Integrated agonist antibody

Abstract

This application relates to an antigen-binding molecule comprising a pair of biparatopic target-binding domains, a pair of cytokine receptor-binding domains, and an Fc domain, wherein the target-binding domains simultaneously bind to the target antigen, and the cytokine receptor-binding domains bind to a subunit of the cytokine receptor complex. The biparatopic assembly of the cytokine receptor-binding domains in the presence of the target antigen enables selective activation of the cytokine receptor and effectively mimics cytokine activity in a targeted manner.

Description

[Technical Field]

[0001] This application relates to an antigen-binding molecule comprising a pair of biparatopic target-binding domains, a pair of cytokine receptor-binding domains, and an Fc domain, wherein the target-binding domains simultaneously bind to the target antigen, and the cytokine receptor-binding domains bind to a subunit of the cytokine receptor complex. Biparatopic binding of the cytokine receptor-binding domains in the presence of the target antigen allows for selective activation of the cytokine receptor, enabling effective mimicry of cytokine activity in a targeted manner. [Background technology]

[0002] In recent years, cancer immunotherapy has grown dramatically, and it is becoming a primary strategy for combating disease. For many cancer types, immune checkpoint modulators such as anti-PD-1 have become the standard treatment. However, despite all the advances made in the field of cancer immunotherapy in recent years, a significant proportion of patients still do not respond to available immunotherapies due to intrinsic or adaptive mechanisms of resistance. Cancer immunotherapy patients with non-inflammatory immune symptoms are more likely to not respond to immunotherapy. Immune cell infiltration in tumors has been shown to correlate with a patient's ability to respond to immunotherapy treatments. The development of new treatment methods aimed at increasing immune cell infiltration and enhancing immunogenicity is essential for patients.

[0003] Alongside the developments mentioned above, cytokines are attracting significant attention as a promising cancer treatment. IFNγ (interferon-gamma or IFN-γ) responds to immune or inflammatory stimuli, and CD4 + and CD8 +IFNγ is a cytokine primarily produced by activated lymphocytes such as T cells, as well as natural killer (NK) cells. IFNγ is a homodimer, and its receptors (IFNγR1 and IFNγR2) are expressed throughout hematopoietic and non-hematopoietic cells. On the cell surface, IFNγR1 is stably expressed, while IFNγR2 is differentially expressed, regulating IFNγ. When IFNγ binds to its receptor, Janus kinases JAK1 and JAK2 are recruited and activated, phosphorylating and activating STAT1. Phosphorylated STAT1 translocates to the nucleus, binds to promoters, and regulates gene transcription under the control of IFNγ.

[0004] In contrast to other cancer treatments currently on the market or under development, IFNγ can act on both tumor cells and immune cells, including T cells and dendritic cells. The effects of IFNγ on various cell types offer several advantages, including: 1) enhanced expression of MHC-I molecules on the surface of both tumor cells and antigen-presenting cells; 2) recruitment of immune cells to tumor sites by inducing CXCL9, CXCL10, and CXCL11 production; and 3) increased tumor antigen cross-presentation, subsequently enhancing the anti-tumor immune response. In addition to these effects, IFNγ promotes the formation of the Th1 environment, monocyte differentiation, macrophage polarization, and angiogenesis. Because the IFNγ receptor is expressed in a wide range of cell types, the sink effect can reduce its activity.

[0005] IFNγ can also exhibit undesirable side effects. Furthermore, problems may arise regarding administration, bioavailability, and short half-life. Therefore, there is a need for novel molecules that can selectively activate the IFNγ pathway at tumor sites.

[0006] Targeting the interleukin-2 (IL-2) pathway as a cancer immunotherapy strategy has a long and divergent history, which attests to both the advantages and complexities of this clinical approach. IL-2 is a cytokine that activates lymphocytes and natural killer (NK) cells. Reports of IL-2-related toxicity associated with peripheral T cell activity and CD25-mediated complications often cast a shadow over its clinical efficacy.

[0007] Cytokines are potent immunomodulators that initiate signal transduction via receptor dimerization, but natural cytokines have several limitations as therapeutic agents. Low stability and difficulties in manufacturing processes are just some of them. It has long been known that antibodies can induce signal transduction on cells and, consequently, replace natural ligands.

[0008] Recent literature provides growing evidence that combining heterodimeric cytokine receptors using both antibody- and non-antibody-based protein scaffolds is a viable strategy for mimicking the activity of native cytokines. For example, Moraga et al. provide an early example of using diabodies as surrogate ligands for the erythropoietin receptor (EpoR) (Moraga et al. Cell, 160, 1196-1208 (2015)). Researchers at Teneobio combined heavy-chain-only antibodies (VHH fused to the Fc domain) against different epitopes on interleukin-2 receptors (IL-2Rβ and IL-2Rγ) with bispecific effectorless IgG4Fc (CH1 deletion) using knob-into-hole technology. Monospecific anti-IL-2Rβ or anti-IL-2Rγ alone or in combination were used with human CD8 +Although it did not induce STAT5 phosphorylation on T cells, the bispecific anti-IL-2Rβγ antibody showed varying levels of agonist activity (Harris, KE, et al. Sci Rep 11(1):10592(2021)). An approach similar to that described by Teneobio was carried out by scientists at Synthekine, which described the functional induction of signaling of two interleukin receptors (IL-2Rβ and IL-2Rγ) via a single-domain antibody (sdAb, International Publication No. 2022 / 032040), and was reviewed by Saxton et al. (Saxton, RA, et al. Nat Rev Drug Discov. 22, 21-37 (2022)). This approach was further extended by researchers at Stanford University, where the authors presented a strategy for discovering cytokine surrogate agonists by using modular ligands such as VHH or scFv human for interleukin-2 / 15, type I interferon, and interleukin-10 receptors. Interestingly, they also identified functional non-native conjugates such as the IL-2Rβ / IL-10Rβ heterodimer (Yen, M. et al. Cell 185(8):1414-1430 e1419(2022)). The same authors also discuss a structure-based approach to decouple the pro-inflammatory and anti-inflammatory functions of interleukin-10 (Saxton, RA et al. Science 371(6535)(2021)). Two academic research groups from the Czech Republic and Israel jointly reported the discovery of non-antibody-based scaffolds mimicking the cytokine IFNλ. Using combinatorial libraries derived from several established small protein scaffolds, they identified variants capable of binding to IFNλR1 or IL-10Rβ and inducing functional signaling (Kolarova, L. et al. FEBS J 289(9):2672-2684(2022)). Researchers at Medikine tackled the challenge of reducing the size of the agonist module. They obtained molecules selected from a peptide library through a screening process designed to identify molecules that simultaneously bind to the Rα and γc subunits of the human IL-7 receptor.These peptides, fused to the IgG1-Fc domain and having a molecular weight of less than 5 kDa, exhibited similar biological properties to IL-7 in vitro when administered to non-human primates (Dower, W., et al. Journal for Immuno Therapy of Cancer 8(Suppl 3):A341-A342(2020)).

[0009] Due to the multifaceted effects of cytokines, there is a need for novel approaches that selectively activate cytokine receptors and effectively mimic cytokine activity. [Overview of the project]

[0010] The present invention relates to a novel antigen-binding molecule comprising a cytokine receptor-binding domain that acts as a cytokine mimetic, selectively activating the receptor pathway under desired conditions of biparatopic binding on a target antigen.

[0011] The present invention relates to an antigen-binding molecule comprising i) a first target-binding domain, ii) a second target-binding domain, iii) a first cytokine receptor-binding domain, iv) a second cytokine receptor-binding domain, and v) an Fc domain, wherein the first target-binding domain can bind to a first epitope on a target antigen, the second target-binding domain can bind to a second epitope on a target antigen, the first and second target-binding domains do not compete for binding to a tumor-associated antigen, the first cytokine receptor-binding domain can bind to a first cytokine receptor subunit, and the second cytokine receptor-binding domain can bind to a second cytokine receptor subunit.

[0012] In one embodiment, the first and second target-binding domains are antibody fragments, particularly Fv, Fab, scFv, scFab molecules, or single-domain antibodies. In one embodiment, the first and second target-binding domains are Fab molecules. In one embodiment, the first target-binding domain includes a heavy chain variable domain (VH1), a light chain variable domain (VL1), a heavy chain constant domain (CH11), and a light chain constant domain (CL1), and the second target-binding domain includes a heavy chain variable domain (VH2), a light chain variable domain (VL2), a heavy chain constant domain (CH12), and a light chain constant domain (CL2). In one embodiment, the first target-binding domain and / or the second target-binding domain are cross-Fab molecules. In one embodiment, the first target-binding domain and / or the second target-binding domain include charge mutations. In one embodiment, the first target-binding domain is cross-Fab and the second target-binding domain contains a charge mutation, or the second target-binding domain is cross-Fab and the first target-binding domain contains a charge mutation.

[0013] In one embodiment, the first target-binding domain and the second target-binding domain specifically bind to tumor-associated antigens or T-cell antigens.

[0014] In one embodiment, the first target-binding domain and the second target-binding domain specifically bind to FAP, PD-1, Her2, Her3, LAG-3, CEA, or EGFR. In one embodiment, a) the first target-binding domain includes VH1 of SEQ ID NO: 20 and VL1 of SEQ ID NO: 21, and the second target-binding domain includes VH2 of SEQ ID NO: 22 and VL2 of SEQ ID NO: 23, or b) the first target-binding domain includes VH1 of SEQ ID NO: 22 and VL1 of SEQ ID NO: 23, and the second target-binding domain includes VH2 of SEQ ID NO: 20 and VL2 of SEQ ID NO: 21, or c) the first target-binding domain includes VH1 of SEQ ID NO: 80 and VL1 of SEQ ID NO: 81, and the second target-binding domain includes VH2 of SEQ ID NO: 82 and VL2 of SEQ ID NO: 83 or d) the first target-binding domain includes VH1 of SEQ ID NO: 82 and VL1 of SEQ ID NO: 83, and the second target-binding domain includes VH2 of SEQ ID NO: 80 and VL2 of SEQ ID NO: 81; e) the first target-binding domain includes VH1 of SEQ ID NO: 82 and VL1 of SEQ ID NO: 83, and the second target-binding domain includes VH2 of SEQ ID NO: 140 and VL2 of SEQ ID NO: 141; or f) the first target-binding domain includes VH1 of SEQ ID NO: 140 and VL1 of SEQ ID NO: 141, and the second target-binding domain includes VH2 of SEQ ID NO: 82 and VL2 of SEQ ID NO: 83.

[0015] In one embodiment, both the first and second cytokine receptor subunits are subunits of an IFNγ receptor complex or an IL-2 receptor complex. In one embodiment, a) the first cytokine receptor binding domain can bind to IFNγR1 and the second cytokine receptor binding domain can bind to IFNγR2, or b) the first cytokine receptor binding domain can bind to IFNγR2 and the second cytokine receptor binding domain can bind to IFNγR1, or c) the first cytokine receptor binding domain can bind to IL-2Rβ and the second cytokine receptor binding domain can bind to IL-2Rγ, or d) the first cytokine receptor binding domain can bind to IL-2Rγ and the second cytokine receptor binding domain can bind to IL-2Rβ.

[0016] In one embodiment, the first and second cytokine receptor-binding domains are antibody fragments, particularly Fv, Fab, scFv, scFab, single-domain antibodies, or VHH domains. In another embodiment, the first and second cytokine receptor-binding domains are VHH domains. In one embodiment, a) the first cytokine receptor binding domain includes 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 includes an amino acid sequence selected from SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 9; b) the first cytokine receptor binding domain includes an amino acid sequence selected from SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 9, and the second cytokine receptor binding domain includes 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 includes the sequences of SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, and SEQ ID NO: 64, and the second cytokine receptor binding domain includes the sequences of SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, and SEQ ID NO: 69; or d) the first cytokine receptor binding domain includes the sequences of SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, and SEQ ID NO: 69, and the second cytokine receptor binding domain includes the sequences of SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, and SEQ ID NO: 64.

[0017] In one embodiment, the Fc domain comprises a first Fc domain subunit and a second Fc domain subunit. In one embodiment, the Fc domain is an IgG Fc domain, particularly an IgG1 Fc domain. In one embodiment, the Fc domain is a human Fc domain. 19. In one embodiment, the antigen-binding molecule according to any one of the preceding claims, wherein the Fc domain comprises modifications that facilitate the association of the first and second subunits of the Fc domain. In one embodiment, the Fc domain comprises one or more amino acid substitutions that reduce binding to and / or effector function of the Fc receptor.

[0018] In one embodiment, the first cytokine receptor binding domain is fused at its C-terminus to the N-terminus of VH1 or VL1 of the first target binding domain, the first target binding domain is fused at its N-terminus to the C-terminus of the first Fc domain subunit, 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, and the second target binding domain is fused at its C-terminus to the N-terminus of the second Fc domain subunit, CH12 or CL1.

[0019] In one embodiment, the antigen-binding molecule comprises, a) a first polypeptide comprising a first Fc domain subunit, VH1 and CH11 in order from the N-terminus to the C-terminus, a second polypeptide comprising a first cytokine receptor-binding domain, VL1 and CL1 in order from the N-terminus to the C-terminus, a third polypeptide comprising VL2, CH12 and a second Fc domain subunit in order from the N-terminus to the C-terminus, and a fourth polypeptide comprising a second cytokine receptor-binding domain, VH2 and CL2 in order from the N-terminus to the C-terminus, or b) from the N-terminus A first polypeptide comprising a first Fc domain subunit, VL1 and CH11 in order toward the C-terminus; a second polypeptide comprising a first cytokine receptor binding domain, VH1 and CL1 in order from the N-terminus toward the C-terminus; a third polypeptide comprising VH2, CH12 and a second Fc domain subunit in order from the N-terminus toward the C-terminus; and a fourth polypeptide comprising a second cytokine receptor binding domain, VL2 and CL2 in order from the N-terminus toward the C-terminus; or c) a first Fc domain subunit in order from the N-terminus toward the C-terminus A first polypeptide comprising a unit, VL1 and CL1, a second polypeptide comprising a first cytokine receptor binding domain, VH1 and CH11 in order from the N-terminus to the C-terminus, a third polypeptide comprising VL2, CH12 and a second Fc domain subunit in order from the N-terminus to the C-terminus, and a fourth polypeptide comprising a second cytokine receptor binding domain, VH2 and CL2 in order from the N-terminus to the C-terminus, or d) a first polypeptide comprising a first Fc domain subunit, VH1 and CL1 in order from the N-terminus to the C-terminus A peptide, a second polypeptide comprising a first cytokine receptor binding domain, VL1 and CH11 in order from the N-terminus to the C-terminus, a third polypeptide comprising VH2, CH12 and a second Fc domain subunit in order from the N-terminus to the C-terminus, and a fourth polypeptide comprising a second cytokine receptor binding domain, VL2 and CL2 in order from the N-terminus to the C-terminus, or e) a first polypeptide comprising a first Fc domain subunit, VH1 and CH11 in order from the N-terminus to the C-terminus,f) A first polypeptide comprising a first cytokine receptor binding domain, VL1 and CL1, a second polypeptide comprising a second cytokine receptor binding domain, VL2, CH12 and a second Fc domain subunit, and a fourth polypeptide comprising VH2 and CL2, in order from the N-terminus to the C-terminus, or g) A first polypeptide comprising a first Fc domain subunit, VL1 and CH11, a second polypeptide comprising a first cytokine receptor binding domain, VH1 and CL1, in order from the N-terminus to the C-terminus, a third polypeptide comprising a second cytokine receptor binding domain, VH2, CH12 and a second Fc domain subunit, and a fourth polypeptide comprising VL2 and CL2, in order from the N-terminus to the C-terminus, or g) A first polypeptide comprising a first Fc domain subunit, VL1 and CL1, in order from the N-terminus to the C-terminus, A second polypeptide comprising the cytokine receptor binding domain, VH1 and CH11, a third polypeptide comprising the second cytokine receptor binding domain, VL2, CH12 and a second Fc domain subunit, and a fourth polypeptide comprising VH2 and CL2, in order from the N-terminus to the C-terminus, or h) a first polypeptide comprising the first Fc domain subunit, VH1 and CL1, in order from the N-terminus to the C-terminus, The polypeptide comprises a second polypeptide containing a cytokine receptor binding domain, VL1, and CH11; a third polypeptide containing a second cytokine receptor binding domain, VH2, CH12, and a second Fc domain subunit, arranged sequentially from the N-terminus to the C-terminus; and a fourth polypeptide containing VL2 and CL2, arranged sequentially from the N-terminus to the C-terminus, where VH1, VL1, CH11, and CL1 form a first target binding domain, and VH2, VL2, CH12, and CL2 form a second target binding domain.

[0020] In one embodiment, the first target-binding domain and the second target-binding domain specifically bind to FAP, and the first and second cytokine receptor subunits are subunits of the IFNγ receptor complex.In one embodiment, the antigen-binding molecule comprises a) a first polypeptide containing the amino acid sequence of SEQ ID NO: 50, a second polypeptide containing the amino acid sequence of SEQ ID NO: 48, a third polypeptide containing the amino acid sequence of SEQ ID NO: 51, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 49, or b) a first polypeptide containing the amino acid sequence of SEQ ID NO: 99, a second polypeptide containing the amino acid sequence of SEQ ID NO: 97, a third polypeptide containing the amino acid sequence of SEQ ID NO: 100, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 98. c) containing the amino acid sequence of SEQ ID NO: 103, the second polypeptide containing the amino acid sequence of SEQ ID NO: 101, the third polypeptide containing the amino acid sequence of SEQ ID NO: 104, and the fourth polypeptide containing the amino acid sequence of SEQ ID NO: 102, or d) containing the amino acid sequence of SEQ ID NO: 107, the second polypeptide containing the amino acid sequence of SEQ ID NO: 105, the third polypeptide containing the amino acid sequence of SEQ ID NO: 108, and the fourth polypeptide containing the amino acid sequence of SEQ ID NO: 106. or, e) comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 99, a second polypeptide containing the amino acid sequence of SEQ ID NO: 109, a third polypeptide containing the amino acid sequence of SEQ ID NO: 100, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 110, or f) comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 107, a second polypeptide containing the amino acid sequence of SEQ ID NO: 111, a third polypeptide containing the amino acid sequence of SEQ ID NO: 113, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 112 or, g) comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 99, a second polypeptide containing the amino acid sequence of SEQ ID NO: 114, a third polypeptide containing the amino acid sequence of SEQ ID NO: 100, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 115, or h) comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 107, a second polypeptide containing the amino acid sequence of SEQ ID NO: 116, a third polypeptide containing the amino acid sequence of SEQ ID NO: 108, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 117.

[0021] In one aspect, the first target binding domain and the second target binding domain specifically bind to PD-1, and the first and second cytokine receptor subunits are subunits of the IL-2 receptor complex.

[0022] In one aspect, the antigen-binding molecule comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 123, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 122, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 124, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 125.

