Antigen binding molecules capable of binding interferon gamma

Abstract

The present disclosure relates to bispecific antibodies directed against interferon gamma (IFN gamma) and tumor-associated antigens to deliver IFN gamma to a tumor, and to medical uses of such antibodies.

Description

Technical Field

[0001] This disclosure relates to antigen-binding molecules targeting interferon-γ (IFNγ) and to the medical use of such antigen-binding molecules. Background Technology

[0002] Treating non-immunogenic or "cold" tumors is more challenging because the lack of immune cells in the tumor area means they respond poorly to immunotherapy.

[0003] A range of targeted therapies have been used to treat cancer, producing varying degrees of immune engagement, including radioactive conjugates, ADC-antibody drug conjugates, and T-cell conjugates.

[0004] IFNγ is a cytokine with defined roles in regulating immunity and inflammation. The interferon family has three main members, now named interferon-α (IFNα), interferon-β (IFNβ), and interferon-γ (IFNγ). IFNγ is generally considered pro-inflammatory.

[0005] Studies have shown that IFNγ is crucial for tumor surveillance by the immune system, and a high correlation has been observed between IFNγ production and tumor regression in immunotherapy. IFNγ also has direct anti-tumor effects because it is anti-angiogenic, inhibits proliferation, sensitizes tumor cells to apoptosis, upregulates MHC class I and II expression, and stimulates anti-tumor immune activity (Clinical uses of Interferon-γ, Ann NY Acad. Sci. 2009 Dec; 1182:69-79).

[0006] Clinical trials have been conducted with Actimmune®, a recombinant form of IFNγ. Due to its pleiotropic effects, administration of IFNγ is associated with systemic and local toxicity, which is related to the range of adverse events reported during the execution of clinical trials and collected in the Actimmune® prescribing information (see HIGHLIGHTS OF PRESCRIBING INFORMATION. ACTIMMUNE® (Interferon γ-1b) injection, for subcutaneous use. Initial US approval: 1990. https: / / www.accessdata.fda.gov / drugsatfda_docs / label / 2015 / 103836s5182lbl.pdf). The most common adverse events are “flu-like,” such as fever, headache, chills, myalgia, or fatigue. However, a number of warnings and precautions are listed in the prescribing information, including hepatotoxicity, nephrotoxicity, and myelotoxicity, cardiovascular and neurological symptoms, and other adverse immune reactions with dose limitations for the approved clinical indication. Local toxicity has resulted in erythema or tenderness at the application site, reported in more than 20% of patients participating in clinical trials, including adverse events such as bleeding and pain at the injection site. Furthermore, local necrotizing lesions of the skin have been described with clinical use of IFNγ (see Krainick U, Kantarjian H, Broussard S, Talpaz M. Local cutaneous necrotizing lesions associated with interferon injections. J Interferon Cytokine Res. Oct 1998;18(10):823-7; and Wang F, Liu JH, Zhao YK, Luo DQ. Interferon-gamma-induced local leukocytoclastic vasculitis at the subcutaneous injection site. An Bras Dermatol. Sep-October 2016;91(5 Supplement 1):76-78). Summary of the Invention

[0007] This disclosure provides an antigen-binding molecule, optionally a separated antigen-binding molecule, capable of binding interferon-γ (IFNγ). The antigen-binding molecule may be an antibody.

[0008] The inventors have recognized that it is possible to utilize the pro-inflammatory / anti-tumorigenic properties of IFNγ while it binds to antigen-binding molecules to obtain various advantages, such as increasing the plasma concentration of endogenous IFNγ, and / or increasing the half-life of exogenously administered IFNγ, and / or targeting IFNγ to target cells or tissues, such as tumors or cancer cells or tissues.

[0009] Therefore, in a first aspect, the present invention relates to a multispecific (e.g., bispecific) antigen-binding molecule comprising an antigen-binding domain for binding interferon-γ (IFNγ) and an antigen-binding domain for binding target antigens other than IFNγ. In some embodiments, the antigen-binding molecule may be a multispecific or bispecific antibody.

