Fc domains of the first and second antigen binding molecules comprising Fabs or scFv molecules as cytokine receptor-binding domains comprise a first Fc domain subunit and a second Fc domain subunit.
  • the Fc domains of the first and second antigen binding molecules are IgG, particularly an IgGi, Fc domains.
  • the Fc domains of the first and second antigen binding molecules are human Fc domains.
  • the Fc domains of the first and second antigen binding molecules comprise a modification promoting the association of the first and the second subunit of the Fc domains.
  • the Fc domains of the first and second antigen binding molecules comprise one or more amino acid substitution that reduces binding to an Fc receptor and/or effector function.
  • the cytokine receptor-binding domains are fused via peptide linkers to their respective fusion points.
  • the peptide linkers comprise an amino acid sequence of SEQ ID NO: 280 or SEQ ID NO: 281.
  • the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 267, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 265 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 266, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 272, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 270 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 271; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 267, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a third polypeptide comprising an amino acid sequence of SEQ ID NO NO:
  • the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 276, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 28 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 275
  • the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 277, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 28 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 278.
  • FIG. 1 Schematic representations of antigens used in llama immunization, phage display and screening for the isolation of human IFNyRl, IFNyR2, IL-2RP and IL-2Ry-specific single domain antibodies.
  • a heterodimer formed of the extracellular domain (ECD) of human IFNyRI fused to biotinylated Fc knob and human IFNyR2 ECD fused to Fc hole (A) and a heterodimer formed of the ECD of human IL-2RP fused to biotinylated Fc knob and human IL-2Ry ECD fused to Fc hole (D) were generated.
  • Figure 2 Workflow for the enrichment of single domain antibodies with binding specificity for human cytokine receptor subunits by phage display.
  • FIG. 3 Specificity screening of single domain antibodies by ELISA.
  • a random set of soluble VHH domains selected for human IFNyRl specificity following three rounds of phage display were tested for binding to immobilized human IFNyRl -Fc and immobilized human Fc (A).
  • VHH domains with human IFNyR2 specificity following three rounds of phage display were also tested for antigen specificity by ELISA (B).
  • VHH domains selected for human IL-2RP specificity following three rounds of phage display were tested for binding to immobilized human IL-2Rp-Fc and immobilized human Fc (C).
  • VHH domains with human IL-2Ry specificity following three rounds of phage display were also tested for antigen specificity by ELISA (D). Absorbance at 450 nm indicating the binding response to each target is represented as a stacked bar chart.
  • FIG. 4 Format conversion of single domain antibody fragments.
  • Round 3 VHH library variants with IFNyRl or IL-2RP specificity were fused to Fc-Knob and cloned into a mammalian cell expression vector using Gibson cloning method (A).
  • Round 3 VHH library variants with IFNyR2 or IL-2Ry specificity were fused to Fc-hole using the same method (B).
  • Bispecific heavy chain antibody comprising IFNyRl -IFNyR2 or IL-2Rp-IL-2Ry VHH domain pairs. VHH domains with IFNyRl or IL-2RP specificity were fused to Fc-knob and VHH domains targeting IFNyR2 or IL-2Ry were fused to Fc-hole. Flexible 5(G4S) linkers were used to fuse VHH moieties to Fc chains. Bispecific heavy chain antibodies were generated via knob-into-hole assembly of Fc chains. Effector functions of the Fc were silenced via incorporation of P329G LALA mutations in the CH2 domain.
  • FIG. 6 Functional screening of bispecific heavy chain antibodies comprising IFNyRl -IFNyR2 or IL- 2Rp-IL-2Ry VHH domain pairs.
  • HEK-Blue IFNy cells were incubated with IFNy agonistic bispecific heavy chain antibodies comprising IFNyRl and IFNyR2 VHH domain pairs generated using a 5x5 bispecificity matrix (A).
  • HEK-Blue IL-2 reporter cells were incubated with IL-2 agonistic bispecific heavy chain antibodies comprising IL-2RP and IL-2Ry VHH pairs generated using a 5x5 bispecificity matrix (B).
  • IFNyR activity and IL-2R activity was quantified by absorbance at 650 nm and fold response/background is shown for each treatment.
  • FIG. 7 Characterisation of dose-dependent response for IFNy agonistic heavy chain antibodies.
  • Previously identified IFNyR agonists (Fig. 6A, Table 1) were screened for dose -dependent IFNyR activity in HEK-Blue IFNy cells, characterized by absorbance at 650 nm.
  • the responses of different IFNy agonistic heavy chain antibodies are grouped according to the anti-IFNyRl VHH clone, i.e.
  • IFNyRl_l clone A
  • IFNyRl_2 clone B
  • IFNyRl_3 clone C
  • IFNyRl_5 clone D
  • Responses were compared to recombinant human IFNy (black dashed line) and FAP-IFNy (P1AF3574; light grey dashed line).
  • FIG. 8 Comparison of EC50 values for IFNy agonistic heavy chain antibodies. EC50 values were derived from HEK-Blue assays (Fig. 7) using GraphPad Prism software. Values are depicted in nanomolar concentration with recombinant human IFNy highlighted as a reference (dotted line).
  • FIG. 9 Characterization of dose -dependent response for IL-2R agonistic heavy chain antibodies.
  • Previously identified IL-2R agonists (Fig. 6B, Table 2) were screened for dose-dependent IL-2R agonism in HEK-Blue IL-2 cells.
  • Anti-PD 1 -IL2v (P1AE4422) was used as a reference molecule and each molecule was incubated with reporter cells at the following concentrations: 20 nM, 0.8 nM, 0.032 nM shown from left to right.
  • IL-2R activity was quantified by absorbance at 650 nm; mean values from technical triplicates are shown with error bars representing standard deviation.
  • FIG. 10 MHC-I and PD-L1 expression on tumor cells following 72h treatment of IFNy agonists.
  • IFNyR agonists P1AH1877-P1AH1890
  • reference molecules recombinant human IFNy and FAP-IFNy
  • 100, 10, 1 and 0.1 nM shown from left to right for each treatment.
  • Measurements were blank- subtracted and normalized to recombinant human IFNy response at the highest concentration (100 nM).
  • MHC-I expression on MKN45 cells A
  • PD-L1 expression on MKN45 cells B
  • MHC-I expression on Bxpc3 cells C
  • D PD-L1 expression on Bxpc3 cells
  • E MHC-I expression on CorL105 cells
  • F PD-L1 expression on CorL105 cells
  • FIG. 11 Concept of split dual-targeted IFNyR agonists. FAP-dependent biparatopic assembly of split IFNy mimetics (A). Note that for illustrative simplification, only one monomer of the FAP dimer is represented in the inset.
  • VHH domains with IFNyRl and IFNyR2 specificity were fused to the N- terminus of two different anti-FAP binding domains, via VH or VL fusion points, resulting in eight configurations within the permutation space: IFNyRl -VHH fused to the VH of FAP binder 1 (B), IFNyR2-VHH fused to the VH of FAP binder 2 (C), IFNyRl -VHH fused to the VL of FAP binder 1 (D), IFNyR2-VHH fused to the VL of FAP binder 2 (E), IFNyR2-VHH fused to the VH of FAP binder 1 (F), IFNyRl-VHH fused to the VH of FAP binder 2 (G), IFNyR2-VHH fused to the VL of FAP binder 1 (H) and IFNyRl-VHH fused to the VL of FAP binder 2 (I). All molecules have the same Fc characteristics as described
  • FIG. 11B-I The eight split IFNy mimetics (Fig. 11B-I) were paired in biparatopic assembly combinations and tested for IFNyR agonism characterized by MHC-I and PD-L1 upregulation.
  • FAP-negative A549 cells were tested for untargeted, non-specific MHC-I (A, B) and PD-L1 upregulation (C, D) by split IFNy mimetics.
  • A549 FAP -positive cells were co-cultured with differentially labeled A549 FAP-negative cells to assess FAP- dependent activity of IFNy mimetics in cis and trans: MHC-I upregulation in cis (E and F), PD-L1 upregulation in cis (G and H), MHC-I upregulation in trans (I and J) and PD-L1 upregulation in trans (K and L).
  • MHC-I upregulation in cis E and F
  • PD-L1 upregulation in cis G and H
  • MHC-I upregulation in trans I and J
  • PD-L1 upregulation in trans K and L
  • Recombinant IFNy and the IFNy agonistic heavy chain antibody Pl AHI 884 (as depicted in Fig. 5) were used as a reference.
  • Median fluorescent intensity (MFI) of MHC-I and PD-L1 expression levels were analyzed in FlowJo; mean values from technical duplicates are shown with error bars representing
  • FIG. 13 Additional formats of split dual-targeted IFNyR agonists.
  • VHH domains with IFNyRl and IFNyR2 specificity were distanced from the anti -FAP binders via fusion to the N-terminus of the opposite Fc chain, resulting in four configurations: IFNyRl and FAP binder 1 (A), IFNyR2 and FAP binder 2 (B), IFNyR2 and FAP binder 1 (C) and IFNyRl and FAP binder 2 (D).
  • the VHH domains with IFNyRl and IFNyR2 specificity were distanced from the anti-FAP binders via fusion to the C-terminus of the same Fc chain, resulting in four configurations: IFNyRl and FAP binder 1 (E), IFNyR2 and FAP binder 2 (F), IFNyR2 and FAP binder 1 (G) and IFNyRl and FAP binder 2 (H).
  • the anti-FAP binders in Fig. 13E-H comprise CrossFab VH/VL engineering (binder 1) and CH1-CL charges (binder 2) to subsequently allow for additional pairings. All molecules have the same Fc characteristics as described in Figure 5.
  • Figure 14 Assessment of IFNyR activity mediated by additional FAP-dependent split IFNyR agonists.
  • Four combinations of molecules depicted in Fig. 13 and reference molecules were tested for IFNyR agonism characterized by MHC-I and PD-L1 upregulation.
  • A549 cells lacking FAP expression were tested for FAP-independent MHC-I upregulation (A) and PD-L1 upregulation (B) of split IFNy mimetics.
  • A549 FAP-positive cells were co-cultured with differentially labeled A549 FAP-negative cells to assess FAP-dependent activity of IFNy mimetics by MHC-I upregulation in cis (C), PD-L1 upregulation in cis (D), MHC-I upregulation in trans (E) and PD-L1 upregulation in trans (F).
  • Recombinant IFNy and an IFNy agonistic heavy chain antibody (P1AH1884) were used as reference molecules.
  • Median fluorescent intensity (MFI) of MHC-I and PD-L1 expression levels were analyzed in FlowJo; mean values from technical duplicates are shown with error bars representing standard deviation.
  • FIG. 15 Concept of split FAP-targeted IFNy agonists with different linker lengths.
  • a single VHH domain with specificity for either IFNyRl or IFNyR2 was engineered to be linked to the hinge region at the N-terminus (top row) or to the C-terminus (bottom row) of the Fc region within an antibody construct containing a FAP-binding Fab domain.
  • the constructs utilized one of two different FAP- binding Fab domains that do not compete for binding.
  • Flexible linkers consisting of 5, 15, or 25 amino acids, denoted by the number following a semicolon after the molecule ID, were used to connect the Fc region to the VHH domain.
  • Figure 16 Assessment of IFNyR activity mediated by additional FAP-dependent split IFNyR agonists. Twelve combinations of molecules depicted in Fig. 15 and reference molecules were tested for IFNyR agonism characterized by MHC-I upregulation. Test compounds were incubated with A549 FAP- negative cells alone (untargeted condition; A and D) or in combination with A549 FAP-positive cells, wherein cis signaling (FAP binding on A549 FAP-positive cells and activity on A549 FAP-positive cells; B and E) and trans signaling (FAP binding on A549 FAP-positive cells and activity on A549 FAP- negative cells, C and F) was assessed.
  • A549 FAP- negative cells alone (untargeted condition; A and D) or in combination with A549 FAP-positive cells, wherein cis signaling (FAP binding on A549 FAP-positive cells and activity on A549 FAP-positive cells; B and E) and trans signaling (FAP binding on A5
  • MFI Median fluorescent intensity
  • FIG. 17 Concept of split EGFR-targeted IFNy agonists.
  • VHH domains with IFNyRl and IFNyR2 specificity were fused to the N-terminus of two different anti-EGFR binding domains, via VH or VL fusion points, resulting in eight individual format configurations (P1AK5514 - P1AK5521) that can be paired to produce the eight functional assemblies depicted: P1AK5520 + P1AK5519 (A), P1AK5516 + P1AK5515 (B), P1AK5521 + P1AK5518 (C), P1AK5517 + P1AK5514 (D), P1AK5520 + P1AK5515 (E), P1AK5516 + P1AK5519 (F), P1AK5521 + P1AK5514 (G) and P1AK5517 + P1AK5518 (H).
  • Figure 18 Functional activity of EGFR-targeted IFNy agonists. Test compounds were incubated with target cells together with PBMCs and CXCL10 production was assessed after 24h stimulation. Recombinant IFNy was used as reference. Shown are mean +/- SD of technical duplicates for each tested molecule concentration. Figure 19. Concept of split dual-targeted IL-2 agonists. PD-1 -dependent biparatopic assembly of split IL-2 mimetics (A).
  • VHH domains with IL-2RP and IL-2Ry specificity were fused to the N-terminus of two different anti-PD-1 binding domains, via VH or VL fusion points: IL-2RP-VHH fused to the VH of PD-1 binder 1 (B), IL-2Ry-VHH fused to the VL of PD-1 binder 2 (C), IL-2RP-VHH fused to the VL of PD-1 binder 1 (D) and IL-2Ry-VHH fused to the VH of PD-1 binder 2 (E).
  • An IL-2 agonistic heavy chain antibody (F) and PD-l-IL2v (G) served as reference molecules. All molecules have the same Fc characteristics as described in Figure 5.
  • FIG. 20 Phosphorylation of STAT5 on CD4 T cells after 15 min and 60 min incubation with IL-2R agonists.
  • Activated T cells expressing PD-1 (PD-1 + subset) and activated T cells preblocked with anti- PD-1 antibodies (PD-1- subset) were differentially labeled and treated with two combinations of split dual-targeted IL-2 mimetics: P1AH6850 + P1AH6813 (A-D), P1AH6814 + P1AI1593 (E-H).
  • the IL-2 mimetic heavy chain antibody (Pl AH 1177) and PDl-IL2v (P1AE4422) were used as reference molecules.
  • MFI and frequency of STAT5-P + cells were measured by FACS and responses were shown for PD-1 + subset (solid line) and PD-T subset (dashed line).
  • Figure 21 Flow cytometry gating strategy and baseline receptor expression. Isolated PBMCs from two healthy donors were stained with viability dye and fluorescent dye -conjugated antibodies against cell surface antigens. Gating strategy to identify five distinct cell types: NK cells and NKT cells (A); CD4 T cells and CD8 T cells (B); and y5 T cells (C). Representative plots showing PD-1 and IL-2RP coexpression on CD8 T cells (D), y5 T cells (E) and NK cells (F).
  • FIG. 22 CD25 expression on PBMC subsets induced by PD-1 targeted IL-2R agonist.
  • Frequency of CD25 expressing CD8 T cells (A), y5 T cells (B) and NK cells (C) were determined by flow cytometry after five days of test compounds co-incubation with freshly isolated CTV -labelled PBMCs.
  • PDl-IL2v (P1AE4422) and Fc-VHH (P1AH1177) were used as reference.
  • a non-binding DP47 antibody (P1AD3966) was used as negative control. Shown are mean values +/- SEM of two PBMC donors combined and technical duplicates for each tested molecule concentration.
  • FIG. 23 Proliferation of PBMC subsets induced by PD-1 targeted IL-2R agonist. Frequency of proliferating CD8 T cells (A), y5 T cells (B) and NK cells (C) were determined by flow cytometry after five days of test compounds co-incubation with freshly isolated CTV -labelled PBMCs. Proliferating cells were identified based on cell division visualized with CTV staining. PDl-IL2v (P1AE4422) and Fc-VHH (P1AH1177) were used as reference. A non-binding DP47 antibody (P1AD3966) was used as negative control. Shown are mean values +/- SEM of two PBMC donors combined and technical duplicates for each tested molecule concentration.
  • Figure 24 Presents the results of an efficacy experiment evaluating PD-1 targeted IL-2R agonist (P1AI1593 and P1AH6814), PDl-IL2v immunoconjugate (P1AE4422) and Pembrolizumab in combination with FOLR1-TCB Mab.
  • the BC004 human breast carcinoma PDX cells were injected intra mammary fat pad in humanized BRGS47 mice to study tumor growth inhibition in a breast orthotopic xenograft model.
  • the amount of antibodies injected per mouse in mg/kg is the following: P1AI1593, P1AH6814 and Pembrolizumab 1 mg/kg, PDl-IL2v 0.1 mg/kg and FOLR1-TCB 0.3 mg/kg.
  • the antibodies were injected i.v. once weekly for 4 weeks.
  • the combination FOLR1-TCB 0.3 mg/kg + PD1- IL2targeted IL-2R agonists (P1AI1593 day 1 + PlAH6814day 2) mediated superior efficacy in terms of tumor growth inhibition compared to FOLR1-TCB single agent treatment and the other combination groups: FOLR1-TCB 0.3 mg/kg + PDl-IL2v and FOLR1-TCB 0.3 mg/kg + Pembrolizumab.
  • FIG. 25 Concept of split PD-1 -targeted IL-2R agonists with alternative anti-PD-1 Fab binders.
  • VHH domains with IL-2RP and IL-2Ry specificity were fused to the N-terminus of two different anti-PD-1 binding domains, via VH or VL fusion points, resulting in eight individual format configurations (P1AK2599; P1AK2798; P1AK2799; P1AK2802; P1AK2803; P1AK2806; P1AK2809; P1AK2810) that can be paired to produce the eight functional assemblies depicted: P1AK2802 + P1AK2599 (A), P1AK2809 + P1AK2799 (B), P1AK2803 + P1AK2806 (C), P1AK2810 + P1AK2798 (D), P1AK2809 + P1AK2599 (E), P1AK2802 P1AK2 799 (F), P1AK2810 + P1AK2806 (G) and P1
  • FIG. 26 Functional activity of PD-1 -targeted IL-2R agonists with alternative anti-PD-1 Fab binders.
  • Test compounds were incubated with HEK Blue IL-2 wt cells (A) or HEK Blue IL-2 human PD-1 cells (B) for 21h at 37°C and 5% CO 2 .
  • PDl-IL2v P1AE4422
  • Fc-VHH P1AH1177
  • a non-binding DP47 antibody (P1AD3966) was used as negative control.
  • IL-2R signaling was measured via absorbance at 650 nm using QUANTI-Blue reagent. Shown are mean absorbance values +/- SEM of technical duplicates for each tested molecule concentration.
  • FIG. 27 Concept of split LAG-3 -targeted IL-2R agonists.
  • VHH domains with IL-2RP and IL-2Ry specificity were fused to the N-terminus of two different anti-LAG-3 binding domains, via VH or VL fusion points, resulting in eight individual format configurations (P1AJ5654 - P1AJ5661) that can be paired to produce the eight functional assemblies depicted: P1AJ5654 + P1AJ5661 (A), P1AJ5658 + P1AJ5657 (B), P1AJ5655 + P1AJ5660 (C), P1AJ5659 + P1AJ5656 (D), P1AJ5658 + P1AJ5661 (E), P1AJ5654 + P1AJ5657 (F), P1AJ5659 (G) and P1AJ5655 + P1AJ5656 (H).
  • FIG. 28 Functional activity of LAG-3 -targeted IL-2R agonists.
  • PDl-IL2v P1AE4422
  • Fc-VHH P1AH1177
  • Frequency (A) and MFI (B) of STAT-5 phosphorylation were determined by flow cytometry after 60 min of test compounds co-incubation with CD4 T cells preactivated for three days with plate-bound anti-CD3 and soluble anti-CD28 antibodies.
  • FIG. 29 Concept of split FAP-targeted IL-2R agonists.
  • VHH domains with IL-2RP and IL-2Ry specificity were fused to the N-terminus of two different anti-FAP binding domains, via VH or VL fusion points, resulting in eight individual format configurations (P1AJ5092 - P1AJ5099) that can be paired to produce the eight functional assemblies: P1AJ5096 + P1AJ5099 (A), P1AJ5092 + P1AJ5095 (B), P1AJ5097 + P1AJ5098 (C), P1AJ5093 + P1AJ5094 (D), P1AJ5096 + P1AJ5095 (E), P1AJ5092 + P1AJ5099 (F), P1AJ5097 + P1AJ5094 (G) and P1AJ5093 + P1AJ5098 (H).
  • Figure 30 Functional activity of FAP-targeted IL-2R agonists.
  • Test compounds were incubated with A549 FAP-negative target cells (A) or A549 FAP-positive target cells (B) and IL-2RPy Bioassay cells for 20h at 37° C and 5% CO 2 .
  • PDl-IL2v P1AE4422
  • FAP-IL2v P1AA5355
  • Fc-VHH Pl AHI 177
  • a non-binding DP47 antibody P1AD3966
  • IL-2R signaling was measured via luminescence using Bio-Gio NL reagent and normalized to background signal. Shown are mean fold change RLU values +/- SEM of technical duplicates for each tested molecule concentration.
  • FIG 31 Concept of split EGFR-targeted IL-2R agonists.
  • VHH domains with IL-2RP and IL-2Ry specificity were fused to the N-terminus of two different anti-EGFR binding domains, via VH or VL fusion points, resulting in eight individual format configurations (P1AJ4165 - P1AJ4172) that can be paired to produce the eight functional assemblies: P1AJ4169 + P1AJ4172 (A), P1AJ4165 + P1AJ4168 (B), P1AJ4170 + P1AJ4171 (C), P1AJ4166 + P1AJ4167 (D), P1AJ4169 + P1AJ4168 (E), P1AJ4165 + P1AJ4172 (F), P1AJ4170 + P1AJ4167 (G) and P1AJ4166 + P1AJ4171 (H).
  • FIG 32 Functional activity of EGFR-targeted IL-2R agonists.
  • EGFR expression on target cells by flow cytometry A.
  • Test compounds were incubated with A431 target cells (B), OE19 target cells (C) or LoVo target cells (D)) and IL-2RPy Bioassay cells for 20h at 37°C and 5% CO 2 .
  • PDl-IL2v P1AE4422
  • Fc-VHH P1AH1177
  • a non-binding DP47 antibody P1AD3966
  • IL-2R signaling was measured via luminescence using Bio- Glo NL reagent and normalized to background signal. Shown are mean fold change RLU values +/- SEM of technical duplicates for each tested molecule concentration.
  • FIG. 33 Concept of split HER2 -targeted and HER3-targeted IL-2R agonists.
  • VHH domains with IL- 2RP and IL-2Ry specificity were fused to the N-terminus of two different anti-HER2 binding domains and two different anti-HER3 binding domains.
  • VH or VL fusion points were selected for each Fab based on structural data.
  • Four format configurations (P1AJ4173 - P1AJ4176) and two paired assemblies were designed for HER2: P1AJ4174 + P1AJ4175 (A) and P1AJ4173 + P1AJ4176 (B).
  • Four format configurations (P1AJ4177 - P1AJ4180) and two paired assemblies were selected for HER3: P1AJ4178
  • FIG. 34 Functional activity of HER2 -targeted and EIER3 -targeted IL-2R agonists.
  • Test compounds were incubated with MDA-MB-231 target cells (A) or OE19 target cells (B) and IL-2RPy Bioassay cells for 20h at 37°C and 5% CO2.
  • PDl-IL2v P1AE4422
  • Fc-VHH P1AH1177
  • a non-binding DP47 antibody P1AD3966
  • IL-2R signaling was measured via luminescence using Bio-Gio NL reagent and normalized to background signal. Shown are mean fold change RLU values +/- SEM of technical duplicates for each tested molecule concentration.
  • FIG. 35 Concept of split PD-l-targeted IL-2R agonists with alternative anti-PD-1 single domain binders.
  • VHH domains with IL-2RP and IL-2Ry specificity were fused to the N-terminus of four different anti-PD-1 single domain binders (P1AI0063; P1AK3046; P1AK3048; P1AK3051 - P1AK3054) that can be paired to produce the six functional assemblies depicted: P1AK3052 + P1AK3048 (A), P1AI0063 + P1AK3051 (B), P1AK6054 + P1AK3048 (C), P1AI0063 + P1AK3053 (D), P1AK3054 + P1AK3046 (E) and P1AK3052 + P1AK3046 (F).
  • Figure 36 Functional activity of PD-l-targeted IL-2R agonists with alternative anti-PD-1 single domain binders.
  • Test compounds were incubated with HEK Blue IL-2 wt cells (A) or HEK Blue IL-2 human PD-1 cells (B) for 20h at 37°C and 5% CO 2 .
  • PDl-IL2v P1AE4422
  • Fc-VHH P1AH1177
  • a non-binding DP47 antibody (P1AD3966) was used as negative control.
  • IL-2R signaling was measured via absorbance at 650 nm using QUANTI-Blue reagent. Shown are mean absorbance values +/- SEM of technical duplicates for each tested molecule concentration.
  • Figure 37 Concept of split PD- 1 -targeted IL-2R agonists with anti-IL2R Fab binders or anti-IL2R scFv binders.
  • Fab fragments with IL-2RP and IL-2Ry specificity were fused to the N-termini of two anti-PDl binding domains with different paratopes, via VH or VL fusion points.
  • P1AL9238 (A) and P1AL9239 (B) comprise the PD1 binder 2 (FV003451 paratope; 1040 binder) and two different IL2Ry binders, FV018863 and FV018864, respectively
  • P1AL9240 (C) and P1AL9241 (D) comprise the PD1 binder 1 (FV000363 paratope; 0376 binder) and two different IL2RP binders, FV002203 and FV018866, respectively.
  • P1AL9310 (E) comprises the PD1 binder 2 (FV003451 paratope) and the IL2Ry binder FV018865
  • P1AL9311 (F) comprises the PD1 binder 1 (FV000363 paratope) and the IL2RP binder FV018866.
  • P1AL9310 + P1AL9311 can be paired to produce a functional assembly: P1AL9310 + P1AL9311.
  • IL-2 agonistic heavy chain antibody Fc-VHH ; P1AH1177
  • P1AD3966 A non-binding DP47 antibody
  • IL-2R signaling was measured via absorbance at 650 nm using QU ANTI -Blue reagent. Shown are fold change in IL-2R signaling over background +/- SEM of technical duplicates for each tested molecule concentration.
  • FIG 39 Schematic representations of antigens and workflow for the isolation of IL-7Ra-specific single domain antibodies.
  • the extracellular domain (ECD) of human IL-7Ra fused to biotinylated Fc knob-into-hole (P1AI1009 represented in A) was generated for alpaca immunization, phage display and screening.
  • Soluble human Fc knob-into-hole (P1AD4290 represented in B) was generated for phage display.
  • Figure 40 Specificity screening of single domain antibodies by ELISA. Soluble VHH domains enriched for IL-7Ra specificity were randomly selected and screened for binding to immobilized human IL-7Ra- Fc and immobilized human Fc. Following three rounds of phage display, VHH from library 1 (A) and library 2 (B) were tested for antigen specificity. Following four rounds, enriched VHH from library 1
  • FIG 41 Format conversion of single domain antibody fragments into bispecific heavy chain antibodies.
  • Round 3 and round 4 IL-7Ra-specific VHH from libraries 1 and 2 were fused to Fc-Knob and cloned into a mammalian cell expression vector using Gibson cloning method (A).
  • Previously identified VHH with IL-2Ry specificity were fused to Fc-hole using the same method (B).
  • Bispecific heavy chain antibodies comprising IL-7Ra-IL-2Ry VHH domain pairs were generated via knob-into- hole assembly of Fc chains and flexible 5(G4S) linkers to fuse VHH moieties to Fc chains. Effector functions of the Fc were silenced via incorporation of P329G LALA mutations in the CH2 domain (C).
  • FIG 42 Functional characterization of dose-dependent response for IL-7R agonists.
  • Bispecific heavy chain antibodies comprising IL-7Ra specificity and IL-2Ry were screened for dose-dependent IL-7R agonism in HEK Blue IL-7 cells.
  • Recombinant IL-7 was used as a reference and an IgG with irrelevant specificity was used as a negative control (P1AA5879).
  • Each molecule was incubated with reporter cells at the following concentrations: 20 nM, 2 nM, 0.2 nM, 0.02 nM shown from left to right.
  • IL-7R activity was quantified by absorbance at 650 nm and fold response/background is shown for each treatment.
  • Graphs were separated according to the four different IL-2Ry-specific VHH clones: IL2Ry_2 (A),
  • IL2Ry_3 B
  • IL2Ry_4 C
  • IL2Ry_5 D
  • FIG 43 Concept of split PD-l-targeted IL-7R agonists.
  • PD-1 -dependent biparatopic assembly of split IL-7 mimetics A.
  • VHH domains with IL-7Ra and IL-2Ry specificity were fused to the N-terminus of two different anti -PD-1 binding domains, via VH or VL fusion points: IL-7Ra-VHH were fused to the VL of PD-1 binder 1 (B), and IL-2Ry-VHH were fused to the VH of PD-1 binder 2 (C).
  • IL-7Ra molecules contained AAA mutations (I253A; H310A; H435 A, numbering according to Kabat EU index) in the CH2 and CH3 domains of the Fc hole to facilitate protein A purification. Effector functions of the Fc were silenced in all molecules via incorporation of P329G LALA mutations in the CH2 domain.
  • FIG 44 Functional activity of PD-l-targeted IL-7R agonists.
  • PDl-IL2v P1AE4422
  • Fc-VHH Pl AHI 177
  • a non-binding DP47 antibody P1AD3966
  • Frequency of STAT-5 phosphorylation was determined by flow cytometry after 60 min of test compounds co-incubation with CD4 T cells pre-activated for three days with plate-bound anti-CD3 and soluble anti-CD28 antibodies for blood donor 1 (A and B) and 2 (C and D).
  • FIG. 45 Concept of split PD-l-targeted IL-2R agonists with monoparatopic assembly onto PD-1.
  • VHH domains with IL-2RP and IL-2Ry specificity were fused to the N-terminus of two different anti- PD-1 binding domains, via VH or VL fusion points, resulting in eight individual format configurations of IL-2R agonist pairs containing the same anti -PD-1 Fab binders (monoparatopic assembly) that can be paired to produce the eight monoparatopic assemblies: P1AK2802 + P1AK2810 (A), P1AK2809 + P1AK2803 (B), P1AK2802 + P1AK2803 (C), P1AK2809 + P1AK2810 (D), P1AK2798 + P1AK2599 (E), P1AK2806 + P1AK2799 (F), P1AK2798 + P1AK2799 (G) and P1AK2806 + P1AK2599 (G) .
  • FIG. 46 Functional activity of monoparatopic vs biparatopic PD-l-targeted IL-2R agonists.
  • Test compounds were incubated with HEK Blue IL-2wt (A) or HEK Blue IL-2 human PD-1 cells (B) for 21h at 37°C and 5% CO2.
  • Two biparatopic PD-l-targeted IL-2R agonists P1AH6814 + P1AI1593 and P1AH6814 + P1AK2802
  • Fc-VHH;P1AH1177 Two biparatopic PD-l-targeted IL-2R agonists (P1AH6814 + P1AI1593 and P1AH6814 + P1AK2802) and the IL-2 mimetic heavy chain antibody (Fc-VHH;P1AH1177) were used as reference.
  • a non-binding DP47 antibody P1AD3966
  • IL-2R signaling was measured via absorbance at 650 nm using QUANTI-Blue reagent.
  • first”, “second”, “third” or “fourth” with respect to binding molecules, epitopes, polypeptides etc. are used for convenience of distinguishing when there is more than one of each type of moiety. Use of these terms is not intended to confer a specific order or orientation of the moiety unless explicitly so stated.
  • antigen binding molecule refers to a polypeptide molecule (composed of one or more polypeptide chains) that is capable of binding to an antigen.
  • An antigen binding molecule may be derived from an antibody, and typically comprises an antigen binding domain.
  • antibody herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies), heavy-chain antibodies, antibody fragments and antigen binding molecules so long as they exhibit the desired antigen-binding activity.
  • heavy chain antibody and “heavy chain-only antibody” and “HCAb” as used herein refer to antibodies devoid of light chains.
  • full-length antibody “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure.
  • an ’’antibody fragment refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
  • antibody fragments include but are not limited to Fv, Fab, cross-Fab, Fab', Fab’-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g. scFv and scFab), single-domain antibodies, and multispecific antibodies formed from antibody fragments.
  • single -domain antibody refers to an antibody fragment consisting of a single monomeric antibody variable domain such as VHHs, nanobodies, VNARs derived from sharks, autonomous VH domains or autonomous VL domains.
  • Single-domain antibodies provide an antigen-binding site which specifically binds to an epitope, i.e. the antigen binding-site is formed solely by the single-domain antibody.
  • VHH or “VHH domain” or “nanobody” refers to a single-domain antibody derived from the variable domains of heavy chain antibodies from camelids, e.g. camel, dromedary, llama, alpaca, etc. (See Nguyen V.K. et al., 2000, The EMBO Journal, 19, 921-930; Muyldermans S., 2001, J Biotechnol., 74, 277-302 and for review Vanlandschoot P. et al., 2011, Antiviral Research 92, 389-407).
  • the antigenbinding site of VHHs is devoid of light chain variable domain.
  • a VHH domain may be humanized.
  • an “antigen binding domain” as used herein refers to a domain that specifically binds to a target antigen.
  • the term in particular refers to an antigen binding domain of an antibody, i.e. the part that comprises the area which binds to and is complementary to part or all of an antigen.
  • an antigen binding domain herein is an antigen binding domain of an antibody.
  • Such an antigen binding domain may be provided by an antibody fragment, for example by a Fab molecule, a singlechain antibody molecule or single-domain antibodies, such as a VHH domain.
  • variable region refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to the antigen.
  • the term includes VHH domains.
  • the variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and complementarity determining regions (CDRs). See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman & Co., page 91 (2007).
  • a single VH or VL domain may be sufficient to confer antigen-binding specificity.
  • antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively.
  • VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively.
  • Portolano et al. J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).
  • Kabat numbering refers to the numbering system set forth by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).
  • amino acid positions of all constant regions and domains of the heavy and light chain are numbered according to the Kabat numbering system described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD
  • Kabat numbering system see pages 647-660 of Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)
  • CL light chain constant domain
  • Kabat EU index numbering system see pages 661-723
  • CHI heavy chain constant domains
  • binding site or “antigen-binding site” as used herein refers to the site, i.e. one or more amino acid residues, of a binding molecule which provides interaction with the antigen.
  • the antigen binding site of an antigen binding domain comprises amino acid residues from the complementarity determining regions (CD Rs).
  • An antigen-binding site may be provided by, for example, one or more variable domains (also called variable regions). In single domain antibodies the antigen-binding site is provided by a single variable domain. Whereas, in a Fab fragment the antigenbinding site is provided by the VH and VL domains.
  • variable region refers to each of the regions of an antigen binding domain which are hypervariable in sequence and which determine antigen binding specificity, for example “complementarity determining regions” (“CDRs”).
  • CDRs complementarity determining regions
  • variable domains comprise three CDRs.
  • antibodies comprising a VH and a VL comprise six CDRs; three in the VH (HCDR1, HCDR2, HCDR3), and three in the VL (LCDR1, LCDR2, LCDR3).
  • Exemplary CDRs herein include:
  • CDRs are determined according to Kabat et al., supra.
  • CDR designations can also be determined according to Chothia, supra, McCallum, supra, or any other scientifically accepted nomenclature system.
  • FR Framework or “FR” refers to variable domain residues other than complementarity determining regions (CDRs).
  • the FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following order in VH and VHH domains (or VL): FR1-HCDR1 (LCDR1)-FR2-HCDR2 (LCDR2)-FR3-HCDR3 (LCDR3)-FR4. Unless otherwise indicated, CDR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.
  • immunoglobulin molecule refers to a protein having the structure of a naturally occurring antibody.
  • immunoglobulins of the IgG class are heterotetrameric glycoproteins of about 150,000 daltons, composed of two light chains and two heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy domain or a heavy chain variable region, followed by three constant domains (CHI, CH2, and CH3), also called a heavy chain constant region.
  • VH variable domain
  • CHI variable heavy domain
  • CH2 constant domains
  • each light chain has a variable domain (VL), also called a variable light domain or a light chain variable region, followed by a constant light (CL) domain, also called a light chain constant region.
  • VL variable domain
  • CL constant light
  • the heavy chain of an immunoglobulin may be assigned to one of five types, called a (IgA), 5 (IgD), a (IgE), y (IgG), or p (IgM), some of which may be further divided into subtypes, e.g. yl (IgGi), y2 (IgG2), y3 (IgG,)- y4 (IgG 4 ), al (IgAl) and a2 (IgA2).
  • the light chain of an immunoglobulin may be assigned to one of two types, called kappa (K) and lambda (X), based on the amino acid sequence of its constant domain.
  • K kappa
  • X lambda
  • An immunoglobulin essentially consists of two Fab molecules and an Fc domain, linked via the immunoglobulin hinge region.
  • the “class” of an antibody or immunoglobulin refers to the type of constant domain or constant region possessed by its heavy chain.
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 5, a, y, and p, respectively.
  • a “Fab molecule” or “Fab” or “Fab fragment” or “Fab domain” refers to a protein consisting of the VH and CHI domain of the heavy chain (the “Fab heavy chain”) and the VL and CL domain of the light chain (the “Fab light chain”) of an immunoglobulin.
  • cross-Fab molecule or “cross-Fab” or “crossover Fab molecule” refers to a Fab molecule, wherein either the variable regions or the constant regions of the heavy and light chain are exchanged.
  • Cross- Fab engineering enables two different chain compositions of a cross-Fab molecules.
  • the variable regions of the Fab heavy and light chain are exchanged, i.e. the cross-Fab molecule comprises a peptide chain composed of the light chain variable region (VL) and the heavy chain constant region (CHI), wherein the CHI may be fused to an Fc domain, and a peptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL).
  • the cross-Fab molecule comprises a peptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL), wherein the CL may be fused to an Fc domain, and a peptide chain composed of the light chain variable region (VL) and the heavy chain constant region (CHI).
  • inventional Fab molecule refers to a Fab molecule composed of a Fab heavy chain comprising a VH and CHI domain and a Fab light chain comprising a VL and CL domain.
  • a “single-chain variable fragment” or “scFv” molecule is a fusion protein of the variable domains of the heavy (VH) and light chain (VL) of an antibody, connected by a linker.
  • the linker is a short polypeptide of 10 to 25 amino acids and is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. This protein retains the specificity of the original antibody, despite removal of the constant regions and the introduction of the linker.
  • multispecific means that a binding molecule (e.g. an antibody) is able to specifically bind to at least two distinct antigens.
  • a multispecific binding molecule e.g. antibody
  • a bispecific binding molecule comprises two antigen binding sites, each of which is specific for a different antigens.
  • the multispecific (e.g. bispecific) binding molecule is capable of simultaneously binding two antigens, particularly two antigens expressed on the same cell, on neighbouring cells, or cells in the same tissue.
  • the term “antigen” refers to a molecule, such as a protein, to which an antigen binding molecule binds.
  • useful antigens can be found, for example, on the surfaces of tumor cells, on the surfaces of tumor stroma cells, on the surfaces of virus-infected cells, on the surfaces of other diseased cells, on the surface of immune cells, free in blood serum, and/or in the extracellular matrix (ECM).
  • ECM extracellular matrix
  • the antigen is a human protein.
  • target antigen refers to an antigen presented on the surface of a target cell, for example an antigen on a cell in a tumor, such as a cancer cell or a cell of the tumor stroma, or an antigen on a T cell.
  • tumor-associated antigen refers to any antigen presented on the surface of a cell in a tumor such as a cancer cell or a cell of the tumor stroma.
  • tumor-associated antigens are CEA, FAP, Her2, Her3 or EGFR.
  • FAP Fibroblast activation protein
  • Prolyl endopeptidase FAP or Seprase EC 3.4.21
  • FAP is expressed on Cancer-associated fibroblasts (CAFs) in the tumor stroma.
  • CAFs Cancer-associated fibroblasts
  • the term also encompasses naturally occurring variants of FAP, e.g., splice variants or allelic variants.
  • the antigen binding molecule of the invention is capable of specific binding to human, mouse and/or cynomolgus FAP.
  • the amino acid sequence of human FAP is shown in UniProt (www.uniprot.org) accession no. Q 12884 (version 149), or NCBI (www.ncbi.nlm.nih.gov/) RefSeq NP 004451.2.
  • the extracellular domain (ECD) of human FAP extends from amino acid position 26 to 760.
  • the amino acid sequence of mouse FAP is shown in UniProt accession no.
  • the extracellular domain (ECD) of mouse FAP extends from amino acid position 26 to 761.
  • an anti-FAP binding molecule binds to the extracellular domain of FAP.
  • Exemplary anti-FAP binding molecules are described in International Patent Application No. WO 2012/020006 A2 and WO 2020/070041 Al .
  • Carcinoembroynic antigen also known as Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), refers to any native CEA from any vertebrate source, including mammals such as primates (e.g. humans) non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice and rats), unless otherwise indicated.
  • the amino acid sequence of human CEA is shown in UniProt accession no. P06731 (version 151).
  • CEA has long been identified as a tumor-associated antigen (Gold and Freedman, J Exp Med., 121:439-462, 1965; Berinstein N.
  • CEA has now been identified in several normal adult tissues. These tissues are primarily epithelial in origin, including cells of the gastrointestinal, respiratory, and urogential tracts, and cells of colon, cervix, sweat glands, and prostate (Nap et al., Tumour Biol., 9(2-3): 145-53, 1988; Nap et al., Cancer Res., 52(8):2329-23339, 1992). Tumors of epithelial origin, as well as their metastases, contain CEA as a tumor associated antigen.
  • CEA While the presence of CEA itself does not indicate transformation to a cancerous cell, the distribution of CEA is indicative.
  • CEA is generally expressed on the apical surface of the cell (Hammarstrom S., Semin Cancer Biol. 9(2):67-81 (1999)), making it inaccessible to antibody in the blood stream.
  • CEA tends to be expressed over the entire surface of cancerous cells (Hammarstrom S., Semin Cancer Biol. 9(2): 67-81 (1999)). This change of expression pattern makes CEA accessible to antibody binding in cancerous cells.
  • CEA expression increases in cancerous cells.
  • CEA expression promotes increased intercellular adhesions, which may lead to metastasis (Marshall J., Semin Oncol., 30(a Suppl. 8):30-6, 2003).
  • CRC colorectal carcinoma
  • NSCLC non-small cell lung cancer
  • HER3 non-small cell lung cancer
  • HER2 refers to any native HER2 from any vertebrate source, including mammals such as primates (e.g. humans), non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice and rats), unless otherwise indicated.
  • the term encompasses “full-length,” unprocessed HER2 as well as any form of HER2 that results from processing in the cell.
  • the term also encompasses naturally occurring variants of HER2, e.g., splice variants or allelic variants.
  • HER2 is human HER2.
  • the amino acid sequence of human HER2 is shown in UniProt (www.uniprot.org) entry no. Q9UK79 (version 95).
  • Epidermal Growth Factor Receptor also named Proto-oncogene c-ErbB-1 or Receptor tyrosine-protein kinase erbB-1, refers to any native EGFR from any vertebrate source, including mammals such as primates (e.g. humans) non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice and rats), unless otherwise indicated.
  • the amino acid sequence of human EGFR is shown in UniProt accession no. P00533.
  • a “T cell antigen” as used herein refers to any antigen present on the surface of a T lymphocyte.
  • PD-1 also known as CD279, PD1 or programmed cell death protein 1
  • CD279 PD1 or programmed cell death protein 1
  • PD-1 refers to any native PD-1 from any vertebrate source, including mammals such as primates (e.g. humans) non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice and rats), in particular to the human protein PD-1 with the amino acid sequence as shown in UniProt (www.uniprot.org) accession no. Q15116.
  • IFNy interferon gamma
  • IFN-y refers to any native IFNy from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated.
  • the term encompasses unprocessed IFNy as well as any form of IFNy that results from processing in the cell.
  • the term also encompasses naturally occurring variants of IFNy, e.g. splice variants or allelic variants.
  • interleukin-2 refers to any native IL-2 from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated.
  • the term encompasses unprocessed IL-2 as well as any form of IL-2 that results from processing in the cell.
  • the term also encompasses naturally occurring variants of IL-2, e.g. splice variants or allelic variants.
  • Cytokine receptors are cell-surface glycoproteins that specifically bind cytokines and transduce their signals. Generally, cytokine receptors function as oligomeric complexes consisting of typically two to four receptor chains, also termed subunits, that may be the same or different. Thus, the term “cytokine receptor complex” refers to cytokine receptors composed of at least two subunits.
  • the IFNy receptor complex comprises the IFNyRl subunit and the IFNyR2 subunit.
  • the term encompasses “full-length”, unprocessed IFNyRl as well as any form of IFNyRl that results from processing in the cell.
  • the term also encompasses naturally occurring variants of IFNyRl, e.g. splice variants or allelic variants.
  • IFNyRl is human IFNyRl.
  • Interferon gamma receptor 2 or “IFNyR2”, also referred to as Interferon gamma receptor - chain (IFNyR ), refers to any native IFNyR2 from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated.
  • the term encompasses “full-length”, unprocessed IFNyR2 as well as any form of IFNyR2 that results from processing in the cell.
  • the term also encompasses naturally occurring variants of IFNyR2, e.g. splice variants or allelic variants.
  • IFNyR2 is human IFNyR2.
  • IL-2Ra or “a-subunit of the IL-2 receptor” also known as CD25, as used herein, refers to any native IL-2Ra from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated.
  • the term encompasses “full-length”, unprocessed IL-2Ra as well as any form of IL-2Ra that results from processing in the cell.
  • the term also encompasses naturally occurring variants of IL-2Ra, e.g. splice variants or allelic variants.
  • IL-2Ra is human IL-2Ra.
  • IL-2RP or “P-subunit of the IL-2 receptor”, also known as CD 122 or p70, refers to any native IL-2RP from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated.
  • the term encompasses “full-length”, unprocessed IL-2RP as well as any form of IL-2RP that results from processing in the cell.
  • the term also encompasses naturally occurring variants of IL-2RP, e.g. splice variants or allelic variants.
  • IL-2RP is human IL-2Rp.
  • IL-2Ry or “y-subunit of the IL-2 receptor”, also known as common cytokine receptor y- subunit, common y-chain, yc, or CD 132, refers to any native IL-2Ry from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated.
  • the term encompasses “full-length”, unprocessed IL-2Ry as well as any form of IL-2Ry that results from processing in the cell.
  • the term also encompasses naturally occurring variants of IL-2Ry, e.g. splice variants or allelic variants.
  • IL-2Ry is human IL-2Ry.
  • the high-affinity IL-2R refers to the heterotrimeric form of the IL-2R, consisting of IL-2Ra, IL-2RP and IL-2Ry.
  • the intermediate-affinity IL-2R refers to the heterodimeric form of the IL-2R, consisting of IL-2RP and IL-2Ry.
  • the low affinity IL-2R refers to the monomeric form of the IL-2R, consisting solely of IL-2Ra (for a review see e.g. Olejniczak and Kasprzak, Med Sci Monit 14, RA179-189 (2008)).
  • IL-2 receptor complex refers to the high-affinity IL-2 receptor or the intermediate -affinity IL-2 receptor.
  • Fc domain or “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain.
  • antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain.
  • an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain, or it may include a cleaved variant of the full-length heavy chain.
  • This may be the case where the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to Kabat EU index). Therefore, the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (Lys447), of the Fc region may or may not be present.
  • a heavy chain including an Fc region (subunit) as specified herein, comprised in a binding molecule according to the invention comprises an additional C-terminal lysine (K447, numbering according to Kabat EU index).
  • a heavy chain including an Fc region (subunit) as specified herein, comprised in a binding molecule according to the invention comprises a C-terminal glycine residue (G446, numbering according to Kabat EU index) and does not comprise a C-terminal lysine (Lys477).