Brief Description of the Drawings

[0023] [Figure 1] Schematic diagram of antigens used for llama immunization, phage display, and screening for isolation of human IFNγR1, IFNγR2, IL-2Rβ, and IL-2Rγ-specific single-domain antibodies. For llama immunization, a heterodimer (A) formed from the extracellular domain (ECD) of human IFNγR1 fused to a biotinylated Fc knob and the human IFNγR2 ECD fused to the Fc hole, and a heterodimer (D) formed from the ECD of human IL-2Rβ fused to a biotinylated Fc knob and the human IL-2Rγ ECD fused to the Fc hole were prepared. For phage display, monovalent human IFNγR1 fused to biotinylated Fc (B), monovalent human IFNγR2 fused to biotinylated Fc (C), monovalent human IL-2Rβ fused to biotinylated Fc (E), monovalent human IL-2Rγ fused to biotinylated Fc (F), and soluble Fc (G) were prepared. [Figure 2] Workflow for enrichment of single-domain antibodies having 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 IFNγR1 specificity after three rounds of phage display was tested for binding to immobilized human IFNγR1-Fc and immobilized human Fc (A). VHH domains with human IFNγR2 specificity after three rounds of phage display were also tested for antigen specificity by ELISA (B). VHH domains selected for human IL-2Rβ specificity after three rounds of phage display were tested for binding to immobilized human IL-2Rβ-Fc and immobilized human Fc (C). VHH domains with human IL-2Rγ specificity after 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 graph. [Figure 4] Format conversion of single-domain antibody fragments. Third-round VHH library variants with IFNγR1 or IL-2Rβ specificity were fused to the Fc knob and cloned into a mammalian cell expression vector using the Gibson cloning method (A). Third-round VHH library variants with IFNγR2 or IL-2Rγ specificity were fused to the Fc hole using the same method (B). [Figure 5] Bispecific heavy-chain antibodies containing VHH domain pairs of IFNγR1-IFNγR2 or IL-2Rβ-IL-2Rγ. A VHH domain with IFNγR1 or IL-2Rβ specificity was fused to the Fc knob, and a VHH domain targeting IFNγR2 or IL-2Rγ was fused to the Fc hole. A flexible 5(G4S) linker was used to fuse the VHH portion to the Fc chain. Bispecific heavy-chain antibodies were generated by knob-into-hole binding of the Fc chains. The effector function of Fc was suppressed by incorporating a P329G LALA mutation into the CH2 domain. [Figure 6]Functional screening of bispecific heavy chain antibodies containing VHH domain pairs of IFNγR1-IFNγR2 or IL-2Rβ-IL-2Rγ. HEK-Blue IFNγ cells were incubated with IFNγ agonist bispecific heavy chain antibodies containing VHH domain pairs of IFNγR1 and IFNγR2, prepared using a 5×5 bispecific matrix (A). HEK-Blue IL-2 reporter cells were incubated with IL-2 agonist bispecific heavy chain antibodies containing VHH pairs of IL-2Rβ and IL-2Rγ, prepared using a 5×5 bispecific matrix (B). IFNγR activity and IL-2R activity were quantified by absorbance at 650 nm, and the response / background ratio is shown for each treatment. [Figure 7] Characterization of dose-dependent responses to IFNγ agonist heavy chain antibodies. Previously identified IFNγR agonists (Figure 6A, Table 1) were screened for dose-dependent IFNγR activity in HEK-Blue IFNγ cells characterized by absorbance at 650 nm. The responses of various IFNγ agonist heavy chain antibodies were grouped according to the anti-IFNγR1 VHH clone; that is, in each graph, the anti-IFNγR2 VHH clones differed, but the anti-IFNγR1 VHH clones remained unchanged as follows: IFNγR1_1 clone (A), IFNγR1_2 clone (B), IFNγR1_3 clone (C), and IFNγR1_5 clone (D). Responses were compared with recombinant human IFNγ (black dashed line) and FAP-IFNγ (P1AF3574, light gray dashed line). [Figure 8] Comparison of EC50 values ​​of IFNγ agonist heavy chain antibodies. EC50 values ​​were obtained from the HEK-Blue assay using GraphPad Prism software (Figure 7). Values ​​are shown in nanomolar concentrations, and recombinant human IFNγ is highlighted as a reference (dotted line). [Figure 9]Characterization of dose-dependent responses to IL-2R agonist heavy chain antibodies. Previously identified IL-2R agonists (Figure 6B, Table 2) were screened for dose-dependent IL-2R agonism in HEK-Blue IL-2 cells. Anti-PD1-IL2v (P1AE4422) was used as the reference molecule, and each molecule was incubated with reporter cells at the following concentrations: 20 nM, 0.8 nM, and 0.032 nM from left to right. IL-2R activity was quantified by absorbance at 650 nm, and the mean value from the technical triplicate is shown with error bars representing the standard deviation. [Figure 10] MHC-I and PD-L1 expression in tumor cells after 72 hours of treatment with IFNγ agonists. Previously identified IFNγR agonists (P1AH1877-P1AH1890) and reference molecules (recombinant human IFNγ and FAP-IFNγ) were incubated with tumor cells at the following treatment concentrations. For each treatment, concentrations of 100, 10, 1, and 0.1 nM are shown from left to right. Measurements were blanked out and normalized to the recombinant human IFNγ response at the highest concentration (100 nM). Responses for MHC-I expression on MKN45 cells (A), PD-L1 expression on MKN45 cells (B), 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) are shown. [Figure 11]Concept of a split-type dual-targeting IFNγR agonist. FAP-dependent biparatopic binding (A) of a split-type IFNγ mimetic. Note that for simplicity of explanation, only one monomer of the FAP dimer is shown in the inset. When a VHH domain with IFNγR1 and IFNγR2 specificity is fused to the N-terminus of two different anti-FAP binding domains via a VH fusion site or a VL fusion site, the following eight structures are generated in the substitution space. IFNγR1-VHH (B) fused to the VH of FAP Binder 1, IFNγR2-VHH (C) fused to the VH of FAP Binder 2, IFNγR1-VHH (D) fused to the VL of FAP Binder 1, IFNγR2-VHH (E) fused to the VL of FAP Binder 2, IFNγR2-VHH (F) fused to the VH of FAP Binder 1, IFNγR1-VHH (G) fused to the VH of FAP Binder 2, IFNγR2-VHH (H) fused to the VL of FAP Binder 1, and IFNγR1-VHH (I) fused to the VL of FAP Binder 2. All molecules have the same Fc properties as shown in Figure 5. [Figure 12] Evaluation of IFNγR activity mediated by FAP-dependent slit-type IFNγR agonists. Eight slit-type IFNγ mimes (Figures 11B-11I) were paired in biparatopic binding combinations and tested for IFNγR agonism characterized by upregulation of MHC-I and PD-L1. FAP-negative A549 cells were tested with slit-type IFNγ mimes for untargeted, nonspecific upregulation of MHC-I (A, B) and PD-L1 (C, D). A549 FAP-positive cells were co-cultured with differentially labeled A549 FAP-negative cells to evaluate the FAP-dependent activity of the following cis and trans IFNγ mimes: upregulation of MHC-I in cis (E and F), upregulation of PD-L1 in cis (G and H), upregulation of MHC-I in trans (I and J), and upregulation of PD-L1 in trans (K and L). (As shown in Figure 5) Recombinant IFNγ and the IFNγ agonist heavy chain antibody P1AH1884 were used as references. The median fluorescence intensity (MFI) of MHC-I and PD-L1 expression levels was analyzed using FlowJo, and the mean values ​​from technical doubles are shown along with error bars representing the standard deviation. [Figure 13] Further formats of split-type dual-targeting IFNγR agonists. By fusing the VHH domain, which has IFNγR1 and IFNγR2 specificity, to the N-terminus of the opposite Fc chain, away from the anti-FAP binder, the following four structures were generated: IFNγR1 and FAP binder 1 (A), IFNγR2 and FAP binder 2 (B), IFNγR2 and FAP binder 1 (C), and IFNγR1 and FAP binder 2 (D). Furthermore, by fusing the VHH domain, which has IFNγR1 and IFNγR2 specificity, to the C-terminus of the same Fc chain, away from the anti-FAP binder, the following four structures were generated. IFNγR1 and FAP binder 1(E), IFNγR2 and FAP binder 2(F), IFNγR2 and FAP binder 1(G), and IFNγR1 and FAP binder 2(H). The anti-FAP binders in Figures 13E to 13H include CrossFab VH / VL operation (binder 1) and CH1-CL charge (binder 2), which then allow for further pair formation. All molecules have the same Fc properties as described in Figure 5. [Figure 14] Further evaluation of IFNγR activity mediated by FAP-dependent splintered IFNγR agonists. Four combinations of molecules and reference molecules shown in Figure 13 were tested for IFNγR agonism characterized by upregulation of MHC-I and PD-L1. A549 cells lacking FAP expression were tested for FAP-independent upregulation of MHC-I (A) and upregulation of PD-L1 (B) by splintered IFNγ mimetic cells. A549 FAP-positive cells were co-cultured with differentially labeled A549 FAP-negative cells, and the FAP-dependent activity of IFNγ mimetic cells was evaluated by upregulation of cis MHC-I (C), cis PD-L1 (D), trans MHC-I (E), and trans PD-L1 (F). Recombinant IFNγ and IFNγ agonist heavy chain antibody (P1AH1884) were used as reference molecules. The median fluorescence intensity (MFI) of MHC-I and PD-L1 expression levels was analyzed using FlowJo, and the mean values ​​from the technical double-digit sequence are shown along with error bars representing the standard deviation. [Figure 15]Concept of a fragmented, dual-targeting IL-2 agonist. PD-1-dependent biparatopic binding of a fragmented IL-2 mimetic (A). VHH domains with IL-2Rβ and IL-2Rγ specificity were fused to the N-terminus of two different anti-PD-1 binding domains via the following VH or VL fusion sites: IL-2Rβ-VHH fused to the VH of PD-1 binder 1 (B), IL-2Rγ-VHH fused to the VL of PD-1 binder 2 (C), IL-2Rβ-VHH fused to the VL of PD-1 binder 1 (D), and IL-2Rγ-VHH fused to the VH of PD-1 binder 2 (E). IL-2 agonist heavy chain antibody (F) and PD-1-IL2v (G) functioned as reference molecules. All molecules have the same Fc properties as shown in Figure 5. [Figure 16] Phosphorylation of STAT5 on CD4 T cells after incubation with an IL-2R agonist for 15 minutes and 60 minutes. Activated T cells expressing PD-1 (PD-1+ subset) and preblocking activated T cells (PD-1- subset) were differentially labeled with an anti-PD-1 antibody and treated with two combinations of fragmented dual-targeted IL-2 mimics: P1AH 6850 + P1AH6813 (AD) and P1AH 6814 + P1AI1593 (EH). IL-2 mimic heavy chain antibody (P1AH1177) and PD1-IL2v (P1AE4422) were used as reference molecules. MFI and frequency of STAT5-P+ cells were measured by FACS, showing responses for the PD-1+ subset (solid line) and PD-1- subset (dashed line). [Figure 17]The concept of a fragmented PD-1-targeted IL-2R agonist using an alternative anti-PD-1 Fab binder. When VHH domains with IL-2Rβ and IL-2Rγ specificity are fused to the N-terminus of two different anti-PD-1 binding domains via a VH fusion site or a VL fusion site, eight distinct format structures (P1AK2599, P1AK2798, P1AK2799, P1AK2802, P1AK2803, P1AK2806, P1AK2809, P1AK2810) are generated, which can pair up to produce the following eight functional bindings. P1AK2802+P1AK2599(A), P1AK2809+P1AK2799(B), P1AK2803+P1AK2806(C), P1AK2810+P1AK2798(D), P1AK2809+P1AK2599(E), P1AK2802 P1AK2799(F), P1AK2810+P1AK2806(G) and P1AK2803+P1AK2798(H). [Figure 18] Functional activity of PD-1-targeted IL-2R agonists using 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) at 37°C and 5% CO2 for 21 hours. PD1-IL2v (P1AE4422) and Fc-VHH (P1AH1177) were used as references. Unbound DP47 antibody (P1AD3966) was used as a negative control. IL-2R signaling was measured by absorbance at 650 nm using QUANTI-Blue reagent. The mean absorbance values ​​+ / - SEM of the technical double series for each molecular concentration tested are shown. [Figure 19]Schematic diagram of the "integrated" format design. A pair of biparatopically separated IFNγ mimetic molecules (P1AI0831 and P1AI0066) exhibiting both potent cis and trans activity were formatted into a single-molecule format to test for FAP-specific activity. One of the anti-FAP binding domains was transplanted to the C-terminus of Fc to achieve sufficient intermolecular spacing between IFNγ mimetic VHH pairs to avoid FAP-independent IFNγR activation. CrossFab VH / VL manipulation and CH1-CL charge were incorporated to generate a tetraspecific format (P1AI5012), in which Fc exhibits the same properties as those described in Figure 5. [Figure 20] Evaluation of FAP-specific IFNγR activity mediated by an "integrated" IFNγ mimite. FAP-negative A549 cells were tested for upregulation of untargeted, nonspecific MHC-I (A) and PD-L1 (B) by an "integrated" IFNγ mimite (P1AI5012). A549 FAP-positive cells were co-cultured with differentially labeled A549 FAP-negative cells and evaluated for upregulation of FAP-dependent MHC-I (C), cis PD-L1 (D), trans MHC-I (E), and trans PD-L1 (F) by cis IFNγ mimite. Recombinant IFNγ and the IFNγ agonist heavy chain antibody P1AH1884 were used as references. Median fluorescence intensity (MFI) of MHC-I and PD-L1 expression was analyzed by FlowJo, and the mean values ​​from technical doubles are shown with error bars representing the standard deviation. [Figure 21] Schematic diagram of further formats of the "integrated" molecule. Variants of the integrated molecule P1AI5012 (A) differ in the agonist VHH pair, the position of each VHH on the VH or VL of the Fab domain, and the different positions of the FAP binder. P1AJ0690 (B), P1AJ0672 (C), P1AJ0700 (D), P1AJ0685 (E), P1AJ0744 (F), P1AJ0735 (G), and P1AJ0747 (H). [Figure 22]MHC-I expression in tumor cells after 72 hours of treatment with IFNγ agonists. Previously identified IFNγR agonists (P1AI5012, P1AJ0690, P1AJ0672, P1AJ0685, P1AJ0700, P1AJ0747, P1AJ0744, and P1AJ0735) and a reference molecule (recombinant human IFNγ) were incubated with tumor cells. A549 FAP-positive cells were co-cultured with differentially labeled A549 FAP-negative cells to evaluate the FAP-dependent activity of the following cis and trans IFNγ mimes: upregulation of MHC-I in cis (A) and upregulation of MHC-I in trans (B). Median fluorescence intensity (MFI) of MHC-I expression levels was analyzed by FlowJo, and the mean value from technical doubles is shown with error bars representing the standard deviation. [Figure 23] Schematic diagram of the "integrated" format design. A pair of biparatopically fragmented IL-2 mimetic molecules (P1AI1593 and P1AH6814) with potent PD1-mediated cis-activity were formatted into a single-molecule format (P1AI5057) to test for PD1 restriction activity. A pair of anti-PD1 binding domains and anti-IL2Rβ binding domains were transplanted to the C-terminus of Fc to achieve sufficient intermolecular spacing between IL-2 mimetic VHH pairs to reduce PD1-independent IL-2R activation. To create a tetraspecific format, crossFab VH / VL manipulation and CH1-CL charge were incorporated into the Fab domain, and a knob-into-hole mutation was introduced into Fc. The effector function of Fc was suppressed by incorporating the P329G LALA mutation into the CH2 domain. [Figure 24]Functional activity of integrated PD-1-targeted IL2R agonists. Test compounds were incubated with HEK Blue IL-2 or HEK Blue IL-2 human PD-1 cells at 37°C and 5% CO2 for 20 hours. PD1-IL2v (P1AE4422) and Fc-VHH (P1AH1177) were used as references. Unbound DP47 antibody (P1AD3966) was used as a negative control. IL2R signaling was measured by absorbance at 650 nm using QUANTI-Blue reagent. The average absorbance values ​​+ / - SEM for each molecular concentration tested are shown. [Figure 25] Concept of a PD-1-targeted IL-2R agonist having monoparatopic binding to PD-1. A VHH domain with IL-2Rβ and IL-2Rγ specificity was fused to the N-terminus of two anti-PD1 binding domains having the same paratope via a VH or VL fusion site. P1AM4293(A) contains two anti-PD1 binding domains (PD1 binder 1, 0376 binder) having the FV000363 paratope, and P1AM4294(B) contains two anti-PD1 binding domains (PD1 binder 2, 1040 binder) having the FV003451 paratope. [Figure 26] Functional activity of monoparatopic PD-1-targeted IL-2R agonists versus biparatopic PD-1-targeted IL-2R agonists. Test compounds were incubated with HEK Blue IL-2 or HEK Blue IL-2 human PD-1 cells at 37°C and 5% CO2 for 18 hours. Fc-VHH (P1AH1177) was used as a reference. Unbound DP47 antibody (P1AD3966) was used as a negative control. IL-2R signaling was measured by absorbance at 650 nm using QUANTI-Blue reagent. The fold change in IL-2R signaling relative to technical dual background + / - SEM for each molecular concentration tested is shown. [Modes for carrying out the invention]

[0024] definition Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have meanings generally understood by those skilled in the art. Furthermore, unless otherwise specified in the context, singular terms shall include plural forms, and plural terms shall include singular forms. The methods and techniques described herein are generally carried out in accordance with conventional methods well known in the art. Generally, the nomenclature and techniques used herein in connection with biochemistry, enzymology, molecular biology, and cell biology, microbiology, genetics, and protein and nucleic acid chemistry, as well as hybridization, are well known and commonly used in the art.

[0025] The terms "a," "an," and "the" generally refer to multiple objects unless the context explicitly indicates otherwise.

[0026] As used herein, the terms “first,” “second,” “third,” or “fourth” with respect to binding molecules, epitopes, polypeptides, etc., are used for convenience to distinguish between two or more parts of each type. The use of these terms is not intended to assign a particular order or orientation to the parts unless expressly stated otherwise.

[0027] As used herein, the term “antigen-binding molecule” refers to a polypeptide molecule (composed of one or more polypeptide chains) that can bind to an antigen. The binding molecule may originate from an antibody and typically contains an antigen-binding domain.

[0028] The term "antibody" as used herein is used in its broadest sense and is not limited to any antibody that exhibits the desired antigen-binding activity, but it encompasses a variety of antibody structures, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), heavy chain antibodies, antibody fragments, and antigen-binding molecules.

[0029] As used herein, the terms “heavy chain antibody,” “heavy chain-only antibody,” and “HCAb” refer to antibodies lacking a light chain.

[0030] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably in this specification to refer to antibodies that have a structure substantially similar to that of a natural antibody.

[0031] An "antibody fragment" refers to a molecule other than an intact antibody, including a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments, though not limited to them, include 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 an overview of specific antibody fragments, see Hollinger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0032] A "single-domain antibody" refers to an antibody fragment consisting of a single monomeric antibody variable domain, such as a VHH, nanobody, shark-derived VNAR, autonomous VH domain, or autonomous VL domain. A single-domain antibody provides an antigen-binding site that specifically binds to an epitope; that is, the antigen-binding site is formed only by a single-domain antibody.

[0033] "VHH," "VHH domain," or "nanobody" refers to a single-domain antibody derived from the variable domain of heavy chain antibodies from camelid animals such as camels, dromedaries, llamas, and alpacas (see Nguyen VKet al., 2000, The EMBO Journal, 19, 921-930; Muyldermans S., 2001, J Biotechnol., 74, 277-302; and for a review, Vanlandschoot P. et., 2011, Antiviral Research 92, 389-407). The antigen-binding site of VHH lacks a light chain variable domain. The VHH domain can be humanized.

[0034] As used herein, “antigen-binding domain” refers to a portion that specifically binds to a target antigen. The term particularly refers to the antigen-binding domain of an antibody, i.e., the portion that binds to part or all of an antigen and is complementary to part or all of the antigen. Therefore, in certain embodiments, the antigen-binding domain as used herein is the antigen-binding domain of an antibody. Such an antigen-binding domain may be provided by an antibody fragment, for example, a Fab molecule, a single-chain antibody molecule, or a single-domain antibody, such as a VHH domain.

[0035] A "variable region" or "variable domain" is a domain in the heavy or light chain of an antibody that is involved in antibody-antigen binding. This term includes the VHH domain. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, and each domain contains four conserved framework regions (FRs) and complementarity-determining regions (CDRs). See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman & Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen can be isolated using the VH or VL domain of the antibody that binds to that antigen, and complementary libraries of VL or VH domains can be screened, respectively. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993) and Clarkson et al., Nature 352:624-628 (1991). Where used herein in relation to variable region sequences, “Kabat numbering” refers to the numbering system described by Kabat et al. Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0036] As used herein, the amino acid positions of all constant regions and domains in the heavy and light chains 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), and are referred to herein as "Kabat numbering" or "Kabat numbering."

[0037] Specifically, the Kabat numbering system (see pp. 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 isotypes, while the Kabat EU index numbering system (see pp. 661-723) is used for the heavy chain constant domains (CH1, hinge, CH2, and CH3). The Kabat EU index numbering system is further clarified herein by the terms "Kabat EU index numbering" or "Kabat EU index numbering."

[0038] As used herein, the terms “binding site” or “antigen-binding site” refer to a site on a binding molecule that provides interaction with an antigen, i.e., one or more amino acid residues. For example, the antigen-binding site of an antigen-binding domain includes amino acid residues from a complementarity-determining region (CDR). The antigen-binding site may also be provided by, for example, one or more variable domains (also called variable regions). In a single-domain antibody, the antigen-binding site is provided by a single variable domain. In contrast, in a Fab fragment, the antigen-binding site is provided by VH and VL domains.

[0039] As used herein, the terms “hypervariable region” or “HVR” refer to each of the regions of the antigen-binding domain, such as “complementarity-determining regions” (CDRs), that are hypervariable within the sequence and determine antigen-binding specificity. Generally, a variable domain contains three CDRs. Therefore, antibodies containing VH and VL contain six CDRs: three in VH (HCDR1, HCDR2, HCDR3) and three in VL (LCDR1, LCDR2, LCDR3). Exemplary CDRs as used herein include: (a) Hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 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 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 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

[0040] (c) Antigen contact occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J.Mol.Biol.262:732-745 (1996)).

[0041] Unless otherwise indicated, CDRs are determined according to Kabat et al. above. Those skilled in the art will understand that CDR designations may also be determined according to Chothia, McCallum, or other scientifically recognized nomenclature.

[0042] "Framework" or "FR" refers to variable domain residues other than the complementarity-determining region (CDR). The variable domain FR generally consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, HVR and FR sequences generally appear in the following order in the 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.

[0043] In this specification, the term “immunoglobulin molecule” refers to a protein having the structure of a naturally occurring antibody. For example, IgG-class immunoglobulins are heterotetrameric glycoproteins with a weight of approximately 150,000 daltons, composed of two disulfide-linked light chains and two heavy chains. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy chain domain or heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3), also called heavy chain constant regions. Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL), also called a variable light chain domain or light chain variable region, followed by a constant light chain (CL) domain, also called a light chain constant region. The heavy chain of an immunoglobulin may be assigned to one of five types called α(IgA), δ(IgD), ε(IgE), γ(IgG), or μ(IgM), some of which may be further divided into subtypes such as γ1(IgG1), γ2(IgG2), γ3(IgG3), γ4(IgG4), α1(IgA1), and α2(IgA2). The light chain of an immunoglobulin may be assigned to one of two types called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain. An immunoglobulin essentially consists of two Fab molecules and an Fc domain, linked via an immunoglobulin hinge region.

[0044] The "class" of an antibody or immunoglobulin refers to the type of constant domain or constant region present in the heavy chain of the antibody or immunoglobulin. Antibodies have five main classes: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0045] "Fab molecule," "Fab," or "Fab fragment" refers to a protein consisting of the VH domain and CH1 domain of the immunoglobulin heavy chain ("Fab heavy chain") and the VL domain and CL domain of the immunoglobulin light chain ("Fab light chain").

[0046] A "cross-Fab molecule," "cross-Fab," or "crossover Fab molecule" refers to a Fab molecule in which either the variable or constant regions of the heavy and light chains are exchanged. The cross-Fab operation allows for two different chain compositions of a cross-Fab molecule. In one case, the variable regions of the heavy and light chains of Fab are exchanged, meaning the cross-Fab molecule includes a peptide chain consisting of a light chain variable region (VL) and a heavy chain constant region (CH1), where CH1 may be fused to an Fc domain, and a peptide chain consisting of a heavy chain variable region (VH) and a light chain constant region (CL). In another case, where the heavy and light chain constant regions of Fab are exchanged, the cross-Fab molecule includes a peptide chain consisting of a heavy chain variable region (VH) and a light chain constant region (CL), where CL may be fused to an Fc domain, and a peptide chain consisting of a light chain variable region (VL) and a heavy chain constant region (CH1).

[0047] The term "conventional Fab molecule" refers to a Fab molecule composed of a Fab heavy chain containing VH and CH1 domains and a Fab light chain containing VL and CL domains.

[0048] The term "multispecificity" means that a binding molecule (e.g., an antibody) can specifically bind to at least two different antigens. A multispecific binding molecule (e.g., an antibody) may be, for example, a bispecific binding molecule. Typically, a bispecific binding molecule contains two antigen-binding sites, each of which is specific to a different antigen. In certain embodiments, a multispecific (e.g., bispecific) binding molecule can simultaneously bind to two antigens, particularly two antigens expressed on the same cell, adjacent cells, or cells within the same tissue.

[0049] As used herein, the term "valence" indicates the presence of a specific number of antigen-binding sites within the binding molecule. In this case, the term "monovalent binding to antigen" indicates the presence of one (and not more than one) antigen-specific antigen-binding sites within the binding molecule.

[0050] As used herein, the term “antigen” refers to a molecule, such as a protein, to which an antigen-binding molecule binds. Useful antigens may be found, for example, on the surface of tumor cells, on the surface of tumor stromal cells, on the surface of virus-infected cells, on the surface of other diseased cells, on the surface of immune cells, free in serum, and / or in the extracellular matrix (ECM). In certain embodiments, the antigen is a human protein.