[0010] The use of the targeted antibody / antigen binding molecule according to the present invention has many advantages compared to other methods used in the art. For example, it engages with the immune system by directly inducing inflammation in tumors; it utilizes endogenous IFNγ to reduce side effects compared to the administration of exogenous agents; and it is able to capture and prolong the local IFNγ burst generated by the tumor, thereby enhancing efficacy. The targeted antibody / antigen binding molecule has a unique three-stage mode of action, including binding to the body's own IFNγ; targeted delivery to cells (e.g., tumor cells); and immune stimulation, including, for example, attracting immune cells and upregulating MHC-1 to generate an immune response against the tumor.

[0011] In some embodiments, the binding of the molecule to IFNγ regulates cellular signaling downstream of the IFNγ receptor.

[0012] In one implementation, the binding of the molecule to IFNγ partially inhibits the pro-inflammatory activity of IFNγ. In some cases, the partial reduction of pro-inflammatory / anti-tumorigenic activity in combination with targeting target cells or tissues (e.g., using bispecific antibodies) can be beneficial in reducing side effects such as systemic inflammation, while accumulating pro-inflammatory / anti-tumorigenic activity at the target site (and thus benefiting from higher concentrations and / or affinity effects at the target site).

[0013] The inventors also unexpectedly discovered that antigen-binding molecules binding to IFNγ can bias the pro-inflammatory and anti-inflammatory activities of IFNγ towards pro-inflammatory activity. This could provide improved treatment for diseases and symptoms in which the pro-inflammatory and / or anti-tumorigenic activities of IFNγ are beneficial, such as in cancer treatment. Although IFNγ is generally considered pro-inflammatory, there is also evidence to support a dual role of IFNγ in inflammation, including activating some anti-inflammatory molecules, regulating the production of pro-inflammatory cytokines, activating apoptosis, and interfering with signal transduction mechanisms by inducing the inhibition of cytokine signaling (SOCS) (Mühl and Pfeilshifter, International Immunopharmacology, Vol. 3, No. 9, September 2003, pp. 1247-1255). (As used herein, the term “pro-inflammatory” is used interchangeably with “anti-tumorigenic”; and the term “anti-inflammatory” is used interchangeably with the term “pro-tumorigenic”.) In one embodiment, the binding of the molecule to IFNγ biases the pro-inflammatory and anti-inflammatory activities of IFNγ towards pro-inflammatory activity. For example, in some embodiments, the antigen-binding molecule may preferentially inhibit anti-inflammatory activity (i.e., it may inhibit anti-inflammatory activity while simultaneously inhibiting pro-inflammatory properties to a lower degree or not inhibiting pro-inflammatory activity at all). In some embodiments, the antigen-binding molecule may partially inhibit the pro-inflammatory activity of IFNγ while more strongly inhibiting its anti-inflammatory activity.

[0014] In some embodiments, the pro-inflammatory activity of cytokines can be measured by their ability to induce the expression of major histocompatibility complex class I (MHC-1) on the surface of cells (e.g., in vitro cells). In some embodiments, the anti-inflammatory activity of cytokines can be measured by their ability to induce the expression of programmed cell death ligand 1 (PD-L1) on the surface of cells (e.g., in vitro cells).

[0015] Therefore, in one embodiment, the molecule is capable of partially inhibiting the induction of MHC-1 expression (e.g., cell surface expression) and / or activity by IFNγ. Alternatively, in some embodiments, the binding of the molecule to IFNγ regulates downstream IFNγ-mediated cell signaling, such that the ratio of MHC-1 expression (e.g., cell surface expression) and / or activity to PD-L1 expression (e.g., cell surface expression) and / or activity is altered to favor MHC-1 expression and / or activity.

[0016] In a second aspect, this disclosure relates to antigen-binding molecules as described below. The antigen-binding molecules described below can bind IFNγ alone, or can bind IFNγ in addition to binding another antigen such as TAA. In some embodiments, any of the antigen-binding molecules described below can be present in multispecific antigen-binding molecules that are also capable of (independently) binding antigens other than interferon γ (target antigen), such as multispecific or bispecific antibodies. That is, in multispecific antigen-binding molecules comprising an antigen-binding domain that binds interferon γ (IFNγ) and an antigen-binding site that binds a target antigen other than IFNγ, as described in the first aspect and according to any embodiment of the embodiments further described herein, the antigen-binding domain that binds interferon γ (IFNγ) can be an antigen-binding domain as follows.