  • a “subunit” of an Fc domain as used herein refers to one of the two polypeptides forming the dimeric Fc domain, i.e. a polypeptide comprising C-terminal constant regions of an immunoglobulin heavy chain, capable of stable self-association.
  • a subunit of an IgG Fc domain comprises an IgG CH2 and an IgG CH3 constant domain.
  • a “modification promoting the association of the first and the second subunit of the Fc domain” is a manipulation of the peptide backbone or the post-translational modifications of an Fc domain subunit that reduces or prevents the association of a polypeptide comprising the Fc domain subunit with an identical polypeptide to form a homodimer.
  • a modification promoting association as used herein preferably includes separate modifications made to each of the two Fc domain subunits desired to associate (i.e. the first and the second subunit of the Fc domain), wherein the modifications are complementary to each other so as to promote association of the two Fc domain subunits.
  • a modification promoting association may alter the structure or charge of one or both of the Fc domain subunits so as to make their association sterically or electrostatically favorable, respectively.
  • (hetero)dimerization occurs between a polypeptide comprising the first Fc domain subunit and a polypeptide comprising the second Fc domain subunit, which may be non-identical in the sense that further components fused to each of the subunits (e.g. antigen binding domains) are not the same.
  • the modification promoting the association of the first and the second subunit of the Fc domain comprises an amino acid mutation in the Fc domain, specifically an amino acid substitution.
  • the modification promoting the association of the first and the second subunit of the Fc domain comprises a separate amino acid mutation, specifically an amino acid substitution, in each of the two subunits of the Fc domain.
  • a heterodimerization approach known in the art is the so-called “knobs-into-holes” technology, which is described in detail providing several examples in e.g. WO 96/027011, Ridgway, J.B., et al., Protein Eng. 9 (1996) 617-621; Merchant, A.M., et al., Nat. Biotechnol. 16 (1998) 677-681; and W098/050431.
  • effector functions refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype.
  • antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell- mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen presenting cells, down regulation of cell surface receptors (e.g. B-cell receptor), and B-cell activation.
  • an “activating Fc receptor” is an Fc receptor that following engagement by an Fc domain of an antibody elicits signaling events that stimulate the receptor-bearing cell to perform effector functions.
  • Human activating Fc receptors include FcyRIIIa (CD 16a), FcyRI (CD64), FcyRIIa (CD32), and FcaRI (CD89).
  • Antibody-dependent cell-mediated cytotoxicity is an immune mechanism leading to the lysis of antibody-coated target cells by immune effector cells.
  • the target cells are cells to which antibodies or derivatives thereof comprising an Fc region specifically bind, generally via the protein part that is N- terminal to the Fc region.
  • reduced ADCC is defined as either a reduction in the number of target cells that are lysed in a given time, at a given concentration of antibody in the medium surrounding the target cells, by the mechanism of ADCC defined above, and/or an increase in the concentration of antibody in the medium surrounding the target cells, required to achieve the lysis of a given number of target cells in a given time, by the mechanism of ADCC.
  • the reduction in ADCC is relative to the ADCC mediated by the same antibody produced by the same type of host cells, using the same standard production, purification, formulation and storage methods (which are known to those skilled in the art), but that has not been engineered.
  • the reduction in ADCC mediated by an antibody comprising in its Fc domain an amino acid substitution that reduces ADCC is relative to the ADCC mediated by the same antibody without this amino acid substitution in the Fc domain.
  • Suitable assays to measure ADCC are well known in the art (see e.g. PCT publication no. WO 2006/082515 or PCT publication no. WO 2012/130831).
  • Reduced binding for example reduced binding to an Fc receptor, refers to a decrease in affinity for the respective interaction, as measured for example by SPR.
  • the term includes also reduction of the affinity to zero (or below the detection limit of the analytic method), i.e. complete abolishment of the interaction.
  • increased binding refers to an increase in binding affinity for the respective interaction.
  • Binding affinity refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen).
  • binding affinity refers to intrinsic binding affinity which reflects a 1: 1 interaction between members of a binding pair (e.g., an antibody and an antigen).
  • KD dissociation constant
  • Affinity can be measured by well-established methods known in the art, including those described herein. A preferred method for measuring affinity is Surface Plasmon Resonance (SPR).
  • engine engineered, engineering
  • engineering includes modifications of the amino acid sequence, of the glycosylation pattern, or of the side chain group of individual amino acids, as well as combinations of these approaches.
  • amino acid mutation as used herein is meant to encompass amino acid substitutions, deletions, insertions, and modifications. Any combination of substitution, deletion, insertion, and modification can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., reduced binding to an Fc receptor, or increased association with another peptide.
  • Amino acid sequence deletions and insertions include amino- and/or carboxy-terminal deletions and insertions of amino acids.
  • Preferred amino acid mutations are amino acid substitutions.
  • nonconservative amino acid substitutions i.e. replacing one amino acid with another amino acid having different structural and/or chemical properties, are particularly preferred.
  • Amino acid substitutions include replacement by non- naturally occurring amino acids or by naturally occurring amino acid derivatives of the twenty standard amino acids (e.g. 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5 -hydroxylysine).
  • Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site directed mutagenesis, PCR, gene synthesis and the like. It is contemplated that methods of altering the side chain group of an amino acid by methods other than genetic engineering, such as chemical modification, may also be useful. Various designations may be used herein to indicate the same amino acid mutation.
  • a substitution from proline at position 329 of the Fc domain to glycine can be indicated as 329G, G329, G329, P329G, or Pro329Gly.
  • fused is meant that the components (e.g. a Fab molecule and an Fc domain subunit) are linked by peptide bonds, either directly or via one or more peptide linkers.
  • the term “fusion point” refers to the site on one component at which said component is fused to another component.
  • a cytokine receptor-binding domain may be fused at its C-terminus to the N-terminus of the VH domain of a targetbinding domain.
  • the fusion point on the cytokine receptor-binding domain is the C-terminus and the fusion point on the target-binding domain is the N-terminus.
  • Percent (%) amino acid sequence identity with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or the FASTA program package.
  • the percent identity values can be generated using the sequence comparison computer program ALIGN-2.
  • the ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087 and is described in WO 2001/007611.
  • % amino acid sequence identity values are generated using the ggsearch program of the FASTA package version 36.3.8c or later with a BLOSUM50 comparison matrix.
  • the FASTA program package was authored by W. R. Pearson and D. J. Lipman (“Improved Tools for Biological Sequence Analysis”, PNAS 85 (1988) 2444-2448), W. R. Pearson (“Effective protein sequence comparison” Meth. Enzymol. 266 (1996) 227-25 258), and Pearson et. al.
  • cytokine receptorbinding domains a pair of antigen binding domains capable of binding different cytokine receptor subunits of a cytokine receptor complex, hereinafter referred to as cytokine receptorbinding domains, can act as cytokine agonists, i.e. mimic a naturally occurring cytokine. Cytokine agonists are also termed cytokine mimetics. This has been shown by combining a pair of cytokine receptor-binding domains capable of binding different cytokine receptor subunits in a single antigen binding molecule. The antibody format of these molecules may provide advantageous properties compared to natural or recombinant cytokines.
  • the pair of cytokine receptor-binding domains were split in two distinct antigen binding molecules, i.e. split molecules or split antibodies. These antigen binding molecules further comprise an antigen binding domain capable of binding a target antigen, hereinafter referred to as target-binding domain.
  • target-binding domain capable of binding a target antigen
  • the molecules can act as cytokine mimetics.
  • Using a target-dependent approach further allows to direct the agonistic activity to a site of interest, such as cells or tissues expressing the target.
  • the target-binding domains of the molecules are capable of binding the same target antigen simultaneously, i.e.
  • Such a pair of target-binding domains are also referred to as biparatopic target-binding domains.
  • the assembly of the cytokine receptor-binding domains by the biparatopic target-binding domains allows to target the cytokine receptor-binding domains specifically to a site of interest, i.e. a cell or environment where the target antigen is expresses, to act as cytokine mimetics.
  • the pair of split antigen binding molecules are thus a biparatopic pair of antigen binding molecules.
  • the present invention provides a pair of antigen binding molecules that specifically bind to a target antigen, comprising a) first antigen binding molecule comprising i) a first target-binding domain, ii) a first cytokine receptor-binding domain and iii) a Fc domain; and a second antigen binding molecule comprising i) a second target-binding domain, ii) a second cytokine receptor-binding domain and iii) a Fc domain; wherein the first target-binding domain is capable of binding a first epitope of the target antigen and the second target-binding domain is capable of binding a second epitope of the target antigen, wherein the first and the second target-binding domains do not compete for binding on the target antigen; and wherein the first cytokine receptor-binding domain is capable of binding a first cytokine receptor subunit and the second cytokine receptor-binding domain is capable of binding a second cytokine receptor
  • each antigen binding molecule of the pair of antigen binding molecules comprises a cytokine receptor-binding domain.
  • the first antigen binding molecule comprises a first cytokine receptor-binding domain and the second antigen binding molecule comprises a second cytokine receptor-binding domain.
  • the cytokine receptor-binding domains comprised in the pair of antigen binding molecules bind different cytokine receptor subunits.
  • Each cytokine receptor-binding domain of the pair of antigen binding molecules binds a distinct cytokine receptor subunit of a cytokine receptor complex.
  • the first antigen binding molecule comprises a first cytokine receptor-binding domain capable of binding a first cytokine receptor subunit and the second antigen binding molecule comprises a second cytokine receptor-binding domain capable of binding a second cytokine receptor subunit.
  • the first and the second cytokine receptor subunit are subunits of a cytokine receptor complex.
  • the cytokine receptor complex may be IFNy receptor complex, IL-2 receptor complex, IL-7 receptor complex, IL- 12 receptor complex or IL- 18 receptor complex.
  • the first and the second cytokine receptor-binding domain may bind different subunits of the IFNy receptor complex, IL-2 receptor complex, IL-7 receptor complex, IL- 12 receptor complex or IL- 18 receptor complex.
  • the first and second cytokine receptor-binding domains bind subunits of the IFNy receptor complex.
  • the first cytokine receptor-binding domain is capable of binding IFNyRl and the second cytokine receptor-binding domain is capable of binding IFNyR2.
  • the first cytokine receptor-binding domain is capable of binding IFNyR2 and the second cytokine receptor-binding domain is capable of binding IFNyRl.
  • the first and second cytokine receptor-binding domains bind subunits of the IL-2 receptor complex.
  • the first cytokine receptor-binding domain is capable of binding IL-2RP and the second cytokine receptor-binding domain is capable of binding IL-2Ry.
  • the first cytokine receptor-binding domain is capable of binding IL-2Ry and the second cytokine receptor-binding domain is capable of binding IL-2Rp.
  • the first and second cytokine receptor-binding domains bind subunits of the IL-7 receptor complex.
  • the first cytokine receptor-binding domain is capable of binding IL-7Ra and the second cytokine receptor-binding domain is capable of binding IL-2Ry.
  • the first cytokine receptor-binding domain is capable of binding IL-2Ry and the second cytokine receptor-binding domain is capable of binding IL-7Ra.
  • the first and second cytokine receptor-binding domains bind subunits of the IL- 12 receptor complex.
  • the first cytokine receptor-binding domain is capable of binding IL-12Rpi and the second cytokine receptor-binding domain is capable of binding IL-12RP2.
  • the first cytokine receptor-binding domain is capable of binding IL-12RP2 and the second cytokine receptor-binding domain is capable of binding IL-12Rpi.
  • the first and/or second cytokine receptor-binding domain may be an antibody fragment, such as Fv, Fab, scFv, scFab or single-domain antibody.
  • the first and/or second cytokine receptor-binding domains are single-domain antibodies.
  • the first and second cytokine receptor-binding domains are VHH domains.
  • the first cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 and the second cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10.
  • the first cytokine receptorbinding domain comprises an amino acid sequence selected from SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, and the second cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.
  • the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 3 and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 7.
  • the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 7 and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 3.
  • the first cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60
  • the second cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65.
  • the first cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65
  • the second cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60.
  • the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 60 and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 62.
  • the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 62 and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 60.
  • the first cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, and the second cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65.
  • the first cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65
  • the second cytokine receptorbinding domain comprises an amino acid sequence selected from SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192.
  • the first cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 62.
  • the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 62 and the second cytokine receptorbinding domain comprises an amino acid sequence selected from SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192.
  • the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 182 and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 62.
  • the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 62 and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 182. In one aspect, the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 186 and the second cytokine receptorbinding domain comprises the sequence of SEQ ID NO: 62. In one aspect, the first cytokine receptorbinding domain comprises the sequence of SEQ ID NO: 62 and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 186.
  • the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 190 and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 62. In one aspect, the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 62 and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 190.
  • each antigen binding molecule of the pair of antigen binding molecules comprises a target-binding domain.
  • the first antigen binding molecule comprises a first target-binding domain and the second antigen binding molecule comprises a second target-binding domain. Both the first and the second target-binding domain bind the same target, i.e. the same antigen. Yet, the first and the second target-binding domains bind distinct epitopes of said target.
  • the first target-binding domain is capable of binding a first epitope on the target antigen and the second target-binding domain is capable of binding a second epitope on the target antigen.
  • the first and the second target-binding domain are capable of binding the target simultaneously, i.e. the target-binding domains are non-competing domains. The first target-binding domain and the second target-binding domain do not compete for binding on the target antigen.
  • the target-binding domain of the first and/or second antigen binding molecule may be an antibody fragment.
  • the first target-binding domain is an antibody fragment and the second target-binding domain is an antibody fragment.
  • the first antigen binding molecule comprises a first target-binding domain, wherein the target binding domain is an antibody fragment
  • the second antigen binding molecule comprises a second target-binding domain, wherein the targetbinding domain is an antibody fragment.
  • the target-binding domain of the first and/or second antigen binding molecule may be a Fv, Fab, scFv, scFab molecule or a single domain antibody.
  • the first antigen binding molecule comprises a first target-binding domain, wherein the target binding domain is a Fv, Fab, scFv, scFab molecule or a single domain antibody
  • the second antigen binding molecule comprises a second target-binding domain, wherein the target-binding domain is a Fv, Fab, scFv, scFab molecule or a single domain antibody.
  • the target-binding domain of the first and/or second antigen binding molecule is a Fab molecule.
  • the first target-binding domain is a Fab molecule and the second target-binding domain is a Fab molecule.
  • the first antigen binding molecule comprises a first target-binding domain, wherein the target binding domain is a Fab molecule, and the second antigen binding molecule comprises a second target-binding domain, wherein the target-binding domain is a Fab molecule.
  • the first target-binding domain comprises a heavy chain variable domain (VHi), a light chain variable domain (VLi), a heavy chain constant domain (CHI i) and a light chain constant domain (CLi).
  • the second target-binding domain comprises a heavy chain variable domain (VH2), a light chain variable domain (VL2), a heavy chain constant domain (CHI 2) and a light chain constant domain (CL2).
  • the target-binding domain may be a cross-Fab.
  • the first target-binding domain is a cross-Fab.
  • the second target-binding domain is a cross-Fab.
  • the first antigen binding molecule comprises a first target-binding domain, wherein the target-binding domain is a Fab molecule, wherein the Fab molecule is a cross-Fab
  • the second antigen binding molecule comprises a second target-binding domain, wherein the target-binding domain is a Fab molecule, wherein the Fab molecule is in not a cross-Fab, i.e. a conventional Fab molecule.
  • the first antigen binding molecule comprises a first target-binding domain, wherein the target-binding domain is Fab molecule, wherein the Fab molecule is a conventional Fab molecule, and the second antigen binding molecule comprises a second target-binding domain, wherein the targetbinding domain is a Fab molecule, wherein the Fab molecule is a cross-Fab. Both the first and the second target-binding domains specifically bind the same target antigen.
  • the target antigen may be a tumor-associated antigen or a T cell antigen.
  • the target antigen may be a tumor-associated antigen such as CEA, FAP, Her2, Her3 or EGFR.
  • the target antigen may be a human tumor-associated antigen.
  • the target antigen may be human CEA, human FAP, human Her2, human Her3 or human EGFR.
  • the first target-binding domain is capable of binding a first epitope of CEA and the second target-binding domain is capable of binding a second epitope of CEA.
  • the first target-binding domain is capable of binding a first epitope of FAP and the second target-binding domain is capable of binding a second epitope of FAP.
  • the first target-binding domain is capable of binding a first epitope of Her2 and the second target-binding domain is capable of binding a second epitope of Her2. In one aspect the first target-binding domain is capable of binding a first epitope of Her3 and the second target-binding domain is capable of binding a second epitope of Her3. In one aspect the first target-binding domain is capable of binding a first epitope of EGFR and the second target-binding domain is capable of binding a second epitope of EGFR.
  • the first target-binding domain comprises a VHi of SEQ ID NO: 20 and a VLi of SEQ ID NO: 21 and the second target-binding domain comprises a VEf of SEQ ID NO: 22 and a VL2 of SEQ ID NO: 23.
  • the first target-binding domain comprises a VHi of SEQ ID NO: 22 and a VLi of SEQ ID NO: 23 and the second target-binding domains comprises a VH2 of SEQ ID NO: 20 and a VL2 of SEQ ID NO: 21.
  • the first target-binding domain comprises a VHi of SEQ ID NO: 114 and a VLi of SEQ ID NO: 115 and the second target-binding domain comprises a VH2 of SEQ ID NO: 116 and a VL2 of SEQ ID NO: 117.
  • the first target-binding domain comprises a VHi of SEQ ID NO: 116 and a VLi of SEQ ID NO: 117 and the second target-binding domains comprises a VH2 of SEQ ID NO: 114 and a VL2 of SEQ ID NO: 115.
  • the first target-binding domain comprises a VHi of SEQ ID NO: 130 and a VLi of SEQ ID NO: 131 and the second target-binding domain comprises a VH2 of SEQ ID NO: 132 and a VL2 of SEQ ID NO: 133.
  • the first target-binding domain comprises a VHi of SEQ ID NO: 132 and a VLi of SEQ ID NO: 133 and the second target-binding domains comprises a VH2 of SEQ ID NO: 130 and a VL2 of SEQ ID NO: 131.
  • the first target-binding domain comprises a VHi of SEQ ID NO: 140 and a VLi of SEQ ID NO: 141 and the second target-binding domain comprises aVEf of SEQ ID NO: 142 and aVL2 of SEQ ID NO: 143.
  • the first target-binding domain comprises a VHi of SEQ ID NO: 142 and a VLi of SEQ ID NO: 143 and the second target-binding domains comprises a VH2 of SEQ ID NO: 140 and a VL 2 of SEQ ID NO: 141.
  • the target antigen may be a T cell antigen such as PD-1 or LAG-3.
  • the target antigen may be human PD-1 or human LAG-3.
  • the first target-binding domain is capable of binding a first epitope of PD-1 and the second target-binding domain is capable of binding a second epitope of PD-1.
  • the first target-binding domain is capable of binding a first epitope of LAG-3 and the second target-binding domain is capable of binding a second epitope of LAG-3.
  • the first target-binding domain comprises a VHi of SEQ ID NO: 76 and a VLi of SEQ ID NO: 77 and the second target-binding domain comprises a VFL of SEQ ID NO: 78 and a VL2 of SEQ ID NO: 79.
  • the first target-binding domain comprises a VHi of SEQ ID NO: 78 and a VLi of SEQ ID NO: 79 and the second target-binding domains comprises a VH2 of SEQ ID NO: 76 and a VL2 of SEQ ID NO: 77.
  • the first target-binding domain comprises a VHi of SEQ ID NO: 93 and a VLi of SEQ ID NO: 94 and the second target-binding domain comprises a VH2 of SEQ ID NO: 78 and a VL2 of SEQ ID NO: 79.
  • the first target-binding domain comprises a VHi of SEQ ID NO: 78 and a VLi of SEQ ID NO: 79 and the second target-binding domains comprises a VH2 of SEQ ID NO: 93 and a VL2 of SEQ ID NO: 94.
  • the first target-binding domain comprises a VHi of SEQ ID NO: 150 and a VLi of SEQ ID NO: 151 and the second target-binding domain comprises a VH2 of SEQ ID NO: 152 and a VL2 of SEQ ID NO: 153.
  • the first target-binding domain comprises a VHi of SEQ ID NO: 152 and a VLi of SEQ ID NO: 153 and the second target-binding domains comprises a VH 2 of SEQ ID NO: 150 and a VL 2 of SEQ ID NO: 151.
  • the first and the second antigen binding molecules comprise an Fc domain.
  • the Fc domain of the binding molecule(s) consists of a pair of polypeptide chains, a first Fc domain subunit and a second Fc domain subunit.
  • the first and second Fc domain subunits may comprise heavy chain domains of an immunoglobulin molecule.
  • the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which comprises the CH2 and CH3 IgG heavy chain constant domains.
  • the two subunits of the Fc domain are capable of stable association with each other.
  • the first antigen binding molecule comprises a Fc domain composed of a first and a second Fc domain subunit
  • the second antigen binding molecule comprises a Fc domain composed of a first and a second Fc domain subunit.
  • the first antigen binding molecule comprises a Fc domain composed of a first and a second Fc domain subunit
  • the second antigen binding molecule comprises a Fc domain composed of a first and a second Fc domain subunit.
  • each of the antigen binding molecules comprises not more than one Fc domain.
  • the Fc domain of the first and/or the second antigen binding molecule may be an IgG Fc domain.
  • the Fc domain is an IgGi Fc domain. In another particular embodiment, the Fc domain is an IgG4 Fc domain. In a more specific aspect, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at position S228 (Kabat EU index numbering), particularly the amino acid substitution S228P. This amino acid substitution reduces in vivo Fab arm exchange of IgG4 antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In further aspects, the Fc domain is ahuman Fc domain. In particular aspects, the Fc domain is a human IgGi Fc domain.
  • the Fc domain comprises a modification promoting the association of the first and the second subunit of the Fc domain.
  • Fc domain modifications promoting heterodimerization are further described hereinbelow.
  • the Fc domain comprises one or more amino acid substitution that reduces binding to an Fc receptor and/or effector function.
  • Fc domain modifications reducing Fc receptor binding and/or effector function are further described hereinbelow.
  • the antigen binding molecule(s) according to the invention comprise a target-binding domain and a cytokine receptor-binding domain, which may be fused to the first and/or the second Fc domain subunit, thus the two Fc domain subunits are typically comprised in two non-identical polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization leads to several possible combinations of the two polypeptides. To improve the yield and purity of the antigen binding molecule(s) in recombinant production, it will thus be advantageous to introduce in the Fc domain of the antigen binding molecule(s) a modification promoting the association of the desired polypeptides.
  • the Fc domain of the antigen binding molecule(s) according to the invention comprises a modification promoting the association of the first and the second subunit of the Fc domain.
  • the site of most extensive protein-protein interaction between the two subunits of a human IgG Fc domain is in the CH3 domain of the Fc domain.
  • said modification is in the CH3 domain of the Fc domain.
  • the CH3 domain of the first subunit of the Fc domain and the CH3 domain of the second subunit of the Fc domain are both engineered in a complementary manner so that each CH3 domain (or the heavy chain comprising it) can no longer homodimerize with itself but is forced to heterodimerize with the complementarily engineered other CH3 domain (so that the first and second CH3 domain heterodimerize and no homdimers between the two first or the two second CH3 domains are formed).
  • said modification promoting the association of the first and the second subunit of the Fc domain is a so-called “knob-into-hole” modification, comprising a “knob” modification in one of the two subunits of the Fc domain and a “hole” modification in the other one of the two subunits of the Fc domain.
  • knob-into-hole technology is described e.g. in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001).
  • the method involves introducing a protuberance (“knob”) at the interface of a first polypeptide and a corresponding cavity (“hole”) in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation.
  • Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g. tyrosine or tryptophan).
  • Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine).
  • an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit which is positionable in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable.
  • said amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W).
  • said amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).
  • the protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g. by site-specific mutagenesis, or by peptide synthesis.
  • the threonine residue at position 366 is replaced with a tryptophan residue (T366W)
  • the CH3 domain of the second subunit of the Fc domain the “hole” subunit
  • the tyrosine residue at position 407 is replaced with a valine residue (Y407V).
  • the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numberings according to Kabat EU index).
  • the serine residue at position 354 is replaced with a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C) (particularly the serine residue at position 354 is replaced with a cysteine residue), and in the second subunit of the Fc domain additionally the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C) (numberings according to Kabat EU index). Introduction of these two cysteine residues results in formation of a disulfide bridge between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).
  • the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W
  • the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to Kabat EU index).
  • CH3 -modification for enforcing the heterodimerization is contemplated as alternatives according to the invention and are described e.g. in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012/058768, WO 2013/157954, WO 2013/096291.
  • heterodimerization approach described in EP 1870459 is used alternatively. This approach is based on the introduction of charged amino acids with opposite charges at specific amino acid positions in the CH3/CH3 domain interface between the two subunits of the Fc domain.
  • a particular aspect for the antigen binding molecule(s) of the invention are amino acid mutations R409D; K370E in one of the two CH3 domains (of the Fc domain) and amino acid mutations D399K; E357K in the other one of the CH3 domains of the Fc domain (numbering according to Kabat EU index).
  • the antigen binding molecule(s) of the invention comprises amino acid mutation T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, and additionally amino acid mutations R409D; K370E in the CH3 domain of the first subunit of the Fc domain and amino acid mutations D399K; E357K in the CH3 domain of the second subunit of the Fc domain (numberings according to Kabat EU index).
  • the antigen binding molecule(s) of the invention comprises amino acid mutations S354C, T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations Y349C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, or said antigen binding molecule(s) comprises amino acid mutations Y349C, T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations S354C, T366S, L368A, Y 407V in the CH3 domains of the second subunit of the Fc domain and additionally amino acid mutations R409D; K370E in the CH3 domain of the first subunit of the Fc domain and amino acid mutations D399K; E357K in the CH3 domain of the second subunit of the Fc domain (all numberings according to Kabat EU index).
  • a first CH3 domain comprises amino acid mutation T366K and a second CH3 domain comprises amino acid mutation L351D (numberings according to Kabat EU index).
  • the first CH3 domain comprises further amino acid mutation L35 IK.
  • the second CH3 domain comprises further an amino acid mutation selected from Y349E, Y349D and L368E (particularly L368E) (numberings according to Kabat EU index).
  • a first CH3 domain comprises amino acid mutations L351Y, Y407A and a second CH3 domain comprises amino acid mutations T366A, K409F.
  • the second CH3 domain comprises a further amino acid mutation at position T411, D399, S400, F405, N390, or K392, e.g.
  • a first CH3 domain comprises amino acid mutations L351Y, Y407A and a second CH3 domain comprises amino acid mutations T366V, K409F.
  • a first CH3 domain comprises amino acid mutation Y 407A and a second CH3 domain comprises amino acid mutations T366A, K409F.
  • the second CH3 domain further comprises amino acid mutations K392E, T41 IE, D399R and S400R (numberings according to Kabat EU index).
  • heterodimerization approach described in WO 2011/143545 is used alternatively, e.g. with the amino acid modification at a position selected from the group consisting of 368 and 409 (numbering according to Kabat EU index).
  • a first CH3 domain comprises amino acid mutation T366W and a second CH3 domain comprises amino acid mutation Y407A.
  • a first CH3 domain comprises amino acid mutation T366Y and a second CH3 domain comprises amino acid mutation Y407T (numberings according to Kabat EU index).
  • the antigen binding molecule(s) or its Fc domain is of IgG2 subclass and the heterodimerization approach described in WO 2010/129304 is used alternatively.
  • a modification promoting association of the first and the second subunit of the Fc domain comprises a modification mediating electrostatic steering effects, e.g. as described in PCT publication WO 2009/089004.
  • this method involves replacement of one or more amino acid residues at the interface of the two Fc domain subunits by charged amino acid residues so that homodimer formation becomes electrostatically unfavorable but heterodimerization electrostatically favorable.
  • a first CH3 domain comprises amino acid substitution of K392 or N392 with a negatively charged amino acid (e.g. glutamic acid (E), or aspartic acid (D), particularly K392D or N392D) and a second CH3 domain comprises amino acid substitution of D399, E356, D356, or E357 with a positively charged amino acid (e.g. lysine (K) or arginine (R), particularly D399K, E356K, D356K, or E357K, and more particularly D399K and E356K).
  • the first CH3 domain further comprises amino acid substitution of K409 or R409 with a negatively charged amino acid (e.g.
  • the first CH3 domain further or alternatively comprises amino acid substitution of K439 and/or K370 with a negatively charged amino acid (e.g. glutamic acid (E), or aspartic acid (D)) (all numberings according to Kabat EU index).
  • a negatively charged amino acid e.g. glutamic acid (E), or aspartic acid (D)
  • a first CH3 domain comprises amino acid mutations K253E, D282K, and K322D and a second CH3 domain comprises amino acid mutations D239K, E240K, and K292D (numberings according to Kabat EU index).
  • the heterodimerization approach described in WO 2007/110205 can be used alternatively.
  • the first subunit of the Fc domain comprises amino acid substitutions K392D and K409D
  • the second subunit of the Fc domain comprises amino acid substitutions D356K and D399K (numbering according to Kabat EU index).
  • the Fc domain confers to the antigen binding molecule(s) favorable pharmacokinetic properties, including a long serum half-life, which contributes to good accumulation in the target tissue and a favorable tissue-blood distribution ratio. At the same time it may, however, lead to undesirable targeting of the binding molecule(s) to cells expressing Fc receptors rather than to the preferred antigen-bearing cells. Moreover, the co-activation of Fc receptor signaling pathways may lead to cytokine release which may result in excessive activation of cytokine receptors and severe side effects upon systemic administration. Activation of (Fc receptor-bearing) immune cells other than T cells may even reduce efficacy of the pair of antigen binding molecules due to the potential destruction of T cells e.g. by NK cells.
  • the Fc domain of the antigen binding molecule(s) according to the invention exhibits reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a native IgGi Fc domain.
  • the Fc domain (or the antigen binding molecule comprising said Fc domain) exhibits less than 50%, particularly less than 20%, more particularly less than 10% and most particularly less than 5% of the binding affinity to an Fc receptor, as compared to a native IgGi Fc domain (or a antigen binding molecule comprising a native IgGi Fc domain), and/or less than 50%, particularly less than 20%, more particularly less than 10% and most particularly less than 5% of the effector function, as compared to a native IgGi Fc domain domain (or a antigen binding molecule comprising a native IgGi Fc domain).
  • the Fc domain domain (or the antigen binding molecule comprising said Fc domain) does not substantially bind to an Fc receptor and/or induce effector function.
  • the Fc receptor is an Fey receptor.
  • the Fc receptor is a human Fc receptor.
  • the Fc receptor is an activating Fc receptor.
  • the Fc receptor is an activating human Fey receptor, more specifically human FcyRIIIa, FcyRI or FcyRIIa, most specifically human FcyRIIIa.
  • the effector function is one or more selected from the group of CDC, ADCC, ADCP, and cytokine secretion.
  • the effector function is ADCC.
  • the Fc domain exhibits substantially similar binding affinity to neonatal Fc receptor (FcRn), as compared to a native IgGi Fc domain domain.
  • FcRn neonatal Fc receptor
  • Substantially similar binding to FcRn is achieved when the Fc domain (or the antigen binding molecule comprising said Fc domain) exhibits greater than about 70%, particularly greater than about 80%, more particularly greater than about 90% of the binding affinity of a native IgGi Fc domain (or the antigen binding molecule comprising a native IgGi Fc domain) to FcRn.
  • the Fc domain is engineered to have reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a non-engineered Fc domain.
  • the Fc domain of the antigen binding molecule(s) comprises one or more amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and/or effector function.
  • the same one or more amino acid mutation is present in each of the two subunits of the Fc domain.
  • the amino acid mutation reduces the binding affinity of the Fc domain to an Fc receptor.
  • the amino acid mutation reduces the binding affinity of the Fc domain to an Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold.
  • the combination of these amino acid mutations may reduce the binding affinity of the Fc domain to an Fc receptor by at least 10-fold, at least 20-fold, or even at least 50-fold.
  • the antigen binding molecule(s) comprising an engineered Fc domain exhibits less than 20%, particularly less than 10%, more particularly less than 5% of the binding affinity to an Fc receptor as compared to a antigen binding molecule comprising a nonengineered Fc domain.
  • the Fc receptor is an Fey receptor.
  • the Fc receptor is a human Fc receptor.
  • the Fc receptor is an activating Fc receptor.
  • the Fc receptor is an activating human Fey receptor, more specifically human FcyRIIIa, FcyRI or FcyRIIa, most specifically human FcyRIIIa.
  • binding to each of these receptors is reduced.
  • binding affinity to a complement component, specifically binding affinity to Clq is also reduced.
  • binding affinity to neonatal Fc receptor (FcRn) is not reduced.
  • Substantially similar binding to FcRn i.e. preservation of the binding affinity of the Fc domain to said receptor, is achieved when the Fc domain (or the antigen binding molecule comprising said Fc domain) exhibits greater than about 70% of the binding affinity of a non-engineered form of the Fc domain (or the antigen binding molecule comprising said non-engineered form of the Fc domain) to FcRn.
  • the Fc domain, or antigen binding molecule(s) of the invention comprising said Fc domain may exhibit greater than about 80% and even greater than about 90% of such affinity.
  • the Fc domain of the antigen binding molecule(s) is engineered to have reduced effector function, as compared to a nonengineered Fc domain.
  • the reduced effector function can include, but is not limited to, one or more of the following: reduced complement dependent cytotoxicity (CDC), reduced antibody dependent cell- mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling inducing apoptosis, reduced crosslinking of target-bound antibodies, reduced dendritic cell maturation, or reduced T cell priming.
  • CDC reduced complement dependent cytotoxicity
  • ADCC reduced antibody dependent cell- mediated cytotoxicity
  • ADCP reduced antibody-dependent cellular phagocytosis
  • reduced immune complex-mediated antigen uptake by antigen-presenting cells reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling induc
  • the reduced effector function is one or more selected from the group of reduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion.
  • the reduced effector function is reduced ADCC.
  • the reduced ADCC is less than 20% of the ADCC induced by a nonengineered Fc domain (or a binding molecule comprising a non-engineered Fc domain).
  • the amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and/or effector function is an amino acid substitution.
  • the Fc domain comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331 and P329 (numberings according to Kabat EU index).
  • the Fc domain comprises an amino acid substitution at a position selected from the group of L234, L235 and P329 (numberings according to Kabat EU index).
  • the Fc domain comprises the amino acid substitutions L234A and L235A (numberings according to Kabat EU index).
  • the Fc domain is an IgGi Fc domain, particularly a human IgGi Fc domain.
  • the Fc domain comprises an amino acid substitution at position P329.
  • the amino acid substitution is P329A or P329G, particularly P329G (numberings according to Kabat EU index).
  • the Fc domain comprises an amino acid substitution at position P329 and a further amino acid substitution at a position selected from E233, L234, L235, N297 and P331 (numberings according to Kabat EU index).
  • the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S.
  • the Fc domain comprises amino acid substitutions at positions P329, L234 and L235 (numberings according to Kabat EU index).
  • the Fc domain comprises the amino acid mutations L234A, L235A and P329G (“P329G LALA”, “PGLALA” or “LALAPG”).
  • each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering), i.e.
  • the leucine residue at position 234 is replaced with an alanine residue (L234A)
  • the leucine residue at position 235 is replaced with an alanine residue (L235A)
  • the proline residue at position 329 is replaced by a glycine residue (P329G) (numbering according to Kabat EU index).
  • the Fc domain is an IgGi Fc domain, particularly a human IgGi Fc domain.
  • the “P329G LALA” combination of amino acid substitutions almost completely abolishes Fey receptor (as well as complement) binding of a human IgGi Fc domain, as described in PCT publication no. WO 2012/130831, which is incorporated herein by reference in its entirety.
  • WO 2012/130831 also describes methods of preparing such mutant Fc domains and methods for determining its properties such as Fc receptor binding or effector functions.
  • the Fc domain of the binding molecule(s) of the invention is an IgG 4 Fc domain, particularly a human IgG 4 Fc domain.
  • the IgG 4 Fc domain comprises an amino acid substitution at position S228, specifically the amino acid substitution S228P (numberings according to Kabat EU index).
  • the IgG4 Fc domain comprises an amino acid substitution at position L235, specifically the amino acid substitution L235E (numberings according to Kabat EU index).
  • the IgG 4 Fc domain comprises an amino acid substitution at position P329, specifically the amino acid substitution P329G (numberings according to Kabat EU index).
  • the IgG 4 Fc domain comprises amino acid substitutions at positions S228, L235 and P329, specifically amino acid substitutions S228P, L235E and P329G (numberings according to Kabat EU index).
  • Such IgG 4 Fc domain mutants and their Fey receptor binding properties are described in PCT publication no. WO 2012/130831, incorporated herein by reference in its entirety.
  • the Fc domain exhibiting reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a native IgGi Fc domain is a human IgGi Fc domain comprising the amino acid substitutions L234A, L235A and optionally P329G, or a human IgG 4 Fc domain comprising the amino acid substitutions S228P, L235E and optionally P329G (numberings according to Kabat EU index).
  • the Fc domain comprises an amino acid mutation at position N297, particularly an amino acid substitution replacing asparagine by alanine (N297A) or aspartic acid (N297D) (numberings according to Kabat EU index).
  • Fc domains with reduced Fc receptor binding and/or effector function also include those with substitution of one or more ofFc domain residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056) (numberings according to Kabat EU index).
  • Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581).
  • Mutant Fc domains can be prepared by amino acid deletion, substitution, insertion or modification using genetic or chemical methods well known in the art. Genetic methods may include site specific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, and the like. The correct nucleotide changes can be verified for example by sequencing.
  • Binding to Fc receptors can be easily determined e.g. by ELISA, or by Surface Plasmon Resonance (SPR) using standard instrumentation such as a BIAcore instrument (GE Healthcare), and Fc receptors such as may be obtained by recombinant expression.
  • binding affinity of Fc domains or binding molecule(s) comprising an Fc domain for Fc receptors may be evaluated using cell lines known to express particular Fc receptors, such as human NK cells expressing Fcyllla receptor.
  • Effector function of an Fc domain, or a binding molecule comprising an Fc domain can be measured by methods known in the art. Examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83, 7059- 7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S. Patent No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987).
  • non-radioactive assays may be employed (see, for example, ACTITM nonradioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)).
  • Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
  • ADCC activity of the molecule of interest may be assessed in vivo, e.g. in an animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).
  • binding of the Fc domain to a complement component, specifically to Clq is reduced.
  • said reduced effector function includes reduced CDC.
  • Clq binding assays may be carried out to determine whether the Fc domain, or the binding molecule comprising the Fc domain, is able to bind Clq and hence has CDC activity.
  • a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)).
  • FcRn binding and in vivo clearance/half life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int’l. Immunol. 18(12): 1759-1769 (2006); WO 2013/120929).
  • the antigen binding molecule(s) according to the present invention may have various molecular configurations, i.e. the domains of the binding molecule(s) may be linked to each other in different ways.
  • the antigen binding molecule(s) may comprise a target-binding domain, a cytokine receptor-binding domain and a Fc domain, wherein the cytokine receptor-binding domain is fused at its C-terminus to the N-terminus of the target-binding domain, and the target-binding domain is fused at its C-terminus to the N-terminus of one of the Fc domain subunits.
  • the target-binding domain may be a Fab molecule.
  • the cytokine receptor-binding domain may be fused to the VH or VL domain of the target bindingdomain.
  • the antigen binding molecule(s) comprise a target-binding domain, a cytokine receptor-binding domain and a Fc domain, wherein the target-binding domain is a Fab molecule, and wherein the cytokine receptor-binding domain is fused at its C-terminus to the N-terminus of the VH domain of the target-binding domain, and the target-binding domain is fused at its C-terminus to the N-terminus of one of the Fc domain subunits (see e.g. Fig. 1 IB, 11C, 1 IF and 11G for schematic illustration).
  • the antigen binding molecule(s) comprise a target-binding domain, a cytokine receptor-binding domain and a Fc domain, wherein the target-binding domain is a Fab molecule, and wherein the cytokine receptor-binding domain is fused at its C-terminus to the N-terminus of the VL domain of the target-binding domain, and the target-binding domain is fused at its C-terminus to the N-terminus of one of the Fc domain subunits (see e.g. Fig. 1 ID, 1 IE, 11H and 1 II for schematic illustration).
  • the antigen binding molecule(s) may comprise a target-binding domain, a cytokine receptor-binding domain and a Fc domain, wherein the target-binding domain is fused at its C-terminus to the N-terminus of the first Fc domain subunit and the cytokine receptor-binding domain is fused at its C-terminus to the N-terminus of the second Fc domain subunit (see e.g. Fig. 13A, 13B, 13C and 13D for schematic illustration).
  • the antigen binding molecule(s) may comprise a target-binding domain, a cytokine receptor-binding domain and a Fc domain, wherein the target-binding domain is fused at its C-terminus to the N-terminus of one of the Fc domain subunits and the cytokine receptor-binding domain is fused at its N-terminus to the C-terminus of the same Fc domain subunit (see e.g. Fig. 13E, 13F, 13G and 13H for schematic illustration).
  • the cytokine-receptor binding domain may be fused via a peptide linker.
  • the peptide linker may comprise an amino acid sequence according to SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 279, SEQ ID NO: 280 or SEQ ID NO: 281.
  • both antigen binding molecules of the pair of antigen binding molecules comprise a targetbinding domain, a cytokine receptor-binding domain and a Fc domain, wherein the cytokine receptorbinding domain is fused at its C-terminus to the N-terminus of the target-binding domain, and the targetbinding domain is fused at its C-terminus to the N-terminus of one of the Fc domain subunits.
  • the target-binding domains of the antigen binding molecules are Fab molecules.
  • the pair of antigen binding molecules comprises a first antigen binding molecule comprising a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a first cytokine receptor-binding domain, VHi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VLi and CLi; and a second antigen binding molecule comprising a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a second cytokine receptor-binding domain, VH2, CHI 2 and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VL2 and CL2.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VHi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus a first cytokine receptor-binding domain, VLi and CLi; and a second antigen binding molecule comprising a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VH2, CHI 2 and a second Fc domain subunit; a third polypeptide comprising in order from the N-terminus to C-terminus a second cytokine receptor-binding domain, VL2 and CL2.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a first cytokine receptor-binding domain, VHi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VLi and CLi; and a second antigen binding molecule comprising a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VH2, CHI 2 and a second Fc domain subunit; a third polypeptide comprising in order from the N-terminus to C-terminus a second cytokine receptor-binding domain, VL2 and CL2.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a first cytokine receptor-binding domain, VLi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VHi and CLi; and a second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a second cytokine receptor-binding domain, VH2, CHI 2 and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VL2 and CL2.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VLi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus a first cytokine receptor-binding domain, VHi and CLi; and a second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VH2, CHI 2 and a second Fc domain subunit; a third polypeptide comprising in order from the N-terminus to C-terminus a second cytokine receptor-binding domain, VL2 and CL2.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a first cytokine receptor-binding domain, VLi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VHi and CLi; and a second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VH2, CHh and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus a second cytokine receptor-binding domain, VL2 and CL2.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a VLi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus a first cytokine receptor-binding domain, VHi and CLi; and a second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a second cytokine receptor-binding domain, VH2, CHI 2 and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VL2 and CL2.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 27 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 30 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 38 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 37 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 31, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 33.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 35, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 36.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 27 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 33.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 30 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 31.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 38 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 36.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 37 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 35.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 260 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 259 and a third polypeptide comprising an amino acid sequence of SEQ
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 119 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 256, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 122 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 255.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 261 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 258 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 127.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 119 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 257, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 112 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 254.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 260 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 112 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 255.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 119 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 256, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 259 and a third polypeptide comprising an amino acid sequence of SEQ
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 261 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 112 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 254.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 119 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 257, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 258 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 127.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 86, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 44 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 83, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 88 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 87.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 92, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 44 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 91.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 96 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 95, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 101 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 99 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 98, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 88 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 102.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 96 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 97, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 103 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 100 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 98, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 88 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 104.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 98 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 99, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 101 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 96 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 95, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 88 and a third polypeptide comprising an amino acid sequence of SEQ ID
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 100 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 98, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 103 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 96 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 97, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 88 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 104.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 110 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 113 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 105, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 109.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 111 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 112 and a third polypeptide comprising an amino acid sequence of SEQ
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 107, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 108.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 110 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 109.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 105, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 113 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 111 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 108.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 107, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 112 and a third polypeptide comprising an amino acid sequence of SEQ
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 125 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 129 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 127.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 119 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 118, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 122 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 123.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 126 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 128 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 127.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 119 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 120, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 122 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 121.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 125 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 122 and a third polypeptide comprising an amino acid sequence of SEQ
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 119 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 118, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 129 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 127.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 126 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 122 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 121.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 119 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 120, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 128 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 127.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 136 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 134, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 138 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 137.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 135 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 134, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 138 and a third polypeptide comprising an amino acid sequence of SEQ
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 146 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 144, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 148 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 147.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 145 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 144, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 148 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 149.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 155 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 154, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 165 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 163.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 161 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 160, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 158 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 159.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 155 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 156, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 164 and a third polypeptide comprising an amino acid sequence of SEQ
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 162 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 160, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 158 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 157.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 161 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 160, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 165 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 163.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 155 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 154, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 158 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 159.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 162 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 160, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 164 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 163.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 155 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 156, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 158 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 157.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 194 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 193, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 195 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 196 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 197 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 198 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 199 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 200 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 201 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 202 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 203 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 204 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 205 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89.
  • both antigen binding molecules of the pair of antigen binding molecules comprise a targetbinding domain, a cytokine receptor-binding domain and a Fc domain, wherein the target-binding domain is fused at its C-terminus to the N-terminus of the first Fc domain subunit and the cytokine receptor-binding domain is fused at its C-terminus to the N-terminus of the second Fc domain subunit.
  • both antigen binding molecules of the pair of antigen binding molecules comprise a targetbinding domain, a cytokine receptor-binding domain and a Fc domain, wherein the target-binding domain is fused at its C-terminus to the N-terminus of the first Fc domain subunit and the cytokine receptor-binding domain is fused at its C-terminus to the N-terminus of the second Fc domain subunit, wherein the target-binding domain is a Fab molecule.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising in order from the N-terminus to C-terminus VHi, CHli and a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VLi and CLi, and athird polypeptide comprising in order from the N-terminus to C-terminus a first cytokine receptor-binding domain and a second Fc domain subunit, and a second antigen binding molecule comprises a first polypeptide comprising in order from the N-terminus to C-terminus VFE, CHE and a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VL2 and CL2, and athird polypeptide comprising in order from the N-terminus to C-terminus a second cytokine receptor-binding domain and a second Fc domain subunit.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising in order from the N-terminus to C-terminus VLi, CHli and a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VHi and CLi, and a third polypeptide comprising in order from the N-terminus to C-terminus a first cytokine receptor-binding domain and a second Fc domain subunit, and a second antigen binding molecule comprising a first polypeptide comprising in order from the N-terminus to C-terminus VH2, CHh and a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VL2 and CL2, and a third polypeptide comprising in order from the N-terminus to C-terminus a second cytokine receptor-binding domain and a second Fc domain subunit.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 32, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 25 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 39, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 34, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 29 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 40.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 34, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 29 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 39, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 32, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 25 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 40.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 237, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 238, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 235, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 236, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 239, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 240, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 235, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 237, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 236, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 235, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 240, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 239, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
  • both antigen binding molecules of the pair of antigen binding molecules comprise a targetbinding domain, a cytokine receptor-binding domain and a Fc domain, wherein the target-binding domain is fused at its C-terminus to the N-terminus of one of the Fc domain subunits and the cytokine receptor-binding domain is fused at its N-terminus to the C-terminus of the same Fc domain subunit.
  • the pair of antigen binding molecules comprises a first antigen binding molecule comprising a first target-binding domain fused at its C-terminus to the N-terminus of one of the Fc domain subunits and a first cytokine receptor-binding domain fused at its N-terminus to the C-terminus of the same Fc domain subunit, and second antigen binding molecule comprising a second target-binding domain fused at its C-terminus to the N-terminus of one of the Fc domain subunits and a second cytokine receptor-binding domain fused at its N-terminus to the C-terminus of the same Fc domain subunit.
  • the target-binding domain is a Fab molecule.