[0051] 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 stretch of consecutive amino acids or conformations consisting of different regions of discontinuous amino acids. Epitopes often contain chemically active surface groups of the antigen, such as amino acids, glycan side chains, phosphoryl or sulfonyl groups, and may have specific three-dimensional structural properties and / or specific charge properties. Two different antigen-binding domains capable of binding to the same antigen may bind to different epitopes of the antigen. Such different antigen-binding domains are said not to compete for binding if both antigen-binding domains can bind to the antigen simultaneously. In this case, the two different antigen-binding domains are non-competitive. Alternatively, the antigen-binding domains may compete for binding, i.e., exhibit competitive binding. This may result from partially overlapping epitopes or from steric hindrance preventing simultaneous binding of the two antigen-binding domains. Assays for determining whether an antigen-binding domain exhibits non-competitive or competitive binding are well known in the art and include competitive binding analyses using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

[0052] As used herein, “target antigen” refers to an antigen presented on the surface of a target cell, such as an antigen on a cell in a tumor, such as a cancer cell or a cell in the tumor stromal tissue, or an antigen on a T cell.

[0053] As used herein, “tumor-associated antigen” refers to any antigen presented on the surface of cells in a tumor, such as cancer cells or cells of the tumor stroma. Specific tumor-associated antigens include CEA, FAP, Her2, Her3, or EGFR.

[0054] The term “fibroblast-activating protein (FAP),” also known as prolyl endopeptidase FAP or seplacase (EC3.4.21), means, unless otherwise indicated, any native form of FAP derived 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). FAP is expressed on cancer-associated fibroblasts (CAFs) in tumor stroma. The term encompasses not only “full-length,” untreated FAP, but also any form of FAP resulting from intracellular processing. The term also encompasses naturally occurring variants of FAP, such as splice variants or allele variants. In one embodiment, the antigen-binding molecule of the present invention can specifically bind to human, mouse, and / or cynomolgus monkey FAP. The amino acid sequence of human FAP is shown in UniProt (www.uniprot.org) accession number Q12884 (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 number 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, the anti-FAP binding molecule binds to the extracellular domain of FAP. Exemplary anti-FAP binding molecules are described in International Publication No. 2012 / 020006 and International Publication No. 2020 / 070041.

[0055] The term "carcinoembryonic antigen (CEA)," also known as carcinoembryonic antigen-associated cell adhesion molecule 5 (CEACAM5), refers, unless otherwise indicated, to any native CEA derived 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). The amino acid sequence of human CEA is shown in UniProt accession number P06731 (version 151). CEA has long been identified as a tumor-associated antigen (Gold and Freedman, J Exp Med., 121:439-462, 1965; Berinstein NL, J Clin Oncol., 20:2197-2207, 2002). Originally classified as a protein expressed only in fetal tissues, CEA has now been identified in several healthy adult tissues. These tissues are primarily epithelial in origin, including cells of the gastrointestinal, respiratory, and urogenital tracts, as well as cells of the colon, cervix, sweat glands, and prostate (Nap et al., Tumor Biol., 9(2-3):145-53, 1988; Nap et al., Cancer Res., 52(8):2329-23339, 1992). Epithelial tumors and their metastases contain CEA as a tumor-associated antigen. While the presence of CEA itself does not necessarily mean transformation into cancerous cells, the distribution of CEA suggests this. In normal tissues, CEA is generally expressed on the apical surface of cells (Hammarstrom S., Semin Cancer Biol. 9(2):67-81(1999)), preventing it from reaching antibodies in the bloodstream. In contrast to normal tissue, CEA tends to be expressed across the entire surface of cancer cells (Hammarstrom S., Semin Cancer Biol. 9(2):67-81 (1999)). This change in expression pattern makes CEA more likely to bind to antibodies within cancer cells. Furthermore, CEA expression is increased in cancer cells. In addition, increased CEA expression promotes increased cell-cell adhesion, 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 line with published data, independent analyses performed on tissue samples confirmed this high prevalence: approximately 95% in colorectal cancer (CRC), 90% in pancreatic cancer, 80% in gastric cancer, 60% in non-small cell lung cancer (NSCLC, co-expressed with HER3), and 40% in breast cancer. Lower expression rates were observed in small cell lung cancer and glioblastoma.

[0056] "HER2" (also known as erbB-2 or CD340) refers, unless otherwise indicated, to any native HER2 derived from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus macaques), and rodents (e.g., mice and rats). The term encompasses "full-length" unprocessed HER2 and any form of HER2 resulting from intracellular processing. The term also encompasses naturally occurring variants of HER2, such as splice variants or allele variants. In one embodiment, HER2 is human HER2. The amino acid sequence of human HER2 is shown in UniProt (www.uniprot.org) entry number Q9UK79 (version 95).

[0057] The epidermal growth factor receptor (EGFR), also known as the proto-oncogene c-ErbB-1 or receptor tyrosine-protein kinase ErbB-1, refers, unless otherwise indicated, to any native EGFR derived 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). The amino acid sequence of human EGFR is shown in UniProt accession number P00533.

[0058] As used herein, “T cell antigen” refers to any antigen present on the surface of a T lymphocyte.

[0059] The term "PD-1," also known as CD279, PD1, or programmed cell death protein 1, refers to any native PD-1 derived 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 human protein PD-1 having the amino acid sequence shown in UniProt (www.uniprot.org) accession number Q15116.

[0060] The terms “interferon-gamma,” “IFNγ,” or “IFN-γ,” as used herein, refer to any native form of IFNγ derived from any vertebrate source, including mammals, e.g., primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The terms encompass both untreated IFNγ and any form of IFNγ resulting from cell processing. The terms also encompass naturally occurring variants of IFNγ, e.g., splice variants or allele variants.

[0061] When used herein, the terms “interleukin-2” or “IL-2” refer to any native form of IL-2 derived from any vertebrate source, including mammals, e.g., primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The terms encompass both untreated IL-2 and any form of IL-2 resulting from cell processing. The terms also encompass naturally occurring variants of IL-2, e.g., splice variants or allele variants.

[0062] Cytokine receptors are cell surface glycoproteins that specifically bind to cytokines and transmit their signals. Generally, cytokine receptors function as oligomeric complexes consisting of 2 to 4 receptor chains, also called subunits, which may be the same or different. Therefore, the term “cytokine receptor complex” refers to a cytokine receptor composed of at least two subunits.

[0063] The IFNγ receptor complex comprises the IFNγR1 subunit and the IFNγR2 subunit. The terms “interferon gamma receptor 1” or “IFNγR1,” also known as CD119 (differentiation cluster 119) or interferon gamma receptor α chain (IFNγRα), refer, unless otherwise indicated, to any native IFNγR1 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). This term includes not only “full-length” untreated IFNγR1 but also any form of IFNγR1 resulting from intracellular processing. This term also encompasses naturally occurring variants of IFNγR1, such as splice variants or allele variants. In certain embodiments, IFNγR1 is human IFNγR1.

[0064] The term “interferon-gamma receptor 2” or “IFNγR2” is also called interferon-gamma receptor β chain (IFNγRβ) and, unless otherwise indicated, refers to any native IFNγR2 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). This term includes not only “full-length” untreated IFNγR2 but also any form of IFNγR2 resulting from intracellular processing. This term also encompasses naturally occurring variants of IFNγR2, such as splice variants or allele variants. In certain embodiments, IFNγR2 is human IFNγR2.

[0065] As used herein, the terms “IL-2Rα” or “the α-subunit of the IL-2 receptor,” also known as CD25, refer to any native form of IL-2Rα derived 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” untreated IL-2Rα and any form of IL-2Rα resulting from intracellular processing. The term also encompasses naturally occurring variants of IL-2Rα, such as splice variants or allele variants. In certain embodiments, IL-2Rα is human IL-2Rα.

[0066] The term “IL-2Rβ” or “the β-subunit of the IL-2 receptor,” also known as CD122 or p70, refers, unless otherwise indicated, to any native form of IL-2Rβ from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses “full-length” untreated IL-2Rβ and any form of IL-2Rβ resulting from intracellular processing. The term also encompasses naturally occurring variants of IL-2Rβ, such as splice variants or allele variants. In certain embodiments, IL-2Rβ is human IL-2Rβ.

[0067] The term “IL-2Rγ” or “the γ subunit of the IL-2 receptor,” also known as the common cytokine receptor γ subunit, common γ chain, γc, or CD132, refers, unless otherwise indicated, to any native IL-2Rγ derived from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses “full-length” untreated IL-2Rγ and any form of IL-2Rγ resulting from intracellular processing. The term also encompasses naturally occurring variants of IL-2Rγ, such as splice variants or allele variants. In certain embodiments, IL-2Rγ is human IL-2Rγ.

[0068] Different associations of individual IL-2R subunits, IL-2Rα, IL-2Rβ, and IL-2Rγ, can produce three IL-2R forms with different affinities to IL-2. High-affinity IL-2R refers to the heterotrimer form of IL-2R consisting of IL-2Rα, IL-2Rβ, and IL-2Rγ. Intermediate-affinity IL-2R refers to the heterodimer form of IL-2R consisting of IL-2Rβ and IL-2Rγ. On the other hand, low-affinity IL-2R refers to the monomer form of IL-2R consisting of IL-2Rα alone (for a review, see, for example, Olejniczak and Kasprzak, Med Sci Monit 14, RA179-189 (2008)).

[0069] As used herein, the term “IL-2 receptor complex” refers to either a high-affinity IL-2 receptor or an intermediate-affinity IL-2 receptor.

[0070] In this specification, the terms “Fc domain” or “Fc region” are used to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. This term includes the native sequence Fc region and the variant Fc region. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more amino acids from the C-terminus of the heavy chain, particularly one or two amino acids. Thus, by expression of certain nucleic acid molecules encoding a full-length heavy chain, antibodies produced by host cells may contain the full-length heavy chain or cleaved variants of the full-length heavy chain. This is the case when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbered according to the Kabat EU index). Therefore, the C-terminal lysine (Lys447) or C-terminal glycine (Gly446) and lysine (Lys447) of the Fc region may or may not be present. In one embodiment, the heavy chain containing the Fc region (subunit) specified herein, which is included in the binding molecule according to the present invention, includes an additional C-terminal lysine (K447, numbering according to the Kabat EU index). In one embodiment, the heavy chain containing the Fc region (subunit) specified herein, which is included in the binding molecule according to the present invention, includes a C-terminal glycine residue (G446, numbering according to the Kabat EU index) but does not include a C-terminal lysine (Lys447). Unless otherwise specified herein, the numbering of amino acid residues within the Fc region or constant region follows the EU numbering system, also known as 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).As used herein, a “subunit” of an Fc domain refers to one of the two polypeptides that form a dimeric Fc domain, i.e., a polypeptide containing the C-terminal constant region of an immunoglobulin heavy chain capable of stable self-assembly. For example, a subunit of an IgG Fc domain includes the IgG CH2 and IgG CH3 constant domains.

[0071] "Modifications that promote the association of the first and second subunits of the Fc domain" are manipulations of the peptide backbone or post-translational modifications of the Fc domain subunits that reduce or prevent the association of a polypeptide containing an Fc domain subunit with an identical polypeptide for homodimer formation. As used herein, association-promoting modifications preferably involve distinct modifications made to each of two Fc domain subunits that are desired to associate (i.e., the first and second subunits of the Fc domain), and these modifications are complementary to each other in order to promote the association of the two Fc domain subunits. For example, the association-promoting modifications may alter the structure or charge of one or both of the Fc domain subunits so that they perform a sterically or electrostatically desired association. Thus, (hetero)dimerization occurs between a polypeptide containing a first Fc domain subunit and a polypeptide containing a second Fc domain subunit, which may not be identical in the sense that the further components fused to each subunit (e.g., antigen-binding domains) are not the same. In one embodiment, the modification that facilitates the association of the first and second subunits of the Fc domain includes an amino acid mutation, specifically an amino acid substitution, within the Fc domain. In a particular embodiment, the modification that facilitates the association of the first and second subunits of the Fc domain includes separate amino acid mutations, specifically amino acid substitutions, in each of the two subunits of the Fc domain.

[0072] One heterodimerization technique known in the art is the so-called "knob-into-hole" technique, which is described in detail with several examples, for example, in International Publication No. 96 / 027011, Ridgway, JB, et al., Protein Eng. 9 (1996) 617-621; Merchant, AM, et al., Nat. Biotechnol. 16 (1998) 677-681, and International Publication No. 98 / 050431. In the "knob-into-hole" technique, specific amino acids on each CH3 domain are manipulated to create a projection ("knob") on one CH3 domain and a cavity ("hole") on the other CH3 domain within the interface formed between two CH3 domains in the tertiary structure of the antibody. In the tertiary structure of a multispecific antibody, the introduced projection of one CH3 domain can be positioned within the introduced cavity of the other CH3 domain.

[0073] The term "effector function" refers to the biological activity resulting from the Fc region of an antibody, which varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cell-mediated cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), cytokine secretion, antigen uptake via immune complexes by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0074] An "activated Fc receptor" is an Fc receptor that, following the binding of an antibody's Fc domain, triggers a signaling event that stimulates receptor-hosting cells to perform effector functions. Examples of human activated Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89).

[0075] Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism in which immune effector cells induce the lysis of antibody-coated target cells. Target cells are cells to which antibodies or derivatives containing an Fc region specifically bind, typically via a protein portion that is N-terminal to the Fc region. As used herein, the term "ADCC reduction" is defined as either a decrease in the number of target cells lysed within a given time at a given antibody concentration in the culture medium surrounding the target cells by the ADCC mechanism as defined above, and / or an increase in the antibody concentration in the culture medium surrounding the target cells required to achieve the lysis of a given number of target cells within a given time by the ADCC mechanism. ADCC reduction is relative to ADCC mediated by the same antibody, produced using the same standard production, purification, formulation, and storage methods (known to those skilled in the art), by the same type of host cells, but without manipulation. For example, ADCC reduction mediated by an antibody containing an amino acid substitution in its Fc domain that reduces ADCC is compared to ADCC mediated by the same antibody without this amino acid substitution in its Fc domain. Appropriate assays for measuring ADCC are well known in the art (see, for example, International Publication No. 2006 / 082515 or International Publication No. 2012 / 130831).

[0076] "Reduced binding," for example, reduced binding to the Fc receptor, refers to a decrease in affinity for each interaction, as measured by SPR. To clarify, this term also includes reducing affinity to zero (or below the detection limit of the analytical method), i.e., complete disappearance of the interaction. Conversely, "increased binding" refers to an increase in binding affinity for each interaction.

[0077] "Affinity" refers to the sum of the non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between the members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be expressed by its 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).

[0078] As used herein, the terms “to manipulate,” “to be manipulated,” and “to manipulate” are understood to include any manipulation or post-translational modification of the peptide backbone of naturally occurring or recombinant polypeptides or fragments thereof. Manipulation includes modification of amino acid sequences, modification of glycosylation patterns, or modification of the side chain groups of individual amino acids, and combinations thereof.

[0079] The term "amino acid mutation," as used herein, encompasses the substitution, deletion, insertion, and modification of amino acids. Any combination of substitutions, deletions, insertions, and modifications can be used to arrive at a final construct, provided that the final construct has the desired characteristics (e.g., decreased binding to the Fc receptor or increased association with another peptide). Deletions and insertions of amino acid sequences include deletions and insertions of the amino-terminus and / or carboxyl-terminus of amino acids. A preferred amino acid mutation is amino acid substitution. For example, non-conservative amino acid substitution, i.e., replacing one amino acid with another amino acid having different structural and / or chemical properties, is particularly preferred for the purpose of altering the binding properties of the Fc region. Amino acid substitutions include substitutions with non-natural amino acids or substitutions with natural amino acid derivatives of 20 standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be induced using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, etc. Methods other than genetic manipulation to alter the side chain groups of amino acids, such as chemical modifications, may also be useful. Various names can be used herein to indicate the same amino acid mutation. For example, a substitution of proline to glycine at position 329 of the Fc domain can be denoted as 329G, G329, G329, P329G, or Pro329Gly.

[0080] "Fused" means that the constituent elements (e.g., the Fab molecule and the Fc domain subunit) are linked directly by peptide bonds or via one or more peptide linkers.

[0081] The terms “linker” or “peptide linker” refer to a peptide containing one or more amino acids, typically about 2 to 30 amino acids. Peptide linkers are known in the art or described herein. A suitable non-immunogenic linker peptide is, for example, a (G4S)n peptide linker, where G = glycine, S = serine, and “n” is generally a number from 1 to 10. Furthermore, the linker may include (part of) an immunoglobulin hinge region. In particular, if the Fab molecule is fused to the N-terminus of an Fc domain subunit, it may be fused via an immunoglobulin hinge region or part thereof.

[0082] The "amino acid sequence identity percentage (%)" for a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after sequence alignment and, if necessary, introducing gaps to achieve the maximum possible sequence identity percentage, without considering any conservative substitutions as part of the sequence identity. Alignment for determining the amino acid sequence identity percentage can be achieved by various methods within the scope of the art, for example, 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 the sequences, including any algorithm necessary to achieve the maximum alignment over the entire length of the sequences to be compared. Alternatively, the identity percentage value can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and its source code, along with user documentation, is filed with the U.S. Copyright Office (Washington, DC, 20559), registered under U.S. Copyright Registration No. TXU510087, and is listed in International Publication No. 2001 / 007611.

[0083] Unless otherwise specified, for the purposes of this specification, amino acid sequence identity percentage values ​​are generated using the ggsearch program in FASTA package version 36.3.8c or later, along with the BLOSUM50 comparison matrix. The FASTA program package was created by WRPearson and DJLipman ("Improved Tools for Biological Sequence Analysis," PNAS 85(1988) 2444-2448), WRPearson ("Effective protein sequence comparison," Meth.Enzymol. 266(1996) 227-25 258), and Pearson et al. (Genomics 46(1997) 24-36), and is publicly available at www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www.ebi.ac.uk / Tools / sss / fasta.

[0084] Alternatively, you can use the public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi to perform a global alignment (not just a local one) and compare sequences using the ggsearch(global protein:protein) program with default options (BLOSUM50;open:-10;ext:-2;Ktup=2). The amino acid identity percentage is shown in the output alignment header.

[0085] Cytokine mimetic The inventors have found that a pair of antigen-binding domains (hereinafter referred to as cytokine receptor-binding domains) capable of binding to different cytokine receptor subunits of a cytokine receptor complex can act as a cytokine agonist, i.e., mimic naturally occurring cytokines. Cytokine agonists are also called cytokine mimes. This is demonstrated by combining a pair of cytokine receptor-binding domains capable of binding to different cytokine receptor subunits in a single antigen-binding molecule. The antibody formats of these molecules may offer advantageous properties compared to natural or recombinant cytokines. To achieve conditional activation of cytokine receptors, the pair of cytokine receptor-binding domains was split into two different antigen-binding molecules, i.e., split molecules or split antibodies. These antigen-binding molecules further include antigen-binding domains (hereinafter referred to as target-binding domains) capable of binding to a target antigen. Target-dependent binding of the pair of cytokine receptor-binding domains allows the molecule to act as a cytokine mime. By using a target-dependent approach, it becomes even possible to direct agonist activity to a desired site, such as cells or tissues expressing the target. The target-binding domains of a molecule can simultaneously bind to the same target antigen; that is, they bind to different epitopes of the antigen and are non-competitive. Such a pair of target-binding domains is also called a biparatopic target-binding domain. Binding of the cytokine receptor-binding domain by the biparatopic target-binding domain allows the cytokine receptor-binding domain to specifically target the site of interest, i.e., the cell or environment in which the target antigen is expressed, enabling it to act as a cytokine mimetic. Therefore, a split antigen-binding molecule pair is a biparatopic pair of antigen-binding molecules. Finally, the cytokine receptor-binding domain pair and the target-binding domain are incorporated into a single molecule (hereinafter referred to as the integrated format). Target-independent binding of the cytokine receptor-binding domain, i.e., binding in the absence of the target antigen, is prevented by keeping the cytokine receptor-binding domains apart from each other.In the presence of the target antigen, biparatopic binding of the cytokine receptor-binding domain leads to activation of the cytokine receptor.

[0086] Integrated format The integrated format provides an antigen-binding molecule comprising i) a first target-binding domain, ii) a second target-binding domain, iii) a first cytokine receptor-binding domain, iv) a second cytokine receptor-binding domain, and v) an Fc domain, wherein the first target-binding domain can bind to a first epitope on a target antigen, the second target-binding domain can bind to a second epitope on a target antigen, the first and second target-binding domains do not compete for binding to the target antigen, the first cytokine receptor-binding domain can bind to a first cytokine receptor subunit, and the second cytokine receptor-binding domain can bind to a second cytokine receptor subunit.

[0087] Cytokine receptor binding domain According to the present invention, an integrated antigen-binding molecule comprises a first cytokine receptor-binding domain and a second cytokine receptor-binding domain. The cytokine receptor-binding domains contained in the antigen-binding molecule bind to different cytokine receptor subunits. Each cytokine receptor-binding domain of the antigen-binding molecule binds to a different cytokine receptor subunit of the cytokine receptor complex. Therefore, the integrated antigen-binding molecule comprises a first cytokine receptor-binding domain that can bind to a first cytokine receptor subunit and a second cytokine receptor-binding domain that can bind to a second cytokine receptor subunit. The first and second cytokine receptor subunits are subunits of the cytokine receptor complex.

[0088] The cytokine receptor complex may be an IFNγ receptor complex, an IL-2 receptor complex, an IL-7 receptor complex, an IL-12 receptor complex, or an IL-18 receptor complex. Therefore, the first and second cytokine receptor binding domains may bind to different subunits of the IFNγ 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 to a subunit of the IFNγ receptor complex. Therefore, in one embodiment, the first cytokine receptor binding domain may bind to IFNγR1 and the second cytokine receptor binding domain may bind to IFNγR2. Alternatively, the first cytokine receptor binding domain may bind to IFNγR2 and the second cytokine receptor binding domain may bind to IFNγR1. In a further embodiment, the first and second cytokine receptor binding domains bind to a subunit of the IL-2 receptor complex. Therefore, in one embodiment, the first cytokine receptor binding domain can bind to IL-2Rβ, and the second cytokine receptor binding domain can bind to IL-2Rγ. In another embodiment, the first and second cytokine receptor binding domains bind to subunits of the IL-7 receptor complex. Therefore, in one embodiment, the first cytokine receptor binding domain can bind to IL-7Ra, and the second cytokine receptor binding domain can bind to IL-2Rγ. In yet another embodiment, the first and second cytokine receptor binding domains bind to subunits of the IL-12 receptor complex. Therefore, in one embodiment, the first cytokine receptor binding domain can bind to IL-12Rβ1, and the second cytokine receptor binding domain can bind to IL-12Rβ2.In one embodiment, the first cytokine receptor binding domain can bind to IL-12Rβ2, and the second cytokine receptor binding domain can bind to IL-12Rβ1.