[0017] In one embodiment, the present invention relates to an antigen-binding molecule comprising an antigen-binding domain targeting IFNγ, wherein the antigen-binding site comprises a heavy chain variable region containing HC-CDR1, HC-CDR2, and HC-CDR3, wherein: a) The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 4 or a variant thereof in which one, two or three amino acids in HC-CDR1 are replaced by another amino acid; b) The heavy chain CDR2 contains the amino acid sequence of SEQ ID NO: 5 or a variant thereof in which one, two or three amino acids in HC-CDR2 are replaced by another amino acid; c) The heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 6 or a variant thereof in which one, two or three amino acids in HC-CDR3 are replaced by another amino acid; And / or wherein the antigen binding site comprises a light chain variable region containing LC-CDR1, LC-CDR2 and LC-CDR3, wherein: d) The light chain CDR1 contains the amino acid sequence of SEQ ID NO: 7 or a variant thereof in which one, two, or three amino acids in LC-CDR1 are replaced by another amino acid; e) The light chain CDR2 contains the amino acid sequence of SEQ ID NO: 8 or a variant thereof in which one, two or three amino acids in LC-CDR2 are replaced by another amino acid; f) The light chain CDR3 contains the amino acid sequence of SEQ ID NO: 9 or a variant thereof in which one, two or three amino acids in LC-CDR3 are replaced by another amino acid.

[0018] In some embodiments, the heavy chain CDR1 comprises a variant of SEQ ID NO: 4 with a substitution at position 30 according to the IMGT scheme number (i.e., GYTXTNYY (SEQ ID NO: 84), where X can be any amino acid), wherein the residue at that position is preferably D, E, N, or Q, most preferably E. For example, the heavy chain CDR1 can be GYTETNYY (SEQ ID NO: 85). IMGT position 30 corresponds to Kabat position 29.

[0019] In another embodiment, the heavy chain CDR2 comprises a variant of SEQ ID NO: 5 with a substitution at position 63 according to the IMGT scheme number (i.e., INPSNXGT (SEQ ID NO: 86), where X can be any amino acid and preferably not D), wherein the residue at this position is preferably selected from E, H, and Q, and most preferably H. Removing D at this position eliminates a potential isomerization site because the residue forms a potential isomerization sequence defect for DG. For example, the heavy chain CDR2 could be INPSNHGT (SEQ ID NO: 87). IMGT position 63 corresponds to Kabat position 55, and this variant is sometimes referred to herein (e.g., in the sequence listing) as the “D55H” variant.

[0020] In another embodiment, the light chain CDR1 comprises a variant of SEQ ID NO: 7 with substitutions at one or both positions 29 and 35 according to the IMGT scheme number (i.e., QSXLYSSNXKNY (SEQ ID NO: 88), where X can be any amino acid), wherein the residue at position 29 is preferably selected from G, A, V, L, or I, and most preferably V, and / or the residue at position 35 is preferably selected from D, E, N, or Q, and most preferably N. For example, LC-CDR1 can be QSVLYSSNNKNY (SEQ ID NO: 89). IMGT positions 29 and 35 correspond to Kabat positions 27B and 29, respectively.

[0021] In some embodiments, the antigen-binding molecule comprises a VH region having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 10, optionally at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or a VL region having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 11, optionally at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0022] An exemplary antigen-binding molecule may have a VH region and a VL region, wherein the VH region has a sequence selected from SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112 and SEQ ID NO: 113 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with such sequences, and the VL region has a sequence selected from SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 and SEQ ID NO: 118 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with such sequences.