  • the pair of antigen binding molecules comprises a first antigen binding molecule comprising a target-binding domain fused at its C-terminus of CHli to the N-terminus of one of the Fc domain subunits and a first cytokine receptor-binding domain is fused at its N-terminus to the C-terminus of the same Fc domain subunit, and a second antigen binding molecule comprising a target-binding domain fused at its C-terminus of CHh to the N-terminus of one of the Fc domain subunits and a second cytokine receptor-binding domain fused at its N-terminus to the C-terminus of the same Fc domain subunit.
  • the target-binding domain of one of the antigen-binding molecules is a cross-Fab molecule.
  • the pair of antigen binding molecules comprises a first antigen binding molecule comprising a target-binding domain fused at its C-terminus of CHli to the N-terminus of one of the Fc domain subunits and a first cytokine receptor-binding domain is fused at its N-terminus to the C-terminus of the same Fc domain subunit, and a second antigen binding molecule comprising a target-binding domain fused at its C-terminus of CL2 to the N-terminus of one of the Fc domain subunits and a second cytokine receptor-binding domain fused at its N-terminus to the C-terminus of the same Fc domain subunit.
  • the pair of antigen binding molecules comprises a first antigen binding molecule comprising a target-binding domain fused at its C-terminus of CLi to the N-terminus of one of the Fc domain subunits and a first cytokine receptor-binding domain is fused at its N-terminus to the C-terminus of the same Fc domain subunit, and a second antigen binding molecule comprising a target-binding domain fused at its C-terminus of CHI 2 to the N-terminus of one of the Fc domain subunits and a second cytokine receptor-binding domain fused at its N-terminus to the C-terminus of the same Fc domain subunit.
  • the pair of antigen binding molecules comprises a first antigen binding molecule comprising a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a VHi, a CHli, a second Fc domain subunit and a first cytokine receptor-binding domain, and a third polypeptide comprising in order from the N-terminus to C-terminus a VLi and a CLi; and a second antigen binding molecule comprising a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a VH2, a CHh, a second Fc domain subunit and a second cytokine receptor-binding domain, and a third polypeptide comprising in order from the N-terminus to C-terminus VL2 and CL2.
  • the pair of antigen binding molecules comprises a first antigen binding molecule comprising a first polypeptide comprising a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a VLi, a CHli, a second Fc domain subunit and a first cytokine receptor-binding domain, and a third polypeptide comprising in order from the N-terminus to C-terminus a VHi and a CLi; and a second antigen binding molecule comprising a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a VH2, a CHI 2, a second Fc domain subunit and a second cytokine receptor-binding domain, and a third polypeptide comprising in order from the N-terminus to C-terminus a VL2 and a CL2.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 42 and a third polypeptide comprising an amino acid sequence of SEQ ID NO : 41 , and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 45, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 44 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 43.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 46 and a third polypeptide comprising an amino acid sequence of SEQ ID NO : 41 , and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 47, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 44 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 43.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 246 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 249 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 242 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 245 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 250 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 253 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 248 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 247 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 244 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 243 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 252 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 251 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 42 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 42, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 245 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
  • the antigen binding molecule(s) may comprise a target-binding domain, a cytokine receptor-binding domain and a Fc domain, wherein the cytokine receptor-binding domain is fused at its C-terminus to the N-terminus of the target-binding domain, and the target-binding domain is fused at its C-terminus to the N-terminus of one of the Fc domain subunits, wherein the target-binding domain is a VHH domain.
  • the antigen binding molecule may comprise a first polypeptide comprising a first Fc domain subunit and a second polypeptide comprising in order from the N-terminus to C-terminus a first cytokine receptor-binding domain, a first target-binding domain and a second Fc domain subunit, and a second antigen binding molecule comprising a first polypeptide comprising a first Fc domain subunit and a second polypeptide comprising in order from the N-terminus to C-terminus a second cytokine receptorbinding domain, a second target-binding domain and a second Fc domain subunit.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28 and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 173, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28 and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 171.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28 and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 176, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28 and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 172.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28 and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 175, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28 and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 171.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28 and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 176, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28 and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 174.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28 and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 175, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28 and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 170.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28 and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 173, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28 and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 170.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28 and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 176, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28 and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 172.
  • the antigen binding molecule(s) may comprise a target-binding domain, a cytokine receptor-binding domain and a Fc domain, wherein the cytokine receptor-binding domain is fused at its C-terminus to the N-terminus of the target-binding domain, and the target-binding domain is fused at its C-terminus to the N-terminus of one of the Fc domain subunits, wherein the cytokine receptor-binding domain is a Fab molecule.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 267, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 268 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 265, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 272, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 270 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 271.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 267, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 268 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 265, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 274, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 270 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 273.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 269, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 265 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 268, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 272, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 270 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 271.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 269, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 265 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 268, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 274, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 270 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 273.
  • the antigen binding molecule(s) may comprise a target-binding domain, a cytokine receptor-binding domain and a Fc domain, wherein the cytokine receptor-binding domain is fused at its C-terminus to the N-terminus of the target-binding domain, and the target-binding domain is fused at its C-terminus to the N-terminus of one of the Fc domain subunits, wherein the cytokine receptor-binding domain is a scFv domain.
  • the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 276 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 275, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 277 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 278.
  • a pair of antigen binding molecules that specifically bind to a target antigen comprising a) a first antigen binding molecule comprising i) a first target-binding domain, ii) a first cytokine receptorbinding domain, and iii) a Fc domain; and b) a second antigen binding molecule comprising i) a second target-binding domain, ii) a second cytokine receptor-binding domain, and iii) a Fc domain; wherein the first target-binding domain is capable of binding a first epitope on the target antigen and the second target-binding domain is capable of binding a second epitope on the target antigen, wherein the first and second target-binding domains do not compete for binding on the target antigen; and wherein the first cytokine receptor-binding domain is capable of binding a first cytokine receptor subunit and the second cytokine receptor-binding domain is capable of
  • first and second targetbinding domains are antibody fragments, such as Fv, Fab, scFv, scFab molecules or single domain antibodies.
  • the first target-binding domain comprises a heavy chain variable domain (VHi), a light chain variable domain (VLi), a heavy chain constant domain (CHli) and a light chain constant domain (CLi)
  • the second targetbinding domain comprises a heavy chain variable domain (VH2), a light chain variable domain (VL2), a heavy chain constant domain (CHI 2) and a light chain constant domain (CL2).
  • the first target-binding domain comprises a VHi of SEQ ID NO: 20 and a VLi of SEQ ID NO: 21 and the second target-binding domain comprises a VH2 of SEQ ID NO: 22 and a VL2 of SEQ ID NO: 23, or b) the first target-binding domain comprises a VHi of SEQ ID NO: 22 and a VLi of SEQ ID NO: 23 and the second target-binding domain comprises a VH2 of SEQ ID NO: 20 and a VL2 of SEQ ID NO: 21, or c) the first target-binding domain comprises a VHi of SEQ ID NO: 76 and a VLi of SEQ ID NO: 77 and the second target-binding domain comprises a VH2 of SEQ ID NO: 78 and a VL2 of SEQ ID NO: 79, or d) the first target-binding domain comprises a VHi of SEQ ID NO: 76 and a VLi of SEQ ID NO: 77 and the second target-
  • both the first and second cytokine receptor subunits are subunits of the IFNy receptor complex or, IL-2 receptor complex or IL-7 receptor complex.
  • the pair of antigen binding molecules according to any one of the preceding embodiments, wherein a) the first cytokine receptor-binding domain is capable of binding IFNyRl and the second cytokine receptor-binding domain is capable of binding IFNyR2, or b) the first cytokine receptor-binding domain is capable of binding IFNyR2 and the second cytokine receptor-binding domain is capable of binding IFNyRl; or c) the first cytokine receptor-binding domain is capable of binding IL-2RP and the second cytokine receptor-binding domain is capable of binding IL-2Ry, or d) the first cytokine receptor-binding domain is capable of binding IL-2Ry and the second cytokine receptor-binding domain is capable of binding IL-2RP; or e) the first cytokine receptor-binding domain is capable of binding IL-2Ry and the second cytokine receptor-binding domain is capable of binding IL-7Ra; or f) the first cyto
  • first and second cytokine receptor-binding domains are antibody fragments, such as Fv, Fab, scFv, scFab or single domain antibodies.
  • the first cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 5 and the second cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, or b) the first cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10 and the second cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 5; or c) the first cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 56, SEQ ID NO
  • the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 3 and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 7, or b) the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 7 and the second cytokine receptorbinding domain comprises the sequence of SEQ ID NO: 3; or c) the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 60 and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 62, or d) the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 62 and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 60; or e) the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 186, and the second cytokine receptor-binding domain comprises the sequence of
  • peptide linkers comprise an amino acid sequence selected from SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 279, SEQ ID NO: 280 or SEQ ID NO: 281.
  • the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus the first cytokine receptorbinding domain, VHi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VLi and CLi; and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus the second cytokine receptor-binding domain, VH2, CHI 2 and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C- terminus VL2 and CL2; or b) the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-
  • the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus the first cytokine receptorbinding domain, VHi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VLi and CLi
  • the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus the second cytokine receptor-binding domain, VH2, CHI 2 and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C- terminus VL2 and CL2; or b) the first antigen binding molecule comprises a first polypeptide comprising a first F
  • the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 27 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 30 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 38 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO:
  • the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 260 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124
  • the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 259 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 127;
  • the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 119 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 256
  • the second antigen binding molecule comprises a
  • the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 119 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 257, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 112 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 254; or e) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 260 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises
  • the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 261 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 112 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 254; or h) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 119 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 257, and the second antigen binding molecule comprises
  • the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 86, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 44 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 83
  • the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 88 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 87;
  • the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89
  • the second antigen binding molecule comprises a first polypeptide comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 86, a second polypeptide
  • first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 155 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 154
  • second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 165 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 163.
  • the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 161 and athird polypeptide comprising an amino acid sequence of SEQ ID NO: 160
  • the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence
  • first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 155 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 154
  • second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 165 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 163
  • the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 161 and athird polypeptide comprising an amino acid sequence of SEQ ID NO: 160
  • the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence
  • the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 110 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 113 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 105, and the second antigen binding molecule comprises a first polypeptide
  • first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 136 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 134
  • second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 138 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 137;
  • the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 135 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 134
  • the second antigen binding molecule comprises a
  • the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 146 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 144
  • the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 148 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 147;
  • the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 145 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 144
  • the second antigen binding molecule comprises
  • the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 194 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 193, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 195 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino
  • the first antigen binding molecule comprises a first polypeptide comprising in order from the N- terminus to C-terminus VHi, CHli and a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VLi and CLi, and a third polypeptide comprising in order from the N-terminus to C-terminus the first cytokine receptor-binding domain and a second Fc domain subunit
  • the second antigen binding molecule comprises a first polypeptide comprising in order from the N-terminus to C-terminus VH2, CH 12 and a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VL2 and CL2, and a third polypeptide comprising in order from the N-terminus to C-terminus the second cytokine receptor-binding domain and a second Fc domain
  • the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 32, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 25 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 39
  • the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 34, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 29 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 40
  • the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 34, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 29 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 39
  • the second antigen binding molecule comprises a first polypeptide comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 34, a second polypeptide comprising an amino acid
  • the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 42 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 41
  • the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 45, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 44 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 43
  • the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 46 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 41
  • the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 41
  • the first cytokine receptor-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 282 and a VL domain comprising the amino acid sequence of SEQ ID NO: 283 or VH domain comprising the amino acid sequence of SEQ ID NO: 284 and a VL domain comprising the amino acid sequence of SEQ ID NO: 285, and the second cytokine receptor domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 286 and a VL domain comprising the amino acid sequence of SEQ ID NO: 287 or VH domain comprising the amino acid sequence of SEQ ID NO: 288 and a VL domain comprising the amino acid sequence of SEQ ID NO: 289; or b) the first cytokine receptor-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 286 and a VL domain comprising the amino acid sequence of SEQ ID NO: 287 or VH
  • the first and second cytokine receptor-binding domains are scFv molecules.
  • 52 The pair of antigen binding molecules according to embodiments 51, wherein a) the first cytokine receptor-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 290 and a VL domain comprising the amino acid sequence of SEQ ID NO: 291, and the second cytokine receptor domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 292 and a VL domain comprising the amino acid sequence of SEQ ID NO: 293; or b) the first cytokine receptorbinding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 292 and a VL domain comprising the amino acid sequence of SEQ ID NO: 293, and the second cytokine receptor domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 290 and a VL domain comprising
  • the pair of antigen binding molecules according to embodiments 58, wherein the peptide linkers comprise an amino acid sequence of SEQ ID NO: 280 or SEQ ID NO: 281.
  • the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 267, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 265 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 266, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 272, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 270 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 271; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 267, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a third
  • Example 1 Generation of binders for IFNy and IL-2 receptor subunits
  • the extracellular domain (ECD) of human IFNyRl subunit and the ECD of human IFNyR2 subunit were co-expressed as an Fc-tagged heterodimer based on the knobs-into-holes technology (Fig. 1A, P1AG0843, SEQ ID NO: 48 and SEQ ID NO: 49).
  • ECD of human IL-2RP (CD 122) subunit and human IL-2Ry (common gamma chain; CD 132) subunit were coexpressed as an Fc-tagged heterodimer (Fig. ID, P1AE2657, SEQ ID NO: 80 and SEQ ID NO: 81).
  • the C-terminus of IFNyRl ECD was fused to Fc knob via a G4SG4 linker.
  • An AviTag was included at the C- terminus of the Fc for site-specific biotinylation during co-expression with BirA biotin ligase.
  • the IFNyR2 ECD containing C174S substitution was fused to Fc hole with a G4SG4 linker and the construct contained a C-terminal Twin-Strep-tag.
  • the C-terminus of IL-2RP ECD was fused to Fc knob via a GAQ linker.
  • An AviTag was included at the C-terminus of the Fc for site-specific biotinylation during co-expression with BirA biotin ligase.
  • the IL-2Ry ECD was C-terminally fused to Fc hole with a GAQ linker.
  • the VHH library was constructed using ⁇ 3xl0 8 peripheral blood mononuclear cells (PBMCs) from bleed 2 and ⁇ 6.6xl0 8 PBMCs bleed 3 of a single llama.
  • the library was provided in the TGI E. coli strain encoded in a derivative of the pADL-23c phagemid vector (Antibody Design Labs, San Diego, CA), incorporated by approximately -IxlO 9 independent electroporation events with >95% insert efficiency. Libraries were provided as ⁇ 3.2xlO 10 cfu/mL E. coli in 2xYT medium containing 20% glycerol.
  • E. coli containing the VHH-encoded phagemid library were inoculated in 2xYT medium containing 100 pg/ml ampicillin and 1% glucose. Following infection with VCSM13 helper phage, E. coli were grown in 2xYT medium containing 100 pg/ml ampicillin and 50 pg/ml kanamycin. Following overnight incubation on a shaker at 30°C, cultures were harvested by centrifugation at 8000 rpm for 20 minutes and supernatants were collected. Phage were precipitated in 20% PEG/2.5 M NaCl solution and incubated on ice for 1 hour.
  • Precipitated phage were centrifuged at 8000 rpm for 20 minutes and pellets were resuspended in ddlEO followed by precipitation in PEG/NaCl solution for 1 hour on ice. Phage were centrifuged at 8000 rpm for 20 minutes and pellets were resuspended in PBS containing 20% glycerol.
  • Fc- competitive soluble selections were performed against human IFNyRl antigen (P1AG1987), human IFNyR2 antigen (P1AG6735), human IL-2RP antigen (P1AF1104) and human IL-2Ry antigen (P1AF1106), in the presence of human Fc (Fig. 1G, P1AD4290, SEQ ID NO: 52 and SEQ ID NO: 53).
  • An aliquot of IxlO 11 phage from the llama-derived VHH library was used per antigen.
  • Phage and magnetic streptavidin beads were independently blocked with 1% BSA, 0.1% Tween-20 in phosphate -buffered saline (PBS) to reduce non-specific interactions. All incubations were performed for one hour at 20°C, unless otherwise stated.
  • the biotinylated, Fc-tagged antigens were added at 100 nM concentration to blocked phage in the presence of excess amounts (500 nM) of non-biotinylated Fc ( Figure 2). Following incubation, preblocked Dynabeads were added to capture biotinylated antigens of interest and bound VHH-expressing phage.
  • Fc-specific and non-specific phage were removed by 5 - 10 wash steps in PBS containing 0.1% Tween-20 followed by PBS using a KingFisher magnetic particle processor (Thermo Fisher Scientific).
  • Antigen-specific phage were eluted in 800 pl of 100 mM trimethylamine (TEA) and neutralized by addition of 400 pl of 1 M Tris pH 7.4.
  • TGI E. coli in 2xYT medium were infected with eluted phage, followed by infection with VCSM13 helper phage. Following addition of 100 pg/ml ampicillin and 50 pg/ml kanamycin, infected E. coli were incubated overnight on a shaker at 30°C.
  • Phage were precipitated with PEG/NaCl and used for the next round. Selections were performed for a total of 3 rounds using decreasing target antigen concentrations (100 nM, 50 nM, 25 nM) while soluble Fc concentrations remained constant (500 nM) (Fig. 2).
  • Round 3 enrichment libraries were screened for target specificity vs Fc specificity by ELISA.
  • Individual VHH clones were expressed in 1 ml E. coli cultures in a 96-well plate and supernatants containing soluble VHH were screened for specificity by ELISA.
  • Supernatants were transferred to neutravidin 96- well plates coated with biotinylated Fc (P1AE6073, SEQ ID NO: 51 and SEQ ID NO: 54) or target Fc- tagged biotinylated antigen (P1AG1987, P1AG6735, P1AF1104 or P1AF1106). Following incubation for 1 hour at 20°C, plates were washed 3 times with PBST and 3 times with PBS.
  • HRP -conjugated anti- His antibody (Sigma, Cat. No: A7058) was added to wells at 1:2000 dilution and plates were incubated for 1 hour at room temperature. Plates were washed 3 times with PBST followed by 3 washes with PBS. Plates were developed by adding 1-Step Ultra TMB-ELISA substrate (Thermo Fisher Scientific, Cat. No: 34028) and reactions were quenched with sulfuric acid. Absorbance at 450 nm was measured using a Tecan infinite M1000 Pro and reference absorbance values were measured at 650 nm. Reference- subtracted absorbance values at 450 nm were represented as stacked bar charts for IFNyRl -specific VHHs (Fig.
  • ELISA data suggested >90% of clones were target-specific and VHH from round 3 enrichment libraries were subsequently cloned into bispecific heavy chain antibody formats.
  • IFNyR activation is achieved by IFNy-mediated cross-linking of the IFNyRl and IFNyR2 receptor chains.
  • a VHH domain with IFNyRl specificity was paired with an IFNyR2 -specific VHH by fusing to knob-into-hole Fc chains via a flexible linker generating IFNyRl -IFNyR2 bispecific heavy chain antibodies.
  • IL-2R activation is achieved by Remediated cross-linking of the IL-2RP and IL-2Ry receptor chains.
  • VHH domain with IL-2RP specificity was paired with an IL-2Ry-specific VHH by fusing to knob-into-hole Fc chains via a flexible linker generating IL-2Rp-IL-2Ry bispecific heavy chain antibodies.
  • the round 3 VHH library enriched for IFNyRl- or IL-2RP- specificity were cloned in bulk using Gibson assembly to achieve format conversion of VHH by fusing to Fc knob via a 5(G 4 S) linker ( Figure 4A).
  • Expi293 Using the Expi293 system, a 5x5 matrix was generated to express 25 unique knob-into-holes bispecific heavy chain antibodies comprising IFNyRl and IFNyR2 VHH pairs and 25 unique knob-into-holes bispecific heavy chain antibodies comprising IL-2RP VHH and IL-2Ry VHH pairs ( Figure 5).
  • Expi293 cells were transiently transfected following manufacturer’s recommendation (Expi293TM Expression System Kit, Thermo Fisher Scientific, Cat. No: A 14635) and supernatants containing secreted soluble bispecific heavy chain antibodies were collected. The bispecific heavy chain antibodies were purified by Protein A resin and further characterized for functional activity using reporter cell assays. 2.2 Functional characterization of bispecific heavy chain antibodies
  • the HEK Blue IFNy reporter cell assay (Invivogen) was used to screen bispecific heavy chain antibodies comprising different IFNyRl and IFNyR2 VHH pairs and identify IFNyR agonists.
  • the HEK Blue IL-2 reporter cell assay (Invivogen) was used to screen bispecific heavy chain antibodies comprising different IL-2RP and IL-2Ry VHH pairs and identify IL-2R agonists.
  • the in vitro HEK Blue IFNy reporter cell system (Invivogen, Cat. No: hkb-ifhg) reports IFNyR signaling via a STAT1- inducible secreted alkaline phosphatase (SEAP) system.
  • SEAP STAT1- inducible secreted alkaline phosphatase
  • IFNyRl-IFNyR2 bispecific heavy chain antibodies (P1AH1877-P1AH1890, see Table 1) were tested for dose -dependent IFNyR signaling and agonistic IL-2Rp-IL-2Ry bispecific heavy chain antibodies were tested for dose-dependent IL-2R signaling (see Table 2).
  • Table 1 IFNyRl-IFNyR2 bispecific heavy chain antibodies generated by the 5x5 expression matrix showing IFNy agonistic activity in Fig. 6A.
  • a concentration range spanning from 100 nM to 0.01 nM with 10-fold serial dilution steps was used with HEK Blue IFNy cells according to the Invivogen HEK Blue protocol. Absorbance at 650 nm was measured and responses were plotted using GraphPad software ( Figure 7). Graphs were separated according to VHH clone with IFNyRl specificity, with recombinant human IFNy (recIFNy; SEQ ID NO: 26) and anti-FAP IgG fused to IFNy homodimer (P1AF3574; SEQ ID NO: 24 and SEQ ID NO: 25) serving as reference molecules.
  • recIFNy recombinant human IFNy
  • P1AF3574 anti-FAP IgG fused to IFNy homodimer
  • Table 2 IL-2Rp-IL-2Ry bispecific heavy chain antibodies generated by the 5x5 expression matrix showing IL-2R activity in Fig. 6B.
  • a concentration range of 20 nM, 0.8 nM and 0.032 nM was used with HEK Blue IL-2 reporter cells according to the Invivogen HEK Blue protocol.
  • Agonistic activity of the IL-2 mimetics was compared with IL-2v fused to anti-PDl IgG (PDl-IL2v; P1AE4422, SEQ ID NO: 83, SEQ ID NO: 84 and SEQ ID NO: 85).
  • the VHH pairs displayed potent IL-2R agonism in a dose-dependent manner ( Figure 9).
  • the use of a VHH pair to activate the receptor provided opportunities for conditionally active IL-2 mimetics via molecular split approaches.
  • MHC-I and PD-L1 were selected as tumor cell surface biomarkers to characterize the TME- modulating potential of functional bispecific VHH pairs.
  • MHC-I upregulation is a downstream response following IFNyR stimulation and may provide an increase in antigenicity on tumor cells, while PD-L1 upregulation is another downstream indicator of functional IFNy signaling.
  • MHC-I and PD-L1 expression levels were quantified by flow cytometry and data were analyzed using FlowJo software.
  • Mean fluorescent intensity (MFI) values were blank-subtracted and normalized to recombinant IFNy responses. Upregulation of MHC-I and PD-L1 was measured in three tumor cell types: MKN45 (Fig. 10A, 10B), BxPC-3 (Fig. 10C, 10D) and COR-L105 (Fig. 10E, 10F).
  • IFNyR signaling via an agonistic IFNyR VHH pair and IL-2R signaling via an agonistic IL-2R VHH pair provided opportunities to engineer conditionally active IFNy and IL-2 mimetics.
  • P1AH1884 was selected as a potent IFNy mimetic (Fig.
  • VHH pairs were further engineered to generate conditionally active agonists.
  • Fibroblast activation protein FAP
  • a serine protease described with high expression in cancer- associated stromal tissue of immune-deserted tumors was selected as a target to achieve cold tumor penetration.
  • FAP Fibroblast activation protein
  • a pair of agonistic VHH domains mimicking IFNy were distributed onto two distinct molecules to inactivate the VHH pair, whilst incorporating a FAP-dependent assembly strategy to restore agonistic VHH activity in the presence of FAP (Fig. 11A).
  • VHH domains of the P1AH1884 heavy chain antibody were fused to the N-terminus of the VH or VL domains of the anti- FAP binders via a flexible 5(G 4 S) linker, resulting in 8 possible format configurations (Fig. 11B-I).
  • upregulation of MHC-I and PD-L1 was quantified in three FAP expression scenarios.
  • the A549 lung adenocarcinoma cell line was used to test for untargeted, FAP-independent activity in a FAP-negative setting.
  • A549 cells engineered for ectopic FAP expression were used to characterize FAP-dependent activity in cis, while both FAP -positive and FAP- negative cell lines were differentially labelled and co-cultured to compare cis and trans activity of FAP- dependent IFNy mimetics.
  • Split dual-targeted IFNy mimetics were serially diluted to span a concentration range from 10 nM to 1 pM. Recombinant IFNy and the intact, parental VHH pair deficient in FAP targeting (Pl AHI 884, format depicted in Fig.
  • VHHs were fused to the N-terminus of the opposite Fc chain via a 3(G4S) linker (Fig. 13A-D) or to the C-terminus of the same Fc chain via a 3(G4S) linker (Fig. 13E-H).
  • the sets were tested in vitro in the three FAP expression scenarios, as described previously. FAP-independent activity was observed with the reference molecules (recombinant IFNy and P1AH1884, Fig.
  • IFNy agonists comprising different lengths of linker between the VHH and the fusion point were explored ( concepts described in Fig. 15).
  • the molecule P1AJ8572 was replaced with P1AI0187 due to a batch failure in production, the activity of IFNy agonists was measured by MHC-I upregulation.
  • activity in an untargeted scenario (A549 FAP -negative cells) as well as in cis-targeted (IFNy activity in A549 FAP-positive cells co-cultured with A549 FAP -negative cells) and trans-targeted (IFNy activity in A549 FAP-negative cells co-cultured with A549 FAP-positive cells) scenarios were assessed.
  • test compounds four dilution series, starting concentration of 50’000 pM were added to the assay plate.
  • the upregulation of chemoattractant CXCL10 was measured following 24h incubation with U87 and PBMCs. Briefly, 20’000 U87 cells per well were seeded in a 96 well plate flat bottom. After overnight incubation at 37°C and 5% CO2 to allow the cells to adhere, 50pL/well of PBMCs at 2 * 10 6 PBMCs/well was added to the wells. Lastly, 50pL/well of control medium or compound dilution was added to the wells. Samples were incubated at 37°C and 5% CO2 for 24h before assay readout with the human IP 10 R&D ELISA KIT (cat# DY266) accordingly to the manufacturer’s instructions. While the degree of upregulation varied between the EGFR-targeted IFNy agonists, all pairs demonstrated upregulation of the chemoattractant CXCL10 (Fig. 18).
  • a strategy was devised to harness cell surface markers, reportedly enriched within the tumor environment, to scaffold the assembly of the IL-2 agonistic VHH domain pair.
  • the aim was to bind two proximal epitopes on a single target using two non-competing anti-target binders (Fig. 19A).
  • PD-1 was selected as the target for several reasons. First, upregulation of PD-1 is observed upon T cell activation, providing an opportunity to enhance selectivity towards the tumor-specific effector T cell population.
  • PD-L1 -competitive anti -PD-1 binders would provide immune checkpoint inhibition mediated by blockade of the PD1/PD-L1 signaling cascade.
  • CD4 T cells were sorted from healthy donor PBMCs with CD4 beads (Miltenyi, Cat. No: 130-045-101) and activated for 3 days in presence of 1 pg/ml plate bound anti-CD3 (overnight precoated, clone OKT3, Cat. No: 317315, BioLegend) and 1 pg/ml of soluble anti-CD28 (clone CD28.2, Cat. No: 302923, BioLegend) antibodies to induce PD1 expression. Three days later, the cells were harvested and washed several times to remove endogenous cytokines and half of the cells were labelled with CTV (5 pM, 5 min at 20°C; Cat.
  • CTV 5 pM, 5 min at 20°C; Cat.
  • the cells were acquired at the FACS BD-LSR Fortessa (BD Bioscience).
  • the frequency of STAT-5P was determined with FlowJo (V10) and plotted with GraphPad Prism.
  • the dose-response curves on PD-1 + T cells provide information on the potency of the PD 1-based IL-2R agonists in signaling through the IL-2R.
  • the dose-response curves on T cells pre-treated with a competing anti-PDl antibody, to prevent the PD1 mediated delivery show the potency of the PD1- based IL-2R agonist molecules in providing IL-2R signaling independently from PD-1 expression.
  • PD1- IL2v showed reduced activity on T cells in absence of PD1 binding (pre-blocked) (Fig.20, black solid and black dashed lines).
  • PBMC proliferation and activation assay In order the study the biological activity of PD-1 targeted IL-2R agonists downstream of STAT-5 phosphorylation, the molecules of interest were tested in a PBMC proliferation and activation assay. Briefly, human PBMCs from healthy donors were isolated from buffy coats (Zurich Blood Donation Center in accordance with the Declaration of Helsinki) using standard density-gradient isolation over LymphoprepTM (STEMCELL Technologies). Freshly isolated PBMCs were stained with CcllTracc TM Violet Cell proliferation Kit (Thermo Fischer, Cat. No. C34557).
  • CD4 clone SK3, BD, Cat. No 563550
  • PD-1 clone EH12.1, BD, Cat. No 612791
  • CD56 clone HCD56, Biolegend, Cat. No 318334
  • CD122 clone 5H4, Biolegend, Cat. No 339006
  • y5 TCR clone 11F2, BD, Cat. No 655410
  • CD25 clone BC96, Biolegend, Cat. No 302610
  • CD3 clone OKT3, Biolegend, Cat. No 317340
  • CD8 clone SKI, BD, Cat.
  • Fig. 21 different cell types were identified according to lineage marker expression on the cell surface by flow cytometry (Fig. 21). Since the goal is to generate PD-1 targeted IL-2R agonists that specifically deliver IL-2 signals to PD-1 expressing cells while sparring PD-1 negative cells, the prevalence of PD-1 + IL-2R + target cells was evaluated within the PBMC fraction. Across the two tested donors, a subset of CD8 T cells and y5 T cells (10-25%) co-expressed PD-1 and IL-2RP (Fig. 21D and E) whereas NK cells expressed high levels of IL-2RP and almost no PD-1 (Fig. 2 IF).
  • PD-1 targeted IL- 2R agonist (P1AH6814+P1AI1593) showed no activity on the phenotype and proliferation ofNK cells with high IL-2R expression and negligible PD-1.
  • the PD-1 targeted IL-2R agonist (PlAH6814+PlAI1593) was further tested in the human breast BC004 patient derived cells orthotopically injected into humanized BRGS47 mice.
  • BC004 PDX material human breast carcinoma
  • Tumor fragments were digested with Collagenase D and DNase I (Roche, Switzerland), and BC004 cells were used for transplantation.
  • IxlO 6 cells per animal were injected intra mammary fat pad in 100 pl of RPMI cell culture medium (Gibco, Germany) into the flank of mice using a 1 ml tuberculin syringe (BD Biosciences, Germany).
  • Fully humanized BRGS47 female mice (Jackson Labs, USA) were maintained under specific-pathogen- firee condition with daily cycles of 12 h light / 12 h darkness according to committed guidelines (GV- Solas; Felasa; TierschG). Continuous health monitoring was carried out on a regular basis.
  • mice were injected intra mammary fat pad on study day 0 with IxlO 6 of BC004 cells, randomized and weighed. Forty days after the tumor cell injection (tumor volume > 200 mm 3 ), mice were injected i.v. with the following molecules: P1AI1593, P1AH6814, Pembrolizumab 1 mg/kg, PDl-IL2v 0.1 mg/kg and FOLR1-TCB 0.3 mg/kg or Vehicle once a week for four weeks.
  • the PD-1 targeted IL-2R agonist split constructs P1AH6814 and P1AI1593 were given once a week but on consecutive days.
  • mice were also tested with Vehicle or FolRl-TCB as single agent or the combination of the PD-1 targeted IL-2R agonist, PDl-IL2v or Pembrolizumab with FOLR1-TCB. Due to the chimeric nature of the BRGS47 humanized mice, no HLA-restricted T cell responses occur in the BC004 breast orthotopic model. In order to see anti-tumor response in this mouse study the test compounds needed to be combined with the bispecific FOLR1 -TCB molecule that triggers signal one first and as such allows the evaluation of the additive effect of immunomodulator or checkpoint inhibitor compounds. All mice were injected i.v. with 200 pl of the appropriate solution.
  • mice in the Vehicle group were injected with Histidine Buffer and the treatment groups with the different constructs.
  • the stock solutions were diluted with Histidine Buffer when necessary (see Table 3).
  • Tumor growth measurements were evaluated with a caliper three times a week and plotted with GrahPad Prism software as volume in mm 3 +/- SEM.
  • Figure 24 shows that the combination FOLR1-TCB 0.3 mg/kg + PDl-IL2v mimetic (PlAI1593day 1 + P1AH6814 day 2) mediated superior efficacy in terms of tumor growth inhibition compared to FOLR1- TCB single agent treatment and the other combination groups: FOLR1-TCB 0.3 mg/kg + PDl-IL2v and FOLR1-TCB 0.3 mg/kg + Pembrolizumab.
  • Table 3 Overview of administered compounds.
  • LAG3 -targeting constructs as another example besides PD-1 targeting of T cell in cis.
  • Two noncompeting LAG3-specific Fabs were selected and engineered to mediate assembly of the agonistic VHH pair.
  • VHH domains of the P1AH1177 heavy chain antibody were fused to the N-terminus of the VH or VL domains of the anti-LAG3 binders via a flexible 5(G4S) linker, resulting in eight possible format configurations (P1AJ5654 - P1AJ5661) which can be paired to produce eight assembly combinations (Fig. 27).
  • IL-2R IL-2R agonists
  • tumor EGFR, HER2, HER3
  • FAP stroma
  • the pair of agonistic VHH domains of heavy chain antibody Pl AHI 177 mimicking IL-2 were distributed onto two distinct molecules to inactivate the VHH pair, whilst incorporating a targetdependent assembly strategy to restore agonistic VHH activity in the presence of each target (FAP, EGFR, HER2, HER3).
  • Two non-competing target-specific Fabs were selected for each target and engineered to mediate assembly of the agonistic VHH pair.
  • VHH domains of the P1AH1177 heavy chain antibody were fused to the N-terminus of the VH or VL domains of the anti- FAP binders via a flexible 5(G4S) linker, resulting in eight possible format configurations (P1AJ5092 - P1AJ5099) which can be paired to produce eight assembly combinations (Fig. 29).
  • molecular concepts targeting EGFR were designed with two non-competing anti-EGFR Fabs.
  • the eight possible format configurations (P1AJ4165 - P1AJ4172) can be paired to produce eight assembly combinations (Fig. 31).
  • IL-2RPy Bioassay cells express physiological levels of IL-2RP and IL-2Ry similar to human T cells, with a STAT5 phosphorylation luminescent reporter system.
  • target cells A549 FAP negative, A549 FAP positive, A-431 EGFR high, OE19 EGFR medium, LoVo EGFR medium, MDA-MB-231 HER2/HER3 negative or OE19 HER2/HER3 high
  • test compounds 4-fold dilution series, starting concentration of 100 nM
  • 10,000 IL-2RPy Bioassay reporter cells per well were added to the assay plate.
  • EGFR expression was assessed by flow cytometry using a PE -conjugated anti-human EGFR antibody (Biolegend, Cat. No. 352904) on A -431, LoVo and OE19 target cell lines (Fig. 32A).
  • a PE -conjugated anti-human EGFR antibody Biolegend, Cat. No. 352904
  • Fig. 32A Of the eight tested EGFR-targeted IL-2R agonists, one pair (P1AJ4165 + P1AJ4172) showed consistent activity in the presence of medium or high EGFR expressing target cells (Fig. 32B, 32C and 32D). High target expression seems to be required for IL-2R signaling in a trans setting.
  • Fig. 38A While none of the tested combinations showed activity in the absence of PD-1 expression (Fig. 38A), they demonstrated varying levels of activity in PD-1 expressing reporter cells (Fig. 38B).
  • the strongest IL-2R signaling was observed with anti-IL2R agonists comprising VHH domains (P1AH6814+P1AI1593) followed by the anti-IL2R agonists comprising scFv domains (P1AL9310+1AL9311).
  • Anti-IL2R Fab agonists displayed the weakest potency, indicating that VHH- based agonists may be ideal candidates to bring IL2RP and IL2Ry receptor chains in close proximity and trigger IL-2R signaling.
  • x- ray crystal structures comprising Fab: target complexes were used to measure the distance between N- terminal residues of two non-competing anti-target Fabs. Distances were measured for PD-1 (Table 4), EGFR (Table 5), HER2 (Table 6) and HER3 (Table 7) targets.
  • Table 4 Distances (A) measured between the N-termini of the heavy chain (HC) and light chain (LC) of two anti -PD-1 Fabs. The distance between the most proximal combination of N-terminal fusion points is highlighted in grey.
  • Table 5 Distances (A) measured between the N-termini of the heavy chain (HC) and light chain (LC) of two anti-EGFR Fabs. The distance between the most proximal combination of N-terminal fusion points is highlighted in grey.
  • Table 6 Distances (A) measured between the N-termini of the heavy chain (HC) and light chain (LC) of two anti-HER2 Fabs. The distance between the most proximal combination of N-terminal fusion points is highlighted in grey.
  • Table 7 Distances (A) measured between the N-termini of the heavy chain (HC) and light chain (LC) of two anti-HER3 Fabs. The distance between the most proximal combination of N-terminal fusion points is highlighted in grey.
  • the extracellular domain (ECD) of human IL-7Ra (CD 127) subunit was expressed as an Fc-tagged monomer based on the knobs-into-holes technology (Fig. 39A, P1AI1009, SEQ ID NO: 208 and SEQ ID NO: 209).
  • the C-terminus of IL-7Ra ECD was fused to Fc knob via an ASGS linker.
  • An AviTag was included at the C-terminus of the Fc for site-specific biotinylation during co-expression with BirA biotin ligase.
  • VHH libraries were constructed using lymphocytes isolated from two alpacas.
  • the libraries were provided in the TGI E. coli strain encoded in a derivative of the pHEN4 phagemid vector (Hassanzadeh- Ghassabeh et al., 2010), yielding 5-7xl0 8 clones per library. Libraries were provided in 2xYT medium containing 20% glycerol.
  • E. coli containing the VHH-encoded phagemid library were inoculated in 2xYT medium containing 100 pg/ml ampicillin and 1% glucose. Following infection with VCSM13 helper phage, E. coli were grown in 2xYT medium containing 100 pg/ml ampicillin and 50 pg/ml kanamycin. Following overnight incubation on a shaker at 30° C, cultures were harvested by centrifugation at 8000 rpm for 20 minutes and supernatants were collected. Phage were precipitated in 20% PEG/2.5 M NaCl solution and incubated on ice for 1 hour.
  • Precipitated phage were centrifuged at 8000 rpm for 20 minutes and pellets were resuspended in ddH2O followed by precipitation in PEG/NaCl solution for 1 hour on ice. Phage were centrifuged at 8000 rpm for 20 minutes and pellets were resuspended in PBS containing 20% glycerol.
  • Fc competitive soluble selections were performed against human IL-7Ra antigen (P1AI1009) in the presence of human Fc (Fig. 39B, P1AD4290).
  • An aliquot of IxlO 12 phage from each of the two alpaca- derived VHH libraries was used per target antigen.
  • Phage and magnetic streptavidin beads (Dynabeads; M-280 Streptavidin, Invitrogen, Cat. No: 11205D) were independently blocked with 1% BSA, 0.1% Tween-20 in phosphate-buffered saline (PBS) to reduce non-specific interactions.
  • Antigen-specific phage were eluted in 800 pl of 100 mM trimethylamine (TEA) and neutralized by addition of 400 pl of 1 M Tris pH 7.4.
  • TGI E. coli in 2xYT medium were infected with eluted phage, followed by infection with VCSM13 helper phage.
  • VCSM13 helper phage Following addition of 100 pg/ml ampicillin and 50 pg/ml kanamycin, infected E. coli were incubated overnight on a shaker at 30° C. Phage were precipitated with PEG/NaCl and used for the next round.
  • alpaca library 1 and alpaca library 2 were performed for a total of four rounds using decreasing target antigen concentrations (100 nM, 50 nM, 25 nM, 12.5 nM) while soluble Fc concentrations remained constant (500 nM) (Fig. 39C).
  • IL-7R activation is achieved by IL7-mediated cross-linking of the IL-7Ra (CD 127) and IL-2Ry (CD 132; common gamma chain) receptor chains.
  • IL-7Ra-specific VHH domain was paired with previously identified VHH domains with IL-2Ry specificity (IL- 2Ry_2, IL-2Ry_3, IL-2Ry_4 and IL-2Ry_5).
  • VHH were fused to knob-into-hole Fc chains via a flexible linker generating IL-7Ra-IL-2Ry bispecific heavy chain antibodies.
  • IL-7Ra-specific VHH Sixteen IL-7Ra-specific VHH were selected from rounds 3 and 4 of phage display enrichment and four previously identified IL-2Ry-specific VHH were selected. Format conversion was performed by Gibson assembly cloning by fusing IL-7Ra- specific VHH to Fc knob via a 5(G4S) linker (Fig. 41 A) and by fusing IL-2Ry-specific VHH to Fc hole via a 5(G4S) linker (Fig. 41B). During the assembly, a suitable signal sequence and vector compatible with mammalian expression were included. Primers, PCR conditions and Gibson assembly reactions were defined as per New England Biolabs’ NEBuilder HiFi DNA Assembly protocol (Cat. No: E5510).
  • Expi293 system was used to express bispecific heavy chain antibodies in a 16x4 matrix to generate 64 unique antibodies comprising IL-7Ra VHH and IL-2Ry VHH pairs (Fig. 41C).
  • Expi293 cells were transiently transfected following manufacturer’s recommendation (Expi293 TM Expression System Kit, Thermo Fisher Scientific, Cat. No: A 14635) and supernatants containing secreted soluble bispecific heavy chain antibodies were collected.
  • the bispecific heavy chain antibodies were purified by Protein A resin and further characterized for functional activity using reporter cell assays.
  • Table 8 IL-7Ra-IL-2Ry bispecific heavy chain antibodies generated by the 16x4 expression matrix. 4.7 Functional characterization of bispecific heavy chain antibodies
  • the HEK Blue IL-7 reporter cell assay (Invivogen) was used to screen bispecific heavy chain antibodies comprising different IL-7Ra and IL-2Ry VHH pairs and identify IL-7R agonists.
  • the in vitro HEK Blue IL-7 reporter cell (Invivogen, Cat. No: hkb-il7) recapitulates the human IL-7R signaling pathway via expression of the IL-7Ra and IL-2Ry chains, effectors of the downstream signaling cascade (JAK3 and STAT5) and a STAT5 -inducible secreted alkaline phosphatase (SEAP) reporter system.
  • QuantiBlue substrate (Invivogen, Cat.
  • absorbance at 650 nm is measured, correlating with SEAP levels and IL-7R activity.
  • the 64 heavy chain antibodies and reference molecules were incubated with HEK Blue IL-7 cells for 20 hours.
  • IL-7R activity was quantified by addition of QuantiBlue reagent, according to the manufacturer's recommendation.
  • the absorbance at 650 nm was measured and fold response/background was plotted as bar charts (Fig 42A-D). All 64 molecules showed IL-7R agonism with a broad range of activity.
  • agonistic VHH pairs (IL-7Ra_l, IL-7Ra_2, IL-7Ra_3, IL-7Ra_5, IL-7Ra_6, IL-7Ra_7, IL-7Ra_10, IL-7Ra_l l, IL-7Ra_13, IL-7Ra_14, IL- 7Ra_15, IL-7Ra_16 in combination with IL2Ry_2) were selected to further engineer for PD-l-targeted biparatopic assembly.
  • the PD-1 cell surface marker was selected to scaffold the assembly of the IL-7 mimetic VHH pair.
  • the aim was to bind two proximal epitopes on PD-1 using two non-competing anti-PD-1 binders to bridge the assembly of a functional IL- 7 mimetic (Fig. 43A).
  • VHH fusion points on the two PD-1 binders were selected according to N-terminal residue proximity of the two Fabs determined by x-ray crystal structures (Table 4).
  • the 12 selected IL- 7Ra-specific VHH were fused to the VL of anti-PD-1 binder 1 via a 5(G4S) linker (P1AL0544 - P1AL0556, Fig. 43B) and the selected VHH domain with IL-2Ry specificity was fused to the VH of anti-PD-1 binder 2 (P1AH6814; Fig. 43C).
  • IL-7R agonists targeted to PD-1
  • the phosphorylation of STAT5 on pre-activated CD4 T cells after 60 min incubation with test compounds was determined as previously described.
  • the IL-2 mimetic heavy chain antibody (P1AH1177), a PD-1 dependent biparatopic IL-2 mimetic (P1AH6814 + P1AI1593) and PDl-IL2v (P1AE4422) were used as reference molecules.
  • a non-binding DP47 antibody (P1AD3966, SEQ ID NO: 230 and SEQ ID NO: 231) was used as negative control.

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Abstract

The application relates to pairs of antigen binding molecules, each molecule comprising a target-binding domain, a cytokine receptor-binding domain and an Fc domain, wherein the target-binding domains simultaneously bind the target antigen and the cytokine receptor-binding domains bind subunits of a cytokine receptor complex. Biparatopic assembly of the cytokine receptor-binding domains in presence of the target antigen allows to selectively activate a cytokine receptor and effectively mimic cytokine activity in a targeted manner.

Description

AGONISTIC SPLIT ANTIBODIES
Field of the Invention
The application relates to pairs of antigen binding molecules, each molecule comprising a target-binding domain, a cytokine receptor-binding domain and an Fc domain, wherein the target-binding domains simultaneously bind the target antigen and the cytokine receptor-binding domains bind subunits of a cytokine receptor complex. Biparatopic assembly of the cytokine receptor-binding domains in presence of the target antigen allows to selectively activate a cytokine receptor and effectively mimic cytokine activity in a targeted manner.
Background of the Invention
In recent years, immunotherapy treatments for cancer have grown dramatically, and cancer immunotherapy is becoming a major strategy for combating disease. For many cancer types, immune checkpoint modulators, including anti-PD-1, have become the standard of care. However, despite all the advances made in the field of cancer immunotherapy treatment in recent years, a significant proportion of patients still fail to respond to available immunotherapies because of intrinsic or adaptive mechanisms of resistance. Cancer immunotherapy patients with non-inflamed immune conditions are more likely to not respond to immunotherapies. Immune cell infiltration in tumors has been shown to correlate with the ability of patients to respond to immunotherapy treatments. Developing new therapies aimed at increasing immune cell infiltration and enhancing immunogenicity is essential for patients.
In parallel with the above developments, cytokines have gained great interest as potential cancer treatments. IFNy (interferon gamma or IFN-y) is a cytokine that is mainly produced by activated lymphocytes, such as CD4+ and CD8+ T cells, and natural killer cells (NK cells) in response to immune or inflammatory stimuli. IFNy is a homodimer and its receptors (IFNyRl and IFNyR2) are expressed across hematopoietic and non-hematopoietic cells. On the cell surface, IFNyRl is stably expressed whereas IFNyR2 is differentially expressed and regulates IFNy. When IFNy binds to its receptors, Janus kinases JAK1 and JAK2 are recruited and activated, which phosphorylate and activate STAT1. A phosphorylated STAT1 translocates to the nucleus, binds promoters, and modulates gene transcription under the control of IFNy. In contrast to other cancer therapies currently on the market or in development, IFNy can act on both tumor cells and immune cells including T cells and dendritic cells. The effects of IFNy on different cell types have several benefits, which include 1) enhanced expression of MHC-I molecules on the surface of both tumor cells and antigen-presenting cells, 2) the recruitment of immune cells to the tumor site through the induction of CXCL9, CXCL10 and CXCL11 production and 3) the ability to increase tumor antigen cross-presentation with a subsequent enhancement of an anti-tumor immune response. In addition to these effects, IFNy promotes the formation of a Th 1 environment, monocyte differentiation, macrophage polarization, and angiogenesis. As IFNy receptors are expressed on a wide range of cell types, sink effects may diminish its activity.
