Interferon gamma variants and antigen-binding molecules containing the same

Abstract

The present invention relates to novel antigen-binding molecules comprising (i) an antibody that specifically binds to a tumor-associated antigen, and (ii) an interferon gamma (IFNG) variant polypeptide that terminates in the C-terminal amino acid sequence KRKRP (SEQ ID NO:1), the novel IFNG variant polypeptides contained therein, methods for producing these molecules, and methods of using them.

Description

[Technical field]

[0001] The present invention relates to (i) an antibody that specifically binds to a tumor-associated antigen, and (ii) a novel antigen-binding molecule comprising a novel interferon gamma (IFNG) variant polypeptide that terminates with the C-terminal amino acid sequence KRKRP (SEQ ID NO: 1), in particular an antibody that specifically binds to fibroblast activation protein (FAP) and an antigen-binding molecule comprising a novel IFNG variant polypeptide. Furthermore, the present invention relates to the novel IFNG variant polypeptide contained in the molecule, as well as polynucleotide molecules encoding the antigen-binding molecule or the IFNG variant polypeptide, and vectors and host cells comprising such polynucleotide molecules. Further aspects of the present invention are methods of producing these molecules and methods of using these molecules. [Background technology]

[0002] In recent years, the application of immunotherapy treatments for cancer has increased dramatically, making cancer immunotherapy a key strategy to fight cancer. Immune checkpoint modulators, including anti-PD-1, have been established as standard treatments for many cancer types. However, despite the progress in cancer immunotherapy treatments over the past few years, some patients still do not respond to available immunotherapies due to intrinsic or adaptive resistance mechanisms. In particular, it is becoming more evident that patients who do not respond to cancer immunotherapy are often characterized by a non-inflammatory immune phenotype. Indeed, a correlation has been shown between immune cell infiltration in tumors and the ability of patients to respond to immunotherapy treatments. Overall, there is a clear medical need for patients to develop new therapies that aim to enhance immunogenicity and increase immune cell infiltration in tumors.

[0003] In parallel with the above developments, there has been a rapid increase in interest in cytokines as potential cancer treatments. Interferon gamma (IFN-γ) is a cytokine that stimulates the CD4 + and CD8 +It is a cytokine produced and secreted primarily by activated lymphocytes such as T cells and natural killer (NK) cells. IFNG is a homodimer and its receptors (IFNGR1 and IFNGR2) are expressed across hematopoietic and non-hematopoietic cells. IFNGR1 is stably expressed on the cell surface, whereas IFNGR2 is differentially expressed and is used to regulate IFNG signaling. Binding of IFNG to its receptor induces the recruitment and activation of JAK1 and JAK2, Janus kinases that phosphorylate and activate STAT1. After phosphorylation, STAT1 translocates to the nucleus, where it binds to specific promoters and regulates the transcription of IFNG-regulated genes.

[0004] In contrast to many cancer treatments on the market and in development, IFNG has the ability to act on both tumor cells and multiple immune cells, including T cells and dendritic cells. The effects of IFNG on various cell types have 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 induction of CXCL9, CXCL10 and CXCL11 production, and 3) increased tumor antigen cross-presentation and subsequent enhancement of antitumor immune responses. In addition to all these effects, IFNG plays a role in generating a Th1 environment, monocyte differentiation, macrophage polarization and angiogenesis. Based on its cytostatic, proapoptotic and antiproliferative functions, IFNG is considered to be potentially useful in the treatment of cancer.

[0005] However, the receptor of IFNG is expressed in many different cell types, and therefore its activity may be reduced by sink effect. IFNG may also show undesirable side effects. In addition, problems may arise regarding administration, bioavailability and short half-life. Therefore, there is a need for new IFNG molecules that can selectively activate T cells in the tumor environment. WO 2017 / 139468 discloses a fusion protein comprising a Her2-binding scFv and an interferon gamma monomer that terminates with the amino acid sequence AKTGKRKRSQ (SEQ ID NO: 127). However, there is still a need to provide a molecule that has high stability to maintain IFNG activity and is more suitable for administration to human patients. Summary of the Invention

[0006] To overcome the current challenges, mainly related to dose-limiting toxicity and sink effects, we developed a tumor-targeted masked IFNG that induces the above benefits, which is inactive in the circulation and in healthy tissues, and active only at the tumor site. By delivering active IFNG directly to the tumor environment, the potential dose-limiting effects of IFNG produced after T and NK cell activation are overcome, providing an opportunity to efficiently address immune desert tumors, where there is currently a greater need for novel cancer immunotherapies.

[0007] Thus, the present invention provides a novel approach to target IFNG variants with advantageous properties for immunotherapy directly to immune effector cells, such as cytotoxic T lymphocytes, rather than tumor cells, by conjugation of IFNG variants to antibodies that bind to tumor-associated antigens, in particular fibroblast activation protein (FAP). This leads to the activation of T cells in the tumor microenvironment. Fibroblast activation protein (FAP) is a serine protease that is highly expressed on the cell surface of cancer-associated stromal cells and on fibroblastic reticular cells of secondary lymphoid organs, but has very limited expression in normal tissues. FAP is very common in various cancer indications, allowing its use as a targeting moiety for drugs that should accumulate in the tumor stroma.

[0008] The molecule of the present invention comprises a homodimer of interferon gamma (IFNG) variant polypeptide that ends with the C-terminal amino acid sequence KRKRP (SEQ ID NO: 1).It is shown herein that the presence of KRKR patch, i.e., the amino acid sequence of KRKR (SEQ ID NO: 78), is important for the activity of IFNG.The inventors have found that in order to avoid proteolysis, the C-terminus of wild-type IFNG must be stabilized by a proline cap, so that the sequence of IFNG variant polypeptide ends with the amino acid sequence KRKRP (SEQ ID NO: 1).

[0009] Thus, as used herein, there is provided an antigen-binding molecule comprising: (i) an antibody that specifically binds to a tumor-associated antigen; (ii) a homodimer of an interferon gamma (IFNG) variant polypeptide, wherein the IFNG variant polypeptide is characterized by the C-terminal amino acid sequence KRKRP (SEQ ID NO: 1), meaning that the C-terminus terminates with the amino acid sequence KRKRP (SEQ ID NO: 1).

[0010] Thus, these antigen binding molecules include antibodies fused with two identical interferon gamma (IFNG) variant polypeptides that, unlike wild-type IFNG, terminate at the amino acid sequence KRKRP (SEQ ID NO:1). In one embodiment, the interferon gamma (IFNG) variant polypeptide comprises the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3. In one embodiment, the interferon gamma (IFNG) variant polypeptide consists of the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3. In a particular embodiment, the IFNG variant polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:2.

[0011] In one aspect, an antigen-binding molecule is provided comprising: (i) an antibody that specifically binds to a tumor-associated antigen; and (ii) a homodimer of interferon gamma (IFNG) variant polypeptides, wherein the IFNG variant polypeptide terminates at its C-terminus with the amino acid sequence KRKRP (SEQ ID NO:1), a first IFNG variant polypeptide is fused at its N-terminus to the C-terminus of a first heavy chain via a first linker, and a second IFNG variant polypeptide is fused at its N-terminus to the C-terminus of a second heavy chain, optionally via a linker.

[0012] In one embodiment, the antibody that specifically binds to a tumor-associated antigen is an antibody that specifically binds to fibroblast activation protein (FAP). In one embodiment, the antibody that specifically binds to a FAP comprises: (a) a heavy chain complementarity determining region (CDR-H1) comprising the amino acid sequence of SEQ ID NO:4, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:5, and a heavy chain variable region (VH1) comprising the amino acid sequence of SEQ ID NO:6; H (iv) a light chain variable region (V FAP) comprising a light chain complementarity determining region CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9; L(b) a heavy chain variable region (V FAP) comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 12, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14. H A light chain variable region (VFAP) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 15, CDR-L2 having the amino acid sequence of SEQ ID NO: 16, and CDR-L3 having the amino acid sequence of SEQ ID NO: 17. L In one embodiment, an antibody that specifically binds to FAP is provided that comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 10. H A light chain variable region (V L A heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 18. H A light chain variable region (V L In one particular embodiment, an antibody that specifically binds to FAP is provided that comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 10. H A light chain variable region (V L An antigen-binding molecule comprising:

[0013] In one embodiment, the antigen binding molecule comprises an Fc domain, in particular an IgG1 Fc domain or an IgG4 Fc domain. In one embodiment, the Fc domain comprises one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antibody to the Fc receptor. In one embodiment, the Fc domain is of the human IgG1 subclass with amino acid mutations L234A, L235A and P329G (EU numbering according to Kabat EU index). In another embodiment, the Fc domain is a mouse Fc domain and comprises amino acid mutations D265A and P329G (EU numbering according to Kabat EU index).

[0014] In one embodiment, the antigen-binding molecule is protease-activatable and comprises a protease recognition site and a masking moiety. Thus, the antigen-binding molecule comprises (i) an antibody that specifically binds to a tumor-associated antigen, (ii) a homodimer of an interferon gamma (IFNG) variant polypeptide, characterized in that the IFNG variant polypeptide terminates at its C-terminus with the amino acid sequence KRKRP (SEQ ID NO: 1), (iii) a protease recognition site, and (iv) a masking moiety.

[0015] In one embodiment, the protease recognition site is a substrate for matriptase. In one embodiment, the protease recognition site comprises or consists of the amino acid sequence PQARK (SEQ ID NO:20) or HQARK (SEQ ID NO:21). In a particular embodiment, the protease recognition site comprises or consists of the amino acid sequence PQARK (SEQ ID NO:20). In one embodiment, the protease recognition site is part of a cleavable peptide linker that connects the masking moiety to the IFNG variant polypeptide.

[0016] In one embodiment, an antigen binding molecule is provided in which a masking moiety is fused at its N-terminus to the C-terminus of an IFNG variant polypeptide via a cleavable peptide linker (mask release format). In one embodiment, the IFNG variant polypeptide is fused at its N-terminus to the C-terminus of an antibody via a stable linker.

[0017] In another embodiment, an antigen binding molecule is provided in which a masking moiety is fused at its N-terminus to the C-terminus of an Fc domain via a stable linker and at its C-terminus to the N-terminus of an IFNG variant polypeptide via a cleavable peptide linker (cytokine release format).

[0018] In one embodiment, the masking moiety is an antibody fragment that specifically binds to IFNG. In a particular embodiment, the masking moiety is an scFv that specifically binds to IFNG.

[0019] In one embodiment, the masking moiety, in particular an scFv, that specifically binds to IFNG is (a) a heavy chain variable region (VH1) comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 22, a CDR-H2 having the amino acid sequence of SEQ ID NO: 23, and a CDR-H3 having the amino acid sequence of SEQ ID NO: 24; H (iv) a light chain variable region (VIFNG) comprising a light chain complementarity determining region CDR-L1 comprising the amino acid sequence of SEQ ID NO: 25, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 27. L IFNG), or (b) a heavy chain variable region (VH1) comprising the amino acid sequence of SEQ ID NO: 30, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 31, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 32; H IFNG), and a light chain variable region (V IFNG) comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 34, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 35. L IFNG).

[0020] In one embodiment, the masking moiety that specifically binds to IFNG, in particular an scFv, comprises: (a) a heavy chain variable region (V H IFNG) and a light chain variable region (V L IFNG), or (b) a heavy chain variable region (V H IFNG) and a light chain variable region (V L In a particular embodiment, the masking moiety that specifically binds to IFNG, in particular the scFv, comprises: (a) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 28. H IFNG) and a light chain variable region (V L IFNG).

[0021] In a specific aspect, there is provided herein an antigen-binding molecule comprising an IFNG variant polypeptide, the antigen-binding molecule comprising: (i) two heavy chains comprising the amino acid sequence of SEQ ID NO: 40 and two light chains comprising the amino acid sequence of SEQ ID NO: 41, or (ii) two heavy chains comprising the amino acid sequence of SEQ ID NO: 42 and two light chains comprising the amino acid sequence of SEQ ID NO: 41; or (iii) two heavy chains comprising the amino acid sequence of SEQ ID NO: 43 and two light chains comprising the amino acid sequence of SEQ ID NO: 44; or (iv) two heavy chains comprising the amino acid sequence of SEQ ID NO: 45 and two light chains comprising the amino acid sequence of SEQ ID NO: 44.

[0022] Further provided herein is an interferon gamma (IFNG) variant polypeptide characterized by the C-terminal amino acid sequence KRKRP (SEQ ID NO: 1). In one embodiment, the IFNG variant polypeptide is a human IFNG variant polypeptide and comprises or consists of the amino acid sequence of SEQ ID NO: 2. In another embodiment, the IFNG variant polypeptide is a mouse IFNG variant polypeptide and comprises or consists of the amino acid sequence of SEQ ID NO: 3.

[0023] According to another aspect of the present invention, one or more isolated polynucleotides are provided that encode the antigen binding molecule described hereinabove. Also provided is an isolated polynucleotide that encodes the IFNG variant polypeptide described hereinabove. The present invention further provides a vector, particularly an expression vector, comprising the isolated polynucleotide of the present invention, and a host cell comprising the isolated nucleic acid or expression vector of the present invention. In some embodiments, the host cell is a eukaryotic cell, particularly a mammalian cell. In some embodiments, the host cell is a prokaryotic cell. In another aspect, a method for producing an antigen binding molecule or an IFNG variant polypeptide described hereinabove is provided, comprising culturing the host cell described above under conditions suitable for expression of the antigen binding molecule or the IFNG variant polypeptide, and isolating the antigen binding molecule or the IFNG variant polypeptide. The present invention also encompasses an antigen binding molecule comprising an IFNG variant polypeptide or an IFNG variant polypeptide produced by the method described herein.

[0024] The present invention further provides a pharmaceutical composition comprising the antigen binding molecule described herein above or the IFNG variant polypeptide described herein above and a pharma- ceutically acceptable carrier.In one embodiment, the pharmaceutical composition comprises an additional therapeutic agent.In one embodiment, the pharmaceutical composition is for treating a disease.In a particular embodiment, the disease is cancer.

[0025] Also encompassed by the present invention is an antigen-binding molecule or an IFNG variant polypeptide as described herein above, or a pharmaceutical composition comprising an antigen-binding molecule or an IFNG variant polypeptide, for use as a medicament.

[0026] In one embodiment, an antigen binding molecule as described herein above or an IFNG variant polypeptide as described herein above or a pharmaceutical composition of the invention is provided for use in the treatment of cancer. In another specific embodiment, the present invention provides an antigen binding molecule or an IFNG variant polypeptide as described herein above for use in the treatment of cancer, wherein the antigen binding molecule or the IFNG variant polypeptide is administered in combination with a chemotherapeutic agent, radiation and / or other agent for use in cancer immunotherapy.

[0027] In a further aspect, the present invention provides a method for inhibiting the proliferation of tumor cells in an individual, comprising administering to the individual an effective amount of an antigen binding molecule or an IFNG variant polypeptide as described herein above, or a pharmaceutical composition of the present invention, thereby inhibiting the proliferation of tumor cells. In another aspect, the present invention provides a method for treating a disease in an individual, comprising administering to the individual a therapeutically effective amount of an antigen binding molecule or an IFNG variant polypeptide as described herein above, or a pharmaceutical composition of the present invention. In a particular aspect, the disease is cancer.

[0028] Also provided is the use of the antigen-binding molecule described herein above for the manufacture of a medicament for the treatment of a disease in an individual in need thereof, in particular for the manufacture of a medicament for the treatment of cancer. In a particular embodiment, the disease is cancer. In any of the above embodiments, the individual is a mammal, in particular a human. [Brief description of the drawings]

[0029] [Figure 1A] FIG. 1 provides an overview of the engineering of the IFNG C-terminal sequence. [Figure 1B] FIG. 1 provides an overview of the engineering of the IFNG C-terminal sequence. [Figure 1C]FIG. 1 provides an overview of the engineering of the IFNG C-terminal sequence. In FIG. 1A, a schematic scheme of the format of the IFNG molecule prepared in Example 1 is shown. IFNG is fused to the C-terminus of the heavy chain of a targeting IgG antibody. The IgG-fused IFNG wild-type C-terminus is prone to proteolysis resulting in truncated variants that are inactive in signal transduction. In FIG. 1B, the IFNG C-terminal sequence with deletion of specific C-terminal amino acids is shown. In the deletion series, the C-terminal residues are gradually removed and then replaced with other amino acids to obtain a cap. The KRKR sequence is important for activity and must be preserved. FIG. 1C shows an overview of the amino acid mutations introduced into the KRKR sequence of the mutation series described in Example 1. [Figure 1D] 1 shows a schematic scheme of the masked release format. IFNG is fused to the C-terminus of the heavy chain of the targeting IgG via a stable linker (Linker 1). To create the masked format, an IFNG-specific scFv domain is fused to the C-terminus of IFNG via a cleavable linker (Linker 2 with a PQARK cleavage site). [Figure 1E] Figure 1 shows a schematic scheme of the cytokine release format: IFNG is fused via a cleavable linker (Linker 2 with a PQARK cleavage site) to the C-terminus of an IFNG-specific scFv domain, which is fused via a stable linker (Linker 1) to the heavy chain of a targeting IgG. [Figure 2A] FIG. 1 shows signaling activity of human IFNG deletion series variants measured in HEK-blue IFNG reporter cells. [Figure 2B] FIG. 1 shows signaling activity of human IFNG deletion series variants measured in HEK-blue IFNG reporter cells. [Figure 2C]Figure 2 shows the signaling activity of human IFNG deletion series variants measured in HEK-blue IFNG reporter cells. Figure 2A compares the signaling activity of molecules with wild-type IFNG (P1AF3568), inactive variant (P1AF3569) and IFNG variants described in the literature (Slodowski et al.). Figure 2B compares wild-type IFNG molecules (P1AF3568) with molecules containing proline-capped IFNG variants. Figure 2C compares wild-type IFNG molecules (P1AF3568) with molecules containing serine-proline-capped IFNG variants. [Figure 3A] FIG. 1 shows signaling activity of human IFNG mutant series variants measured in HEK-blue IFNG reporter cells. [Figure 3B] FIG. 1 shows signaling activity of human IFNG mutant series variants measured in HEK-blue IFNG reporter cells. [Figure 3C] Figure 3 shows the signaling activity of human IFNG mutant series variants measured in HEK-blue IFNG reporter cells. In Figure 3A, the signaling activity of IFNG variant (P1AF3574) with C-terminal KRKRP is compared with IFNG variant molecules with one or two amino acids in the KRKR patch replaced by glutamic acid. Figure 3B compares the signaling activity of IFNG variant (P1AF3574) with C-terminal KRKRP and IFNG variant molecules with one or two amino acids in the KRKR patch replaced by proline. Figure 3C compares the signaling activity of IFNG variant (P1AF3574) with C-terminal KRKRP and IFNG variant molecules with one or two amino acids in the KRKR patch replaced by serine. [Figure 4A]Figure 1 shows a multiple sequence alignment of human and mouse wild-type IFNG and engineered IFNG variants. The aligned sequences shown are the consensus sequence (SEQ ID NO: 125), human IFNG KRKRP 1-132 (SEQ ID NO: 2), mouse IFNG KRKRP 22-153 (SEQ ID NO: 3), human wild-type IFNG (SEQ ID NO: 60) and mouse wild-type IFNG (SEQ ID NO: 61). [Figure 4B] FIG. 1 shows induction of MHC-I in mouse MC38-huCEA tumor cell line in response to 2 days of treatment with FAP-targeted anti-mouse IFNG-scFv masked IFNG compound containing a cleavable PQARK linker (with and without matriptase) compared to the activity of FAP-targeted unmasked IFNG. [Figure 4C] FIG. 1 shows induction of PD-L1 in mouse MC38-huCEA tumor cell line in response to 2 days of treatment with FAP-targeted anti-mouse IFNG-scFv masked IFNG compounds containing a cleavable PQARK linker (with and without matriptase) compared to the activity of FAP-targeted unmasked IFNG. [Diagram 5] Figure 1 shows the study layout highlighting key events of the in vivo study conducted to test the activity of P1AG3755 in vivo. The KPC4662-huCEA tumor model is an exclusion tumor model, meaning that CD8 T cells are only present at the margins of the tumor. MHC-I and PD-L1 expression are low at baseline. P1AG3755 was administered once at two different doses to observe changes in MHC-I, PD-L1 expression and CD8 infiltration. [Figure 6A] FIG. 6 shows MHC-I expression in cancer cells (FIG. 6A) and fibroblasts (FIG. 6B) at different time points of the study. [Figure 6B] Figure 6A shows MHC-I expression in cancer cells (Figure 6A) and fibroblasts (Figure 6B) at different time points of the study. MHC-I expression, as measured by flow cytometry, is statistically significantly increased in cancer cells and fibroblasts only at early and late times (days 3 and 7) after treatment administration. [Figure 6C] Figure 6C shows PD-L1 expression in cancer cells (Figure 6C) and fibroblast cells (Figure 6D) at different time points of the study. [Figure 6D] Figure 6C shows PD-L1 expression in cancer cells (Figure 6C) and fibroblast cells (Figure 6D) at different time points of the study. PD-L1 expression is also statistically significantly increased in both cell types at the higher dose, but only at day 3 and not at day 7 post-treatment. [Figure 7] Figure 1 shows the results of histological analysis by immunofluorescence (3DIP), illustrating CD8 T cell infiltration and MHCI expression in tumor sections. MHCI expression increased in a dose-dependent manner 3 and 7 days after P1AG3755 administration, supporting the flow cytometry results. The increase in MHCI expression was localized and not the same throughout the tumor. The MHCI expression pattern correlates with the presence of CD8 T cells in the tumor core. [Figure 8] Figure 1 shows that CD8 infiltration into tumors, as measured by flow cytometry, was not statistically significantly different from the vehicle group. This was expected, as CD8 T cell infiltration follows highly localized MHCI expression. Immunofluorescence imaging reveals that CD8 T cells are more strongly present in MHC-I positive areas on day 3 post-treatment compared to day 7 post-treatment. [Figure 9] FIG. 14 shows cytokine analysis showing that the T cell-attracting chemokine CXCL9 is statistically significantly increased in serum only 7 days after therapeutic injection (10 mg / kg) but not earlier, at lower doses, or in tumor tissue. [Figure 10A] Figure 1 shows a schematic diagram of sterically masked IFNG (P1AG8692) as described in Example 6. The IFNG variant is inserted between the upper and lower hinge regions of an IgG1 antibody. [Figure 10B] FIG. 1 shows a schematic diagram of an unmasked control compound (P1AG8697) in which the IFNG variant is fused to a Fab via a linker (Linker 4). [Figure 10C] Graph showing activity of sterically masked IFNG variants measured in HEK-blue IFNG reporter cells as relative absorbance. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] definition Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly used in the art to which this invention belongs. For purposes of interpreting this specification, the following definitions shall apply, and whenever appropriate, terms used in the singular shall also include the plural and vice versa.

[0031] As used herein, the term "antigen-binding molecule" in its broadest sense refers to a molecule that specifically binds to an antigenic determinant. Examples of antigen-binding molecules are antibodies, bispecific or multispecific antibodies, immunoconjugates, antibody fragments and scaffold antigen-binding proteins.

[0032] As used herein, the term "antibody that specifically binds to a tumor-associated antigen" or "moiety that specifically binds to a tumor-associated antigen" refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, an antigen-binding domain is capable of activating signaling through its target cell antigen. In a particular embodiment, an antigen-binding domain is capable of directing the entity to which it is bound (e.g., an IFNG variant polypeptide) to a target site, e.g., a particular type of tumor cell or tumor stroma that bears the antigenic determinant. Antigen-binding domains capable of specific binding to tumor-associated antigens include antibodies and fragments thereof as further defined herein. Furthermore, antibodies capable of specific binding to tumor-associated antigens include scaffold antigen-binding proteins as further defined herein, such as binding domains based on designed repeat proteins or designed repeat domains (see, e.g., WO 2002 / 020565). In particular, an antibody capable of specific binding to a tumor-associated antigen is an antibody capable of specific binding to fibroblast activation protein (FAP). With respect to an antibody or a fragment thereof, the term "antibody that specifically binds to a tumor-associated antigen" refers to a part of a molecule that comprises a region that specifically binds to and is complementary to part or all of an antigen. An antigen-binding domain capable of specific antigen binding may for example be provided by one or more antibody variable domains (also called antibody variable regions). In particular, an antigen-binding domain capable of specific antigen binding comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). In another embodiment, an "antigen-binding domain capable of specific binding to a tumor-associated antigen" may be a Fab fragment or a cross-Fab fragment.

[0033] The term "antibody" herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0034] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies in the population are identical and / or bind to the same epitope, except for possible variant antibodies, including, for example, naturally occurring mutations or mutations that arise during production of a monoclonal antibody preparation, such variants will generally be present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.