[0089] The first and second cytokine receptor-binding domains may be antibody fragments. In one embodiment, the first and second cytokine receptor-binding domains are single-domain antibodies. In a specific embodiment, the first and second cytokine receptor-binding domains are VHH domains.

[0090] In one embodiment, the first cytokine receptor binding domain includes 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 includes 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 embodiment, the first cytokine receptor binding domain includes 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 includes 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 embodiment, the first cytokine receptor binding domain includes the sequence of SEQ ID NO: 3, and the second cytokine receptor binding domain includes the sequence of SEQ ID NO: 7. In one embodiment, the first cytokine receptor binding domain includes the sequence of SEQ ID NO: 7, and the second cytokine receptor binding domain includes the sequence of SEQ ID NO: 3.

[0091] In one embodiment, the first cytokine receptor binding domain includes an amino acid sequence selected from SEQ ID NOs. 60, 61, 62, 63, and 64, and the second cytokine receptor binding domain includes an amino acid sequence selected from SEQ ID NOs. 65, 66, 67, 68, and 69. In another embodiment, the first cytokine receptor binding domain includes an amino acid sequence selected from SEQ ID NOs. 65, 66, 67, 68, and 69, and the second cytokine receptor binding domain includes an amino acid sequence selected from SEQ ID NOs. 60, 61, 62, 63, and 64. In yet another embodiment, the first cytokine receptor binding domain includes the sequence of SEQ ID NO. 64, and the second cytokine receptor binding domain includes the sequence of SEQ ID NO. 66. In yet another embodiment, the first cytokine receptor binding domain includes the sequence of SEQ ID NO. 66, and the second cytokine receptor binding domain includes the sequence of SEQ ID NO. 64.

[0092] Target antigen binding domain According to the present invention, the antigen-binding molecule comprises a first target-binding domain and a second target-binding domain. Both the first and second target-binding domains bind to the same target, i.e., the same antigen. However, the first and second target-binding domains bind to different epitopes of the target. The first target-binding domain can bind to a first epitope on the target antigen, and the second target-binding domain can bind to a second epitope on the target antigen. The first and second target-binding domains can bind to the target simultaneously; that is, the target-binding domains are non-competitive domains. The first and second target-binding domains do not compete for binding to the target antigen.

[0093] The target-binding domain of the 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. The first and / or second target-binding domains may be Fv, Fab, scFv, scFab molecules, or single-domain antibodies. In one embodiment, the target-binding domain is Fv, Fab, scFv, scFab molecules, or single-domain antibodies, and the target-binding domain is Fv, Fab, scFv, scFab molecules, or single-domain antibodies.

[0094] In one embodiment, the first and second target-binding domains are Fab molecules. 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 target-binding domain includes a heavy-chain variable domain (VH1), a light-chain variable domain (VL1), a heavy-chain constant domain (CH11), and a light-chain constant domain (CL1). In one embodiment, the second target-binding domain includes a heavy-chain variable domain (VH2), a light-chain variable domain (VL2), a heavy-chain constant domain (CH12), and a light-chain constant domain (CL2).

[0095] The target-binding domain may be cross-Fab. In one embodiment, the first target-binding domain is cross-Fab. In another embodiment, the second target-binding domain is cross-Fab. In one embodiment, the first target-binding domain is a Fab molecule, the Fab molecule is cross-Fab, and the second target-binding domain is a Fab molecule, the Fab molecule is not cross-Fab. In one embodiment, the first target-binding domain is a Fab molecule, the Fab molecule is a conventional Fab molecule, the second target-binding domain is a Fab molecule, the Fab molecule is cross-Fab.

[0096] In one embodiment, the Fab molecule includes charge modification. To reduce mispairing of heavy and light chains from different Fab molecules, and thus increase the purity and yield of the desired antigen-binding molecule, the Fab molecule may include different charged amino acid substitutions (so-called "charge modifications"). These modifications are introduced into the cross-Fab CH1 and CL domains, or into the conventional Fab CH1 and CL domains. In a particular embodiment, the Fab molecule has the amino acid at position 123 (EU numbering) replaced with arginine (R) and the amino acid at position 124 (EU numbering) replaced with lysine (K) in the CL domain, and the amino acids at positions 147 (EU numbering) and / or 213 (EU numbering) replaced with glutamic acid (E) in the CH1 domain. Preferably, the charge modification is carried out in the conventional Fab molecule.

[0097] Charge modification can be introduced into the CH1 domain and CL domain of either the Fab molecule of the first target-binding domain or the Fab molecule of the second target-binding domain. In one embodiment, the first target-binding domain includes charge modification. In another embodiment, the second target-binding domain includes charge modification. In yet another embodiment, the first target-binding domain includes charge modification and the second target-binding domain includes cross-Fab. In yet another embodiment, the first target-binding domain includes cross-Fab and the second target-binding domain includes charge modification.

[0098] Both the first and second target-binding domains specifically bind to 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. Thus, in one embodiment, the first target-binding domain can bind to a first epitope of CEA, and the second target-binding domain can bind to a second epitope of CEA. In one embodiment, the first target-binding domain can bind to a first epitope of FAP, and the second target-binding domain can bind to a second epitope of FAP. In one embodiment, the first target-binding domain can bind to a first epitope of Her2, and the second target-binding domain can bind to a second epitope of Her2. In one embodiment, the first target-binding domain can bind to a first epitope of Her3, and the second target-binding domain can bind to a second epitope of Her3. In one embodiment, the first target-binding domain can bind to a first epitope of EGFR, and the second target-binding domain can bind to a second epitope of EGFR.

[0099] In one embodiment, the first target-binding domain includes VH1 of SEQ ID NO: 20 and VL1 of SEQ ID NO: 21, and the second target-binding domain includes VH2 of SEQ ID NO: 22 and VL2 of SEQ ID NO: 23. In another embodiment, the first target-binding domain includes VH1 of SEQ ID NO: 22 and VL1 of SEQ ID NO: 23, and the second target-binding domain includes VH2 of SEQ ID NO: 20 and VL2 of SEQ ID NO: 21.

[0100] 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. In one embodiment, the first target-binding domain can bind to a first epitope of PD-1, and the second target-binding domain can bind to a second epitope of PD-1. In one embodiment, the first target-binding domain can bind to a first epitope of LAG-3, and the second target-binding domain can bind to a second epitope of LAG-3. In one embodiment, the first target-binding domain includes VH1 of SEQ ID NO: 80 and VL1 of SEQ ID NO: 81, and the second target-binding domain includes VH2 of SEQ ID NO: 82 and VL2 of SEQ ID NO: 83. In one embodiment, the first target-binding domain includes VH1 of SEQ ID NO: 82 and VL1 of SEQ ID NO: 83, and the second target-binding domain includes VH2 of SEQ ID NO: 80 and VL2 of SEQ ID NO: 81. In one embodiment, the first target-binding domain includes VH1 of SEQ ID NO: 82 and VL1 of SEQ ID NO: 83, and the second target-binding domain includes VH2 of SEQ ID NO: 140 and VL2 of SEQ ID NO: 141. In another embodiment, the first target-binding domain includes VH1 of SEQ ID NO: 140 and VL1 of SEQ ID NO: 141, and the second target-binding domain includes VH2 of SEQ ID NO: 82 and VL2 of SEQ ID NO: 83.

[0101] FC Domain According to the present invention, the antigen-binding molecule includes an Fc domain.

[0102] The Fc domain of the binding molecule 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 contain the heavy chain domains of the immunoglobulin molecule. For example, the Fc domain of the immunoglobulin G (IgG) molecule is a dimer, and each subunit contains the CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain can stably associate with each other.

[0103] In one embodiment, the antigen-binding molecule includes an Fc domain composed of a first and a second Fc domain subunit. In a particular embodiment, the antigen-binding molecule includes one or fewer Fc domains.

[0104] In one embodiment, the Fc domain of the antigen-binding molecule is an IgG Fc domain. In a specific embodiment, the Fc domain is an IgG1 Fc domain. In another embodiment, the Fc domain is an IgG4 Fc domain.

[0105] In a more specific embodiment, the Fc domain is an IgG4Fc domain containing an amino acid substitution at position S228 (Kabat EU index numbering), particularly the amino acid substitution S228P. This amino acid substitution reduces Fab arm exchange of IgG4 antibodies in vivo (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In yet another embodiment, the Fc domain is a human Fc domain. In a particular embodiment, the Fc domain is a human IgG1Fc domain.

[0106] In one embodiment, the Fc domain includes modifications that promote the association of the first and second subunits of the Fc domain. Further Fc domain modifications that promote heterodimerization are described below.

[0107] In one embodiment, the Fc domain includes one or more amino acid substitutions that reduce Fc receptor binding and / or effector function. Fc domain modifications that reduce Fc receptor binding and / or effector function are further described below herein.

[0108] Fc domain modification that promotes heterodimerization The antigen-binding molecule according to the present invention comprises two target-binding domains and two cytokine receptor-binding domains that can be fused to a first or second Fc domain subunit, and thus the two Fc domain subunits are typically contained in two non-identical polypeptide chains. Several combinations of the two polypeptides are possible through recombinant co-expression of these polypeptides and subsequent dimerization. To increase the yield and purity of the antigen-binding molecule in recombinant production, it is advantageous to introduce modifications to the Fc domain of the antigen-binding molecule that promote the association of the desired polypeptide.

[0109] Therefore, in certain embodiments, the Fc domain of the antigen-binding molecule according to the present invention includes modifications that facilitate the association of the first and second subunits of the Fc domain. The site of the most extensive protein-protein interaction between the two subunits of the human IgG Fc domain is located within the CH3 domain of the Fc domain. Therefore, in one embodiment, the modifications are located within the CH3 domain of the Fc domain.

[0110] To enhance heterodimerization, several methods exist for modifying the CH3 domain within the Fc domain, which are well described, for example, in International Publications 96 / 27011, 98 / 050431, European Patent No. 1870459, 2007 / 110205, 2007 / 147901, 2009 / 089004, 2010 / 129304, 2011 / 90754, 2011 / 143545, 2012058768, 2013157954, and 2013096291. Typically, in all such methods, 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 designed complementaryly so that each CH3 domain (or the heavy chain comprising it) cannot homodimerize on its own, but is forced to heterodimerize with other complementaryly engineered CH3 domains (so that the first and second CH3 domains heterodimerize and homodimers are not formed between two first or two second CH3 domains).

[0111] In certain embodiments, the modification that facilitates the association of the first and second subunits of the Fc domain is a so-called "knob-into-hole" modification, which includes a "knob" modification on one of the two subunits of the Fc domain and a "hole" modification on the other of the two subunits of the Fc domain.

[0112] The knob-into-hole technique is described, for example, in U.S. Patents 5,731,168, 7,695,936, Ridgway et al., Prot Eng 9,617-621 (1996), and Carter, J Immunol Meth 248,7-15 (2001). Generally, this method involves introducing a projection ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, so that the projection can be positioned within the cavity to promote heterodimerization and inhibit homodimerization. The projection is constructed by substituting a smaller amino acid side chain from the contact surface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan).

[0113] A compensatory cavity of the same or similar size as the protrusion is formed at the interface of the second polypeptide by replacing a larger amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).

[0114] Therefore, in a preferred embodiment, in the CH3 domain of the first subunit of the Fc domain of the antigen-binding molecule, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a projection within the CH3 domain of the first subunit that can be accommodated 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 that can accommodate the projection within the CH3 domain of the first subunit.

[0115] Preferably, the 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, the 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 protrusions and cavities can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis.

[0116] In a specific embodiment, in the CH3 domain of the first subunit of the Fc domain ("knob" 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 ("whole" subunit), the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In another embodiment, in the second subunit of the Fc domain, the threonine residue at position 366 is further replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering according to the Kabat EU index).

[0117] In a further embodiment, in the first subunit of the Fc domain, the serine residue at position 354 is further 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, the tyrosine residue at position 349 is further replaced with a cysteine ​​residue (Y349C) (numbered according to the Kabat EU index).

[0118] The introduction of these two cysteine ​​residues creates a disulfide bridge between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0119] In certain embodiments, the first subunit of the Fc domain includes the amino acid substitutions S354C and T366W, and the second subunit of the Fc domain includes the amino acid substitutions Y349C, T366S, L368A and Y407V (numbered according to the Kabat EU index).

[0120] Other techniques for CH3 modification to enhance heterodimerization are envisioned as alternatives to the present invention and are described, for example, in International Publication Nos. 96 / 27011, 98 / 050431, European Patent No. 1870459, International Publication Nos. 2007 / 110205, 2007 / 147901, 2009 / 089004, 2010 / 129304, 2011 / 90754, 2011 / 143545, 2012 / 058768, 2013 / 157954, and 2013 / 096291.

[0121] In one embodiment, the heterodimerization method described in European Patent No. 1870459 is used instead. This method is based on the introduction of oppositely charged amino acids to specific amino acid positions at the CH3 / CH3 domain interface between two subunits of the Fc domain.

[0122] A particular embodiment of the antigen-binding molecule of the present invention is characterized by an amino acid mutation R409D;K370E in one of the two CH3 domains (of the Fc domain) and an amino acid mutation D399K;E357K in the other CH3 domain of the Fc domain (numbered according to the Kabat EU index).

[0123] In one embodiment, the antigen-binding molecule of the present invention contains the amino acid mutation T366W in the CH3 domain of the first subunit of the Fc domain, the amino acid mutations T366S, L368A, and Y407V in the CH3 domain of the second subunit of the Fc domain, and further amino acid mutations R409D and K370E in the CH3 domain of the first subunit of the Fc domain, and amino acid mutations D399K and E357K in the CH3 domain of the second subunit of the Fc domain (numbered according to the Kabat EU index).

[0124] In one embodiment, the antigen-binding molecule of the present invention contains amino acid mutations S354C and T366W in the CH3 domain of the first subunit of the Fc domain, and amino acid mutations Y349C, T366S, L368A, and Y407V in the CH3 domain of the second subunit of the Fc domain; or the antigen-binding molecule contains amino acid mutations Y349C and T366W in the CH3 domain of the first subunit of the Fc domain, and amino acid mutations S354C, T366S, L368A, and Y407V in the CH3 domain of the second subunit of the Fc domain, and further contains amino acid mutations R409D and K370E in the CH3 domain of the first subunit of the Fc domain, and amino acid mutations D399K and E357K in the CH3 domain of the second subunit of the Fc domain (all numbered according to the Kabat EU index).

[0125] In one embodiment, the heterodimerization method described in International Publication No. 2013 / 157953 is used instead. In one embodiment, the first CH3 domain contains the amino acid mutation T366K, and the second CH3 domain contains the amino acid mutation L351D (numbered according to the Kabat EU index). In a further embodiment, the first CH3 domain contains a further amino acid mutation L351K. In a further embodiment, the second CH3 domain further contains amino acid mutations selected from Y349E, Y349D, and L368E (especially L368E) (numbered according to the Kabat EU index).

[0126] In one embodiment, the heterodimerization method described in International Publication No. 2012 / 058768 is used instead. In one embodiment, the first CH3 domain contains amino acid mutations L351Y, Y407A, and the second CH3 domain contains amino acid mutations T366A, K409F. In a further embodiment, the second CH3 domain contains further amino acid mutations at positions T411, D399, S400, F405, N390, or K392, for example, a) T411N, T411R, T411Q, T411K, T411D, T411E, or T411W, b) D399R, D399W, D399Y Alternatively, selected from 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 (numbered according to the Kabat EU index). In a further embodiment, the first CH3 domain contains amino acid mutations L351Y, Y407A, and the second CH3 domain contains amino acid mutations T366V, K409F. In a further embodiment, the first CH3 domain contains amino acid mutation Y407A, and the second CH3 domain contains amino acid mutations T366A, K409F. In a further embodiment, the second CH3 domain further includes amino acid mutations K392E, T411E, D399R, and S400R (numbered according to the Kabat EU index).

[0127] In one embodiment, the heterodimerization method described in International Publication No. 2011 / 143545 is used as an alternative by using amino acid modifications at positions selected from the group consisting of, for example, 368 and 409 (numbering based on the Kabat EU index).

[0128] In one embodiment, the heterodimerization method described in International Publication No. 2011 / 090762, which also uses the knob-into-hole technique described above, is used as an alternative. In one embodiment, the first CH3 domain contains the amino acid mutation T366W and the second CH3 domain contains the amino acid mutation Y407A. In another embodiment, the first CH3 domain contains the amino acid mutation T366Y and the second CH3 domain contains the amino acid mutation Y407T (numbered according to the Kabat EU index).

[0129] In one embodiment, the antigen-binding molecule or its Fc domain is an IgG2 subclass, and the heterodimerization method described in International Publication No. 2010 / 129304 is used instead.

[0130] In alternative embodiments, modifications that facilitate the association of the first and second subunits of the Fc domain include modifications that intervene in the electrostatic maneuvering effect, as described, for example, in International Publication No. 2009 / 089004. Generally, this method involves the substitution of one or more amino acid residues with charged amino acid residues at the interface of the two Fc domain subunits such that homodimerization is electrostatically undesirable, but heterodimerization is electrostatically desirable. In some such embodiments, the first CH3 domain includes an 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 the second CH3 domain includes an 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, more specifically D399K and E356K). In further embodiments, the first CH3 domain further includes an 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 a further embodiment, the first CH3 domain further or alternatively comprises amino acid substitutions of K439 and / or K370 with negatively charged amino acids (e.g., glutamic acid (E) or aspartic acid (D)) (all numbering is by Kabat EU index).

[0131] In one embodiment, the heterodimerization method described in International Publication No. 2007 / 147901 is used instead. In one embodiment, the first CH3 domain contains amino acid mutations K253E, D282K, and K322D, and the second CH3 domain contains amino acid mutations D239K, E240K, and K292D (numbered according to the Kabat EU index). In one embodiment, the heterodimerization method described in International Publication No. 2007 / 110205 is used instead.

[0132] In one embodiment, the first subunit of the Fc domain includes amino acid substitutions K392D and K409D, and the second subunit of the Fc domain includes amino acid substitutions D356K and D399K (numbered according to the Kabat EU index).

[0133] Fc domain modification that reduces Fc receptor binding and / or effector function. The Fc domain confers desirable pharmacokinetic properties to antigen-binding molecules, including a long serum half-life and a desirable tissue-to-blood distribution ratio, contributing to good accumulation in target tissues. However, it can also lead to undesirable targeting of the binding molecule to cells expressing the Fc receptor rather than the preferred antigen-containing cells. Furthermore, co-activation of the Fc receptor signaling pathway can lead to cytokine release, potentially resulting in excessive activation of cytokine receptors and serious side effects during systemic administration. Activation of immune cells other than T cells (those possessing the Fc receptor) can even reduce the effectiveness of antigen-binding molecule pairs, for example, due to the potential destruction of T cells by NK cells.

[0134] Therefore, in certain embodiments, the Fc domain of the antigen-binding molecule according to the present invention exhibits reduced binding affinity to the Fc receptor and / or reduced effector function compared to the native IgG1Fc domain. In some such embodiments, the Fc domain (or the antigen-binding molecule containing the Fc domain) exhibits binding affinity to the Fc receptor of less than 50%, particularly less than 20%, more specifically less than 10%, and most specifically less than 5%, compared to the native IgG1Fc domain (or the antigen-binding molecule containing the native IgG1Fc domain). In one embodiment, the Fc domain (or the antigen-binding molecule containing the Fc domain) does not substantially bind to the Fc receptor and / or does not induce effector function. In certain embodiments, the Fc receptor is the Fcγ receptor. In one embodiment, the Fc receptor is the human Fc receptor. In one embodiment, the Fc receptor is an activated Fc receptor. In a particular embodiment, the Fc receptor is an activated human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, most specifically human FcγRIIIa. In one embodiment, the effector function is one or more selected from the group of CDC, ADCC, ADCP, and cytokine secretion. In a particular embodiment, the effector function is ADCC. In one embodiment, the Fc domain exhibits substantially similar binding affinity to the neonatal Fc receptor (FcRn) compared to the native IgG1Fc domain. Substantially similar binding to FcRn is achieved when the Fc domain (or an antigen-binding molecule containing the Fc domain) exhibits a binding affinity to FcRn that is greater than about 70%, particularly greater than about 80%, and more specifically greater than about 90%, compared to the native IgG1Fc domain (or an antigen-binding molecule containing the native IgG1Fc domain).

[0135] In certain embodiments, the Fc domain is engineered to have reduced binding affinity and / or effector function to the Fc receptor compared to an unengineered Fc domain. In certain embodiments, the Fc domain of an antigen-binding molecule contains one or more amino acid mutations that reduce the binding affinity and / or effector function of the Fc domain to the Fc receptor. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. In one embodiment, the amino acid mutation reduces the binding affinity of the Fc domain to the Fc receptor. In one embodiment, the amino acid mutation reduces the binding affinity of the Fc domain to the Fc receptor by at least half, at least one-fifth, or at least one-tenth. In embodiments 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 the Fc receptor by at least one-tenth, at least one-twentieth, or even at least one-fiftieth. In one embodiment, an antigen-binding molecule containing an engineered Fc domain exhibits a binding affinity to the Fc receptor of less than 20%, particularly less than 10%, and more specifically less than 5%, compared to an antigen-binding molecule containing an unengineered Fc domain. In a specific embodiment, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activated Fc receptor. In a specific embodiment, the Fc receptor is an activated human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, most specifically human FcγRIIIa. Preferably, binding to each of these receptors is reduced. In one embodiment, binding affinity to complement components, particularly to C1q, is also reduced. In one embodiment, binding affinity to the neonatal Fc receptor (FcRn) is not reduced.

[0136] Substantially similar binding to FcRn (i.e., protection of the binding affinity of the Fc domain to the receptor) is achieved when the Fc domain (or the antigen-binding molecule containing the Fc domain) exhibits a binding affinity greater than approximately 70% of the binding affinity of the unmodified form of the Fc domain (or the antigen-binding molecule containing this unmodified form of Fc) to FcRn. The Fc domain, or the antigen-binding molecule of the present invention containing the Fc domain, may exhibit an affinity greater than approximately 80% and even greater than approximately 90% of such affinity. In certain embodiments, the Fc domain of the antigen-binding molecule is modified to have reduced effector function compared to the unmodified Fc domain. Reduced effector function may include, but is not limited to, one or more of the following: reduced complement-dependent cell-mediated cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent 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-induced apoptosis, reduced cross-linking with target-binding antibodies, reduced dendritic cell maturation, or reduced T cell priming. In one embodiment, reduced effector function is one or more selected from the group of reduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion. In a particular embodiment, reduced effector function is reduced ADCC. In one embodiment, reduced ADCC is less than 20% of ADCC induced by an unmodified Fc domain (or a binding molecule containing an unmodified Fc domain).