IFNy can also show undesirable side effects. In addition, problems with administration, bioavailabilty and short half-life may arise. Thus, there is a need for new molecules able to selectively activate the IFNy pathway at the tumor site.
Targeting the Interleukin 2 (IL-2) pathway as a cancer immunotherapy strategy has a long and ramified history, serving as testament to both the triumphs and the complexities of this clinical approach. IL-2 is a cytokine that activates lymphocytes and natural killer (NK) cells. Efficacy in the clinic is often overshadowed by reports of IL-2 -associated toxicity linked to peripheral T cell activity and CD25- mediated complications.
Cytokines are powerful immune modulators that initiate signaling through receptor dimerization, but natural cytokines have some limitations as therapeutics. Low stability and difficulties in the production process are just some of them. It is known for some time that antibodies can induce signaling on cells, and therefore substitute a natural ligand.
In the recent literature there is growing evidence that bringing together heterodimeric cytokine receptors using both antibody and non-antibody based protein scaffolds is a viable strategy to mimic the activity of native cytokines. For instance, Moraga and colleagues provide an early example of using diabodies as surrogate ligands for Erythropoietin receptor (EpoR) (Moraga et al. Cell 160, 1196-1208 (2015)). Researchers at Teneobio combined heavy chain-only antibodies (VHHs fused to an Fc domain) against different epitopes on the Interleukin-2 receptors (IL-2RP and IL-2Ry) into a bispecific effectorless IgG4 Fc (CHI deleted) using knob-in-hole technology. While monospecific anti-IL-2Rp or anti-IL-2Ry alone or in mixture did not induce STAT5 phosphorylation on human CD8+ T cells, bispecific anti-IL-2RPy antibodies showed varying levels of agonist activity (Harris, K. E., et al. Sci Rep 11(1): 10592 (2021)). A similar approach as described by Teneobio, was undertaken by scientists at Synthekine, which described the functional induction of signaling of the two interleukin receptors (IL-2RP and IL-2Ry) via single domain antibodies (sdAbs; WO 2022/032040 Al), and reviewed by Saxton and colleagues (Saxton, R. A., et al. Nat Rev Drug Discov. 22, 21-37 (2022)). This approach was further expanded by researchers at Stanford University where the authors presented a strategy to discovery cytokine surrogate agonists by using modular ligands like VHH or scFv human for interleukin-2/15, type-I interferon, and interleukin- 10 receptors. Interestingly, they also identified functional, non-natural assemblies like the IL-2Rp/IL-10Rp heterodimer (Yen, M., et al. Cell 185(8): 1414-1430 el419 (2022)). The same authors also discuss a structure-based approach for the decoupling of the pro- and anti-inflammatory functions of interleukin- 10 (Saxton, R. A., et al. Science 371(6535) (2021)). Two academic research groups from Czech Republic and Israel reported together the discovery of non-antibody based scaffolds that mimic the cytokine IFNZ. Combinatorial libraries derived from several established small protein scaffolds were used to identify variants capable of binding to the IFNXR1 or IL-10RP and induce functional signaling (Kolarova, L., et al. FEBS J 289(9): 2672-2684 (2022)). Reducing the size of the agonistic modules was addressed by researchers at Medikine. They obtained molecules selected from peptide libraries by screens designed to identify molecules binding simultaneously to the Ra and yc subunits of the human IL-7 receptor. Those peptides, with an molecular weight of less than 5 kDa fused to an IgGi-Fc domain exhibits biological properties similar to those of IL-7 in vitro, and when administered to non-human primates (Dower, W., et al. Journal for Immuno Therapy of Cancer 8 (Suppl 3): A341-A342 (2020)).
Due to the pleiotropic effects of cytokines, there is a need for novel approaches to selectively activate cytokine receptors and effectively mimic cytokine activity.
Summary of the Invention
The present invention relates to novel antigen binding molecules comprising cytokine receptor-binding domains, which, under the desired conditions of biparatopic assembly on a target antigen, serve as cytokine mimetics selectively activating the receptor pathway.
The present invention relates to a pair of antigen binding molecules that specifically bind to a target antigen, comprising a) a first antigen binding molecule comprising i) a first target-binding domain, ii) a first cytokine receptor-binding domain, and iii) a Fc domain; and b) a second antigen binding molecule comprising i) a second target-binding domain, ii) a second cytokine receptor-binding domain, and iii) a Fc domain;wherein the first target-binding domain is capable of binding a first epitope on the target antigen and the second target-binding domain is capable of binding a second epitope on the target antigen, wherein the first and second target-binding domains do not compete for binding on the target antigen; and wherein the first cytokine receptor-binding domain is capable of binding a first cytokine receptor subunit and the second cytokine receptor-binding domain is capable of binding a second cytokine receptor subunit. In one aspect, the first and second target-binding domains are antibody fragments, such as Fv, Fab, scFv, scFab molecules or single domain antibodies. In one aspect, the first and second target-binding domains are Fab molecules. In one aspect, the first target-binding domain comprises a heavy chain variable domain (VHi), a light chain variable domain (VLi), a heavy chain constant domain (CHli) and a light chain constant domain (CLi), and wherein the second target-binding domain comprises a heavy chain variable domain (VH2), a light chain variable domain (VL2), a heavy chain constant domain (CHI2) and a light chain constant domain (CL2). In one aspect, the first and/or second target-binding domain is a cross-Fab molecule.
In one aspect, the first and second target-binding domains specifically bind to a tumor-associated antigen or a T-cell antigen. In one aspect, the first and second target-binding domains specifically bind to FAP, PD-1, Her2, Her3, LAG-3 or EGFR. In one aspect, a) the first target-binding domain comprises a VHi of SEQ ID NO: 20 and a VLi of SEQ ID NO: 21 and the second target-binding domain comprises a VH2 of SEQ ID NO: 22 and a VL2 of SEQ ID NO: 23, or b) the first target-binding domain comprises a VHi of SEQ ID NO: 22 and a VLi of SEQ ID NO: 23 and the second target-binding domain comprises a VH2 of SEQ ID NO: 20 and a VL2 of SEQ ID NO: 21, or c) the first target-binding domain comprises a VHi of SEQ ID NO: 76 and a VLi of SEQ ID NO: 77 and the second target-binding domain comprises a VH2 of SEQ ID NO: 78 and a VL2 of SEQ ID NO: 79, or d) the first target-binding domain comprises a VHi of SEQ ID NO: 78 and a VLi of SEQ ID NO: 79 and the second target-binding domain comprises a VH2 of SEQ ID NO: 76 and a VL2 of SEQ ID NO: 77; or e) the first target-binding domain comprises a VHi of SEQ ID NO: 93 and a VLi of SEQ ID NO: 94 and the second target-binding domain comprises a VH2 of SEQ ID NO: 78 and a VL2 of SEQ ID NO: 79; or f) the first target-binding domain comprises a VHi of SEQ ID NO: 78 and a VLi of SEQ ID NO: 79 and the second target-binding domains comprises a VH2 of SEQ ID NO: 93 and a VL2 of SEQ ID NO: 94, or g) the first target-binding domain comprises a VHi ofSEQ ID NO: 130 and aVLi ofSEQ ID NO: 131 and the second target-binding domain comprises a VH2 of SEQ ID NO: 132 and a VL2 of SEQ ID NO: 133; or h) the first target-binding domain comprises a VHi of SEQ ID NO: 132 and a VLi of SEQ ID NO: 133 and the second target-binding domains comprises a VH2 of SEQ ID NO: 130 and a VL2 of SEQ ID NO: 131 ; or i) the first target-binding domain comprises a VHi of SEQ ID NO: 140 and a VLi of SEQ ID NO: 141 and the second target-binding domain comprises a VH2 of SEQ ID NO: 142 and a VL2 of SEQ ID NO: 143; or j) the first targetbinding domain comprises a VHi of SEQ ID NO: 142 and a VLi of SEQ ID NO: 143 and the second target-binding domains comprises a VH2 of SEQ ID NO: 140 and a VL2 of SEQ ID NO: 141; or k) the first target-binding domain comprises a VHi of SEQ ID NO: 114 and a VLi of SEQ ID NO: 115 and the second target-binding domain comprises a VH2 of SEQ ID NO: 116 and a VL2 of SEQ ID NO: 117; or 1) the first target-binding domain comprises a VHi of SEQ ID NO: 116 and a VLi of SEQ ID NO: 117 and the second target-binding domains comprises a VH2 of SEQ ID NO: 114 and a VL2 of SEQ ID NO: 115; or m) the first target-binding domain comprises a VHi of SEQ ID NO: 150 and a VLi of SEQ ID NO: 151 and the second target-binding domain comprises a VH2 of SEQ ID NO: 152 and a VL2 of SEQ ID NO: 153; or n) the first target-binding domain comprises a VHi of SEQ ID NO: 152 and a VLi of SEQ ID NO: 153 and the second target-binding domains comprises a VH2 of SEQ ID NO: 150 and a VL2 of SEQ ID NO: 151.
In one aspect, both the first and second cytokine receptor subunits are subunits of the IFNy receptor complex or, IL-2 receptor complex or IL-7 receptor complex. In one aspect, a) the first cytokine receptor-binding domain is capable of binding IFNyRl and the second cytokine receptor-binding domain is capable of binding IFNyR2, or b) the first cytokine receptor-binding domain is capable of binding IFNyR2 and the second cytokine receptor-binding domain is capable of binding IFNyRl ; or c) the first cytokine receptor-binding domain is capable of binding IL-2RP and the second cytokine receptor-binding domain is capable of binding IL-2Ry, or d) the first cytokine receptor-binding domain is capable of binding IL-2Ry and the second cytokine receptor-binding domain is capable of binding IL- 2RP; or e) the first cytokine receptor-binding domain is capable of binding IL-2Ry and the second cytokine receptor-binding domain is capable of binding IL-7Ra; or f) the first cytokine receptor-binding domain is capable of binding IL-7Ra and the second cytokine receptor-binding domain is capable of binding IL-2Ry.
In one aspect, the first and second cytokine receptor-binding domains are antibody fragments, such as Fv, Fab, scFv, scFab or single domain antibodies. In one aspect, the first and second cytokine receptorbinding domains are VHH domains. In one aspect, a) the first cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 5 and the second cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, or b) the first cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10 and the second cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 5; c) the first cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58 SEQ ID NO: 60, and the second cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64 and SEQ ID NO: 65; or d) the first cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64 and SEQ ID NO: 65 and the second cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58 SEQ ID NO: 60; or e) the first cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, and the second cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65; or f) the first cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, and the second cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192.
In one aspect, a) the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 3 and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 7, or b) the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 7 and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 3;or c) the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 60 and the second cytokine receptorbinding domain comprises the sequence of SEQ ID NO: 62, or d) the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 62 and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 60; or e) the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 186, and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 62, or f) the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 62, and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 186.
In one aspect, the Fc domains of the first and second antigen binding molecules comprise a first Fc domain subunit and a second Fc domain subunit. In one aspect, the Fc domains of the first and second antigen binding molecules are IgG, particularly an IgGi, Fc domains. In one aspect, the Fc domains of the first and second antigen binding molecules are human Fc domains. In one aspect, the Fc domains of the first and second antigen binding molecules comprise a modification promoting the association of the first and the second subunit of the Fc domains. In one aspect, the Fc domains of the first and second antigen binding molecules comprise one or more amino acid substitution that reduces binding to an Fc receptor and/or effector function.
In one aspect, the cytokine receptor-binding domains are fused via peptide linkers to their respective fusion points. In one aspect, the peptide linkers comprise an amino acid sequence selected from SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 279, SEQ ID NO: 280 or SEQ ID NO: 281. In one aspect, the first cytokine receptor-binding domain is fused at its C-terminus to the N-terminus of VHi or VLi of the first target-binding domain and the second cytokine receptor-binding domain is fused at its C-terminus to the N-terminus of VH2 or VL2 of the second target-binding domain. In one aspect, a) the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus the first cytokine receptorbinding domain, VHi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VLi and CLi; and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus the second cytokine receptor-binding domain, VH2, CHI 2 and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C- terminus VL2 and CL2; or b) the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VHi, CH li and a second Fc domain subunit, and a third polypeptide comprising in order from the N- terminus to C-terminus the first cytokine receptor-binding domain, VLi and CLi; and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VH2, CHI 2 and a second Fc domain subunit, a third polypeptide comprising in order from the N-terminus to C-terminus the second cytokine receptor-binding domain, VL2 and CL2; or c) the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus the first cytokine receptor-binding domain, VHi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VLi and CLi ; and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VH2, CHI 2 and a second Fc domain subunit, a third polypeptide comprising in order from the N-terminus to C-terminus the second cytokine receptor-binding domain, VL2 and CL2; wherein VHi, VLi, CHli and CLi form the first target-binding domain and VH2, VL2, CHI 2, and CL2, form the second target-binding domain.
In one aspect, a) the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus the first cytokine receptor-binding domain, VHi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VLi and CLi, and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus the second cytokine receptor-binding domain, VH2, CHI 2 and a second Fc domain subunit, and a third polypeptide comprising in order from the N- terminus to C-terminus VL2 and CL2; or b) the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VHi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus the first cytokine receptor-binding domain, VLi and CLi, and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VH2, CHI 2 and a second Fc domain subunit, a third polypeptide comprising in order from the N-terminus to C-terminus the second cytokine receptor-binding domain, VL2 and CL2; or c) the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus the first cytokine receptor-binding domain, VHi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VLi and CLi, and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VH2, CHI 2 and a second Fc domain subunit, a third polypeptide comprising in order from the N-terminus to C-terminus the second cytokine receptor-binding domain, VL2 and CL2; or d) the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus the first cytokine receptor-binding domain, VLi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VHi and CLi, and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus the second cytokine receptor-binding domain, VH2, CHh and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VL2 and CL2; or e) the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VLi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus the first cytokine receptor-binding domain, VHi and CLi, and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VH2, CHh and a second Fc domain subunit, a third polypeptide comprising in order from the N-terminus to C-terminus the second cytokine receptorbinding domain, VL2 and CL2; or f) the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus the first cytokine receptor-binding domain, VLi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VHi and CLi, and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VH2, CHI 2 and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus the second cytokine receptor-binding domain, VL2 and CL2 ; or g) the first antigen binding molecule comprising a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a VLi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus a first cytokine receptor-binding domain, VHi and CLi, and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a second cytokine receptor-binding domain, VH2, CHI 2 and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VL2 and CL2; wherein VHi, VLi, CHli and CLi form the first target-binding domain and VH2, VL2, CHI 2, and CL2, form the second target-binding domain.
In one aspect, the first and the second target-binding domains specifically bind to FAP, and the first and second cytokine receptor subunits are subunits of the IFNy receptor complex. Thus, In one aspect, a) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 27 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 30 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 38 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 37 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or c) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO : 31 , and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 33; or d) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 35, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 36; or e) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 27 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 33; or f) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 30 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 31; or g) the first antigen binding molecule comprising a first polypeptide comprises an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 38 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 36; or h) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 37 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 35.
In one aspect, first and the second target-binding domains specifically bind to EGFR, and the first and second cytokine receptor subunits are subunits of the IFNy receptor complex. In one aspect, a) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 260 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 259 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 127; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 119 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 256, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 122 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 255; or c) the first antigen binding molecule comprises a first polypeptide comprising -I lan amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 261 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO:
258 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 127; or d) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 119 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 257, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 112 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 254; or e) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 260 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 112 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 255; or f) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 119 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 256, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO:
259 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 127; or g) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 261 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 112 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 254; or h) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 119 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 257, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 258 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 127. In one aspect, the first and the second target-binding domains specifically bind to PD-1, and the first and second cytokine receptor subunits are subunits of the IL-2 receptor complex. In one aspect, a) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 86, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 44 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 83, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 88 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 87; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 92, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 44 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 91; or c) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 96 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 95, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 101 and athird polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or d) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 99 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 98, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 88 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 102; or e) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 96 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 97, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 103 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or f) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 100 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 98, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 88 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 104; or g) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 98 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 99, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 101 and athird polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or h) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 96 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 95, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 88 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 102; or i) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 100 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 98, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 103 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or j) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 96 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 97, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 88 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 104.
In one aspect, the first and the second target-binding domains specifically bind to LAG-3, and the first and second cytokine receptor subunits are subunits of the IL-2 receptor complex. In one aspect, a) first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 155 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 154, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 165 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 163.; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 161 and athird polypeptide comprising an amino acid sequence of SEQ ID NO: 160, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 158 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 159; or c) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 155 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 156, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 164 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 163; or d) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 162 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 160, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 158 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 157; or e) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 161 and athird polypeptide comprising an amino acid sequence of SEQ ID NO: 160, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 165 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 163; or f) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 155 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 154, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 158 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 159; or g) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 162 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 160, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 164 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 163; or h) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 155 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 156, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 158 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 157.
In one aspect, the first and the second target-binding domains specifically bind to EGFR, and the first and second cytokine receptor subunits are subunits of the IL-2 receptor complex. In one aspect, a) first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 155 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 154, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 165 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 163; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 161 and athird polypeptide comprising an amino acid sequence of SEQ ID NO: 160, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 158 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 159.; or c) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 155 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 156, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 164 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 163; or d)the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 162 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 160, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 158 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 157; or e) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 161 and athird polypeptide comprising an amino acid sequence of SEQ ID NO: 160, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 165 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 163; or f) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 155 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 154, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 158 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 159; or g)the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 162 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 160, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 164 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 163; or h) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 155 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 156, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 158 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 157.
In one aspect, the first and the second target-binding domains specifically bind to FAP, and the first and second cytokine receptor subunits are subunits of the IL-2 receptor complex. In one aspect, a) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 110 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 113 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 105, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 109; or c) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 111 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 112 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or d) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 107, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 108; or e) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 110 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 109; or f) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 105, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 113 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or g) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 111 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 108; or h) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and athird polypeptide comprising an amino acid sequence of SEQ ID NO: 107, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 112 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
In one aspect, the first and the second target-binding domains specifically bind to Her2, and the first and second cytokine receptor subunits are subunits of the IL-2 receptor complex. In one aspect, a) first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 136 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 134, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 138 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 137; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 135 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 134, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 138 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 139.
In one aspect, the first and the second target-binding domains specifically bind to Her3, and the first and second cytokine receptor subunits are subunits of the IL-2 receptor complex. In one aspect, a) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 146 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 144, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 148 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 147; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 145 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 144, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 148 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 149.
In one aspect, the first and the second target-binding domains specifically bind to PD-1, and the first and second cytokine receptor subunits are subunits of the IL-7 receptor complex. In one aspect, a) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 194 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 193, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 195 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or c) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 196 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or d) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 197 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or d)the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 198 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or e) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 199 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or f) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 200 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or g) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 201 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or h) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 202 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or i) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 203 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or j) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 204 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or k) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 205 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89.
In one aspect, the first target-binding domain is fused at its C-terminus of CHI i to the N-terminus of the first Fc domain subunit and the first cytokine receptor-binding domain is fused at its C-terminus to the N-terminus of the second Fc domain subunit and the second target-binding domain is fused at its C- terminus of CHE to the N-terminus of the first Fc domain subunit and the second cytokine receptorbinding domain is fused at its C-terminus to the N-terminus of the second Fc domain subunit. In one aspect, a) the first antigen binding molecule comprises a first polypeptide comprising in order from the N-terminus to C-terminus VHi, CHli and a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VLi and CLi, and a third polypeptide comprising in order from the N-terminus to C-terminus the first cytokine receptor-binding domain and a second Fc domain subunit, and the second antigen binding molecule comprises a first polypeptide comprising in order from the N-terminus to C-terminus VH2, CH 12 and a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VL2 and CL2, and a third polypeptide comprising in order from the N-terminus to C-terminus the second cytokine receptor-binding domain and a second Fc domain subunit; or b) the first antigen binding molecule comprises a first polypeptide comprising in order from the N-terminus to C-terminus VLi, CHI 1 and a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VHi and CLi, and a third polypeptide comprising in order from the N-terminus to C-terminus the first cytokine receptor-binding domain and a second Fc domain subunit, and the second antigen binding molecule comprises a first polypeptide comprising in order from the N-terminus to C-terminus VH2, CHh and a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VL2 and CL2, and a third polypeptide comprising in order from the N-terminus to C-terminus the second cytokine receptorbinding domain and a second Fc domain subunit; wherein VHi, VLi, CHI 1 and CLi form the first targetbinding domain and VH2, VL2, CHh, and CL2, form the second target-binding domain. In one aspect, a) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 32, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 25 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 39, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 34, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 29 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 40; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 34, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 29 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 39, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 32, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 25 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 40; or c) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 237, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 238, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or d) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 235, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 236, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or e) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 239, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 240, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or f) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 235, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 237, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or g) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 236, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 235, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or h) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 240, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 239, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
In one aspect, the first target-binding domain is fused at its C-terminus of CHli to the N-terminus of one of the Fc domain subunits and the first cytokine receptor-binding domain is fused at its N-terminus to the C-terminus of the same Fc domain subunit and the second target-binding domain is fused at its C- terminus of CHE to the N-terminus of one of the Fc domain subunits and the second cytokine receptorbinding domain is fused at its N-terminus to the C-terminus of the same Fc domain subunit. In one aspect, a) the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a VHi, a CHli, a second Fc domain subunit and a the first cytokine receptor-binding domain, and a third polypeptide comprising in order from the N-terminus to C-terminus a VLi and a CLi ; and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a VH2, a CHE, a second Fc domain subunit and a the second cytokine receptor-binding domain, and a third polypeptide comprising in order from the N- terminus to C-terminus VL2 and CL2; or b) the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a VLi, a CHE, a second Fc domain subunit and a the first cytokine receptorbinding domain, and a third polypeptide comprising in order from the N-terminus to C-terminus a VHi and a CLi; and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a VH2, a CHh, a second Fc domain subunit and the second cytokine receptor-binding domain, and a third polypeptide comprising in order from the N-terminus to C-terminus a VL2 and a CL2; or c) the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a VLi, a CHli, a second Fc domain subunit and a the first cytokine receptor-binding domain, and a third polypeptide comprising in order from the N-terminus to C-terminus a VHi and a CLi ; and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a VH2, a CHI 2, a second Fc domain subunit and a the second cytokine receptor-binding domain, and a third polypeptide comprising in order from the N-terminus to C- terminus VL2 and CL2; wherein VHi, VLi, CHli and CLi form the first target-binding domain and VH2, VL2, CHh, and CL2, form the second target-binding domain. In one aspect, a) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 42 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 41, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 45, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 44 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 43; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 46 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 41, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 47, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 44 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 43; or c) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 246 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 249 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or d) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 242 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 245 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or e) the first antigen binding molecule comprising a first polypeptide comprises an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 250 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 253 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or f) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 248 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 247 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or g) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 244 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 243 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or h) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 252 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 251 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or i) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 42 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 42, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 245 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
In one aspect, the first and second cytokine receptor-binding domains of the pair of antigen binding molecules are Fab molecules. In one aspect, a) the first cytokine receptor-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 282 and a VL domain comprising the amino acid sequence of SEQ ID NO: 283 or VH domain comprising the amino acid sequence of SEQ ID NO: 284 and a VL domain comprising the amino acid sequence of SEQ ID NO: 285, and the second cytokine receptor domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 286 and a VL domain comprising the amino acid sequence of SEQ ID NO: 287 or VH domain comprising the amino acid sequence of SEQ ID NO: 288 and a VL domain comprising the amino acid sequence of SEQ ID NO: 289; or b) the first cytokine receptor-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 286 and a VL domain comprising the amino acid sequence of SEQ ID NO: 287 or VH domain comprising the amino acid sequence of SEQ ID NO: 288 and a VL domain comprising the amino acid sequence of SEQ ID NO: 289, and the second cytokine receptor domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 282 and a VL domain comprising the amino acid sequence of SEQ ID NO: 283 or VH domain comprising the amino acid sequence of SEQ ID NO: 284 and a VL domain comprising the amino acid sequence of SEQ ID NO: 285.16.
In one aspect, the first and second cytokine receptor-binding domains of the pair of antigen binding molecules are scFv molecules. In one aspect, a) the first cytokine receptor-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 290 and a VL domain comprising the amino acid sequence of SEQ ID NO: 291, and the second cytokine receptor domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 292 and a VL domain comprising the amino acid sequence of SEQ ID NO: 293; or b) the first cytokine receptor-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 292 and a VL domain comprising the amino acid sequence of SEQ ID NO: 293, and the second cytokine receptor domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 290 and a VL domain comprising the amino acid sequence of SEQ ID NO: 291.
In one aspect, the Fc domains of the first and second antigen binding molecules comprising Fabs or scFv molecules as cytokine receptor-binding domains comprise a first Fc domain subunit and a second Fc domain subunit. In one aspect, the Fc domains of the first and second antigen binding molecules are IgG, particularly an IgGi, Fc domains. In one aspect, the Fc domains of the first and second antigen binding molecules are human Fc domains. In one aspect, the Fc domains of the first and second antigen binding molecules comprise a modification promoting the association of the first and the second subunit of the Fc domains. In one aspect, the Fc domains of the first and second antigen binding molecules comprise one or more amino acid substitution that reduces binding to an Fc receptor and/or effector function. In one aspect, the cytokine receptor-binding domains are fused via peptide linkers to their respective fusion points. In one aspect, the peptide linkers comprise an amino acid sequence of SEQ ID NO: 280 or SEQ ID NO: 281. In one aspect, a) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 267, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 265 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 266, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 272, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 270 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 271; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 267, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 265 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 266, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 274, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 270 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 273; or c) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 269, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 265 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 268, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 268, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 270 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 271; or d) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 269, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 265 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 268, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 274, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 270 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 273. In one aspect, the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 276, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 28 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 275, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 277, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 28 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 278.
Brief Description of the Figures
Figure 1. Schematic representations of antigens used in llama immunization, phage display and screening for the isolation of human IFNyRl, IFNyR2, IL-2RP and IL-2Ry-specific single domain antibodies. For llama immunization a heterodimer formed of the extracellular domain (ECD) of human IFNyRI fused to biotinylated Fc knob and human IFNyR2 ECD fused to Fc hole (A) and a heterodimer formed of the ECD of human IL-2RP fused to biotinylated Fc knob and human IL-2Ry ECD fused to Fc hole (D) were generated. For phage display monovalent human IFNyRl fused to biotinylated Fc (B), monovalent human IFNyR2 fused to biotinylated Fc (C), monovalent human IL-2RP fused to biotinylated Fc (E), monovalent human IL-2Ry fused to biotinylated Fc (F) and soluble Fc (G) were generated.
Figure 2. Workflow for the enrichment of single domain antibodies with binding specificity for human cytokine receptor subunits by phage display.
Figure 3. Specificity screening of single domain antibodies by ELISA. A random set of soluble VHH domains selected for human IFNyRl specificity following three rounds of phage display were tested for binding to immobilized human IFNyRl -Fc and immobilized human Fc (A). VHH domains with human IFNyR2 specificity following three rounds of phage display were also tested for antigen specificity by ELISA (B). VHH domains selected for human IL-2RP specificity following three rounds of phage display were tested for binding to immobilized human IL-2Rp-Fc and immobilized human Fc (C). VHH domains with human IL-2Ry specificity following three rounds of phage display were also tested for antigen specificity by ELISA (D). Absorbance at 450 nm indicating the binding response to each target is represented as a stacked bar chart.
Figure 4. Format conversion of single domain antibody fragments. Round 3 VHH library variants with IFNyRl or IL-2RP specificity were fused to Fc-Knob and cloned into a mammalian cell expression vector using Gibson cloning method (A). Round 3 VHH library variants with IFNyR2 or IL-2Ry specificity were fused to Fc-hole using the same method (B).
Figure 5. Bispecific heavy chain antibody comprising IFNyRl -IFNyR2 or IL-2Rp-IL-2Ry VHH domain pairs. VHH domains with IFNyRl or IL-2RP specificity were fused to Fc-knob and VHH domains targeting IFNyR2 or IL-2Ry were fused to Fc-hole. Flexible 5(G4S) linkers were used to fuse VHH moieties to Fc chains. Bispecific heavy chain antibodies were generated via knob-into-hole assembly of Fc chains. Effector functions of the Fc were silenced via incorporation of P329G LALA mutations in the CH2 domain.
Figure 6. Functional screening of bispecific heavy chain antibodies comprising IFNyRl -IFNyR2 or IL- 2Rp-IL-2Ry VHH domain pairs. HEK-Blue IFNy cells were incubated with IFNy agonistic bispecific heavy chain antibodies comprising IFNyRl and IFNyR2 VHH domain pairs generated using a 5x5 bispecificity matrix (A). HEK-Blue IL-2 reporter cells were incubated with IL-2 agonistic bispecific heavy chain antibodies comprising IL-2RP and IL-2Ry VHH pairs generated using a 5x5 bispecificity matrix (B). IFNyR activity and IL-2R activity was quantified by absorbance at 650 nm and fold response/background is shown for each treatment.
Figure 7. Characterisation of dose-dependent response for IFNy agonistic heavy chain antibodies. Previously identified IFNyR agonists (Fig. 6A, Table 1) were screened for dose -dependent IFNyR activity in HEK-Blue IFNy cells, characterized by absorbance at 650 nm. The responses of different IFNy agonistic heavy chain antibodies, are grouped according to the anti-IFNyRl VHH clone, i.e. within each graph the anti-IFNyR2 VHH clone is variable while the anti-IFNyRl VHH clone remains constant: IFNyRl_l clone (A), IFNyRl_2 clone (B), IFNyRl_3 clone (C) and IFNyRl_5 clone (D). Responses were compared to recombinant human IFNy (black dashed line) and FAP-IFNy (P1AF3574; light grey dashed line).
Figure 8. Comparison of EC50 values for IFNy agonistic heavy chain antibodies. EC50 values were derived from HEK-Blue assays (Fig. 7) using GraphPad Prism software. Values are depicted in nanomolar concentration with recombinant human IFNy highlighted as a reference (dotted line).
Figure 9. Characterization of dose -dependent response for IL-2R agonistic heavy chain antibodies. Previously identified IL-2R agonists (Fig. 6B, Table 2) were screened for dose-dependent IL-2R agonism in HEK-Blue IL-2 cells. Anti-PD 1 -IL2v (P1AE4422) was used as a reference molecule and each molecule was incubated with reporter cells at the following concentrations: 20 nM, 0.8 nM, 0.032 nM shown from left to right. IL-2R activity was quantified by absorbance at 650 nm; mean values from technical triplicates are shown with error bars representing standard deviation.
Figure 10. MHC-I and PD-L1 expression on tumor cells following 72h treatment of IFNy agonists. Previously identified IFNyR agonists (P1AH1877-P1AH1890) and reference molecules (recombinant human IFNy and FAP-IFNy) were incubated with tumor cells at the following concentration of treatment: 100, 10, 1 and 0.1 nM shown from left to right for each treatment. Measurements were blank- subtracted and normalized to recombinant human IFNy response at the highest concentration (100 nM). Responses are depicted for MHC-I expression on MKN45 cells (A), PD-L1 expression on MKN45 cells (B), for MHC-I expression on Bxpc3 cells (C), PD-L1 expression on Bxpc3 cells (D), MHC-I expression on CorL105 cells (E) and PD-L1 expression on CorL105 cells (F).
Figure 11. Concept of split dual-targeted IFNyR agonists. FAP-dependent biparatopic assembly of split IFNy mimetics (A). Note that for illustrative simplification, only one monomer of the FAP dimer is represented in the inset. VHH domains with IFNyRl and IFNyR2 specificity were fused to the N- terminus of two different anti-FAP binding domains, via VH or VL fusion points, resulting in eight configurations within the permutation space: IFNyRl -VHH fused to the VH of FAP binder 1 (B), IFNyR2-VHH fused to the VH of FAP binder 2 (C), IFNyRl -VHH fused to the VL of FAP binder 1 (D), IFNyR2-VHH fused to the VL of FAP binder 2 (E), IFNyR2-VHH fused to the VH of FAP binder 1 (F), IFNyRl-VHH fused to the VH of FAP binder 2 (G), IFNyR2-VHH fused to the VL of FAP binder 1 (H) and IFNyRl-VHH fused to the VL of FAP binder 2 (I). All molecules have the same Fc characteristics as described in Figure 5.
Figure 12. Assessment of IFNyR activity mediated by FAP-dependent split IFNyR agonists. The eight split IFNy mimetics (Fig. 11B-I) were paired in biparatopic assembly combinations and tested for IFNyR agonism characterized by MHC-I and PD-L1 upregulation. FAP-negative A549 cells were tested for untargeted, non-specific MHC-I (A, B) and PD-L1 upregulation (C, D) by split IFNy mimetics. A549 FAP -positive cells were co-cultured with differentially labeled A549 FAP-negative cells to assess FAP- dependent activity of IFNy mimetics in cis and trans: MHC-I upregulation in cis (E and F), PD-L1 upregulation in cis (G and H), MHC-I upregulation in trans (I and J) and PD-L1 upregulation in trans (K and L). Recombinant IFNy and the IFNy agonistic heavy chain antibody Pl AHI 884 (as depicted in Fig. 5) were used as a reference. Median fluorescent intensity (MFI) of MHC-I and PD-L1 expression levels were analyzed in FlowJo; mean values from technical duplicates are shown with error bars representing standard deviation.
Figure 13. Additional formats of split dual-targeted IFNyR agonists. VHH domains with IFNyRl and IFNyR2 specificity were distanced from the anti -FAP binders via fusion to the N-terminus of the opposite Fc chain, resulting in four configurations: IFNyRl and FAP binder 1 (A), IFNyR2 and FAP binder 2 (B), IFNyR2 and FAP binder 1 (C) and IFNyRl and FAP binder 2 (D). Alternatively, the VHH domains with IFNyRl and IFNyR2 specificity were distanced from the anti-FAP binders via fusion to the C-terminus of the same Fc chain, resulting in four configurations: IFNyRl and FAP binder 1 (E), IFNyR2 and FAP binder 2 (F), IFNyR2 and FAP binder 1 (G) and IFNyRl and FAP binder 2 (H). The anti-FAP binders in Fig. 13E-H comprise CrossFab VH/VL engineering (binder 1) and CH1-CL charges (binder 2) to subsequently allow for additional pairings. All molecules have the same Fc characteristics as described in Figure 5.
Figure 14. Assessment of IFNyR activity mediated by additional FAP-dependent split IFNyR agonists. Four combinations of molecules depicted in Fig. 13 and reference molecules were tested for IFNyR agonism characterized by MHC-I and PD-L1 upregulation. A549 cells lacking FAP expression were tested for FAP-independent MHC-I upregulation (A) and PD-L1 upregulation (B) of split IFNy mimetics. A549 FAP-positive cells were co-cultured with differentially labeled A549 FAP-negative cells to assess FAP-dependent activity of IFNy mimetics by MHC-I upregulation in cis (C), PD-L1 upregulation in cis (D), MHC-I upregulation in trans (E) and PD-L1 upregulation in trans (F). Recombinant IFNy and an IFNy agonistic heavy chain antibody (P1AH1884) were used as reference molecules. Median fluorescent intensity (MFI) of MHC-I and PD-L1 expression levels were analyzed in FlowJo; mean values from technical duplicates are shown with error bars representing standard deviation.
Figure 15. Concept of split FAP-targeted IFNy agonists with different linker lengths. A single VHH domain with specificity for either IFNyRl or IFNyR2 was engineered to be linked to the hinge region at the N-terminus (top row) or to the C-terminus (bottom row) of the Fc region within an antibody construct containing a FAP-binding Fab domain. The constructs utilized one of two different FAP- binding Fab domains that do not compete for binding. Flexible linkers, consisting of 5, 15, or 25 amino acids, denoted by the number following a semicolon after the molecule ID, were used to connect the Fc region to the VHH domain. This resulted in a series of 24 unique configurations which can be paired to produce 12 functional FAP-targeted IFNy agonists: P1AJ8564 + P1AJ8567, P1AJ8560 + P1AJ8563 and P1AJ8568 + P1AJ8571 (A); P1AJ8566 + P1AJ8565, P1AJ8562 + P1AJ8561 and P1AJ8570 + P1AJ8569 (B); P1AJ8576 + P1AJ8579, P1AI0187 + P1AJ8575 and P1AJ8580 + P1AJ8583 (C); P1AJ8578 + P1AJ8577, P1AJ8574 + P1AJ8573 and P1AJ8582 + P1AJ8581 (C).
Figure 16. Assessment of IFNyR activity mediated by additional FAP-dependent split IFNyR agonists. Twelve combinations of molecules depicted in Fig. 15 and reference molecules were tested for IFNyR agonism characterized by MHC-I upregulation. Test compounds were incubated with A549 FAP- negative cells alone (untargeted condition; A and D) or in combination with A549 FAP-positive cells, wherein cis signaling (FAP binding on A549 FAP-positive cells and activity on A549 FAP-positive cells; B and E) and trans signaling (FAP binding on A549 FAP-positive cells and activity on A549 FAP- negative cells, C and F) was assessed. Recombinant IFNy and an IFNy agonistic heavy chain antibody (P1AH1884) were used as reference molecules. Median fluorescent intensity (MFI) of MHC-I expression levels were analyzed in FlowJo; mean values from technical duplicates are shown with error bars representing standard deviation.
Figure 17. Concept of split EGFR-targeted IFNy agonists. VHH domains with IFNyRl and IFNyR2 specificity were fused to the N-terminus of two different anti-EGFR binding domains, via VH or VL fusion points, resulting in eight individual format configurations (P1AK5514 - P1AK5521) that can be paired to produce the eight functional assemblies depicted: P1AK5520 + P1AK5519 (A), P1AK5516 + P1AK5515 (B), P1AK5521 + P1AK5518 (C), P1AK5517 + P1AK5514 (D), P1AK5520 + P1AK5515 (E), P1AK5516 + P1AK5519 (F), P1AK5521 + P1AK5514 (G) and P1AK5517 + P1AK5518 (H).
Figure 18. Functional activity of EGFR-targeted IFNy agonists. Test compounds were incubated with target cells together with PBMCs and CXCL10 production was assessed after 24h stimulation. Recombinant IFNy was used as reference. Shown are mean +/- SD of technical duplicates for each tested molecule concentration. Figure 19. Concept of split dual-targeted IL-2 agonists. PD-1 -dependent biparatopic assembly of split IL-2 mimetics (A). VHH domains with IL-2RP and IL-2Ry specificity were fused to the N-terminus of two different anti-PD-1 binding domains, via VH or VL fusion points: IL-2RP-VHH fused to the VH of PD-1 binder 1 (B), IL-2Ry-VHH fused to the VL of PD-1 binder 2 (C), IL-2RP-VHH fused to the VL of PD-1 binder 1 (D) and IL-2Ry-VHH fused to the VH of PD-1 binder 2 (E). An IL-2 agonistic heavy chain antibody (F) and PD-l-IL2v (G) served as reference molecules. All molecules have the same Fc characteristics as described in Figure 5.
Figure 20. Phosphorylation of STAT5 on CD4 T cells after 15 min and 60 min incubation with IL-2R agonists. Activated T cells expressing PD-1 (PD-1+ subset) and activated T cells preblocked with anti- PD-1 antibodies (PD-1- subset) were differentially labeled and treated with two combinations of split dual-targeted IL-2 mimetics: P1AH6850 + P1AH6813 (A-D), P1AH6814 + P1AI1593 (E-H). The IL-2 mimetic heavy chain antibody (Pl AH 1177) and PDl-IL2v (P1AE4422) were used as reference molecules. MFI and frequency of STAT5-P+ cells were measured by FACS and responses were shown for PD-1+ subset (solid line) and PD-T subset (dashed line).
Figure 21. Flow cytometry gating strategy and baseline receptor expression. Isolated PBMCs from two healthy donors were stained with viability dye and fluorescent dye -conjugated antibodies against cell surface antigens. Gating strategy to identify five distinct cell types: NK cells and NKT cells (A); CD4 T cells and CD8 T cells (B); and y5 T cells (C). Representative plots showing PD-1 and IL-2RP coexpression on CD8 T cells (D), y5 T cells (E) and NK cells (F).
Figure 22. CD25 expression on PBMC subsets induced by PD-1 targeted IL-2R agonist. Frequency of CD25 expressing CD8 T cells (A), y5 T cells (B) and NK cells (C) were determined by flow cytometry after five days of test compounds co-incubation with freshly isolated CTV -labelled PBMCs. PDl-IL2v (P1AE4422) and Fc-VHH (P1AH1177) were used as reference. A non-binding DP47 antibody (P1AD3966) was used as negative control. Shown are mean values +/- SEM of two PBMC donors combined and technical duplicates for each tested molecule concentration.
Figure 23. Proliferation of PBMC subsets induced by PD-1 targeted IL-2R agonist. Frequency of proliferating CD8 T cells (A), y5 T cells (B) and NK cells (C) were determined by flow cytometry after five days of test compounds co-incubation with freshly isolated CTV -labelled PBMCs. Proliferating cells were identified based on cell division visualized with CTV staining. PDl-IL2v (P1AE4422) and Fc-VHH (P1AH1177) were used as reference. A non-binding DP47 antibody (P1AD3966) was used as negative control. Shown are mean values +/- SEM of two PBMC donors combined and technical duplicates for each tested molecule concentration. Figure 24. Presents the results of an efficacy experiment evaluating PD-1 targeted IL-2R agonist (P1AI1593 and P1AH6814), PDl-IL2v immunoconjugate (P1AE4422) and Pembrolizumab in combination with FOLR1-TCB Mab. The BC004 human breast carcinoma PDX cells were injected intra mammary fat pad in humanized BRGS47 mice to study tumor growth inhibition in a breast orthotopic xenograft model. The amount of antibodies injected per mouse in mg/kg is the following: P1AI1593, P1AH6814 and Pembrolizumab 1 mg/kg, PDl-IL2v 0.1 mg/kg and FOLR1-TCB 0.3 mg/kg. The antibodies were injected i.v. once weekly for 4 weeks. The combination FOLR1-TCB 0.3 mg/kg + PD1- IL2targeted IL-2R agonists (P1AI1593 day 1 + PlAH6814day 2) mediated superior efficacy in terms of tumor growth inhibition compared to FOLR1-TCB single agent treatment and the other combination groups: FOLR1-TCB 0.3 mg/kg + PDl-IL2v and FOLR1-TCB 0.3 mg/kg + Pembrolizumab.
Figure 25. Concept of split PD-1 -targeted IL-2R agonists with alternative anti-PD-1 Fab binders. VHH domains with IL-2RP and IL-2Ry specificity were fused to the N-terminus of two different anti-PD-1 binding domains, via VH or VL fusion points, resulting in eight individual format configurations (P1AK2599; P1AK2798; P1AK2799; P1AK2802; P1AK2803; P1AK2806; P1AK2809; P1AK2810) that can be paired to produce the eight functional assemblies depicted: P1AK2802 + P1AK2599 (A), P1AK2809 + P1AK2799 (B), P1AK2803 + P1AK2806 (C), P1AK2810 + P1AK2798 (D), P1AK2809 + P1AK2599 (E), P1AK2802 P1AK2 799 (F), P1AK2810 + P1AK2806 (G) and P1AK2803 + P1AK2798 (H).
Figure 26. Functional activity of PD-1 -targeted IL-2R agonists with alternative anti-PD-1 Fab binders. Test compounds were incubated with HEK Blue IL-2 wt cells (A) or HEK Blue IL-2 human PD-1 cells (B) for 21h at 37°C and 5% CO2. PDl-IL2v (P1AE4422) and Fc-VHH (P1AH1177) were used as reference. A non-binding DP47 antibody (P1AD3966) was used as negative control. IL-2R signaling was measured via absorbance at 650 nm using QUANTI-Blue reagent. Shown are mean absorbance values +/- SEM of technical duplicates for each tested molecule concentration.
Figure 27. Concept of split LAG-3 -targeted IL-2R agonists. VHH domains with IL-2RP and IL-2Ry specificity were fused to the N-terminus of two different anti-LAG-3 binding domains, via VH or VL fusion points, resulting in eight individual format configurations (P1AJ5654 - P1AJ5661) that can be paired to produce the eight functional assemblies depicted: P1AJ5654 + P1AJ5661 (A), P1AJ5658 + P1AJ5657 (B), P1AJ5655 + P1AJ5660 (C), P1AJ5659 + P1AJ5656 (D), P1AJ5658 + P1AJ5661 (E), P1AJ5654 + P1AJ5657 (F), P1AJ5659 (G) and P1AJ5655 + P1AJ5656 (H).
Figure 28. Functional activity of LAG-3 -targeted IL-2R agonists. PDl-IL2v (P1AE4422) and Fc-VHH (P1AH1177) were used as reference. Frequency (A) and MFI (B) of STAT-5 phosphorylation were determined by flow cytometry after 60 min of test compounds co-incubation with CD4 T cells preactivated for three days with plate-bound anti-CD3 and soluble anti-CD28 antibodies.
Figure 29. Concept of split FAP-targeted IL-2R agonists. VHH domains with IL-2RP and IL-2Ry specificity were fused to the N-terminus of two different anti-FAP binding domains, via VH or VL fusion points, resulting in eight individual format configurations (P1AJ5092 - P1AJ5099) that can be paired to produce the eight functional assemblies: P1AJ5096 + P1AJ5099 (A), P1AJ5092 + P1AJ5095 (B), P1AJ5097 + P1AJ5098 (C), P1AJ5093 + P1AJ5094 (D), P1AJ5096 + P1AJ5095 (E), P1AJ5092 + P1AJ5099 (F), P1AJ5097 + P1AJ5094 (G) and P1AJ5093 + P1AJ5098 (H).
Figure 30. Functional activity of FAP-targeted IL-2R agonists. Test compounds were incubated with A549 FAP-negative target cells (A) or A549 FAP-positive target cells (B) and IL-2RPy Bioassay cells for 20h at 37° C and 5% CO2. PDl-IL2v (P1AE4422), FAP-IL2v (P1AA5355) and Fc-VHH (Pl AHI 177) were used as reference. A non-binding DP47 antibody (P1AD3966) was used as negative control. IL-2R signaling was measured via luminescence using Bio-Gio NL reagent and normalized to background signal. Shown are mean fold change RLU values +/- SEM of technical duplicates for each tested molecule concentration.
Figure 31. Concept of split EGFR-targeted IL-2R agonists. VHH domains with IL-2RP and IL-2Ry specificity were fused to the N-terminus of two different anti-EGFR binding domains, via VH or VL fusion points, resulting in eight individual format configurations (P1AJ4165 - P1AJ4172) that can be paired to produce the eight functional assemblies: P1AJ4169 + P1AJ4172 (A), P1AJ4165 + P1AJ4168 (B), P1AJ4170 + P1AJ4171 (C), P1AJ4166 + P1AJ4167 (D), P1AJ4169 + P1AJ4168 (E), P1AJ4165 + P1AJ4172 (F), P1AJ4170 + P1AJ4167 (G) and P1AJ4166 + P1AJ4171 (H).
Figure 32. Functional activity of EGFR-targeted IL-2R agonists. EGFR expression on target cells by flow cytometry (A). Test compounds were incubated with A431 target cells (B), OE19 target cells (C) or LoVo target cells (D)) and IL-2RPy Bioassay cells for 20h at 37°C and 5% CO2. PDl-IL2v (P1AE4422) and Fc-VHH (P1AH1177) were used as reference. A non-binding DP47 antibody (P1AD3966) was used as negative control. IL-2R signaling was measured via luminescence using Bio- Glo NL reagent and normalized to background signal. Shown are mean fold change RLU values +/- SEM of technical duplicates for each tested molecule concentration.
Figure 33. Concept of split HER2 -targeted and HER3-targeted IL-2R agonists. VHH domains with IL- 2RP and IL-2Ry specificity were fused to the N-terminus of two different anti-HER2 binding domains and two different anti-HER3 binding domains. VH or VL fusion points were selected for each Fab based on structural data. Four format configurations (P1AJ4173 - P1AJ4176) and two paired assemblies were designed for HER2: P1AJ4174 + P1AJ4175 (A) and P1AJ4173 + P1AJ4176 (B). Four format configurations (P1AJ4177 - P1AJ4180) and two paired assemblies were selected for HER3: P1AJ4178
+ P1AJ4179 (C) and P1AJ4177 + P1AJ4180 (D).