[0023] In one embodiment, the humanized antibody / antigen binding molecule may have a VH region and a VL region, wherein the VH region has the sequence of SEQ ID NO: 109 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with it, and the VL region has the sequence of SEQ ID NO: 116 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with it. In another embodiment, the humanized antibody / antigen binding molecule may have a VH region and a VL region, wherein the VH region has the sequence of SEQ ID NO: 110 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with it, and the VL region has the sequence of SEQ ID NO: 117 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with it. In another embodiment, the humanized antibody / antigen binding molecule may have a VH region and a VL region, wherein the VH region has the sequence of SEQ ID NO: 111 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with it, and the VL region has the sequence of SEQ ID NO: 118 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with it. In another embodiment, the humanized antibody / antigen binding molecule may have a VH region and a VL region, wherein the VH region has the sequence of SEQ ID NO: 110 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with it, and the VL region has the sequence of SEQ ID NO: 118 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with it. In another embodiment, the humanized antibody / antigen binding molecule may have a VH region and a VL region, wherein the VH region has the sequence of SEQ ID NO: 111 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with it, and the VL region has the sequence of SEQ ID NO: 117 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with it.In another embodiment, the humanized antibody / antigen binding molecule may have a VH region and a VL region, wherein the VH region has the sequence of SEQ ID NO: 112 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with it, and the VL region has the sequence of SEQ ID NO: 117 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with it. In another embodiment, the humanized antibody / antigen binding molecule may have a VH region and a VL region, wherein the VH region has the sequence of SEQ ID NO: 112 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with it, and the VL region has the sequence of SEQ ID NO: 118 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with it. In another embodiment, the humanized antibody / antigen binding molecule may have a VH region and a VL region, wherein the VH region has the sequence of SEQ ID NO: 113 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with it, and the VL region has the sequence of SEQ ID NO: 117 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with it. In another embodiment, the humanized antibody / antigen binding molecule may have a VH region and a VL region, wherein the VH region has the sequence of SEQ ID NO: 113 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with it, and the VL region has the sequence of SEQ ID NO: 118 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with it.

[0024] In another embodiment, the present invention relates to an antigen-binding molecule comprising an antigen-binding domain targeting IFNγ, wherein the antigen-binding site comprises a heavy chain variable region containing HC-CDR1, HC-CDR2, and HC-CDR3, wherein: a) The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 12 or a variant thereof in which one, two or three amino acids in HC-CDR1 are replaced by another amino acid; b) The heavy chain CDR2 contains the amino acid sequence of SEQ ID NO: 13 or a variant thereof in which one, two or three amino acids in HC-CDR2 are replaced by another amino acid; c) The heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 14 or its variants in which one, two or three amino acids in HC-CDR3 are replaced by another amino acid; And / or wherein the antigen binding site comprises a light chain variable region containing LC-CDR1, LC-CDR2 and LC-CDR3, wherein: d) The light chain CDR1 contains the amino acid sequence of SEQ ID NO: 15 or a variant thereof in which one, two, or three amino acids in LC-CDR1 are replaced by another amino acid; e) The light chain CDR2 contains the amino acid sequence of SEQ ID NO: 16 or a variant thereof in which one, two or three amino acids in LC-CDR2 are replaced by another amino acid; f) The light chain CDR3 contains the amino acid sequence of SEQ ID NO: 17 or a variant thereof in which one, two or three amino acids in LC-CDR3 are replaced by another amino acid.

[0025] In some embodiments, the antibody / antigen binding molecule comprises a VH region having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 18, optionally at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or a VL region having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 19, optionally at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0026] In another embodiment, the present invention relates to an antigen-binding molecule comprising an antigen-binding domain targeting IFNγ, wherein the antigen-binding site comprises a heavy chain variable region containing HC-CDR1, HC-CDR2, and HC-CDR3, wherein: a) The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 20 or a variant thereof in which one, two or three amino acids in HC-CDR1 are replaced by another amino acid; b) The heavy chain CDR2 contains the amino acid sequence of SEQ ID NO: 21 or a variant thereof in which one, two or three amino acids in HC-CDR2 are replaced by another amino acid; c) The heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 22 or a variant thereof in which one, two or three amino acids in HC-CDR3 are replaced by another amino acid; And / or wherein the antigen binding site comprises a light chain variable region containing LC-CDR1, LC-CDR2 and LC-CDR3, wherein: d) The light chain CDR1 contains the amino acid sequence of SEQ ID NO: 23 or a variant thereof in which one, two, or three amino acids in LC-CDR1 are replaced by another amino acid; e) The light chain CDR2 contains the amino acid sequence of SEQ ID NO: 24 or a variant thereof in which one, two or three amino acids in LC-CDR2 are replaced by another amino acid; f) The light chain CDR3 contains the amino acid sequence of SEQ ID NO: 25 or a variant thereof in which one, two or three amino acids in LC-CDR3 are replaced by another amino acid.