[0137] In one embodiment, the amino acid mutation that reduces the binding affinity and / or effector function of the Fc domain to the Fc receptor is an amino acid substitution. In one embodiment, the Fc domain contains an amino acid substitution at a position selected from the group E233, L234, L235, N297, P331, and P329 (numbered according to the Kabat EU index). In a more specific embodiment, the Fc domain contains an amino acid substitution at a position selected from the group L234, L235, and P329 (numbered according to the Kabat EU index). In one embodiment, the Fc domain contains amino acid substitutions L234A and L235A (numbered according to the Kabat EU index). In some such embodiments, the Fc domain is an IgG1Fc domain, particularly a human IgG1Fc domain. In one embodiment, the Fc domain contains an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, particularly P329G (numbered according to the Kabat EU Index). In one embodiment, the Fc domain contains an amino acid substitution at position P329 and further amino acid substitutions at positions selected from E233, L234, L235, N297, and P331 (numbered according to the Kabat EU Index). In a more specific embodiment, the further amino acid substitutions are E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In a particular embodiment, the Fc domain contains amino acid substitutions at positions P329, L234, and L235 (numbered according to the Kabat EU Index). In a more specific embodiment, the Fc domain includes amino acid mutations L234A, L235A, and P329G ("P329G LALA", "PGLALA", or "LALAPG").Specifically, in certain embodiments, each subunit of the Fc domain contains amino acid substitutions L234A, L235A, and P329G (Kabat EU index numbering), namely, in the first and second subunits 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 with a glycine residue (P329G) (Kabat EU index numbering).

[0138] In some such embodiments, the Fc domain is an IgG1Fc domain, particularly a human IgG1Fc domain. The amino acid substitution combination "P329G LALA" almost completely eliminates the Fcγ receptor (and complement) binding of the human IgG1Fc domain, as described in International Publication 2012 / 130831, which is incorporated herein by reference in its entirety. International Publication 2012 / 130831 also describes methods for preparing such mutant Fc domains and determining their properties, such as Fc receptor binding or effector function.

[0139] IgG4 antibodies exhibit reduced binding affinity to Fc receptors and reduced effector function compared to IgG1 antibodies. Therefore, in one embodiment, the Fc domain of the binding molecule of the present invention is an IgG4Fc domain, particularly a human IgG4Fc domain. In one embodiment, the IgG4Fc domain includes an amino acid substitution at position S228, specifically amino acid substitution S228P (numbered according to the Kabat EU index). To further reduce its binding affinity to Fc receptors and / or its effector function, in one embodiment, the IgG4Fc domain includes an amino acid substitution at position L235, specifically amino acid substitution L235E (numbered according to the Kabat EU index). In one embodiment, the IgG4Fc domain includes an amino acid substitution at position P329, specifically amino acid substitution P329G (numbered according to the Kabat EU index). In a preferred embodiment, the IgG4Fc domain includes amino acid substitutions at positions S228, L235, and P329, specifically amino acid substitutions S228P, L235E, and P329G (numbered according to the Kabat EU index). Such IgG4Fc domain variants and their Fcγ receptor binding properties are described in PCT Publication No. 2012 / 130831 (in whole, incorporated herein by reference).

[0140] In certain embodiments, Fc domains exhibiting reduced binding affinity to the Fc receptor and / or reduced effector function compared to the native IgG1Fc domain are human IgG1Fc domains containing amino acid substitutions L234A, L235A and optionally P329G, or human IgG4Fc domains containing amino acid substitutions S228P, L235E and optionally P329G (numbered according to the Kabat EU index).

[0141] In certain embodiments, N-glycosylation of the Fc domain is excluded. In some such embodiments, the Fc domain contains amino acid mutations at position N297, particularly amino acid substitutions in which asparagine is replaced with alanine (N297A) or aspartic acid (N297D) (numbered according to the Kabat EU index).

[0142] In addition to the Fc domains described herein and in PCT Publication No. 2012 / 130831, Fc domains with reduced Fc receptor binding and / or effector function also include those having one or more substitutions at Fc domain residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056) (numbered by the Kabat EU Index). Such Fc variants include the so-called "DANA" Fc variant, which has substitutions at alanine residues 265 and 297, as well as Fc variants having substitutions at two or more of the amino acids 265, 269, 270, 297, and 327 (U.S. Patent No. 7,332,581).

[0143] 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-directed mutagenesis of coding DNA sequences, PCR, gene synthesis, etc. Correct nucleotide changes can be confirmed, for example, by sequencing.

[0144] Binding to the Fc receptor can be readily identified, for example, by ELISA or by surface plasmon resonance (SPR) using standard equipment such as a BIAcore instrument (GE Healthcare), and the Fc receptor can be obtained by recombinant expression. Alternatively, the binding affinity of the Fc domain or a binding molecule containing the Fc domain to the Fc receptor may be evaluated using cell lines known to express a specific Fc receptor, such as human NK cells expressing the FcγIIIa receptor.

[0145] The effector function of the Fc domain, or a binding molecule containing an Fc domain, can be measured by methods known in the art. Examples of in vitro assays for evaluating the ADCC activity of the 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).

[0146] Alternatively, non-radioactive assays may be used (e.g., ACTI® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc., Mountain View, California), and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wisconsin)). Effector cells useful for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells.

[0147] Alternatively, or furthermore, the ADCC activity of the molecule of interest may be evaluated in vivo in an animal model, for example, as disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

[0148] In one embodiment, the binding of the Fc domain to complement components, particularly C1q, is reduced. Therefore, in one embodiment where the Fc domain is designed to have reduced effector function, this reduction in effector function includes a reduction in CDC activity. C1q binding assays may be performed to determine whether an Fc domain or a binding molecule containing an Fc domain can bind to C1q and therefore possess CDC activity. See, for example, the C1q and C3c binding ELISAs in International Publication Nos. 2006 / 029879 and International Publication Nos. 2005 / 100402. A CDC assay may be performed to assess complement activation (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)).

[0149] The determination of FcRn binding and in vivo clearance / half-life can also be performed using methods known in the art (see, for example, Petkova, SB et al., Int'l.Immunol. 18(12):1759-1769 (2006), International Publication No. 2013 / 120929).

[0150] Integrated format configuration The antigen-binding molecule according to the present invention may have various molecular structures; that is, the domains of the binding molecule may be linked to each other in different ways.

[0151] An integrated antigen-binding molecule comprises first and second target-binding domains, first and second cytokine receptor-binding domains, and first and second Fc domain subunits. In certain embodiments, one target-binding domain and one cytokine receptor domain are fused to one of the Fc domain subunits, and the other target-binding domain and cytokine receptor-binding domain are fused to the other Fc domain subunit. The cytokine receptor-binding domain may be fused at its C-terminus to the N-terminus of the target-binding domain. The target-binding domain may be fused at its N-terminus or C-terminus to the C-terminus or N-terminus of one of the Fc domain subunits, respectively.

[0152] In one embodiment, the target-binding domain is a Fab molecule. Therefore, the first target-binding domain may include a heavy chain variable domain (VH1), a light chain variable domain (VL1), a heavy chain constant domain (CH11), and a light chain constant domain (CL1), and the second target-binding domain may include a heavy chain variable domain (VH2), a light chain variable domain (VL2), a heavy chain constant domain (CH12), and a light chain constant domain (CL2). In one embodiment, the first and / or second target-binding domains are cross-Fab molecules. In one embodiment, the first target-binding domain is a cross-Fab molecule. In one embodiment, the second target-binding domain is a cross-Fab molecule. In one embodiment, the antigen-binding molecule includes first and second target-binding domains, the first target-binding domain is cross-Fab, and the second target-binding domain is a conventional Fab molecule. In one embodiment, the antigen-binding molecule includes a first and a second target-binding domain, the first target-binding domain being a conventional Fab molecule, and the second target-binding domain being a cross-Fab molecule.

[0153] In one embodiment, the antigen-binding molecule includes a first cytokine receptor-binding domain fused at its C-terminus to the N-terminus of VH1 or VL1 of a first target-binding domain, the first target-binding domain being fused at its N-terminus to the C-terminus of a first Fc domain subunit at its VH1 or VL1, the second cytokine receptor-binding domain being fused at its C-terminus to the N-terminus of VH2 or VL2 of a second target-binding domain, and the second target-binding domain being fused at its C-terminus to the N-terminus of a second Fc domain subunit at its CH12.

[0154] In one embodiment, the antigen-binding molecule comprises, in order from the N-terminus to the C-terminus, a first polypeptide comprising a first Fc domain subunit, VH1, and CH11; a second polypeptide comprising, in order from the N-terminus to the C-terminus, a first cytokine receptor-binding domain, VL1, and CL1; a third polypeptide comprising, in order from the N-terminus to the C-terminus, VL2, CH12, and a second Fc domain subunit; and a fourth polypeptide comprising, in order from the N-terminus to the C-terminus, a second cytokine receptor-binding domain, VH2, and CL2.

[0155] In one embodiment, the antigen-binding molecule comprises, in order from the N-terminus to the C-terminus, a first polypeptide comprising a first Fc domain subunit, VL1, and CH11; a second polypeptide comprising, in order from the N-terminus to the C-terminus, a first cytokine receptor-binding domain, VH1, and CL1; a third polypeptide comprising, in order from the N-terminus to the C-terminus, VH2, CH12, and a second Fc domain subunit; and a fourth polypeptide comprising, in order from the N-terminus to the C-terminus, a second cytokine receptor-binding domain, VL2, and CL2.

[0156] In one embodiment, the antigen-binding molecule comprises, in order from the N-terminus to the C-terminus, a first polypeptide comprising a first Fc domain subunit, VL1, and CL1; a second polypeptide comprising, in order from the N-terminus to the C-terminus, a first cytokine receptor-binding domain, VH1, and CH11; a third polypeptide comprising, in order from the N-terminus to the C-terminus, VL2, CH12, and a second Fc domain subunit; and a fourth polypeptide comprising, in order from the N-terminus to the C-terminus, a second cytokine receptor-binding domain, VH2, and CL2.

[0157] In one embodiment, the antigen-binding molecule comprises, in order from the N-terminus to the C-terminus, a first polypeptide comprising a first Fc domain subunit, VH1, and CL1; a second polypeptide comprising, in order from the N-terminus to the C-terminus, a first cytokine receptor-binding domain, VL1, and CH11; a third polypeptide comprising, in order from the N-terminus to the C-terminus, VH2, CH12, and a second Fc domain subunit; and a fourth polypeptide comprising, in order from the N-terminus to the C-terminus, a second cytokine receptor-binding domain, VL2, and CL2.

[0158] In one embodiment, the antigen-binding molecule comprises, in order from the N-terminus to the C-terminus, a first polypeptide comprising a first Fc domain subunit, VH1, and CH11; a second polypeptide comprising, in order from the N-terminus to the C-terminus, a first cytokine receptor-binding domain, VL1, and CL1; a third polypeptide comprising, in order from the N-terminus to the C-terminus, a second cytokine receptor-binding domain, VL2, CH12, and a second Fc domain subunit; and a fourth polypeptide comprising, in order from the N-terminus to the C-terminus, VH2, and CL2.

[0159] In one embodiment, the antigen-binding molecule comprises, in order from the N-terminus to the C-terminus, a first polypeptide comprising a first Fc domain subunit, VL1, and CH11; a second polypeptide comprising, in order from the N-terminus to the C-terminus, a first cytokine receptor-binding domain, VH1, and CL1; a third polypeptide comprising, in order from the N-terminus to the C-terminus, a second cytokine receptor-binding domain, VH2, CH12, and a second Fc domain subunit; and a fourth polypeptide comprising, in order from the N-terminus to the C-terminus, VL2, and CL2.

[0160] In one embodiment, the antigen-binding molecule comprises, in order from the N-terminus to the C-terminus, a first polypeptide comprising a first Fc domain subunit, VL1, and CL1; a second polypeptide comprising, in order from the N-terminus to the C-terminus, a first cytokine receptor-binding domain, VH1, and CH11; a third polypeptide comprising, in order from the N-terminus to the C-terminus, a second cytokine receptor-binding domain, VL2, CH12, and a second Fc domain subunit; and a fourth polypeptide comprising, in order from the N-terminus to the C-terminus, VH2, and CL2.

[0161] In one embodiment, the antigen-binding molecule comprises, in order from the N-terminus to the C-terminus, a first polypeptide comprising a first Fc domain subunit, VH1, and CL1; a second polypeptide comprising, in order from the N-terminus to the C-terminus, a first cytokine receptor-binding domain, VL1, and CH11; and a third polypeptide comprising, in order from the N-terminus to the C-terminus, a second cytokine receptor-binding domain, VH2, CH12, and a second Fc domain subunit.

[0162] In one embodiment, the antigen-binding molecule comprises first and second target-binding domains that specifically bind to FAP, and the first and second cytokine receptor subunits are subunits of an IFNγ receptor complex. In another embodiment, the antigen-binding molecule comprises first and second target-binding domains that specifically bind to FAP, the first cytokine receptor-binding domain can bind to IFNγR1, and the second cytokine receptor-binding domain can bind to IFNγR2. In yet another embodiment, the antigen-binding molecule comprises first and second target-binding domains that specifically bind to FAP, the first cytokine receptor-binding domain can bind to IFNγR2, and the second cytokine receptor-binding domain can bind to IFNγR1.

[0163] In one embodiment, the antigen-binding molecule comprises, in order from the N-terminus to the C-terminus, a first polypeptide containing a first Fc domain subunit, VH1, and CH11; a second polypeptide containing a first cytokine receptor-binding domain, VL1, and CL1, in order from the N-terminus to the C-terminus; a third polypeptide containing VL2, CH12, and a second Fc domain subunit, in order from the N-terminus to the C-terminus; and a fourth polypeptide containing a second cytokine receptor-binding domain, VH2, and CL2, in order from the N-terminus to the C-terminus, wherein VH1, VL1, CH11, and CL1 form a first target-binding domain, and VH2, VL2, CH12, and CL2 form a second target-binding domain, the first and second target-binding domains bind to FAP, and the first and second cytokine receptor-binding domains bind to subunits of the IFNγ receptor complex.

[0164] In one embodiment, the antigen-binding molecule comprises, in order from the N-terminus to the C-terminus, a first polypeptide containing a first Fc domain subunit, VL1, and CH11; a second polypeptide containing a first cytokine receptor-binding domain, VH1, and CL1, in order from the N-terminus to the C-terminus; a third polypeptide containing VH2, CH12, and a second Fc domain subunit, in order from the N-terminus to the C-terminus; and a fourth polypeptide containing a second cytokine receptor-binding domain, VL2, and CL2, in order from the N-terminus to the C-terminus, wherein VH1, VL1, CH11, and CL1 form a first target-binding domain, and VH2, VL2, CH12, and CL2 form a second target-binding domain, the first and second target-binding domains bind to FAP, and the first and second cytokine receptor-binding domains bind to subunits of the IFNγ receptor complex.

[0165] In one embodiment, the antigen-binding molecule comprises, in order from the N-terminus to the C-terminus, a first polypeptide containing a first Fc domain subunit, VL1, and CL1; a second polypeptide containing a first cytokine receptor-binding domain, VH1, and CH11; a third polypeptide containing VL2, CH12, and a second Fc domain subunit; and a fourth polypeptide containing a second cytokine receptor-binding domain, VH2, and CL2; VH1, VL1, CH11, and CL1 form a first target-binding domain, VH2, VL2, CH12, and CL2 form a second target-binding domain; the first and second target-binding domains bind to FAP; and the first and second cytokine receptor-binding domains bind to subunits of the IFNγ receptor complex.

[0166] In one embodiment, the antigen-binding molecule comprises, in order from the N-terminus to the C-terminus, a first polypeptide containing a first Fc domain subunit, VH1, and CL1; a second polypeptide containing a first cytokine receptor-binding domain, VL1, and CH11, in order from the N-terminus to the C-terminus; a third polypeptide containing VH2, CH12, and a second Fc domain subunit, in order from the N-terminus to the C-terminus; and a fourth polypeptide containing a second cytokine receptor-binding domain, VL2, and CL2, in order from the N-terminus to the C-terminus, wherein VH1, VL1, CH11, and CL1 form a first target-binding domain, and VH2, VL2, CH12, and CL2 form a second target-binding domain, the first and second target-binding domains bind to FAP, and the first and second cytokine receptor-binding domains bind to subunits of the IFNγ receptor complex.

[0167] In one embodiment, the antigen-binding molecule comprises, in order from the N-terminus to the C-terminus, a first polypeptide containing a first Fc domain subunit, VH1, and CH11; a second polypeptide containing a first cytokine receptor-binding domain, VL1, and CL1, in order from the N-terminus to the C-terminus; a third polypeptide containing a second cytokine receptor-binding domain, VL2, CH12, and a second Fc domain subunit, in order from the N-terminus to the C-terminus; and a fourth polypeptide containing VH2 and CL2, in order from the N-terminus to the C-terminus, wherein VH1, VL1, CH11, and CL1 form a first target-binding domain, and VH2, VL2, CH12, and CL2 form a second target-binding domain, the first and second target-binding domains bind to FAP, and the first and second cytokine receptor-binding domains bind to subunits of the IFNγ receptor complex.

[0168] In one embodiment, the antigen-binding molecule comprises, in order from the N-terminus to the C-terminus, a first polypeptide comprising a first Fc domain subunit, VL1, and CH11; a second polypeptide comprising, in order from the N-terminus to the C-terminus, a first cytokine receptor-binding domain, VH1, and CL1; a third polypeptide comprising, in order from the N-terminus to the C-terminus, a second cytokine receptor-binding domain, VH2, CH12, and a second Fc domain subunit; and a fourth polypeptide comprising, in order from the N-terminus to the C-terminus, VL2, CL2, VH1, CH11, and CL1, forming a first target-binding domain; VH2, VL2, CH12, and CL2, forming a second target-binding domain; the first and second target-binding domains bind to FAP; and the first and second cytokine receptor-binding domains bind to subunits of the IFNγ receptor complex.

[0169] In one embodiment, the antigen-binding molecule comprises, in order from the N-terminus to the C-terminus, a first polypeptide containing a first Fc domain subunit, VL1, and CL1; a second polypeptide containing a first cytokine receptor-binding domain, VH1, and CH11; a third polypeptide containing a second cytokine receptor-binding domain, VL2, CH12, and a second Fc domain subunit; and a fourth polypeptide containing VH2 and CL2, in order from the N-terminus to the C-terminus, wherein VH1, VL1, CH11, and CL1 form a first target-binding domain, and VH2, VL2, CH12, and CL2 form a second target-binding domain, the first and second target-binding domains bind to FAP, and the first and second cytokine receptor-binding domains bind to subunits of the IFNγ receptor complex.

[0170] In one embodiment, the antigen-binding molecule comprises, in order from the N-terminus to the C-terminus, a first polypeptide comprising a first Fc domain subunit, VH1, and CL1; a second polypeptide comprising, in order from the N-terminus to the C-terminus, a first cytokine receptor-binding domain, VL1, and CH11; a third polypeptide comprising, in order from the N-terminus to the C-terminus, a second cytokine receptor-binding domain, VH2, CH12, and a second Fc domain subunit; and a fourth polypeptide comprising, in order from the N-terminus to the C-terminus, VL2, CL2, VH1, CH11, and CL1, forming a first target-binding domain; VH2, VL2, CH12, and CL2, forming a second target-binding domain; the first and second target-binding domains bind to FAP; and the first and second cytokine receptor-binding domains bind to subunits of the IFNγ receptor complex.

[0171] In one embodiment, the antigen-binding molecule comprises first and second target-binding domains that specifically bind to PD-1, and the first and second cytokine receptor subunits are subunits of the IL-2 receptor complex. In another embodiment, the antigen-binding molecule comprises first and second target-binding domains that specifically bind to FAP, the first cytokine receptor-binding domain can bind to IL-2Rβ, and the second cytokine receptor-binding domain can bind to IL-2Rγ. In yet another embodiment, the antigen-binding molecule comprises first and second target-binding domains that specifically bind to FAP, the first cytokine receptor-binding domain can bind to IL-2Rγ, and the second cytokine receptor-binding domain can bind to IL-2Rβ.

[0172] In one embodiment, the antigen-binding molecule comprises, in order from the N-terminus to the C-terminus, a first polypeptide containing a first Fc domain subunit, VL1, and CL1; a second polypeptide containing a first cytokine receptor-binding domain, VH1, and CH11; a third polypeptide containing VL2, CH12, and a second Fc domain subunit; and a fourth polypeptide containing a second cytokine receptor-binding domain, VH2, and CL2; VH1, VL1, CH11, and CL1 form a first target-binding domain, VH2, VL2, CH12, and CL2 form a second target-binding domain; the first and second target-binding domains bind to PD-1; and the first and second cytokine receptor-binding domains bind to subunits of the IL-2 receptor complex.

[0173] In one embodiment, the antigen-binding molecule comprises, in order from the N-terminus to the C-terminus, a first polypeptide containing a first Fc domain subunit, VH1, and CL1; a second polypeptide containing a first cytokine receptor-binding domain, VL1, and CH11, in order from the N-terminus to the C-terminus; a third polypeptide containing VH2, CH12, and a second Fc domain subunit, in order from the N-terminus to the C-terminus; and a fourth polypeptide containing a second cytokine receptor-binding domain, VL2, and CL2, in order from the N-terminus to the C-terminus, wherein VH1, VL1, CH11, and CL1 form a first target-binding domain, and VH2, VL2, CH12, and CL2 form a second target-binding domain, the first and second target-binding domains bind to PD-1, and the first and second cytokine receptor-binding domains bind to subunits of the IL-2 receptor complex.

[0174] In one embodiment, the antigen-binding molecule comprises, in order from the N-terminus to the C-terminus, a first polypeptide comprising a first Fc domain subunit, VH1, and CH11; a second polypeptide comprising, in order from the N-terminus to the C-terminus, a first cytokine receptor-binding domain, VL1, and CL1; a third polypeptide comprising, in order from the N-terminus to the C-terminus, a second cytokine receptor-binding domain, VL2, CH12, and a second Fc domain subunit; and a fourth polypeptide comprising, in order from the N-terminus to the C-terminus, VH2, VL1, CH11, and CL1, forming a first target-binding domain; VH2, VL2, CH12, and CL2, forming a second target-binding domain; the first and second target-binding domains bind to PD-1; and the first and second cytokine receptor-binding domains bind to subunits of the IL-2 receptor complex.