Figure 34. Functional activity of HER2 -targeted and EIER3 -targeted IL-2R agonists. Test compounds were incubated with MDA-MB-231 target cells (A) or OE19 target cells (B) and IL-2RPy Bioassay cells for 20h at 37°C and 5% CO2. PDl-IL2v (P1AE4422) and Fc-VHH (P1AH1177) were used as reference. A non-binding DP47 antibody (P1AD3966) was used as negative control. IL-2R signaling was measured via luminescence using Bio-Gio NL reagent and normalized to background signal. Shown are mean fold change RLU values +/- SEM of technical duplicates for each tested molecule concentration.
Figure 35. Concept of split PD-l-targeted IL-2R agonists with alternative anti-PD-1 single domain binders. VHH domains with IL-2RP and IL-2Ry specificity were fused to the N-terminus of four different anti-PD-1 single domain binders (P1AI0063; P1AK3046; P1AK3048; P1AK3051 - P1AK3054) that can be paired to produce the six functional assemblies depicted: P1AK3052 + P1AK3048 (A), P1AI0063 + P1AK3051 (B), P1AK6054 + P1AK3048 (C), P1AI0063 + P1AK3053 (D), P1AK3054 + P1AK3046 (E) and P1AK3052 + P1AK3046 (F).
Figure 36. Functional activity of PD-l-targeted IL-2R agonists with alternative anti-PD-1 single domain binders. Test compounds were incubated with HEK Blue IL-2 wt cells (A) or HEK Blue IL-2 human PD-1 cells (B) for 20h at 37°C and 5% CO2. PDl-IL2v (P1AE4422) and Fc-VHH (P1AH1177) were used as reference. A non-binding DP47 antibody (P1AD3966) was used as negative control. IL-2R signaling was measured via absorbance at 650 nm using QUANTI-Blue reagent. Shown are mean absorbance values +/- SEM of technical duplicates for each tested molecule concentration.
Figure 37. Concept of split PD- 1 -targeted IL-2R agonists with anti-IL2R Fab binders or anti-IL2R scFv binders. Fab fragments with IL-2RP and IL-2Ry specificity were fused to the N-termini of two anti-PDl binding domains with different paratopes, via VH or VL fusion points. P1AL9238 (A) and P1AL9239 (B) comprise the PD1 binder 2 (FV003451 paratope; 1040 binder) and two different IL2Ry binders, FV018863 and FV018864, respectively, whereas P1AL9240 (C) and P1AL9241 (D) comprise the PD1 binder 1 (FV000363 paratope; 0376 binder) and two different IL2RP binders, FV002203 and FV018866, respectively. These can be paired to produce the four functional assemblies: P1AL9238 + P1AL9240, P1AL9238 + P1AL9241, P1AL9239 + P1AL9240, P1AL9239 + P1AL9241. Alternatively, scFv fragments with IL-2RP and IL-2Ry specificity were fused to the N-termini of two anti-PD 1 binding domains with different paratopes, via VH orVL fusion points. P1AL9310 (E) comprises the PD1 binder 2 (FV003451 paratope) and the IL2Ry binder FV018865, whereas in P1AL9311 (F) comprises the PD1 binder 1 (FV000363 paratope) and the IL2RP binder FV018866. These can be paired to produce a functional assembly: P1AL9310 + P1AL9311. Figure 38. Functional activity of PD-1 -targeted IL-2R agonists with anti-IL2R Fab and scFv binders. Test compounds were incubated with HEK Blue IL-2 wt cells (A) or HEK Blue IL-2 human PD-1 cells
(B) for 18h at 37°C and 5% CO2. An IL-2 agonistic heavy chain antibody (Fc-VHH ; P1AH1177) was used as reference. A non-binding DP47 antibody (P1AD3966) was used as negative control. IL-2R signaling was measured via absorbance at 650 nm using QU ANTI -Blue reagent. Shown are fold change in IL-2R signaling over background +/- SEM of technical duplicates for each tested molecule concentration.
Figure 39. Schematic representations of antigens and workflow for the isolation of IL-7Ra-specific single domain antibodies. The extracellular domain (ECD) of human IL-7Ra fused to biotinylated Fc knob-into-hole (P1AI1009 represented in A) was generated for alpaca immunization, phage display and screening. Soluble human Fc knob-into-hole (P1AD4290 represented in B) was generated for phage display. Workflow for the enrichment of single domain antibodies with binding specificity for IL-7Ra by phage display (C).
Figure 40. Specificity screening of single domain antibodies by ELISA. Soluble VHH domains enriched for IL-7Ra specificity were randomly selected and screened for binding to immobilized human IL-7Ra- Fc and immobilized human Fc. Following three rounds of phage display, VHH from library 1 (A) and library 2 (B) were tested for antigen specificity. Following four rounds, enriched VHH from library 1
(C) and library 2 (D) were also tested for specificity by ELISA. Absorbance at 450 nm indicating the binding response to each target is represented as a stacked bar chart.
Figure 41. Format conversion of single domain antibody fragments into bispecific heavy chain antibodies. Round 3 and round 4 IL-7Ra-specific VHH from libraries 1 and 2 were fused to Fc-Knob and cloned into a mammalian cell expression vector using Gibson cloning method (A). Previously identified VHH with IL-2Ry specificity were fused to Fc-hole using the same method (B). Bispecific heavy chain antibodies comprising IL-7Ra-IL-2Ry VHH domain pairs were generated via knob-into- hole assembly of Fc chains and flexible 5(G4S) linkers to fuse VHH moieties to Fc chains. Effector functions of the Fc were silenced via incorporation of P329G LALA mutations in the CH2 domain (C).
Figure 42. Functional characterization of dose-dependent response for IL-7R agonists. Bispecific heavy chain antibodies comprising IL-7Ra specificity and IL-2Ry were screened for dose-dependent IL-7R agonism in HEK Blue IL-7 cells. Recombinant IL-7 was used as a reference and an IgG with irrelevant specificity was used as a negative control (P1AA5879). Each molecule was incubated with reporter cells at the following concentrations: 20 nM, 2 nM, 0.2 nM, 0.02 nM shown from left to right. IL-7R activity was quantified by absorbance at 650 nm and fold response/background is shown for each treatment. Graphs were separated according to the four different IL-2Ry-specific VHH clones: IL2Ry_2 (A),
IL2Ry_3 (B), IL2Ry_4 (C) and IL2Ry_5(D).
Figure 43. Concept of split PD-l-targeted IL-7R agonists. PD-1 -dependent biparatopic assembly of split IL-7 mimetics (A). VHH domains with IL-7Ra and IL-2Ry specificity were fused to the N-terminus of two different anti -PD-1 binding domains, via VH or VL fusion points: IL-7Ra-VHH were fused to the VL of PD-1 binder 1 (B), and IL-2Ry-VHH were fused to the VH of PD-1 binder 2 (C). IL-7Ra molecules contained AAA mutations (I253A; H310A; H435 A, numbering according to Kabat EU index) in the CH2 and CH3 domains of the Fc hole to facilitate protein A purification. Effector functions of the Fc were silenced in all molecules via incorporation of P329G LALA mutations in the CH2 domain.
Figure 44. Functional activity of PD-l-targeted IL-7R agonists. PDl-IL2v (P1AE4422) and Fc-VHH (Pl AHI 177) were used as reference. A non-binding DP47 antibody (P1AD3966) was used as negative control. Frequency of STAT-5 phosphorylation was determined by flow cytometry after 60 min of test compounds co-incubation with CD4 T cells pre-activated for three days with plate-bound anti-CD3 and soluble anti-CD28 antibodies for blood donor 1 (A and B) and 2 (C and D).
Figure 45. Concept of split PD-l-targeted IL-2R agonists with monoparatopic assembly onto PD-1. VHH domains with IL-2RP and IL-2Ry specificity were fused to the N-terminus of two different anti- PD-1 binding domains, via VH or VL fusion points, resulting in eight individual format configurations of IL-2R agonist pairs containing the same anti -PD-1 Fab binders (monoparatopic assembly) that can be paired to produce the eight monoparatopic assemblies: P1AK2802 + P1AK2810 (A), P1AK2809 + P1AK2803 (B), P1AK2802 + P1AK2803 (C), P1AK2809 + P1AK2810 (D), P1AK2798 + P1AK2599 (E), P1AK2806 + P1AK2799 (F), P1AK2798 + P1AK2799 (G) and P1AK2806 + P1AK2599 (G) .
Figure 46. Functional activity of monoparatopic vs biparatopic PD-l-targeted IL-2R agonists. Test compounds were incubated with HEK Blue IL-2wt (A) or HEK Blue IL-2 human PD-1 cells (B) for 21h at 37°C and 5% CO2. Two biparatopic PD-l-targeted IL-2R agonists (P1AH6814 + P1AI1593 and P1AH6814 + P1AK2802) and the IL-2 mimetic heavy chain antibody (Fc-VHH;P1AH1177) were used as reference. A non-binding DP47 antibody (P1AD3966) was used as negative control. IL-2R signaling was measured via absorbance at 650 nm using QUANTI-Blue reagent. Shown are mean absorbance values +/- SEM of technical duplicates for each tested molecule concentration. Detailed Description of the Invention
Definitions
Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art. Generally, nomenclatures used in connection with, and techniques of biochemistry, enzymology, molecular, and cellular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art.
The terms “a”, “an” and “the” generally include plural referents, unless the context clearly indicates otherwise.
As used herein, the terms “first”, “second”, “third” or “fourth” with respect to binding molecules, epitopes, polypeptides etc., are used for convenience of distinguishing when there is more than one of each type of moiety. Use of these terms is not intended to confer a specific order or orientation of the moiety unless explicitly so stated.
The term “antigen binding molecule” as used herein refers to a polypeptide molecule (composed of one or more polypeptide chains) that is capable of binding to an antigen. An antigen binding molecule may be derived from an antibody, and typically comprises an antigen binding domain.
The term “antibody” herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies), heavy-chain antibodies, antibody fragments and antigen binding molecules so long as they exhibit the desired antigen-binding activity.
The term “heavy chain antibody” and “heavy chain-only antibody” and “HCAb” as used herein refer to antibodies devoid of light chains.
The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure.
An ’’antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, cross-Fab, Fab', Fab’-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g. scFv and scFab), single-domain antibodies, and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Hollinger and Hudson, Nature Biotechnology 23: 1126-1136 (2005).
A "single -domain antibody" refers to an antibody fragment consisting of a single monomeric antibody variable domain such as VHHs, nanobodies, VNARs derived from sharks, autonomous VH domains or autonomous VL domains. Single-domain antibodies provide an antigen-binding site which specifically binds to an epitope, i.e. the antigen binding-site is formed solely by the single-domain antibody.
A “VHH” or “VHH domain” or “nanobody” refers to a single-domain antibody derived from the variable domains of heavy chain antibodies from camelids, e.g. camel, dromedary, llama, alpaca, etc. (See Nguyen V.K. et al., 2000, The EMBO Journal, 19, 921-930; Muyldermans S., 2001, J Biotechnol., 74, 277-302 and for review Vanlandschoot P. et al., 2011, Antiviral Research 92, 389-407). The antigenbinding site of VHHs is devoid of light chain variable domain. A VHH domain may be humanized.
An “antigen binding domain” as used herein refers to a domain that specifically binds to a target antigen. The term in particular refers to an antigen binding domain of an antibody, i.e. the part that comprises the area which binds to and is complementary to part or all of an antigen. Accordingly, in particular aspects, an antigen binding domain herein is an antigen binding domain of an antibody. Such an antigen binding domain may be provided by an antibody fragment, for example by a Fab molecule, a singlechain antibody molecule or single-domain antibodies, such as a VHH domain.
The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to the antigen. The term includes VHH domains. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and complementarity determining regions (CDRs). See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman & Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991). As used herein in connection with variable region sequences, "Kabat numbering" refers to the numbering system set forth by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).
As used herein, the amino acid positions of all constant regions and domains of the heavy and light chain are numbered according to the Kabat numbering system described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD
(1991), referred to as “numbering according to Kabaf ’ or “Kabat numbering” herein.
Specifically the Kabat numbering system (see pages 647-660 of Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)) is used for the light chain constant domain CL of kappa and lambda isotype and the Kabat EU index numbering system (see pages 661-723) is used for the heavy chain constant domains (CHI, hinge, CH2 and CH3), which is herein further clarified by referring to “numbering according to Kabat EU index” or “Kabat EU index numbering” in this case.
The terms “binding site” or “antigen-binding site” as used herein refers to the site, i.e. one or more amino acid residues, of a binding molecule which provides interaction with the antigen. For example, the antigen binding site of an antigen binding domain comprises amino acid residues from the complementarity determining regions (CD Rs). An antigen-binding site may be provided by, for example, one or more variable domains (also called variable regions). In single domain antibodies the antigen-binding site is provided by a single variable domain. Whereas, in a Fab fragment the antigenbinding site is provided by the VH and VL domains.
The term “hypervariable region” or “HVR”, as used herein, refers to each of the regions of an antigen binding domain which are hypervariable in sequence and which determine antigen binding specificity, for example “complementarity determining regions” (“CDRs”). Generally, variable domains comprise three CDRs. Thus antibodies comprising a VH and a VL comprise six CDRs; three in the VH (HCDR1, HCDR2, HCDR3), and three in the VL (LCDR1, LCDR2, LCDR3). Exemplary CDRs herein include:
(a) hypervariable loops occurring at amino acid residues 26-32 (LI), 50-52 (L2), 91-96 (L3), 26-32 (Hl), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));
(b) CDRs occurring at amino acid residues 24-34 (LI), 50-56 (L2), 89-97 (L3), 3 l-35b (Hl), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and
(c) antigen contacts occurring at amino acid residues 27c-36 (LI), 46-55 (L2), 89-96 (L3), 30-35b (Hl), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)).
Unless otherwise indicated, the CDRs are determined according to Kabat et al., supra. One of skill in the art will understand that the CDR designations can also be determined according to Chothia, supra, McCallum, supra, or any other scientifically accepted nomenclature system.
"Framework" or "FR" refers to variable domain residues other than complementarity determining regions (CDRs). The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following order in VH and VHH domains (or VL): FR1-HCDR1 (LCDR1)-FR2-HCDR2 (LCDR2)-FR3-HCDR3 (LCDR3)-FR4. Unless otherwise indicated, CDR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.
The term “immunoglobulin molecule” herein refers to a protein having the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are heterotetrameric glycoproteins of about 150,000 daltons, composed of two light chains and two heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy domain or a heavy chain variable region, followed by three constant domains (CHI, CH2, and CH3), also called a heavy chain constant region. Similarly, from N- to C-terminus, each light chain has a variable domain (VL), also called a variable light domain or a light chain variable region, followed by a constant light (CL) domain, also called a light chain constant region. The heavy chain of an immunoglobulin may be assigned to one of five types, called a (IgA), 5 (IgD), a (IgE), y (IgG), or p (IgM), some of which may be further divided into subtypes, e.g. yl (IgGi), y2 (IgG2), y3 (IgG,)- y4 (IgG4), al (IgAl) and a2 (IgA2). The light chain of an immunoglobulin may be assigned to one of two types, called kappa (K) and lambda (X), based on the amino acid sequence of its constant domain. An immunoglobulin essentially consists of two Fab molecules and an Fc domain, linked via the immunoglobulin hinge region.
The “class” of an antibody or immunoglobulin refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several ofthese maybe further divided into subclasses (isotypes), e.g., IgGi, IgG2, IgG;. IgG4, IgAl, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 5, a, y, and p, respectively.
A “Fab molecule” or “Fab” or “Fab fragment” or “Fab domain” refers to a protein consisting of the VH and CHI domain of the heavy chain (the “Fab heavy chain”) and the VL and CL domain of the light chain (the “Fab light chain”) of an immunoglobulin.
A “cross-Fab molecule” or “cross-Fab” or “crossover Fab molecule” refers to a Fab molecule, wherein either the variable regions or the constant regions of the heavy and light chain are exchanged. Cross- Fab engineering enables two different chain compositions of a cross-Fab molecules. On the one hand, the variable regions of the Fab heavy and light chain are exchanged, i.e. the cross-Fab molecule comprises a peptide chain composed of the light chain variable region (VL) and the heavy chain constant region (CHI), wherein the CHI may be fused to an Fc domain, and a peptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL). On the other hand, when the constant regions of the Fab heavy and light chain are exchanged, the cross-Fab molecule comprises a peptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL), wherein the CL may be fused to an Fc domain, and a peptide chain composed of the light chain variable region (VL) and the heavy chain constant region (CHI).
The term “conventional Fab molecule” refers to a Fab molecule composed of a Fab heavy chain comprising a VH and CHI domain and a Fab light chain comprising a VL and CL domain.
A “single-chain variable fragment” or “scFv” molecule is a fusion protein of the variable domains of the heavy (VH) and light chain (VL) of an antibody, connected by a linker. In particular, the linker is a short polypeptide of 10 to 25 amino acids and is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. This protein retains the specificity of the original antibody, despite removal of the constant regions and the introduction of the linker.
The term “multispecific” means that a binding molecule (e.g. an antibody) is able to specifically bind to at least two distinct antigens. A multispecific binding molecule (e.g. antibody) can be, for example, a bispecific binding molecule. Typically, a bispecific binding molecule comprises two antigen binding sites, each of which is specific for a different antigens. In certain aspects, the multispecific (e.g. bispecific) binding molecule is capable of simultaneously binding two antigens, particularly two antigens expressed on the same cell, on neighbouring cells, or cells in the same tissue.
The term “valent” as used herein denotes the presence of a specified number of antigen binding sites in a binding molecule. As such, the term “monovalent binding to an antigen” denotes the presence of one (and not more than one) antigen binding site specific for the antigen in the binding molecule.
As used herein, the term “antigen” refers to a molecule, such as a protein, to which an antigen binding molecule binds. Useful antigens can be found, for example, on the surfaces of tumor cells, on the surfaces of tumor stroma cells, on the surfaces of virus-infected cells, on the surfaces of other diseased cells, on the surface of immune cells, free in blood serum, and/or in the extracellular matrix (ECM). In particular aspects, the antigen is a human protein.
As used herein, the term “epitope” refers to a site on an antigen to which an antigen-binding site binds. Epitopes can be formed from a contiguous stretch of amino acids or a conformational configuration made up of different regions of noncontiguous amino acids. Epitopes often include chemically active surface groupings of antigens such as amino acids, glycan side chains, phosphoryl, or sulfonyl, and may have specific three dimensional structural characteristics, and/or specific charge characteristics. Two distinct antigen-binding domains capable of binding the same antigen may bind different epitopes of said antigen. Such distinct antigen-binding domains are said to not compete for binding if both antigenbinding domains can simultaneously bind to the antigen. In this case two distinct antigen binding domains are non-competing. Alternatively, antigen-binding domains may compete for binding, i.e. show competitive binding. This may be due to partially overlapping epitopes or simultaneous binding of the two antigen binding domains is hampered due to steric hindrance. Assays to determine whether antigenbinding domains show non-competitive or competitive binding are well known in the art, for example competitive binding analysis using ELISA, RIA, surface plasmon resonance, flow cytometry or any other quantitative or qualitative antibody-binding assay available in the art.
A “target antigen” as used herein refers to an antigen presented on the surface of a target cell, for example an antigen on a cell in a tumor, such as a cancer cell or a cell of the tumor stroma, or an antigen on a T cell.
A “tumor-associated antigen” as used herein refers to any antigen presented on the surface of a cell in a tumor such as a cancer cell or a cell of the tumor stroma. Particular tumor-associated antigens are CEA, FAP, Her2, Her3 or EGFR.
The term “Fibroblast activation protein (FAP)”, also known as Prolyl endopeptidase FAP or Seprase (EC 3.4.21), refers to any native FAP from any vertebrate source, including mammals such as primates (e.g. humans) non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice and rats), unless otherwise indicated. FAP is expressed on Cancer-associated fibroblasts (CAFs) in the tumor stroma. The term encompasses “full-length,” unprocessed FAP as well as any form of FAP which results from processing in the cell. The term also encompasses naturally occurring variants of FAP, e.g., splice variants or allelic variants. In one embodiment, the antigen binding molecule of the invention is capable of specific binding to human, mouse and/or cynomolgus FAP. The amino acid sequence of human FAP is shown in UniProt (www.uniprot.org) accession no. Q 12884 (version 149), or NCBI (www.ncbi.nlm.nih.gov/) RefSeq NP 004451.2. The extracellular domain (ECD) of human FAP extends from amino acid position 26 to 760. The amino acid sequence of mouse FAP is shown in UniProt accession no. P97321 (version 126), or NCBI RefSeq NP_032012.1. The extracellular domain (ECD) of mouse FAP extends from amino acid position 26 to 761. Preferably, an anti-FAP binding molecule binds to the extracellular domain of FAP. Exemplary anti-FAP binding molecules are described in International Patent Application No. WO 2012/020006 A2 and WO 2020/070041 Al .
The term “Carcinoembroynic antigen (CEA)”, also known as Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), refers to any native CEA from any vertebrate source, including mammals such as primates (e.g. humans) non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice and rats), unless otherwise indicated. The amino acid sequence of human CEA is shown in UniProt accession no. P06731 (version 151). CEA has long been identified as a tumor-associated antigen (Gold and Freedman, J Exp Med., 121:439-462, 1965; Berinstein N. L., J Clin Oncol., 20:2197-2207, 2002). Originally classified as a protein expressed only in fetal tissue, CEA has now been identified in several normal adult tissues. These tissues are primarily epithelial in origin, including cells of the gastrointestinal, respiratory, and urogential tracts, and cells of colon, cervix, sweat glands, and prostate (Nap et al., Tumour Biol., 9(2-3): 145-53, 1988; Nap et al., Cancer Res., 52(8):2329-23339, 1992). Tumors of epithelial origin, as well as their metastases, contain CEA as a tumor associated antigen. While the presence of CEA itself does not indicate transformation to a cancerous cell, the distribution of CEA is indicative. In normal tissue, CEA is generally expressed on the apical surface of the cell (Hammarstrom S., Semin Cancer Biol. 9(2):67-81 (1999)), making it inaccessible to antibody in the blood stream. In contrast to normal tissue, CEA tends to be expressed over the entire surface of cancerous cells (Hammarstrom S., Semin Cancer Biol. 9(2): 67-81 (1999)). This change of expression pattern makes CEA accessible to antibody binding in cancerous cells. In addition, CEA expression increases in cancerous cells. Furthermore, increased CEA expression promotes increased intercellular adhesions, which may lead to metastasis (Marshall J., Semin Oncol., 30(a Suppl. 8):30-6, 2003). The prevalence of CEA expression in various tumor entities is generally very high. In concordance with published data, own analyses performed in tissue samples confirmed its high prevalence, with approximately 95% in colorectal carcinoma (CRC), 90% in pancreatic cancer, 80% in gastric cancer, 60% in non-small cell lung cancer (NSCLC, where it is co-expressed with HER3), and 40% in breast cancer; low expression was found in small cell lung cancer and glioblastoma.
“HER2” (also known as erbB-2 or CD340) refers to any native HER2 from any vertebrate source, including mammals such as primates (e.g. humans), non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed HER2 as well as any form of HER2 that results from processing in the cell. The term also encompasses naturally occurring variants of HER2, e.g., splice variants or allelic variants. In one aspect, HER2 is human HER2. The amino acid sequence of human HER2 is shown in UniProt (www.uniprot.org) entry no. Q9UK79 (version 95).
“Epidermal Growth Factor Receptor (EGFR)”, also named Proto-oncogene c-ErbB-1 or Receptor tyrosine-protein kinase erbB-1, refers to any native EGFR from any vertebrate source, including mammals such as primates (e.g. humans) non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice and rats), unless otherwise indicated. The amino acid sequence of human EGFR is shown in UniProt accession no. P00533.
A “T cell antigen” as used herein refers to any antigen present on the surface of a T lymphocyte. The term “PD-1”, also known as CD279, PD1 or programmed cell death protein 1, refers to any native PD-1 from any vertebrate source, including mammals such as primates (e.g. humans) non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice and rats), in particular to the human protein PD-1 with the amino acid sequence as shown in UniProt (www.uniprot.org) accession no. Q15116.
The term “interferon gamma” or “IFNy” or “IFN-y” as used herein, refers to any native IFNy from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses unprocessed IFNy as well as any form of IFNy that results from processing in the cell. The term also encompasses naturally occurring variants of IFNy, e.g. splice variants or allelic variants.
The term “interleukin-2” or “IL-2” as used herein, refers to any native IL-2 from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses unprocessed IL-2 as well as any form of IL-2 that results from processing in the cell. The term also encompasses naturally occurring variants of IL-2, e.g. splice variants or allelic variants.
Cytokine receptors are cell-surface glycoproteins that specifically bind cytokines and transduce their signals. Generally, cytokine receptors function as oligomeric complexes consisting of typically two to four receptor chains, also termed subunits, that may be the same or different. Thus, the term “cytokine receptor complex” refers to cytokine receptors composed of at least two subunits.
The IFNy receptor complex comprises the IFNyRl subunit and the IFNyR2 subunit. The term “Interferon gamma receptor 1” or “IFNyRl”, also referred to as CD119 (Cluster of differentiation 119) or Interferon gamma receptor a-chain (IFNyRa), refers to any native IFNyRl from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length”, unprocessed IFNyRl as well as any form of IFNyRl that results from processing in the cell. The term also encompasses naturally occurring variants of IFNyRl, e.g. splice variants or allelic variants. In certain embodiments IFNyRl is human IFNyRl.
The term “Interferon gamma receptor 2” or “IFNyR2”, also referred to as Interferon gamma receptor - chain (IFNyR ), refers to any native IFNyR2 from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length”, unprocessed IFNyR2 as well as any form of IFNyR2 that results from processing in the cell. The term also encompasses naturally occurring variants of IFNyR2, e.g. splice variants or allelic variants. In certain embodiments IFNyR2 is human IFNyR2. The term “IL-2Ra” or “a-subunit of the IL-2 receptor” also known as CD25, as used herein, refers to any native IL-2Ra from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length”, unprocessed IL-2Ra as well as any form of IL-2Ra that results from processing in the cell. The term also encompasses naturally occurring variants of IL-2Ra, e.g. splice variants or allelic variants. In certain embodiments IL-2Ra is human IL-2Ra.
The term “IL-2RP” or “P-subunit of the IL-2 receptor”, also known as CD 122 or p70, refers to any native IL-2RP from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length”, unprocessed IL-2RP as well as any form of IL-2RP that results from processing in the cell. The term also encompasses naturally occurring variants of IL-2RP, e.g. splice variants or allelic variants. In certain embodiments IL-2RP is human IL-2Rp.
The term “IL-2Ry” or “y-subunit of the IL-2 receptor”, also known as common cytokine receptor y- subunit, common y-chain, yc, or CD 132, refers to any native IL-2Ry from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length”, unprocessed IL-2Ry as well as any form of IL-2Ry that results from processing in the cell. The term also encompasses naturally occurring variants of IL-2Ry, e.g. splice variants or allelic variants. In certain embodiments IL-2Ry is human IL-2Ry.
Different associations of the individual IL-2R subunits IL-2Ra, IL-2RP and IL-2Ry, can produce three IL-2R forms that differ in their affinity to IL-2. The high-affinity IL-2R refers to the heterotrimeric form of the IL-2R, consisting of IL-2Ra, IL-2RP and IL-2Ry. The intermediate-affinity IL-2R refers to the heterodimeric form of the IL-2R, consisting of IL-2RP and IL-2Ry. Whereas, the low affinity IL-2R refers to the monomeric form of the IL-2R, consisting solely of IL-2Ra (for a review see e.g. Olejniczak and Kasprzak, Med Sci Monit 14, RA179-189 (2008)).
The term “IL-2 receptor complex” as used herein refers to the high-affinity IL-2 receptor or the intermediate -affinity IL-2 receptor.
The term “Fc domain” or “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one aspect, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Therefore, an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain, or it may include a cleaved variant of the full-length heavy chain. This may be the case where the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to Kabat EU index). Therefore, the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (Lys447), of the Fc region may or may not be present. In one aspect, a heavy chain including an Fc region (subunit) as specified herein, comprised in a binding molecule according to the invention, comprises an additional C-terminal lysine (K447, numbering according to Kabat EU index). In one aspect, a heavy chain including an Fc region (subunit) as specified herein, comprised in a binding molecule according to the invention, comprises a C-terminal glycine residue (G446, numbering according to Kabat EU index) and does not comprise a C-terminal lysine (Lys477). Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991 (see also above). A “subunit” of an Fc domain as used herein refers to one of the two polypeptides forming the dimeric Fc domain, i.e. a polypeptide comprising C-terminal constant regions of an immunoglobulin heavy chain, capable of stable self-association. For example, a subunit of an IgG Fc domain comprises an IgG CH2 and an IgG CH3 constant domain.
A “modification promoting the association of the first and the second subunit of the Fc domain” is a manipulation of the peptide backbone or the post-translational modifications of an Fc domain subunit that reduces or prevents the association of a polypeptide comprising the Fc domain subunit with an identical polypeptide to form a homodimer. A modification promoting association as used herein preferably includes separate modifications made to each of the two Fc domain subunits desired to associate (i.e. the first and the second subunit of the Fc domain), wherein the modifications are complementary to each other so as to promote association of the two Fc domain subunits. For example, a modification promoting association may alter the structure or charge of one or both of the Fc domain subunits so as to make their association sterically or electrostatically favorable, respectively. Thus, (hetero)dimerization occurs between a polypeptide comprising the first Fc domain subunit and a polypeptide comprising the second Fc domain subunit, which may be non-identical in the sense that further components fused to each of the subunits (e.g. antigen binding domains) are not the same. In one aspect, the modification promoting the association of the first and the second subunit of the Fc domain comprises an amino acid mutation in the Fc domain, specifically an amino acid substitution. In particular aspects, the modification promoting the association of the first and the second subunit of the Fc domain comprises a separate amino acid mutation, specifically an amino acid substitution, in each of the two subunits of the Fc domain. One heterodimerization approach known in the art is the so-called “knobs-into-holes” technology, which is described in detail providing several examples in e.g. WO 96/027011, Ridgway, J.B., et al., Protein Eng. 9 (1996) 617-621; Merchant, A.M., et al., Nat. Biotechnol. 16 (1998) 677-681; and W098/050431. In the “knobs-into-holes” technology, within the interface formed between two CH3 domains in the tertiary structure of the antibody, particular amino acids on each CH3 domain are engineered to produce a protuberance (“knob”) in one of the CH3 domains and a cavity (“hole”) in the other one of the CH3 domains, respectively. In the tertiary structure of the multispecific antibody the introduced protuberance in the one CH3 domain is positionable in the introduced cavity in the other CH3 domain.
The term “effector functions” refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell- mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen presenting cells, down regulation of cell surface receptors (e.g. B-cell receptor), and B-cell activation.
An “activating Fc receptor” is an Fc receptor that following engagement by an Fc domain of an antibody elicits signaling events that stimulate the receptor-bearing cell to perform effector functions. Human activating Fc receptors include FcyRIIIa (CD 16a), FcyRI (CD64), FcyRIIa (CD32), and FcaRI (CD89).
Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism leading to the lysis of antibody-coated target cells by immune effector cells. The target cells are cells to which antibodies or derivatives thereof comprising an Fc region specifically bind, generally via the protein part that is N- terminal to the Fc region. As used herein, the term “reduced ADCC” is defined as either a reduction in the number of target cells that are lysed in a given time, at a given concentration of antibody in the medium surrounding the target cells, by the mechanism of ADCC defined above, and/or an increase in the concentration of antibody in the medium surrounding the target cells, required to achieve the lysis of a given number of target cells in a given time, by the mechanism of ADCC. The reduction in ADCC is relative to the ADCC mediated by the same antibody produced by the same type of host cells, using the same standard production, purification, formulation and storage methods (which are known to those skilled in the art), but that has not been engineered. For example, the reduction in ADCC mediated by an antibody comprising in its Fc domain an amino acid substitution that reduces ADCC, is relative to the ADCC mediated by the same antibody without this amino acid substitution in the Fc domain. Suitable assays to measure ADCC are well known in the art (see e.g. PCT publication no. WO 2006/082515 or PCT publication no. WO 2012/130831). “Reduced binding”, for example reduced binding to an Fc receptor, refers to a decrease in affinity for the respective interaction, as measured for example by SPR. For clarity, the term includes also reduction of the affinity to zero (or below the detection limit of the analytic method), i.e. complete abolishment of the interaction. Conversely, “increased binding” refers to an increase in binding affinity for the respective interaction.
“Affinity” refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1: 1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by well-established methods known in the art, including those described herein. A preferred method for measuring affinity is Surface Plasmon Resonance (SPR).
As used herein, the terms “engineer, engineered, engineering”, are considered to include any manipulation of the peptide backbone or the post-translational modifications of a naturally occurring or recombinant polypeptide or fragment thereof. Engineering includes modifications of the amino acid sequence, of the glycosylation pattern, or of the side chain group of individual amino acids, as well as combinations of these approaches.
The term “amino acid mutation” as used herein is meant to encompass amino acid substitutions, deletions, insertions, and modifications. Any combination of substitution, deletion, insertion, and modification can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., reduced binding to an Fc receptor, or increased association with another peptide. Amino acid sequence deletions and insertions include amino- and/or carboxy-terminal deletions and insertions of amino acids. Preferred amino acid mutations are amino acid substitutions. For the purpose of altering e.g. the binding characteristics of an Fc region, nonconservative amino acid substitutions, i.e. replacing one amino acid with another amino acid having different structural and/or chemical properties, are particularly preferred. Amino acid substitutions include replacement by non- naturally occurring amino acids or by naturally occurring amino acid derivatives of the twenty standard amino acids (e.g. 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5 -hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site directed mutagenesis, PCR, gene synthesis and the like. It is contemplated that methods of altering the side chain group of an amino acid by methods other than genetic engineering, such as chemical modification, may also be useful. Various designations may be used herein to indicate the same amino acid mutation. For example, a substitution from proline at position 329 of the Fc domain to glycine can be indicated as 329G, G329, G329, P329G, or Pro329Gly. By “fused” is meant that the components (e.g. a Fab molecule and an Fc domain subunit) are linked by peptide bonds, either directly or via one or more peptide linkers. The term “fusion point” refers to the site on one component at which said component is fused to another component. For example a cytokine receptor-binding domain may be fused at its C-terminus to the N-terminus of the VH domain of a targetbinding domain. Thus, the fusion point on the cytokine receptor-binding domain is the C-terminus and the fusion point on the target-binding domain is the N-terminus.
“Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or the FASTA program package. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Alternatively, the percent identity values can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087 and is described in WO 2001/007611.
Unless otherwise indicated, for purposes herein, % amino acid sequence identity values are generated using the ggsearch program of the FASTA package version 36.3.8c or later with a BLOSUM50 comparison matrix. The FASTA program package was authored by W. R. Pearson and D. J. Lipman (“Improved Tools for Biological Sequence Analysis”, PNAS 85 (1988) 2444-2448), W. R. Pearson (“Effective protein sequence comparison” Meth. Enzymol. 266 (1996) 227-25 258), and Pearson et. al. (Genomics 46 (1997) 24-36) and is publicly available from www.fasta.bioch.virginia.edu/fasta_www2/fasta_down.shtml or www.ebi.ac.uk/Tools/sss/fasta.
Alternatively, a public server accessible at fasta.bioch.virginia.edu/fasta_www2/index.cgi can be used to compare the sequences, using the ggsearch (global proteimprotein) program and default options (BLOSUM50; open: -10; ext: -2; Ktup = 2) to ensure a global, ratherthan local, alignment is performed. Percent amino acid identity is given in the output alignment header. Cytokine mimetics
The present inventors have found that a pair of antigen binding domains capable of binding different cytokine receptor subunits of a cytokine receptor complex, hereinafter referred to as cytokine receptorbinding domains, can act as cytokine agonists, i.e. mimic a naturally occurring cytokine. Cytokine agonists are also termed cytokine mimetics. This has been shown by combining a pair of cytokine receptor-binding domains capable of binding different cytokine receptor subunits in a single antigen binding molecule. The antibody format of these molecules may provide advantageous properties compared to natural or recombinant cytokines. To achieve conditional activation of a cytokine receptor, the pair of cytokine receptor-binding domains were split in two distinct antigen binding molecules, i.e. split molecules or split antibodies. These antigen binding molecules further comprise an antigen binding domain capable of binding a target antigen, hereinafter referred to as target-binding domain. By targetdependent assembly of the pair of cytokine receptor-binding domains, the molecules can act as cytokine mimetics. Using a target-dependent approach further allows to direct the agonistic activity to a site of interest, such as cells or tissues expressing the target. The target-binding domains of the molecules are capable of binding the same target antigen simultaneously, i.e. they bind different epitopes of said antigen and are non-competing. Such a pair of target-binding domains are also referred to as biparatopic target-binding domains. The assembly of the cytokine receptor-binding domains by the biparatopic target-binding domains allows to target the cytokine receptor-binding domains specifically to a site of interest, i.e. a cell or environment where the target antigen is expresses, to act as cytokine mimetics. The pair of split antigen binding molecules are thus a biparatopic pair of antigen binding molecules.
Split biparatopic antigen binding molecule pairs
In one aspect, the present invention provides a pair of antigen binding molecules that specifically bind to a target antigen, comprising a) first antigen binding molecule comprising i) a first target-binding domain, ii) a first cytokine receptor-binding domain and iii) a Fc domain; and a second antigen binding molecule comprising i) a second target-binding domain, ii) a second cytokine receptor-binding domain and iii) a Fc domain; wherein the first target-binding domain is capable of binding a first epitope of the target antigen and the second target-binding domain is capable of binding a second epitope of the target antigen, wherein the first and the second target-binding domains do not compete for binding on the target antigen; and wherein the first cytokine receptor-binding domain is capable of binding a first cytokine receptor subunit and the second cytokine receptor-binding domain is capable of binding a second cytokine receptor subunit.
The pair of antigen binding molecules described above and herein, may incorporate, singly or in combination, any of the features described in the following (unless the context dictates otherwise). Cytokine receptor-binding domain
According to the invention, each antigen binding molecule of the pair of antigen binding molecules comprises a cytokine receptor-binding domain. The first antigen binding molecule comprises a first cytokine receptor-binding domain and the second antigen binding molecule comprises a second cytokine receptor-binding domain. The cytokine receptor-binding domains comprised in the pair of antigen binding molecules bind different cytokine receptor subunits. Each cytokine receptor-binding domain of the pair of antigen binding molecules binds a distinct cytokine receptor subunit of a cytokine receptor complex. Thus, the first antigen binding molecule comprises a first cytokine receptor-binding domain capable of binding a first cytokine receptor subunit and the second antigen binding molecule comprises a second cytokine receptor-binding domain capable of binding a second cytokine receptor subunit. The first and the second cytokine receptor subunit are subunits of a cytokine receptor complex.
The cytokine receptor complex may be IFNy receptor complex, IL-2 receptor complex, IL-7 receptor complex, IL- 12 receptor complex or IL- 18 receptor complex. Thus, the first and the second cytokine receptor-binding domain may bind different subunits of the IFNy receptor complex, IL-2 receptor complex, IL-7 receptor complex, IL- 12 receptor complex or IL- 18 receptor complex. In one embodiment, the first and second cytokine receptor-binding domains bind subunits of the IFNy receptor complex. Thus, in one embodiment the first cytokine receptor-binding domain is capable of binding IFNyRl and the second cytokine receptor-binding domain is capable of binding IFNyR2. Alternatively, the first cytokine receptor-binding domain is capable of binding IFNyR2 and the second cytokine receptor-binding domain is capable of binding IFNyRl. In a further embodiment, the first and second cytokine receptor-binding domains bind subunits of the IL-2 receptor complex. Thus, in one embodiment the first cytokine receptor-binding domain is capable of binding IL-2RP and the second cytokine receptor-binding domain is capable of binding IL-2Ry. In one embodiment, the first cytokine receptor-binding domain is capable of binding IL-2Ry and the second cytokine receptor-binding domain is capable of binding IL-2Rp. In another embodiment, the first and second cytokine receptor-binding domains bind subunits of the IL-7 receptor complex. Thus, in one embodiment the first cytokine receptor-binding domain is capable of binding IL-7Ra and the second cytokine receptor-binding domain is capable of binding IL-2Ry. In one embodiment, the first cytokine receptor-binding domain is capable of binding IL-2Ry and the second cytokine receptor-binding domain is capable of binding IL-7Ra. In a further embodiment, the first and second cytokine receptor-binding domains bind subunits of the IL- 12 receptor complex. Thus, in one embodiment the first cytokine receptor-binding domain is capable of binding IL-12Rpi and the second cytokine receptor-binding domain is capable of binding IL-12RP2. In one embodiment, the first cytokine receptor-binding domain is capable of binding IL-12RP2 and the second cytokine receptor-binding domain is capable of binding IL-12Rpi. The first and/or second cytokine receptor-binding domain may be an antibody fragment, such as Fv, Fab, scFv, scFab or single-domain antibody. In one embodiment, the first and/or second cytokine receptor-binding domains are single-domain antibodies. In a particular embodiment, the first and second cytokine receptor-binding domains are VHH domains.
In one aspect, the first cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 and the second cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10. In one aspect, the first cytokine receptorbinding domain comprises an amino acid sequence selected from SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, and the second cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. In one aspect, the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 3 and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 7. In one aspect, the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 7 and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 3.
In one aspect, the first cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, and the second cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65. In one aspect, the first cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, and the second cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60. In one aspect, the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 60 and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 62. In one aspect, the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 62 and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 60.
In one aspect, the first cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, and the second cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65. In one aspect, the first cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, and the second cytokine receptorbinding domain comprises an amino acid sequence selected from SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192. In one aspect, the first cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 62. In one aspect, the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 62 and the second cytokine receptorbinding domain comprises an amino acid sequence selected from SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192. In one aspect, the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 182 and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 62. In one aspect, the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 62 and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 182. In one aspect, the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 186 and the second cytokine receptorbinding domain comprises the sequence of SEQ ID NO: 62. In one aspect, the first cytokine receptorbinding domain comprises the sequence of SEQ ID NO: 62 and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 186. In one aspect, the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 190 and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 62. In one aspect, the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 62 and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 190.
Target antigen-binding domain
According to the invention, each antigen binding molecule of the pair of antigen binding molecules comprises a target-binding domain. The first antigen binding molecule comprises a first target-binding domain and the second antigen binding molecule comprises a second target-binding domain. Both the first and the second target-binding domain bind the same target, i.e. the same antigen. Yet, the first and the second target-binding domains bind distinct epitopes of said target. The first target-binding domain is capable of binding a first epitope on the target antigen and the second target-binding domain is capable of binding a second epitope on the target antigen. The first and the second target-binding domain are capable of binding the target simultaneously, i.e. the target-binding domains are non-competing domains. The first target-binding domain and the second target-binding domain do not compete for binding on the target antigen.
The target-binding domain of the first and/or second antigen binding molecule may be an antibody fragment. In one embodiment, the first target-binding domain is an antibody fragment and the second target-binding domain is an antibody fragment. In one embodiment, the first antigen binding molecule comprises a first target-binding domain, wherein the target binding domain is an antibody fragment, and the second antigen binding molecule comprises a second target-binding domain, wherein the targetbinding domain is an antibody fragment. The target-binding domain of the first and/or second antigen binding molecule may be a Fv, Fab, scFv, scFab molecule or a single domain antibody. In one embodiment, the first antigen binding molecule comprises a first target-binding domain, wherein the target binding domain is a Fv, Fab, scFv, scFab molecule or a single domain antibody, and the second antigen binding molecule comprises a second target-binding domain, wherein the target-binding domain is a Fv, Fab, scFv, scFab molecule or a single domain antibody.
In one embodiment, the target-binding domain of the first and/or second antigen binding molecule is a Fab molecule. In one embodiment, the first target-binding domain is a Fab molecule and the second target-binding domain is a Fab molecule. In one embodiment, the first antigen binding molecule comprises a first target-binding domain, wherein the target binding domain is a Fab molecule, and the second antigen binding molecule comprises a second target-binding domain, wherein the target-binding domain is a Fab molecule. In one embodiment, the first target-binding domain comprises a heavy chain variable domain (VHi), a light chain variable domain (VLi), a heavy chain constant domain (CHI i) and a light chain constant domain (CLi). In one embodiment, the second target-binding domain comprises a heavy chain variable domain (VH2), a light chain variable domain (VL2), a heavy chain constant domain (CHI 2) and a light chain constant domain (CL2).
The target-binding domain may be a cross-Fab. In one embodiment, the first target-binding domain is a cross-Fab. In another embodiment, the second target-binding domain is a cross-Fab. In one embodiment, the first antigen binding molecule comprises a first target-binding domain, wherein the target-binding domain is a Fab molecule, wherein the Fab molecule is a cross-Fab, and the second antigen binding molecule comprises a second target-binding domain, wherein the target-binding domain is a Fab molecule, wherein the Fab molecule is in not a cross-Fab, i.e. a conventional Fab molecule. In one embodiment, the first antigen binding molecule comprises a first target-binding domain, wherein the target-binding domain is Fab molecule, wherein the Fab molecule is a conventional Fab molecule, and the second antigen binding molecule comprises a second target-binding domain, wherein the targetbinding domain is a Fab molecule, wherein the Fab molecule is a cross-Fab. Both the first and the second target-binding domains specifically bind the same target antigen. The target antigen may be a tumor-associated antigen or a T cell antigen.
The target antigen may be a tumor-associated antigen such as CEA, FAP, Her2, Her3 or EGFR. The target antigen may be a human tumor-associated antigen. Thus, the target antigen may be human CEA, human FAP, human Her2, human Her3 or human EGFR. Thus, in one aspect the first target-binding domain is capable of binding a first epitope of CEA and the second target-binding domain is capable of binding a second epitope of CEA. In one aspect the first target-binding domain is capable of binding a first epitope of FAP and the second target-binding domain is capable of binding a second epitope of FAP. In one aspect the first target-binding domain is capable of binding a first epitope of Her2 and the second target-binding domain is capable of binding a second epitope of Her2. In one aspect the first target-binding domain is capable of binding a first epitope of Her3 and the second target-binding domain is capable of binding a second epitope of Her3. In one aspect the first target-binding domain is capable of binding a first epitope of EGFR and the second target-binding domain is capable of binding a second epitope of EGFR.
In one aspect, the first target-binding domain comprises a VHi of SEQ ID NO: 20 and a VLi of SEQ ID NO: 21 and the second target-binding domain comprises a VEf of SEQ ID NO: 22 and a VL2 of SEQ ID NO: 23. In one aspect, the first target-binding domain comprises a VHi of SEQ ID NO: 22 and a VLi of SEQ ID NO: 23 and the second target-binding domains comprises a VH2 of SEQ ID NO: 20 and a VL2 of SEQ ID NO: 21. In one aspect, the first target-binding domain comprises a VHi of SEQ ID NO: 114 and a VLi of SEQ ID NO: 115 and the second target-binding domain comprises a VH2 of SEQ ID NO: 116 and a VL2 of SEQ ID NO: 117. In one aspect, the first target-binding domain comprises a VHi of SEQ ID NO: 116 and a VLi of SEQ ID NO: 117 and the second target-binding domains comprises a VH2 of SEQ ID NO: 114 and a VL2 of SEQ ID NO: 115. In one aspect, the first target-binding domain comprises a VHi of SEQ ID NO: 130 and a VLi of SEQ ID NO: 131 and the second target-binding domain comprises a VH2 of SEQ ID NO: 132 and a VL2 of SEQ ID NO: 133. In one aspect, the first target-binding domain comprises a VHi of SEQ ID NO: 132 and a VLi of SEQ ID NO: 133 and the second target-binding domains comprises a VH2 of SEQ ID NO: 130 and a VL2 of SEQ ID NO: 131. In one aspect, the first target-binding domain comprises a VHi of SEQ ID NO: 140 and a VLi of SEQ ID NO: 141 and the second target-binding domain comprises aVEf of SEQ ID NO: 142 and aVL2 of SEQ ID NO: 143. In one aspect, the first target-binding domain comprises a VHi of SEQ ID NO: 142 and a VLi of SEQ ID NO: 143 and the second target-binding domains comprises a VH2 of SEQ ID NO: 140 and a VL2 of SEQ ID NO: 141.