[0035] As used herein, the term "monospecific" antibody refers to an antibody having one or more binding sites, each of which binds to the same epitope of the same antigen. The term "bispecific" means that an antigen-binding molecule can specifically bind to at least two different antigenic determinants. Typically, a bispecific antigen-binding molecule contains two antigen-binding sites, each of which is specific for a different antigenic determinant. In certain embodiments, a bispecific antigen-binding molecule can simultaneously bind to two antigenic determinants, particularly two antigenic determinants expressed on two different cells. The bispecific antigen-binding molecules described herein can also form part of a multispecific antibody.

[0036] The term "valent" as used within this application refers to the presence of a designated number of binding sites specific for one different antigenic determinant in an antigen-binding molecule specific for one different antigenic determinant. Thus, the terms "bivalent", "tetravalent" and "hexavalent" refer to the presence of two binding sites, four binding sites and six binding sites, respectively, specific for a particular antigenic determinant in an antigen-binding molecule. In certain aspects of the invention, a bispecific antigen-binding molecule according to the invention may be monovalent for a particular antigenic determinant, meaning that it has only one binding site for that antigenic determinant, or bivalent or tetravalent for a particular antigenic determinant, meaning that it has two binding sites or four binding sites, respectively, for that antigenic determinant.

[0037] The terms "full-length antibody", "intact antibody" and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a natural antibody structure. "Native antibody" refers to naturally occurring immunoglobulin molecules with various structures. For example, a natural IgG class antibody is a heterotetrameric glycoprotein of about 150,000 daltons, composed of two light chains and two heavy chains disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3), also called heavy chain constant region. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called variable light domain or light chain variable domain, followed by a light chain constant domain (CL), also called light chain constant region. The heavy chain of an antibody 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, e.g., γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1) and α2 (IgA2). The light chain of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0038] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include Fv, Fab, Fab', Fab'-SH, F(ab') 2Antibody fragments include, but are not limited to, diabodies, triabodies, tetrabodies, cross-Fab fragments, linear antibodies, single-chain antibody molecules (e.g., scFv), and single domain antibodies. For a review of certain antibody fragments, see Hudson et al., Nat Med. 9, 129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994), and also WO 93 / 16185 and U.S. Pat. Nos. 5,571,894 and 5,587,458. For a description of Fab and F(ab')2 fragments that contain salvage receptor binding epitope residues and have increased half-life in vivo, see U.S. Pat. No. 5,869,046. Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific, see, for example, EP 404097; WO 1993 / 01161; Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). Single-domain antibodies are antibody fragments that contain all or a portion of an antibody heavy chain variable domain or all or a portion of an antibody light chain variable domain. In certain embodiments, single-domain antibodies are human single-domain antibodies (Domantis, Inc., Waltham, MA; see, for example, U.S. Pat. No. 6,248,516). Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phages), as described herein.

[0039] Papain digestion of an intact antibody produces two identical antigen-binding fragments, called "Fab" fragments, each of which contains the heavy and light chain variable domains, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Thus, as used herein, the term "Fab fragment" refers to an antibody fragment that contains the VL domain and constant domain of the light chain (CL) and the VH domain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residues of the constant domains bear a free thiol group. Pepsin treatment produces F(ab') fragments with two antigen-binding sites (two Fab fragments) and part of the Fc region. 2 According to the present invention, the term "Fab fragment" also includes "cross-Fab fragments" or "crossover Fab fragments" as defined below.

[0040] The term "cross-Fab fragment" or "xFab fragment" or "crossover Fab fragment" refers to a Fab fragment in which either the variable or constant regions of the heavy and light chains have been exchanged. Two different chain compositions of crossover Fab molecules are possible and are included in the bispecific antibodies of the present invention. On the other hand, the variable regions of the Fab heavy and light chains have been exchanged, i.e. the crossover Fab molecule comprises a peptide chain composed of the light chain variable region (VL) and the heavy chain constant region (CH1) and a peptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL). This crossover Fab molecule is called CrossFab (VLVH) On the other hand, when the constant regions of the Fab heavy and light chains are exchanged, the crossover Fab molecule contains a peptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL) and a peptide chain composed of the light chain variable region (VL) and the heavy chain constant region (CH1). This crossover Fab molecule is called CrossFab. (CLCH1) It is also called.

[0041] A "single-chain Fab fragment" or "scFab" is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, the antibody domains and the linker having one of the following orders from N-terminus to C-terminus: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL, the linker being a polypeptide of at least 30 amino acids, preferably 32-50 amino acids. The single-chain Fab fragment is stabilized by a native disulfide bond between the CL and CH1 domains. In addition, these single-chain Fab molecules may be further stabilized by the insertion of cysteine ​​residues (e.g., at position 44 of the variable heavy chain and position 100 of the variable light chain according to the Kabat numbering) to create an interchain disulfide bond.

[0042] A "crossover single chain Fab fragment" or "x-scFab" is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, the antibody domains and the linker having one of the following orders from N-terminus to C-terminus: a) VH-CL-linker-VL-CH1 and b) VL-CH1-linker-VH-CL, where VH and VL together form an antigen binding site that specifically binds to an antigen, and the linker is a polypeptide of at least 30 amino acids. Furthermore, these x-scFab molecules may be further stabilized by the creation of an interchain disulfide bond by the insertion of cysteine ​​residues (e.g., at position 44 of the variable heavy chain and position 100 of the variable light chain according to the Kabat numbering).

[0043] A "single-chain variable fragment (scFv)" is a fragment of an antibody heavy chain (V) connected by a short linker peptide of 10 to about 25 amino acids. H ) and light chain (V L) variable region. The linker is usually rich in glycine for flexibility, as well as rich in serine or threonine for solubility, and the V H N-terminus of V L The scFv antibody can be linked to the C-terminus of a VH domain or vice versa. This protein retains the properties of the original antibody despite the removal of the constant region and the introduction of the linker. scFv antibodies are described, for example, in Houston, JS, Methods in Enzymol. 203 (1991) 46-96. Furthermore, antibody fragments include single-chain polypeptides characterized by a VH domain, i.e. capable of assembling with a VL domain, or characterized by a VL domain, i.e. capable of assembling with a VH domain into a functional antigen-binding site, thereby providing the antigen-binding properties of a full-length antibody.

[0044] "Scaffold antigen-binding proteins" are known in the art, for example, fibronectin and designed ankyrin repeat proteins (DARPins) have been used as alternative scaffolds for antigen-binding domains, see, e.g., Gebauer and Skerra, Engineered protein scaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol 13:245-255 (2009) and Stumpp et al., Darpins: A new generation of protein therapeutics. Drug Discovery Today 13:695-701 (2008). In one embodiment of the invention, the scaffold antigen binding protein is selected from the group consisting of CTLA-4 (Evibody), lipocalin (Anticalin), protein A derived molecules such as the Z-domain of protein A (Affibody), A-domain (Avimer / Maxibody), serum transferrin (Transbody); designed ankyrin repeat proteins (DARPins), variable domains of antibody light or heavy chains (single domain antibodies, sdAb), variable domains of antibody heavy chains (nanobodies, aVH), V NARFragment, Fibronectin (Adnectin), C-type lectin domain (Tetranectin); Variable domain of novel antigen receptor beta-lactamase (V NAR fragments), human gamma-crystallin or ubiquitin (Affilin molecules); Kunitz-type domains of human protease inhibitors, microbodies, e.g. proteins from the knottin family, peptide aptamers and fibronectin (adnectins). CTLA-4 (Cytotoxic T-lymphocyte-associated antigen 4) is a cytotoxic T-lymphocyte-associated antigen that is primarily expressed by CD4 +It is a CD28 family receptor expressed on T cells. Its extracellular domain has a variable domain-like Ig fold. The loops corresponding to the CDRs of an antibody can be replaced with heterologous sequences to confer different binding properties. CTLA-4 molecules engineered to have different binding specificities are also known as Evibodies (e.g., U.S. Pat. No. 7,166,697). Evibodies are approximately the same size as the isolated variable regions of an antibody (e.g., domain antibodies). For further details, see Journal of Immunological Methods 248(1-2), 31-45(2001). Lipocalins are a family of extracellular proteins that transport small hydrophobic molecules such as steroids, villins, retinoids and lipids. They have a rigid beta sheet secondary structure with several loops at the open end of a conical structure that can be engineered to bind different target antigens. Anticalins are 160-180 amino acids in size and are derived from lipocalins. For further details, see Biochim Biophys Acta 1482:337-350 (2000), US Patent No. 7,250,297 and US Patent Publication No. 20070224633. Affibodies are scaffolds derived from Staphylococcus aureus Protein A that can be engineered to bind antigens. The domains consist of three helical bundles of about 58 amino acids. Libraries are created by randomization of surface residues. For further details, see Protein Eng. Des. Sel. 2004, 17, 455-462 and EP 1641818. Avimers are multi-domain proteins derived from the A-domain scaffold family. Natural domains of about 35 amino acids adopt defined disulfide-bonded structures. Diversity is generated by shuffling of the natural variations exhibited by the family of A-domains.For further details, see Nature Biotechnology 23(12), 1556-1561 (2005) and Expert Opinion on Investigational Drugs 16(6), 909-917 (June 2007). Transferrin is a monomeric serum transport glycoprotein. Transferrin can be engineered to bind different target antigens by inserting peptide sequences into permissive surface loops. Examples of engineered transferrin scaffolds include transbodies. For further details, see J.Biol.Chem 274, 24066-24073 (1999). Designed ankyrin repeat proteins (DARPins) are derived from ankyrins, a family of proteins that mediate the attachment of integral membrane proteins to the cytoskeleton. A single ankyrin repeat is a 33-residue motif consisting of two α-helices and a β-turn. Single ankyrin repeats can be engineered to bind different target antigens by randomizing residues in the first α-helix and β-turn of each repeat. The binding interface can be increased by increasing the number of modules (affinity maturation method). For further details, see J.Mol.Biol.332, 489-503 (2003), PNAS 100(4), 1700-1705 (2003) and J.Mol.Biol.369, 1015-1028 (2007), and US Patent Publication No. 20040132028. Single domain antibodies are antibody fragments consisting of one monomeric variable antibody domain. The first single domain was derived from the variable domain of an antibody heavy chain from camelid (nanobody or V. H Furthermore, the term single domain antibody can refer to an antibody that contains an autonomous human heavy chain variable domain (aVH) or a shark-derived VH. NARAdnectins include fragments. Fibronectin is a scaffold that can be engineered to bind antigens. Adnectins consist of a backbone with the natural amino acid sequence of the 10th domain of the 15 repeating units of human fibronectin type III (FN3). Three loops at one end of the β-sandwich can be engineered to allow Adnectins to specifically recognize therapeutic targets of interest. For further details, see Protein Eng. Des. Sel. 18, 435-444 (2005), US Patent Publication No. 20080139791, WO 2005056764 and US Patent No. 6,818,418. Peptide aptamers are combinatorial recognition molecules that consist of a constant scaffold protein, typically thioredoxin (TrxA), that contains a constrained variable peptide loop that is inserted into the active site. For further details, see Expert Opin. Biol. Ther. 5, 783-797 (2005). Microbodies are derived from naturally occurring microproteins that are 25-50 amino acids long and contain 3-4 cysteine ​​bridges; examples of microproteins include KalataBI and conotoxins and knottins. Microproteins have loops that can be engineered to contain up to 25 amino acids without affecting the overall folding of the microprotein. For further details of engineered knottin domains, see WO2008098796.

[0045] An "antibody that binds to the same epitope" as a reference molecule refers to an antigen-binding molecule that blocks the binding of the reference molecule to its antigen by 50% or more in a competitive assay, and conversely, the reference molecule blocks the binding of the antigen-binding molecule to its antigen by 50% or more in a competitive assay. An "antibody that does not bind to the same epitope" as a reference molecule refers to an antigen-binding molecule that does not block the binding of the reference molecule to its antigen by 50% or more in a competitive assay, and conversely, the reference molecule does not block the binding of the antigen-binding molecule to its antigen by 50% or more in a competitive assay.

[0046] The term "antigen-binding domain" or "antigen-binding site" refers to a portion of an antigen-binding molecule that contains an area that specifically binds to an antigen and is partially or completely complementary to the antigen. If the antigen is large, the antigen-binding molecule may bind only to a specific portion of the antigen, which portion is called an epitope. An antigen-binding domain may, for example, be provided by one or more variable domains (also called variable regions). Preferably, the antigen-binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0047] As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope" and refers to a site on a polypeptide macromolecule to which an antigen-binding moiety binds (e.g., a contiguous stretch of amino acids or a conformational structure composed of different regions of non-contiguous amino acids) to form an antigen-binding moiety-antigen complex. Useful antigenic determinants can be found, for example, on tumor cell surfaces, on virus-infected cell surfaces, on other diseased cell surfaces, on immune cell surfaces, free in serum, and / or within the extracellular matrix (ECM). Unless otherwise specified, proteins useful as antigens herein may be proteins in any natural form from any vertebrate source, including mammals, e.g., primates (e.g., humans) and rodents (e.g., mice and rats). In certain embodiments, the antigen is a human protein. When a particular protein is referred to herein, the term encompasses not only the "full-length" unprocessed protein, but any type of protein resulting from processing within the cell. The term also encompasses naturally occurring protein variants, e.g., splice variants or allelic variants.

[0048] By "specific binding" it is meant that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The binding ability of an antigen-binding molecule to a particular antigen can be measured by either enzyme-linked immunosorbent assay (ELISA) or other techniques well known to those skilled in the art, such as surface plasmon resonance (SPR) techniques (analysis on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and classical binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the extent of binding of the antigen-binding molecule to an unrelated protein is less than about 10% of the binding of the antigen-binding molecule to the antigen, as measured, for example, by SPR. In certain embodiments, a molecule that binds to an antigen has a dissociation constant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 M).

[0049] "Affinity" or "binding affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the inherent binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its binding partner Y can generally be represented by a dissociation constant (Kd), which is the ratio of the desorption rate constant and the dissociation rate constant (koff and kon, respectively). Thus, equivalent affinities can include different rate constants as long as the ratio of the rate constants remains the same. Affinity can be measured by methods common in the art, including those described herein. A particular method of measuring affinity is surface plasmon resonance (SPR).

[0050] An "affinity matured" antibody refers to an antibody that has one or more alterations in one or more hypervariable regions (HVRs) relative to a parent antibody that does not possess such alterations, which alterations improve the affinity of the antibody for antigen.

[0051] As used herein, "tumor-associated antigen" or TAA refers to an antigenic determinant presented on the surface of a target cell, e.g., a cell within a tumor, such as a cancer cell or a tumor stromal cell. In certain embodiments, the target cell antigen is an antigen on the surface of a tumor cell. In one embodiment, the TAA is selected from the group consisting of fibroblast activation protein (FAP), carcinoembryonic antigen (CEA), folate receptor alpha (FolR1), melanoma-associated chondroitin sulfate proteoglycan (MCSP), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2) and p95HER2. In particular, the tumor-associated antigen is a fibroblast activation protein (FAP).

[0052] The term "fibroblast activation protein (FAP)", also known as prolyl endopeptidase FAP or seprase (EC 3.4.21), refers to any naturally occurring FAP from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length" unprocessed FAPs, as well as any form of FAP resulting from processing within a cell. The term also encompasses naturally occurring variants of FAPs, such as splice variants or allelic variants. In one embodiment, the antigen-binding molecule of the present invention is capable of specifically binding to human, mouse and / or cynomolgus monkey FAPs. The amino acid sequence of human FAP is shown in UniProt (www.uniprot.org) Accession No. Q12884 (Version 149, SEQ ID NO: 46) 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 position 760. The amino acid sequence of mouse FAP is shown in UniProt Accession No. P97321 (Version 126, SEQ ID NO: 47) or NCBI RefSeq NP_032012.1. The extracellular domain (ECD) of mouse FAP extends from amino acid position 26 to position 761. Preferably, the anti-FAP binding molecule of the invention binds to the extracellular domain of FAP.

[0053] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in the binding of an antigen-binding molecule to an antigen. The variable domains of heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, and each domain contains four conserved framework regions (FR) and three hypervariable regions (HVR). See, for example, Kindt et al., Kuby Immunology, 6th Edition, WH Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0054] As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain that are hypervariable in sequence and that determine antigen-binding specificity, e.g., the "complementarity determining regions" (CDRs).

[0055] Generally, an antibody comprises six CDRs, three in the VH (CDR-H1, CDR-H2, CDR-H3) and three in the VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs herein include the following: (a) hypervariable loops located 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 located 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 (c) Antigen contact sites present 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)).

[0056] Unless otherwise specified, CDRs are determined according to Kabat et al., supra. Those skilled in the art will understand that the designations of CDRs can also be determined according to Chothia, supra, McCallum, supra, or any other scientifically accepted nomenclature system.

[0057] "Framework" or "FR" refers to variable domain residues other than the complementarity determining regions (CDRs). The FRs of a variable domain generally consist of four FR domains, FR1, FR2, FR3, and FR4. Thus, the CDR and FR sequences generally appear in the VH (or VL) in the following sequence: FR1-CDR-H1 (CDR-L1)-FR2-CDR-H2 (CDR-L2)-FR3-CDR-H3 (CDR-L3)-FR4.

[0058] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0059] The "class" of an antibody refers to the type of constant domain or constant region that its heavy chain has. There are five major classes of antibodies, IgA, IgD, IgE, IgG, and IgM, and some of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0060] A "humanized" antibody refers to a chimeric antibody that comprises amino acid residues from a non-human HVR and amino acid residues from a human FR. In certain embodiments, a humanized antibody comprises substantially all of at least one, typically two, variable domains, with all or substantially all of the HVRs (e.g., CDRs) corresponding to a non-human antibody and all or substantially all of the FRs corresponding to a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization. Other forms of "humanized antibodies" encompassed by the present invention are those in which the constant regions have been further modified or altered from the constant regions of the original antibody to generate properties according to the invention, particularly with respect to C1q binding and / or Fc receptor (FcR) binding.

[0061] The term "CH1 domain" refers to a portion of an antibody heavy chain polypeptide extending from approximately EU position 118 to EU position 215 (EU numbering system according to Kabat). In one aspect, the CH1 domain has the amino acid sequence of ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKV (SEQ ID NO: 48). Typically, a segment having the amino acid sequence of EPKSC (SEQ ID NO: 49) then links the CH1 domain to the hinge region.

[0062] The term "hinge region" refers to a portion of an antibody heavy chain polypeptide that connects the CH1 and CH2 domains in a wild-type antibody heavy chain (e.g., from about position 216 to about position 230, or from about position 226 to about position 230 according to the EU numbering system of Kabat). Hinge regions of other IgG subclasses can be determined by aligning with the hinge region cysteine ​​residues of an IgG1 subclass sequence. Hinge regions are usually dimeric molecules consisting of two polypeptides with identical amino acid sequences. Hinge regions generally contain up to 25 amino acid residues and are flexible, allowing the associated target binding sites to move independently. Hinge regions can be subdivided into three domains: upper, middle, and lower hinge domains (see, e.g., Roux et al., J. Immunol. 161 (1998) 4083). In one embodiment, the hinge region has the amino acid sequence DKTHTCPXCP (SEQ ID NO: 50), where X is either S or P. In one aspect, the hinge region has the amino acid sequence HTCPXCP (SEQ ID NO:51), where X is either S or P. In one aspect, the hinge region has the amino acid sequence CPXCP (SEQ ID NO:52), where X is either S or P.

[0063] The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an antibody heavy chain that includes at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. The IgG Fc region includes the IgG CH2 and IgG CH3 domains. The "CH2 domain" of a human IgG Fc region typically extends from about amino acid residue 231 to about amino acid residue 340. (EU numbering system according to Kabat). In one embodiment, the CH2 domain has the amino acid sequence APELLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQESTYRW SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAK (SEQ ID NO: 53). The CH2 domain is unique in that it is not closely paired with other domains. Rather, two N-linked branched carbohydrate chains are sandwiched between the two CH2 domains of an intact native Fc region. It has been speculated that carbohydrates may provide an alternative domain-domain pairing and help stabilize the CH2 domain. Burton, Mol. Immunol. 22 (1985) 161-206. In one embodiment, the carbohydrate chains are attached to the CH2 domain. The CH2 domain herein may be a native sequence CH2 domain or a variant CH2 domain. The "CH3 domain" comprises a stretch of residues C-terminal to the CH2 domain in the Fc region (i.e., from about amino acid residue 341 to about amino acid residue 447 according to the EU numbering system of Kabat for IgG). In one aspect, the CH3 domain has the amino acid sequence of GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG (SEQ ID NO:54).The CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain with a "protrusion" ("knob") introduced in one chain and a corresponding "cavity" ("hole") introduced in the other chain; see U.S. Pat. No. 5,821,333, expressly incorporated herein by reference). Such variant CH3 domains may be used to promote heterodimerization of two non-identical antibody heavy chains as described herein. In one embodiment, the human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to 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.

[0064] The term "wild-type Fc domain" refers to an amino acid sequence identical to the amino acid sequence of an Fc domain found in nature. Wild-type human Fc domains include native human IgG1 Fc regions (non-A and A allotypes), native human IgG2 Fc regions, native human IgG3 Fc regions and native human IgG4 Fc regions and naturally occurring variants thereof. Wild-type Fc regions are shown in SEQ ID NO: 55 (IgG1, Caucasian allotype), SEQ ID NO: 56 (IgG1, Afro-American allotype), SEQ ID NO: 57 (IgG2), SEQ ID NO: 58 (IgG3) and SEQ ID NO: 59 (IgG4). The term "variant (human) Fc domain" refers to an amino acid sequence that differs from the "wild-type" (human) Fc domain amino acid sequence by at least one "amino acid mutation". In one embodiment, the variant Fc region has at least one amino acid mutation compared to the native Fc region, for example about 1 to about 10 amino acid mutations, in one embodiment about 1 to about 5 amino acid mutations in the native Fc region. In one aspect, the (variant) Fc region has at least about 95% homology to the wild-type Fc region.

[0065] The "knob-into-hole" technique has been described, for example, in U.S. Patent Nos. 5,731,168; 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). In general, the method involves introducing a protrusion ("knob") into the interface of a first polypeptide and a corresponding cavity ("hole") into the interface of a second polypeptide, such that the protrusion is positioned within the cavity, thereby promoting heterodimer formation and preventing homodimer formation. The protrusion is constructed by replacing a small amino acid side chain at the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A compensatory cavity of identical or similar size to the protrusion is created at the interface of the second polypeptide by replacing the large amino acid side chain with a small amino acid side chain (e.g., alanine or threonine). The protrusion and cavity can be created by modifying the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis. In a specific embodiment, the knob modification comprises the amino acid substitution T366W in one of the two subunits of the Fc domain, and the hole modification comprises the amino acid substitutions T366S, L368A and Y407V in the other of the two subunits of the Fc domain. In a more specific embodiment, the subunit of the Fc domain that comprises the knob modification further comprises the amino acid substitution S354C, and the subunit of the Fc domain that comprises the hole modification further comprises the amino acid substitution Y349C. The introduction of these two cysteine ​​residues forms a disulfide bridge between the two subunits of the Fc region, thereby further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0066] "A region corresponding to the Fc region of an immunoglobulin" is intended to include naturally occurring allelic variants of the Fc region of an immunoglobulin, as well as variants that have modifications that produce substitutions, additions, or deletions, but do not substantially reduce the ability of the immunoglobulin to mediate effector functions (such as antibody-dependent cellular cytotoxicity). For example, one or more amino acids can be deleted from the N-terminus or C-terminus of the Fc region of an immunoglobulin without substantially impairing biological function. Such variants can be selected according to general rules known in the art to have minimal effect on activity (see, for example, Bowie, JU et al., Science 247:1306-10 (1990)).

[0067] The term "effector function" refers to the biological activities attributable to the Fc region of an antibody, which vary depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and activation of B cells.

[0068] Fc receptor binding-dependent effector functions can be mediated by the interaction of the Fc region of an antibody with Fc receptors (FcRs), specialized cell surface receptors on hematopoietic cells. Fc receptors belong to the immunoglobulin superfamily and have been shown to mediate both the removal of antibody-coated pathogens by phagocytosis of immune complexes via antibody-dependent cell-mediated cytotoxicity (ADCC) and the lysis of corresponding antibody-coated red blood cells and various other cellular targets (e.g., tumor cells) (see, e.g., Van de Winkel, JG and Anderson, CL, J. Leukoc. Biol. 49 (1991) 511-524). FcRs are defined by their specificity for immunoglobulin isotypes. Fc receptors for IgG antibodies are called FcγRs. Fc receptor binding is described, for example, in Ravetch, JV and Kinet, JP, Annu. Rev. Immunol. 9 (1991) 457-492; Capel, PJ et al., Immunomethods 4 (1994) 25-34; de Haas, M. et al., J. Lab. Clin. Med. 126 (1995) 330-341; and Gessner, JE et al., Ann. Hematol. 76 (1998) 231-248.