[0175] In one embodiment, the antigen-binding molecule comprises, in order from the N-terminus to the C-terminus, a first polypeptide comprising a first Fc domain subunit, VL1, and CH11; a second polypeptide comprising, in order from the N-terminus to the C-terminus, a first cytokine receptor-binding domain, VH1, and CL1; a third polypeptide comprising, in order from the N-terminus to the C-terminus, a second cytokine receptor-binding domain, VH2, CH12, and a second Fc domain subunit; and a fourth polypeptide comprising, in order from the N-terminus to the C-terminus, VL2, CL2, VH1, CH11, and CL1, forming a first target-binding domain; VH2, VL2, CH12, and CL2, forming a second target-binding domain; the first and second target-binding domains bind to PD-1; and the first and second cytokine receptor-binding domains bind to subunits of the IL-2 receptor complex.

[0176] In one embodiment, the antigen-binding molecule comprises, in order from the N-terminus to the C-terminus, a first polypeptide containing a first Fc domain subunit, VL1, and CL1; a second polypeptide containing a first cytokine receptor-binding domain, VH1, and CH11; a third polypeptide containing a second cytokine receptor-binding domain, VL2, CH12, and a second Fc domain subunit; and a fourth polypeptide containing VH2 and CL2, in order from the N-terminus to the C-terminus, wherein VH1, VL1, CH11, and CL1 form a first target-binding domain, and VH2, VL2, CH12, and CL2 form a second target-binding domain, the first and second target-binding domains bind to PD-1, and the first and second cytokine receptor-binding domains bind to subunits of the IL-2 receptor complex.

[0177] In one embodiment, the antigen-binding molecule comprises, in order from the N-terminus to the C-terminus, a first polypeptide containing a first Fc domain subunit, VH1, and CL1; a second polypeptide containing a first cytokine receptor-binding domain, VL1, and CH11, in order from the N-terminus to the C-terminus; a third polypeptide containing a second cytokine receptor-binding domain, VH2, CH12, and a second Fc domain subunit, in order from the N-terminus to the C-terminus; and a fourth polypeptide containing VL2 and CL2, in order from the N-terminus to the C-terminus, wherein VH1, VL1, CH11, and CL1 form a first target-binding domain, and VH2, VL2, CH12, and CL2 form a second target-binding domain, the first and second target-binding domains bind to PD-1, and the first and second cytokine receptor-binding domains bind to subunits of the IL-2 receptor complex.

[0178] The domains of the binding molecule (target antigen-binding domain, cytokine receptor-binding domain, and Fc domain) can be fused to one or more peptide linkers, particularly (G4S)n peptide linkers. The cytokine receptor-binding domain can be linked to the target antigen-binding domain via a (G4S)1 peptide linker (GGGGS, SEQ ID NO: 118), a (G4S)2 peptide linker (GGGGGSGGGGS, SEQ ID NO: 119), a (G4S)3 peptide linker (GGGGSGGGGSGGGGS, SEQ ID NO: 120), or a (G4S)5 peptide linker (GGGGSGGGGSGGGGSGGGGSGGGGS, SEQ ID NO: 121). The target-binding domain can be fused to the C-terminus of a single Fc domain subunit via a (G4S)1 peptide linker, a (G4S)2 peptide linker, a (G4S)3 peptide linker, or a (G4S)5 peptide linker at its N-terminus of its VH or VL.

[0179] In one embodiment, the antigen-binding molecule comprises a first polypeptide containing the amino acid sequence of SEQ ID NO: 50, a second polypeptide containing the amino acid sequence of SEQ ID NO: 48, a third polypeptide containing the amino acid sequence of SEQ ID NO: 51, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 49. In another embodiment, the antigen-binding molecule comprises a first polypeptide containing the amino acid sequence of SEQ ID NO: 99, a second polypeptide containing the amino acid sequence of SEQ ID NO: 97, a third polypeptide containing the amino acid sequence of SEQ ID NO: 100, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 98. In yet another embodiment, the antigen-binding molecule comprises a first polypeptide containing the amino acid sequence of SEQ ID NO: 103, a second polypeptide containing the amino acid sequence of SEQ ID NO: 101, a third polypeptide containing the amino acid sequence of SEQ ID NO: 104, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 102. In yet another embodiment, the antigen-binding molecule comprises a first polypeptide containing the amino acid sequence of SEQ ID NO: 107, a second polypeptide containing the amino acid sequence of SEQ ID NO: 105, a third polypeptide containing the amino acid sequence of SEQ ID NO: 108, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 106. In one embodiment, the antigen-binding molecule comprises a first polypeptide containing the amino acid sequence of SEQ ID NO: 99, a second polypeptide containing the amino acid sequence of SEQ ID NO: 109, a third polypeptide containing the amino acid sequence of SEQ ID NO: 100, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 110. In another embodiment, the antigen-binding molecule comprises a first polypeptide containing the amino acid sequence of SEQ ID NO: 107, a second polypeptide containing the amino acid sequence of SEQ ID NO: 111, a third polypeptide containing the amino acid sequence of SEQ ID NO: 113, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 112. In yet another embodiment, the antigen-binding molecule comprises a first polypeptide containing the amino acid sequence of SEQ ID NO: 99, a second polypeptide containing the amino acid sequence of SEQ ID NO: 114, a third polypeptide containing the amino acid sequence of SEQ ID NO: 100, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 115.In one embodiment, the antigen-binding molecule comprises a first polypeptide containing the amino acid sequence of SEQ ID NO: 107, a second polypeptide containing the amino acid sequence of SEQ ID NO: 116, a third polypeptide containing the amino acid sequence of SEQ ID NO: 108, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 117.

[0180] In one embodiment, the antigen-binding molecule comprises a first polypeptide containing the amino acid sequence of SEQ ID NO: 123, a second polypeptide containing the amino acid sequence of SEQ ID NO: 122, a third polypeptide containing the amino acid sequence of SEQ ID NO: 124, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 125.

[0181] Specific embodiments of the present invention The following lists specific embodiments of the present invention.

[0182] 1. An antigen-binding molecule, i) The first target-binding domain, ii) The second target-binding domain, iii) The first cytokine receptor binding domain, iv) The second cytokine receptor binding domain, v) Including the Fc domain, The first target-binding domain can bind to a first epitope on the target antigen, the second target-binding domain can bind to a second epitope on the target antigen, and the first and second target-binding domains do not compete for binding to tumor-associated antigens. The first cytokine receptor binding domain can bind to the first cytokine receptor subunit, and the second cytokine receptor binding domain can bind to the second cytokine receptor subunit. Antigen-binding molecules.

[0183] 2. The antigen-binding molecule according to Embodiment 1, wherein the first and second target-binding domains are antibody fragments, particularly Fv, Fab, scFv, scFab, or single-domain antibodies.

[0184] 3. The antigen-binding molecule according to Embodiment 1 or 2, wherein the first and second target-binding domains are Fab molecules.

[0185] 4. An antigen-binding molecule according to any one of Embodiments 1 to 3, wherein the first target-binding domain comprises a heavy chain variable domain (VH1), a light chain variable domain (VL1), a heavy chain constant domain (CH11), and a light chain constant domain (CL1), and the second target-binding domain comprises a heavy chain variable domain (VH2), a light chain variable domain (VL2), a heavy chain constant domain (CH12), and a light chain constant domain (CL2).

[0186] 5. An antigen-binding molecule according to any one of Embodiments 1 to 4, wherein the first target-binding domain and / or the second target-binding domain is a cross-Fab molecule.

[0187] 6. An antigen-binding molecule according to any one of Embodiments 1 to 5, wherein the first target-binding domain and / or the second target-binding domain includes a charge mutation.

[0188] 7. An antigen-binding molecule according to any one of Embodiments 1 to 6, wherein the first target-binding domain is cross-Fab and the second target-binding domain contains a charge mutation, or the second target-binding domain is cross-Fab and the first target-binding domain contains a charge mutation.

[0189] 8. An antigen-binding molecule according to any one of Embodiments 1 to 7, wherein the first target-binding domain and the second target-binding domain specifically bind to a tumor-associated antigen or a T cell antigen.

[0190] 9. An antigen-binding molecule according to any one of Embodiments 1 to 8, wherein the first target-binding domain and the second target-binding domain specifically bind to FAP, PD-1, Her2, Her3, LAG-3, CEA, or EGFR.

[0191] 10. An antigen-binding molecule according to any one of Embodiments 1 to 9, wherein the first target-binding domain and the second target-binding domain specifically bind to FAP or PD-1.

[0192] 11.a) The first target-binding domain includes VH1 of SEQ ID NO: 20 and VL1 of SEQ ID NO: 21, and the second target-binding domain includes VH2 of SEQ ID NO: 22 and VL2 of SEQ ID NO: 23, or b) The first target-binding domain includes VH1 of SEQ ID NO: 22 and VL1 of SEQ ID NO: 23, and the second target-binding domain includes VH2 of SEQ ID NO: 20 and VL2 of SEQ ID NO: 21, or c) The first target-binding domain includes VH1 of SEQ ID NO: 80 and VL1 of SEQ ID NO: 81, and the second target-binding domain includes VH2 of SEQ ID NO: 82 and VL2 of SEQ ID NO: 83, or d) The first target-binding domain includes VH1 of SEQ ID NO: 82 and VL1 of SEQ ID NO: 83, and the second target-binding domain includes VH2 of SEQ ID NO: 80 and VL2 of SEQ ID NO: 81, or e) The first target-binding domain includes VH1 of SEQ ID NO: 82 and VL1 of SEQ ID NO: 83, and the second target-binding domain includes VH2 of SEQ ID NO: 140 and VL2 of SEQ ID NO: 141, or f) The first target-binding domain includes VH1 of SEQ ID NO: 140 and VL1 of SEQ ID NO: 141, and the second target-binding domain includes VH2 of SEQ ID NO: 82 and VL2 of SEQ ID NO: 83. An antigen-binding molecule according to any one of Embodiments 1 to 10.

[0193] 12. An antigen-binding molecule according to any one of embodiments 1 to 11, wherein both the first and second cytokine receptor subunits are subunits of an IFNγ receptor complex or an IL-2 receptor complex.

[0194] 13.a) The first cytokine receptor binding domain can bind to IFNγR1 and the second cytokine receptor binding domain can bind to IFNγR2, or b) The first cytokine receptor binding domain can bind to IFNγR2, and the second cytokine receptor binding domain can bind to IFNγR1, c) The first cytokine receptor binding domain can bind to IL-2Rβ and the second cytokine receptor binding domain can bind to IL-2Rγ, or d) The first cytokine receptor binding domain can bind to IL-2Rγ, and the second cytokine receptor binding domain can bind to IL-2Rβ. An antigen-binding molecule according to any one of Embodiments 1 to 12.

[0195] 14. An antigen-binding molecule according to any one of Embodiments 1 to 13, wherein the first and second cytokine receptor-binding domains are antibody fragments, particularly Fv, Fab, scFv, scFab, single-domain antibody, or VHH domain.

[0196] 15. An antigen-binding molecule according to any one of embodiments 1 to 14, wherein the first and second cytokine receptor-binding domains are VHH domains.

[0197] 16.a) The first cytokine receptor binding domain contains 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 contains an amino acid sequence selected from SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 9, or b) The first cytokine receptor binding domain comprises an amino acid sequence selected from SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 9, 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 includes the sequences of SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, and SEQ ID NO: 64, and the second cytokine receptor binding domain includes the sequences of SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, and SEQ ID NO: 69, or d) The first cytokine receptor binding domain includes the sequences of SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68 and SEQ ID NO: 69, and the second cytokine receptor binding domain includes the sequences of SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62 and SEQ ID NO: 64, An antigen-binding molecule according to any one of Embodiments 1 to 15.

[0198] 17. An antigen-binding molecule according to any one of Embodiments 1 to 16, wherein the Fc domain comprises a first Fc domain subunit and a second Fc domain subunit.

[0199] 18. An antigen-binding molecule according to any one of Embodiments 1 to 17, wherein the Fc domain is IgG, particularly the IgG1Fc domain.

[0200] 19. An antigen-binding molecule according to any one of Embodiments 1 to 18, wherein the Fc domain is a human Fc domain.

[0201] 20. An antigen-binding molecule according to any one of Embodiments 1 to 19, wherein the Fc domain includes a modification that promotes the association of the first and second subunits of the Fc domain.

[0202] 21. An antigen-binding molecule according to any one of Embodiments 1 to 20, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to and / or effector function of the Fc receptor.

[0203] 22. An antigen-binding molecule according to any one of Embodiments 1 to 21, wherein a first cytokine receptor-binding domain is fused at its C-terminus to the N-terminus of VH1 or VL1 of a first target-binding domain, the first target-binding domain is fused at its N-terminus to the C-terminus of a first Fc domain subunit, the second cytokine receptor-binding domain is fused at its C-terminus to the N-terminus of VH2 or VL2 of a second target-binding domain, and the second target-binding domain is fused at its C-terminus to the N-terminus of a second Fc domain subunit, wherein the CH12 or CL1 of the second target-binding domain is fused at its C-terminus to the N-terminus of a second Fc domain subunit.

[0204] 23. An antigen-binding molecule according to any one of Embodiments 1 to 22, a) comprising, in order from the N-terminus to the C-terminus, a first polypeptide containing a first Fc domain subunit, VH1, and CH11; a second polypeptide containing a first cytokine receptor binding domain, VL1, and CL1, in order from the N-terminus to the C-terminus; a third polypeptide containing VL2, CH12, and a second Fc domain subunit, in order from the N-terminus to the C-terminus; and a fourth polypeptide containing a second cytokine receptor binding domain, VH2, and CL2, in order from the N-terminus to the C-terminus, or b) comprising, in order from the N-terminus to the C-terminus, a first polypeptide containing a first Fc domain subunit, VL1, and CH11; a second polypeptide containing a first cytokine receptor binding domain, VH1, and CL1, in order from the N-terminus to the C-terminus; a third polypeptide containing VH2, CH12, and a second Fc domain subunit, in order from the N-terminus to the C-terminus; and a fourth polypeptide containing a second cytokine receptor binding domain, VL2, and CL2, in order from the N-terminus to the C-terminus, or c) comprising, in order from the N-terminus to the C-terminus, a first polypeptide containing a first Fc domain subunit, VL1, and CL1; a second polypeptide containing a first cytokine receptor binding domain, VH1, and CH11, in order from the N-terminus to the C-terminus; a third polypeptide containing VL2, CH12, and a second Fc domain subunit, in order from the N-terminus to the C-terminus; and a fourth polypeptide containing a second cytokine receptor binding domain, VH2, and CL2, in order from the N-terminus to the C-terminus, or d) comprising, in order from the N-terminus to the C-terminus, a first polypeptide containing a first Fc domain subunit, VH1, and CL1; a second polypeptide containing, in order from the N-terminus to the C-terminus, a first cytokine receptor binding domain, VL1, and CH11; a third polypeptide containing, in order from the N-terminus to the C-terminus, VH2, CH12, and a second Fc domain subunit; and a fourth polypeptide containing, in order from the N-terminus to the C-terminus, a second cytokine receptor binding domain, VL2, and CL2; or e) comprising, in order from the N-terminus to the C-terminus, a first polypeptide containing a first Fc domain subunit, VH1, and CH11; a second polypeptide containing a first cytokine receptor binding domain, VL1, and CL1, in order from the N-terminus to the C-terminus; a third polypeptide containing a second cytokine receptor binding domain, VL2, CH12, and a second Fc domain subunit, in order from the N-terminus to the C-terminus; and a fourth polypeptide containing VH2 and CL2, in order from the N-terminus to the C-terminus, or f) A polypeptide comprising, in order from the N-terminus to the C-terminus, a first polypeptide containing a first Fc domain subunit, VL1, and CH11; a second polypeptide comprising, in order from the N-terminus to the C-terminus, a first cytokine receptor binding domain, VH1, and CL1; a third polypeptide comprising, in order from the N-terminus to the C-terminus, a second cytokine receptor binding domain, VH2, CH12, and a second Fc domain subunit; and a fourth polypeptide comprising, in order from the N-terminus to the C-terminus, VL2, and CL2; or g) comprising, in order from the N-terminus to the C-terminus, a first polypeptide containing a first Fc domain subunit, VL1 and CL1; a second polypeptide containing, in order from the N-terminus to the C-terminus, a first cytokine receptor binding domain, VH1 and CH11; a third polypeptide containing, in order from the N-terminus to the C-terminus, a second cytokine receptor binding domain, VL2, CH12 and a second Fc domain subunit; and a fourth polypeptide containing, in order from the N-terminus to the C-terminus, VH2 and CL2; or h) A first polypeptide comprising, in order from the N-terminus to the C-terminus, a first Fc domain subunit, VH1, and CL1; a second polypeptide comprising, in order from the N-terminus to the C-terminus, a first cytokine receptor binding domain, VL1, and CH11; a third polypeptide comprising, in order from the N-terminus to the C-terminus, a second cytokine receptor binding domain, VH2, CH12, and a second Fc domain subunit; and a fourth polypeptide comprising, in order from the N-terminus to the C-terminus, VL2, and CL2. Antigen-binding molecules in which VH1, VL1, CH11, and CL1 form a first target-binding domain, and VH2, VL2, CH12, and CL2 form a second target-binding domain.

[0205] 24. An antigen-binding molecule according to any one of Embodiments 1 to 23, wherein the first target-binding domain and the second target-binding domain specifically bind to FAP, and the first and second cytokine receptor subunits are subunits of an IFNγ receptor complex.

[0206] 25. An antigen-binding molecule according to any one of Embodiments 1 to 24, a) A first polypeptide containing the amino acid sequence of SEQ ID NO: 50, a second polypeptide containing the amino acid sequence of SEQ ID NO: 48, a third polypeptide containing the amino acid sequence of SEQ ID NO: 51, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 49. b) A first polypeptide containing the amino acid sequence of SEQ ID NO: 99, a second polypeptide containing the amino acid sequence of SEQ ID NO: 97, a third polypeptide containing the amino acid sequence of SEQ ID NO: 100, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 98. c) A first polypeptide containing the amino acid sequence of SEQ ID NO: 103, a second polypeptide containing the amino acid sequence of SEQ ID NO: 101, a third polypeptide containing the amino acid sequence of SEQ ID NO: 104, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 102. d) A first polypeptide containing the amino acid sequence of SEQ ID NO: 107, a second polypeptide containing the amino acid sequence of SEQ ID NO: 105, a third polypeptide containing the amino acid sequence of SEQ ID NO: 108, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 106. e) A first polypeptide containing the amino acid sequence of SEQ ID NO: 99, a second polypeptide containing the amino acid sequence of SEQ ID NO: 109, a third polypeptide containing the amino acid sequence of SEQ ID NO: 100, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 110. f) A first polypeptide containing the amino acid sequence of SEQ ID NO: 107, a second polypeptide containing the amino acid sequence of SEQ ID NO: 111, a third polypeptide containing the amino acid sequence of SEQ ID NO: 113, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 112. g) A first polypeptide containing the amino acid sequence of SEQ ID NO: 99, a second polypeptide containing the amino acid sequence of SEQ ID NO: 114, a third polypeptide containing the amino acid sequence of SEQ ID NO: 100, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 115. h) A first polypeptide comprising the amino acid sequence of SEQ ID NO: 107, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 116, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 108, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 117. Antigen-binding molecules.

[0207] 26. An antigen-binding molecule according to any one of Embodiments 1 to 23, wherein the first target-binding domain and the second target-binding domain specifically bind to PD-1, and the first and second cytokine receptor subunits are subunits of the IL-2 receptor complex.

[0208] 27. The antigen-binding molecule according to Embodiment 25, comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 123, a second polypeptide containing the amino acid sequence of SEQ ID NO: 122, a third polypeptide containing the amino acid sequence of SEQ ID NO: 124, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 125. [Examples]

[0209] Example 1: Production of a binder for cytokine receptor subunits 1.1 Antigen production for immunization and phage display For llama immunization, the extracellular domain (ECD) of the human IFNγR1 subunit and the ECD of the human IFNγR2 subunit were co-expressed as Fc-tagged heterodimers based on knob-into-hole technology (Figure 1A, P1AG0843). Similarly, the ECDs of the human IL-2Rβ (CD122) subunit and the human IL-2Rγ (common gamma chain, CD132) subunit were co-expressed as Fc-tagged heterodimers (Figure 1D, P1AE2657). The C-terminus of the IFNγR1 ECD was fused to the Fc knob via a G4SG4 linker. For site-specific biotinylation during co-expression with BirA biotin ligase, AviTag was included at the C-terminus of Fc. The IFNγR2 ECD containing a C174S substitution was fused to the Fc hole with a G4SG4 linker, and the construct contained a C-terminal Twin-Strep tag. The C-terminus of IL-2RβECD was fused to the Fc knob via a GAQ linker. AviTag was included at the C-terminus of Fc for site-directed biotinylation during co-expression with BirA biotin ligase. IL-2RγECD was C-fused to the Fc hole using a GAQ linker.

[0210] For phage display, monovalent IFNγR1 ECD fused to a biotinylated Fc knob paired with an empty Fc hole (Figure 1B, P1AG1987), monovalent IFNγR2 ECD C174S fused to an Fc hole paired with an empty biotinylated Fc knob (Figure 1C, P1AG6735), and monovalent IL-2Rβ ECD fused to a biotinylated Fc knob paired with an empty Fc hole (Figure 1E, P1AF1104) and monovalent IL-2Rγ ECD fused to an Fc hole paired with an empty biotinylated Fc knob (Figure 1F, P1AF1106). All antigens were produced using the Expi293 mammalian expression system (Expi293® Expression System Kit, Thermo Fisher Scientific, catalog number: A14635) according to the manufacturer's recommendations. The supernatant was collected by centrifugation, filtered, and purified by Protein A affinity chromatography (Protein A MabSelect® SuRe®, Cytiva, catalog number: 17543803) according to the manufacturer's recommendations, followed by size exclusion chromatography (SEC) (HiLoad S200 16 / 600, Cytiva, catalog number: 28989335).