[0027] In some embodiments, the antibody / antigen binding molecule comprises a VH region having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 26, optionally at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or a VL region having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 27, optionally at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0028] In another embodiment, the present invention relates to an antigen-binding molecule comprising an antigen-binding domain targeting IFNγ, wherein the antigen-binding site comprises a heavy chain variable region containing HC-CDR1, HC-CDR2, and HC-CDR3, wherein: a) The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 28 or a variant thereof in which one, two or three amino acids in HC-CDR1 are replaced by another amino acid; b) The heavy chain CDR2 contains the amino acid sequence of SEQ ID NO: 29 or a variant thereof in which one, two or three amino acids in HC-CDR2 are replaced by another amino acid; c) The heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 30 or a variant thereof in which one, two or three amino acids in HC-CDR3 are replaced by another amino acid; And / or wherein the antigen binding site comprises a light chain variable region containing LC-CDR1, LC-CDR2 and LC-CDR3, wherein: d) The light chain CDR1 contains the amino acid sequence of SEQ ID NO: 31 or a variant thereof in which one, two or three amino acids in LC-CDR1 are replaced by another amino acid; e) The light chain CDR2 contains the amino acid sequence of SEQ ID NO: 32 or a variant thereof in which one, two or three amino acids in LC-CDR2 are replaced by another amino acid; f) The light chain CDR3 contains the amino acid sequence of SEQ ID NO: 33 or its variants in which one, two or three amino acids in LC-CDR3 are replaced by another amino acid.

[0029] In some embodiments, the antibody / antigen binding molecule comprises a VH region having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 34, optionally at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or a VL region having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 35, optionally at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0030] In one embodiment, the present invention relates to an antigen-binding molecule comprising an antigen-binding domain targeting IFNγ, wherein the antigen-binding site comprises a heavy chain variable region containing HC-CDR1, HC-CDR2, and HC-CDR3, wherein: a) The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 36 or a variant thereof in which one, two or three amino acids in HC-CDR1 are replaced by another amino acid; b) The heavy chain CDR2 contains the amino acid sequence of SEQ ID NO: 37 or a variant thereof in which one, two or three amino acids in HC-CDR2 are replaced by another amino acid; c) The heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 38 or a variant thereof in which one, two or three amino acids in HC-CDR3 are replaced by another amino acid; And / or wherein the antigen binding site comprises a light chain variable region containing LC-CDR1, LC-CDR2 and LC-CDR3, wherein: d) The light chain CDR1 contains the amino acid sequence of SEQ ID NO: 39 or a variant thereof in which one, two, or three amino acids in LC-CDR1 are replaced by another amino acid; e) The light chain CDR2 contains the amino acid sequence of SEQ ID NO: 40 or a variant thereof in which one, two or three amino acids in LC-CDR2 are replaced by another amino acid; f) The light chain CDR3 contains the amino acid sequence of SEQ ID NO: 41 or a variant thereof in which one, two or three amino acids in LC-CDR3 are replaced by another amino acid.

[0031] In some embodiments, the antibody / antigen binding molecule comprises a VH region having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 42, optionally at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or a VL region having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 43, optionally at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0032] Antigen-binding molecules can possess any of the functional properties described above. For example, it can be able to bind IFNγ and bias the pro-inflammatory and anti-inflammatory activities of IFNγ towards pro-inflammatory activity, and / or it can be non-inhibitory or partially inhibitory of the pro-inflammatory activity of IFNγ. It can be able to bind interferon-γ (IFNγ) and modulate downstream IFNγ cell signaling such that the ratio of IFNγ-induced PD-L1 expression (e.g., cell surface expression) and / or activity to MHC-1 expression (e.g., cell surface expression) and / or activity is altered to favor MHC-1 expression and / or activity, and / or it can be non-inhibitory or partially inhibitory of IFNγ-induced MHC-1 expression (e.g., cell surface expression) or activity.

[0033] In a third aspect, the present invention relates to an antigen-binding molecule comprising an antigen-binding domain targeting IFNγ, the antigen-binding domain binding to the same IFNγ epitope as one of the aforementioned antibodies, for example, binding to the same epitope as an antibody having the VH and VL sequences as described above. In some embodiments, according to any embodiment of the above embodiments and according to any embodiment of the embodiments further described herein, the antigen-binding molecule may be a multispecific or bispecific antigen-binding molecule.