[0069] Cross-linking of receptors to the Fc region of IgG antibodies (FcγR) triggers a wide variety of effector functions, including phagocytosis, antibody-dependent cellular cytotoxicity, and release of inflammatory mediators, as well as control of immune complex clearance and antibody production. In humans, three classes of FcγR have been characterized:

[0070] FcγRI (CD64) binds monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils and eosinophils. Modification of the Fc region IgG at at least one of the amino acid residues E233-G236, P238, D265, N297, A327, and P329 (numbering according to the Kabat EU index) reduces binding to FcγRI. Substitution of IgG2 residues at positions 233-236 into IgG1 and IgG4 reduces binding to FcγRI by 103-fold and abolishes the human monocyte response to antibody-sensitized erythrocytes (Armour, KL, et al., Eur. J. Immunol. 29 (1999) 2613-2624), -FcγRII (CD32) binds complexed IgG with moderate to low affinity and is widely expressed. This receptor can be divided into two subtypes, FcγRIIA and FcγRIIB. FcγRIIA is found on many cells involved in killing (e.g., macrophages, monocytes, neutrophils) and appears to be able to activate the killing process. FcγRIIB appears to play a role in inhibitory processes and is found on B cells, macrophages, as well as mast cells and eosinophils. On B cells, FcγRIIB appears to function to suppress the production of further immunoglobulins and isotype switching, for example to the IgE class. On macrophages, FcγRIIB acts to inhibit phagocytosis mediated by FcγRIIA. On eosinophils and mast cells, the B form may serve to suppress activation of these cells through IgE binding to its other receptors. Reduced binding to FcγRIIA is seen, for example, in antibodies comprising an IgG Fc region having mutations in at least one of amino acid residues E233 to G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292 and K414 (numbering according to the Kabat EU index).

[0071] FcγRIII (CD16) binds IgG with moderate to low affinity and exists as two types. FcγRIIIA is found on NK cells, macrophages, eosinophils, and some monocytes and T cells, and mediates ADCC. FcγRIIIB is highly expressed on neutrophils. Reduced binding to FcγRIIIA is seen, for example, in antibodies that contain an IgG Fc region with mutations at least one of the amino acid residues E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338, and D376 (numbering according to Kabat EU index).

[0072] Mapping of the binding sites on human IgG1 for Fc receptors, the mutation sites described above, and methods for measuring binding to FcγRI and FcγRIIA are described in Shields, RL et al., J. Biol. Chem. 276 (2001) 6591-6604.

[0073] The term "ADCC" or "antibody-dependent cellular cytotoxicity" refers to the lysis of target cells by antibodies as reported herein, a function mediated by Fc receptor binding, in the presence of effector cells. The ability of an antibody to trigger the initial step of mediating ADCC is investigated by measuring the binding of the antibody to cells expressing Fcγ receptors, such as recombinantly expressing FcγRI and / or FcγRIIA or NK cells (which inherently express FcγRIIIA). In particular, binding to FcγR on NK cells is measured.

[0074] An "activating Fc receptor" is an Fc receptor that, upon engagement by the Fc region of an antibody, triggers a signaling event that stimulates the receptor-containing cell to exert an effector function. Activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32) and FcαRI (CD89). A particular activating Fc receptor is human FcγRIIIa (see UniProt Accession No. P08637, version 141).

[0075] As used herein, the term "IFNG" refers to any interferon gamma polypeptide, including recombinantly produced polypeptides, synthetically produced polypeptides, and IFNG isolated from cells or tissues, such as from T lymphocytes and NK cells and other sources. When isolated from any source or produced, IFNG polypeptides may be heterogeneous in length, typically ranging from 124 to 146 amino acids in length. Heterogeneity is typically observed at both ends, at the N-terminus due to post-translational removal of Cys-Tyr-Cys amino acids, and at the C-terminus due to variable proteolytic processing. Heterogeneity may also arise due to N-glycosylation of the polypeptide. Heterogeneity of IFNG polypeptides may vary depending on the source of the IFNG polypeptide. Thus, reference to IFNG polypeptides refers to the heterogeneous population produced or isolated.

[0076] IFNG is a cytokine secreted in response to viral infection or cancerous growth. IFNG regulates MHC antigen expression of T cell class I and II, Fc receptors, and macrophages. IFNG signals are transmitted through a multimeric receptor complex consisting of two different chains, the IFNG receptor binding subunit (IFNGR, IFNGR1) and the transmembrane cofactor (IFNGR2). The IFNG signaling complex is formed upon ligand-driven dimerization of the IFNG receptor. As used herein, the term "IFNG polypeptide" may include its monomeric or dimeric form, as appropriate. As used herein, the term "human IFNG" (huIFNG) includes IFNG, allelic variant isoforms, synthetic molecules, proteins isolated from human tissues and cells, and modified forms thereof, with an exemplary human wild-type IFNG polypeptide shown in SEQ ID NO:60. As used herein, the term "mouse IFNG" (muIFNG) includes IFNG, allelic variant isoforms, synthetic molecules, proteins isolated from mouse tissues and cells, and modified forms thereof, with an exemplary mouse wild-type IFNG polypeptide being set forth in SEQ ID NO:61.

[0077] As used herein, the term "IFNG variant polypeptide" refers to a monomeric IFNG polypeptide in which one or more amino acids have been truncated and / or there are amino acid substitutions, deletions or insertions compared to the amino acid sequence of a wild-type IFNG polypeptide.

[0078] The terms "anti-IFNG antibody", "anti-IFNG", "IFNG antibody" and "antibody that specifically binds to IFNG" refer to an antibody or antibody fragment that specifically binds to IFNG with sufficient affinity such that the antibody or antibody fragment is useful as a diagnostic and / or therapeutic agent in targeting IFNG. When associated with a molecule that contains IFNG, the anti-IFNG antibody can function as a masking moiety of the molecule. Specifically disclosed herein are scFvs specific for IFNG that can block the activity of the cytokine. In one aspect, the extent of binding of the anti-IFNG antibody to an unrelated non-IFNG protein is less than about 10% of the binding of the antibody to IFNG, as measured, for example, by radioimmunoassay (RIA) or flow cytometry (FACS). In certain embodiments, the antibody that binds to IFNG has a specific binding affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -6 M or less, e.g. 10 -68 M~10 -13 M, for example 10 -8 M~10 -10 Dissociation constant (K D In one embodiment, the anti-IFNG antibody is an antibody fragment, in particular an scFv.

[0079] The term "linker" or "peptide linker" refers to a peptide that includes one or more amino acids, typically about 2-30 amino acids. Peptide linkers are known in the art or described herein. Suitable non-immunogenic linker peptides include, for example, (G 4 S) n , (S.G. 4 ) n or G 4 (SG 4 ) n is a peptide linker, where "n" is generally a number from 1 to 10, typically from 2 to 5, and particularly 3, i.e., the peptide may be GGGGS (SEQ ID NO: 62), GGGGSGGGGS (SEQ ID NO: 63), SGGGGSGGGG (SEQ ID NO: 64), GGGGSGGGGSGGGG (SEQ ID NO: 65), (GS) 3(SEQ ID NO: 66), (G4S) 4 (SEQ ID NO: 67) and (G4S) 5 (SEQ ID NO:68), GGGGSGGGGSGGGGSGGGGSGGGGSGGGGG (SEQ ID NO:69), and GGGGSGGG (SEQ ID NO:72), but also including peptide linkers such as LEVLFQG (SEQ ID NO:73) or GGGGSGGGGSGGGGSGGGGSGLEVLFQGPGGGGGSGGGGG (SEQ ID NO:74). A particularly interesting peptide linker is the protease cleavable linker ("cleavable linker") GGGGSGGGGSGGGPQARKGGGGGGSGGGGG (SEQ ID NO:70) or GGGGSGGGGSGGGHQARKGGGGGGSGGGGG (SEQ ID NO:71). A "non-cleavable linker" is a peptide linker that is not recognized by proteases and is stable to proteolysis.

[0080] As used in this application, the term "amino acid" refers to the group of naturally occurring carboxy α-amino acids including alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y) and valine (val, V).

[0081] By "fused" or "linked" is meant that the components (e.g., an antibody heavy chain and a Fab fragment) are linked by a peptide bond, either directly or via one or more peptide linkers.

[0082] "Percent (%) amino acid sequence identity" to a reference polypeptide (protein) 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 aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity, without considering conservative substitutions as part of sequence identity for the purpose of alignment. Alignment to determine percent amino acid sequence identity can be achieved using a variety of methods within the skill of the art, for example, publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or FASTA program package. Those skilled in the art can determine the appropriate parameters for sequence alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. Alternatively, percent identity values ​​can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code has been filed together with user documentation in the U.S. Copyright Office, Washington DC, 20559, where it is registered under U.S. Copyright Registration No. TXU510087, and is set forth in WO 2001 / 007611. Unless otherwise specified, for purposes herein, percent amino acid sequence identity values ​​are generated using the ggsearch program of the FASTA package version 36.3.8c, or subsequent use of the BLOSUM50 comparison matrix.The FASTA program package is written by WR Pearson and DJ Lipman (1988), "Improved Tools for Biological Sequence Analysis", PNAS 85:2444-2448; WR Pearson (1996) "Effective protein sequence comparison" Meth. Enzymol. 266:227-258; and Pearson et al. (1997) Genomics 46:24-36, and is publicly available at www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www.ebi.ac.uk / Tools / sss / fasta. Alternatively, sequences can be compared using the public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi using the ggsearch(global protein:protein) program and default options (BLOSUM50; open:-10; ext:-2; Ktup=2) to ensure a global, rather than local, alignment. The percent amino acid identity is given in the output alignment header.

[0083] In certain embodiments, amino acid sequence variants of the bispecific antigen-binding molecules provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the TNF ligand trimer-containing antigen-binding molecules. Amino acid sequence variants of the TNF ligand trimer-containing antigen-binding molecules can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the molecule or by peptide synthesis. Such modifications include, for example, deletions from and / or insertions into and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions and substitutions can be made to arrive at the final construct, so long as the final construct has the desired properties, e.g., antigen binding. Sites of interest for substitutional mutagenesis include HVRs and frameworks (FRs). Conservative substitutions are provided in Table B under the heading of "preferred substitutions" and are further described below with reference to amino acid side chain classes (1)-(6). Amino acid substitutions may be introduced into the molecule of interest and the products screened for a desired activity, e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0084] [Table 1]

[0085] Amino acids can be classified according to common side chain properties. (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln (3) Acidic: Asp, Glu (4) Basic: His, Lys, Arg (5) Residues that affect chain orientation: Gly, Pro (6) Aromatic: Trp, Tyr, Phe

[0086] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0087] The term "amino acid sequence variant" includes substantial variants in which there is an amino acid substitution in one or more hypervariable region residues of a parent antigen-binding molecule (e.g., a humanized or human antibody). Generally, the resulting variant selected for further testing has a modification (e.g., an improvement) of a particular biological property (e.g., increased affinity, decreased immunogenicity) compared to the parent antigen-binding molecule, and / or substantially retains a particular biological property of the parent antigen-binding molecule. An exemplary substitutional variant is an affinity-matured antibody, which can be conveniently generated, for example, using phage display-based affinity maturation techniques as described herein. Briefly, one or more CDR residues are mutated, and the variant antigen-binding molecule is displayed on phage and screened for a particular biological activity (e.g., binding affinity). In certain embodiments, substitutions, insertions, or deletions can be made within one or more HVRs, so long as such changes do not substantially reduce the ability of the antigen-binding molecule to bind to the antigen. For example, conservative changes (e.g., conservative substitutions provided herein) that do not substantially reduce binding affinity can be made in the CDRs. A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues, e.g., Arg, Asp, His, Lys, and Glu) is identified and replaced by neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions may be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of the antigen-antigen binding molecule complex to identify contact points between the antibody and the antigen. Such contact and adjacent residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they have the desired properties.

[0088] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is a bispecific antigen-binding molecule of the invention with an N-terminal methionyl residue. Other insertion variants of the molecule include fusions to polypeptides at the N- or C-terminus that increase the serum half-life of the bispecific antigen-binding molecule.

[0089] In certain embodiments, the bispecific antigen-binding molecules provided herein are modified to increase or decrease the extent to which the antibody is glycosylated. Glycosylation variants of the molecule may be conveniently obtained by altering the amino acid sequence so that one or more glycosylation sites are created or removed. If the TNF ligand trimer-containing antigen-binding molecule comprises an Fc region, the carbohydrate attached thereto may vary. Natural antibodies produced by mammalian cells typically comprise branched biantennary oligosaccharides that are generally attached to Asn297 of the CH2 domain of the Fc region by N-linkage. See, for example, Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc of the "stem" of the biantennary oligosaccharide structure. In some embodiments, modification of oligosaccharides in TNF family ligand trimer-containing antigen binding molecules can be performed to generate variants with specific improved properties. In one aspect, variants of bispecific antigen binding molecules or antibodies of the present invention are provided that have carbohydrate structures that lack fucose attached (directly or indirectly) to the Fc region. Such fucosylated variants may have improved ADCC function, see, for example, US Patent Application Publication No. 2003 / 0157108 (Presta, L.) or US Patent Application Publication No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). In another aspect, variants of bispecific antigen binding molecules or antibodies of the present invention are provided that have bisected oligosaccharides, for example, biantennary oligosaccharides attached to the Fc region are bisected by GlcNAc. Such variants may have reduced fucosylation and / or improved ADCC function, see, e.g., WO 2003 / 011878 (Jean-Mairet et al.), U.S. Patent No. 6,602,684 (Umana et al.) and U.S. Patent Application Publication No. 2005 / 0123546 (Umana et al.). Variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided.Such antibody variants may have improved CDC function and are described, for example, in WO 1997 / 30087 (Patel et al.), WO 1998 / 58964 (Raju, S.) and WO 1999 / 22764 (Raju, S.).

[0090] In certain embodiments, it may be desirable to generate cysteine ​​engineered variants of the bispecific antigen-binding molecules of the present invention, such as "thioMAbs" in which one or more residues of the molecule are replaced with cysteine ​​residues. In certain embodiments, the replaced residues occur at accessible sites of the molecule. By replacing these residues with cysteine, reactive thiol groups are thereby placed at accessible sites of the antibody, which can be used to conjugate the antibody to other sites, such as drug moieties or linker-drug moieties, to generate immunoconjugates. In certain embodiments, any of one or more of the following residues may be replaced with cysteine: V205 (Kabat numbering) of the light chain, A118 (EU numbering) of the heavy chain, and S400 (EU numbering) of the heavy chain Fc region. Cysteine ​​engineered antigen-binding molecules may be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0091] As used herein, with respect to an antigen-binding molecule comprising an interferon gamma (IFNG) variant, "protease-activatable" refers to a molecule comprising an IFNG variant that has reduced or abrogated ability to bind to an IFNG receptor due to a masking moiety that reduces or abrogates the ability of the IFNG variant to bind to the IFNG receptor. When the masking moiety is released by proteolytic cleavage, for example by proteolytic cleavage of a linker that connects the masking moiety to the molecule comprising the IFNG variant, binding to the IFNG receptor can be restored, thereby activating the IFNG variant.

[0092] As used herein, "reversibly masking" refers to binding of a masking moiety to an IFNG variant polypeptide such that the IFNG variant polypeptide is prevented from binding to its receptor. This masking is reversible in that the masking moiety can be released from the IFNG variant polypeptide, for example by protease cleavage, thereby freeing the IFNG variant polypeptide to bind to its receptor.

[0093] As used herein, "protease" or "proteolytic enzyme" refers to any proteolytic enzyme expressed by a target cell that cleaves a linker at a recognition site. Such proteases may be secreted by the target cell or may remain associated with the target cell, for example, on the target cell surface. Examples of proteases include, but are not limited to, metalloproteinases, such as matrix metalloproteinases 1-28 and A disintegrin and metalloproteinase (ADAM) 2, 7-12, 15, 17-23, 28-30 and 33, serine proteases, such as urokinase-type plasminogen activator and matriptase, cysteine ​​proteases (e.g., cathepsin S) and matrix metalloproteinases (e.g., MMP-2 and MMP-9). Matriptase, matrix metalloproteinase 2 (MMP-2, gelatinase A) and matrix metalloproteinase 9 (MMP-9, gelatinase B) are overexpressed, for example, in breast and ovarian cancer. MMP-2 and MMP-9 activity was detected in cervical, breast and ovarian cancer and in the ascites of epithelial ovarian cancer (EOC) patients, but not in the serum of these patients (Demeter, A. et al., Anticancer Res. 2005, 25, 2885-2889). Matriptase can be detected in normal epithelial cells, but matriptase activity is mainly detected in cancer (LeBeau, A. M. et al., Proc. Natl. Acad. Sci. USA 2013, 110, 93-98).

[0094] The term "nucleic acid" or "polynucleotide" includes any compound and / or substance that includes a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Nucleic acid molecules are often described by the sequence of bases, which represent the primary (linear) structure of the nucleic acid molecule. The sequence of bases is typically represented from 5' to 3'. As used herein, the term nucleic acid molecule encompasses, for example, deoxyribonucleic acid (DNA), including complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers containing two or more of these molecules. Nucleic acid molecules may be linear or circular. Furthermore, the term nucleic acid molecule includes both sense and antisense strands, and both single-stranded and double-stranded forms. Furthermore, the nucleic acid molecules described herein can include naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules that are suitable as vectors for directly expressing the antibodies of the present invention in vitro and / or in vivo, for example in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule, such that the mRNA can be injected into a subject to produce antibodies in vivo (see, for example, Stadler et al., Nature Medicine 2017, doi:10.1038 / nm.4356, published online June 12, 2017, or European Patent No. 2101823).

[0095] An "isolated" nucleic acid refers to a nucleic acid molecule that is separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0096] An "isolated nucleic acid encoding a bispecific antigen-binding molecule or antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of a bispecific antigen-binding molecule or antibody, comprising such nucleic acid molecules on a single vector or on separate vectors, and such nucleic acid molecules are present at one or more locations within a host cell.

[0097] A nucleic acid or polynucleotide having a nucleotide sequence that is, for example, at least 95% "identical" to a reference nucleotide sequence of the present invention means that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that it may contain up to 5 point mutations per 100 nucleotides of the reference nucleotide sequence.In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or replaced with another nucleotide, or up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.Such modifications of the reference sequence may occur at the 5' or 3' terminal position of the reference nucleotide sequence, or at any position between these terminal positions, either individually interspersed among the residues in the reference sequence or interspersed in one or more consecutive groups in the reference sequence. As a practical matter, whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention can be conventionally determined using known computer programs, such as those described above for polypeptides (e.g., ALIGN-2).

[0098] The term "expression cassette" refers to a polynucleotide produced recombinantly or synthetically with a set of specific nucleic acid elements that allow transcription of a specific nucleic acid in a target cell. A recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter. In certain embodiments, an expression cassette of the present invention comprises a polynucleotide sequence that encodes a bispecific antigen-binding molecule of the present invention or a fragment thereof.

[0099] The term "vector" or "expression vector" is synonymous with "expression construct" and refers to a DNA molecule used to introduce and induce expression of a particular gene to which it is operably linked in a target cell. This term includes the vector as a self-replicating nucleic acid structure and the vector integrated into the genome of the host cell to which it is introduced. The expression vector of the present invention comprises an expression cassette. The expression vector allows for stable transcription of large amounts of mRNA. Once inside the target cell, the ribonucleic acid molecule or protein encoded by the gene is produced by the cellular transcription and / or translation machinery. In one embodiment, the expression vector of the present invention comprises an expression cassette comprising a polynucleotide sequence encoding the bispecific antigen-binding molecule of the present invention or a fragment thereof.

[0100] The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", including the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected in the originally transformed cell are included herein. Host cells are any type of cell line that can be used to make the bispecific antigen-binding molecules of the present invention. Host cells include cultured cells, such as cultured mammalian cells, such as CHO cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insect cells, and plant cells, including transgenic animals, transgenic plants, or cells contained in cultured plants or animal tissues, to name just a few.

[0101] An "effective amount" of an agent refers to the amount necessary to induce a certain physiological change in a cell or tissue to which the agent is administered.

[0102] A "therapeutically effective amount" of an agent, e.g., a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of an agent, for example, eliminates, reduces, delays, minimizes, or prevents the deleterious effects of a disease.

[0103] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, an individual or subject is a human.

[0104] The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a preparation in which the biological activity of the active ingredient contained therein is effective and which does not contain additional ingredients that are unacceptably toxic to the subject to which the pharmaceutical composition is administered.

[0105] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition or formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, additives, stabilizers, or preservatives.

[0106] The term "package insert" is used to refer to instructions typically included in the commercial packaging of a therapeutic product that contain information regarding the indications, use, dosage, administration, concomitant therapy, contraindications and / or warnings concerning the use of such therapeutic product.

[0107] As used herein, "treatment" (and grammatical variants thereof, e.g., "treat" or "treating") refers to clinical intervention in an attempt to change the natural course in the individual being treated, and can be performed for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of direct or indirect pathological consequences of disease, prevention of metastasis, slowing the rate of disease progression, amelioration or alleviation of disease state, and remission or improvement of prognosis. In some embodiments, the molecules of the present invention are used to delay the onset of disease or to slow the progression of disease.

[0108] The term "cancer", as used herein, refers to a proliferative disease, such as, for example, lymphoma, lymphocytic leukemia, lung cancer, non-small cell lung (NSCL) cancer, alveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastrointestinal cancer, colon cancer, breast cancer, uterine cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal gland cancer, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, renal pelvis carcinoma, mesothelioma, hepatocellular carcinoma, cholangiocarcinoma, neoplasms of the central nervous system (CNS), spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, and Ewing's sarcoma (including refractory forms of any of the above cancers), or a combination of one or more of the above cancers.

[0109] As used herein, the term "chemotherapeutic agent" refers to a chemical compound useful in the treatment of cancer. In one aspect, the chemotherapeutic agent is an antimetabolite. In one aspect, the antimetabolite is selected from the group consisting of aminopterin, methotrexate, pemetrexed, raltitrexed, cladribine, clofarabine, fludarabine, mercaptopurine, pentostatin, thioguanine, capecitabine, cytarabine, fluorouracil, floxuridine, and gemcitabine. In a particular aspect, the antimetabolite is capecitabine or gemcitabine. In another aspect, the antimetabolite is fluorouracil. In one aspect, the chemotherapeutic agent is an agent that affects microtubule formation. In one aspect, the agent that affects microtubule formation is selected from the group consisting of paclitaxel, docetaxel, vincristine, vinblastine, vindesine, vinorelbine, taxotere, etoposide, and teniposide. In another embodiment, the chemotherapeutic agent is an alkylating agent, such as cyclophosphamide. In one embodiment, the chemotherapeutic agent is a cytotoxic antibiotic, such as a topoisomerase II inhibitor. In one embodiment, the topoisomerase II inhibitor is doxorubicin.

[0110] The term "agent for use in cancer immunotherapy" refers to any substance, including monoclonal antibodies, that affects the immune system. The antigen-binding molecules described herein may be considered as such agents. Agents for use in cancer immunotherapy can be used as antitumor agents to treat cancer. In one embodiment, the agent includes, but is not limited to, anti-CTLA4 antibodies (e.g., ipilimumab), anti-PD1 antibodies (e.g., nivolumab or pembrolizumab), PD-L1 antibodies (e.g., atezolizumab, avelumab or durvalumab), LAG3 antibodies (e.g., relatlimab), PD1-LAG3 bispecific antibodies and TIGIT antibodies (e.g., tiracolumab).

[0111] ANTIGEN-BINDING MOLECULES CONTAINING INTERFERON GAMA (IFNG) VARIANT POLYPEPTIDES - Patent application The present invention provides a new antigen-binding molecule that contains an interferon gamma (IFNG) variant polypeptide that can specifically bind to tumor-associated antigens, particularly fibroblast activation proteins (FAPs), and thus combines an antibody that specifically binds to FAPs with a new IFNG variant polypeptide homodimer. The antigen-binding molecule described herein has particularly advantageous properties, such as productivity, stability, binding affinity, biological activity, targeting efficiency, reduced sink effect, excellent pharmacokinetic (PK) properties, reduced toxicity, extended therapeutic window and therefore potentially enhanced efficacy.

[0112] Exemplary antigen-binding molecules In one aspect, an antigen binding molecule is disclosed herein that is characterized by comprising a homodimer of an interferon gamma (IFNG) variant polypeptide, the IFNG variant polypeptide being characterized by a C-terminal amino acid sequence KRKRP (SEQ ID NO: 1). It is shown herein that the presence of the KRKR patch, i.e., the amino acid sequence of KRKR (SEQ ID NO: 78), is important for the activity of IFNG. Recently, it has been confirmed that the KRKR patch is involved in the binding of IFNG to the heparan sulfate moiety of the extracellular matrix, thereby preventing lethal systemic toxicity (J. Kemna et al., Nature Immunology, 2023, 24, 414-422). To avoid proteolysis, the C-terminus of wild-type IFNG is stabilized by a proline cap so that the sequence of the IFNG variant polypeptide is terminated with the amino acid sequence KRKRP (SEQ ID NO: 1).