[0211] 1.2 Building a VHH Library The VHH library was approximately 3 × 10 from the second blood draw of a single llama. 8 Individual peripheral blood mononuclear cells (PBMCs), and approximately 6.6 × 10⁶ cells from the third blood sample. 8 Constructed by ProSci Inc. using PBMCs. The library is provided in TG1 E. coli strain encoded in a derivative of the pADL-23c phagemide vector (Antibody Design Labs, San Diego, California), and contains approximately 1 × 10⁶ cells. 9 The libraries were incorporated with an insertion efficiency of over 95% by independent electroporation events. The libraries were approximately 3.2 × 10¹⁶ in 2 × YT medium containing 20% ​​glycerol. 10 The E. coli was provided as cfu / mL.

[0212] 1.3 Preparation of a VHH phage library E. coli containing a VHH-coded phagemid library was inoculated into 2xYT medium containing 100 μg / ml ampicillin and 1% glucose. After infection with VCSM13 helper phage, E. coli was grown in 2xYT medium containing 100 μg / ml ampicillin and 50 μg / ml kanamycin. After incubation overnight on a 30°C shaker, the culture was collected by centrifugation at 8000 rpm for 20 minutes, and the supernatant was collected. The phages were precipitated in 20% PEG / 2.5M NaCl solution and incubated on ice for 1 hour. The precipitated phages were centrifuged at 8000 rpm for 20 minutes, the pellet was resuspended in ddH2O, and then precipitated in PEG / NaCl solution on ice for 1 hour. The phages were centrifuged at 8000 rpm for 20 minutes, and the pellet was resuspended in PBS containing 20% ​​glycerol.

[0213] 1.4 Isolation of IFNγR1-specific, IFNγR2-specific, IL-2Rβ-specific, and IL-2Rγ-specific VHH by phage display To selectively enrich antigen-specific binders and prevent enrichment of Fc-specific binders, competitive solubility selection of human IFNγR1 antigen (P1AG1987), human IFNγR2 antigen (P1AG6735), human IL-2Rβ antigen (P1AF1104), and human IL-2Rγ antigen (P1AF1106) was performed in the presence of human Fc (Figure 1G, P1AD4290). 1 × 10⁶ samples were taken from a llama-derived VHH library. 11Phage aliquots were used for each antigen. The phages and magnetic streptavidin beads (Dynabeads, M-280 streptavidin, Invitrogen, catalog number: 11205D) were independently blocked in phosphate-buffered saline (PBS) with 1% BSA and 0.1% Tween-20 to reduce nonspecific interactions. Unless otherwise specified, all incubations were performed at 20°C for 1 hour. Biotinylated Fc-tagged antigens were added to the blocked phages at a concentration of 100 nM in the presence of an excess amount (500 nM) of non-biotinylated Fc (Figure 2). After incubation, pre-blocking Dynabeads were added to capture the target biotinylated antigen and the bound VHH-expressing phages. Fc-specific and non-specific phages were removed by 5-10 washes in PBS containing 0.1% Tween-20, followed by PBS treatment using a KingFisher magnetic particle processor (Thermo Fisher Scientific). Antigen-specific phages were eluted with 800 μl of 100 mM trimethylamine (TEA) and neutralized by adding 400 μl of 1 M Tris pH 7.4. TG1 E. coli in the mid-logarithmic growth phase in 2×YT medium were infected with the eluted phages, followed by infection with VCSM13 helper phages. After adding 100 μg / ml ampicillin and 50 μg / ml kanamycin, the infected E. coli were incubated overnight on a shaker at 30°C. The phages were precipitated with PEG / NaCl and used in the next round. While maintaining a constant soluble Fc concentration (500 nM), the target antigen concentration (100 nM, 50 nM, 25 nM) was decreased, and a total of three rounds of selection were performed (Figure 2).

[0214] 1.5 Verification of target specificity using ELISA The third round of concentrated libraries was screened for target specificity versus Fc specificity by ELISA. Individual VHH clones were expressed in 1 ml of E. coli culture in a 96-well plate, and the supernatant containing soluble VHH was screened for specificity by ELISA. The supernatant was transferred to a 96-well plate coated with biotinylated Fc (P1AE6073) or targeted Fc-tagged biotinylated antigen (P1AG1987, P1AG6735, P1AF1104, or P1AF1106) using Nutraavidin. After incubation at 20°C for 1 hour, the plate was washed three times with PBST and three times with PBS. HRP-conjugated anti-His antibody (Sigma, catalog number: A7058) was added to the wells at a 1:2000 dilution, and the plate was incubated at room temperature for 1 hour. The plate was washed three times with PBST and then three times with PBS. The plates were color-developed by adding the 1-Step Ultra TMB-ELISA substrate (Thermo Fisher Scientific, catalog number: 34028), and the reaction mixture was quenched with sulfuric acid. Absorbance at 450 nm was measured using a Tecan infinite M1000 Pro, and the reference absorbance value was measured at 650 nm. The reference subtracted absorbance values ​​at 450 nm were represented as stacked bar graphs for IFNγR1-specific VHH (Figure 3A), IFNγR2-specific VHH (Figure 3B), IL-2Rβ-specific VHH (Figure 3C), and IL-2Rγ-specific VHH (Figure 3D). The ELISA data suggested that over 90% of the clones were target-specific, and subsequently, VHH from the third-round enrichment library was cloned into a bispecific heavy-chain antibody format.

[0215] Example 2: Evaluation of agonist activity against IFNγ and IL-2 receptors 2.1 Binding of enriched VHH libraries to heavy chain antibody format IFNγR activation is achieved by IFNγ-mediated crosslinking of IFNγR1 and IFNγR2 receptor chains. To mimic binding in an IFNγ-independent manner, a VHH domain with IFNγR1 specificity was paired with IFNγR2-specific VHH by fusing it to a knob-into-hole Fc chain via a flexible linker that generates an IFNγR1-IFNγR2 bispecific heavy chain antibody. IL-2R activation is achieved by IL2-mediated crosslinking of IL-2Rβ and IL-2Rγ receptor chains. Therefore, to mimic binding in an IL2-independent manner, a VHH domain with IL-2Rβ specificity was paired with IL-2Rγ-specific VHH by fusing it to a knob-into-hole Fc chain via a flexible linker that generates an IL-2Rβ-IL-2Rγ bispecific heavy chain antibody. For this purpose, third-round VHH libraries enriched with IFNγR1 specificity or IL-2Rβ specificity were cloned in bulk using Gibson binding to achieve VHH format conversion by fusing them to the Fc knob via a 5(G4S) linker (Figure 4A). Similarly, third-round IFNγR2-specific VHH libraries or IL-2Rγ-specific VHH libraries were fusing to the Fc hole via a 5(G4S) linker using Gibson binding (Figure 4B). Appropriate signal sequences and vectors suitable for mammalian expression were included during binding. Primers, PCR conditions, and Gibson binding reactions were defined according to the New England Biolabs NEBuilder HiFi DNA Assembly protocol (catalog number: E5510). After bacterial transformation, colonies were randomly selected, and five unique sequences were chosen from each specificity pool. Using the Expi293 system, a 5×5 matrix was constructed to express 25 unique knob-into-hole bispecific heavy chain antibodies containing VHH pairs of IFNγR1 and IFNγR2, as well as 25 unique knob-into-hole bispecific heavy chain antibodies containing VHH pairs of IL-2Rβ and IL-2Rγ (Figure 5).Expi293 cells were transiently transfected according to the manufacturer's recommendations (Expi293® Expression System Kit, Thermo Fisher Scientific, catalog number: A14635), and the supernatant containing secreted soluble bispecific heavy chain antibodies was collected. The bispecific heavy chain antibodies were purified using protein A resin and further characterized for their functional activity using a reporter cell assay.

[0216] 2.2 Functional Characterization of Bispecific Heavy Chain Antibodies Using the HEK Blue IFNγ reporter cell assay (Invivogen), bispecific heavy chain antibodies containing different IFNγR1 and IFNγR2 VHH pairs were screened to identify IFNγR agonists. Conversely, using the HEK Blue IL-2 reporter cell assay (Invivogen), bispecific heavy chain antibodies containing different IL-2Rβ and IL-2RγVHH pairs were screened to identify IL-2R agonists. The in vitro HEK Blue IFNγ reporter cell line (Invivogen, catalog number: hkb-ifng) reports IFNγR signaling via the STAT1-inducible secreted alkaline phosphatase (SEAP) system. On the other hand, in vitro HEK Blue IL-2 reporter cells (Invivogen, catalog number: hkb-il2) replicate the human IL-2R signaling pathway through the expression of IL-2R chains (IL-2Rα, β, and γ subunits), downstream signaling cascade effectors (JAK3 and STAT5), and a STAT5-inducible secreted alkaline phosphatase (SEAP) reporter system. Upon addition of QuantiBlue substrate (Invivogen, catalog number: rep-qbs), absorbance at 650 nm was measured, which correlated with SEAP levels and IFNγR and IL-2R activity, respectively. In the initial screening, supernatants from Expi293 5×5 expression matrices containing soluble IFNγR1-IFNγR2 or IL-2Rβ-IL-2Rγ bispecific heavy chain antibodies were incubated with HEK Blue IFNγ or HEK Blue IL-2 reporter cells, respectively. After a 20-hour incubation, IFNγR and IL-2 activity were quantified by adding QuantiBlue reagent (Invivogen, catalog number: rep-qbs) according to the manufacturer's recommendations. Absorbance at 650 nm was measured, and higher absorbance levels indicated increased IFNγR or IL-2 signaling.

[0217] Of the 25 tested IFNγR1-IFNγR2 bispecific heavy chain antibodies (in the format shown in Figure 5), 14 showed IFNγR activity (Figure 6A). Thus, 14 pairs of IFNγR1-VHH domains and IFNγR2-VHH domains showed IFNγ agonist activity. Of the 25 tested IL-2Rβ-IL-2Rγ bispecific heavy chain antibodies, 9 pairs containing nonspecific VHH domains (IL2Rβ_4 and IL2Rγ_1) did not induce IL-2R activity. The other 16 pairs formed from functional IL-2Rβ and IL-2Rγ binders all showed IL-2R agonism (Figure 6B). Thus, 16 pairs of IL-2Rβ-VHH domains and IL-2Rγ-VHH domains showed IL-2 agonist activity.

[0218] Next, purified agonist IFNγR1-IFNγR2 bispecific heavy chain antibodies (P1AH1877-P1AH1890, see Table 1) were tested for dose-dependent IFNγR signaling, and agonist IL-2Rβ-IL-2Rγ bispecific heavy chain antibodies were tested for dose-dependent IL-2R signaling (see Table 2). [Table 1]

[0219] Following the Invivogen HEK Blue protocol, HEK Blue IFNγ cells were treated with concentrations ranging from 100 nM to 0.01 nM via 10-fold serial dilutions. Absorbance at 650 nm was measured, and responses were plotted using GraphPad software (Figure 7). The graphs were separated according to VHH clones with IFNγR1 specificity, and recombinant human IFNγ (recIFNγ, SEQ ID NO: 26) and anti-FAP IgG (P1AF3574, SEQ ID NO: 24 and SEQ ID NO: 25) fused to IFNγ homodimers were used as reference molecules. The diverse responses observed in all graphs, ranging from potent IFNγ mimes to weak agonists, demonstrated interaction between both IFNγR1 and IFNγR2 binders. EC50 values ​​were derived using GraphPad Prism software (Figure 8), and these values ​​indicated a range of agonist activity, highlighting the potential of VHH pairs to achieve potent yet tunable cytokine mimes. [Table 2]

[0220] Concentration ranges of 20 nM, 0.8 nM, and 0.032 nM were used for HEK Blue IL-2 reporter cells according to the Invivogen HEK Blue protocol. The agonist activity of IL-2 mimetic was compared to IL-2v fused to anti-PD1 IgG (PD1-IL2v, P1AE4422). The VHH pair exhibited potent IL-2R agonism in a dose-dependent manner (Figure 9). Furthermore, in contrast to innate cytokines whose agonism is mediated by a single molecule, the use of the VHH pair to activate the receptor provided an opportunity for conditionally active IL-2 mimetic via a molecular splitting approach.

[0221] To further characterize the agonist activity of 14 functional IFNγR1- and IFNγR2-VHH pairs, we selected various tumor cell lines (MKN45, BxPC-3, COR-L105, data not shown) whose responsiveness to IFNγ had been previously identified, and investigated the therapeutic downstream effects of IFNγR signaling that may have the ability to modulate the tumor microenvironment (TME). First, we selected MHC-I and PD-L1 as tumor cell surface biomarkers to characterize the TME modulating ability of functional bispecific VHH pairs. Upregulation of MHC-I is a downstream response after IFNγR stimulation and may result in increased antigenicity on tumor cells, while upregulation of PD-L1 is another downstream indicator of functional IFNγ signaling. To evaluate the ability of VHH pairs to induce IFNγ signaling in vitro, tumor cells were incubated with 14 pre-tested heavy-chain antibodies (P1AH1877-P1AH1890) in a 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 the data were analyzed using FlowJo software. Mean fluorescence intensity (MFI) values ​​were blank-subtracted and normalized for recombinant IFNγ response. Upregulation of MHC-I and PD-L1 was measured in the following three tumor cell types: MKN45 (Figure 10A, Figure 10B), BxPC-3 (Figure 10C, Figure 10D), and COR-L105 (Figure 10E, Figure 10F). A variety of responses were observed, supporting the broad agonist activity previously observed in HEK Blue IFNγ reporter cells. One notable finding is the frequent decrease in PD-L1 expression compared to MHC-I expression induced by specific VHH pairs. This suggests the complexity of IFNγ signaling, which may involve different pathways, and highlights the possibility that agonist VHH pairs may induce bias toward the desired phenotypic effect.

[0222] Induction of IFNγR signaling via agonist-mediated IFNγR VHH pairs and IL-2R signaling via agonist-mediated IL-2R VHH pairs provided an opportunity to manipulate conditionally active IFNγ and IL-2 mimetic compounds. As a starting point for conditional IFNγR agonists, P1AH1884 was selected as a potent IFNγ mimetic (Figure 8), exhibiting activity closely correlated with recombinant IFNγ response across all tumor lines tested (Figure 9). P1AH1177 was selected as a conditional IL-2R agonist.

[0223] The corresponding VHH pairs were further manipulated to generate conditionally active agonists.

[0224] Example 3: Manipulation of conditionally active IFNγ and IL-2 mimetic 3.1 FAP-dependent biparatopic binding of split-type dual-targeted IFNγ mimetic To achieve conditional immunomodulation within the immune-deficient tumor microenvironment, we further investigated cell surface markers reported to be expressed in non-inflammatory tumor phenotypes. Fibroblast-activating protein (FAP), a serine protease reported to be highly expressed in cancer-associated stromal tissue of immune-deficient tumors, was selected as a target for achieving cold tumor permeability. As a next step, we incorporated an FAP-dependent binding strategy to inactivate the VHH pair by distributing a pair of IFNγ-mimicking agonist VHH domains to two distinct molecules, while simultaneously restoring agonist VHH activity in the presence of FAP (Figure 11A). For this purpose, we selected two proximal epitopes on the FAP antigen and engineered corresponding non-competitive anti-FAP binders to mediate the binding of the agonist VHH pair. First, fusion of the VHH domain of the P1AH1884 heavy chain antibody to the N-terminus of the VH or VL domain of the anti-FAP binder via a flexible 5 (G4S) linker resulted in eight possible format structures (Figure 11B-I). To investigate FAP-dependent IFNγR signaling, the upregulation of MHC-I and PD-L1 was quantified in three FAP expression scenarios. Initially, the FAP-independent activity of untargeted IFNγ mimics in a FAP-negative state was tested using the A549 lung adenocarcinoma cell line. Next, the FAP-dependent activity in the cis state was characterized using A549 cells engineered for ectopic FAP expression, while both FAP-positive and FAP-negative cell lines were differentially labeled and co-cultured to compare the cis and trans activities of FAP-dependent IFNγ mimics. Separated dual-targeted IFNγ mimics were serially diluted to a concentration range of 10 nM to 1 pM. An intact parental VHH pair (P1AH1884, in the format shown in Figure 5) lacking recombinant IFNγ and FAP targeting was used as a reference molecule. After 72 hours of incubation under three FAP expression scenarios, MHC-I and PD-L1 expression levels were quantified by flow cytometry. In the absence of FAP expression, upregulation of MHC-I and PD-L1 was induced by the reference molecule, while none of the combinations of fragmented dual-targeting molecules exhibited FAP-independent activity (Figures 12A and 12B).In the presence of FAP expression, both the cis configuration (Figures 12C and 12D) and the trans configuration (Figures 12E and 12F) exhibited FAP-specific activity. In particular, the cis configuration highlighted the importance of geometric shape for achieving potent activity, with slight format adjustments (i.e., swapping the fusion site in the anti-FAP binder from VH to VL) leading to significant differences in IFNγR signaling. The P1AI0831+P1AI0066 pair yielded the most potent FAP-dependent IFNγR activity. Interestingly, geometric constraints did not apply to the same extent in the trans scenario, where all format combinations exhibited FAP-dependent activity.

[0225] To further explore format options and broaden our understanding of the constraints imposed by molecular geometry, we designed additional molecular sets in which VHH is further away from the FAP-binding domain. VHH was fused to the N-terminus of the opposite Fc chain via a 3(G4S) linker (Figures 13A-13D) or to the C-terminus of the same Fc chain via a 3(G4S) linker (Figures 13E-H). These sets were tested in vitro in three FAP expression scenarios, as previously described. FAP-independent activity was observed with the reference molecules (recombinant IFNγ and P1AH1884, Figures 14A and 14B), while the split-type dual-targeting combination did not exhibit IFNγR agonism except at high concentrations for one combination (P1AI5306+P1AI5307, Figure 14A). Next, FAP-dependent activity was tested in FAP-expressing cells (cis-targeting). Upregulation of MHC-I (Figure 14C) and PD-L1 (Figure 14D) was observed with the reference molecule and the P1AI5306+P1AI5307 combination, while all other combinations remained inactive. Finally, these combinations were tested in a trans scenario to evaluate their activity against FAP-negative cells in the presence of FAP-expressing cells. All combinations exhibited FAP-dependent trans activity, inducing both MHC-1 upregulation (Figure 14E) and PD-L1 upregulation (Figure 14F). Due to previously identified geometric constraints imposed in the cis scenario by cleaving VHH from the FAP-binding region, little FAP-dependent cis activity was observed. However, in the trans setting, all combinations, regardless of format, exhibited IFNγR agonism.

[0226] 3.2 Design of PD1-dependent biparatopic coupling of IL-2 mimetic To incorporate tumor selectivity while maintaining conditional agonist activity, a strategy was devised that utilizes cell surface markers reported to be concentrated within the tumor environment to scaffold the binding of an IL-2 agonistic VHH domain pair. The goal was to bind two proximal epitopes on a single target using two non-competing anti-target binders (Figure 15A). First, 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 for tumor-specific effector T cell populations. Second, PD-L1-competitive anti-PD-1 binders result in immune checkpoint inhibition mediated by blockade of the PD1 / PD-L1 signaling cascade. Third, recent evidence has highlighted the synergistic effect of IL-2R agonism in combination with cis-targeted PD-1 blockade, resulting in “superior” T cell differentiation (Codarri Deak et al. Nature 610;161-172 (2022)).

[0227] In this experiment, the potency and cis-delivery of two pairs of IL-2 mimics (Figures 15B and 15C), as well as PD1-IL2v (Figure 15E, P1AE4422) as a reference molecule and a potent IL-2 agonist heavy chain antibody (Figure 15D, P1AH117), were measured based on their IL-2R signaling in activated PD-1 + and PD-1 - (anti-PD1 pre-treated) CD4 T cells treated with increasing concentrations of the molecules. This was done to examine the dependence of PD-1-based IL-2R agonists on PD1 expression in T cells to deliver IL-2R signaling.

[0228] For this purpose, CD4 T cells were selected from healthy donor PBMCs using CD4 beads (Miltenyi, catalog number 130-045-101), and activated for 3 days in the presence of 1 μg / ml plate-bound anti-CD3 (overnight pre-coated, clone OKT3, catalog number 317315, BioLegend) and 1 μg / ml soluble anti-CD28 (clone CD28.2, catalog number 302923, BioLegend) antibodies to induce PD-1 expression. After 3 days, the cells were harvested, washed several times to remove endogenous cytokines, and half of the cells were labeled with CTV (5 μM, 5 min at 20°C, catalog number: C34557, Thermo Scientific), while the other half were left unlabeled.

[0229] Next, unlabeled cells were incubated with a saturated concentration of competitive anti-PD-1 antibody (in-house molecule, 10 μg / ml) at 20°C for 30 minutes, followed by several washing steps to remove excess unbound anti-PD-1 antibody. Subsequently, PD1 preblocking unlabeled cells (25 μl, 6 × 10⁶) were prepared. 6 Cells / ml) in a V-bottom plate with PD1 + CTV-labeled cells (25 μl, 6 x 10 6 After co-culturing cells ( / ml) in a 1:1 ratio, the cells were treated at 37°C for 15 or 60 minutes, increasing the concentration of the treatment molecule (50 μl, 1:10 dilution step). To preserve the phosphorylation state, an equal volume of Phosphoflow Fixation Buffer I (100 μl, catalog no.: 557870, BD) was added after incubation with the various treatment molecules. The cells were then incubated at 37°C for a further 30 minutes, followed by permeabilization overnight at -80°C using Phosphoflow PermBuffer III (catalog no. 558050, BD). The following day, phosphorylated STAT-5 was stained at 4°C for 30 minutes using anti-STAT-5P antibody (47 / Stat5(pY694) clone, catalog no. 562076, BD).

[0230] Cells were acquired using FACS BD-LSR Fortessa (BD Bioscience). The frequency of STAT-5P was determined using FlowJo (V10) and plotted using GraphPad Prism.

[0231] PD-1 + Dose-response curves on T cells provide information about the efficacy of PD1-based IL-2R agonists in IL-2R-mediated signaling. Furthermore, dose-response curves on T cells pretreated with competing anti-PD1 antibodies to prevent PD1-mediated delivery demonstrate the efficacy of PD1-based IL-2R agonist molecules that provide IL-2R signaling independently of PD-1 expression. PD1-IL2v showed reduced activity against T cells in the absence of PD1 binding (preblock) (Figure 16, solid and dashed black lines). On the other hand, in the absence of PD1 binding, no activity was observed for two combinations of fragmented IL-2 mimetic (P1AH6850+P1AH6813, P1AH6814+P1AI1593) (Figure 16, light gray dashed line). A decrease in IL-2 signaling was not observed with non-targeted IL-2 agonist bispecific heavy chain antibodies (P1AH1177, Figure 16, dark gray solid and dark gray dashed lines).