[0034] In a fourth aspect, the present invention provides an antigen-binding molecule that binds to interferon-γ (IFNγ), wherein the binding of the molecule to IFNγ biases the pro-inflammatory and anti-inflammatory activities of IFNγ toward pro-inflammatory activity. For example, in some embodiments, the antigen-binding molecule may preferentially inhibit anti-inflammatory activity (i.e., it may inhibit anti-inflammatory activity while inhibiting pro-inflammatory properties to a low degree or not inhibiting pro-inflammatory activity at all). In some embodiments, the antigen-binding molecule may partially inhibit the pro-inflammatory activity of IFNγ while more strongly inhibiting the anti-inflammatory activity of IFNγ. Optionally, the antibody is human or humanized.

[0035] As described above, the pro-inflammatory activity of cytokines can be measured by their ability to induce the expression of major histocompatibility complex class I (MHC-1) on the surface of cells (e.g., in vitro cells). In some embodiments, the anti-inflammatory activity of cytokines can be measured by their ability to induce the expression of programmed cell death ligand 1 (PD-L1) on the surface of cells (e.g., in vitro cells). Therefore, the present invention can provide an antigen-binding molecule, optionally an antibody, capable of binding interferon-γ (IFNγ) and modulating downstream IFNγ cell signaling such that the ratio of IFNγ-induced PD-L1 expression (e.g., cell surface expression) and / or activity to MHC-1 expression and / or activity is altered to favor MHC-1 expression (e.g., cell surface expression) and / or activity.

[0036] In any aspect or embodiment described herein, an antigen-binding molecule capable of binding interferon-γ (alone, or in addition to binding another antigen such as TAA) may comprise an antibody Fc region and / or may be modified by conjugation to another half-life-extending portion. This can result in the antigen-binding molecule having a significantly extended half-life, thereby extending the half-life of the antigen bound by the molecule.

[0037] In any of the foregoing aspects and implementation methods, the antibody may be human or humanized.

[0038] In another aspect, the present invention relates to a pharmaceutical composition comprising any of the antigen-binding molecules as described herein.

[0039] In another aspect, this disclosure relates to a method of treating a disease or condition in which the pro-inflammatory and / or anti-tumorigenic activity of IFNγ is beneficial, comprising administering to a subject an antigen-binding molecule or pharmaceutical composition of any of the foregoing aspects or embodiments. In another aspect, this disclosure relates to a method of treating cancer in a subject in need, comprising administering to the subject an antigen-binding molecule or pharmaceutical composition of any of the foregoing aspects or embodiments.

[0040] This treatment can be combined with one or more other anticancer therapies, as further discussed herein. In some embodiments, the treatment can be combined with one or more checkpoint inhibitors known in the art. Methods for sensitizing a subject with cancer to checkpoint inhibitor treatment are also disclosed, comprising administering to the subject a composition containing an antigen-binding molecule or pharmaceutical composition disclosed herein.

[0041] This disclosure also relates to antigen-binding molecules or pharmaceutical compositions of any of the foregoing aspects or embodiments for use in treatment methods, such as any treatment method described herein. For example, in one aspect, the invention relates to an antigen-binding molecule as described above for use in treating a condition in which pro-inflammatory and / or anti-tumorigenic activity of IFNγ is beneficial. More particularly, antigen-binding molecules or pharmaceutical compositions of any of the foregoing aspects or embodiments are provided for use in methods of treating cancer. Antigen-binding molecules or pharmaceutical compositions of any of the foregoing aspects or embodiments are also disclosed for use in methods of sensitizing a subject with cancer to checkpoint inhibitor therapy. Antigen-binding molecules or pharmaceutical compositions of any of the foregoing aspects or embodiments are also disclosed for use in methods of treating cancer, the methods further comprising administering one or more additional anticancer therapies. In some embodiments, the one or more additional anticancer therapies may include checkpoint inhibitors. Antigen-binding molecules or pharmaceutical compositions of any of the foregoing aspects or embodiments, as well as one or more additional anticancer therapies, are also disclosed for use in methods of treating cancer. In one embodiment, the one or more additional anticancer therapies may include checkpoint inhibitors.