The target antigen may be a T cell antigen such as PD-1 or LAG-3. The target antigen may be human PD-1 or human LAG-3. Thus, in one aspect the first target-binding domain is capable of binding a first epitope of PD-1 and the second target-binding domain is capable of binding a second epitope of PD-1. In one aspect the first target-binding domain is capable of binding a first epitope of LAG-3 and the second target-binding domain is capable of binding a second epitope of LAG-3. In one aspect, the first target-binding domain comprises a VHi of SEQ ID NO: 76 and a VLi of SEQ ID NO: 77 and the second target-binding domain comprises a VFL of SEQ ID NO: 78 and a VL2 of SEQ ID NO: 79. In one aspect, the first target-binding domain comprises a VHi of SEQ ID NO: 78 and a VLi of SEQ ID NO: 79 and the second target-binding domains comprises a VH2 of SEQ ID NO: 76 and a VL2 of SEQ ID NO: 77. In one aspect, the first target-binding domain comprises a VHi of SEQ ID NO: 93 and a VLi of SEQ ID NO: 94 and the second target-binding domain comprises a VH2 of SEQ ID NO: 78 and a VL2 of SEQ ID NO: 79. In one aspect, the first target-binding domain comprises a VHi of SEQ ID NO: 78 and a VLi of SEQ ID NO: 79 and the second target-binding domains comprises a VH2 of SEQ ID NO: 93 and a VL2 of SEQ ID NO: 94. In one aspect, the first target-binding domain comprises a VHi of SEQ ID NO: 150 and a VLi of SEQ ID NO: 151 and the second target-binding domain comprises a VH2 of SEQ ID NO: 152 and a VL2 of SEQ ID NO: 153. In one aspect, the first target-binding domain comprises a VHi of SEQ ID NO: 152 and a VLi of SEQ ID NO: 153 and the second target-binding domains comprises a VH2 of SEQ ID NO: 150 and a VL2 of SEQ ID NO: 151.
Fc domain
According to the invention, the first and the second antigen binding molecules comprise an Fc domain.
The Fc domain of the binding molecule(s) consists of a pair of polypeptide chains, a first Fc domain subunit and a second Fc domain subunit. The first and second Fc domain subunits may comprise heavy chain domains of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which comprises the CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain are capable of stable association with each other.
In one aspect, the first antigen binding molecule comprises a Fc domain composed of a first and a second Fc domain subunit, and/or the second antigen binding molecule comprises a Fc domain composed of a first and a second Fc domain subunit. In one aspect, the first antigen binding molecule comprises a Fc domain composed of a first and a second Fc domain subunit, and the second antigen binding molecule comprises a Fc domain composed of a first and a second Fc domain subunit. In one aspect, each of the antigen binding molecules comprises not more than one Fc domain. one aspectThe Fc domain of the first and/or the second antigen binding molecule may be an IgG Fc domain. In a particular embodiment, the Fc domain is an IgGi Fc domain. In another particular embodiment, the Fc domain is an IgG4 Fc domain. In a more specific aspect, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at position S228 (Kabat EU index numbering), particularly the amino acid substitution S228P. This amino acid substitution reduces in vivo Fab arm exchange of IgG4 antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In further aspects, the Fc domain is ahuman Fc domain. In particular aspects, the Fc domain is a human IgGi Fc domain.
In one aspect, the Fc domain comprises a modification promoting the association of the first and the second subunit of the Fc domain. Fc domain modifications promoting heterodimerization are further described hereinbelow.
In one aspect, the Fc domain comprises one or more amino acid substitution that reduces binding to an Fc receptor and/or effector function. Fc domain modifications reducing Fc receptor binding and/or effector function are further described hereinbelow.
Fc domain modifications promoting heterodimerization
The antigen binding molecule(s) according to the invention comprise a target-binding domain and a cytokine receptor-binding domain, which may be fused to the first and/or the second Fc domain subunit, thus the two Fc domain subunits are typically comprised in two non-identical polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization leads to several possible combinations of the two polypeptides. To improve the yield and purity of the antigen binding molecule(s) in recombinant production, it will thus be advantageous to introduce in the Fc domain of the antigen binding molecule(s) a modification promoting the association of the desired polypeptides.
Accordingly, in particular aspects, the Fc domain of the antigen binding molecule(s) according to the invention comprises a modification promoting the association of the first and the second subunit of the Fc domain. The site of most extensive protein-protein interaction between the two subunits of a human IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in one aspect, said modification is in the CH3 domain of the Fc domain.
There exist several approaches for modifications in the CH3 domain of the Fc domain in order to enforce heterodimerization, which are well described e.g. in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012058768, WO 2013157954, WO 2013096291. Typically, in all such approaches the CH3 domain of the first subunit of the Fc domain and the CH3 domain of the second subunit of the Fc domain are both engineered in a complementary manner so that each CH3 domain (or the heavy chain comprising it) can no longer homodimerize with itself but is forced to heterodimerize with the complementarily engineered other CH3 domain (so that the first and second CH3 domain heterodimerize and no homdimers between the two first or the two second CH3 domains are formed).
In specific aspects, said modification promoting the association of the first and the second subunit of the Fc domain is a so-called “knob-into-hole” modification, comprising a “knob” modification in one of the two subunits of the Fc domain and a “hole” modification in the other one of the two subunits of the Fc domain.
The knob-into-hole technology is described e.g. in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a protuberance (“knob”) at the interface of a first polypeptide and a corresponding cavity (“hole”) in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g. tyrosine or tryptophan).
Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine).
Accordingly, in preferred aspects, in the CH3 domain of the first subunit of the Fc domain of the antigen binding molecule(s) an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit which is positionable in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable.
Preferably said amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably said amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V). The protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g. by site-specific mutagenesis, or by peptide synthesis.
In specific aspects, in (the CH3 domain of) the first subunit of the Fc domain (the “knobs” subunit) the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in (the CH3 domain of) the second subunit of the Fc domain (the “hole” subunit) the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one aspect, in the second subunit of the Fc domain additionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numberings according to Kabat EU index).
In yet further aspects, in the first subunit of the Fc domain additionally the serine residue at position 354 is replaced with a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C) (particularly the serine residue at position 354 is replaced with a cysteine residue), and in the second subunit of the Fc domain additionally the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C) (numberings according to Kabat EU index). Introduction of these two cysteine residues results in formation of a disulfide bridge between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).
In particular aspects, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W, and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to Kabat EU index).
Other techniques of CH3 -modification for enforcing the heterodimerization are contemplated as alternatives according to the invention and are described e.g. in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012/058768, WO 2013/157954, WO 2013/096291.
In one aspect, the heterodimerization approach described in EP 1870459, is used alternatively. This approach is based on the introduction of charged amino acids with opposite charges at specific amino acid positions in the CH3/CH3 domain interface between the two subunits of the Fc domain.
A particular aspect for the antigen binding molecule(s) of the invention are amino acid mutations R409D; K370E in one of the two CH3 domains (of the Fc domain) and amino acid mutations D399K; E357K in the other one of the CH3 domains of the Fc domain (numbering according to Kabat EU index).
In one aspect, the antigen binding molecule(s) of the invention comprises amino acid mutation T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, and additionally amino acid mutations R409D; K370E in the CH3 domain of the first subunit of the Fc domain and amino acid mutations D399K; E357K in the CH3 domain of the second subunit of the Fc domain (numberings according to Kabat EU index).
In one aspect, the antigen binding molecule(s) of the invention comprises amino acid mutations S354C, T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations Y349C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, or said antigen binding molecule(s) comprises amino acid mutations Y349C, T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations S354C, T366S, L368A, Y 407V in the CH3 domains of the second subunit of the Fc domain and additionally amino acid mutations R409D; K370E in the CH3 domain of the first subunit of the Fc domain and amino acid mutations D399K; E357K in the CH3 domain of the second subunit of the Fc domain (all numberings according to Kabat EU index).
In one aspect, the heterodimerization approach described in WO 2013/157953 is used alternatively. In one aspect, a first CH3 domain comprises amino acid mutation T366K and a second CH3 domain comprises amino acid mutation L351D (numberings according to Kabat EU index). In further aspects, the first CH3 domain comprises further amino acid mutation L35 IK. In further aspects, the second CH3 domain comprises further an amino acid mutation selected from Y349E, Y349D and L368E (particularly L368E) (numberings according to Kabat EU index).
In one aspect, the heterodimerization approach described in WO 2012/058768 is used alternatively. In one aspect, a first CH3 domain comprises amino acid mutations L351Y, Y407A and a second CH3 domain comprises amino acid mutations T366A, K409F. In further aspects, the second CH3 domain comprises a further amino acid mutation at position T411, D399, S400, F405, N390, or K392, e.g. selected from a) T41 IN, T411R, T41 IQ, T41 IK, T41 ID, T41 IE or T411W, b) D399R, D399W, D399Y or D399K, c) S400E, S400D, S400R, or S400K, d) F405I, F405M, F405T, F405S, F405V or F405W, e) N390R, N390K or N390D, f) K392V, K392M, K392R, K392L, K392F or K392E (numberings according to Kabat EU index). In further aspects, a first CH3 domain comprises amino acid mutations L351Y, Y407A and a second CH3 domain comprises amino acid mutations T366V, K409F. In further aspects, a first CH3 domain comprises amino acid mutation Y 407A and a second CH3 domain comprises amino acid mutations T366A, K409F. In further aspects, the second CH3 domain further comprises amino acid mutations K392E, T41 IE, D399R and S400R (numberings according to Kabat EU index).
In one aspect, the heterodimerization approach described in WO 2011/143545 is used alternatively, e.g. with the amino acid modification at a position selected from the group consisting of 368 and 409 (numbering according to Kabat EU index).
In one aspect, the heterodimerization approach described in WO 2011/090762, which also uses the knobs-into-holes technology described above, is used alternatively. In one aspect, a first CH3 domain comprises amino acid mutation T366W and a second CH3 domain comprises amino acid mutation Y407A. In one aspect, a first CH3 domain comprises amino acid mutation T366Y and a second CH3 domain comprises amino acid mutation Y407T (numberings according to Kabat EU index).
In one aspect, the antigen binding molecule(s) or its Fc domain is of IgG2 subclass and the heterodimerization approach described in WO 2010/129304 is used alternatively. In an alternative aspect, a modification promoting association of the first and the second subunit of the Fc domain comprises a modification mediating electrostatic steering effects, e.g. as described in PCT publication WO 2009/089004. Generally, this method involves replacement of one or more amino acid residues at the interface of the two Fc domain subunits by charged amino acid residues so that homodimer formation becomes electrostatically unfavorable but heterodimerization electrostatically favorable. In some such aspects, a first CH3 domain comprises amino acid substitution of K392 or N392 with a negatively charged amino acid (e.g. glutamic acid (E), or aspartic acid (D), particularly K392D or N392D) and a second CH3 domain comprises amino acid substitution of D399, E356, D356, or E357 with a positively charged amino acid (e.g. lysine (K) or arginine (R), particularly D399K, E356K, D356K, or E357K, and more particularly D399K and E356K). In further aspects, the first CH3 domain further comprises amino acid substitution of K409 or R409 with a negatively charged amino acid (e.g. glutamic acid (E), or aspartic acid (D), particularly K409D or R409D). In further aspects, the first CH3 domain further or alternatively comprises amino acid substitution of K439 and/or K370 with a negatively charged amino acid (e.g. glutamic acid (E), or aspartic acid (D)) (all numberings according to Kabat EU index).
In one aspect, the heterodimerization approach described in WO 2007/147901 is used alternatively. In one aspect, a first CH3 domain comprises amino acid mutations K253E, D282K, and K322D and a second CH3 domain comprises amino acid mutations D239K, E240K, and K292D (numberings according to Kabat EU index). In one aspect, the heterodimerization approach described in WO 2007/110205 can be used alternatively.
In one aspect, the first subunit of the Fc domain comprises amino acid substitutions K392D and K409D, and the second subunit of the Fc domain comprises amino acid substitutions D356K and D399K (numbering according to Kabat EU index).
Fc domain modifications reducing Fc receptor binding and/or effector function
The Fc domain confers to the antigen binding molecule(s) favorable pharmacokinetic properties, including a long serum half-life, which contributes to good accumulation in the target tissue and a favorable tissue-blood distribution ratio. At the same time it may, however, lead to undesirable targeting of the binding molecule(s) to cells expressing Fc receptors rather than to the preferred antigen-bearing cells. Moreover, the co-activation of Fc receptor signaling pathways may lead to cytokine release which may result in excessive activation of cytokine receptors and severe side effects upon systemic administration. Activation of (Fc receptor-bearing) immune cells other than T cells may even reduce efficacy of the pair of antigen binding molecules due to the potential destruction of T cells e.g. by NK cells. Accordingly, in particular aspects, the Fc domain of the antigen binding molecule(s) according to the invention exhibits reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a native IgGi Fc domain. In some such aspects, the Fc domain (or the antigen binding molecule comprising said Fc domain) exhibits less than 50%, particularly less than 20%, more particularly less than 10% and most particularly less than 5% of the binding affinity to an Fc receptor, as compared to a native IgGi Fc domain (or a antigen binding molecule comprising a native IgGi Fc domain), and/or less than 50%, particularly less than 20%, more particularly less than 10% and most particularly less than 5% of the effector function, as compared to a native IgGi Fc domain domain (or a antigen binding molecule comprising a native IgGi Fc domain). In one aspect, the Fc domain domain (or the antigen binding molecule comprising said Fc domain) does not substantially bind to an Fc receptor and/or induce effector function. In particular aspects, the Fc receptor is an Fey receptor. In one aspect, the Fc receptor is a human Fc receptor. In one aspect, the Fc receptor is an activating Fc receptor. In specific aspects, the Fc receptor is an activating human Fey receptor, more specifically human FcyRIIIa, FcyRI or FcyRIIa, most specifically human FcyRIIIa. In one aspect, the effector function is one or more selected from the group of CDC, ADCC, ADCP, and cytokine secretion. In particular aspects, the effector function is ADCC. In one aspect, the Fc domain exhibits substantially similar binding affinity to neonatal Fc receptor (FcRn), as compared to a native IgGi Fc domain domain. Substantially similar binding to FcRn is achieved when the Fc domain (or the antigen binding molecule comprising said Fc domain) exhibits greater than about 70%, particularly greater than about 80%, more particularly greater than about 90% of the binding affinity of a native IgGi Fc domain (or the antigen binding molecule comprising a native IgGi Fc domain) to FcRn.
In certain aspects, the Fc domain is engineered to have reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a non-engineered Fc domain. In particular aspects, the Fc domain of the antigen binding molecule(s) comprises one or more amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and/or effector function. Typically, the same one or more amino acid mutation is present in each of the two subunits of the Fc domain. In one aspect, the amino acid mutation reduces the binding affinity of the Fc domain to an Fc receptor. In one aspect, the amino acid mutation reduces the binding affinity of the Fc domain to an Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In aspects where there is more than one amino acid mutation that reduces the binding affinity of the Fc domain to the Fc receptor, the combination of these amino acid mutations may reduce the binding affinity of the Fc domain to an Fc receptor by at least 10-fold, at least 20-fold, or even at least 50-fold. In one aspect, the antigen binding molecule(s) comprising an engineered Fc domain exhibits less than 20%, particularly less than 10%, more particularly less than 5% of the binding affinity to an Fc receptor as compared to a antigen binding molecule comprising a nonengineered Fc domain. In particular aspects, the Fc receptor is an Fey receptor. In one aspect, the Fc receptor is a human Fc receptor. In one aspect, the Fc receptor is an activating Fc receptor. In specific aspects, the Fc receptor is an activating human Fey receptor, more specifically human FcyRIIIa, FcyRI or FcyRIIa, most specifically human FcyRIIIa. Preferably, binding to each of these receptors is reduced. In one aspect, binding affinity to a complement component, specifically binding affinity to Clq, is also reduced. In one aspect, binding affinity to neonatal Fc receptor (FcRn) is not reduced.
Substantially similar binding to FcRn, i.e. preservation of the binding affinity of the Fc domain to said receptor, is achieved when the Fc domain (or the antigen binding molecule comprising said Fc domain) exhibits greater than about 70% of the binding affinity of a non-engineered form of the Fc domain (or the antigen binding molecule comprising said non-engineered form of the Fc domain) to FcRn. The Fc domain, or antigen binding molecule(s) of the invention comprising said Fc domain, may exhibit greater than about 80% and even greater than about 90% of such affinity. In certain aspects, the Fc domain of the antigen binding molecule(s) is engineered to have reduced effector function, as compared to a nonengineered Fc domain. The reduced effector function can include, but is not limited to, one or more of the following: reduced complement dependent cytotoxicity (CDC), reduced antibody dependent cell- mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling inducing apoptosis, reduced crosslinking of target-bound antibodies, reduced dendritic cell maturation, or reduced T cell priming. In one aspect, the reduced effector function is one or more selected from the group of reduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion. In particular aspects, the reduced effector function is reduced ADCC. In one aspect, the reduced ADCC is less than 20% of the ADCC induced by a nonengineered Fc domain (or a binding molecule comprising a non-engineered Fc domain).
In one aspect, the amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and/or effector function is an amino acid substitution. In one aspect, the Fc domain comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331 and P329 (numberings according to Kabat EU index). In a more specific aspect, the Fc domain comprises an amino acid substitution at a position selected from the group of L234, L235 and P329 (numberings according to Kabat EU index). In one aspect, the Fc domain comprises the amino acid substitutions L234A and L235A (numberings according to Kabat EU index). In some such aspects, the Fc domain is an IgGi Fc domain, particularly a human IgGi Fc domain. In one aspect, the Fc domain comprises an amino acid substitution at position P329. In a more specific aspect, the amino acid substitution is P329A or P329G, particularly P329G (numberings according to Kabat EU index). In one aspect, the Fc domain comprises an amino acid substitution at position P329 and a further amino acid substitution at a position selected from E233, L234, L235, N297 and P331 (numberings according to Kabat EU index). In a more specific aspect, the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In particular aspects, the Fc domain comprises amino acid substitutions at positions P329, L234 and L235 (numberings according to Kabat EU index). In more particular aspects, the Fc domain comprises the amino acid mutations L234A, L235A and P329G (“P329G LALA”, “PGLALA” or “LALAPG”). Specifically, in particular aspects, each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering), i.e. in each of the first and the second subunit of the Fc domain the leucine residue at position 234 is replaced with an alanine residue (L234A), the leucine residue at position 235 is replaced with an alanine residue (L235A) and the proline residue at position 329 is replaced by a glycine residue (P329G) (numbering according to Kabat EU index).
In some such aspects, the Fc domain is an IgGi Fc domain, particularly a human IgGi Fc domain. The “P329G LALA” combination of amino acid substitutions almost completely abolishes Fey receptor (as well as complement) binding of a human IgGi Fc domain, as described in PCT publication no. WO 2012/130831, which is incorporated herein by reference in its entirety. WO 2012/130831 also describes methods of preparing such mutant Fc domains and methods for determining its properties such as Fc receptor binding or effector functions.
IgG4 antibodies exhibit reduced binding affinity to Fc receptors and reduced effector functions as compared to IgGi antibodies. Hence, in one aspect, the Fc domain of the binding molecule(s) of the invention is an IgG4 Fc domain, particularly a human IgG4 Fc domain. In one aspect, the IgG4 Fc domain comprises an amino acid substitution at position S228, specifically the amino acid substitution S228P (numberings according to Kabat EU index). To further reduce its binding affinity to an Fc receptor and/or its effector function, in one aspect, the IgG4 Fc domain comprises an amino acid substitution at position L235, specifically the amino acid substitution L235E (numberings according to Kabat EU index). In one aspect, the IgG4 Fc domain comprises an amino acid substitution at position P329, specifically the amino acid substitution P329G (numberings according to Kabat EU index). In a preferred aspect, the IgG4 Fc domain comprises amino acid substitutions at positions S228, L235 and P329, specifically amino acid substitutions S228P, L235E and P329G (numberings according to Kabat EU index). Such IgG4 Fc domain mutants and their Fey receptor binding properties are described in PCT publication no. WO 2012/130831, incorporated herein by reference in its entirety.
In particular aspects, the Fc domain exhibiting reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a native IgGi Fc domain, is a human IgGi Fc domain comprising the amino acid substitutions L234A, L235A and optionally P329G, or a human IgG4 Fc domain comprising the amino acid substitutions S228P, L235E and optionally P329G (numberings according to Kabat EU index).
In certain aspects, N-glycosylation of the Fc domain has been eliminated. In some such aspects, the Fc domain comprises an amino acid mutation at position N297, particularly an amino acid substitution replacing asparagine by alanine (N297A) or aspartic acid (N297D) (numberings according to Kabat EU index).
In addition to the Fc domains described hereinabove and in PCT publication no. WO 2012/130831, Fc domains with reduced Fc receptor binding and/or effector function also include those with substitution of one or more ofFc domain residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056) (numberings according to Kabat EU index). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581).
Mutant Fc domains can be prepared by amino acid deletion, substitution, insertion or modification using genetic or chemical methods well known in the art. Genetic methods may include site specific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, and the like. The correct nucleotide changes can be verified for example by sequencing.
Binding to Fc receptors can be easily determined e.g. by ELISA, or by Surface Plasmon Resonance (SPR) using standard instrumentation such as a BIAcore instrument (GE Healthcare), and Fc receptors such as may be obtained by recombinant expression. Alternatively, binding affinity of Fc domains or binding molecule(s) comprising an Fc domain for Fc receptors may be evaluated using cell lines known to express particular Fc receptors, such as human NK cells expressing Fcyllla receptor.
Effector function of an Fc domain, or a binding molecule comprising an Fc domain, can be measured by methods known in the art. Examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83, 7059- 7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S. Patent No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987).
Alternatively, non-radioactive assays may be employed (see, for example, ACTI™ nonradioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g. in an animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998). In one aspect, binding of the Fc domain to a complement component, specifically to Clq, is reduced. Accordingly, in one aspect wherein the Fc domain is engineered to have reduced effector function, said reduced effector function includes reduced CDC. Clq binding assays may be carried out to determine whether the Fc domain, or the binding molecule comprising the Fc domain, is able to bind Clq and hence has CDC activity. See e.g., Clq and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)).
FcRn binding and in vivo clearance/half life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int’l. Immunol. 18(12): 1759-1769 (2006); WO 2013/120929).
Configuration of the antigen binding molecules
The antigen binding molecule(s) according to the present invention may have various molecular configurations, i.e. the domains of the binding molecule(s) may be linked to each other in different ways.
The antigen binding molecule(s) may comprise a target-binding domain, a cytokine receptor-binding domain and a Fc domain, wherein the cytokine receptor-binding domain is fused at its C-terminus to the N-terminus of the target-binding domain, and the target-binding domain is fused at its C-terminus to the N-terminus of one of the Fc domain subunits. The target-binding domain may be a Fab molecule. Thus, the cytokine receptor-binding domain may be fused to the VH or VL domain of the target bindingdomain. In one embodiment, the antigen binding molecule(s) comprise a target-binding domain, a cytokine receptor-binding domain and a Fc domain, wherein the target-binding domain is a Fab molecule, and wherein the cytokine receptor-binding domain is fused at its C-terminus to the N-terminus of the VH domain of the target-binding domain, and the target-binding domain is fused at its C-terminus to the N-terminus of one of the Fc domain subunits (see e.g. Fig. 1 IB, 11C, 1 IF and 11G for schematic illustration). In one embodiment, the antigen binding molecule(s) comprise a target-binding domain, a cytokine receptor-binding domain and a Fc domain, wherein the target-binding domain is a Fab molecule, and wherein the cytokine receptor-binding domain is fused at its C-terminus to the N-terminus of the VL domain of the target-binding domain, and the target-binding domain is fused at its C-terminus to the N-terminus of one of the Fc domain subunits (see e.g. Fig. 1 ID, 1 IE, 11H and 1 II for schematic illustration).
The antigen binding molecule(s) may comprise a target-binding domain, a cytokine receptor-binding domain and a Fc domain, wherein the target-binding domain is fused at its C-terminus to the N-terminus of the first Fc domain subunit and the cytokine receptor-binding domain is fused at its C-terminus to the N-terminus of the second Fc domain subunit (see e.g. Fig. 13A, 13B, 13C and 13D for schematic illustration).
The antigen binding molecule(s) may comprise a target-binding domain, a cytokine receptor-binding domain and a Fc domain, wherein the target-binding domain is fused at its C-terminus to the N-terminus of one of the Fc domain subunits and the cytokine receptor-binding domain is fused at its N-terminus to the C-terminus of the same Fc domain subunit (see e.g. Fig. 13E, 13F, 13G and 13H for schematic illustration).
The cytokine-receptor binding domain may be fused via a peptide linker. The peptide linker may comprise an amino acid sequence according to SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 279, SEQ ID NO: 280 or SEQ ID NO: 281.
Pairs of antigen binding molecules
In one aspect, both antigen binding molecules of the pair of antigen binding molecules comprise a targetbinding domain, a cytokine receptor-binding domain and a Fc domain, wherein the cytokine receptorbinding domain is fused at its C-terminus to the N-terminus of the target-binding domain, and the targetbinding domain is fused at its C-terminus to the N-terminus of one of the Fc domain subunits.
In one aspect, the target-binding domains of the antigen binding molecules are Fab molecules. Thus, in one aspect the pair of antigen binding molecules comprises a first antigen binding molecule comprising a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a first cytokine receptor-binding domain, VHi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VLi and CLi; and a second antigen binding molecule comprising a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a second cytokine receptor-binding domain, VH2, CHI 2 and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VL2 and CL2.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VHi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus a first cytokine receptor-binding domain, VLi and CLi; and a second antigen binding molecule comprising a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VH2, CHI 2 and a second Fc domain subunit; a third polypeptide comprising in order from the N-terminus to C-terminus a second cytokine receptor-binding domain, VL2 and CL2. In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a first cytokine receptor-binding domain, VHi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VLi and CLi; and a second antigen binding molecule comprising a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VH2, CHI 2 and a second Fc domain subunit; a third polypeptide comprising in order from the N-terminus to C-terminus a second cytokine receptor-binding domain, VL2 and CL2.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a first cytokine receptor-binding domain, VLi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VHi and CLi; and a second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a second cytokine receptor-binding domain, VH2, CHI 2 and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VL2 and CL2.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VLi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus a first cytokine receptor-binding domain, VHi and CLi; and a second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VH2, CHI 2 and a second Fc domain subunit; a third polypeptide comprising in order from the N-terminus to C-terminus a second cytokine receptor-binding domain, VL2 and CL2.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a first cytokine receptor-binding domain, VLi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VHi and CLi; and a second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VH2, CHh and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus a second cytokine receptor-binding domain, VL2 and CL2. In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a VLi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus a first cytokine receptor-binding domain, VHi and CLi; and a second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a second cytokine receptor-binding domain, VH2, CHI 2 and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VL2 and CL2.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 27 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 30 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 38 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 37 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 31, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 33.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 35, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 36.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 27 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 33.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 30 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 31.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 38 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 36.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 37 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 35.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 260 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 259 and a third polypeptide comprising an amino acid sequence of SEQ
ID NO: 127.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 119 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 256, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 122 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 255.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 261 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 258 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 127.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 119 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 257, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 112 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 254.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 260 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 112 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 255.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 119 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 256, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 259 and a third polypeptide comprising an amino acid sequence of SEQ
ID NO: 127.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 261 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 112 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 254.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 119 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 257, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 258 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 127.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 86, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 44 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 83, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 88 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 87.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 92, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 44 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 91.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 96 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 95, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 101 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 99 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 98, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 88 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 102.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 96 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 97, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 103 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 100 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 98, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 88 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 104.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 98 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 99, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 101 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 96 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 95, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 88 and a third polypeptide comprising an amino acid sequence of SEQ ID
NO: 102.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 100 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 98, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 103 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 96 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 97, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 88 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 104.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 110 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 113 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 105, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 109.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 111 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 112 and a third polypeptide comprising an amino acid sequence of SEQ
ID NO: 29.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 107, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 108.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 110 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 109.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 105, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 113 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 111 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 108.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 107, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 112 and a third polypeptide comprising an amino acid sequence of SEQ
ID NO: 29.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 125 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 129 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 127.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 119 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 118, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 122 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 123.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 126 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 128 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 127.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 119 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 120, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 122 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 121.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 125 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 122 and a third polypeptide comprising an amino acid sequence of SEQ
ID NO: 123.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 119 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 118, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 129 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 127.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 126 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 122 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 121.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 119 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 120, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 128 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 127.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 136 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 134, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 138 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 137.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 135 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 134, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 138 and a third polypeptide comprising an amino acid sequence of SEQ
ID NO: 139.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 146 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 144, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 148 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 147.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 145 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 144, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 148 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 149.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 155 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 154, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 165 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 163.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 161 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 160, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 158 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 159.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 155 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 156, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 164 and a third polypeptide comprising an amino acid sequence of SEQ
ID NO: 163.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 162 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 160, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 158 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 157.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 161 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 160, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 165 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 163.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 155 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 154, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 158 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 159.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 162 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 160, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 164 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 163.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 155 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 156, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 158 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 157.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 194 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 193, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 195 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 196 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 197 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 198 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID
NO: 89.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 199 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 200 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 201 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 202 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 203 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 204 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 205 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89.
In one aspect, both antigen binding molecules of the pair of antigen binding molecules comprise a targetbinding domain, a cytokine receptor-binding domain and a Fc domain, wherein the target-binding domain is fused at its C-terminus to the N-terminus of the first Fc domain subunit and the cytokine receptor-binding domain is fused at its C-terminus to the N-terminus of the second Fc domain subunit. In one aspect, both antigen binding molecules of the pair of antigen binding molecules comprise a targetbinding domain, a cytokine receptor-binding domain and a Fc domain, wherein the target-binding domain is fused at its C-terminus to the N-terminus of the first Fc domain subunit and the cytokine receptor-binding domain is fused at its C-terminus to the N-terminus of the second Fc domain subunit, wherein the target-binding domain is a Fab molecule. In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising in order from the N-terminus to C-terminus VHi, CHli and a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VLi and CLi, and athird polypeptide comprising in order from the N-terminus to C-terminus a first cytokine receptor-binding domain and a second Fc domain subunit, and a second antigen binding molecule comprises a first polypeptide comprising in order from the N-terminus to C-terminus VFE, CHE and a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VL2 and CL2, and athird polypeptide comprising in order from the N-terminus to C-terminus a second cytokine receptor-binding domain and a second Fc domain subunit. In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising in order from the N-terminus to C-terminus VLi, CHli and a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VHi and CLi, and a third polypeptide comprising in order from the N-terminus to C-terminus a first cytokine receptor-binding domain and a second Fc domain subunit, and a second antigen binding molecule comprising a first polypeptide comprising in order from the N-terminus to C-terminus VH2, CHh and a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VL2 and CL2, and a third polypeptide comprising in order from the N-terminus to C-terminus a second cytokine receptor-binding domain and a second Fc domain subunit.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 32, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 25 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 39, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 34, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 29 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 40.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 34, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 29 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 39, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 32, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 25 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 40.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 237, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 238, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 235, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 236, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 239, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 240, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 235, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 237, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 236, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 235, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 240, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 239, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
In one aspect, both antigen binding molecules of the pair of antigen binding molecules comprise a targetbinding domain, a cytokine receptor-binding domain and a Fc domain, wherein the target-binding domain is fused at its C-terminus to the N-terminus of one of the Fc domain subunits and the cytokine receptor-binding domain is fused at its N-terminus to the C-terminus of the same Fc domain subunit. Thus in one aspect, the pair of antigen binding molecules comprises a first antigen binding molecule comprising a first target-binding domain fused at its C-terminus to the N-terminus of one of the Fc domain subunits and a first cytokine receptor-binding domain fused at its N-terminus to the C-terminus of the same Fc domain subunit, and second antigen binding molecule comprising a second target-binding domain fused at its C-terminus to the N-terminus of one of the Fc domain subunits and a second cytokine receptor-binding domain fused at its N-terminus to the C-terminus of the same Fc domain subunit.
In one aspect, the target-binding domain is a Fab molecule. Thus in one embodiment, the pair of antigen binding molecules comprises a first antigen binding molecule comprising a target-binding domain fused at its C-terminus of CHli to the N-terminus of one of the Fc domain subunits and a first cytokine receptor-binding domain is fused at its N-terminus to the C-terminus of the same Fc domain subunit, and a second antigen binding molecule comprising a target-binding domain fused at its C-terminus of CHh to the N-terminus of one of the Fc domain subunits and a second cytokine receptor-binding domain fused at its N-terminus to the C-terminus of the same Fc domain subunit.
In one aspect, the target-binding domain of one of the antigen-binding molecules is a cross-Fab molecule. Thus, in one embodiment, the pair of antigen binding molecules comprises a first antigen binding molecule comprising a target-binding domain fused at its C-terminus of CHli to the N-terminus of one of the Fc domain subunits and a first cytokine receptor-binding domain is fused at its N-terminus to the C-terminus of the same Fc domain subunit, and a second antigen binding molecule comprising a target-binding domain fused at its C-terminus of CL2 to the N-terminus of one of the Fc domain subunits and a second cytokine receptor-binding domain fused at its N-terminus to the C-terminus of the same Fc domain subunit. In one embodiment, the pair of antigen binding molecules comprises a first antigen binding molecule comprising a target-binding domain fused at its C-terminus of CLi to the N-terminus of one of the Fc domain subunits and a first cytokine receptor-binding domain is fused at its N-terminus to the C-terminus of the same Fc domain subunit, and a second antigen binding molecule comprising a target-binding domain fused at its C-terminus of CHI 2 to the N-terminus of one of the Fc domain subunits and a second cytokine receptor-binding domain fused at its N-terminus to the C-terminus of the same Fc domain subunit.
In one aspect, the pair of antigen binding molecules comprises a first antigen binding molecule comprising a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a VHi, a CHli, a second Fc domain subunit and a first cytokine receptor-binding domain, and a third polypeptide comprising in order from the N-terminus to C-terminus a VLi and a CLi; and a second antigen binding molecule comprising a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a VH2, a CHh, a second Fc domain subunit and a second cytokine receptor-binding domain, and a third polypeptide comprising in order from the N-terminus to C-terminus VL2 and CL2.
In one aspect, the pair of antigen binding molecules comprises a first antigen binding molecule comprising a first polypeptide comprising a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a VLi, a CHli, a second Fc domain subunit and a first cytokine receptor-binding domain, and a third polypeptide comprising in order from the N-terminus to C-terminus a VHi and a CLi; and a second antigen binding molecule comprising a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a VH2, a CHI 2, a second Fc domain subunit and a second cytokine receptor-binding domain, and a third polypeptide comprising in order from the N-terminus to C-terminus a VL2 and a CL2.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 42 and a third polypeptide comprising an amino acid sequence of SEQ ID NO : 41 , and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 45, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 44 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 43.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 46 and a third polypeptide comprising an amino acid sequence of SEQ ID NO : 41 , and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 47, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 44 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 43.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 246 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 249 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 242 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 245 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 250 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 253 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 248 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 247 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 244 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 243 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 252 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 251 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 42 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 42, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 245 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
Alternative configurations of the antigen binding molecules
The antigen binding molecule(s) may comprise a target-binding domain, a cytokine receptor-binding domain and a Fc domain, wherein the cytokine receptor-binding domain is fused at its C-terminus to the N-terminus of the target-binding domain, and the target-binding domain is fused at its C-terminus to the N-terminus of one of the Fc domain subunits, wherein the target-binding domain is a VHH domain. Thus, the antigen binding molecule may comprise a first polypeptide comprising a first Fc domain subunit and a second polypeptide comprising in order from the N-terminus to C-terminus a first cytokine receptor-binding domain, a first target-binding domain and a second Fc domain subunit, and a second antigen binding molecule comprising a first polypeptide comprising a first Fc domain subunit and a second polypeptide comprising in order from the N-terminus to C-terminus a second cytokine receptorbinding domain, a second target-binding domain and a second Fc domain subunit.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28 and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 173, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28 and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 171.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28 and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 176, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28 and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 172.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28 and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 175, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28 and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 171.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28 and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 176, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28 and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 174.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28 and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 175, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28 and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 170.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28 and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 173, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28 and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 170.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28 and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 176, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28 and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 172.
The antigen binding molecule(s) may comprise a target-binding domain, a cytokine receptor-binding domain and a Fc domain, wherein the cytokine receptor-binding domain is fused at its C-terminus to the N-terminus of the target-binding domain, and the target-binding domain is fused at its C-terminus to the N-terminus of one of the Fc domain subunits, wherein the cytokine receptor-binding domain is a Fab molecule.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 267, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 268 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 265, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 272, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 270 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 271.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 267, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 268 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 265, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 274, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 270 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 273.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 269, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 265 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 268, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 272, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 270 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 271.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 269, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 265 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 268, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 274, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 270 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 273.
The antigen binding molecule(s) may comprise a target-binding domain, a cytokine receptor-binding domain and a Fc domain, wherein the cytokine receptor-binding domain is fused at its C-terminus to the N-terminus of the target-binding domain, and the target-binding domain is fused at its C-terminus to the N-terminus of one of the Fc domain subunits, wherein the cytokine receptor-binding domain is a scFv domain.
In one aspect, the pair of antigen binding molecules comprises first antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 276 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 275, and a second antigen binding molecule comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 277 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 278.
Specific embodiments of the invention
In the following specific embodiments of the invention are listed. 1. A pair of antigen binding molecules that specifically bind to a target antigen, comprising a) a first antigen binding molecule comprising i) a first target-binding domain, ii) a first cytokine receptorbinding domain, and iii) a Fc domain; and b) a second antigen binding molecule comprising i) a second target-binding domain, ii) a second cytokine receptor-binding domain, and iii) a Fc domain; wherein the first target-binding domain is capable of binding a first epitope on the target antigen and the second target-binding domain is capable of binding a second epitope on the target antigen, wherein the first and second target-binding domains do not compete for binding on the target antigen; and wherein the first cytokine receptor-binding domain is capable of binding a first cytokine receptor subunit and the second cytokine receptor-binding domain is capable of binding a second cytokine receptor subunit.
2. The pair of antigen binding molecules according to embodiment 1, wherein the first and second targetbinding domains are antibody fragments, such as Fv, Fab, scFv, scFab molecules or single domain antibodies.
3. The pair of antigen binding molecules according to embodiment 1 or 2, wherein the first and second target-binding domains are Fab molecules.
4. The pair of antigen binding molecules according to embodiment 3, wherein the first target-binding domain comprises a heavy chain variable domain (VHi), a light chain variable domain (VLi), a heavy chain constant domain (CHli) and a light chain constant domain (CLi), and wherein the second targetbinding domain comprises a heavy chain variable domain (VH2), a light chain variable domain (VL2), a heavy chain constant domain (CHI 2) and a light chain constant domain (CL2).
5. The pair of antigen binding molecules according to embodiment 3 or 4, wherein the first and/or second target-binding domain is a cross-Fab molecule.
6. The pair of antigen binding molecules according to any one of the preceding embodiments, wherein the first and second target-binding domains specifically bind to a tumor-associated antigen or a T-cell antigen.
7. The pair of antigen binding molecules according to any one of the preceding embodiments, wherein the first and second target-binding domains specifically bind to FAP, PD-1, Her2, Her3, LAG-3 or EGFR.
8. The pair of antigen binding molecules according to any one of the preceding embodiments, wherein a) the first target-binding domain comprises a VHi of SEQ ID NO: 20 and a VLi of SEQ ID NO: 21 and the second target-binding domain comprises a VH2 of SEQ ID NO: 22 and a VL2 of SEQ ID NO: 23, or b) the first target-binding domain comprises a VHi of SEQ ID NO: 22 and a VLi of SEQ ID NO: 23 and the second target-binding domain comprises a VH2 of SEQ ID NO: 20 and a VL2 of SEQ ID NO: 21, or c) the first target-binding domain comprises a VHi of SEQ ID NO: 76 and a VLi of SEQ ID NO: 77 and the second target-binding domain comprises a VH2 of SEQ ID NO: 78 and a VL2 of SEQ ID NO: 79, or d) the first target-binding domain comprises a VHi of SEQ ID NO: 78 and a VLi of SEQ ID NO: 79 and the second target-binding domain comprises a VH2 of SEQ ID NO: 76 and a VL2 of SEQ ID NO: 77; or e) the first target-binding domain comprises a VHi of SEQ ID NO: 93 and a VLi of SEQ ID NO: 94 and the second target-binding domain comprises a VH2 of SEQ ID NO: 78 and a VL2 of SEQ ID NO: 79; or f) the first target-binding domain comprises a VHi of SEQ ID NO: 78 and a VLi of SEQ ID NO: 79 and the second target-binding domains comprises a VH2 of SEQ ID NO: 93 and a VL2 of SEQ ID NO: 94, or g) the first target-binding domain comprises a VHi of SEQ ID NO: 130 and a VLi of SEQ ID NO: 131 and the second target-binding domain comprises a VH2 of SEQ ID NO: 132 and a VL2 of SEQ ID NO: 133; or h) the first target-binding domain comprises a VHi of SEQ ID NO: 132 and a VLi of SEQ ID NO: 133 and the second target-binding domains comprises a VH2 of SEQ ID NO: 130 and a VL2 of SEQ ID NO: 131; or i) the first target-binding domain comprises a VHi of SEQ ID NO: 140 and a VLi of SEQ ID NO: 141 and the second target-binding domain comprises a VH2 of SEQ ID NO: 142 and a VL2 of SEQ ID NO: 143; or j) the first target-binding domain comprises a VHi of SEQ ID NO: 142 and a VLi of SEQ ID NO: 143 and the second target-binding domains comprises a VH2 of SEQ ID NO: 140 and a VL2 of SEQ ID NO: 141; or k) the first target-binding domain comprises a VHi of SEQ ID NO: 114 and a VLi of SEQ ID NO: 115 and the second target-binding domain comprises a VH2 of SEQ ID NO: 116 and a VL2 of SEQ ID NO: 117; or 1) the first target-binding domain comprises a VHi of SEQ ID NO: 116 and a VLi of SEQ ID NO: 117 and the second target-binding domains comprises a VH2 of SEQ ID NO: 114 and a VL2 of SEQ ID NO: 115; or m) the first target-binding domain comprises a VHi of SEQ ID NO: 150 and a VLi of SEQ ID NO: 151 and the second target-binding domain comprises a VH2 of SEQ ID NO: 152 and a VL2 of SEQ ID NO: 153; or n) the first target-binding domain comprises a VHi of SEQ ID NO: 152 and a VLi of SEQ ID NO: 153 and the second targetbinding domains comprises a VH2 of SEQ ID NO: 150 and a VL2 of SEQ ID NO: 151.
9. The pair of antigen binding molecules according to any one of the preceding embodiments, wherein both the first and second cytokine receptor subunits are subunits of the IFNy receptor complex or, IL-2 receptor complex or IL-7 receptor complex.
10. The pair of antigen binding molecules according to any one of the preceding embodiments, wherein a) the first cytokine receptor-binding domain is capable of binding IFNyRl and the second cytokine receptor-binding domain is capable of binding IFNyR2, or b) the first cytokine receptor-binding domain is capable of binding IFNyR2 and the second cytokine receptor-binding domain is capable of binding IFNyRl; or c) the first cytokine receptor-binding domain is capable of binding IL-2RP and the second cytokine receptor-binding domain is capable of binding IL-2Ry, or d) the first cytokine receptor-binding domain is capable of binding IL-2Ry and the second cytokine receptor-binding domain is capable of binding IL-2RP; or e) the first cytokine receptor-binding domain is capable of binding IL-2Ry and the second cytokine receptor-binding domain is capable of binding IL-7Ra; or f) the first cytokine receptor- binding domain is capable of binding IL-7Ra and the second cytokine receptor-binding domain is capable of binding IL-2Ry.
11. The pair of antigen binding molecules according to any one of the preceding embodiments, wherein the first and second cytokine receptor-binding domains are antibody fragments, such as Fv, Fab, scFv, scFab or single domain antibodies.
12. The pair of antigen binding molecules according to any one of the preceding embodiments, wherein the first and second cytokine receptor-binding domains are VHH domains.
13. The pair of antigen binding molecules according to any one of the preceding embodiments, wherein a) the first cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 5 and the second cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, or b) the first cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10 and the second cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 5; or c) the first cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58 SEQ ID NO: 60, and the second cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64 and SEQ ID NO: 65; or d) the first cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64 and SEQ ID NO: 65 and the second cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58 SEQ ID NO: 60; or e) the first cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, and the second cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65; or f) the first cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, and the second cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192.
14. The pair of antigen binding molecules according to any one of the preceding embodiments, wherein a) the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 3 and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 7, or b) the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 7 and the second cytokine receptorbinding domain comprises the sequence of SEQ ID NO: 3; or c) the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 60 and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 62, or d) the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 62 and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 60; or e) the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 186, and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 62, or f) the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 62, and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 186.
15. The pair of antigen binding molecules according to any one of the preceding embodiments, wherein the Fc domains of the first and second antigen binding molecules comprise a first Fc domain subunit and a second Fc domain subunit.
16. The pair of antigen binding molecules according to any one of the preceding embodiments, wherein the Fc domains of the first and second antigen binding molecules are IgG, particularly an IgGi, Fc domains.
17. The pair of antigen binding molecules according to any one of the preceding embodiments, wherein the Fc domains of the first and second antigen binding molecules are human Fc domains.
18. The pair of antigen binding molecules according to any one of the preceding embodiments, wherein the Fc domains of the first and second antigen binding molecules comprise a modification promoting the association of the first and the second subunit of the Fc domains.
19. The pair of antigen binding molecules according to any one of the preceding embodiments, wherein the Fc domains of the first and second antigen binding molecules comprise one or more amino acid substitution that reduces binding to an Fc receptor and/or effector function.
20. The pair of antigen binding molecules according to any one of the preceding embodiments, wherein the cytokine receptor-binding domains are fused via peptide linkers to their respective fusion points.
21. The pair of antigen binding molecules according to embodiments 20, wherein the peptide linkers comprise an amino acid sequence selected from SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 279, SEQ ID NO: 280 or SEQ ID NO: 281.
22. The pair of antigen binding molecules according to any one of the preceding embodiments, wherein the first cytokine receptor-binding domain is fused at its C-terminus to the N-terminus of VHi or VLi of the first target-binding domain and the second cytokine receptor-binding domain is fused at its C- terminus to the N-terminus of VH2 or VL2 of the second target-binding domain.
23. The pair of antigen binding molecules according to any one of the preceding embodiments, wherein a) the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus the first cytokine receptorbinding domain, VHi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VLi and CLi; and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus the second cytokine receptor-binding domain, VH2, CHI 2 and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C- terminus VL2 and CL2; or b) the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VHi, CH li and a second Fc domain subunit, and a third polypeptide comprising in order from the N- terminus to C-terminus the first cytokine receptor-binding domain, VLi and CLi; and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VH2, CHI 2 and a second Fc domain subunit, a third polypeptide comprising in order from the N-terminus to C-terminus the second cytokine receptor-binding domain, VL2 and CL2; or c) the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus the first cytokine receptor-binding domain, VHi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VLi and CLi ; and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VH2, CHI 2 and a second Fc domain subunit, a third polypeptide comprising in order from the N-terminus to C-terminus the second cytokine receptor-binding domain, VL2 and CL2; wherein VHi, VLi, CHli and CLi form the first target-binding domain and VH2, VL2, CHI 2, and CL2, form the second target-binding domain.