[0113] In addition, the antigen binding molecule comprises a targeting moiety for tumor-associated antigens, particularly FAP.In addition, the antigen binding molecule described herein comprises an Fc region that comprises a mutation that reduces effector function.The use of an Fc region that comprises a mutation that reduces or abolishes effector function prevents non-specific agonism by cross-linking via Fc receptors, and prevents ADCC of FAP-expressing cells.The antigen binding molecule described herein has the advantage over conventional IFNG in that it selectively induces immune response in target cells that are typically in the tumor stroma, i.e., in close proximity to tumors.

[0114] In the presence of FAP-expressing cells, the antigen binding molecules described herein can upregulate the expression of MHCl and PDL1 (Example 5). Following the upregulation of MHCI expression, the antigen binding molecules increase the amount of CD8 T cells in tumor tissue (Example 5). Furthermore, treatment with the antigen binding molecules described herein results in an increase in the level of CXCL9 (a T cell-attracting chemokine) after treatment, which is important for the immune infiltration cascade.

[0115] As used herein, there is provided an antigen-binding molecule, comprising: (i) an antibody that specifically binds to a tumor-associated antigen; (ii) a homodimer of an interferon gamma (IFNG) variant polypeptide, wherein the IFNG variant polypeptide is characterized by the C-terminal amino acid sequence KRKRP (SEQ ID NO: 1).

[0116] Thus, there is provided an antigen-binding molecule comprising: (i) an antibody that specifically binds to a tumor-associated antigen; and (ii) a homodimer of an interferon gamma (IFNG) variant polypeptide, wherein the IFNG variant polypeptide is characterized in that its C-terminus terminates with the amino acid sequence KRKRP (SEQ ID NO: 1).

[0117] The IFNG variant polypeptides are truncated at the C-terminus and protected with a proline at the C-terminus to protect against proteolysis.

[0118] In one embodiment, the antibody that specifically binds to a tumor-associated antigen is an antibody that specifically binds to fibroblast activation protein (FAP). In one embodiment, the antibody that specifically binds to a FAP comprises: (a) a heavy chain complementarity determining region (CDR-H1) comprising the amino acid sequence of SEQ ID NO:4, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:5, and a heavy chain variable region (VH1) comprising the amino acid sequence of SEQ ID NO:6; H (iv) a light chain variable region (V FAP) comprising a light chain complementarity determining region CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9; L (b) a heavy chain variable region (V FAP) comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 12, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14. HA light chain variable region (VFAP) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 15, CDR-L2 having the amino acid sequence of SEQ ID NO: 16, and CDR-L3 having the amino acid sequence of SEQ ID NO: 17. L In one embodiment, an antibody that specifically binds to FAP is provided that comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 10. H A light chain variable region (V L A heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 18. H A light chain variable region (V L In one particular embodiment, an antibody that specifically binds to FAP is provided that comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 10. H A light chain variable region (V L An antigen-binding molecule comprising:

[0119] In one embodiment, the antigen binding molecule comprises an Fc domain, in particular an IgG1 Fc domain or an IgG4 Fc domain. In one embodiment, the Fc domain comprises one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antibody to the Fc receptor. In one embodiment, the Fc domain is of the human IgG1 subclass with amino acid mutations L234A, L235A and P329G (EU numbering according to Kabat EU index). In another embodiment, the Fc domain is a mouse Fc domain and comprises amino acid mutations D265A and P329G (EU numbering according to Kabat EU index).

[0120] In one embodiment, the antigen-binding molecule is protease-activatable and comprises a protease recognition site and a masking moiety. Thus, the antigen-binding molecule comprises (i) an antibody that specifically binds to a tumor-associated antigen, (ii) a homodimer of an interferon gamma (IFNG) variant polypeptide, characterized in that the IFNG variant polypeptide terminates at its C-terminus with the amino acid sequence KRKRP (SEQ ID NO: 1), (iii) a protease recognition site, and (iv) a masking moiety.

[0121] Protease recognition site In one embodiment, the protease recognition site is a substrate for matriptase. In one embodiment, the protease recognition site comprises or consists of the amino acid sequence PQARK (SEQ ID NO:20) or HQARK (SEQ ID NO:21). In a particular embodiment, the protease recognition site comprises or consists of the amino acid sequence PQARK (SEQ ID NO:20). In one embodiment, the protease recognition site is part of a cleavable peptide linker that connects the masking moiety to the IFNG variant polypeptide. In one embodiment, the cleavable peptide linker comprises the amino acid sequence of SEQ ID NO:70 or SEQ ID NO:71, and in a particular embodiment, the cleavable peptide linker comprises the amino acid sequence of SEQ ID NO:70.

[0122] In one embodiment, an antigen binding molecule is provided in which a masking moiety is fused at its N-terminus to the C-terminus of an IFNG variant polypeptide via a cleavable peptide linker (mask release format). In one embodiment, the IFNG variant polypeptide is fused at its N-terminus to the C-terminus of an antibody via a stable linker.

[0123] In another embodiment, an antigen binding molecule is provided in which a masking moiety is fused at its N-terminus to the C-terminus of an Fc domain via a stable linker and at its C-terminus to the N-terminus of an IFNG variant polypeptide via a cleavable peptide linker (cytokine release format).

[0124] Masking part The protease-activatable antigen binding molecule comprises a masking moiety, in particular two masking moieties for IFNG variant homodimers.In one embodiment, the masking moiety is an antibody fragment that can specifically bind and mask IFNG variant polypeptide.In a particular embodiment, the masking moiety is an scFv that specifically binds to IFNG variant polypeptide.

[0125] In one embodiment, the masking moiety that specifically binds to the IFNG variant polypeptide, in particular an scFv, comprises: (a) a heavy chain variable region (VH1) comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 22, a CDR-H2 having the amino acid sequence of SEQ ID NO: 23, and a CDR-H3 having the amino acid sequence of SEQ ID NO: 24; H (iv) a light chain variable region (VIFNG) comprising a light chain complementarity determining region CDR-L1 comprising the amino acid sequence of SEQ ID NO: 25, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 27. L IFNG), or (b) a heavy chain variable region (VH1) comprising the amino acid sequence of SEQ ID NO: 30, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 31, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 32; H IFNG), and a light chain variable region (V IFNG) comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 34, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 35. L IFNG).

[0126] In one embodiment, a masking moiety that specifically binds to an IFNG variant polypeptide, in particular an scFv, comprises: (a) a heavy chain variable region (V H IFNG) and a light chain variable region (V L IFNG), or (b) a heavy chain variable region (V HIFNG) and a light chain variable region (V L In a particular embodiment, the masking moiety that specifically binds to an IFNG variant polypeptide, in particular an scFv, comprises: (a) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 28. H IFNG) and a light chain variable region (V L IFNG).

[0127] In one embodiment, the masking moiety that specifically binds to and masks the IFNG variant polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 38. In one embodiment, the masking moiety that specifically binds to the IFNG variant polypeptide comprises the amino acid sequence of SEQ ID NO:38.

[0128] In another embodiment, the masking moiety that specifically binds to and masks the IFNG variant polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 39. In one embodiment, the masking moiety that specifically binds to the IFNG variant polypeptide comprises the amino acid sequence of SEQ ID NO:39.

[0129] Mask-releasing and cytokine-releasing formats In one embodiment, an antigen binding molecule is provided in which the masking moiety is fused at its N-terminus to the C-terminus of the IFNG variant polypeptide via a cleavable peptide linker (mask release format).In one embodiment, the IFNG variant polypeptide is fused at its N-terminus to the C-terminus of the antibody via a stable linker.The schematic scheme of the format is shown in Figure 1D.

[0130] In a specific aspect, there is provided herein an antigen-binding molecule comprising an IFNG variant polypeptide, the antigen-binding molecule comprising: and two heavy chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 40, and two light chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 41. In one embodiment, the antigen-binding molecule comprises two heavy chains comprising the amino acid sequence of SEQ ID NO: 40 and two light chains comprising the amino acid sequence of SEQ ID NO: 41.

[0131] In another specific aspect, there is provided herein an antigen-binding molecule comprising an IFNG variant polypeptide, the antigen-binding molecule comprising: and two heavy chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 43, and two light chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 44. In one embodiment, the antigen-binding molecule comprises two heavy chains comprising the amino acid sequence of SEQ ID NO: 43 and two light chains comprising the amino acid sequence of SEQ ID NO: 44.

[0132] In another embodiment, an antigen-binding molecule is provided in which a masking moiety is fused at its N-terminus to the C-terminus of an Fc domain via a stable linker, and at its C-terminus to the N-terminus of an IFNG variant polypeptide via a cleavable peptide linker (cytokine release format). A schematic diagram of the format is shown in Figure 1E.

[0133] In a specific aspect, there is provided herein an antigen-binding molecule comprising an IFNG variant polypeptide, the antigen-binding molecule comprising: and two heavy chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 42, and two light chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 41. In one embodiment, the antigen-binding molecule comprises two heavy chains comprising the amino acid sequence of SEQ ID NO: 42 and two light chains comprising the amino acid sequence of SEQ ID NO: 41.

[0134] In another specific aspect, there is provided herein an antigen-binding molecule comprising an IFNG variant polypeptide, the antigen-binding molecule comprising: and two heavy chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 45, and two light chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 44. In one embodiment, the antigen-binding molecule comprises two heavy chains comprising the amino acid sequence of SEQ ID NO: 45 and two light chains comprising the amino acid sequence of SEQ ID NO: 44.

[0135] Formats containing sterically masked IFNG variant polypeptides Also, as used herein, there is provided an antigen-binding molecule, (i) an antibody that specifically binds to a tumor-associated antigen; (ii) a homodimer of an interferon gamma (IFNG) variant polypeptide, wherein the IFNG variant polypeptide is C-terminally terminated with the amino acid sequence KRKRP (SEQ ID NO: 1) and the IFNG variant polypeptide is inserted between the upper and lower hinge regions of an antibody.

[0136] These antigen-binding molecules are characterized in that they contain a sterically hidden, i.e., sterically masked, IFNG variant polypeptide.

[0137] In one embodiment, an antigen binding molecule is provided, wherein IFNG variant polypeptide is fused at its N-terminus via a first linker to the C-terminus of the VHCH1 chain of the Fab domain that specifically binds to tumor-associated antigen, particularly FAP.Furthermore, IFNG variant polypeptide is fused at its C-terminus via a second linker to the lower hinge region of Fc domain at its N-terminus.The schematic scheme of the format is shown in Figure 10A.

[0138] In one embodiment, the first linker is a glycine-serine linker. In a particular embodiment, the first linker has the amino acid sequence of SEQ ID NO:69.

[0139] In one embodiment, the second linker is a glycine-serine linker or a cleavable linker. In a particular embodiment, the second linker has an amino acid sequence selected from the group consisting of SEQ ID NO: 72, SEQ ID NO: 73 or SEQ ID NO: 75.

[0140] In one embodiment, the lower hinge region has the amino acid sequence of SEQ ID NO: 76. In another embodiment, the lower hinge region is truncated and has the amino acid sequence of SEQ ID NO: 77.

[0141] In one aspect, the present specification provides an antigen-binding molecule comprising an IFNG variant polypeptide, the antigen-binding molecule comprising: and two heavy chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 119, and two light chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 41. In one embodiment, the antigen-binding molecule comprises two heavy chains comprising the amino acid sequence of SEQ ID NO: 119 and two light chains comprising the amino acid sequence of SEQ ID NO: 41.

[0142] In another aspect, there is provided herein an antigen-binding molecule comprising an IFNG variant polypeptide, the antigen-binding molecule comprising: and two heavy chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 120, and two light chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 41. In one embodiment, the antigen-binding molecule comprises two heavy chains comprising the amino acid sequence of SEQ ID NO: 120 and two light chains comprising the amino acid sequence of SEQ ID NO: 41.

[0143] In another aspect, there is provided herein an antigen-binding molecule comprising an IFNG variant polypeptide, the antigen-binding molecule comprising: and two heavy chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 121, and two light chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 41. In one embodiment, the antigen-binding molecule comprises two heavy chains comprising the amino acid sequence of SEQ ID NO: 121 and two light chains comprising the amino acid sequence of SEQ ID NO: 41.

[0144] In yet another aspect, there is provided herein an antigen-binding molecule comprising an IFNG variant polypeptide, the antigen-binding molecule comprising: and two heavy chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 122, and two light chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 41. In one embodiment, the antigen-binding molecule comprises two heavy chains comprising the amino acid sequence of SEQ ID NO: 122 and two light chains comprising the amino acid sequence of SEQ ID NO: 41.

[0145] In another aspect, there is provided herein an antigen-binding molecule comprising an IFNG variant polypeptide, the antigen-binding molecule comprising: and two heavy chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 123, and two light chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 41. In one embodiment, the antigen-binding molecule comprises two heavy chains comprising the amino acid sequence of SEQ ID NO: 124 and two light chains comprising the amino acid sequence of SEQ ID NO: 41.

[0146] Fc domain modifications that reduce Fc receptor binding and / or effector function The antigen-binding molecule of the present invention further comprises an Fc domain composed of the first and second subunits and containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule to the Fc receptor. Thus, one or more amino acid modifications can be introduced into the Fc region of the antibody provided herein, thereby generating an Fc region variant. The Fc region variant can include a human Fc region sequence (e.g., the Fc region of human IgG1, IgG2, IgG3 or IgG4) that contains an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0147] The Fc domain confers desirable pharmacokinetic properties to the bispecific antibodies of the invention, including a long serum half-life contributing to good accumulation in target tissues, a desirable tissue-blood distribution ratio. However, at the same time, it may result in undesirable targeting of the bispecific antibodies of the invention to cells expressing Fc receptors rather than to cells carrying the preferred antigen. Thus, in a particular embodiment, the Fc domain of the bispecific antibodies of the invention exhibits reduced binding affinity to Fc receptors and / or reduced effector function compared to natural IgG Fc domains, in particular IgG1 Fc domains or IgG4 domains. More particularly, the Fc domain is an IgG1 Fc domain.

[0148] In one such embodiment, the Fc domain (or the bispecific antigen-binding molecule of the invention comprising said Fc domain) exhibits less than 50%, preferably less than 20%, more preferably less than 10%, most preferably less than 5% of the binding affinity to an Fc receptor compared to a native IgG1 Fc domain (or a bispecific antigen-binding molecule of the invention comprising a native IgG1 Fc domain) and / or exhibits less than 50%, preferably less than 20%, more preferably less than 10%, most preferably less than 5% of the effector function compared to a native IgG1 Fc domain (or a bispecific antigen-binding molecule of the invention comprising a native IgG1 Fc domain). In one embodiment, the Fc domain (or the bispecific antigen-binding molecule of the invention comprising said Fc domain) does not substantially bind to an Fc receptor and / or does not induce effector function. In a particular 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 activating Fc receptor. In a particular embodiment, the Fc receptor is an activating human Fcγ receptor, more particularly human FcγRIIIa, FcγRI or FcγRIIa, most particularly human FcγRIIIa. In one embodiment, the Fc receptor is an inhibitory Fc receptor. In a particular embodiment, the Fc receptor is an inhibitory human Fcγ receptor, more particularly human FcγRIIB. In one embodiment, the effector function is one or more of CDC, ADCC, ADCP, and cytokine secretion. In a particular embodiment, the effector function is ADCC. In one embodiment, the domain of the Fc domain exhibits substantially similar binding affinity to the neonatal Fc receptor (FcRn) compared to the natural IgG1 Fc domain. Substantially similar binding to FcRn is achieved when the Fc domain (or a bispecific antigen-binding molecule of the invention comprising said Fc domain) exhibits a binding affinity to FcRn that is greater than about 70%, particularly greater than about 80%, and more particularly greater than about 90%, compared to a native IgG1 Fc domain (or a bispecific antigen-binding molecule of the invention comprising a native IgG1 Fc domain).

[0149] In certain embodiments, the Fc domain is engineered to have a reduced binding affinity to the Fc receptor and / or reduced effector function compared to a non-engineered Fc domain. In certain embodiments, the Fc domain of the bispecific antigen-binding molecule of the present invention comprises 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 mutations reduce the binding affinity of the Fc domain to the Fc receptor. In another embodiment, the amino acid mutations reduce the binding affinity of the Fc domain to the Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In one embodiment, the bispecific antigen-binding molecule of the present invention comprising an engineered Fc domain exhibits less than 20%, particularly less than 10%, more particularly less than 5% of the binding affinity to the Fc receptor compared to a bispecific antibody of the present invention comprising a non-engineered Fc domain. In certain embodiments, the Fc receptor is an Fcγ receptor. In another embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an inhibitory Fc receptor. In a particular embodiment, the Fc receptor is an inhibitory human Fcγ receptor, more specifically human FcγRIIB. In some embodiments, the Fc receptor is an activating Fc receptor. In a particular embodiment, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, most specifically human FcγRIIIa. Preferably, the binding to each of these receptors is reduced. In some embodiments, the binding affinity to complement components (specifically to C1q) is also reduced. In one embodiment, the binding affinity to neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn (i.e., preservation of the binding affinity of the Fc domain to said receptor) is achieved when the Fc domain (or a bispecific antigen-binding molecule of the invention comprising said Fc domain) exhibits a binding affinity to FcRn that is greater than about 70% of the binding affinity of an unengineered form of the Fc domain (or a bispecific antigen-binding molecule of the invention comprising this unengineered form of Fc).The Fc domain or the bispecific antigen-binding molecule of the present invention comprising the Fc domain may exhibit greater than about 80% and even greater than about 90% of such affinity. In certain embodiments, the Fc domain of the bispecific antigen-binding molecule of the present invention is engineered to have reduced effector function compared to a non-engineered Fc domain. Reduced effector function includes, but is not limited to, one or more of reduced complement-dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling to induce apoptosis, reduced maturation of dendritic cells, or reduced T cell priming.

[0150] Antibodies with reduced effector function include those with substitutions at one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (US Pat. No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc variants with substitutions at residues 265 and 297 to alanine (US Pat. No. 7,332,581). Certain antibody variants with improved or reduced binding to FcR have been described (e.g., US Pat. No. 6,737,056; WO 2004 / 056312; and Shields, RL et al., J. Biol. Chem. 276 (2001) 6591-6604).

[0151] In one aspect of the invention, the Fc domain comprises amino acid substitutions at positions E233, L234, L235, N297, P331 and P329. In some aspects, the Fc domain comprises the amino acid substitutions L234A and L235A ("LALA"). In one such embodiment, the Fc domain is an IgG1 Fc domain, in particular a human IgG1 Fc domain. In one aspect, the Fc domain comprises an amino acid substitution at position P329. In a more specific aspect, the amino acid substitution is P329A or P329G, in particular P329G. In one embodiment, the Fc domain comprises an amino acid substitution at position P329 and further amino acid substitutions selected from the group consisting of E233P, L234A, L235A, L235E, N297A, N297D or P331S. In a more preferred embodiment, the Fc domain comprises the amino acid mutations L234A, L235A and P329G ("P329G LALA"). The amino acid substitution combination "P329G LALA" almost completely abolishes Fcγ receptor binding of human IgG1 Fc domains, as described in PCT Application WO 2012 / 130831A1. The document also describes methods for preparing such mutant Fc domains and for determining properties such as Fc receptor binding or effector function. Such antibodies are IgG1 with the mutations L234A and L235A, or the mutations L234A, L235A and P329G (numbering according to the EU index of Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0152] In one aspect, the Fc domain is an IgG4 Fc domain. In a more specific embodiment, the Fc domain is an IgG4 Fc domain that comprises an amino acid substitution at position S228 (Kabat numbering), in particular the amino acid substitution S228P. In a more specific embodiment, the Fc domain is an IgG4 Fc domain that comprises the amino acid substitutions L235E and S228P and P329G. 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)).

[0153] Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), responsible for the transfer of maternal IgG to the fetus (Guyer, RL et al., J. Immunol. 117 (1976) 587-593, and Kim, JK et al., J. Immunol. 24 (1994) 2429-2434) have been described in US Patent Application Publication No. 2005 / 0014934. These antibodies comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of Fc region residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, such as a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826). For other examples of Fc region variants, see also Duncan, AR and Winter, G., Nature 322 (1988) 738-740; U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351.

[0154] Binding to Fc receptors can be easily determined, for example, by ELISA or by surface plasmon resonance (SPR) using standard equipment such as a BIAcore instrument (GE Healthcare), and Fc receptors can be obtained by recombinant expression. Suitable such binding assays are described herein. Alternatively, the binding affinity of Fc domains or cell-activating bispecific antigen-binding molecules comprising Fc domains to Fc receptors can be evaluated using cell lines known to express specific Fc receptors, such as human NK cells expressing FcγIIIa receptors. The effector function of Fc domains or bispecific antigen-binding molecules of the present invention comprising Fc domains can be measured by methods known in the art. Suitable assays for measuring ADCC are described herein. Other examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362, Hellstrom et al., Proc Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985), U.S. Patent No. 5,821,337, Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively, non-radioactive assay methods may be used (e.g., ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc., Mountain View, CA) and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes, R. et al., Proc. Natl. Acad. Sci. USA 95, 652-656 (1998).

[0155] The following sections describe preferred embodiments of the antigen-binding molecules described herein that contain Fc domain modifications that reduce Fc receptor binding and / or effector function. In one embodiment, an antigen-binding molecule is provided that comprises: (i) an antibody that specifically binds to a tumor-associated antigen; and (ii) an interferon gamma (IFNG) variant polypeptide homodimer, the IFNG variant polypeptide being characterized by the C-terminal amino acid sequence KRKRP (SEQ ID NO: 1), the antigen-binding molecule comprising an IgG1 Fc domain or an IgG4 Fc domain, the Fc domain comprising one or more amino acid substitutions that reduce the binding affinity of the antibody to an Fc receptor and / or effector function. In another aspect, there is provided an antigen binding molecule comprising: (i) an antibody that specifically binds to a tumor-associated antigen; and (ii) a homodimer of an interferon gamma (IFNG) variant polypeptide, wherein the IFNG variant polypeptide is characterized in that its C-terminus terminates with the amino acid sequence KRKRP (SEQ ID NO:1), wherein the Fc domain is of the human IgG1 subclass with the amino acid mutations L234A, L235A and P329G (EU numbering according to Kabat EU index).

[0156] Exemplary IFNG Variant Polypeptides In one aspect, an IFNG variant polypeptide is provided, characterized in that its C-terminus ends with the amino acid sequence KRKRP (SEQ ID NO: 1).It is shown herein that the presence of KRKR patch, i.e., the amino acid sequence of KRKR (SEQ ID NO: 78), is important for the activity of IFNG.To avoid proteolysis, the C-terminus of wild-type IFNG is stabilized by a proline cap, so that the sequence of IFNG variant polypeptide ends with the amino acid sequence KRKRP (SEQ ID NO: 1).

[0157] In one embodiment, an IFNG variant polypeptide is provided that comprises or consists of the amino acid sequence of SEQ ID NO: 2. In another embodiment, an IFNG variant polypeptide is provided that comprises or consists of the amino acid sequence of SEQ ID NO: 3.

[0158] Also provided herein are mutational or insertional variants of IFNG variant polypeptides characterized in that they terminate C-terminally with the amino acid sequence KRKRP (SEQ ID NO:1).

[0159] In one aspect, provided herein is an IFNG variant polypeptide, characterized in that it terminates at its C-terminus with the amino acid sequence KRKRP (SEQ ID NO: 1), in which one or two of the amino acids in SEQ ID NO: 1 are replaced with an amino acid selected from the group consisting of serine (S), proline (P) and glutamic acid (E). Thus, provided herein are AKTGSRKRP (SEQ ID NO: 137), AKTGKSKRP (SEQ ID NO: 138), AKTGKRSRP (SEQ ID NO: 139), AKTGKRKSP (SEQ ID NO: 134), AKTGPRKRP (SEQ ID NO: 140), AKTGKPKRP (SEQ ID NO: 141), AKTGKRPRP (SEQ ID NO: 142), AKTGKRPPP (SEQ ID NO: 143), AKTGERKRP (SEQ ID NO: 144), AKTGKEKRP (SEQ ID NO: 145), AKTGKRERP (SEQ ID NO: 146), AKTGKRKEP (SEQ ID NO: 147), AKTGSRSRP (SEQ ID NO: 148 ...TGKRKSP ( IFNG variant polypeptides are provided, characterized in that their C-terminus terminates with an amino acid sequence selected from the group consisting of TGPRPRP (SEQ ID NO: 149), AKTGERERP (SEQ ID NO: 150), AKTGKSKSP (SEQ ID NO: 151), AKTGKPKPP (SEQ ID NO: 152), AKTGKEKEP (SEQ ID NO: 153), AKTGSSKRP (SEQ ID NO: 154), AKTGPPKRP (SEQ ID NO: 155), AKTGEEKRP (SEQ ID NO: 156), AKTGKRSSP (SEQ ID NO: 157), AKTGKRPPP (SEQ ID NO: 158) and AKTGKREEP (SEQ ID NO: 159).