[0232] Further investigation of the biparatopic binding concept was conducted using an alternative anti-PD-1 Fab binder (Figure 17). IL-2 signaling was evaluated using HEK Blue IL-2 wt cells (PD-1 negative) and HEK Blue IL-2 cells expressing human PD-1.

[0233] As observed with the previously mentioned 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 (Figure 18A). However, replacing the PD-1 binder 0376 with 7G12 (P1AK2802+P1AK2599) also showed strong efficacy against PD-1-expressing cells (Figure 18B). Of the eight tested PD-1-targeted IL-2R agonists (7G12 and 1040) containing the same set of PD-1 binders, three showed strong dose-response IL-2R activation.

[0234] 3.3 FAP-dependent biparatopic binding of IFNγ mimetic using a single-molecule format To determine whether a single molecule retaining all the characteristics of a split bimolecule approach could be achieved, we designed a four-specificity format incorporating the requirements of a split bimolecule targeting molecule. The integrated format provides biparatopic binding of cytokine receptor-activating VHH domain pairs by combining two functionally split biparatopic molecules into a single molecule. First, we selected the most potent split structure of an IFNγ agonist, which was shown to yield both cis and trans activity (P1AI0831+P1AI0066, Figure 12). Next, the two selected VHH-Fab fusions were transplanted into a single molecule using Fc as a spacer to separate the VHH domains and reduce the likelihood of VHH-mediated IFNγR activation in an FAP-independent manner. Finally, we incorporated knob-into-hole and CrossFab techniques to generate a four-specificity format while maintaining all the optimal fusion structures defined by the P1AI0831+P1AI0066 combination (Figure 19). In detail, a VHH domain with IFNγR1 specificity was fused to the N-terminus of the anti-FAP binder 1 VL domain using a 5(G4S) linker. The N-terminus of the anti-FAP binder 1 VH domain was fused to the C-terminus of the Fc knob chain via a 3(G4S) linker, modifying the CH1 / CL domain to have an additional attractive charge. Anti-FAP binder 2 was located upstream of the hinge on the hole chain and contained a crossed VH / VL domain. A VHH domain with IFNγR2 specificity was fused to the N-terminus of the VH domain currently located on the light chain using a 5(G4S) linker.

[0235] The resulting integrated format, referred to as P1AI5012, was tested in vitro for FAP-specific IFNγR activation using the three FAP expression scenarios described earlier. In the absence of FAP expression, the reference molecule showed upregulation of MHC-I (Figure 20A, dashed line) and PD-L1 (Figure 20B, dashed line), while P1AI5012 did not induce IFNγR activation (Figures 20A and 20B, solid lines). In FAP-expressing cells, P1AI5012 exhibited FAP-dependent IFNγR agonism and showed activity similar to recombinant IFNγ at a low concentration of 25 pM (Figures 20C and 20D). In settings that reproduced trans-activity, potent IFNγR agonism was observed in the presence of P1AI5012 (Figures 20E and 20F).

[0236] 3.4 Further Formats of "Integrated" Molecules To determine whether the mechanism of action is specific to the particular format in which it is produced, the inventors designed several variants of the integrated format (P1AI5012) with different agonist VHH pairs (VHH pair 1: IFNγR1_3 VHH and IFNγR2_2 derived from P1AH1884, VHH pair 2: IFNγR1_5 VHH and IFNγR2_1 VHH derived from P1AH1886), different positions of each VHH on the VH or VL of the Fab domain, different positions on the FAP binder, and different linker lengths between the Fab domain and the VHH (5, 10, 15, and 25 amino acid) linker lengths (Figure 21). P1AI5012, P1AJ0690, P1AJ0672, P1AJ0700, and P1AJ0685 contain VHH pair 1. P1AJ0744, P1AJ0735, and P1AJ0747 include VHH pair 2.

[0237] The resulting integrated format was tested in vitro for FAP-specific IFNγR activation using the three FAP expression scenarios described above.

[0238] Based on the upregulation of MHC-I, the tested integrated mutants can be divided into three distinct groups. Specifically, mutants that can induce high levels of MHC-I belong to this group, including integrated mutants of recIFNγ, the reference molecule (P1AI5012), and P1AJ0690; mutants that can induce moderate MHC-I upregulation belong to this group, including integrated mutants of P1AJ0672, P1AJ0685, P1AJ0744, and P1AJ0735; and finally, mutants that can induce little / no MHC-I upregulation belong to this group, including the following mutants, P1AJ0700, and P1AJ0747 (Figure 22).

[0239] 3.5 PD-1-dependent biparatopic binding of IL-2 mimetic using a single-molecule format To determine whether a single molecule retaining all the features of a fragmented IL-2 mimic approach could be achieved, we designed a four-specificity format incorporating the requirements of a fragmented dual-targeting molecule (Figure 23). The integrated format provided biparatopic binding of cytokine-mimicking VHH pairs by combining two functionally fragmented biparatopic molecules into a single molecule. First, we selected the most potent fragmented structures of the IL-2 mimic, which were shown to induce PD1-mediated cis-activity (P1AI1593 and P1AH6814). Next, the two selected VHH-Fab fusions were transplanted into a single molecule using Fc as a spacer to separate the VHH domain and reduce the likelihood of VHH-mediated IL-2R activation in a PD1-independent manner. Finally, we incorporated knob-into-hole and CrossFab technologies to generate a four-specificity format while maintaining all the optimal fusion structures defined by the P1AI1593+P1AH6814 combination. In detail, a VHH domain with IL-2Rβ specificity was fused to the N-terminus of the anti-PD1 binder 1 VL domain using a 5(G4S) linker. The N-terminus of the anti-PD1 binder 1 VH domain was fused to the C-terminus of the Fc knob chain via a 2(G4S) linker, modifying the CH1 / CL domain to have an additional attractive charge. Anti-PD1 binder 2 was located upstream of the hinge on the hole chain and contained a crossed VH / VL domain. A VHH domain with IL-2Rγ specificity was fused to the N-terminus of the VH domain currently located on the light chain using a 5(G4S) linker.

[0240] To measure the ability of PD-1-targeted IL-2R agonists to induce IL-2R signaling, HEK Blue IL-2 cells (Invivogen) expressing or not expressing human PD-1 were used. The reporter system measures secreted SEAP levels by absorbance readout as a substitute for STAT-5 phosphorylation. Briefly, 12,000 cells / well were seeded into 384-well flat-bottom tissue culture processed plates (Corning, catalog no. 3701). The test compound (4-fold dilution series, starting concentration 100 nM) was added to the assay plate. After incubation of the samples at 37°C and 5% CO2 for 20 hours, the assay was readout using QUANTI-Blue solution (Invivogen, catalog no. Rep-qbs3) according to the manufacturer's instructions. Absorbance readout was performed using a Tecan Spark 10M plate reader. The dose-response of test compounds versus IL2R signaling (SEAP level at A650) was plotted using GraphPad Prism software. Fc-fusion IL2R agonists showed activity in the presence and absence of PD-1, but the ability of the integrated PD-1-targeted IL-2R agonist (P1AI5057) was strongly dependent on PD-1 (Figure 24).

[0241] 3.6. Functional Characterization of Monoparatopic and Biparatopic Cis-Targeted IL-2R Integrated Agonists To understand the necessity of biparatopic binding of IL-2R agonists, we tested the monoparatopic concept of IL-2R signaling via binding to the same PD-1 epitope (Figure 25). IL-2 signaling was evaluated using HEK Blue IL-2 wt cells and HEK Blue IL-2 human PD-1 cells. As previously demonstrated, biparatopic binding of the PD-1-targeted IL-2R agonist (P1AI5057) resulted in strong dose-responsive IL-2R signaling in a reporter assay (Figure 26B), while preferential PD-1-dependent activity was not observed with the two monoparatopic compounds tested (Figures 26A and 26B), highlighting the necessity of biparatopic binding for functional IL-2R signaling. Notably, the residual PD-1-independent activity of the PD-1-targeted IL-2R agonist was similar to that of the monoparatopic and biparatopic molecules (Figure 26A).

[0242] In vitro data provided robust evidence that IFNγ and IL-2 mimics can be manipulated for conditional activity under desired conditions using a biparatopic molecular binding strategy. As a first approach, it was demonstrated that VHH pairs mimicking IFNγ and IL-2 could be rendered non-functional by distribution onto two separate molecules, thereby mediating the binding of functional IFNγ and IL-2 mimics under desired conditions. As an extension of this approach, the possibility of designing a single-molecule format that retains conditional cis and trans activity was explored. An integrated format was achieved by inactivating the cytokine mimic by increasing the distance between the VHH pairs, while conforming the molecular structure to satisfy the geometric requirements for conditional cis and trans agonist activity. It was demonstrated that the format and geometric shape can be utilized to fine-tune the activity of the conditional cytokine mimic. TIFF2026515641000003.tif254170TIFF2026515641000004.tif255170TIFF2026515641000005.tif255170TIFF2026515641000006.tif253170TIFF2026515641000007.tif255170TIFF2026515641000008.tif253170TIFF2026515641000009.tif255170TIFF2026515641000010.tif253170TIFF2026515641000011.tif255170TIFF2026515641000012.tif252170TIFF2026515641000013.tif255170TIFF2026515641000014.tif255170TIFF2026515641000015.tif253170TIFF2026515641000016.tif253170TIFF2026515641000017.tif255170TIFF2026515641000018.tif255170TIFF2026515641000019.tif255170TIFF2026515641000020.tif255170TIFF2026515641000021.tif255170TIFF2026515641000022.tif255170TIFF2026515641000023.tif255170TIFF2026515641000024.tif255170TIFF2026515641000025.tif136170

Claims

1. It is an antigen-binding molecule, i) The first target-binding domain and ii) The second target-binding domain, iii) The first cytokine receptor binding domain, iv) The second cytokine receptor binding domain, v) Including the Fc domain, The first target-binding domain can bind to a first epitope on the target antigen, the second target-binding domain can bind to a second epitope on the target antigen, and the first and second target-binding domains do not compete for binding to tumor-associated antigens. The first cytokine receptor binding domain can bind to a first cytokine receptor subunit, and the second cytokine receptor binding domain can bind to a second cytokine receptor subunit. Antigen-binding molecules.

2. The antigen-binding molecule according to claim 1, wherein the first and second target-binding domains are antibody fragments, particularly Fv, Fab, scFv, scFab, or single-domain antibodies.

3. The antigen-binding molecule according to claim 1 or 2, wherein the first and second target-binding domains are Fab molecules.

4. The first target-binding domain is a heavy chain variable domain (VH １ ), light chain variable domain (VL １ ), heavy chain constant domain (CH1 １ ) and light chain constant domain (CL １ ) includes, and the second target binding domain is a heavy chain variable domain (VH ２ ), light chain variable domain (VL ２ ), heavy chain constant domain (CH1 ２ ) and light chain constant domain (CL ２ An antigen-binding molecule according to any one of claims 1 to 3, comprising )

5. The antigen-binding molecule according to any one of claims 1 to 4, wherein the first target-binding domain and / or the second target-binding domain is a cross-Fab molecule.

6. The antigen-binding molecule according to any one of claims 1 to 5, wherein the first target-binding domain and / or the second target-binding domain includes a charge mutation.

7. The antigen-binding molecule according to any one of claims 1 to 6, wherein the first target-binding domain is cross-Fab and the second target-binding domain contains a charge mutation, or the second target-binding domain is cross-Fab and the first target-binding domain contains a charge mutation.

8. The antigen-binding molecule according to any one of claims 1 to 7, wherein the first target-binding domain and the second target-binding domain specifically bind to a tumor-associated antigen or a T cell antigen.

9. The antigen-binding molecule according to any one of claims 1 to 8, wherein the first target-binding domain and the second target-binding domain specifically bind to FAP, PD-1, Her2, Her3, LAG-3, CEA, or EGFR.

10. a) the first target binding domain comprises VH of SEQ ID NO: 20 １ and VL of SEQ ID NO: 21 １ and the second target binding domain comprises VH of SEQ ID NO: 22 ２ and VL of SEQ ID NO: 23 ２ or b) The first target-binding domain is VH of SEQ ID NO: 22 １ and VL of Sequence ID No. 23 １ The second target-binding domain is VH of Sequence ID No.

20. ２ and VL of sequence number 21 ２ Includes or c) The first target-binding domain is VH of Sequence ID No. 80 １ and VL of sequence number 81 １ The second target-binding domain is VH of Sequence ID No.

82. ２ and VL of sequence number 83 ２ Includes or d) The first target-binding domain is VH of Sequence ID No. 82 １ and VL of sequence number 83 １ The second target-binding domain is VH of Sequence ID No.

80. ２ and VL of sequence number 81 ２ Includes or e) The first target-binding domain is VH of Sequence ID No. 82 １ and VL of sequence number 83 １ The second target-binding domain is VH of sequence number 140. ２ and VL of sequence number 141 ２ Includes or f) The first target-binding domain is VH of Sequence ID No. 140 １ and VL of sequence number 141 １ The second target-binding domain is VH of Sequence ID No.

82. ２ and VL of sequence number 83 ２ including, The antigen-binding molecule according to any one of claims 1 to 9.

11. The antigen-binding molecule according to any one of claims 1 to 10, wherein both the first and second cytokine receptor subunits are subunits of an IFNγ receptor complex or an IL-2 receptor complex.

12. a) The first cytokine receptor binding domain can bind to IFNγR1, and the second cytokine receptor binding domain can bind to IFNγR2, or b) The first cytokine receptor binding domain can bind to IFNγR2, and the second cytokine receptor binding domain can bind to IFNγR1, c) The first cytokine receptor binding domain can bind to IL-2Rβ, and the second cytokine receptor binding domain can bind to IL-2Rγ, or d) The first cytokine receptor binding domain can bind to IL-2Rγ, and the second cytokine receptor binding domain can bind to IL-2Rβ, The antigen-binding molecule according to any one of claims 1 to 11.

13. The antigen-binding molecule according to any one of claims 1 to 12, wherein the first and second cytokine receptor-binding domains are antibody fragments, particularly Fv, Fab, scFv, scFab, single-domain antibodies, or VHH domains.

14. The antigen-binding molecule according to any one of claims 1 to 13, wherein the first and second cytokine receptor-binding domains are VHH domains.

15. 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, and SEQ ID NO: 9, or b) The first cytokine receptor binding domain comprises an amino acid sequence selected from SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 9, 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 includes the sequences of SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, and SEQ ID NO: 64, and the second cytokine receptor binding domain includes the sequences of SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, and SEQ ID NO: 69, or d) The first cytokine receptor binding domain includes the sequences of SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68 and SEQ ID NO: 69, and the second cytokine receptor binding domain includes the sequences of SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62 and SEQ ID NO: 64, The antigen-binding molecule according to any one of claims 1 to 14.

16. The antigen-binding molecule according to any one of claims 1 to 15, wherein the Fc domain comprises a first Fc domain subunit and a second Fc domain subunit.

17. The aforementioned Fc domain is IgG, and more specifically IgG １ An antigen-binding molecule according to any one of claims 1 to 16, wherein the Fc domain.

18. The antigen-binding molecule according to any one of claims 1 to 17, wherein the Fc domain is a human Fc domain.

19. The antigen-binding molecule according to any one of claims 1 to 18, wherein the Fc domain includes a modification that promotes the association of the first and second subunits of the Fc domain.

20. The antigen-binding molecule according to any one of claims 1 to 19, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to and / or effector function of the Fc receptor.

21. The first cytokine receptor binding domain has a VH of the first target binding domain at its C-terminus. １ or VL １ It is fused to the N-terminus, and the first target-binding domain is its VH １ or VL １ The N-terminus of the second cytokine receptor-binding domain is fused to the C-terminus of the first Fc domain subunit, and the VH of the second target-binding domain is fused to the C-terminus of the second target-binding domain. ２ or VL ２ It is fused to the N-terminus, and the second target-binding domain is its CH1 ２ or CL １ The antigen-binding molecule according to any one of claims 1 to 20, wherein the C-terminus of the second Fc domain subunit is fused to the N-terminus of the second Fc domain subunit.

22. a) Starting from the N-terminus and moving toward the C-terminus, the first Fc domain subunit, VH １ and CH1 １ A first polypeptide comprising, in order from the N-terminus to the C-terminus, the first cytokine receptor binding domain, VL １ and CL １ A second polypeptide containing, and sequentially from the N-terminus to the C-terminus, VL ２ CH1 ２ and a third polypeptide comprising a second Fc domain subunit, and sequentially from the N-terminus to the C-terminus, the second cytokine receptor binding domain, VH ２ and CL ２ A fourth polypeptide comprising, or b) Starting from the N-terminus and moving toward the C-terminus, the first Fc domain subunit, VL １ and CH1 １ A first polypeptide comprising, in order from the N-terminus to the C-terminus, the first cytokine receptor binding domain, VH １ and CL １ A second polypeptide containing, and sequentially from the N-terminus to the C-terminus, VH ２ CH1 ２ and a third polypeptide comprising a second Fc domain subunit, and sequentially from the N-terminus to the C-terminus, the second cytokine receptor binding domain, VL ２ and CL ２ A fourth polypeptide comprising, or c) From the N-terminus to the C-terminus, in order: the first Fc domain subunit, VL １ and CL １ A first polypeptide comprising, in order from the N-terminus to the C-terminus, the first cytokine receptor binding domain, VH １ and CH1 １ A second polypeptide containing, and sequentially from the N-terminus to the C-terminus, VL ２ CH1 ２ and a third polypeptide comprising a second Fc domain subunit, and sequentially from the N-terminus to the C-terminus, the second cytokine receptor binding domain, VH ２ and CL ２ A fourth polypeptide comprising, or d) Starting from the N-terminus and moving toward the C-terminus, the first Fc domain subunit, VH １ and CL １ A first polypeptide comprising, in order from the N-terminus to the C-terminus, the first cytokine receptor binding domain, VL １ and CH1 １ A second polypeptide containing, and sequentially from the N-terminus to the C-terminus, VH ２ CH1 ２ and a third polypeptide comprising a second Fc domain subunit, and sequentially from the N-terminus to the C-terminus, the second cytokine receptor binding domain, VL ２ and CL ２ A fourth polypeptide comprising, or e) Starting from the N-terminus and moving toward the C-terminus, the first Fc domain subunit, VH １ and CH1 １ A first polypeptide comprising, in order from the N-terminus to the C-terminus, the first cytokine receptor binding domain, VL １ and CL １ A second polypeptide containing, and sequentially from the N-terminus to the C-terminus, the second cytokine receptor binding domain, VL ２ CH1 ２ and a third polypeptide comprising a second Fc domain subunit, and sequentially from the N-terminus to the C-terminus, VH ２ and CL ２ A fourth polypeptide comprising, or f) Starting from the N-terminus and moving toward the C-terminus, the first Fc domain subunit, VL １ and CH1 １ A first polypeptide comprising, in order from the N-terminus to the C-terminus, the first cytokine receptor binding domain, VH １ and CL １ A second polypeptide containing, and sequentially from the N-terminus to the C-terminus, the second cytokine receptor binding domain, VH ２ CH1 ２ and a third polypeptide including a second Fc domain subunit, and sequentially from the N-terminus to the C-terminus, VL ２ and CL ２ A fourth polypeptide comprising, or g) In order from the N-terminus to the C-terminus, a first Fc domain subunit, VL １ and CL １ a first polypeptide comprising, and in order from the N-terminus to the C-terminus, the first cytokine receptor binding domain, VH １ and CH1 １ a second polypeptide comprising, and in order from the N-terminus to the C-terminus, the second cytokine receptor binding domain, VL ２ CH1 ２ and a third polypeptide comprising a second Fc domain subunit, and in order from the N-terminus to the C-terminus, VH ２ and CL ２ a fourth polypeptide comprising, or h) Starting from the N-terminus and moving toward the C-terminus, the first Fc domain subunit, VH １ and CL １ A first polypeptide comprising, in order from the N-terminus to the C-terminus, the first cytokine receptor binding domain, VL １ and CH1 １ A second polypeptide containing, and sequentially from the N-terminus to the C-terminus, the second cytokine receptor binding domain, VH ２ CH1 ２ and a third polypeptide including a second Fc domain subunit, and sequentially from the N-terminus to the C-terminus, VL ２ and CL ２ A fourth polypeptide comprising, VH １ , VL １ CH1 １ and CL １ This forms the first target-binding domain, VH ２ , VL ２ CH1 ２ and CL ２ This forms the second target-binding domain, The antigen-binding molecule according to any one of claims 1 to 21.

23. The antigen-binding molecule according to any one of claims 1 to 22, wherein the first target-binding domain and the second target-binding domain specifically bind to FAP, and the first and second cytokine receptor subunits are subunits of an IFNγ receptor complex.

24. An antigen-binding molecule according to any one of claims 1 to 23, a) comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 50, a second polypeptide containing the amino acid sequence of SEQ ID NO: 48, a third polypeptide containing the amino acid sequence of SEQ ID NO: 51, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 49, or b) comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 99, a second polypeptide containing the amino acid sequence of SEQ ID NO: 97, a third polypeptide containing the amino acid sequence of SEQ ID NO: 100, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 98, or c) comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 103, a second polypeptide containing the amino acid sequence of SEQ ID NO: 101, a third polypeptide containing the amino acid sequence of SEQ ID NO: 104, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 102, or d) comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 107, a second polypeptide containing the amino acid sequence of SEQ ID NO: 105, a third polypeptide containing the amino acid sequence of SEQ ID NO: 108, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 106, or e) comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 99, a second polypeptide containing the amino acid sequence of SEQ ID NO: 109, a third polypeptide containing the amino acid sequence of SEQ ID NO: 100, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 110, or f) comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 107, a second polypeptide containing the amino acid sequence of SEQ ID NO: 111, a third polypeptide containing the amino acid sequence of SEQ ID NO: 113, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 112, or g) comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 99, a second polypeptide containing the amino acid sequence of SEQ ID NO: 114, a third polypeptide containing the amino acid sequence of SEQ ID NO: 100, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 115, or h) A first polypeptide comprising the amino acid sequence of SEQ ID NO: 107, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 116, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 108, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO:

117. Antigen-binding molecules.

25. The antigen-binding molecule according to any one of claims 1 to 22, wherein the first target-binding domain and the second target-binding domain specifically bind to PD-1, and the first and second cytokine receptor subunits are subunits of the IL-2 receptor complex.

26. An antigen-binding molecule according to any one of claims 1 to 22 or 25, comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 123, a second polypeptide containing the amino acid sequence of SEQ ID NO: 122, a third polypeptide containing the amino acid sequence of SEQ ID NO: 124, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 125.

Images