24. The pair of antigen binding molecules according to any one of the preceding embodiments, wherein a) the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus the first cytokine receptorbinding domain, VHi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VLi and CLi, and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus the second cytokine receptor-binding domain, VH2, CHI 2 and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C- terminus VL2 and CL2; or b) the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VHi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N- terminus to C-terminus the first cytokine receptor-binding domain, VLi and CLi, and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VH2, CHI 2 and a second Fc domain subunit, a third polypeptide comprising in order from the N-terminus to C-terminus the second cytokine receptor-binding domain, VL2 and CL2; or c) the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus the first cytokine receptor-binding domain, VHi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VLi and CLi, and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VH2, CHI 2 and a second Fc domain subunit, a third polypeptide comprising in order from the N-terminus to C-terminus the second cytokine receptor-binding domain, VL2 and CL2; or d) the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus the first cytokine receptor-binding domain, VLi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VHi and CLi, and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus the second cytokine receptor-binding domain, VH2, CHI 2 and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VL2 and CL2; or e) the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VLi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus the first cytokine receptor-binding domain, VHi and CLi, and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VH2, CHh and a second Fc domain subunit, a third polypeptide comprising in order from the N-terminus to C-terminus the second cytokine receptor-binding domain, VL2 and CL2; or f) the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus the first cytokine receptor-binding domain, VLi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VHi and CLi, and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VH2, CHh and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus the second cytokine receptor-binding domain, VL2 and CL2; or g) the first antigen binding molecule comprising a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a VLi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus a first cytokine receptor-binding domain, VHi and CLi, and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a second cytokine receptor-binding domain, VH2, CHI 2 and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VL2 and CL2; wherein VHi, VLi, CHli and CLi form the first target-binding domain and VH2, VL2, CHI 2, and CL2, form the second target-binding domain.
25. The pair of antigen binding molecules according to any one of the preceding embodiments, wherein the first and the second target-binding domains specifically bind to FAP, and the first and second cytokine receptor subunits are subunits of the IFNy receptor complex.
26. The pair of antigen binding molecules according to any one of the preceding embodiments, wherein a) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 27 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 30 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 38 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 37 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or c) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO : 31 , and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 33; or d) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 35, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 36; or e) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 27 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 33; or f) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 30 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 31; or g) the first antigen binding molecule comprising a first polypeptide comprises an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 38 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 36; or h) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 37 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 35.
27. The pair of antigen binding molecules according to any one of embodiments 1 to 24, wherein the first and the second target-binding domains specifically bind to EGFR, and the first and second cytokine receptor subunits are subunits of the IFNy receptor complex.
28. The pair of antigen binding molecules according to any one of embodiments 1 to 24 or 27, wherein a) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 260 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 259 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 127; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 119 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 256, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 122 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 255; or c) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 261 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO:
258 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 127; or d) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 119 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 257, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 112 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 254; or e) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 260 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 112 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 255; or f) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 119 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 256, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO:
259 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 127; or g) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 261 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 112 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 254; or h) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 119 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 257, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 258 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 127. 29. The pair of antigen binding molecules according to any one of embodiments 1 to 24, wherein the first and the second target-binding domains specifically bind to PD-1, and the first and second cytokine receptor subunits are subunits of the IL-2 receptor complex.
30. The pair of antigen binding molecules according to any one of embodiments 1 to 24 or 29, wherein a) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 86, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 44 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 83, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 88 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 87; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 92, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 44 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 91; or c) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 96 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 95, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 101 and athird polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or d) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 99 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 98, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 88 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 102; or e) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 96 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 97, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 103 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or f) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 100 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 98, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 88 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 104; or g) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 98 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 99, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 101 and athird polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or h) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 96 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 95, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 88 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 102; or i) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 100 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 98, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 103 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or j) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 96 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 97, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 88 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 104.
31. The pair of antigen binding molecules according to any one of embodiments 1 to 24, wherein the first and the second target-binding domains specifically bind to LAG-3, and the first and second cytokine receptor subunits are subunits of the IL-2 receptor complex.
32. The pair of antigen binding molecules according to any one of embodiments 1 to 24 or 31, wherein a) first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 155 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 154, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 165 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 163.; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 161 and athird polypeptide comprising an amino acid sequence of SEQ ID NO: 160, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 158 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 159; or c) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 155 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 156, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 164 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 163; or d) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 162 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 160, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 158 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 157; or e) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 161 and athird polypeptide comprising an amino acid sequence of SEQ ID NO: 160, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 165 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 163; or f) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 155 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 154, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 158 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 159; or g) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 162 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 160, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 164 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 163; or h) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 155 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 156, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 158 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 157.
33. The pair of antigen binding molecules according to any one of embodiments 1 to 24, wherein the first and the second target-binding domains specifically bind to EGFR, and the first and second cytokine receptor subunits are subunits of the IL-2 receptor complex.
34. The pair of antigen binding molecules according to any one of embodiments 1 to 24 or 33, wherein a) first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 155 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 154, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 165 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 163; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 161 and athird polypeptide comprising an amino acid sequence of SEQ ID NO: 160, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 158 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 159.; or c) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 155 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 156, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 164 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 163; or d)the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 162 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 160, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 158 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 157; or e) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 161 and athird polypeptide comprising an amino acid sequence of SEQ ID NO: 160, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 165 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 163; or f) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 155 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 154, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 158 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 159; or g)the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 162 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 160, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 164 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 163; or h) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 155 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 156, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 158 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 157.
35. The pair of antigen binding molecules according to any one of embodiments 1 to 24, wherein the first and the second target-binding domains specifically bind to FAP, and the first and second cytokine receptor subunits are subunits of the IL-2 receptor complex.
36. The pair of antigen binding molecules according to any one of embodiments 1 to 24 or 35, wherein a) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 110 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 113 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 105, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 109; or c) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 111 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 112 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or d) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 107, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 108; or e) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 110 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 109; or f) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 105, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 113 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or g) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 111 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 108; or h) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and athird polypeptide comprising an amino acid sequence of SEQ ID NO: 107, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 112 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29. 37. The pair of antigen binding molecules according to any one of embodiments 1 to 24, wherein the first and the second target-binding domains specifically bind to Her2, and the first and second cytokine receptor subunits are subunits of the IL-2 receptor complex.
38. The pair of antigen binding molecules according to any one of embodiments 1 to 24 or 37, wherein a) first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 136 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 134, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 138 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 137; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 135 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 134, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 138 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 139.
39. The pair of antigen binding molecules according to any one of embodiments 1 to 24, wherein the first and the second target-binding domains specifically bind to Her3, and the first and second cytokine receptor subunits are subunits of the IL-2 receptor complex.
40. The pair of antigen binding molecules according to any one of embodiments 1 to 24 or 39, wherein a) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 146 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 144, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 148 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 147; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 145 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 144, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 148 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 149.
41. The pair of antigen binding molecules according to any one of embodiments 1 to 24, wherein the first and the second target-binding domains specifically bind to PD-1, and the first and second cytokine receptor subunits are subunits of the IL-7 receptor complex. 42. The pair of antigen binding molecules according to any one of embodiments 1 to 24 and 41, wherein a) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 194 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 193, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 195 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or c) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 196 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or d) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 197 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or d)the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 198 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or e) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 199 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or f) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 200 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or g) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 201 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or h) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 202 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or i) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 203 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or j) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 204 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or k) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 205 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89. 43. The pair of antigen binding molecules according to any one of embodiments 1 to 21, wherein the first target-binding domain is fused at its C-terminus of CHli to the N-terminus of the first Fc domain subunit and the first cytokine receptor-binding domain is fused at its C-terminus to the N-terminus of the second Fc domain subunit and the second target-binding domain is fused at its C-terminus of CHI 2 to the N-terminus of the first Fc domain subunit and the second cytokine receptor-binding domain is fused at its C-terminus to the N-terminus of the second Fc domain subunit.
44. The pair of antigen binding molecules according to any one of embodiments 1 to 21 or 43, wherein a) the first antigen binding molecule comprises a first polypeptide comprising in order from the N- terminus to C-terminus VHi, CHli and a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VLi and CLi, and a third polypeptide comprising in order from the N-terminus to C-terminus the first cytokine receptor-binding domain and a second Fc domain subunit, and the second antigen binding molecule comprises a first polypeptide comprising in order from the N-terminus to C-terminus VH2, CH 12 and a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VL2 and CL2, and a third polypeptide comprising in order from the N-terminus to C-terminus the second cytokine receptor-binding domain and a second Fc domain subunit; or b) the first antigen binding molecule comprises a first polypeptide comprising in order from the N-terminus to C-terminus VLi, CHI 1 and a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VHi and CLi, and a third polypeptide comprising in order from the N-terminus to C-terminus the first cytokine receptor-binding domain and a second Fc domain subunit, and the second antigen binding molecule comprises a first polypeptide comprising in order from the N-terminus to C-terminus VH2, CHh and a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VL2 and CL2, and a third polypeptide comprising in order from the N-terminus to C-terminus the second cytokine receptorbinding domain and a second Fc domain subunit; wherein VHi, VLi, CHI 1 and CLi form the first targetbinding domain and VH2, VL2, CHh, and CL2, form the second target-binding domain.
45. The pair of antigen binding molecules according to any one of embodiments 1 to 21 or 43 to 44, wherein a) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 32, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 25 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 39, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 34, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 29 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 40; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 34, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 29 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 39, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 32, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 25 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 40; or c) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 237, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 238, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or d) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 235, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 236, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or e) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 239, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 240, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or f) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 235, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 237, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or g) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 236, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 235, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or h) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 240, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 239, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
46. The pair of antigen binding molecules according to any one of embodiments 1 to 21, wherein the first target-binding domain is fused at its C-terminus of CH 11 to the N-terminus of one of the Fc domain subunits and the first cytokine receptor-binding domain is fused at its N-terminus to the C-terminus of the same Fc domain subunit and the second target-binding domain is fused at its C-terminus of CHE to the N-terminus of one of the Fc domain subunits and the second cytokine receptor-binding domain is fused at its N-terminus to the C-terminus of the same Fc domain subunit.
47. The pair of antigen binding molecules according to any one of embodiments 1 to 21 or 46, wherein a) the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a VHi, a CHli, a second Fc domain subunit and a the first cytokine receptor-binding domain, and a third polypeptide comprising in order from the N-terminus to C-terminus a VLi and a CLi ; and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a VH2, a CHE, a second Fc domain subunit and a the second cytokine receptor-binding domain, and a third polypeptide comprising in order from the N-terminus to C-terminus VL2 and CL2; or b) the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a VLi, a CH , a second Fc domain subunit and a the first cytokine receptor-binding domain, and a third polypeptide comprising in order from the N-terminus to C-terminus a VHi and a CLi; and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a VH2, a CHE, a second Fc domain subunit and the second cytokine receptor-binding domain, and a third polypeptide comprising in order from the N-terminus to C-terminus a VL2 and a CL2; or c) the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a VLi, a CHE, a second Fc domain subunit and a the first cytokine receptor-binding domain, and a third polypeptide comprising in order from the N-terminus to C-terminus a VHi and a CLi; and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a VH2, a CHE, a second Fc domain subunit and a the second cytokine receptor-binding domain, and a third polypeptide comprising in order from the N-terminus to C-terminus VL2 and CL2; wherein VHi, VLi, CHE and CLi form the first target-binding domain and VH2, VL2, CHE, and CL2, form the second target-binding domain.
48. The pair of antigen binding molecules according to any one of embodiments 1 to 21 or 46 to 47, wherein a) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 42 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 41, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 45, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 44 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 43; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 46 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 41, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 47, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 44 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 43; or c) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 246 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 249 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or d) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 242 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 245 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or e) the first antigen binding molecule comprising a first polypeptide comprises an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 250 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 253 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or f) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 248 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 247 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or g) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 244 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 243 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or h) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 252 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 251 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or i) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 42 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 42, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 245 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
49. The pair of antigen binding molecules according to any one of embodiments 1 to 11, wherein the first and second cytokine receptor-binding domains are Fab molecules.
50. The pair of antigen binding molecules according to embodiments 49, wherein a) the first cytokine receptor-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 282 and a VL domain comprising the amino acid sequence of SEQ ID NO: 283 or VH domain comprising the amino acid sequence of SEQ ID NO: 284 and a VL domain comprising the amino acid sequence of SEQ ID NO: 285, and the second cytokine receptor domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 286 and a VL domain comprising the amino acid sequence of SEQ ID NO: 287 or VH domain comprising the amino acid sequence of SEQ ID NO: 288 and a VL domain comprising the amino acid sequence of SEQ ID NO: 289; or b) the first cytokine receptor-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 286 and a VL domain comprising the amino acid sequence of SEQ ID NO: 287 or VH domain comprising the amino acid sequence of SEQ ID NO: 288 and a VL domain comprising the amino acid sequence of SEQ ID NO: 289, and the second cytokine receptor domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 282 and a VL domain comprising the amino acid sequence of SEQ ID NO: 283 or VH domain comprising the amino acid sequence of SEQ ID NO: 284 and a VL domain comprising the amino acid sequence of SEQ ID NO: 285. 16.
51. The pair of antigen binding molecules according to any one of embodiments 1 to 11, wherein the first and second cytokine receptor-binding domains are scFv molecules. 52. The pair of antigen binding molecules according to embodiments 51, wherein a) the first cytokine receptor-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 290 and a VL domain comprising the amino acid sequence of SEQ ID NO: 291, and the second cytokine receptor domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 292 and a VL domain comprising the amino acid sequence of SEQ ID NO: 293; or b) the first cytokine receptorbinding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 292 and a VL domain comprising the amino acid sequence of SEQ ID NO: 293, and the second cytokine receptor domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 290 and a VL domain comprising the amino acid sequence of SEQ ID NO: 291.
53. The pair of antigen binding molecules according to any one of embodiments 49 to 52, wherein the Fc domains of the first and second antigen binding molecules comprise a first Fc domain subunit and a second Fc domain subunit.
54. The pair of antigen binding molecules according to any one of embodiments 49 to 53, wherein the Fc domains of the first and second antigen binding molecules are IgG, particularly an IgGi, Fc domains.
55. The pair of antigen binding molecules according to any one of embodiments 49 to 54, wherein the Fc domains of the first and second antigen binding molecules are human Fc domains.
56. The pair of antigen binding molecules according to any one of embodiments 49 to 55, wherein the Fc domains of the first and second antigen binding molecules comprise a modification promoting the association of the first and the second subunit of the Fc domains.
57. The pair of antigen binding molecules according to any one of embodiments 49 to 56, wherein the Fc domains of the first and second antigen binding molecules comprise one or more amino acid substitution that reduces binding to an Fc receptor and/or effector function.
58. The pair of antigen binding molecules according to any one of embodiments 49 to 57, wherein the cytokine receptor-binding domains are fused via peptide linkers to their respective fusion points.
59. The pair of antigen binding molecules according to embodiments 58, wherein the peptide linkers comprise an amino acid sequence of SEQ ID NO: 280 or SEQ ID NO: 281.
60. The pair of antigen binding molecules according to embodiments 49, wherein, a) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 267, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 265 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 266, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 272, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 270 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 271; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 267, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 265 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 266, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 274, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 270 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 273; or c) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 269, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 265 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 268, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 268, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 270 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 271 ; or d) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 269, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 265 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 268, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 274, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 270 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 273.
61. The pair of antigen binding molecules according to embodiments 51, wherein the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 276, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 28 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 275, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 277, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 28 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 278.
Examples
Example 1: Generation of binders for IFNy and IL-2 receptor subunits
1.1 Generation of antigens for immunization and phage display
For llama immunization, the extracellular domain (ECD) of human IFNyRl subunit and the ECD of human IFNyR2 subunit were co-expressed as an Fc-tagged heterodimer based on the knobs-into-holes technology (Fig. 1A, P1AG0843, SEQ ID NO: 48 and SEQ ID NO: 49). Likewise, the ECD of human IL-2RP (CD 122) subunit and human IL-2Ry (common gamma chain; CD 132) subunit were coexpressed as an Fc-tagged heterodimer (Fig. ID, P1AE2657, SEQ ID NO: 80 and SEQ ID NO: 81). The C-terminus of IFNyRl ECD was fused to Fc knob via a G4SG4 linker. An AviTag was included at the C- terminus of the Fc for site-specific biotinylation during co-expression with BirA biotin ligase. The IFNyR2 ECD containing C174S substitution was fused to Fc hole with a G4SG4 linker and the construct contained a C-terminal Twin-Strep-tag. The C-terminus of IL-2RP ECD was fused to Fc knob via a GAQ linker. An AviTag was included at the C-terminus of the Fc for site-specific biotinylation during co-expression with BirA biotin ligase. The IL-2Ry ECD was C-terminally fused to Fc hole with a GAQ linker.
For phage display, monovalent IFNyRl ECD fused to biotinylated Fc knob paired with empty Fc hole (Fig. IB, P1AG1987, SEQ ID NO: 48 and SEQ ID NO: 50) and monovalent IFNyR2 ECD C174S fused to Fc hole paired with empty biotinylated Fc knob (Fig, 1C, P1AG6735, SEQ ID NO: 49 and SEQ ID NO: 51), as well as monovalent IL-2RP ECD fused to biotinylated Fc knob paired with empty Fc hole (Fig. IE, P1AF1104, SEQ ID NO: 80 and SEQ ID NO: 82) and monovalent IL-2Ry ECD fused to Fc hole paired with empty biotinylated Fc knob (Fig. IF, P1AF1106, SEQ ID NO: 81 and SEQ ID NO: 51), were generated. All antigens were produced in the Expi293 mammalian expression system (Expi293™ Expression System Kit, Thermo Fisher Scientific, Cat. No: A 14635) following manufacturer’s recommendation. Supernatants were harvested by centrifugation, filtered and purified by Protein A affinity chromatography (Protein A MabSclcctI SuRc ™. Cytiva, Cat. No: 17543803) followed by size exclusion chromatography (SEC) (HiLoad S200 16/600, Cytiva, Cat. No: 28989335) according to manufacturer’s recommendation.
1.2 VHH library construction
The VHH library was constructed using ~3xl08 peripheral blood mononuclear cells (PBMCs) from bleed 2 and ~6.6xl08 PBMCs bleed 3 of a single llama. The library was provided in the TGI E. coli strain encoded in a derivative of the pADL-23c phagemid vector (Antibody Design Labs, San Diego, CA), incorporated by approximately -IxlO9 independent electroporation events with >95% insert efficiency. Libraries were provided as ~3.2xlO10 cfu/mL E. coli in 2xYT medium containing 20% glycerol.
1.3 Generation of VHH phage library
E. coli containing the VHH-encoded phagemid library were inoculated in 2xYT medium containing 100 pg/ml ampicillin and 1% glucose. Following infection with VCSM13 helper phage, E. coli were grown in 2xYT medium containing 100 pg/ml ampicillin and 50 pg/ml kanamycin. Following overnight incubation on a shaker at 30°C, cultures were harvested by centrifugation at 8000 rpm for 20 minutes and supernatants were collected. Phage were precipitated in 20% PEG/2.5 M NaCl solution and incubated on ice for 1 hour. Precipitated phage were centrifuged at 8000 rpm for 20 minutes and pellets were resuspended in ddlEO followed by precipitation in PEG/NaCl solution for 1 hour on ice. Phage were centrifuged at 8000 rpm for 20 minutes and pellets were resuspended in PBS containing 20% glycerol.
1.4 Isolation of IFNyRl-specific, IFNyR2-specific, IL-2RB-specific and IL-2Ry-specific VHHs by phage display
To selectively enrich for antigen-specific binders and prevent the enrichment of Fc-specific binders, Fc- competitive soluble selections were performed against human IFNyRl antigen (P1AG1987), human IFNyR2 antigen (P1AG6735), human IL-2RP antigen (P1AF1104) and human IL-2Ry antigen (P1AF1106), in the presence of human Fc (Fig. 1G, P1AD4290, SEQ ID NO: 52 and SEQ ID NO: 53). An aliquot of IxlO11 phage from the llama-derived VHH library was used per antigen. Phage and magnetic streptavidin beads (Dynabeads; M-280 Streptavidin, Invitrogen, Cat. No: 11205D) were independently blocked with 1% BSA, 0.1% Tween-20 in phosphate -buffered saline (PBS) to reduce non-specific interactions. All incubations were performed for one hour at 20°C, unless otherwise stated. The biotinylated, Fc-tagged antigens were added at 100 nM concentration to blocked phage in the presence of excess amounts (500 nM) of non-biotinylated Fc (Figure 2). Following incubation, preblocked Dynabeads were added to capture biotinylated antigens of interest and bound VHH-expressing phage. Fc-specific and non-specific phage were removed by 5 - 10 wash steps in PBS containing 0.1% Tween-20 followed by PBS using a KingFisher magnetic particle processor (Thermo Fisher Scientific). Antigen-specific phage were eluted in 800 pl of 100 mM trimethylamine (TEA) and neutralized by addition of 400 pl of 1 M Tris pH 7.4. Mid-logarithmic phase TGI E. coli in 2xYT medium were infected with eluted phage, followed by infection with VCSM13 helper phage. Following addition of 100 pg/ml ampicillin and 50 pg/ml kanamycin, infected E. coli were incubated overnight on a shaker at 30°C. Phage were precipitated with PEG/NaCl and used for the next round. Selections were performed for a total of 3 rounds using decreasing target antigen concentrations (100 nM, 50 nM, 25 nM) while soluble Fc concentrations remained constant (500 nM) (Fig. 2).
1.5 Verification of target specificity by ELISA
Round 3 enrichment libraries were screened for target specificity vs Fc specificity by ELISA. Individual VHH clones were expressed in 1 ml E. coli cultures in a 96-well plate and supernatants containing soluble VHH were screened for specificity by ELISA. Supernatants were transferred to neutravidin 96- well plates coated with biotinylated Fc (P1AE6073, SEQ ID NO: 51 and SEQ ID NO: 54) or target Fc- tagged biotinylated antigen (P1AG1987, P1AG6735, P1AF1104 or P1AF1106). Following incubation for 1 hour at 20°C, plates were washed 3 times with PBST and 3 times with PBS. HRP -conjugated anti- His antibody (Sigma, Cat. No: A7058) was added to wells at 1:2000 dilution and plates were incubated for 1 hour at room temperature. Plates were washed 3 times with PBST followed by 3 washes with PBS. Plates were developed by adding 1-Step Ultra TMB-ELISA substrate (Thermo Fisher Scientific, Cat. No: 34028) and reactions were quenched with sulfuric acid. Absorbance at 450 nm was measured using a Tecan infinite M1000 Pro and reference absorbance values were measured at 650 nm. Reference- subtracted absorbance values at 450 nm were represented as stacked bar charts for IFNyRl -specific VHHs (Fig. 3A), IFNyR2-specific VHHs (Fig. 3B), IL-2Rp-specific VHHs (Fig. 3C) and IL-2Ry- specific VHHs (Fig. 3D). ELISA data suggested >90% of clones were target-specific and VHH from round 3 enrichment libraries were subsequently cloned into bispecific heavy chain antibody formats.
Example 2: Evaluation of agonist activity towards IFNy and IL-2 receptors
2.1 Assembly of enriched VHH libraries into heavy chain antibody formats
IFNyR activation is achieved by IFNy-mediated cross-linking of the IFNyRl and IFNyR2 receptor chains. To mimic the assembly in an IFNy-independent manner, a VHH domain with IFNyRl specificity was paired with an IFNyR2 -specific VHH by fusing to knob-into-hole Fc chains via a flexible linker generating IFNyRl -IFNyR2 bispecific heavy chain antibodies. IL-2R activation is achieved by Remediated cross-linking of the IL-2RP and IL-2Ry receptor chains. Thus, to mimic the assembly in an IL2 -independent manner, a VHH domain with IL-2RP specificity was paired with an IL-2Ry-specific VHH by fusing to knob-into-hole Fc chains via a flexible linker generating IL-2Rp-IL-2Ry bispecific heavy chain antibodies. To this end, the round 3 VHH library enriched for IFNyRl- or IL-2RP- specificity were cloned in bulk using Gibson assembly to achieve format conversion of VHH by fusing to Fc knob via a 5(G4S) linker (Figure 4A). In the same way, the round 3 IFNyR2- or IL-2Ry-specific VHH library were fused to Fc hole via a 5(G4S) linker using Gibson assembly (Figure 4B). During the assembly, a suitable signal sequence and vector compatible with mammalian expression were included. Primers, PCR conditions and Gibson assembly reactions were defined as per New England Biolabs’ NEBuilder HiFi DNA Assembly protocol (Cat. No: E5510). Following bacterial transformation, colonies were selected at random and five unique sequences from each specificity pool were selected. Using the Expi293 system, a 5x5 matrix was generated to express 25 unique knob-into-holes bispecific heavy chain antibodies comprising IFNyRl and IFNyR2 VHH pairs and 25 unique knob-into-holes bispecific heavy chain antibodies comprising IL-2RP VHH and IL-2Ry VHH pairs (Figure 5). Expi293 cells were transiently transfected following manufacturer’s recommendation (Expi293™ Expression System Kit, Thermo Fisher Scientific, Cat. No: A 14635) and supernatants containing secreted soluble bispecific heavy chain antibodies were collected. The bispecific heavy chain antibodies were purified by Protein A resin and further characterized for functional activity using reporter cell assays. 2.2 Functional characterization of bispecific heavy chain antibodies
The HEK Blue IFNy reporter cell assay (Invivogen) was used to screen bispecific heavy chain antibodies comprising different IFNyRl and IFNyR2 VHH pairs and identify IFNyR agonists. Whereas, the HEK Blue IL-2 reporter cell assay (Invivogen) was used to screen bispecific heavy chain antibodies comprising different IL-2RP and IL-2Ry VHH pairs and identify IL-2R agonists. The in vitro HEK Blue IFNy reporter cell system (Invivogen, Cat. No: hkb-ifhg) reports IFNyR signaling via a STAT1- inducible secreted alkaline phosphatase (SEAP) system. While the in vitro HEK Blue IL-2 reporter cell (Invivogen, Cat. No: hkb-il2) recapitulates the human IL-2R signaling pathway via expression of the IL-2R chains (IL-2Ra, P and y subunits) and effectors of the downstream signaling cascade (JAK3 and STAT5) and a STAT5 -inducible secreted alkaline phosphatase (SEAP) reporter system. Upon addition of QuantiBlue substrate (Invivogen, Cat. No: rep-qbs), absorbance at 650 nm is measured, correlating with SEAP levels and IFNyR and IL-2R activity, respectively. In the initial screening, supernatants from the Expi293 5x5 expression matrix containing soluble IFNyRl -IFNyR2 or IL-2Rp-IL-2Ry bispecific heavy chain antibodies were incubated with HEK Blue IFNy or HEK Blue IL-2 reporter cells, respectively. Following a 20 hour incubation, IFNyR and IL-2 activity was quantified by addition of QuantiBlue reagent (Invivogen, Cat. No: rep-qbs), according to the manufacturer's recommendation. The absorbance at 650 nm was measured, with higher absorbance levels indicating increased IFNyR or IL-2 signaling.
Of the 25 tested IFNyRl -IFNyR2 bispecific heavy chain antibodies (format depicted in Figure 5), 14 showed IFNyR activity (Figure 6A). Thus, 14 pairs of IFNyRl- and IFNyR2-VHH domains demonstrated IFNy agonistic activity. Of the 25 tested IL-2Rp-IL-2Ry bispecific heavy chain antibodies, 9 pairs containing non-specific VHH domains (IL2Rp_4 and IL2Ry_l) did not induce IL-2R activity. The other 16 pairs, which were formed of functional IL-2RP and IL-2Ry binders, all showed IL-2R agonism (Figure 6B). Thus, 16 pairs of IL-2RP- and IL-2Ry-VHH domains demonstrated IL-2 agonistic activity.
Next, purified agonistic IFNyRl-IFNyR2 bispecific heavy chain antibodies (P1AH1877-P1AH1890, see Table 1) were tested for dose -dependent IFNyR signaling and agonistic IL-2Rp-IL-2Ry bispecific heavy chain antibodies were tested for dose-dependent IL-2R signaling (see Table 2). Table 1: IFNyRl-IFNyR2 bispecific heavy chain antibodies generated by the 5x5 expression matrix showing IFNy agonistic activity in Fig. 6A.
A concentration range spanning from 100 nM to 0.01 nM with 10-fold serial dilution steps was used with HEK Blue IFNy cells according to the Invivogen HEK Blue protocol. Absorbance at 650 nm was measured and responses were plotted using GraphPad software (Figure 7). Graphs were separated according to VHH clone with IFNyRl specificity, with recombinant human IFNy (recIFNy; SEQ ID NO: 26) and anti-FAP IgG fused to IFNy homodimer (P1AF3574; SEQ ID NO: 24 and SEQ ID NO: 25) serving as reference molecules. The variety of responses observed in all graphs, ranging from potent IFNy mimetics to weak agonists, demonstrated an interplay between both the IFNyRl and IFNyR2 binders. EC50 values were derived using GraphPad Prism software (Figure 8), illustrating the breadth of agonistic activities and underlining the potential for VHH pairs to achieve potent yet tunable cytokine mimetics.
Table 2: IL-2Rp-IL-2Ry bispecific heavy chain antibodies generated by the 5x5 expression matrix showing IL-2R activity in Fig. 6B.
A concentration range of 20 nM, 0.8 nM and 0.032 nM was used with HEK Blue IL-2 reporter cells according to the Invivogen HEK Blue protocol. Agonistic activity of the IL-2 mimetics was compared with IL-2v fused to anti-PDl IgG (PDl-IL2v; P1AE4422, SEQ ID NO: 83, SEQ ID NO: 84 and SEQ ID NO: 85). The VHH pairs displayed potent IL-2R agonism in a dose-dependent manner (Figure 9). Moreover, in contrast to the natural cytokine in which agonism is mediated by a single molecule, the use of a VHH pair to activate the receptor provided opportunities for conditionally active IL-2 mimetics via molecular split approaches.
To further characterize the agonistic activity of the 14 functional IFNyRl- and IFNyR2-VHH pairs, various tumor cell lines with previously identified responsiveness to IFNy (MKN45, BxPC-3, COR- L105; data not shown) were selected to explore therapeutically relevant downstream effects of IFNyR signaling which may have the capacity to modulate the tumor microenvironment (TME). In the first instance, MHC-I and PD-L1 were selected as tumor cell surface biomarkers to characterize the TME- modulating potential of functional bispecific VHH pairs. MHC-I upregulation is a downstream response following IFNyR stimulation and may provide an increase in antigenicity on tumor cells, while PD-L1 upregulation is another downstream indicator of functional IFNy signaling. To assess the capacity of VHH pairs to induce IFNy signaling in vitro, tumor cells were incubated with the 14 previously tested heavy chain antibodies (P1AH1877-P1AH1890) in concentration series ranging from 100 nM to 0.1 nM. After 72 hours, MHC-I and PD-L1 expression levels were quantified by flow cytometry and data were analyzed using FlowJo software. Mean fluorescent intensity (MFI) values were blank-subtracted and normalized to recombinant IFNy responses. Upregulation of MHC-I and PD-L1 was measured in three tumor cell types: MKN45 (Fig. 10A, 10B), BxPC-3 (Fig. 10C, 10D) and COR-L105 (Fig. 10E, 10F). A variety of responses was observed, corroborating the broad range of agonistic activities observed previously in HEK Blue IFNy reporter cells. One notable observation was an often decreased PD-L1 expression compared to MHC-I expression induced by certain VHH pairs, suggesting a complexity in IFNy signaling that may engage different pathways and highlighting the potential for agonistic VHH pairs to elicit a bias towards desired phenotypic effects.
The induction of IFNyR signaling via an agonistic IFNyR VHH pair and IL-2R signaling via an agonistic IL-2R VHH pair provided opportunities to engineer conditionally active IFNy and IL-2 mimetics. As a starting point for a conditional IFNyR agonist, P1AH1884 was selected as a potent IFNy mimetic (Fig.
8) with activity closely correlating with recombinant IFNy responses across all tumor lines tested (Fig.
9). For a conditional IL-2R agonist P1AH1177 was selected.
The corresponding VHH pairs were further engineered to generate conditionally active agonists.
Example 3: Engineering of conditionally active IFNy and IL-2 mimetics
3.1 FAP-dependent biparatopic assembly of split dual-targeted IFNy mimetics
To achieve conditional immunomodulation within an immune-deserted tumor microenvironment, cell surface markers reportedly expressed in non-inflamed tumor phenotypes were further explored. Fibroblast activation protein (FAP), a serine protease described with high expression in cancer- associated stromal tissue of immune-deserted tumors, was selected as a target to achieve cold tumor penetration. As a next step, a pair of agonistic VHH domains mimicking IFNy were distributed onto two distinct molecules to inactivate the VHH pair, whilst incorporating a FAP-dependent assembly strategy to restore agonistic VHH activity in the presence of FAP (Fig. 11A). To this end, two proximal epitopes on the FAP antigen were selected and the corresponding non-competing anti-FAP binders were engineered to mediate assembly of an agonistic VHH pair. In the first instance, VHH domains of the P1AH1884 heavy chain antibody were fused to the N-terminus of the VH or VL domains of the anti- FAP binders via a flexible 5(G4S) linker, resulting in 8 possible format configurations (Fig. 11B-I). To test for FAP-dependent IFNyR signaling, upregulation of MHC-I and PD-L1 was quantified in three FAP expression scenarios. First, the A549 lung adenocarcinoma cell line was used to test for untargeted, FAP-independent activity in a FAP-negative setting. Next, A549 cells engineered for ectopic FAP expression were used to characterize FAP-dependent activity in cis, while both FAP -positive and FAP- negative cell lines were differentially labelled and co-cultured to compare cis and trans activity of FAP- dependent IFNy mimetics. Split dual-targeted IFNy mimetics were serially diluted to span a concentration range from 10 nM to 1 pM. Recombinant IFNy and the intact, parental VHH pair deficient in FAP targeting (Pl AHI 884, format depicted in Fig. 5) were used as reference molecules. Following a 72h incubation in the three FAP expression scenarios, MHC-I and PD-L1 expression levels were quantified by flow cytometry. In the absence of FAP expression, MHC-I and PD-L1 upregulation was induced by reference molecules while none of the combinations of split dual-targeted molecules exhibited FAP-independent activity (Fig. 12A and 12B). In the presence of FAP expression, both the recapitulation of a cis setting (Fig. 12C and 12D) and the recapitulation of a trans setting (Fig. 12E and 12F) showed FAP-specific activity. In particular, the cis setting underscored the impact of geometry to achieve potent activity, with minor format adjustments (i.e. swapping of fusion points from VH to VL on anti -FAP binders) translating into differences in IFNyR signaling. The P1AI0831 + P1AI0066 pair provided the most potent FAP -dependent IFNyR activity. Interestingly, the geometrical constraints did not apply to the same degree in a trans scenario, with all format combinations displaying FAP -dependent activity.
To further explore format options and extend our understanding of the constraints imposed by molecular geometry, we designed additional sets of molecules in which the VHHs were further distanced from the FAP binding domains. The VHHs were fused to the N-terminus of the opposite Fc chain via a 3(G4S) linker (Fig. 13A-D) or to the C-terminus of the same Fc chain via a 3(G4S) linker (Fig. 13E-H). The sets were tested in vitro in the three FAP expression scenarios, as described previously. FAP-independent activity was observed with the reference molecules (recombinant IFNy and P1AH1884, Fig. 14A and 14B) while split dual -targeted combinations did not show IFNyR agonism except at high concentration for one combination (P1AI5306 + P1AI5307; Fig. 14A) in the FAP-negative scenario. Next, FAP- dependent activity was tested in cells expressing FAP (cis targeting). MHC-I upregulation (Fig. 14C) and PD-L1 upregulation (Fig. 14D) was observed with the reference molecules and the P1AI5306 + P1AI5307 combination, but all other combinations remained inactive. Finally, the combinations were tested in a trans scenario, to assess activity on FAP-negative cells in the presence of FAP-expressing cells. All combinations showed FAP-dependent trans activity, inducing both MHC-1 (Fig. 14E) and PD-L1 upregulation (Fig. 14F). By disconnecting the VHHs from the FAP binding moiety, FAP- dependent cis activity was rarely observed due to the previously identified geometrical constraints imposed in a cis scenario. However, in a trans setting, all combinations displayed IFNyR agonism regardless of the format.
Moreover, FAP-dependent IFNy agonists comprising different lengths of linker between the VHH and the fusion point were explored ( concepts described in Fig. 15). The molecule P1AJ8572 was replaced with P1AI0187 due to a batch failure in production, the activity of IFNy agonists was measured by MHC-I upregulation. Specifically, activity in an untargeted scenario (A549 FAP -negative cells) as well as in cis-targeted (IFNy activity in A549 FAP-positive cells co-cultured with A549 FAP -negative cells) and trans-targeted (IFNy activity in A549 FAP-negative cells co-cultured with A549 FAP-positive cells) scenarios were assessed. Briefly, for the untargeted condition 7’000 A549 FAP-negative per well were seeded, while for assessing cis and trans activity a combination of 3'500 A549 FAP-negative and 3'500 A549 FAP-positive cells per well were respectively seeded in a 96 well flat-bottom plate. After overnight incubation at 37°C and 5% CO2 to allow the cells to adhere, test compounds (four dilution series, starting concentration of 50’000 pM) were added to the assay plate. Samples were incubated at 37°C and 5% CO2 for 72h before the assay readout using a MHC-I antibody from BioLegend clone W6/32 (Cat No: 311430) measured on a BD FACS Fortessa.
In the absence of FAP expression, MHC-I upregulation was induced by recombinant IFNy and by the IFNy agonistic heavy chain antibody (P1AH1884), while none ofthe combinations of split dual-targeted molecules exhibited FAP-independent activity (Fig. 16A and 16D). In the cis targeting scenario, MHC- I upregulation was observed with recombinant IFNy and the IFNy agonistic heavy chain antibody, yet all combinations of split dual-targeted molecules remained inactive (Fig. 16B and E). Finally, in the trans scenario all combinations of FAP-dependent split IFNRy agonists showed FAP-dependent trans activity upregulation of MHC-I (Fig. 16C and 16F).
3.2 Functional characterization of EGFR-dependent trans-targeted IFNyR agonists
To further extend the application for biparatopic assembly of cytokine receptor agonists, the concept was tested with IFNyR agonists and EGFR-targeted assembly. A pair of agonistic VHH domains mimicking IFNy were distributed onto two separate molecules to inactivate the VHH pair, whilst incorporating an EGFR-dependent assembly via two non-competing anti-EGFR Fabs. The VHH domains ofthe Pl AHI 884 heavy chain antibody were fused to the N-terminus ofthe VH or VL domains of the anti-EGFR binders via a flexible 5(G4S) linker, resulting in eight possible format configurations (P1AK5514 - P1AK5521) which can be paired to produce eight assembly combinations (Fig 17). The activity of the eight assembly combination was measured assessing the upregulation of the chemoattractant CXCL10 following 24h stimulation (Fig. 18).
In order to assess the activity of the eight assembly combinations, the upregulation of chemoattractant CXCL10 was measured following 24h incubation with U87 and PBMCs. Briefly, 20’000 U87 cells per well were seeded in a 96 well plate flat bottom. After overnight incubation at 37°C and 5% CO2 to allow the cells to adhere, 50pL/well of PBMCs at 2 * 106 PBMCs/well was added to the wells. Lastly, 50pL/well of control medium or compound dilution was added to the wells. Samples were incubated at 37°C and 5% CO2 for 24h before assay readout with the human IP 10 R&D ELISA KIT (cat# DY266) accordingly to the manufacturer’s instructions. While the degree of upregulation varied between the EGFR-targeted IFNy agonists, all pairs demonstrated upregulation of the chemoattractant CXCL10 (Fig. 18).
3.3 PDl-dependent biparatopic assembly of split dual-targeted IL-2 mimetics
In a bid to maintain conditional agonistic activity whilst incorporating tumor selectivity, a strategy was devised to harness cell surface markers, reportedly enriched within the tumor environment, to scaffold the assembly of the IL-2 agonistic VHH domain pair. The aim was to bind two proximal epitopes on a single target using two non-competing anti-target binders (Fig. 19A). In the first instance, PD-1 was selected as the target for several reasons. First, upregulation of PD-1 is observed upon T cell activation, providing an opportunity to enhance selectivity towards the tumor-specific effector T cell population. Second, PD-L1 -competitive anti -PD-1 binders would provide immune checkpoint inhibition mediated by blockade of the PD1/PD-L1 signaling cascade. Third, recent evidence highlighted the synergistic effects of IL-2R agonism combined with cis-targeted PD-1 blockade, resulting in the differentiation of ‘better effector’ T cells (Codarri Deak et al. Nature 610; 161— 172 (2022)).
In this experiment, the potency and the cis-delivery of two pairs of IL-2 mimetics (Fig. 19B and 19C; Fig. 19D and 19E), as well as PDl-IL2v (Fig. 19G, P1AE4422) and a potent IL-2 agonistic heavy chain antibody (Fig. 19F, P1AH117) as reference molecules, were measured based on their IL-2R signaling by treating activated PD-1+ and PD-1" (anti-PDl pre-treated) CD4 T cells with increasing concentrations of molecules. The purpose was to determine the dependency of the PD-l-based IL-2R agonists on the PD1 expression of the T cells in order to deliver an IL-2R signaling.
For this purpose CD4 T cells were sorted from healthy donor PBMCs with CD4 beads (Miltenyi, Cat. No: 130-045-101) and activated for 3 days in presence of 1 pg/ml plate bound anti-CD3 (overnight precoated, clone OKT3, Cat. No: 317315, BioLegend) and 1 pg/ml of soluble anti-CD28 (clone CD28.2, Cat. No: 302923, BioLegend) antibodies to induce PD1 expression. Three days later, the cells were harvested and washed several times to remove endogenous cytokines and half of the cells were labelled with CTV (5 pM, 5 min at 20°C; Cat. No: C34557, Thermo Scientific) and the other half left unlabelled. Then, the unlabelled cells were incubated with a saturating concentration of a competing anti -PD-1 antibody (in-house molecule, 10 pg/ml) for 30 min at 20°C followed by several washing steps to remove the excess unbound anti-PDl antibody. Thereafter the PD1 pre-blocked unlabelled cells (25 pl, 6xl06 cells/ml) were co-cultured 1: 1 with the PD1+ CTV labelled cells (25 pl, 6xl06 cells/ml) in a V-bottom plate before being treated for 15 or 60 min at 37°C with increasing concentrations of treatment molecules (50 pl, 1: 10 dilution steps). To preserve the phosphorylation state, an equal amount of Phosphoflow Fix Buffer I (100 pl, Cat. No: 557870, BD) was added after incubation with the various treatment molecules. The cells were then incubated for an additional 30 min at 37°C before being permeabilized overnight at -80°C with Phosphoflow PermBuffer III (Cat. No: 558050, BD). On the next day STAT-5 in its phosphorylated form was stained for 30 min at 4°C by using an anti-STAT-5P antibody
(47/Stat5(pY694) clone, Cat. No: 562076, BD).
The cells were acquired at the FACS BD-LSR Fortessa (BD Bioscience). The frequency of STAT-5P was determined with FlowJo (V10) and plotted with GraphPad Prism.
The dose-response curves on PD-1+ T cells provide information on the potency of the PD 1-based IL-2R agonists in signaling through the IL-2R. In addition, the dose-response curves on T cells pre-treated with a competing anti-PDl antibody, to prevent the PD1 mediated delivery, show the potency of the PD1- based IL-2R agonist molecules in providing IL-2R signaling independently from PD-1 expression. PD1- IL2v showed reduced activity on T cells in absence of PD1 binding (pre-blocked) (Fig.20, black solid and black dashed lines). While no activity was seen for the two combinations of split IL-2 mimetics (P1AH6850 + P1AH6813, P1AH6814 + P1AI1593) in absence of PD1 binding (Fig. 20, light grey dashed lines). No reduction in IL-2 signaling was seen for the non-targeted IL-2 agonistic bispecific heavy chain antibody (P1AH1177, Fig. 20, dark gray solid and dark grey dashed lines).
In order the study the biological activity of PD-1 targeted IL-2R agonists downstream of STAT-5 phosphorylation, the molecules of interest were tested in a PBMC proliferation and activation assay. Briefly, human PBMCs from healthy donors were isolated from buffy coats (Zurich Blood Donation Center in accordance with the Declaration of Helsinki) using standard density-gradient isolation over Lymphoprep™ (STEMCELL Technologies). Freshly isolated PBMCs were stained with CcllTracc ™ Violet Cell proliferation Kit (Thermo Fischer, Cat. No. C34557). 200,000 CTV labelled PBMCs per well were added to U-bottom 96-well plate (TPP) and co-incubated with test compounds at 5 concentrations ranging from 50,000 to 5 pM in assay medium (RPMI 1640 (Gibco) containing 10% FBS (Gibco) and IX Glutamax (Gibco)). Assay plates were incubated at 37°C and 5% CO2 for five days before flow cytometry readout. Cells were stained with Zombie Aqua™ Fixable Viability Kit (Biolegend, Cat. No. 423102) in PBS containing Human TruStain FcX™ (Biolegend, Cat. No. 422302) for 10 minutes at room temperature. Cells were washed with FACS buffer (PBS containing 0.1% BSA) and stained with fluorescent dye-conjugated antibodies against human CD4 (clone SK3, BD, Cat. No 563550), PD-1 (clone EH12.1, BD, Cat. No 612791), CD56 (clone HCD56, Biolegend, Cat. No 318334), CD122 (clone 5H4, Biolegend, Cat. No 339006), y5 TCR (clone 11F2, BD, Cat. No 655410), CD25 (clone BC96, Biolegend, Cat. No 302610), CD3 (clone OKT3, Biolegend, Cat. No 317340) and CD8 (clone SKI, BD, Cat. No 560179) in FACS buffer for 30 minutes at 4°C.. After two washing steps, cells were resuspended in FACS buffer and acquired with a 5-laser A3 Symphony instrument (BD). Data analysis was performed with FlowJo vl0.8. 1 and Prism 8 (GraphPad software).
At baseline, different cell types were identified according to lineage marker expression on the cell surface by flow cytometry (Fig. 21). Since the goal is to generate PD-1 targeted IL-2R agonists that specifically deliver IL-2 signals to PD-1 expressing cells while sparring PD-1 negative cells, the prevalence of PD-1+ IL-2R+ target cells was evaluated within the PBMC fraction. Across the two tested donors, a subset of CD8 T cells and y5 T cells (10-25%) co-expressed PD-1 and IL-2RP (Fig. 21D and E) whereas NK cells expressed high levels of IL-2RP and almost no PD-1 (Fig. 2 IF). Upregulation of the activation marker CD25 and the proliferative capacity of several cell types were used as functionality readouts at assay endpoint. An untargeted IL-2 agonistic heavy chain antibody (Fc-VHH; P1AH1177) showed a strong CD25 expression on a large fraction of CD8 T cells (Fig. 22A), y5 T cells (Fig. 22B) and NK cells (Fig. 22C), confirming the strong potency of IL-2R agonists. PD-1 targeted IL-2R agonist (P1AH6814+P1AI1593) demonstrated a dose-dependent induction of CD25 expression on a subset of CD8 T cells (Fig. 22A) and y5 T cells (Fig. 22B) but not on NK cells (Fig. 22C). A similar pattern was observed with the frequency of proliferating cells (Fig. 23) highlighting that the biparatopic assembly of IL-2R agonist onto PD-1 ensures the specific targeting of PD-1 expressing cells. PD-1 targeted IL- 2R agonist (P1AH6814+P1AI1593) showed no activity on the phenotype and proliferation ofNK cells with high IL-2R expression and negligible PD-1.
The PD-1 targeted IL-2R agonist (PlAH6814+PlAI1593)was further tested in the human breast BC004 patient derived cells orthotopically injected into humanized BRGS47 mice.BC004 PDX material (human breast carcinoma) were originally obtained from OncoTest (Freiburg, Germany) and after in vivo expansion deposited in the Roche-Glycart internal cell bank. Tumor fragments were digested with Collagenase D and DNase I (Roche, Switzerland), and BC004 cells were used for transplantation. IxlO6 cells per animal were injected intra mammary fat pad in 100 pl of RPMI cell culture medium (Gibco, Germany) into the flank of mice using a 1 ml tuberculin syringe (BD Biosciences, Germany).
Fully humanized BRGS47 female mice (Jackson Labs, USA) were maintained under specific-pathogen- firee condition with daily cycles of 12 h light / 12 h darkness according to committed guidelines (GV- Solas; Felasa; TierschG). Continuous health monitoring was carried out on a regular basis.