[0160] In a preferred aspect, there is provided herein an IFNG variant polypeptide characterized in that it terminates at its C-terminus with the amino acid sequence KRKRP (SEQ ID NO: 1), wherein one or two of the amino acids in SEQ ID NO: 1 are substituted with an amino acid selected from serine (S) or proline (P). This includes IFNG variant polypeptides characterized in that the C-terminus terminates with an amino acid sequence selected from the group consisting of AKTGSRKRP (SEQ ID NO: 137), AKTGKSKRP (SEQ ID NO: 138), AKTGKRSRP (SEQ ID NO: 139), AKTGKRKSP (SEQ ID NO: 134), AKTGPRKRP (SEQ ID NO: 140), AKTGKPKRP (SEQ ID NO: 141), AKTGKRPRP (SEQ ID NO: 142), AKTGKRPPP (SEQ ID NO: 143), AKTGSRSRP (SEQ ID NO: 148), AKTGPRPRP (SEQ ID NO: 149), AKTGKSKSP (SEQ ID NO: 151), AKTGKPKPP (SEQ ID NO: 152), AKTGSSKRP (SEQ ID NO: 154), AKTGPPKRP (SEQ ID NO: 155), AKTGKRSSP (SEQ ID NO: 157) and AKTGKRPPP (SEQ ID NO: 158).

[0161] Also, as used herein, there is provided an antigen-binding molecule, (i) an antibody that specifically binds to a tumor-associated antigen; (ii) a homodimer of an interferon gamma (IFNG) variant polypeptide, wherein the IFNG variant polypeptide is characterized in that its C-terminus terminates with the amino acid sequence KRKRP (SEQ ID NO: 1), and one or two of the amino acids in SEQ ID NO: 1 are substituted with an amino acid selected from the group consisting of serine (S), proline (P) and glutamic acid (E).

[0162] In a particular aspect, the antigen binding molecule comprises: (i) an antibody that specifically binds to a tumor-associated antigen; (ii) a homodimer of an interferon gamma (IFNG) variant polypeptide, wherein the IFNG variant polypeptide is characterized in that its C-terminus terminates with the amino acid sequence KRKRP (SEQ ID NO: 1), and one or two of the amino acids in SEQ ID NO: 1 are substituted with an amino acid selected from serine (S) or proline (P).

[0163] In one aspect, an antigen binding molecule comprising: (i) an antibody that specifically binds to a tumor-associated antigen; (ii) a homodimer of an interferon gamma (IFNG) variant polypeptide, the IFNG variant polypeptide being selected from the group consisting of AKTGSRKRP (SEQ ID NO: 137), AKTGKSKRP (SEQ ID NO: 138), AKTGKRSRP (SEQ ID NO: 139), AKTGKRKSP (SEQ ID NO: 134), AKTGPRKRP (SEQ ID NO: 140), AKTGKPKRP (SEQ ID NO: 141), AKTGKRPRP (SEQ ID NO: 142), AKTGKRPPP (SEQ ID NO: 143), AKTGSRS and a homodimer of an IFNG variant polypeptide, characterized by a C-terminal amino acid sequence selected from the group consisting of RP (SEQ ID NO: 148), AKTGPRPRP (SEQ ID NO: 149), AKTGKSKSP (SEQ ID NO: 151), AKTGKPKPP (SEQ ID NO: 152), AKTGSSKRP (SEQ ID NO: 154), AKTGPPKRP (SEQ ID NO: 155), AKTGKRSSP (SEQ ID NO: 157) and AKTGKRPPP (SEQ ID NO: 158).

[0164] In one aspect, the present specification provides an antigen-binding molecule comprising an IFNG variant polypeptide, the antigen-binding molecule comprising: and two heavy chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 91, and two light chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 41. In one embodiment, the antigen-binding molecule comprises two heavy chains comprising the amino acid sequence of SEQ ID NO: 91 and two light chains comprising the amino acid sequence of SEQ ID NO: 41.

[0165] In one aspect, the present specification provides an antigen-binding molecule comprising an IFNG variant polypeptide, the antigen-binding molecule comprising: and two heavy chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 92, and two light chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 41. In one embodiment, the antigen-binding molecule comprises two heavy chains comprising the amino acid sequence of SEQ ID NO: 92 and two light chains comprising the amino acid sequence of SEQ ID NO: 41.

[0166] In one aspect, the present specification provides an antigen-binding molecule comprising an IFNG variant polypeptide, the antigen-binding molecule comprising: and two heavy chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 93, and two light chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 41. In one embodiment, the antigen-binding molecule comprises two heavy chains comprising the amino acid sequence of SEQ ID NO: 93 and two light chains comprising the amino acid sequence of SEQ ID NO: 41.

[0167] In one aspect, the present specification provides an antigen-binding molecule comprising an IFNG variant polypeptide, the antigen-binding molecule comprising: and two heavy chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 94, and two light chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 41. In one embodiment, the antigen-binding molecule comprises two heavy chains comprising the amino acid sequence of SEQ ID NO: 94 and two light chains comprising the amino acid sequence of SEQ ID NO: 41.

[0168] In one aspect, the present specification provides an antigen-binding molecule comprising an IFNG variant polypeptide, the antigen-binding molecule comprising: and two heavy chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 95, and two light chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 41. In one embodiment, the antigen-binding molecule comprises two heavy chains comprising the amino acid sequence of SEQ ID NO: 95 and two light chains comprising the amino acid sequence of SEQ ID NO: 41.

[0169] In one aspect, the present specification provides an antigen-binding molecule comprising an IFNG variant polypeptide, the antigen-binding molecule comprising: and two heavy chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 96, and two light chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 41. In one embodiment, the antigen-binding molecule comprises two heavy chains comprising the amino acid sequence of SEQ ID NO: 96 and two light chains comprising the amino acid sequence of SEQ ID NO: 41.

[0170] In one aspect, the present specification provides an antigen-binding molecule comprising an IFNG variant polypeptide, the antigen-binding molecule comprising: and two heavy chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 97, and two light chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 41. In one embodiment, the antigen-binding molecule comprises two heavy chains comprising the amino acid sequence of SEQ ID NO: 97 and two light chains comprising the amino acid sequence of SEQ ID NO: 41.

[0171] In one aspect, the present specification provides an antigen-binding molecule comprising an IFNG variant polypeptide, the antigen-binding molecule comprising: and two heavy chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 98, and two light chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 41. In one embodiment, the antigen-binding molecule comprises two heavy chains comprising the amino acid sequence of SEQ ID NO: 98 and two light chains comprising the amino acid sequence of SEQ ID NO: 41.

[0172] In one aspect, the present specification provides an antigen-binding molecule comprising an IFNG variant polypeptide, the antigen-binding molecule comprising: and two heavy chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 103, and two light chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 41. In one embodiment, the antigen-binding molecule comprises two heavy chains comprising the amino acid sequence of SEQ ID NO: 103 and two light chains comprising the amino acid sequence of SEQ ID NO: 41.

[0173] In one aspect, the present specification provides an antigen-binding molecule comprising an IFNG variant polypeptide, the antigen-binding molecule comprising: and two heavy chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 104, and two light chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 41. In one embodiment, the antigen-binding molecule comprises two heavy chains comprising the amino acid sequence of SEQ ID NO: 104 and two light chains comprising the amino acid sequence of SEQ ID NO: 41.

[0174] In one aspect, the present specification provides an antigen-binding molecule comprising an IFNG variant polypeptide, the antigen-binding molecule comprising: and two heavy chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 106, and two light chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 41. In one embodiment, the antigen-binding molecule comprises two heavy chains comprising the amino acid sequence of SEQ ID NO: 106 and two light chains comprising the amino acid sequence of SEQ ID NO: 41.

[0175] In one aspect, the present specification provides an antigen-binding molecule comprising an IFNG variant polypeptide, the antigen-binding molecule comprising: and two heavy chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 107, and two light chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 41. In one embodiment, the antigen-binding molecule comprises two heavy chains comprising the amino acid sequence of SEQ ID NO: 107 and two light chains comprising the amino acid sequence of SEQ ID NO: 41.

[0176] In one aspect, the present specification provides an antigen-binding molecule comprising an IFNG variant polypeptide, the antigen-binding molecule comprising: and two heavy chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 109, and two light chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 41. In one embodiment, the antigen-binding molecule comprises two heavy chains comprising the amino acid sequence of SEQ ID NO: 109 and two light chains comprising the amino acid sequence of SEQ ID NO: 41.

[0177] In one aspect, the present specification provides an antigen-binding molecule comprising an IFNG variant polypeptide, the antigen-binding molecule comprising: and two heavy chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 110, and two light chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 41. In one embodiment, the antigen-binding molecule comprises two heavy chains comprising the amino acid sequence of SEQ ID NO: 110 and two light chains comprising the amino acid sequence of SEQ ID NO: 41.

[0178] In one aspect, the present specification provides an antigen-binding molecule comprising an IFNG variant polypeptide, the antigen-binding molecule comprising: and two heavy chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 112, and two light chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 41. In one embodiment, the antigen-binding molecule comprises two heavy chains comprising the amino acid sequence of SEQ ID NO: 112 and two light chains comprising the amino acid sequence of SEQ ID NO: 41.

[0179] In one aspect, the present specification provides an antigen-binding molecule comprising an IFNG variant polypeptide, the antigen-binding molecule comprising: and two heavy chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 113, and two light chains comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 41. In one embodiment, the antigen-binding molecule comprises two heavy chains comprising the amino acid sequence of SEQ ID NO: 113 and two light chains comprising the amino acid sequence of SEQ ID NO: 41.

[0180] Polynucleotides Also provided herein is an isolated polynucleotide encoding an antigen-binding molecule or fragment thereof described herein, or an isolated polynucleotide encoding an IFNG variant polypeptide described herein.

[0181] The isolated polynucleotides encoding the antigen-binding molecules disclosed herein can be expressed as a single polynucleotide encoding the entire antigen-binding molecule, or as multiple (e.g., two or more) polynucleotides that are coexpressed. The polypeptides encoded by the coexpressed polynucleotides can associate, for example, via disulfide bonds or other means, to form a functional antigen-binding molecule. For example, the light chain portion of an immunoglobulin can be encoded by a polynucleotide separate from the heavy chain portion of an immunoglobulin. When coexpressed, the heavy chain polypeptide associates with the light chain polypeptide to form an immunoglobulin.

[0182] In some embodiments, the isolated polynucleotide encodes a polypeptide comprised in the antigen-binding molecule described herein. In one embodiment, an isolated polynucleotide is provided that encodes an IFNG variant polypeptide, wherein the IFNG variant polypeptide is characterized by the C-terminal amino acid sequence KRKRP (SEQ ID NO: 1).

[0183] In one aspect, an isolated polynucleotide is provided that encodes an antigen-binding molecule comprising: (i) an antibody that specifically binds to a tumor-associated antigen; and (ii) a homodimer of an IFNG variant polypeptide, wherein the IFNG variant polypeptide is characterized by the C-terminal amino acid sequence KRKRP (SEQ ID NO: 1). In one aspect, one or more isolated polynucleotides are provided that encode an antigen-binding molecule, comprising: (i) an antibody that specifically binds to a tumor-associated antigen; and (ii) a homodimer of an interferon gamma (IFNG) variant polypeptide, wherein the IFNG variant polypeptide is characterized by the C-terminal amino acid sequence KRKRP (SEQ ID NO: 1), (iii) a protease recognition site, and (iv) a masking moiety.

[0184] In certain embodiments, the polynucleotide or nucleic acid is DNA. In other embodiments, the polynucleotide of the present invention is RNA, for example in the form of messenger RNA (mRNA). The RNA of the present invention may be single-stranded or double-stranded.

[0185] Recombinant methods The bispecific antigen-binding molecules described herein may be obtained, for example, by recombinant production. For recombinant production, one or more polynucleotides encoding the antigen-binding molecule or a polypeptide fragment thereof are provided. One or more polynucleotides encoding the bispecific antigen-binding molecule are isolated and inserted into one or more vectors for cloning and / or expression in a host cell. Such polynucleotides can be easily isolated and sequenced using conventional procedures. In one aspect of the present invention, a vector, preferably an expression vector, is provided that comprises one or more polynucleotides of the present invention. Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequence of the antigen-binding molecule (fragment) according to appropriate transcriptional / translational regulatory signals. Such methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination / genetic recombination. See, for example, the techniques described in Maniatis et al., MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, NY (1989); and Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience, NY (1989). An expression vector can be part of a plasmid, a virus, or can be a nucleic acid fragment. An expression vector contains an expression cassette into which a polynucleotide encoding an antigen-binding molecule or a polypeptide fragment thereof (i.e., a coding region) is cloned in operable association with a promoter and / or other transcription or translation control elements. As used herein, a "coding region" is a portion of a nucleic acid consisting of codons that are translated into amino acids. A "stop codon" (TAG, TGA, or TAA), although not translated into amino acids, may be considered to be part of the coding region if present, although any adjacent sequences, such as promoters, ribosome binding sites, transcription terminators, introns, 5' and 3' untranslated regions, etc., are not part of the coding region.Two or more coding regions may be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. Furthermore, any vector may contain a single coding region or may contain two or more coding regions, e.g., a vector of the present invention may encode one or more polypeptides that are post-translationally or co-translationally separated into a final protein via proteolytic cleavage. Furthermore, a vector, polynucleotide, or nucleic acid of the present invention may encode a heterologous coding region, fused or unfused to a polynucleotide encoding an antigen-binding molecule disclosed herein or a polypeptide fragment thereof, or a variant or derivative thereof. Heterologous coding regions include, but are not limited to, specialized elements or motifs, such as secretory signal peptides or heterologous functional domains. An operably associated is when a coding region of a gene product, such as a polypeptide, is associated with one or more regulatory sequences such that expression of the gene product is under the influence or control of the regulatory sequences. Two DNA fragments (such as a polypeptide coding region and its associated promoter) are "operably associated" if induction of promoter function results in transcription of an mRNA encoding a desired gene product, and if the nature of the binding between the two DNA fragments does not interfere with the ability of the expression control sequence to direct expression of the gene product or does not interfere with the ability of the DNA template to transcribe. Thus, if the promoter is capable of effecting transcription of a nucleic acid encoding a polypeptide, the promoter region will be operably associated with the nucleic acid encoding the polypeptide. The promoter may be a cell-specific promoter that directs substantial transcription of the DNA only in a given cell. Other transcriptional regulatory elements other than the promoter, such as enhancers, operators, repressors, and transcription termination signals, may be operably associated with the polynucleotide to direct cell-specific transcription.

[0186] Suitable promoters and other transcriptional regulatory regions are disclosed herein. Various transcriptional regulatory regions are known to those skilled in the art. These include, but are not limited to, transcriptional regulatory regions that function in vertebrate cells, such as, but are not limited to, promoters and enhancer segments from cytomegalovirus (e.g., immediate early promoter and intron A), Simian Virus 40 (e.g., immediate early promoter), and retroviruses (e.g., Rous sarcoma virus, etc.). Other transcriptional regulatory regions include those derived from vertebrate genes, such as actin, heat shock proteins, bovine growth hormone and rabbit a-globin, and other sequences capable of controlling gene expression in eukaryotic cells. Further suitable transcriptional regulatory regions include tissue-specific promoters and enhancers, as well as inducible promoters (e.g., tetracycline promoter-inducible). Similarly, various translational control elements are known to those skilled in the art. These include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (particularly, internal ribosome entry sites or IRES, also called CITE sequences). The expression cassette may also include other features, such as an origin of replication and / or chromosomal integration elements, such as the retroviral long terminal repeat (LTR) or the adeno-associated viral (AAV) inverted terminal repeat (ITR).

[0187] The polynucleotide and nucleic acid coding regions of the present invention may be associated with additional coding regions encoding secretory or signal peptides that direct the secretion of the polypeptides encoded by the polynucleotides provided herein. For example, if secretion of an antigen-binding molecule or a polypeptide fragment thereof is desired, DNA encoding a signal sequence may be placed upstream of the nucleic acid encoding the antigen-binding molecule or a polypeptide fragment thereof. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein once transport of the growing protein chain across the rough endoplasmic reticulum has begun. Those skilled in the art are aware that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to generate the secreted or "mature" form of the polypeptide. In certain embodiments, a native signal peptide, such as an immunoglobulin heavy or light chain signal peptide, is used, or a functional derivative of that sequence that retains the ability to induce secretion of a polypeptide operably associated therewith is used. Alternatively, a heterologous mammalian signal peptide or a functional derivative thereof may be used. For example, the wild-type leader sequence may be substituted with the leader sequence of human tissue plasminogen activator (TPA) or mouse β-glucuronidase.

[0188] DNA encoding short protein sequences that can be used to facilitate later purification (e.g., histidine tags) or to aid in labeling of the fusion protein may be included within or at the end of the polynucleotide encoding the bispecific antigen-binding molecule of the invention or a polypeptide fragment thereof.

[0189] In a further aspect, a host cell is provided that comprises one or more polynucleotides of the invention. In a particular aspect, a host cell is provided that comprises one or more vectors. The polynucleotides and vectors can incorporate any of the features described herein in connection with the polynucleotides and vectors, respectively, alone or in combination. In one aspect, the host cell comprises a vector (e.g., transformed or transfected with a vector) that comprises a polynucleotide encoding (a part of) an antigen-binding molecule described herein. Host cells suitable for supporting the replication and expression of antigen-binding molecules are well known in the art. Such cells may be transfected or transduced with a particular expression vector as needed, and cells containing large amounts of the vector can be grown to inoculate large-scale fermenters to obtain sufficient amounts of antigen-binding molecules for clinical applications. Suitable host cells include prokaryotic microorganisms such as E. coli, or various eukaryotic cells such as Chinese hamster ovary cells (CHO), insect cells, etc. For example, the polypeptides can be produced in bacteria, especially when glycosylation is not required. After expression, the polypeptides can be isolated from the bacterial cell paste in a soluble fraction and further purified. In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for polypeptide-encoding vectors, including fungal and yeast strains in which the glycosylation pathway has been "humanized," resulting in the production of polypeptides with partially or fully human glycosylation patterns. See Gerngross, Nat Biotech 22, 1409-1414 (2004), and Li et al., Nat Biotech 24, 210-215 (2006).

[0190] Suitable host cells for the expression of (glycosylated) polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified that can be used in combination with insect cells, particularly for the transfection of Spodoptera frugiperda cells. Plant cell cultures can also be utilized as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (PLANTIBODIES FOR PRODUCING ANTIBODIES IN TRANSGENIC PLANTS). (商標)See, for example, the description of the art. Vertebrate cells can also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension can be useful. Other examples of useful mammalian host cell lines include monkey kidney CV1 line transformed by SV40 (COS-7), human embryonic kidney lines (e.g., 293 or 293T cells as described in Graham et al., J Gen Virol 36, 59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells as described in Mather, Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT 060562), TRI cells (e.g., Mather et al., Annals NY Acad Sci 383, 44-68 (1982)), TRI cells, MRC5 cells, and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including dhfr-CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77, 4216 (1980)), and myeloma cell lines, such as YO, NS0, P3X63, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for protein production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (ed. BKC Lo, Humana Press, Totowa, NJ), pp. 255-268 (2003). Host cells include cultured cells, such as cultured mammalian cells, yeast cells, insect cells, bacterial cells and plants, including cells contained in transgenic animals, transgenic plants or cultured plants or animal tissues, to name just a few. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell, such as a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell).Standard techniques for expressing foreign genes in these systems are known in the art. Cells that express a polypeptide containing either an immunoglobulin heavy or light chain can be engineered to also express the other of the immunoglobulin chains, such that the expression product is an immunoglobulin having both a heavy and a light chain.

[0191] In one aspect, a method for producing an antigen-binding molecule or a polypeptide fragment thereof disclosed herein is provided, the method comprising the steps of culturing a host cell comprising a polynucleotide encoding the antigen-binding molecule or polypeptide fragment thereof provided herein under conditions suitable for expression of the antigen-binding molecule or polypeptide fragment thereof, and recovering the antigen-binding molecule or polypeptide fragment thereof of the present invention from the host cell (or host cell culture medium).

[0192] The antigen-binding molecules prepared as described herein can be purified by techniques known in the art, such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The actual conditions used to purify a particular protein will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to one of skill in the art. For affinity chromatography purification, an antibody, ligand, receptor, or antigen to which the antigen-binding molecule binds can be used. For example, for affinity chromatography purification of the fusion protein of the present invention, a matrix with protein A or protein G can be used. Sequential protein A or G affinity chromatography and size exclusion chromatography can be used to isolate the antigen-binding molecules essentially as described in the Examples. The purity of the antigen-binding molecule or a fragment thereof can be measured by any of a wide variety of well-known analytical techniques, including gel electrophoresis, high pressure liquid chromatography, etc. For example, the antigen-binding molecules expressed as described in the Examples were shown to be intact and properly assembled, as demonstrated by reducing and non-reducing SDS-PAGE.

[0193] Assay The antigen-binding molecules provided herein can be characterized for their binding properties and / or biological activity by various assays known in the art. In particular, they are characterized by the assays described in more detail in the Examples.

[0194] 1. Binding Assay The binding of the antigen binding molecules and IFNG variant polypeptides provided herein to corresponding target-expressing cells can be evaluated, for example, by using a mouse fibroblast cell line expressing human fibroblast activation protein (FAP) and flow cytometry (FACS) analysis. The binding behavior of IFNG variants to IFNGR1 and IFNGR1 / 2 can be evaluated by surface plasmon resonance (SPR) as described in the Examples.

[0195] 2. Activity Assay The antigen-binding molecules and IFNG variant polypeptides described herein are tested for biological activity. Biological activity can include the efficacy and specificity of bispecific antigen-binding molecules. The activity of IFNG variant polypeptides can be measured by HEK Blue IFNG reporter cell assay as described in the Examples. In addition, the activity of IFNG variant polypeptides and antigen-binding molecules can be measured by upregulation of MHC-I and PD-L1 on mouse MC38-huCEA tumor cell line, as well as upregulation of chemoattractant (CXCL9), as described in the Examples.

[0196] Pharmaceutical Compositions, Formulations, and Routes of Administration In a further aspect, a pharmaceutical composition comprising any of the antigen-binding molecules provided herein is provided, for example, for use in any of the following therapeutic methods. In one aspect, the pharmaceutical composition comprises any of the antigen-binding molecules provided herein and at least one pharma- ceutically acceptable excipient. In another aspect, the pharmaceutical composition comprises any of the antigen-binding molecules provided herein and at least one additional therapeutic agent, for example, as described below.

[0197] The pharmaceutical compositions described herein comprise a therapeutically effective amount of one or more antigen-binding molecules dissolved or dispersed in a pharma- ceutically acceptable carrier. The phrase "pharmaceutical acceptable or pharmacologically acceptable" refers to molecular entities and compositions that are generally non-toxic to recipients at the dosages and concentrations used, i.e., do not produce adverse allergic or other untoward reactions when administered to animals, e.g., humans, as appropriate. Preparation of pharmaceutical compositions containing at least one antigen-binding molecule disclosed herein, and optionally additional active ingredients, will be known to those skilled in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, which is incorporated herein by reference. In particular, the compositions are lyophilized formulations or aqueous solutions. As used herein, "pharmaceutical acceptable excipients" include any solvent, buffer, dispersion medium, coating, surfactant, antioxidant, preservative (e.g., antibacterial, antifungal), isotonic agent, salt, stabilizer, and combinations thereof, as known to those skilled in the art.

[0198] Parenteral compositions include those designed for administration by injection (e.g., subcutaneous, intradermal, intralesional, intravenous, intraarterial, intramuscular, intrathecal or intraperitoneal injection). For injection, the antigen-binding molecules disclosed herein may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. The solutions may contain formulating agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the antigen-binding molecules may be in powder form for constitution with a suitable vehicle, e.g., pyrogen-free sterile water, before use. Sterile injectable solutions are prepared by incorporating the antigen-binding molecules of the present invention in the required amount in an appropriate solvent, with various other ingredients as listed below, as needed. Sterility can be easily achieved, for example, by filtration through a sterile filtration membrane. In general, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and / or other ingredients. In the case of sterile powders for preparing sterile injectable solutions, suspensions or emulsions, the preferred preparation method is vacuum drying or freeze-drying technology, which yields a powder of the active ingredient and any additional desired ingredients from a previously sterile filtered liquid medium. The liquid medium should be appropriately buffered if necessary, and the liquid diluent is first rendered isotonic with sufficient saline or glucose prior to injection. The composition must be stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi. It is understood that endotoxin contamination should be kept to a minimum at a safe level, for example, less than 0.5ng / mg protein.Suitable pharma- ceutically acceptable additives include, but are not limited to, buffers such as phosphate, citrate and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (e.g., octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides, proteins, For example, serum albumin, gelatin or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine, monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose or sorbitol, salt forming counterions such as sodium, metal complexes (e.g. Zn-protein complexes), and / or non-ionic surfactants such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, dextran, and the like. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Additionally, suspensions of the active compounds may be prepared as appropriate oil injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.