[0042] The use of any of the foregoing aspects or embodiments of the antigen-binding molecule or pharmaceutical composition in the manufacture of a medicament for treating diseases or conditions (e.g., cancer) as described herein is also provided. Cancer treatment may be combined with one or more other anticancer therapies, as further discussed herein.

[0043] The present invention includes combinations of the described aspects and preferred features, except where such combinations are explicitly not permitted or should be explicitly avoided. Attached Figure Description

[0044] The embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying drawings, in which: Figure 1 A to Figure 1 E is a graph illustrating the binding of the exemplary bivalent anti-IFNγ antibody of this disclosure to human recombinant IFNγ (hrIFNγ). Biacore sensor plots and fitted curves were used to determine the binding of the exemplary antibody 4F4 ( Figure 1 A) 3G1 ( Figure 1 B), 16D2 ( Figure 1 C), 9F1 Figure 1 D) and 2C1 ( Figure 1 E) Binding affinity with hrIFNγ. The sensor plot shows the binding curves of hrIFNγ and mAb at two different dilution series of hrIFNγ (lower curve: 1.11 to 10 nM series, upper curve: 10 to 90 nM series), which were fitted together (black line) to calculate the affinity. The affinity values ​​in nM are shown on the right side of the plot.

[0045] Figure 1 F illustrates the ability of anti-IFNγ antibodies to inhibit IFNγ-mediated heterotetramerization of the IFNγR1 and IFNγR2 subunits, thereby neutralizing the IFNγ response. Commercial neutralizing antibodies and non-neutralizing antibodies are shown for reference. Figure 1 G illustrates the ability of a truncated monovalent form of antibody 4F4 prepared via Duobody technology (4F4-VH(K409R) (SEQ ID NO: 75): Truncated Fc-monovalent mAb IgG1.1 Fc(H-CH2-CH3)(F405L) (SEQ ID NO: 83) to increase endogenous IFNγ plasma concentrations over time following a single intravenous administration to cynomolgus monkeys. Mean values ​​(including SEM) for two cynomolgus monkeys at each time point are shown.

[0046] Figure 2 A to Figure 2 E shows the IFNγ antibody (4F4) of this disclosure preconjugated with 1 ng / mL (0.06 nM) recombinant IFNγ. Figure 2 A) 3G1 ( Figure 2 B), 16D2 ( Figure 2 C), 9F1 Figure 2 D) and 2C1 ( Figure 2 Normalized surface expression of major histocompatibility complex class I (MHC-1) and programmed death-ligand 1 (PD-L1) proteins on PC-3 prostate cancer cells treated with E)). Surface expression levels of MHC-1 and PD-L1 were normalized to the surface expression levels of corresponding control cells treated with only 1 ng / mL (0.06 nM) IFNγ. Figure 2 F and Figure 2 A to Figure 2 Similarly, normalized surface expression of MHC-1 and PD-L1 proteins on A549 lung cancer cells pre-conjugated with 1 ng / mL (0.06 nM) recombinant IFNγ is shown. The surface expression levels of MHC-1 and PD-L1 were normalized against the surface expression levels of corresponding control cells treated only with 1 ng / mL (0.06 nM) IFNγ. Figure 2 The left panel (G) is a bar chart showing the dependence of MHC-1 surface expression on IFNγ concentration in the presence or absence of 50 nM (7,300 ng / mL) 4F4 antibody. Figure 2 (G, left figure). Figure 2 The right side of the graph (G) is a bar chart showing the dependence of PD-L1 surface expression on IFNγ concentration in the presence or absence of 50 nM (7,300 ng / mL) 4F4 antibody. Figure 2 (G, right figure).

[0047] Figure 3 The binding saturation curves of LDC-1 with PSMA-positive LNCaP cells and LDC-2, LDC-3, LDC-4, and LDC-5 with PSMA-transfected PC-3 cells, obtained by flow cytometry, are shown. LDC-1, LDC-2, LDC-3, LDC-4, and LDC-5 are bispecific forms of certain antibodies disclosed herein and also possess a PSMA-binding domain, as described in more detail in Example 7. The binding affinity of each LDC in nM is shown in the lower right corner of each graph.