Mice were injected intra mammary fat pad on study day 0 with IxlO6 of BC004 cells, randomized and weighed. Forty days after the tumor cell injection (tumor volume > 200 mm3), mice were injected i.v. with the following molecules: P1AI1593, P1AH6814, Pembrolizumab 1 mg/kg, PDl-IL2v 0.1 mg/kg and FOLR1-TCB 0.3 mg/kg or Vehicle once a week for four weeks. The PD-1 targeted IL-2R agonist split constructs P1AH6814 and P1AI1593 were given once a week but on consecutive days. Groups of mice were also tested with Vehicle or FolRl-TCB as single agent or the combination of the PD-1 targeted IL-2R agonist, PDl-IL2v or Pembrolizumab with FOLR1-TCB. Due to the chimeric nature of the BRGS47 humanized mice, no HLA-restricted T cell responses occur in the BC004 breast orthotopic model. In order to see anti-tumor response in this mouse study the test compounds needed to be combined with the bispecific FOLR1 -TCB molecule that triggers signal one first and as such allows the evaluation of the additive effect of immunomodulator or checkpoint inhibitor compounds. All mice were injected i.v. with 200 pl of the appropriate solution. The mice in the Vehicle group were injected with Histidine Buffer and the treatment groups with the different constructs. To obtain the proper amount of immuno-conjugate per 200 pl, the stock solutions were diluted with Histidine Buffer when necessary (see Table 3). Tumor growth measurements were evaluated with a caliper three times a week and plotted with GrahPad Prism software as volume in mm3 +/- SEM.
Figure 24 shows that the combination FOLR1-TCB 0.3 mg/kg + PDl-IL2v mimetic (PlAI1593day 1 + P1AH6814 day 2) mediated superior efficacy in terms of tumor growth inhibition compared to FOLR1- TCB single agent treatment and the other combination groups: FOLR1-TCB 0.3 mg/kg + PDl-IL2v and FOLR1-TCB 0.3 mg/kg + Pembrolizumab.
Table 3: Overview of administered compounds.
3.4 Functional characterization of further cis-targeted IL-2R agonists
Further investigations of the biparatopic assembly concept were undertaken with alternative anti-PD-1 Fab binders (Fig. 25). HEK Blue IL-2 wt cells (PD-1 -negative) and HEK Blue IL-2 cells expressing human PD-1 were used to assess IL-2 signaling. As observed with the previously described PD-1- targeted IL-2R agonists (P1AH6814 + P1AI1593 and P1AH6850 + P1AH6813), none of the tested combinations showed IL-2R signaling in the absence of PD-1 expression (Fig. 26A) whereas replacing PD-1 binder 0376 with 7G12 (P1AK2802 + P1AK2599) also demonstrated strong potency on PD-1 expressing cells (Fig. 26B). Of the eight tested PD-l-targeted IL-2R agonists containing the same set of PD-1 binders (7G12 and 1040), three demonstrated strong dose-response IL-2R activation, highlighting the impact of the geometry on biparatopic assembly and delivery of IL-2 signals.
To further explore the biparatopic assembly of cis-targeted IL-2R agonists, the concept was tested with LAG3 -targeting constructs, as another example besides PD-1 targeting of T cell in cis. Two noncompeting LAG3-specific Fabs were selected and engineered to mediate assembly of the agonistic VHH pair. VHH domains of the P1AH1177 heavy chain antibody were fused to the N-terminus of the VH or VL domains of the anti-LAG3 binders via a flexible 5(G4S) linker, resulting in eight possible format configurations (P1AJ5654 - P1AJ5661) which can be paired to produce eight assembly combinations (Fig. 27). In order to assess the cis activity of IL-2R agonists targeted to LAG3, the phosphorylation of STAT5 on pre-activated CD4 T cells after 60 min incubation with test compounds was determined as previously described. Of note, the activation protocol yields comparable frequencies of PD-1 and LAG3 expressing T cells (data not shown). One pair of LAG3-targeted IL-2R agonists (P1AJ5658 + P1AJ5657) led to IL-2R signaling (Fig. 28A and 28B).
3.5 Functional characterization of trans-targeted IL-2R agonists
To further explore the biparatopic assembly of IL-2R agonists, the concept was tested with tumor (EGFR, HER2, HER3) or stroma (FAP) targets to measure delivery of IL-2R signaling in a trans setting. The pair of agonistic VHH domains of heavy chain antibody Pl AHI 177 mimicking IL-2 were distributed onto two distinct molecules to inactivate the VHH pair, whilst incorporating a targetdependent assembly strategy to restore agonistic VHH activity in the presence of each target (FAP, EGFR, HER2, HER3). Two non-competing target-specific Fabs were selected for each target and engineered to mediate assembly of the agonistic VHH pair. In the first instance, VHH domains of the P1AH1177 heavy chain antibody were fused to the N-terminus of the VH or VL domains of the anti- FAP binders via a flexible 5(G4S) linker, resulting in eight possible format configurations (P1AJ5092 - P1AJ5099) which can be paired to produce eight assembly combinations (Fig. 29). In addition, molecular concepts targeting EGFR were designed with two non-competing anti-EGFR Fabs. The eight possible format configurations (P1AJ4165 - P1AJ4172) can be paired to produce eight assembly combinations (Fig. 31). Moreover, two pairs of Her2 -targeted IL-2R agonists (P1AJ4174 + P1AJ4175 and P1AJ4173 + P1AJ4176) as well as two pairs of Her3-targeted IL-2R agonists (P1AJ4178 + P1AJ4179 and P1AJ4177 + P1AJ4180) were analyzed (Fig. 33).
To measure the capacity of FAP, EGFR, HER2 and HER3 targeted IL-2R agonists to induce IL-2R signaling, a co-culture system of target positive or negative cell lines and IL-2RPy Bioassay cells (Promega, CS2018K02) was used. IL-2RPy Bioassay cells express physiological levels of IL-2RP and IL-2Ry similar to human T cells, with a STAT5 phosphorylation luminescent reporter system. Briefly, 10,000 target cells (A549 FAP negative, A549 FAP positive, A-431 EGFR high, OE19 EGFR medium, LoVo EGFR medium, MDA-MB-231 HER2/HER3 negative or OE19 HER2/HER3 high) per well were seeded in a white 384-well flat bottom tissue culture treated plate (Falcon, Cat. No. 353988). After 5-6h of incubation at 37°C and 5% CO2 to allow the cells to adhere, test compounds (4-fold dilution series, starting concentration of 100 nM) and 10,000 IL-2RPy Bioassay reporter cells per well were added to the assay plate. Samples were incubated at 37°C and 5% CO2 for 20h before assay readout with Bio- Glo-NL Luciferase Assay System (Promega, Cat. No. J3081) according to the manufacturer’s instructions. Luminescence readout of luciferase activity was performed using Tecan Spark 10M plate reader (500ms acquisition time). Dose-response of test compounds vs fold change IL-2R signaling (RLU value/background) were plotted in GraphPad Prism software.
While the activity of PDl-IL2v was comparable in the presence and absence of FAP on A549 target cells, there was a clear activity gain of FAP-IL2v in A549 FAP+ cells compared to FAP- cells, demonstrating the validity of the experimental conditions to study trans IL-2R signaling (Fig. 30). A bell-shaped trans activity of FAP-targeted IL-2R agonists was observed, with the strongest IL-2R signaling for the pairs that contain one VHH fused to the heavy chain of a FAP binder and the other VHH fused to the light chain of the second FAP binder (e.g P1AJ5098 + P1AJ5093, Fig. 30B). Importantly, there was no activity of the FAP-targeted IL-2R agonists in the absence of FAP (Fig. 30A), highlighting the biparatopic assembly requirement for IL-2R signaling.
A similar experimental setup was applied to characterize the activity of EGFR-targeted IL-2R agonists. EGFR expression was assessed by flow cytometry using a PE -conjugated anti-human EGFR antibody (Biolegend, Cat. No. 352904) on A -431, LoVo and OE19 target cell lines (Fig. 32A). Of the eight tested EGFR-targeted IL-2R agonists, one pair (P1AJ4165 + P1AJ4172) showed consistent activity in the presence of medium or high EGFR expressing target cells (Fig. 32B, 32C and 32D). High target expression seems to be required for IL-2R signaling in a trans setting. Moreover, only one HER2- targeted IL-2R agonist (P1AJ4174 + P1AJ4175) displayed a weak ability to trigger IL-2R signaling in the presence of OE19 target cells expressing high levels of HER2 (Fig. 34B).
3.6 Evaluation of different antibody fragments as target binding domains or cytokine receptor binding domains
In order to understand if the small size of single domain antibodies would be favorable for optimal assembly of IL-2R agonists, we tested four anti-PD-1 VHH binders as target-binding domains as shown in Figure 35. None of the tested combinations showed IL-2R signaling in the absence ofPD-1 expression (Fig. 36A). While the six tested combinations demonstrated IL-2R signaling in reporter assays (Fig. 36B), the magnitude of the response was weaker than with anti-PD-1 Fabs (P1AH6814 + P1AI1593). In order to understand if the small size of single domain antibodies (VHH) as cytokine receptor-binding domains is beneficial for optimal IL-2R signaling, four pairs of anti-IL2R Fabs (Fig. 37A-D) and one pair of anti-IL2R scFv (Fig. 37E and F) using the biparatopic assembly concept were tested in a HEK Blue IL-2 reporter assay.
While none of the tested combinations showed activity in the absence of PD-1 expression (Fig. 38A), they demonstrated varying levels of activity in PD-1 expressing reporter cells (Fig. 38B). The strongest IL-2R signaling was observed with anti-IL2R agonists comprising VHH domains (P1AH6814+P1AI1593) followed by the anti-IL2R agonists comprising scFv domains (P1AL9310+1AL9311). Anti-IL2R Fab agonists displayed the weakest potency, indicating that VHH- based agonists may be ideal candidates to bring IL2RP and IL2Ry receptor chains in close proximity and trigger IL-2R signaling.
3.7 Role of distance and geometry in biparatopic assembly
To further investigate the role of geometry in biparatopic assembly of cytokine mimetics, available x- ray crystal structures comprising Fab: target complexes were used to measure the distance between N- terminal residues of two non-competing anti-target Fabs. Distances were measured for PD-1 (Table 4), EGFR (Table 5), HER2 (Table 6) and HER3 (Table 7) targets.
Table 4: Distances (A) measured between the N-termini of the heavy chain (HC) and light chain (LC) of two anti -PD-1 Fabs. The distance between the most proximal combination of N-terminal fusion points is highlighted in grey.
Table 5: Distances (A) measured between the N-termini of the heavy chain (HC) and light chain (LC) of two anti-EGFR Fabs. The distance between the most proximal combination of N-terminal fusion points is highlighted in grey.
Table 6: Distances (A) measured between the N-termini of the heavy chain (HC) and light chain (LC) of two anti-HER2 Fabs. The distance between the most proximal combination of N-terminal fusion points is highlighted in grey. Table 7: Distances (A) measured between the N-termini of the heavy chain (HC) and light chain (LC) of two anti-HER3 Fabs. The distance between the most proximal combination of N-terminal fusion points is highlighted in grey.
The measurements confirmed the impact of geometry in targeted biparatopic assembly of cytokine mimetics. In conclusion, the shorter the distance between two non-competing anti -target binders, the greater the potency of the resulting mimetic assembly.
Example 4. Identification of IL-7R agonists
4.1 Generation of antigen for immunization and phage display
For alpaca immunization and phage display, the extracellular domain (ECD) of human IL-7Ra (CD 127) subunit was expressed as an Fc-tagged monomer based on the knobs-into-holes technology (Fig. 39A, P1AI1009, SEQ ID NO: 208 and SEQ ID NO: 209). The C-terminus of IL-7Ra ECD was fused to Fc knob via an ASGS linker. An AviTag was included at the C-terminus of the Fc for site-specific biotinylation during co-expression with BirA biotin ligase.
All antigens were produced in the Expi293 mammalian expression system (Expi293™ Expression System Kit, Thermo Fisher Scientific, Cat. No: A14635) following manufacturer’s recommendation. Supernatants were harvested by centrifugation, filtered and purified by Protein A affinity chromatography (Protein A MabSelectTM SuReTM 15, Cytiva, Cat. No: 17543803) followed by size exclusion chromatography (SEC) (HiLoad S200 16/600, Cytiva, Cat. No: 28989335) according to the manufacturer's recommendation.
4.2. VHH library construction
The VHH libraries were constructed using lymphocytes isolated from two alpacas. The libraries were provided in the TGI E. coli strain encoded in a derivative of the pHEN4 phagemid vector (Hassanzadeh- Ghassabeh et al., 2010), yielding 5-7xl08 clones per library. Libraries were provided in 2xYT medium containing 20% glycerol.
4.3 Generation of VHH phage library
E. coli containing the VHH-encoded phagemid library were inoculated in 2xYT medium containing 100 pg/ml ampicillin and 1% glucose. Following infection with VCSM13 helper phage, E. coli were grown in 2xYT medium containing 100 pg/ml ampicillin and 50 pg/ml kanamycin. Following overnight incubation on a shaker at 30° C, cultures were harvested by centrifugation at 8000 rpm for 20 minutes and supernatants were collected. Phage were precipitated in 20% PEG/2.5 M NaCl solution and incubated on ice for 1 hour. Precipitated phage were centrifuged at 8000 rpm for 20 minutes and pellets were resuspended in ddH2O followed by precipitation in PEG/NaCl solution for 1 hour on ice. Phage were centrifuged at 8000 rpm for 20 minutes and pellets were resuspended in PBS containing 20% glycerol.
4.4 Isolation of IL-7Rct -specific VHHs by phage display
To selectively enrich antigen-specific binders and prevent the enrichment of Fc-specific binders, Fc competitive soluble selections were performed against human IL-7Ra antigen (P1AI1009) in the presence of human Fc (Fig. 39B, P1AD4290). An aliquot of IxlO12 phage from each of the two alpaca- derived VHH libraries was used per target antigen. Phage and magnetic streptavidin beads (Dynabeads; M-280 Streptavidin, Invitrogen, Cat. No: 11205D) were independently blocked with 1% BSA, 0.1% Tween-20 in phosphate-buffered saline (PBS) to reduce non-specific interactions. All incubations were performed for one hour at 20° C, unless otherwise stated. The biotinylated, Fc-tagged IL-7Ra antigen was added at 100 nM concentration to blocked phage in the presence of excess amounts (500 nM) of non-biotinylated Fc (Fig. 39C). Following incubation, pre-blocked Dynabeads were added to capture biotinylated antigen of interest and bound VHH-expressing phage. Fc-specific and non-specific phage were removed by 5 - 10 wash steps in PBS containing 0.1% Tween-20 followed by PBS using a KingFisher magnetic particle processor (Thermo Fisher Scientific). Antigen-specific phage were eluted in 800 pl of 100 mM trimethylamine (TEA) and neutralized by addition of 400 pl of 1 M Tris pH 7.4. Mid-logarithmic phase TGI E. coli in 2xYT medium were infected with eluted phage, followed by infection with VCSM13 helper phage. Following addition of 100 pg/ml ampicillin and 50 pg/ml kanamycin, infected E. coli were incubated overnight on a shaker at 30° C. Phage were precipitated with PEG/NaCl and used for the next round. Selections with alpaca library 1 and alpaca library 2 were performed for a total of four rounds using decreasing target antigen concentrations (100 nM, 50 nM, 25 nM, 12.5 nM) while soluble Fc concentrations remained constant (500 nM) (Fig. 39C).
4.5 Verification of target specificity by ELISA
Round 3 and round 4 enrichment libraries were screened for target specificity vs Fc specificity by ELISA. Individual VHH clones were expressed in 1 ml E. coli cultures in a 96-well plate and supernatants containing soluble VHH were screened for specificity by ELISA. Supernatants were transferred to neutravidin 96-well plates coated with biotinylated Fc (P1AE6073) or target Fc-tagged biotinylated antigen (P1AI1009). Following incubation for 1 hour at 20° C, plates were washed 3 times with PBST and 3 times with PBS. HRP-conjugated anti-His antibody (Sigma, Cat. No: A7058) was added to wells at 1:2000 dilution and plates were incubated for 1 hour at 20° C. Plates were washed 3 times with PBST followed by 3 washes with PBS. Plates were developed by adding 1-Step Ultra TMB- ELISA substrate (Thermo Fisher Scientific, Cat. No: 34028) and reactions were quenched with sulfuric acid. Absorbance at 450 nm was measured using a Tecan infinite M1000 Pro and reference absorbance values were measured at 650 nm. Reference-subtracted absorbance values at 450 nm were represented as stacked bar charts for round 3 library 1 and library 2 (Fig. 40A and B, respectively) and round 4 library 1 and library 2 (Fig. 40C and D, respectively). ELISA data suggested that library 2 showed a greater enrichment of target-specific clones. 16 target-specific VHH were selected and subsequently cloned into bispecific heavy chain antibody formats.
4.6 Format conversion of VHH into bispecific heavy chain antibodies
IL-7R activation is achieved by IL7-mediated cross-linking of the IL-7Ra (CD 127) and IL-2Ry (CD 132; common gamma chain) receptor chains. To mimic receptor assembly in the absence of IL-7, an IL-7Ra- specific VHH domain was paired with previously identified VHH domains with IL-2Ry specificity (IL- 2Ry_2, IL-2Ry_3, IL-2Ry_4 and IL-2Ry_5). VHH were fused to knob-into-hole Fc chains via a flexible linker generating IL-7Ra-IL-2Ry bispecific heavy chain antibodies. Sixteen IL-7Ra-specific VHH were selected from rounds 3 and 4 of phage display enrichment and four previously identified IL-2Ry-specific VHH were selected. Format conversion was performed by Gibson assembly cloning by fusing IL-7Ra- specific VHH to Fc knob via a 5(G4S) linker (Fig. 41 A) and by fusing IL-2Ry-specific VHH to Fc hole via a 5(G4S) linker (Fig. 41B). During the assembly, a suitable signal sequence and vector compatible with mammalian expression were included. Primers, PCR conditions and Gibson assembly reactions were defined as per New England Biolabs’ NEBuilder HiFi DNA Assembly protocol (Cat. No: E5510). Next, the Expi293 system was used to express bispecific heavy chain antibodies in a 16x4 matrix to generate 64 unique antibodies comprising IL-7Ra VHH and IL-2Ry VHH pairs (Fig. 41C). Expi293 cells were transiently transfected following manufacturer’s recommendation (Expi293 ™ Expression System Kit, Thermo Fisher Scientific, Cat. No: A 14635) and supernatants containing secreted soluble bispecific heavy chain antibodies were collected. The bispecific heavy chain antibodies were purified by Protein A resin and further characterized for functional activity using reporter cell assays.
Table 8: IL-7Ra-IL-2Ry bispecific heavy chain antibodies generated by the 16x4 expression matrix. 4.7 Functional characterization of bispecific heavy chain antibodies
The HEK Blue IL-7 reporter cell assay (Invivogen) was used to screen bispecific heavy chain antibodies comprising different IL-7Ra and IL-2Ry VHH pairs and identify IL-7R agonists. The in vitro HEK Blue IL-7 reporter cell (Invivogen, Cat. No: hkb-il7) recapitulates the human IL-7R signaling pathway via expression of the IL-7Ra and IL-2Ry chains, effectors of the downstream signaling cascade (JAK3 and STAT5) and a STAT5 -inducible secreted alkaline phosphatase (SEAP) reporter system. Following addition of QuantiBlue substrate (Invivogen, Cat. No: rep-qbs), absorbance at 650 nm is measured, correlating with SEAP levels and IL-7R activity. The 64 heavy chain antibodies and reference molecules were incubated with HEK Blue IL-7 cells for 20 hours. IL-7R activity was quantified by addition of QuantiBlue reagent, according to the manufacturer's recommendation. The absorbance at 650 nm was measured and fold response/background was plotted as bar charts (Fig 42A-D). All 64 molecules showed IL-7R agonism with a broad range of activity. 12 agonistic VHH pairs (IL-7Ra_l, IL-7Ra_2, IL-7Ra_3, IL-7Ra_5, IL-7Ra_6, IL-7Ra_7, IL-7Ra_10, IL-7Ra_l l, IL-7Ra_13, IL-7Ra_14, IL- 7Ra_15, IL-7Ra_16 in combination with IL2Ry_2) were selected to further engineer for PD-l-targeted biparatopic assembly.
4.8 Design of PD-l-targeted biparatopic assembly of IL-7 mimetics
In a bid to bias agonistic activity towards tumor-reactive T cells, the PD-1 cell surface marker was selected to scaffold the assembly of the IL-7 mimetic VHH pair. The aim was to bind two proximal epitopes on PD-1 using two non-competing anti-PD-1 binders to bridge the assembly of a functional IL- 7 mimetic (Fig. 43A). VHH fusion points on the two PD-1 binders were selected according to N-terminal residue proximity of the two Fabs determined by x-ray crystal structures (Table 4). The 12 selected IL- 7Ra-specific VHH were fused to the VL of anti-PD-1 binder 1 via a 5(G4S) linker (P1AL0544 - P1AL0556, Fig. 43B) and the selected VHH domain with IL-2Ry specificity was fused to the VH of anti-PD-1 binder 2 (P1AH6814; Fig. 43C).
4.9 Functional characterization of PD-l-targeted IL-7 mimetics
To assess the cis activity of twelve IL-7R agonists targeted to PD-1, the phosphorylation of STAT5 on pre-activated CD4 T cells after 60 min incubation with test compounds was determined as previously described. The IL-2 mimetic heavy chain antibody (P1AH1177), a PD-1 dependent biparatopic IL-2 mimetic (P1AH6814 + P1AI1593) and PDl-IL2v (P1AE4422) were used as reference molecules. A non-binding DP47 antibody (P1AD3966, SEQ ID NO: 230 and SEQ ID NO: 231) was used as negative control. All PD-l-targeted IL-7R agonists showed dose-dependent induction of STAT-5 phosphorylation with single-digit nM or sub-nM EC50 activity (Fig 44). Example 5. Functional characterization of monoparatopic and biparatopic cis-targeted cytokine receptor agonists
To understand the requirement for biparatopic assembly of cytokine receptor agonists, we tested the monoparatopic concept of IL-2R signaling via binding to the same PD-1 epitope (Fig. 45). The previously described HEK Blue IL-2 reporter cell assay was used to assess IL-2 signaling. As previously demonstrated, biparatopic assembly of PD-l-targeted IL-2R agonists (P1AH6814 + P1AI1593 and P1AH6814 + P1AK2802) showed no signaling in PD-l-negative cells (Fig. 46A) and strong doseresponse IL-2R signaling in PD-l-positive cells (Fig. 46B). Whereas no activity was observed with any of the eight monoparatopic pairs tested, neither in PD-l-negative nor in PD-l-positive cells (Fig. 46A and B), highlighting the biparatopic assembly requirement for functional cytokine receptor signalling.
Sequences

Claims

Claims
1. A pair of antigen binding molecules that specifically bind to a target antigen, comprising a) a first antigen binding molecule comprising i) a first target-binding domain, ii) a first cytokine receptor-binding domain, and iii) a Fc domain; and b) a second antigen binding molecule comprising i) a second target-binding domain, ii) a second cytokine receptor-binding domain, and iii) a Fc domain; wherein the first target-binding domain is capable of binding a first epitope on the target antigen and the second target-binding domain is capable of binding a second epitope on the target antigen, wherein the first and second target-binding domains do not compete for binding on the target antigen; and wherein the first cytokine receptor-binding domain is capable of binding a first cytokine receptor subunit and the second cytokine receptor-binding domain is capable of binding a second cytokine receptor subunit.
2. The pair of antigen binding molecules according to claim 1, wherein the first and second targetbinding domains are antibody fragments, such as Fv, Fab, scFv, scFab molecules or single domain antibodies.
3. The pair of antigen binding molecules according to claim 1 or 2, wherein the first and second targetbinding domains are Fab molecules.
4. The pair of antigen binding molecules according to claim 3, wherein the first target-binding domain comprises a heavy chain variable domain (VHi), a light chain variable domain (VLi), a heavy chain constant domain (CHli) and a light chain constant domain (CLi), and wherein the second targetbinding domain comprises a heavy chain variable domain (VH2), a light chain variable domain (VL2), a heavy chain constant domain (CHI 2) and a light chain constant domain (CL2).
5. The pair of antigen binding molecules according to claim 3 or 4, wherein the first and/or second target-binding domain is a cross-Fab molecule.
6. The pair of antigen binding molecules according to any one of the preceding claims, wherein the first and second target-binding domains specifically bind to a tumor-associated antigen or a T-cell antigen.
7. The pair of antigen binding molecules according to any one of the preceding claims, wherein the first and second target-binding domains specifically bind to FAP, PD-1, Her2, Her3, LAG-3 or EGFR.
8. The pair of antigen binding molecules according to any one of the preceding claims, wherein a) the first target-binding domain comprises a VHi of SEQ ID NO: 20 and a VLi of SEQ ID NO:
21 and the second target-binding domain comprises a VH2 of SEQ ID NO: 22 and a VL2 of SEQ ID NO: 23, or b) the first target-binding domain comprises a VHi of SEQ ID NO: 22 and a VLi of SEQ ID NO:
23 and the second target-binding domain comprises a VH2 of SEQ ID NO: 20 and a VL2 of SEQ ID NO: 21, or c) the first target-binding domain comprises a VHi of SEQ ID NO: 76 and a VLi of SEQ ID NO:
77 and the second target-binding domain comprises a VH2 of SEQ ID NO: 78 and a VL2 of SEQ ID NO: 79, or d) the first target-binding domain comprises a VHi of SEQ ID NO: 78 and a VLi of SEQ ID NO:
79 and the second target-binding domain comprises a VH2 of SEQ ID NO: 76 and a VL2 of SEQ ID NO: 77; or e) the first target-binding domain comprises a VHi of SEQ ID NO: 93 and a VLi of SEQ ID NO:
94 and the second target-binding domain comprises a VH2 of SEQ ID NO: 78 and a VL2 of SEQ ID NO: 79; or f) the first target-binding domain comprises a VHi of SEQ ID NO: 78 and a VLi of SEQ ID NO:
79 and the second target-binding domains comprises a VH2 of SEQ ID NO: 93 and a VL2 of SEQ ID NO: 94, or g) the first target-binding domain comprises a VHi of SEQ ID NO: 130 and a VLi of SEQ ID NO: 131 and the second target-binding domain comprises a VH2 of SEQ ID NO: 132 and a VL2 of SEQ ID NO: 133; or h) the first target-binding domain comprises a VHi of SEQ ID NO: 132 and a VLi of SEQ ID NO: 133 and the second target-binding domains comprises a VH2 of SEQ ID NO: 130 and a VL2 of SEQ ID NO: 131; or i) the first target-binding domain comprises a VHi of SEQ ID NO: 140 and a VLi of SEQ ID NO: 141 and the second target-binding domain comprises a VH2 of SEQ ID NO: 142 and a VL2 of SEQ ID NO: 143; or j) the first target-binding domain comprises a VHi of SEQ ID NO: 142 and a VLi of SEQ ID NO: 143 and the second target-binding domains comprises a VH2 of SEQ ID NO: 140 and a VL2 of SEQ ID NO: 141; or k) the first target-binding domain comprises a VHi of SEQ ID NO: 114 and a VLi of SEQ ID NO: 115 and the second target-binding domain comprises a VH2 of SEQ ID NO: 116 and a VL2 of SEQ ID NO: 117; or l) the first target-binding domain comprises a VHi of SEQ ID NO: 116 and a VLi of SEQ ID NO: 117 and the second target-binding domains comprises a VH2 of SEQ ID NO: 114 and a VL2 of
SEQ ID NO: 115; or m) the first target-binding domain comprises a VHi of SEQ ID NO: 150 and a VLi of SEQ ID NO: 151 and the second target-binding domain comprises a VH2 of SEQ ID NO: 152 and a VL2 of SEQ ID NO: 153; or n) the first target-binding domain comprises a VHi of SEQ ID NO: 152 and a VLi of SEQ ID NO: 153 and the second target-binding domains comprises a VH2 of SEQ ID NO: 150 and a VL2 of SEQ ID NO: 151.
9. The pair of antigen binding molecules according to any one of the preceding claims, wherein both the first and second cytokine receptor subunits are subunits of the IFNy receptor complex or, IL-2 receptor complex or IL-7 receptor complex.
10. The pair of antigen binding molecules according to any one of the preceding claims, wherein a) the first cytokine receptor-binding domain is capable of binding IFNyRl and the second cytokine receptor-binding domain is capable of binding IFNyR2, or b) the first cytokine receptor-binding domain is capable of binding IFNyR2 and the second cytokine receptor-binding domain is capable of binding IFNyRl; or c) the first cytokine receptor-binding domain is capable of binding IL-2RP and the second cytokine receptor-binding domain is capable of binding IL-2Ry, or d) the first cytokine receptor-binding domain is capable of binding IL-2Ry and the second cytokine receptor-binding domain is capable of binding IL-2RP; or e) the first cytokine receptor-binding domain is capable of binding IL-2Ry and the second cytokine receptor-binding domain is capable of binding IL-7Ra; or f) the first cytokine receptor-binding domain is capable of binding IL-7Ra and the second cytokine receptor-binding domain is capable of binding IL-2Ry.
11. The pair of antigen binding molecules according to any one of the preceding claims, wherein the first and second cytokine receptor-binding domains are antibody fragments, such as Fv, Fab, scFv, scFab or single domain antibodies.
12. The pair of antigen binding molecules according to any one of the preceding claims, wherein the first and second cytokine receptor-binding domains are VHH domains.
13. The pair of antigen binding molecules according to any one of the preceding claims, wherein a) the first cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 5 and the second cytokine receptorbinding domain comprises an amino acid sequence selected from SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, or b) the first cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10 and the second cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 5; or c) the first cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58 SEQ ID NO: 60, and the second cytokine receptorbinding domain comprises an amino acid sequence selected from SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64 and SEQ ID NO: 65; or d) the first cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64 and SEQ ID NO: 65 and the second cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58 SEQ ID NO: 60; or e) the first cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, and the second cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65; or f) the first cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, and the second cytokine receptor-binding domain comprises an amino acid sequence selected from SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192.
14. The pair of antigen binding molecules according to any one of the preceding claims, wherein a) the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 3 and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 7, or b) the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 7 and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 3; or c) the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 60 and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 62, or d) the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 62 and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 60; or e) the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 186, and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 62, or f) the first cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 62, and the second cytokine receptor-binding domain comprises the sequence of SEQ ID NO: 186.
15. The pair of antigen binding molecules according to any one of the preceding claims, wherein the Fc domains of the first and second antigen binding molecules comprise a first Fc domain subunit and a second Fc domain subunit.
16. The pair of antigen binding molecules according to any one of the preceding claims, wherein the Fc domains of the first and second antigen binding molecules are IgG, particularly an IgGi, Fc domains.
17. The pair of antigen binding molecules according to any one of the preceding claims, wherein the Fc domains of the first and second antigen binding molecules are human Fc domains.
18. The pair of antigen binding molecules according to any one of the preceding claims, wherein the Fc domains of the first and second antigen binding molecules comprise a modification promoting the association of the first and the second subunit of the Fc domains.
19. The pair of antigen binding molecules according to any one of the preceding claims, wherein the Fc domains of the first and second antigen binding molecules comprise one or more amino acid substitution that reduces binding to an Fc receptor and/or effector function.
20. The pair of antigen binding molecules according to any one of the preceding claims, wherein the cytokine receptor-binding domains are fused via peptide linkers to their respective fusion points.
21. The pair of antigen binding molecules according to claim 20, wherein the peptide linkers comprise an amino acid sequence selected from SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 279, SEQ ID NO: 280 or SEQ ID NO: 281.
22. The pair of antigen binding molecules according to any one of the preceding claims, wherein the first cytokine receptor-binding domain is fused at its C-terminus to the N-terminus of VHi or VLi of the first target-binding domain and the second cytokine receptor-binding domain is fused at its C-terminus to the N-terminus of VH2 or VL2 of the second target-binding domain.
23. The pair of antigen binding molecules according to any one of the preceding claims, wherein a) the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus the first cytokine receptor-binding domain, VHi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VLi and CLi ; and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus the second cytokine receptor-binding domain, VH2, CHI 2 and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VL2 and CL2; or b) the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VHi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus the first cytokine receptor-binding domain, VLi and CLi; and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VH2, CHh and a second Fc domain subunit, a third polypeptide comprising in order from the N-terminus to C-terminus the second cytokine receptor-binding domain, VL2 and CL2; or c) the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus the first cytokine receptor-binding domain, VHi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VLi and CLi ; and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VH2, CHh and a second Fc domain subunit, a third polypeptide comprising in order from the N-terminus to C- terminus the second cytokine receptor-binding domain, VL2 and CL2; wherein VHi, VLi, CHli and CLi form the first target-binding domain and VH2, VL2, CHI 2, and CL2, form the second target-binding domain.
24. The pair of antigen binding molecules according to any one of the preceding claims, wherein a) the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus the first cytokine receptor-binding domain, VHi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VLi and CLi, and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus the second cytokine receptor-binding domain, VH2, CHI 2 and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VL2 and CL2; or b) the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VHi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus the first cytokine receptor-binding domain, VLi and CLi, and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VH2, CHfr and a second Fc domain subunit, a third polypeptide comprising in order from the N-terminus to C-terminus the second cytokine receptor-binding domain, VL2 and CL2; or c) the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus the first cytokine receptor-binding domain, VHi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VLi and CLi, and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VH2, CHfr and a second Fc domain subunit, a third polypeptide comprising in order from the N-terminus to C- terminus the second cytokine receptor-binding domain, VL2 and CL2; or d) the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus the first cytokine receptor-binding domain, VLi, CHli and a second Fc domain subunit, and athird polypeptide comprising in order from the N-terminus to C-terminus VHi and CLi, and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus the second cytokine receptor-binding domain, VH2, CHI 2 and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VL2 and CL2; or e) the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VLi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus the first cytokine receptor-binding domain, VHi and CLi, and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VH2, CHfr and a second Fc domain subunit, athird polypeptide comprising in order from the N-terminus to C-terminus the second cytokine receptor-binding domain, VL2 and CL2; or f) the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus the first cytokine receptor-binding domain, VLi, CHli and a second Fc domain subunit, and athird polypeptide comprising in order from the N-terminus to C-terminus VHi and CLi, and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VH2, CHfr and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C- terminus the second cytokine receptor-binding domain, VL2 and CL2 ; or g) the first antigen binding molecule comprising a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a VLi, CHli and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus a first cytokine receptor-binding domain, VHi and CLi, and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a second cytokine receptorbinding domain, VH2, CHI 2 and a second Fc domain subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VL2 and CL2; wherein VHi, VLi, CHli and CLi form the first target-binding domain and VH2, VL2, CHI 2, and CL2, form the second target-binding domain.
25. The pair of antigen binding molecules according to any one of the preceding claims, wherein the first and the second target-binding domains specifically bind to FAP, and the first and second cytokine receptor subunits are subunits of the IFNy receptor complex.
26. The pair of antigen binding molecules according to any one of the preceding claims, wherein a) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 27 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 30 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 38 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 37 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or c) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 31, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 33; or d) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 35, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 36; or e) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 27 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 33; or f) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 30 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 31; or g) the first antigen binding molecule comprising a first polypeptide comprises an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 38 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 36; or h) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 37 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 35.
27. The pair of antigen binding molecules according to any one of claims 1 to 24, wherein the first and the second target-binding domains specifically bind to EGFR, and the first and second cytokine receptor subunits are subunits of the IFNy receptor complex.
28. The pair of antigen binding molecules according to any one of claims 1 to 24 or 27, wherein a) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 260 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 259 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 127; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 119 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 256, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 122 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 255; or c) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 261 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 258 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 127; or d) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 119 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 257, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 112 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 254; or e) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 260 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 112 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 255; or f) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 119 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 256, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 259 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 127; or g) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 261 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 112 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 254; or h) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 119 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 257, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 258 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 127.
29. The pair of antigen binding molecules according to any one of claims 1 to 24, wherein the first and the second target-binding domains specifically bind to PD-1, and the first and second cytokine receptor subunits are subunits of the IL-2 receptor complex.
30. The pair of antigen binding molecules according to any one of claims 1 to 24 or 29, wherein a) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 86, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 44 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 83, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 88 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 87; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 92, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 44 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 91; or c) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 96 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 95, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 101 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or d) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 99 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 98, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 88 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 102; or e) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 96 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 97, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 103 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or f) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 100 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 98, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 88 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 104; or g) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 98 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 99, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 101 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or h) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 96 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 95, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 88 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 102; or i) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 100 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 98, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 103 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or j) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 96 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 97, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 88 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 104.
31. The pair of antigen binding molecules according to any one of claims 1 to 24, wherein the first and the second target-binding domains specifically bind to LAG-3, and the first and second cytokine receptor subunits are subunits of the IL-2 receptor complex.
32. The pair of antigen binding molecules according to any one of claims 1 to 24 or 31, wherein a) first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 155 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 154, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 165 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 163.; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 161 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 160, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 158 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 159; or c) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 155 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 156, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 164 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 163; or d) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 162 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 160, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 158 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 157; or e) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 161 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 160, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 165 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 163; or f) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 155 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 154, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 158 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 159; or g) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 162 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 160, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 164 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 163; or h) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 155 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 156, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 158 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 157.
33. The pair of antigen binding molecules according to any one of claims 1 to 24, wherein the first and the second target-binding domains specifically bind to EGFR, and the first and second cytokine receptor subunits are subunits of the IL-2 receptor complex.
34. The pair of antigen binding molecules according to any one of claims 1 to 24 or 33, wherein a) first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 155 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 154, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 165 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 163; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 161 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 160, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 158 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 159.; or c) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 155 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 156, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 164 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 163; or d)the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 162 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 160, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 158 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 157; or e) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 161 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 160, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 165 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 163; or f) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 155 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 154, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 158 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 159; or g)the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 162 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 160, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 164 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 163; or h) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 155 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 156, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 158 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 157.
35. The pair of antigen binding molecules according to any one of claims 1 to 24, wherein the first and the second target-binding domains specifically bind to FAP, and the first and second cytokine receptor subunits are subunits of the IL-2 receptor complex.
36. The pair of antigen binding molecules according to any one of claims 1 to 24 or 35, wherein a) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 110 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 113 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 105, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 109; or c) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 111 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 112 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or d) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 107, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 108; or e) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 110 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 109; or f) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 105, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 113 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or g) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 111 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 108; or h) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 107, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 112 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
37. The pair of antigen binding molecules according to any one of claims 1 to 24, wherein the first and the second target-binding domains specifically bind to Her2, and the first and second cytokine receptor subunits are subunits of the IL-2 receptor complex.
38. The pair of antigen binding molecules according to any one of claims 1 to 24 or 37, wherein a) first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 136 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 134, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 138 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 137; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 135 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 134, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 138 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 139.
39. The pair of antigen binding molecules according to any one of claims 1 to 24, wherein the first and the second target-binding domains specifically bind to Her3, and the first and second cytokine receptor subunits are subunits of the IL-2 receptor complex.
40. The pair of antigen binding molecules according to any one of claims 1 to 24 or 39, wherein a) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 146 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 144, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 148 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 147; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 145 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 144, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 148 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 149.
41. The pair of antigen binding molecules according to any one of claims 1 to 24, wherein the first and the second target-binding domains specifically bind to PD-1, and the first and second cytokine receptor subunits are subunits of the IL-7 receptor complex.
42. The pair of antigen binding molecules according to any one of claims 1 to 24 and 41, wherein a) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 194 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 193, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 195 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or c) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 196 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or d) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 197 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or d)the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 198 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or e) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 199 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or f) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 200 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or g) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 201 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or h) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 202 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or i) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 203 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or j) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 204 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89; or k) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 106, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 205 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 124, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 90 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 89.
43. The pair of antigen binding molecules according to any one of claims 1 to 21, wherein the first target-binding domain is fused at its C-terminus of CHli to the N-terminus of the first Fc domain subunit and the first cytokine receptor-binding domain is fused at its C-terminus to the N-terminus of the second Fc domain subunit and the second target-binding domain is fused at its C-terminus of CH 12 to the N-terminus of the first Fc domain subunit and the second cytokine receptor-binding domain is fused at its C-terminus to the N-terminus of the second Fc domain subunit.
44. The pair of antigen binding molecules according to any one of claims 1 to 21 or 43, wherein a) the first antigen binding molecule comprises a first polypeptide comprising in order from the N- terminus to C-terminus VHi, CHli and a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VLi and CLi, and a third polypeptide comprising in order from the N-terminus to C-terminus the first cytokine receptor-binding domain and a second Fc domain subunit, and the second antigen binding molecule comprises a first polypeptide comprising in order from the N-terminus to C-terminus VH2, CHI 2 and a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VL2 and CL2, and a third polypeptide comprising in order from the N-terminus to C-terminus the second cytokine receptor-binding domain and a second Fc domain subunit; or b) the first antigen binding molecule comprises a first polypeptide comprising in order from the N- terminus to C-terminus VLi, CHli and a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VHi and CLi, and a third polypeptide comprising in order from the N-terminus to C-terminus the first cytokine receptor-binding domain and a second Fc domain subunit, and the second antigen binding molecule comprises a first polypeptide comprising in order from the N-terminus to C-terminus VH2, CHI 2 and a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VL2 and CL2, and a third polypeptide comprising in order from the N-terminus to C-terminus the second cytokine receptor-binding domain and a second Fc domain subunit; wherein VHi, VLi, CHli and CLi form the first target-binding domain and VH2, VL2, CHI 2, and CL2, form the second target-binding domain.
45. The pair of antigen binding molecules according to any one of claims 1 to 21 or 43 to 44, wherein a) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 32, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 25 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 39, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 34, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 29 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 40; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 34, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 29 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 39, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 32, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 25 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 40; or c) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 237, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 238, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or d) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 235, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 236, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or e) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 239, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 240, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or f) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 235, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 237, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or g) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 236, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 235, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or h) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 240, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 32 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 239, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 34 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
46. The pair of antigen binding molecules according to any one of claims 1 to 21, wherein the first target-binding domain is fused at its C-terminus of CHli to the N-terminus of one of the Fc domain subunits and the first cytokine receptor-binding domain is fused at its N-terminus to the C-terminus of the same Fc domain subunit and the second target-binding domain is fused at its C-terminus of CH 12 to the N-terminus of one of the Fc domain subunits and the second cytokine receptor-binding domain is fused at its N-terminus to the C-terminus of the same Fc domain subunit.
47. The pair of antigen binding molecules according to any one of claims 1 to 21 or 46, wherein a) the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a VHi, a CHli, a second Fc domain subunit and a the first cytokine receptor-binding domain, and athird polypeptide comprising in order from the N-terminus to C-terminus a VLi and a CLi; and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a VH2, a CHE, a second Fc domain subunit and a the second cytokine receptor-binding domain, and athird polypeptide comprising in order from the N-terminus to C-terminus VL2 and CL2; or b) the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a VLi, a CHli, a second Fc domain subunit and a the first cytokine receptor-binding domain, and athird polypeptide comprising in order from the N-terminus to C-terminus a VHi and a CLi ; and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a VH2, a CHE, a second Fc domain subunit and the second cytokine receptor-binding domain, and a third polypeptide comprising in order from the N-terminus to C-terminus a VL2 and a CL2; or c) the first antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a VLi, a CHli, a second Fc domain subunit and a the first cytokine receptor-binding domain, and athird polypeptide comprising in order from the N-terminus to C-terminus a VHi and a CLi ; and the second antigen binding molecule comprises a first polypeptide comprising a first Fc domain subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a VH2, a CH E. a second Fc domain subunit and a the second cytokine receptor-binding domain, and a third polypeptide comprising in order from the N-terminus to C-terminus VL2 and CL2; wherein VHi, VLi, CHli and CLi form the first target-binding domain and VH2, VL2, CHI 2, and CL2, form the second target-binding domain.
48. The pair of antigen binding molecules according to any one of claims 1 to 21 or 46 to 47, wherein a) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 42 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 41, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 45, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 44 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 43; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 46 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 41, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 47, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 44 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 43; or c) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 246 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 249 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or d) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 242 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 245 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or e) the first antigen binding molecule comprising a first polypeptide comprises an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 250 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 253 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or f) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 248 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 247 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or g) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 244 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 243 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or h) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 252 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 25, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 251 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29; or i) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 42 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 42, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 241, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 245 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 29.
49. The pair of antigen binding molecules according to any one of claims 1 to 11, wherein the first and second cytokine receptor-binding domains are Fab molecules.
50. The pair of antigen binding molecules according to claim 49, wherein a) the first cytokine receptor-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 282 and a VL domain comprising the amino acid sequence of SEQ ID NO: 283 or VH domain comprising the amino acid sequence of SEQ ID NO: 284 and a VL domain comprising the amino acid sequence of SEQ ID NO: 285, and the second cytokine receptor domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 286 and a VL domain comprising the amino acid sequence of SEQ ID NO: 287 or VH domain comprising the amino acid sequence of SEQ ID NO: 288 and a VL domain comprising the amino acid sequence of
SEQ ID NO: 289; or b) the first cytokine receptor-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 286 and a VL domain comprising the amino acid sequence of SEQ ID NO: 287 or VH domain comprising the amino acid sequence of SEQ ID NO: 288 and a VL domain comprising the amino acid sequence of SEQ ID NO: 289, and the second cytokine receptor domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 282 and a VL domain comprising the amino acid sequence of SEQ ID NO: 283 or VH domain comprising the amino acid sequence of SEQ ID NO: 284 and a VL domain comprising the amino acid sequence of SEQ ID NO: 285.16.
51. The pair of antigen binding molecules according to any one of claims 1 to 11, wherein the first and second cytokine receptor-binding domains are scFv molecules.
52. The pair of antigen binding molecules according to claim 51, wherein a) the first cytokine receptor-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 290 and a VL domain comprising the amino acid sequence of SEQ ID NO: 291, and the second cytokine receptor domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 292 and a VL domain comprising the amino acid sequence of SEQ ID NO: 293; or b) the first cytokine receptor-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 292 and a VL domain comprising the amino acid sequence of SEQ ID NO: 293, and the second cytokine receptor domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 290 and a VL domain comprising the amino acid sequence of SEQ ID NO: 291.
53. The pair of antigen binding molecules according to any one of claims 49 to 52, wherein the Fc domains of the first and second antigen binding molecules comprise a first Fc domain subunit and a second Fc domain subunit.
54. The pair of antigen binding molecules according to any one of claims 49 to 53, wherein the Fc domains of the first and second antigen binding molecules are IgG, particularly an IgGi, Fc domains.
55. The pair of antigen binding molecules according to any one of claims 49 to 54, wherein the Fc domains of the first and second antigen binding molecules are human Fc domains.
56. The pair of antigen binding molecules according to any one of claims 49 to 55, wherein the Fc domains of the first and second antigen binding molecules comprise a modification promoting the association of the first and the second subunit of the Fc domains.
57. The pair of antigen binding molecules according to any one of claims 49 to 56, wherein the Fc domains of the first and second antigen binding molecules comprise one or more amino acid substitution that reduces binding to an Fc receptor and/or effector function.
58. The pair of antigen binding molecules according to any one of claims 49 to 57, wherein the cytokine receptor-binding domains are fused via peptide linkers to their respective fusion points.
59. The pair of antigen binding molecules according to claim 58, wherein the peptide linkers comprise an amino acid sequence of SEQ ID NO: 280 or SEQ ID NO: 281.
60. The pair of antigen binding molecules according to claim 49, wherein, a) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 267, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 265 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 266, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 272, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 270 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 271; or b) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 267, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 265 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 266, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 274, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 270 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 273; or c) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 269, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 265 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 268, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 268, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 270 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 271; or d) the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 269, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 265 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 268, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 274, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 28, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 270 and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 273. 1. The pair of antigen binding molecules according to claim 51 , wherein the first antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 276, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 28 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 275, and the second antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 277, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 28 and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 278.
EP24714516.2A 2023-04-03 2024-04-02 Agonistic split antibodies Pending EP4688840A1 (en)

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