[0199] The active ingredient can be encapsulated in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions, e.g., hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, respectively, prepared by coacervation techniques or by interfacial polymerization. Such techniques are disclosed in Remington's Pharmaceutical Sciences (18th Ed. Mack Printing Company, 1990). Sustained-release preparations may also be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, e.g., films or microcapsules. In certain embodiments, sustained absorption of injectable compositions can be brought about by the use in the composition of agents that delay absorption, such as aluminum monostearate, gelatin or a combination thereof.

[0200] In addition to the above-mentioned compositions, antigen-binding molecules can be formulated as depot preparations.Such long-acting preparations can be administered by implantation (e.g., subcutaneous or intramuscular) or intramuscular injection.Thus, for example, antigen-binding molecules can be formulated into suitable polymeric or hydrophobic materials (e.g., as an emulsion in acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as sparingly soluble salts.

[0201] Pharmaceutical compositions comprising the antigen-binding molecules described herein can be prepared by conventional mixing, dissolving, emulsifying, encapsulating, entrapment or lyophilization processes. Pharmaceutical compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients or adjuvants that facilitate the processing of proteins into preparations that can be used pharmaceuticals. Appropriate formulations depend on the selected route of administration.

[0202] The antigen-binding molecules disclosed herein may be formulated into compositions in free acid or free base, neutral or salt form. Pharmaceutically acceptable salts are those that substantially retain the biological activity of the free acid or free base. Pharmaceutically acceptable salts include acid addition salts, such as those formed with free amino groups of proteinaceous compositions, or those formed with inorganic acids, such as hydrochloric acid or phosphoric acid, or those formed with organic acids, such as acetic acid, oxalic acid, tartaric acid or mandelic acid. Salts formed with free carboxyl groups may also be derived from inorganic bases, such as sodium, potassium, ammonium, calcium or ferric hydroxide, or organic bases, such as isopropylamine, trimethylamine, histidine or procaine. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than the corresponding free base forms. The compositions of the present invention may also contain two or more active ingredients as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts effective for the intended purpose. Formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0203] Therapeutic Methods and Compositions Any of the antigen-binding molecules provided herein can be used in therapeutic methods.For use in therapeutic methods, the antigen-binding molecules described herein can be formulated, dosed and administered in a manner consistent with good medical practice.The factors to be considered in this context include the specific disorder to be treated, the specific mammal to be treated, the clinical symptoms of individual patients, the cause of disorder, the delivery site of the drug, the method of administration, administration schedule and other factors known to medical practitioners.

[0204] In one aspect, there is provided an antigen binding molecule or an IFNG variant polypeptide disclosed herein for use as a medicament.

[0205] In a further embodiment, the antigen binding molecule or IFNG variant polypeptide described herein is provided for use in upregulating CXCL9.Furthermore, the antigen binding molecule described herein is provided for (i) treating cancer, (ii) delaying the progression of cancer, and (iii) prolonging the survival of patients suffering from cancer, particularly in the presence of FAP-expressing cells.In a particular embodiment, the antigen binding molecule or IFNG variant polypeptide disclosed herein is provided for use in treating disease, particularly for treating cancer.

[0206] In certain embodiments, the antigen-binding molecule or IFNG variant polypeptide described herein is provided for use in a treatment method. In one embodiment, the antigen-binding molecule or IFNG variant polypeptide described herein is provided for use in the treatment of a disease in an individual in need of the treatment. In certain embodiments, the antigen-binding molecule or IFNG variant polypeptide is provided for use in a method for treating an individual with a disease, comprising administering a therapeutically effective amount of a bispecific antigen-binding molecule to the individual. In certain embodiments, the disease to be treated is cancer. The subject, patient or "individual" in need of treatment is typically a mammal, more specifically a human.

[0207] In a further aspect, the present invention provides the use of an antigen binding molecule or an IFNG variant polypeptide described herein in the manufacture or preparation of a medicament for treating a disease in an individual in need of such treatment. In one aspect, the medicament is for use in a method of treating a disease, comprising administering a therapeutically effective amount of the medicament to an individual having the disease. In a particular aspect, the disease to be treated is a proliferative disorder, in particular cancer. Examples of cancer include, but are not limited to, bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, anal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer. Other examples of cancer include carcinoma, lymphoma (e.g., Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. Other cell proliferative diseases that can be treated using the bispecific antigen-binding molecules or antibodies of the present invention include, but are not limited to, neoplasms located in the abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testes, ovaries, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, chest region, and genitourinary system. Precancerous conditions or lesions and cancer metastases are also included. In certain embodiments, the cancer is selected from the group consisting of renal cell carcinoma, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, and head and neck cancer. Those skilled in the art will readily recognize that in many cases, the antigen-binding molecules or IFNG variant polypeptides as described herein may not provide a cure, but may provide a benefit. In some aspects, physiological changes that provide some benefit are also considered to be therapeutically beneficial. Thus, in some embodiments, the amount of an antigen binding molecule or IFNG variant polypeptide that produces a physiological change is considered an "effective amount" or a "therapeutically effective amount."

[0208] The appropriate dosage of the antigen-binding molecules described herein (when used alone or in combination with one or more other additional therapeutic agents) for preventing or treating a disease depends on the type of disease to be treated, the route of administration, the patient's weight, the specific molecule, the severity and course of the disease, whether the antigen-binding molecules described herein are administered for prophylactic or therapeutic purposes, previous or concurrent therapeutic interventions, the patient's medical history and response to the bispecific antigen-binding molecules, and the discretion of the attending physician. The physician responsible for administration will in any case determine the concentration of active ingredient in the composition and the appropriate dose for each individual subject. Various dosing schedules are contemplated herein, including but not limited to single administration or multiple administrations over various time periods, bolus administration, and pulse infusion.

[0209] The antigen-binding molecule or IFNG variant polypeptide disclosed herein is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, for example, about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) of the antigen-binding molecule or IFNG variant polypeptide can be the initial candidate dosage for administration to the patient, whether by one or multiple individual administrations or continuous infusion. Depending on the factors mentioned above, a typical daily dosage can range from about 1 μg / kg to 100 mg / kg or more. In the case of repeated administration over several days or more, treatment is usually continued until a desired suppression of disease symptoms occurs, depending on the condition. An exemplary dosage of the antigen-binding molecule or IFNG variant polypeptide ranges from about 0.005 mg / kg to about 10 mg / kg. In other examples, dosages may also include about 1 μg / kg body weight, about 5 μg / kg body weight, about 10 μg / kg body weight, about 50 μg / kg body weight, about 100 μg / kg body weight, about 200 μg / kg body weight, about 350 μg / kg body weight, about 500 μg / kg body weight, about 1 mg / kg body weight, about 5 mg / kg body weight, about 10 mg / kg body weight, about 50 mg / kg body weight, about 100 mg / kg body weight, about 200 mg / kg body weight, about 350 mg / kg body weight, about 500 mg / kg body weight to about 1000 mg / kg body weight or more per administration, and any range derivable therebetween. In examples of ranges derivable from the numbers recited herein, ranges such as about 0.1 mg / kg / body weight to about 20 mg / kg / body weight, about 5 μg / kg / body weight to about 1 mg / kg / body weight, based on the above numbers, may be administered. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 5.0 mg / kg or 10 mg / kg (or any combination thereof) can be administered to the patient. Such doses may be administered intermittently, for example, weekly or every three weeks (e.g., the patient receives about 2 to about 20 doses, or for example about 6 doses of the fusion protein). In certain embodiments, the bispecific antigen-binding molecule is administered every three weeks. An initial higher loading dose can be administered, followed by one or more lower doses. However, other dosing regimens may be useful.The progress of this therapy is easily monitored by conventional techniques and assays.

[0210] The antigen-binding molecules or IFNG variant polypeptides described herein are generally used in an amount effective to achieve the intended purpose.For use in treating or preventing disease symptoms, the antigen-binding molecules or IFNG variant polypeptides or pharmaceutical compositions thereof are administered or applied in a therapeutically effective amount.Determining a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.In the case of systemic administration, the therapeutically effective dose can be estimated first from an in vitro assay, such as a cell culture assay.The IC as determined in cell culture can then be used to determine the therapeutically effective amount. 50 Dosage may be set in animal models to achieve a circulating concentration range that includes. Such information can be used to more accurately determine useful doses in humans. Initial dosages can also be estimated from in vivo data, for example, from animal models, using techniques well known in the art. Those skilled in the art can easily optimize administration to humans based on animal data.

[0211] Dosage and dosing intervals can be individually adjusted to provide plasma levels of the antigen-binding molecules or IFNG variant polypeptides disclosed herein that are sufficient to maintain therapeutic efficacy. Usual patient dosages for administration by injection range from about 0.1 to 50 mg / kg / day, typically about 0.1 to 1 mg / kg / day. Therapeutically effective plasma levels can be achieved by multiple daily administrations. Levels in plasma can be measured, for example, by HPLC. In the case of local administration or selective uptake, the effective local concentration of the antigen-binding molecules or IFNG variant polypeptides may not be related to the plasma concentration. One of skill in the art can optimize the therapeutically effective local dosage without undue experimentation.

[0212] The therapeutically effective dose of the antigen-binding molecule or IFNG variant polypeptide described herein generally provides therapeutic benefit without causing substantial toxicity. The toxicity and therapeutic efficacy of the fusion protein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. Using cell culture assays and animal experiments, LD 50 (a dose lethal to 50% of the population) and ED 50 The dose ratio between toxic and therapeutic effects is the therapeutic index, and the LD 50 / ED 50 The therapeutic index of the antigen-binding molecule is preferably expressed as: 1 / 2, 1 / 2, 1 / 1 ...1, 1 / 2, 1 / 1, 1 / 2, 1 / 1, 1 / 1, 1 / 2, 1 / 1, 1 / 2, 1 / 1, 1 / 1, 1 / 2, 1 / 1, 1 / 1, 1 / 2, 1 / 1, 1 / 2, 1 / 1, 1 / 1, 1 / 2, 1 / 1, 1 / 1, 1 / 2, 1 / 1, 1 / 1, 1 / 2, 1 / 1, 1 / 1, 1 / 2, 1 / 1, 1 / 1, 1 / 2, 1 / 1, 1 / 1, 1 / 2, 1 / 1, 1 / 1, 1 / 2, 1 / 1, 1 / 1, 1 / 2, 1 / 1, 1 / 1, 1 / 2, 1 / 1, 1 / 1, 1

[0213] The attending physician of the patient treated with the antigen-binding molecule knows how and when to discontinue, interrupt or adjust the administration due to toxicity, organ failure, etc. Conversely, the attending physician also knows to adjust the treatment to a higher level if the clinical response is not adequate (excluding toxicity). The size of the administered dose in the management of the target disorder will vary depending on the severity of the condition being treated, the route of administration, etc. The severity of the condition can be evaluated, for example, in part, by standard prognostic evaluation methods. Furthermore, the dose and perhaps the frequency of administration will also vary according to the age, weight, and response of the individual patient.

[0214] Other drugs and treatments The antigen binding molecules or IFNG variant polypeptides disclosed herein may be administered in combination with one or more other agents in therapy. For example, the antigen binding molecules or IFNG variant polypeptides may be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" encompasses any agent that can be administered to treat a condition or disease in an individual in need of such treatment. Such additional therapeutic agents may include any active ingredient suitable for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. In certain embodiments, the additional therapeutic agent is another anti-cancer agent, such as a microtubule-disrupting agent, antimetabolite, topoisomerase inhibitor, DNA intercalator, alkylating agent, hormone therapy, kinase inhibitor, receptor antagonist, activator of tumor cell apoptosis, or antiangiogenic agent. In certain aspects, the additional therapeutic agent is an immunomodulator, a cytostatic agent, an inhibitor of cell adhesion, a cytotoxic or cytostatic agent, an activator of cell apoptosis, or an agent that increases the sensitivity of cells to apoptosis inducers.

[0215] Thus, there is provided an antigen binding molecule or IFNG variant polypeptide described herein or a pharmaceutical composition comprising same for use in the treatment of cancer, wherein the antigen binding molecule or IFNG variant polypeptide is administered in combination with chemotherapeutic agents, radiation and / or other agents for use in cancer immunotherapy.

[0216] Such other agents are suitably present in combination in an amount effective for the intended purpose. The effective amount of such other agents depends on the amount of bispecific antibody used, the type of disorder or treatment, and other factors discussed above. The bispecific antigen-binding molecules or antibodies of the present invention are generally used in the same dosages and routes of administration described herein, or 1-99% of the dosages described herein, or any dosage and any route determined experimentally / clinically appropriate. Such combination therapy as described above encompasses combined administration (where two or more therapeutic agents are included in the same composition or separate compositions) and separate administration, where administration of the antigen-binding molecule or IFNG variant polypeptide can be performed before, simultaneously and / or after administration of the additional therapeutic agent and / or adjuvant.

[0217] In a further aspect, there is provided an antigen binding molecule or an IFNG variant polypeptide as described herein above for use in the treatment of cancer, wherein the antigen binding molecule or the IFNG variant polypeptide is administered in combination with another immunomodulatory agent.

[0218] The term "immunomodulator" refers to any substance, including monoclonal antibodies, that affects the immune system. The molecules described herein can be considered as immunomodulators. Immunomodulators can be used as antitumor agents for the treatment of cancer. In one embodiment, immunomodulators include, but are not limited to, anti-CTLA4 antibodies (e.g., ipilimumab), anti-PD1 antibodies (e.g., nivolumab or pembrolizumab), PD-L1 antibodies (e.g., atezolizumab, avelumab or durvalumab), LAG3 antibodies (e.g., relatlimab), PD1-LAG3 bispecific antibodies or TIGIT antibodies (e.g., tiragolumab). In a further embodiment, an antigen binding molecule or IFNG variant polypeptide described herein for use in the treatment of cancer is provided, wherein the antigen binding molecule or IFNG variant polypeptide is administered in combination with an agent that blocks PD-L1 / PD-1 interaction. In one embodiment, the agent that blocks PD-L1 / PD-1 interaction is an anti-PD-L1 antibody or an anti-PD1 antibody. More specifically, the agent that blocks PD-L1 / PD-1 interaction is an antibody selected from the group consisting of atezolizumab, durvalumab, pembrolizumab and nivolumab. In a particular embodiment, the agent that blocks PD-L1 / PD-1 interaction is atezolizumab (MPDL3280A, RG7446). In another embodiment, the agent that blocks PD-L1 / PD-1 interaction is an anti-PD1 antibody, in particular an anti-PD1 antibody selected from pembrolizumab or nivolumab. In another embodiment, the agent that blocks PD-L1 / PD-1 interaction is an anti-PD1 / anti-LAG3 bispecific antibody. Such other agents are suitably present in combination in amounts effective for the intended purpose. The effective amount of such other agents depends on the amount of antigen-binding molecule used, the type of disorder or treatment, and other factors discussed above. The antigen-binding molecules or IFNG variant polypeptides described herein above are generally used in the same doses and routes of administration as described herein, or 1-99% of the dosages described herein, or any dosage and any route determined experimentally / clinically appropriate.

[0219] Such combination therapy as described above encompasses co-administration (wherein two or more therapeutic agents are included in the same composition or in separate compositions), as well as separate administration, where administration of the bispecific antigen-binding molecule may occur prior to, simultaneously with and / or after administration of the additional therapeutic agent and / or adjuvant.

[0220] manufactured goods In another aspect of the present invention, an article of manufacture is provided that contains materials useful for the treatment, prevention and / or diagnosis of the above-mentioned disorders. The article of manufacture includes a container and a label or package insert that is attached to or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container can be made of various materials, such as glass or plastic. The container holds a composition that is effective for treating, preventing, and / or diagnosing a condition, either alone or in combination with other compositions, and can have a sterile access port (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is an antigen-binding molecule as described herein.

[0221] The label or package insert indicates that the composition is used to treat a selected condition.Furthermore, the article of manufacture may include (a) a first container containing a composition comprising the antigen-binding molecule described herein, and (b) a second container containing the composition, the second container containing an additional cytotoxic agent or other therapeutic agent.The article of manufacture in this embodiment of the present invention may further include a package insert indicating that the composition can be used to treat a specific condition.

[0222] Alternatively, or in addition, the article of manufacture may further comprise a second (or third) container containing a pharma- ceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0223] [Table 2] TIFF2025511000000003.tif241170TIFF2025511000000004.tif245170TIFF2025511000000005.tif24 5170TIFF2025511000000006.tif245170TIFF2025511000000007.tif242170TIFF2025511000000008.t if243170TIFF2025511000000009.tif243170TIFF2025511000000010.tif246170TIFF20255110000000 11.tif241170TIFF2025511000000012.tif246170TIFF2025511000000013.tif241170TIFF2025511000 000014.tif246170TIFF2025511000000015.tif241170TIFF2025511000000016.tif246170TIFF202551 1000000017.tif241170TIFF2025511000000018.tif246170TIFF2025511000000019.tif241170TIFF20 25511000000020.tif245170TIFF2025511000000021.tif245170TIFF2025511000000022.tif246170TI FF2025511000000023.tif245170TIFF2025511000000024.tif239170TIFF2025511000000025.tif71170

[0224] The following paragraphs are aspects of the present invention.

[0225] 1. An antigen-binding molecule comprising: (i) an antibody that specifically binds to a tumor-associated antigen; (ii) a homodimer of an interferon gamma (IFNG) variant polypeptide, wherein the IFNG variant polypeptide is characterized by the C-terminal amino acid sequence KRKRP (SEQ ID NO: 1).

[0226] 2. The antigen-binding molecule of clause 1, wherein the IFNG variant polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3.

[0227] 3. The antigen-binding molecule according to item 1 or 2, wherein the antibody that specifically binds to a tumor-associated antigen is an antibody that specifically binds to fibroblast activation protein (FAP).

[0228] 4.An antibody that specifically binds to FAP, (a) a heavy chain variable region (V) comprising a heavy chain complementarity determining region (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 4, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6; H (iv) a light chain variable region (V FAP) comprising a light chain complementarity determining region CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9; L FAP), or (b) a heavy chain variable region (VH1) comprising the amino acid sequence of SEQ ID NO: 12, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14; H A light chain variable region (VFAP) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 15, CDR-L2 having the amino acid sequence of SEQ ID NO: 16, and CDR-L3 having the amino acid sequence of SEQ ID NO: 17. L FAP) The antigen-binding molecule according to any one of items 1 to 3, comprising:

[0229] 5. An antibody that specifically binds to FAP has a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 10. HA light chain variable region (V L A heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 18. H A light chain variable region (V L The antigen-binding molecule according to any one of items 1 to 4, comprising:

[0230] 6. The antigen-binding molecule according to any one of items 1 to 5, wherein the antigen-binding molecule comprises an IgG1 Fc domain or an IgG4 Fc domain, and the Fc domain contains one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antibody for an Fc receptor.

[0231] 7. The antigen-binding molecule according to any one of items 1 to 6, wherein the Fc domain is of the human IgG1 subclass having amino acid mutations L234A, L235A and P329G (EU numbering according to the Kabat EU index).

[0232] 8. The antigen-binding molecule of any one of items 1 to 5, wherein the antigen-binding molecule is protease-activatable and comprises a protease recognition site and a masking moiety.

[0233] 9. The antigen-binding molecule of item 8, wherein the protease recognition site is a substrate for matriptase.

[0234] 10. The antigen-binding molecule of clause 8 or 9, wherein the protease recognition site comprises or consists of PQARK (SEQ ID NO: 20) or HQARK (SEQ ID NO: 21), in particular PQARK (SEQ ID NO: 20).

[0235] 11. The antigen-binding molecule of any one of items 8 to 10, wherein the protease recognition site is part of a cleavable peptide linker that connects the masking moiety to the IFNG variant polypeptide.

[0236] 12. The antigen-binding molecule of any one of items 8 to 11, wherein the masking moiety is fused at its N-terminus to the C-terminus of the IFNG variant polypeptide via a cleavable peptide linker.

[0237] 13. The antigen-binding molecule according to any one of items 8 to 11, wherein the masking moiety is fused at its N-terminus to the C-terminus of the Fc domain via a stable linker and is fused at its C-terminus to the N-terminus of the IFNG variant polypeptide via a cleavable peptide linker.

[0238] 14. The antigen-binding molecule of any one of items 8 to 13, wherein the masking moiety is an antibody or antibody fragment that specifically binds to IFNG.

[0239] 15. The antigen-binding molecule of any one of items 8 to 14, wherein the masking moiety is an scFv that specifically binds to IFNG.

[0240] 16. An scFv that specifically binds to IFNG, (a) a heavy chain variable region (VH1) comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 22, a CDR-H2 having the amino acid sequence of SEQ ID NO: 23, and a CDR-H3 having the amino acid sequence of SEQ ID NO: 24; H (iv) a light chain variable region (VIFNG) comprising a light chain complementarity determining region CDR-L1 comprising the amino acid sequence of SEQ ID NO: 25, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 27. L IFNG), or (b) a heavy chain variable region (VH1) comprising the amino acid sequence of SEQ ID NO: 30, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 31, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 32; H IFNG), and a light chain variable region (V IFNG) comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 34, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 35. L IFNG).

[0241] 17. An scFv that specifically binds to IFNG, (a) a heavy chain variable region (V H IFNG) and a light chain variable region (V L IFNG), or (b) a heavy chain variable region (V H IFNG) and a light chain variable region (V L IFNG) The bispecific antigen-binding molecule according to any one of items 1 to 16, comprising:

[0242] 18. An antigen-binding molecule containing an IFNG variant polypeptide, the antigen-binding molecule comprising: (i) two heavy chains comprising the amino acid sequence of SEQ ID NO: 40 and two light chains comprising the amino acid sequence of SEQ ID NO: 41, or (ii) two heavy chains comprising the amino acid sequence of SEQ ID NO: 42 and two light chains comprising the amino acid sequence of SEQ ID NO: 41; or (iii) two heavy chains comprising the amino acid sequence of SEQ ID NO: 43 and two light chains comprising the amino acid sequence of SEQ ID NO: 44; or (iv) two heavy chains comprising the amino acid sequence of SEQ ID NO: 45 and two light chains comprising the amino acid sequence of SEQ ID NO: 44.

[0243] 19. An interferon gamma (IFNG) variant polypeptide, wherein the IFNG variant polypeptide is characterized by the C-terminal amino acid sequence KRKRP (SEQ ID NO:1).

[0244] 20. The IFNG variant polypeptide of claim 19, wherein the IFNG variant polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3.

[0245] 21. One or more isolated polynucleotides encoding the antigen-binding molecule of any one of clauses 1 to 18 or the IFNG variant polypeptide of clause 19 or 20.

[0246] 22. An expression vector comprising one or more isolated polynucleotides according to paragraph 21.

[0247] 23. A prokaryotic or eukaryotic host cell comprising one or more of the isolated polynucleotides of paragraph 21 or the expression vector of paragraph 22.

[0248] 24. A method for producing an antigen-binding molecule or an IFNG variant polypeptide, comprising: a) culturing a prokaryotic or eukaryotic host cell according to claim 23 under conditions suitable for expression of the antigen-binding molecule or the IFNG variant polypeptide; and b) optionally recovering the antigen-binding molecule or the IFNG variant polypeptide.

[0249] 25. A pharmaceutical composition comprising the antigen-binding molecule according to any one of items 1 to 18 or the IFNG variant polypeptide according to item 19 or 20, and a pharma- ceutically acceptable excipient.

[0250] 26. The antigen-binding molecule of any one of clauses 1 to 17 or the IFNGA variant polypeptide of clause 19 or 20 for use as a pharmaceutical.

[0251] 27. An antigen-binding molecule according to any one of clauses 1 to 17 or an IFNG variant polypeptide according to clause 19 or 20 for use in the treatment of cancer.

[0252] 28. The antigen-binding molecule of any one of clauses 1 to 17 or the IFNG variant polypeptide of clause 19 or 20 for use in the treatment of cancer, wherein the antigen-binding molecule or IFNG variant polypeptide is administered in combination with chemotherapy, radiation and / or other agents for use in cancer immunotherapy.

[0253] 29. Use of an antigen-binding molecule according to any one of clauses 1 to 17 or an IFNG variant polypeptide according to clause 19 or 20 for the manufacture of a medicament for use in the treatment of cancer.

[0254] 30. A method for treating a disease in an individual, comprising administering to the individual a therapeutically effective amount of an antigen-binding molecule according to any one of clauses 1 to 17 or an IFNG variant polypeptide according to clause 19 or 20 in a pharma- ceutically acceptable form.

[0255] 31. The method according to item 30, wherein the disease is cancer. EXAMPLES

[0256] The following are examples of methods and compositions of the present invention. It will be understood that various other embodiments may be practiced in light of the general description provided herein.

[0257] Recombinant DNA techniques and sequencing Standard methods were used to manipulate DNA as described by Sambrook et al., Molecular Cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biology reagents were used according to the manufacturer's instructions. General information regarding the nucleotide sequences of human immunoglobulin light and heavy chains is available in Kabat, EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Ed., NIH Publication No91-3242. DNA sequences were determined by double-stranded sequencing.

[0258] Gene synthesis Desired gene segments were generated by PCR using appropriate templates or synthesized by automated gene synthesis from synthetic oligonucleotides and PCR products by GenScript (China). Gene segments flanked by specific restriction endonuclease cleavage sites were cloned into standard cloning / sequencing vectors. Plasmid DNA was purified from transformed bacteria and concentrations were measured by UV spectroscopy. DNA sequences of subcloned gene fragments were confirmed by DNA sequencing. Gene segments with appropriate restriction sites were designed to allow subcloning into the respective expression vectors. All constructs were designed with a 5'-end DNA sequence encoding a leader peptide that targets the protein for secretion in eukaryotic cells.

[0259] Cell culture technology Standard cell culture techniques were used as described in Current Protocols in Cell Biology (2000), Bonifacino, JS, Dasso, M., Harford, JB, Lippincott-Schwartz, J. and Yamada, KM (eds.), John Wiley & Sons, Inc.

[0260] Protein purification Proteins were purified from the filtered cell culture supernatant with reference to standard protocols. Briefly, Fc-containing proteins were purified from cell culture supernatant by Protein A-affinity chromatography (equilibration buffer: 20 mM sodium citrate, 20 mM sodium phosphate, pH 7.5; elution buffer: 20 mM sodium citrate, pH 3.0). Elution was achieved at pH 3.0, followed by immediate neutralization of the pH of the sample. Proteins were concentrated by centrifugation (Millipore Amicon® ULTRA-15 (Art. Nr.: UFC903096) and aggregated proteins were separated from monomeric proteins by size exclusion chromatography (Superdex 200, GE Healthcare) in 20 mM histidine, 140 mM sodium chloride, pH 6.0. Monomeric compound fractions were pooled, concentrated (if necessary) using e.g. MILLIPORE Amicon Ultra (30 MWCO) centrifugal concentrators, frozen and stored at -80°C. Part of the samples was submitted for subsequent protein analysis and analytical characterization by CE-SDS, size exclusion chromatography (SE-HPLC) and mass spectrometry (LC-MS).

[0261] Alternatively, gene synthesis, cloning, transfection and recovery were outsourced to evitria AG (Schlieren, Switzerland). The corresponding cDNA was cloned into evitria's vector system using conventional (non-PCR-based) cloning techniques. The evitria vector plasmids were gene synthesized. Plasmid DNA was prepared under low-endotoxin conditions based on anion-exchange chromatography. DNA concentration was determined by measuring absorbance at 260 nm wavelength. Sequence accuracy was verified by Sanger sequencing (two sequencing reactions per plasmid). Suspension-adapted CHO K1 cells (received from ATCC and adapted at evitria for serum-free growth in suspension culture) were used for production. Seeds were grown in eviGrow medium, a chemically defined, animal-component-free, serum-free medium. Cells were transfected with eviFect (evitria's custom-made proprietary transfection reagent) and, after transfection, grown in eviMake2, an animal-component-free, serum-free medium. The supernatant was collected by centrifugation and subsequent filtration (0.2 μm filter).

[0262] Alternatively, compounds of interest were prepared by WuXi Biologics using a proprietary vector system with conventional (non-PCR-based) cloning techniques, using suspension-adapted HEK293 cells. Expression of all genes was under the control of the human CMV promoter. For production, WuXi Biologics used commercially available chemically defined media and cultured cells after transfection at 36.5°C and 6% carbon dioxide. The supernatant was harvested by centrifugation and subsequent filtration (0.2 μm filter), and proteins were purified from the harvested supernatant by standard methods.

[0263] Quantification of Fc-containing constructs in the supernatants was performed by Protein A-HPLC on an Agilent HPLC system equipped with a UV detector. Supernatants were injected into a POROS 20 A (Applied Biosystems). Elution peak areas at 280 nm were integrated and converted to concentrations using a calibration curve with standards analyzed in the same run.

[0264] Proteins were purified from filtered cell culture supernatants with reference to standard protocols. Briefly, Fc-containing proteins were purified from cell culture supernatants by Protein A affinity chromatography. After elution, the pH of the sample was immediately neutralized. Proteins were concentrated by centrifugation (Millipore Amicon® ULTRA-15 (Art.Nr.:UFC903096) and aggregated proteins were separated from monomeric proteins by size exclusion chromatography (Akta Pure&HiLoad 26 / 600 Superdex 200; both from Cytiva) in 20 mM histidine, 140 mM sodium chloride, pH 6.0.

[0265] The concentration of purified proteins was determined by measuring the absorbance at 280 nm using the mass extinction coefficient calculated based on the amino acid sequence according to Pace et al., Protein Sci. 1995, 4(11), 2411-2423 (Little Lunatic, Unchained labs). Protein purity and molecular weight were analyzed by CE-SDS in the presence and absence of reducing agents using a LabChipGXII (Perkin Elmer). Aggregate content determination was performed by HPLC chromatography at 25°C using an analytical size exclusion column (TSKgel G3000 SW XL).

[0266] Analysis of the composition of IgG-like proteins The concentration of purified proteins was determined by measuring the absorbance at 280 nm using the mass extinction coefficient calculated based on the amino acid sequence according to Pace et al., Protein Science, 1995, 4(11), 2411-1423. Protein purity and molecular weight were analyzed by CE-SDS in the presence and absence of reducing agents using LabChipGXII or LabChip GX Touch (Perkin Elmer) (Perkin Elmer) with or without pretreatment with rapid PNGase F according to the manufacturer's protocol. Determination of aggregate content was performed by HPLC using running buffer (200 mM KH 2 PO 4 , 250mM KCl (pH6.2), 0.02%NaN 3 Analytical size-exclusion chromatography (TSKgel G3000 SW XL column or UP-SW3000 column) equilibrated in 1000 mL / min was performed by SE-HPLC chromatography at 25° C.

[0267] Mass determination by ESI-MS Samples were analyzed by mass spectrometry to determine the intact / deglycosylated and deglycosylated / reduced mass of purified compounds. For compounds with human Fc and normal Fc glycosylation, 25 μg of protein was diluted to a volume of 40 μL with Milli-Q Type 1 Ultrapure Water. Then, 9 μL of 250 mM sodium phosphate pH 7.5 and 1 μL of PNGase F PRIME™ (N-Zyme Scientifics, NZPP550, Lot No. NZ-2019-0123, 0.2 g / L) were added and the mixture was incubated overnight at 37°C. For proteins with non-human Fc and / or compounds containing more than normal N-glycosites, 10 μg of protein was diluted to 8 μL using Milli-Q Type 1 Ultrapure Water, mixed with 2 μL of "non-reducing" buffer (Rapid PNGaseF non-reducing buffer, NEB#P0711S, lot 10085473, 10 / 21) and incubated at 70 °C for 5 min. Then, 1 μL of Rapid PNGase F was added and incubated at 50 °C for 10 min. After deglycosylation, the samples were diluted to 20 μL using Milli-Q Type 1 Ultrapure Water. To reduce cysteine ​​bonds, 20 μL of human Fc protein was mixed with 20 μL of freshly prepared reducing agent (7 M guanidine, 0.4 M Tris pH 8.0, 0.5 M DTT). For samples digested by rapid PNGase F, 7 μL was mixed with 7 μL of reducing agent. For all sample types, reduction and denaturation were performed at 70 °C for 5 min. Samples were desalted by reversed-phase chromatography on a divinylbenzene column (Agilent, PLRP-S 1000 Å, 2.1 × 150 mm, 8 μm, 0.7 mL / min, 75 °C, 1 μg on column) and mass spectra were recorded using a QTOF mass spectrometer (Agilent 6545XT AdvanceBio LC / Q-TOF). The mass spectrometer was calibrated before the sequence of each sample and lock mass correction was applied to obtain high mass accuracy. For data analysis, a Roche MassAnalyzer was used to sum the mass spectra of the chromatographic peaks and interpret the detected masses.The determined mass was then compared to the calculated theoretical mass.

[0268] Alternatively, LC-MS characterization was performed at NMI Technology Transfer (Reutlingen, Germany). To determine the intact mass, 12.5–25 μg per sample was diluted 1:4 (v / v) with the "non-reducing" buffer included in the rapid PNGase F enzyme kit (Rapid PNGaseF non-reducing, NEB#P0711S, lot 10085472, 10 / 21) and denatured at 80 °C for 2 min. Subsequently, 0.3 μL of rapid PNGase F ("non-reducing") was added and the compounds were deglycosylated at 50 °C for 10 min. Afterwards, samples were diluted with double distilled water to a final volume of 31.25–62.5 μL. To determine the mass of the reduced strand, 12.5–25 μg per sample was diluted with 1:4 (v / v) "reducing" buffer included in the rapid PNGase F enzyme kit (Rapid PNGaseF reducing, NEB#P0710S, lot 10079163 07 / 21) and denatured at 80 °C for 2 min. Subsequently, 0.3 μL of rapid PNGase F ("reducing") was added and the compounds were deglycosylated at 50 °C for 10 min. Afterwards, samples were diluted with double distilled water to a final volume of 31.25–62.5 μL. Samples were desalted by reversed-phase chromatography on a C4 column (Acquity BEH300 C4, 1 mm 50 mm, 1.7 μm Charge 133380461; 150 μL / min, 75° C., 1.6 μg on column) and mass spectra were recorded using a QTOF mass spectrometer (MAXIS, Bruker Daltonics). The mass spectrometer was calibrated before the sequence of each sample and lock mass correction was applied to obtain high mass accuracy. Data analysis was performed by summing the mass spectra of the chromatographic peaks and deconvoluting them with MaxEnt. Identity and completeness were checked by comparing the experimental and theoretical masses.

[0269] Surface plasmon resonance The binding behavior of IFNG variants to IFNGR1 and IFNGR1 / 2 was evaluated by surface plasmon resonance (SPR). All SPR experiments were performed at 25 °C on a Biacore 8K device using HBS-EP (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20, Biacore, Freiburg / Germany) as running buffer. To determine the binding affinity of IFNG variants to IFNG receptors, biotinylated Fc(kih)-IFNG compounds (50 nM) were captured on a flow cell of a streptavidin (SA) sensor chip (contact time 80 s, flow rate 10 uL / min). IFNG mutant series compounds were immobilized at a concentration of 25 nM, contact time 80 s and flow rate 10 μL / min. Immobilization levels up to 450 resonance units (RU) were used. Subsequently, decreasing concentrations of Fc(kih)-IFNGR1 monomer or Fc(kih)-IFNGR1 / 2 heterodimer (800-3.13 nM) were injected as the second analyte over 120 s at a flow rate of 30 µL / min, and dissociation was monitored for 500 s. Bulk refractive index differences were corrected by subtracting the response obtained with a reference flow cell where no protein was immobilized. Affinity constants were derived from the rate constants by fitting to 1:1 Langmuir binding.

[0270] HEK Blue reporter assay The HEK Blue IFNG reporter cell assay allows to specifically measure the activity of IFNG. HEK blue detection medium is used to detect the production of SEAP over time. HEK-Blue™ detection medium was prepared by placing the contents of one HEK-Blue™ detection pouch in a 50 mL Falcon tube and solubilizing the powder in 50 mL of endotoxin-free water. The solution was homogenized by vortexing or swirling. The reconstituted HEK-Blue™ detection was warmed to 37°C for 20-60 min. The medium was sterile filtered (0.2 μm membrane placed in a sterile vial / bottle). HEK-Blue™ detection medium was kept at 37°C before use and stored at 2-8°C for up to 2 weeks. HEKblue cells were detached by vigorous pipetting to create a homogenous cell suspension and cell number and viability were determined using the ViCell Cell Cell Viability Analyzer. The required amount of HEKblue cells was centrifuged and resuspended in 0.55 Mio / mL of HEKblue detection medium. 180 μL of cells (corresponding to 100,000 cells) were distributed per well according to the plating scheme, and 20 μL of test compound (diluted in PBS) was added to each well. The plates were incubated at 4°C for 24 h at 4°C for 30 min at 2°C. 2 The mixture was incubated at 37°C in an incubator for 20-24 hours, and the SEAP levels over time were analyzed using a spectrophotometer at 620-655 nm.

[0271] Activity assay This assay was used in combination with or instead of the HEK blue reporter cell assay to evaluate the activity of IFNG compounds. The induction of MHC-I and PD-L1 on mouse MC38-huCEA tumor cell lines was evaluated in response to 2 days of treatment with anti-mouse IFNG scFv-masked FAP-IFNG compounds containing cleavable linker 2 (PQARK cleavage site) and compared to the activity of unmasked IgG-fused IFNG. Prior to treatment, the constructs were incubated with recombinant matriptase for 2 hours at 37°C. The FAP-IFNG scFv-masked PQARK construct induced MHC-I and PD-L1 in tumor cell lines when the PQARK linker was digested with matriptase. In contrast, the FAP-IFNG scFv-masked construct without preincubation with recombinant matriptase did not induce MHC-I or PD-L1 upregulation.

[0272] MC38-huCEA cells were cultured in DMEM 10% FCS and harvested using cell dissociation buffer. Cells were washed with DMEM 10% FCS and resuspended in DMEM 10% FCS, followed by assessment of cell viability and cell number using an Eve cell counter. Cells were diluted to a concentration of 50,000 / mL in DMEM 10% FCS, and 100 μL of this cell suspension was seeded into cell culture-treated 96F-well plates. Cells were incubated at 37°C, 5% CO 2The cells were incubated overnight at 37°C to ensure cell attachment. The selected concentrations of FAP-IFNg constructs were digested in matriptase buffer (50 mM Tris, 50 mM NaCl, 0.01% Tween 20, pH 9.0) with or without 163 nM / 4.4 ng of recombinant matriptase (4735-SE, lot RIK071951, 0.44 mg / ml) for 2 h at 37°C. After incubation, the digested cytokine Fc fusion solution was diluted to a concentration of 30 nM in DMEM 10% FCS and 50 μL was added per well of cells pre-seeded in 100 μL DMEM 10% FCS, resulting in a final maximum concentration of 10 nM per well. The cytokine Fc fusion solution was serially diluted in a 1:10 ratio until the final minimum concentration per well was 0.1 pM. The cells were incubated in the incubator for 48 h. After 48 hours, cells were washed with PBS and subsequently incubated with 50 μL Trypsin EDTA for 10 min. Detachable cells were collected in DMEM 10% FCS and transferred to round-bottom 96-well plates. Cells were centrifuged (500g, 2 min), the supernatant was discarded, and 150 μL PBS was added per well followed by centrifugation (500g, 2 min). Cells were resuspended in 50 μL staining mixture containing Zombie Near IR fixable Viability Dye (Invitrogen, L10119). Cells were then washed with FACS buffer and 50 μL antibody staining mixture containing anti-muH-2Kb / H-2D-PE (BioLegend, 114608) and anti-muCD274-APC (BioLegend, 124312) was added for 20 min at 4°C. Cells were then washed with PBS, resuspended in 100 μL PFA and incubated for 25 min at RT. Cells were then washed with 100 μL of FACS buffer, resuspended in 100 μL of FCS buffer, and measured on a BD FACS Canto.

[0273] Example 1 1.1 Engineering the C-terminus of IFNG to modulate homogeneity and activity IFNG is an important immunomodulatory cytokine used to treat various immunological diseases. However, its clinical application as an anticancer drug is limited, especially due to the distribution of IFNG receptor 1 (IFNGR1), which traps IFNG throughout the body and prevents its enrichment at tumor sites. Another challenge we observed is the inherent susceptibility of the IFNG C-terminus to proteases present in the producing cell line, which results in a significant amount of partially cleaved by-products with truncated C-terminal sequences that lead to heterogeneity of the final product (Figure 1A). To increase the homogeneity and stability of the compound, we applied sequence engineering to the IFNG C-terminus and identified IFNG variants with differentiated by-product profiles and selective functional behaviors (Figure 1B). We first designed a series of IFNG C-terminal deletion variants to identify the shortest C-terminal sequence of IFNG that maintained functional activity in signaling assays (Table 1A). Several C-terminal variants of IFNG have already been described, highlighting the importance of KRKR residues at the C-terminus of IFNG (Figure 1). Deletion of the KRKR sequence (SEQ ID NO: 78) renders IFNG inactive in downstream signaling (Dobeli et al., Journal of Biotechnology, 1988, 7(3), 199-216). However, truncated IFNG variants have been shown to have activity similar to wild-type IFNG (Slodowski et al., Eur. J. Biochem. 1991, 202, 1133-1140). Therefore, we combined systematic truncation of the KRKR patch with proline or serine-proline capping to maximize C-terminal stability and sequence uniformity, similar to the method described for the C-terminus of IgG (van den Bremer et al., mAbs 2015, 7:4, 672-680). The desired candidate should show the fewest number of by-products and signaling activity comparable to wild-type IFNG.

[0274] [Table 3]

[0275] The second series is based on P1AF3574 from the deletion series, and we introduced additional point mutations into the KRKR sequence to further increase proteolytic stability (Table 1B and Figure 1B). In the systematic sequence variation approach, one or two positions of the KRKR sequence were replaced with serine, proline or glutamic acid. Serine was selected as a neutral amino acid, proline was introduced to sterically hinder the proteolytic recognition sequence, and glutamic acid was introduced to partially reverse the charge of the KRKR sequence (SEQ ID NO: 78).

[0276] [Table 4]

[0277] 1.2 Generation and purification of IgG-fused IFNG deletion and mutation variants All IFNG deletion and mutation series variants were generated, purified, and the effects of engineered changes on by-product profile and signaling activity were characterized (Example 2). IFNG sequence variants were fused via a glycine-serine linker to the C-terminus of the heavy chain of an IgG antibody specific for fibroblast activation protein (FAP), i.e., anti-FAP clone 4B9 (disclosed in WO 2012 / 020006) and the Fc domain containing the P329G LALA mutation (Schlothauer et al., Protein Eng Des Sel. 2016, 29(10), 457-466) (Figure 1A). Compounds were expressed in evitria using a transient CHO expression system. Compounds from the deletion series (Table 1A) reached a median titer of 118 mg / L, and compounds from the mutation series (Table 1B) were expressed at a median of 45.5 mg / L. Tables 2A and 2B list the individual titers obtained for each compound. Compounds were then captured by MabSelectSure HP and eluted with a pH gradient up to pH 3.0. Fractions were neutralized and analyzed for composition by CE-SDS and HMW content by SE-HPLC. Fractions with the highest monomer content were pooled and further purified by SEC. SEC fractions with the highest monomer content were pooled as the final batch. The median monomer content determined by SE-HPLC for the deletion series of compounds was 88.9%, and the mutant series of compounds was purified to a median homogeneity of 99.8%. LC-MS characterization confirmed sequence identity for both series. Detailed LC-MS quality profiles of the compounds are provided in Example 2, Table 3. The expressed titers and monomer content of each compound are listed in Tables 2A and 2B.

[0278] [Table 5]

[0279] [Table 6]

[0280] Example 2 2.1 Identification of C-terminal variants of IFNG with improved quality profiles To maximize the sequence integrity of the C-terminus of IFNG, we identified IFNG sequences with favorable heavy chain by-product profiles. Therefore, we combined proline or serine-proline capping with systematic truncation of the KRKR sequence (Figure 1B, Table 1A). All compounds were expressed, purified, and the final batches were analyzed by LC-MS to identify and quantify the heavy chain by-products (Table 3). Compounds containing wild-type human IFNG contained 7% intact heavy chain and eight truncated heavy chain by-products in various amounts. Published IFNG variants (included in P1AF3570 and P1AF3571) also showed heavy chain sequence heterogeneity due to proteolysis with three and four by-products, respectively. The number of by-products correlated with the length of the IFNG C-terminal sequence, truncation of the C-terminus reduced the number of truncated heavy chains, and compounds without the KRKR sequence were 100% accurate. C-terminal proline capping resulted in fewer heavy chain by-products compared to the serine-proline capped equivalents (Table 3).

[0281] [Table 7]

[0282] 2.2 Signaling activity of IFNG deletion variants The benchmark compounds and other compounds in the deletion series were further characterized for their ability to induce IFNG signaling using the HEKblue assay. Functional activity correlated with the length of the C-terminal sequence, with only compounds containing the entire KRKR sequence retaining activity levels comparable to wild-type IFNG (Figure 2 and Table 4), whereas systematic truncation of the KRKR sequence progressively reduced functional activity.

[0283] [Table 8]

[0284] Combined with the quality data (Table 3), the best profile, i.e., the highest activity combined with the least amount of by-products, was observed for the proline-capped IFNG variant (P1AF3574) terminating in KRKRP. This compound contained 95% correct heavy chain, 5% truncated by-products, and showed 84% activity compared to wild-type IFNG. As a result, this sequence was selected as the basis for further stability engineering in the mutant series (Example 2.4).

[0285] 2.3 IFNG-KRKRP and wild-type IFNG show equivalent binding behavior to the IFNG receptor IFNG is a constitutive homodimer that initiates the formation of a signaling complex with the IFNG receptor to activate the JAK / STAT signaling pathway. The signaling complex consists of a symmetric arrangement of two IFNGR1 / 2 heterodimers with an IFNG homodimer in the center. Each IFNG monomer interacts with one IFNGR1 / 2 heterodimer that contacts both receptor subunits. It has been suggested that the assembly of the signaling complex is triggered by an initial interaction of IFNG with IFNGR1, which creates a binding surface for IFNGR2 (Mendoza et al., Nature, 2019, 567(7746), 56-60). Although the assembly of the signaling complex is primarily driven by the structured domains of IFNG, the unstructured C-terminus of IFNG also contributes to active signaling, possibly by interacting with a site in IFNGR1 / 2 closer to the cell membrane. However, the exact binding mode is unknown and has not been resolved in the available crystal structures.

[0286] To further evaluate the similar properties of wild-type IFNG and our engineered compounds with C-terminal KRKRP (SEQ ID NO:1), we determined the binding affinity of IFNG variants to the IFNG receptor by SPR. To do so, wild-type IFNG (P1AG2651) and IFNG KRKRP (P1AG1550) were produced and purified as C-terminal Fc (kih) fusion proteins and immobilized on a flow cell. Knob-into-hole (kih) mutations are specific mutations in the Fc domain (mutations S354C and T366W EU numbering in the "knob" chain and mutations Y349C, T366S, L368A and Y410V EU numbering in the "hole" chain) that allow enhanced heterodimerization of two different heavy chains (Merchant et al., Nature Biotechnology, 1998, 16(7), 677-681). As a second analyte, either IFNGR1 monomer (P1AF7104) or IFNGR1 / 2 heterodimer (P1AF8126) was passed over the flow cell at different concentrations, and dissociation constants (KD) were calculated based on the observed association and dissociation behavior. Fc(kih)-IFNG KRKRP binds IFNGR1 with an apparent KD of 154 nM. D It binds with a K of 177 nM. D The binding affinity of Fc(kih)-IFNG-KRKRP(P1AG1550) was in the same range as wild-type Fc(kih)-IFNG with a K D and wild-type Fc(kih)-IFNG (P1AF2651) bound with a K of 73.8 nM. D was bonded with. 2.4 Mutations in the IFNG KRKR sequence differentially affect IFNG receptor binding and signaling

[0287] Having identified the minimum C-terminal sequence length, additional point mutations were introduced into the KRKR sequence to further improve stability against proteases. All compounds in the mutant series were expressed and purified as IgG fusion proteins (see Example 1). Analysis of the quality profile by LC-MS confirmed the beneficial effect of the C-terminal proline cap, since no proteolytic by-products were detected in the compounds in this mutant series.

[0288] Mutations in the KRKR sequence affected receptor binding as determined by SPR. To measure binding affinity, the mutant series of compounds was immobilized on a flow cell. As a second analyte, Fc(kih)-IFNGR1 monomer or Fc(kih)-IFNGR1 / R2 heterodimer was passed through the flow cell, and binding affinity (KD) was calculated based on the observed association and dissociation constants. Already single point mutations reduced the apparent binding affinity to the receptor compared to compounds with C-terminal KRKRP. Double point mutations had an additional negative effect on receptor binding. The binding behavior of the mutant series of compounds to IFNGR1 and IFNGR1 / 2 is summarized in Table 5.

[0289] [Table 9]

[0290] The functional impairment of the mutant series variants was further evident in reporter cell assays (Figure 3). Functional characterization revealed that either single serine or proline mutations were well tolerated regardless of their position in the KRKR sequence. In contrast, already single glutamic acid mutations rendered the compound inactive, as did all double mutations regardless of the amino acid exchanged.

[0291] 2.5 Mutations in the IFNG KRKR sequence protect against proteolysis by matriptase To evaluate the effect of mutations on the proteolytic stability of the KRKR motif, all compounds of the mutant series were treated with matriptase (custom purified according to published protocol by Cepter Biopartners) at an enzyme to substrate molar ratio of 1:3500 for 1 h at 37 °C. Subsequently, the release of the main proteolytic by-product (IgG-IFNG cleaved between IFNG-K / RKRP) was detected and quantified by LC-MS in deglycosylated reduced conditions. Point mutations at positions 1 or 2 had a positive effect on the proteolytic stability, where no proteolysis was observed in the selected conditions, whereas a 35% cleavage of the K / RKR motif was detected for the reference molecule (Table 6). Serine, proline or glutamic acid mutations at positions 3 or 4 interfered with proteolysis less efficiently, where 2–11% proteolytic products were detected. Double mutations involving positions 1 or 2 also efficiently prevented proteolysis, whereas double mutations at positions 3 and 4 were tolerated. Nevertheless, given the lower functional activity of the mutant series compounds in the absence of target binding, the IFNG KRKRP remains the preferred sequence and was used to generate masked IFNG compounds (Example 3).

[0292] [Table 10]

[0293] Example 3 3.1 Design of tumor-targeting masked IFNG compounds To develop IFNG with greater therapeutic potential, we designed tumor-targeted IFNG compounds in which the binding of IFNG to IFNGR1 was hindered by an IFNG-specific masking domain. We hypothesized that efficient masking would counteract the associated sink effect and allow the concentration of the compound at the tumor site, ultimately inducing the desired downstream effect. The masking domain was based on an IFNG-specific scFv domain and was introduced either at the C-terminus (mask-releasing format) or N-terminus (cytokine-releasing format) of IFNG-KRKRP (Figure 1C). The masked compounds were designed to be activated in the tumor microenvironment by proteolysis of a cleavable linker connecting IFNG with its mask.

[0294] To create the masked release format, IFNG-KRKRP was fused to the C-terminus of a targeting antibody, an IgG antibody specific for fibroblast activation protein (FAP), namely anti-FAP clone 4B9 (disclosed in WO 2012 / 020006) and an Fc domain containing the P329G LALA mutation (Schlothauer et al., Protein Eng Des Sel. 2016, 29(10), 457-466), via a glycine-serine linker (linker 1, GGGGSGGGGSGGGGSGGGGGSGGGGG, SEQ ID NO: 69).Anti-human IFNG scFv-mask (based on US Patent No. 6,329,511 B1) was fused to the C-terminus of IFNG-KRKRP via a second glycine-serine linker (linker 2, GGGGSGGGGSGGGGSGGGGGSGGGGG, SEQ ID NO: 69). To generate the cytokine release format, the scFv mask was fused to the C-terminus of the targeting IgG antibody via a glycine-serine linker (linker 1) and IFNG-KRKRP was linked to the C-terminus via a second glycine-serine linker (linker 2). To allow proteolytic activation of the masked compound in the tumor microenvironment, a matriptase cleavage site (PQAR / K, SEQ ID NO: 20) was introduced into linker 2 in both formats. This cleavable linker has the amino acid sequence GGGGSGGGGSGGGPQARKGGGGGGSGGGGG (linker 2', PQARK linker, SEQ ID NO: 70). Proteolysis of linker 2' in the mask release format yields IgG-fused unmasked IFNG, while proteolysis of linker 2' in the cytokine release format yields free IFNG-KRKRP homodimers.

[0295] [Table 11]

[0296] 3.2 Production and purification of masked and cytokine-releasing formats Masked formats with cleavable (P1AG7568, P1AG7571) and non-cleavable (P1AG7567, P1AG7570) linker sequences were expressed and purified using WuXi Biologics' transient expression system. An unmasked positive control compound (P1AG1310) was expressed in evitria AG and purified in-house. All compounds were captured by MabSelectSure HP and eluted with a pH gradient up to pH 3.0. Fractions were neutralized and analyzed for composition by CE-SDS and HMW content by SE-HPLC. The eluted fractions with the highest monomer content were pooled and further purified by SEC. The SEC fractions with the highest monomer content were pooled as the final batch (Table 7B). Depending on the format, different expression titers and quality profiles were reached, with the highest production titer observed for the unmasked compound (152 mg / L). The masked format was expressed at lower levels (6.9-54.6 mg / L). LC-MS confirmed the correct chain composition and no proteolytic by-products were detected.

[0297] [Table 12]

[0298] Example 4 4.1 Generation of masked mouse IFNG format as a mouse surrogate To evaluate the therapeutic potential of the masked format in vivo, we focused on a mouse surrogate model (Example 5). The masking domain is based on the scFv domain specific for mouse IFNG, and the mask is introduced either at the C-terminus (mask release format) or at the N-terminus (cytokine release format) of mouse IFNG-KRKRP (Figure 1, Figure 4A). As for the human counterpart, the masked mouse compound was designed to be activated in the tumor microenvironment by proteolysis of the cleavable linker that connects IFNG and its mask.

[0299] A surrogate molecule was designed similar to the human masked compound, and to generate a mask release format (P1AG3766), mouse IFNG-KRKRP was fused to the C-terminus of the mouse IgG1 heavy chain of a targeting IgG antibody specific for fibroblast activation protein (FAP), i.e., anti-FAP clone 28H1 (disclosed in WO 2012 / 020006), via a glycine-serine-linker (linker 1, GGGGSGGGGSGGGGSGGGGGSGGGGG, SEQ ID NO: 69). DAPG mutations were introduced into the constant region of the heavy chain to abolish binding to mouse Fc gamma receptors, for example according to the methods described in Baudino et al., J. Immunol. (2008), 181, 6664-6669, or in WO 2016 / 030350. The anti-mouse IFNG scFv mask was fused to the C-terminus of mouse IFNG-KRKRP via a second glycine-serine linker (Linker 2, GGGGSGGGGSGGGGSGGGGGGSGGGG, SEQ ID NO: 69). To generate the cytokine-releasing format (P1AG3755), the scFv mask was fused to the C-terminus of the targeting IgG via a glycine-serine linker (Linker 1) and mouse IFNG-KRKRP was linked to the C-terminus via a second glycine-serine linker (Linker 2). A matriptase cleavage site (PQAR / K) was introduced into Linker 2 (Linker 2') of both formats to allow proteolytic activation in the tumor microenvironment. The cleavable linker has the amino acid sequence GGGGSGGGGSGGGGPQARKGGGGGGSGGGGSGGGG (Linker 2', PQARK Linker, SEQ ID NO: 70). Proteolysis of linker 2' in the mask release format yields unmasked mouse IFNG-KRKRP fused to IgG, whereas proteolysis of linker 2' in the cytokine release format yields free mouse IFNG-KRKRP homodimer. As a positive control, mouse IFNG-KRKRP was also expressed as a C-terminal IgG fusion without the masking domain (P1AF9672).

[0300] [Table 13]

[0301] 4.2 Generation and purification of mouse surrogates All mouse surrogate compounds were expressed using a transient CHO expression system in evitria. Immediately after harvest, cOmplete protease inhibitor cocktail (Roche) was added at 0.5x concentration and compounds were captured using MabSelectSure HP and eluted with a pH gradient up to pH 3.0. Fractions were neutralized and analyzed for composition by CE-SDS and HMW content by SE-HPLC. Fractions with the highest monomer content were pooled and further purified by SEC. After each SEC purification, the respective peak fractions were analyzed by SE-HPLC and CE-SDS. SEC fractions with the highest monomer content were pooled as the final batch. SE-HPLC revealed >95% monomer content for both formats (P1AG3766 and P1AG3755) as well as the unmasked control (P1AF9672), and CE-SDS and LC-MS confirmed the correct chain composition (Table 8B). For the cytokine releasing format (P1AG3755), LC-MS revealed that in 10% of the compounds, one of the two cleavable linkers was already cleaved at the intended cleavage site (PQAR / K, SEQ ID NO: 20).

[0302] [Table 14]

[0303] 4.3 Functional characterization of mouse surrogates To evaluate the in vitro activity of FAP-targeting scFv-masked FAP anti-mouse IFNG compounds containing cleavable linkers, MC38-huCEA cells were treated in vitro with the aforementioned surrogate molecules. Anti-mouse IFNG scFv+masked FAP-muIFNG compounds were incubated for 2 hours at 37°C in the absence or presence of 163 nM recombinant mouse matriptase. After a 48-hour culture period, MHC-I and PD-L1 expression on MC38-huCEA cells was quantified using flow cytometry. Treatment with unmasked FAP-muIFNG compounds served as a positive control, demonstrating that MHC-I and PD-L1 expression on MC38-huCEA cells was induced upon treatment (Figures 4A and 4B).

[0304] Anti-mouse IFNG scFv-masked FAP-muIFNG molecules that were not pretreated with recombinant mouse matriptase did not enhance MHC-I or PD-L1 levels (Figures 4A and 4B). Preincubation of anti-mouse IFNG scFv-masked FAP-muIFNG with matriptase partially restored activity, as demonstrated by increased expression of MHC-I and PD-L1 on MC38-huCEA cells (Figures 4A and 4B).

[0305] Example 5 5.1 In vivo characterization of mouse surrogate molecules For in vivo experiments, one dose of P1AG3755 at 1.25 or 10 mg / kg was injected intravenously into C57Bl / 6 mice expressing human CEA bearing KPC-4662 huCEA tumors (an exclusion tumor model with low CD8 T cell infiltration) that were injected subcutaneously as a cell suspension 21 days prior to the start of treatment (Figure 5).

[0306] KPC4662 cells were obtained from the University of Pennsylvania and engineered in-house to express human CEA. Cells were cultured in DMEM + 10% FBS + hygromycin 500 μg / ml, counted, and 300,000 cells were injected subcutaneously into the flank of mice in a 1:1 mixture of RPMI and matrigel in a total volume of 100 μL. Tumor growth was measured at least twice weekly using calipers, and tumor volume was calculated as follows: Tumor volume = (W 2 / 2)×L (W: width, L: length).

[0307] After 21 days of initial tumor growth in mice, the tumor size was approximately 225 mm 3 Upon reaching 100 mg / kg / day, mice were randomized into three groups and treated with different doses of P1AG3755 or histidine buffer (vehicle) three times a week. All mice were intravenously injected with 200 μL of the appropriate solution. Stock solutions were diluted in histidine buffer (20 mM histidine, 140 mM NaCl pH 6.0) to obtain the appropriate amount of compound per 200 μL. At least five mice were sacrificed on days 3 and 7 after treatment injection (day 7 = termination). Of these mice, tumors and blood were collected for downstream analysis.

[0308] 5.2 Ex vivo readout Half of the tumors dedicated to flow cytometry analysis were weighed and digested with Liberase and DNAse. Single cell suspensions were then stained with DAPI, podoplanin PE-Cy7, E-cadherin BV421, CD45 AF700, TCRb PE-Cy5 and CD8a BV711, among others, and analyzed on a BD Fortessa Flow Cytometer. Plots and statistical tests were performed in GraphPad PRISM 8.

[0309] For histological analysis by immunofluorescence (3DIP), half of the tumors were fixed for approximately 20 h in BD Cytofix solution diluted 1:4 in PBS. After washing and transferring to PBS, tumors were embedded in 4% low gelling temperature agarose. Tumor sections (70 μm thick) were cut from these blocks using a Leica VT1200s vibratome equipped with a common razor blade. Sections were subsequently permeabilized (TBS + 0.3% Triton-X) and blocked for 2 h using BSA and mouse serum (1% each) before staining overnight (approximately 15 h) at room temperature with the following antibodies, among others: CD8a BV421, E-cadherin CF488, MHCI PE. Image acquisition was performed on a Leica SP8 inverted confocal microscope.

[0310] For cytokine analysis, serum samples were collected from 5 mice per group (on days 3 and 7 after treatment injection again) and stored at -20°C until required for assay. Tumor pieces from 5 mice per group were collected in liquid nitrogen on the same day and stored at -80°C until further use. Assays were performed using 5 μL of serum (1 / 10 dilution) and 40 μg of protein from tumor lysate. Tumor lysate was obtained using Bio-plex cell lysis kit (171304012, BioRad) and Precellys evolution homogenizer lysis device (Bertin instruments). Tumor lysate was centrifuged and the supernatant was retained and then stored at -80°C. Protein levels were determined using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) and ELISA reader (PerkinElmer, EnVision 2104 multi-label reader). Tumor lysate was diluted accordingly with sample diluent from the kit to obtain a final amount of 40ug of protein per sample. The assay was performed using the ProcartaPlex Simplex Kit (reference EPX010-26061-901, Thermo Fisher) on a Luminex Flexmap 3D instrument according to the manufacturer's protocol.

[0311] statistical analysis For flow cytometry and cytokine analysis, one-way ANOVA tests with multiple comparison analysis of treated versus vehicle groups were performed with Dunnett's correction for multiple testing. Plots and statistical tests were performed in GraphPad PRISM 8.

[0312] 5.3 Observations The proximal biomarker of IFNG signaling is the upregulation of MHCI and PDL1 expression. When using mouse surrogate molecules to treat KPC4662-huCEA tumors (Figure 5), we could easily observe the upregulation of MHCl and PDL1 by flow cytometry in the high-dose group on cancer cells and fibroblasts 3 days after treatment (Figure 6). An increase in MHCI expression was also observable 7 days after treatment, but PDL1 levels were not statistically significantly different from the vehicle group 7 days after treatment.

[0313] Next, we observed by immunofluorescence microscopy that the increased MHCI expression in tumor tissues was highly heterogeneous and that the presence of CD8 T cells correlated with MHCI-positive areas in tumor tissues (Figure 7).

[0314] Although the overall CD8 T cell presence was not statistically significantly different between treatment and vehicle groups by flow cytometry, we again showed by imaging that increased amounts of CD8 T cells were present in MHCI-expressing areas of the tumor, especially early after treatment (Figure 8). As with this tumor, CD8 T cells were present at the edge of the tumor at baseline and in the vehicle group, so non-local quantification by flow cytometry failed to pick up statistically significant differences between treatment and vehicle groups.

[0315] Finally, we could observe that treatment with mouse surrogates led to an increase in CXCL9 (a T cell-attracting chemokine) levels in the serum of mice 7 days after treatment administration (Figure 9), indicating that not only is the cancer cell altered and the tumor immune environment modified, but the cytokine milieu is also influenced by tumor-targeted IFNG.

[0316] Example 6 6.1 Design of spatially masked IFNG Given the species specificity of IFNG, a species-specific masking domain is also required to prevent receptor binding outside the tumor microenvironment. Therefore, we investigated species-independent masking and designed a series of sterically masked targeted IFNG compounds. Based on the available crystal structure of the IFNG signaling complex (Mendoza et al., Nature, 2019, 567(7746), 56-60), we hypothesized that in the assembled complex, the C-terminus of IFNG is located close to the membrane, and therefore fusion of a bulky domain to the C-terminus may interfere with IFNGR1 binding. To create a targeted, masked molecule, we inserted an IFNG domain between the upper and lower hinge regions of IgG1. At the N- and C-termini, IFNG was linked using a glycine-serine linker. Furthermore, we investigated several hinge variants.

[0317] [Table 15]

[0318] The amino acid sequences of the different linkers are as follows: linker 1 is GGGGSGGGGSGGGGSGGGGGSGGGGG (SEQ ID NO: 69), linker 2 is GGGGSGGG (SEQ ID NO: 72), linker 3 is LEVLFQGP (SEQ ID NO: 73), linker 4 is GGGGSGGGGSGGGGSGGGGSGLEVLFQGPGGGGGSGGGGG (SEQ ID NO: 74), and linker 5 is QARK (SEQ ID NO: 75). The amino acid sequence of hinge 1 is TCPPCP (SEQ ID NO: 76) and the amino acid sequence of hinge 2 is PCP (SEQ ID NO: 77).

[0319] 6.2 Generation and purification of sterically masked IFNG Sterically masked compounds were expressed in evitria using a transient CHO expression system, captured by MabSelectSure HP, and eluted with a pH gradient up to pH 3.0. Fractions were neutralized and analyzed for composition by CE-SDS and HMW content by SE-HPLC. Fractions with the highest monomer content were pooled and further purified by SEC. In some cases, a second and third SEC step was required to remove all HMW impurities. After each SEC purification, the respective peak fractions were analyzed by SE-HPLC and CE-SDS. Fractions with the highest monomer content were pooled as the final batch. Median monomer content was >98% for all compounds (100% by SE-HPLC, CE-SDS) and sequence identity was confirmed by LC-MS. Detailed quality attributes are summarized in Table 9B.

[0320] [Table 16]

[0321] 6.3 Blocking efficiency of sterically masked IFNG To evaluate the in vitro blocking efficiency of sterically masked IFNG, HEK Blue IFNG reporter cells were stimulated for 20 h to 24 h with unmasked IFNG (P1AG8697) and sterically masked IFNG molecules (P1AG8692), used as a positive control, respectively. SEAP reporter activity in response to IFNG was measured at 620–655 nm using a spectrophotometer. The results obtained showed that sterically masked IFNG exhibited reduced IFNG activity compared to the positive control (Figure 10B).

[0322] The foregoing invention has been described in some detail by way of illustration and example for clarity of understanding, but the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entireties.

Claims

1. It is an antigen-binding molecule, (i) an antibody that specifically binds to tumor-associated antigens, (ii) A homodimer of an interferon-gamma (IFNG) variant polypeptide, characterized in that the IFNG variant polypeptide is terminated at the C-terminus with the amino acid sequence KRKRP (SEQ ID NO: 1), and Antigen-binding molecules containing these molecules.

2. The antigen-binding molecule according to claim 1, wherein the IFNG variant polypeptide comprises or comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO:

3.

3. The antigen-binding molecule according to claim 1 or 2, wherein the antibody that specifically binds to a tumor-associated antigen is an antibody that specifically binds to fibroblast-activating protein (FAP).

4. The antibody that specifically binds to FAP, (a) Heavy chain complementarity determining region (CDR-H1) containing the amino acid sequence of SEQ ID NO: 4, CDR-H2 containing the amino acid sequence of SEQ ID NO: 5, and heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 6 Ｈ FAP), and the light chain complementarity determining region (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO: 7, CDR-L2 containing the amino acid sequence of SEQ ID NO: 8, and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 9 Ｌ FAP), or (b) Heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 12, CDR-H2 containing the amino acid sequence of SEQ ID NO: 13, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 14 Ｈ The light chain variable region (V) includes FAP), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 15, CDR-L2 containing the amino acid sequence of SEQ ID NO: 16, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

17. Ｌ FAP) The antigen-binding molecule according to claim 1 or 2, comprising:

5. The antibody that specifically binds to FAP has a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO:

10. Ｈ FAP) and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 11 Ｌ A heavy chain variable region (V) containing FAP or the amino acid sequence of SEQ ID NO: 18 Ｈ FAP) and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 19 Ｌ An antigen-binding molecule according to claim 1 or 2, comprising FAP.

6. The antigen-binding molecule according to claim 1 or 2, wherein the antigen-binding molecule comprises an IgG1 Fc domain or an IgG4 Fc domain, and the Fc domain comprises one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antibody to the Fc receptor.

7. The antigen-binding molecule according to claim 6, wherein the Fc domain is a human IgG1 subclass having amino acid mutations L234A, L235A and P329G (EU numbering according to the Kabat EU Index).

8. The antigen-binding molecule according to claim 1 or 2, wherein a first IFNG variant polypeptide is fused at its N-terminus to the C-terminus of a first heavy chain via a first linker, and a second IFNG variant polypeptide is fused at its N-terminus to the C-terminus of a second heavy chain via a second linker.

9. The antigen-binding molecule according to claim 8, wherein the first linker and the second linker are peptide linkers.

10. The antigen-binding molecule according to claim 1 or 2, wherein the antigen-binding molecule is protease-activatable and includes a protease recognition site and a masking site.

11. The antigen-binding molecule according to claim 10, wherein the protease recognition site is a substrate of matryptase.

12. The antigen-binding molecule according to claim 10, wherein the protease recognition site includes or comprises PQARK (SEQ ID NO: 20) or HQARK (SEQ ID NO: 21), particularly PQARK (SEQ ID NO: 20).

13. The antigen-binding molecule according to claim 10, wherein the protease recognition site is part of a cleavable peptide linker that connects the masking portion to the IFNG variant polypeptide.

14. The antigen-binding molecule according to claim 10, wherein the masking portion is fused at its N-terminus to the C-terminus of the IFNG variant polypeptide via a cleavable peptide linker.

15. The antigen-binding molecule according to claim 10, wherein the masking portion is fused at its N-terminus to the C-terminus of the Fc domain via a stable linker, and at its C-terminus to the N-terminus of the IFNG variant polypeptide via a cleavable peptide linker.

16. The antigen-binding molecule according to claim 10, wherein the masking portion is an antibody or antibody fragment that specifically binds to IFNG.

17. The antigen-binding molecule according to claim 10, wherein the masking portion is an scFv that specifically binds to IFNG.

18. scFv, which specifically binds to IFNG, The heavy chain variable region (V Ｈ IFNG) containing CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24, and the light chain variable region (V Ｌ IFNG) containing CDR-L1 comprising the amino acid sequence of SEQ ID NO: 25, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 27, or (b) Heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 30, CDR-H2 containing the amino acid sequence of SEQ ID NO: 31, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 32 Ｈ The light chain variable region (V) includes IFNG), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 33, CDR-L2 containing the amino acid sequence of SEQ ID NO: 34, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

35. Ｌ The antigen-binding molecule according to claim 10, comprising IFNG.

19. scFv, which specifically binds to IFNG, (a) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 28 (V Ｈ IFNG) and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 29 Ｌ IFNG), or (b) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 36 (V Ｈ IFNG) and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 37 Ｌ IFNG) The antigen-binding molecule according to claim 10, comprising:

20. The aforementioned antigen-binding molecule (i) Two heavy chains containing the amino acid sequence of SEQ ID NO: 40 and two light chains containing the amino acid sequence of SEQ ID NO: 41, or (ii) Two heavy chains containing the amino acid sequence of SEQ ID NO: 42 and two light chains containing the amino acid sequence of SEQ ID NO: 41, or (iii) Two heavy chains containing the amino acid sequence of SEQ ID NO: 43 and two light chains containing the amino acid sequence of SEQ ID NO: 44, or (iv) The antigen-binding molecule according to claim 1 or 2, comprising two heavy chains containing the amino acid sequence of SEQ ID NO: 45 and two light chains containing the amino acid sequence of SEQ ID NO:

44.

21. An interferon-gamma (IFNG) variant polypeptide, characterized in that the IFNG variant polypeptide terminates with the C-terminal amino acid sequence KRKRP (SEQ ID NO: 1).

22. The IFNG variant polypeptide according to claim 21, wherein the IFNG variant polypeptide comprises or is derived from the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO:

3.

23. One or more isolated polynucleotides encoding the antigen-binding molecule described in claim 1 or the IFNG variant polypeptide described in claim 21.

24. An expression vector comprising one or more isolated polynucleotides as described in claim 23.

25. A prokaryotic host cell or eukaryotic host cell comprising one or more isolated polynucleotides as described in claim 23.

26. A method for producing an antigen-binding molecule or an IFNG variant polypeptide, comprising: a) culturing a prokaryotic host cell or a eukaryotic host cell as described in claim 25 under conditions suitable for the expression of the antigen-binding molecule or the IFNG variant polypeptide; and b) optionally recovering the antigen-binding molecule or the IFNG variant polypeptide.

27. A pharmaceutical composition comprising an antigen-binding molecule according to claim 1 or an IFNG variant polypeptide according to claim 21, and a pharmaceutically acceptable excipient.

28. An antigen-binding molecule according to claim 1 or an IFNG variant polypeptide according to claim 21, for use as a pharmaceutical product.

29. An antigen-binding molecule according to claim 1 or an IFNG variant polypeptide according to claim 21 for use in cancer treatment.

30. The antigen-binding molecule or IFNG variant polypeptide according to claim 1 or claim 21, for use in the treatment of cancer, wherein the antigen-binding molecule or IFNG variant polypeptide is administered in combination with other agents for use in chemotherapy, radiotherapy and / or cancer immunotherapy.

31. Use of the antigen-binding molecule according to claim 1 or the IFNG variant polypeptide according to claim 21 for manufacturing a pharmaceutical product for the treatment of cancer.

32. A method for treating a disease in an individual, comprising administering to the individual a therapeutically effective amount of the antigen-binding molecule described in claim 1 or the IFNG variant polypeptide described in claim 21 in a pharmaceutically acceptable form.

33. The method according to claim 32, wherein the disease is cancer.

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