[0048] Figure 4 A shows the flow cytometry results of IFNγ binding to PC-3 cells (PSMA negative) and LNCaP (PSMA positive) with and without LDC-1 pre-incubation. IFNγ binding to cells was detected using an anti-IFNγ antibody. Figure 4 B shows the binding of fluorescently labeled IFNγ to GFP-PSMA-positive or negative PC-3 cells pre-incubated with increasing concentrations of LDC-2. Figure 4 C shows the expression levels (mean fluorescence intensity, MFI) of MHC-1 and PD-L1 in GFP-PSMA-positive or negative PC-3 cells stimulated for 48 hours with 0.025 nM IFNγ alone or in combination with 0.1, 1, 10, or 100 nM LDC-4 or LDC-6. The expression levels of MHC-1 and PD-L1 in GFP-PSMA-expressing and PSMA-negative PC-3 cells were measured using an Attune flow cytometry apparatus.

[0049] Figure 5 This study demonstrates that IFNγ binding to KiH-3 and KiH-6 preserves pro-inflammatory signaling across different cell lines. Normalized surface expression of major histocompatibility complex class I (MHC-1) and programmed death-ligand 1 (PD-L1) proteins, as assessed by flow cytometry, is shown. HT29 colorectal cancer cells were treated with the bispecific antibodies (KiH-3 or KiH-6) pre-conjugated with 1 ng / mL (0.06 nM) of recombinant IFNγ. Figure 5 A) PC3-FLU prostate cancer cells ( Figure 5 B) and A549 lung cancer cells ( Figure 5 C). Surface expression levels of MHC-1 and PD-L1 were normalized against the surface expression levels of corresponding control cells treated with only 1 ng / mL (0.06 nM) IFNγ. A549 cells were also treated with an IFNγ neutralizing control antibody (MT111W) pre-conjugated with 1 ng / mL (0.06 nM) recombinant IFNγ. Figure 5 C). Indicates the IC50 range of the MT111W (gray bar).

[0050] Figure 6 PC3 PIP cells expressing PSMA, as determined by flow cytometry at 6, 24, 48, and 72 hours, are shown. Figure 6 AB) or A549 cells that do not express PSMA ( Figure 6 MHC-1 and PD-L1 expression levels (mean fluorescence intensity, MFI) in CD). Cells were treated with a single bispecific antibody of this disclosure (KiH-3 or KiH-6), a single human IFNγ (0.1 nM), or a bispecific antibody of this disclosure preconjugated with human IFNγ (0.1 nM). An IFNγ neutralizing antibody MT111W preconjugated with human IFNγ (0.1 nM) was used as a positive control.

[0051] Figure 7 Flow cytometry results are shown for PC3-PIP cells (expressing PSMA) stimulated with IFNγ (0.025 nM or 5.95 nM), and stimulated with 1 nM or 10 nM of the bispecific antibody KiH-3 with or without IFNγ stimulation. MHC-1 ( Figure 7 A) and PD-L1 ( Figure 7 C) Expression level (mean fluorescence intensity, MFI), and for MHC-I+ ( Figure 7 B) and PD-L1+ ( Figure 7The percentage of cells in D).

[0052] Figure 8 This demonstrates the targeted delivery of IFNγ PSMA to tumors using KIH-3. Figure 8 A is a schematic diagram of a PC-3 prostate cancer xenograft tumor model expressing PSMA. Tumors containing PC-3 prostate cancer cells were established in mice by subcutaneous injection of cells on day 0. On day 21, mice were intravenously injected with the bispecific antibody (KiH-3) disclosed herein, with or without injection of hIFNγ. On day 24, tumor resection was performed. Figure 8 B illustrates the use of staining with anti-IFNγ antibody. Figure 8 Immunohistochemical staining of paraffin-embedded sections obtained by the method shown in Figure A. Figure 8 C shows the results of an in vivo imaging experiment in which mice containing prostate cancer xenograft tumors were intravenously injected with an Alexa680-labeled KiH-3 antibody and imaged using a far-infrared camera.

[0053] Figure 9 This study demonstrates that KIH-3 activates IFNγ receptors and upregulates MHC-I in cold prostate cancer. Figure 9 A is a schematic diagram of a PC-3 prostate cancer xenograft tumor model expressing PSMA. Tumors containing PC-3 prostate cancer cells were established in mice by subcutaneous injection of cells on day 0. On day 21, mice were intravenously injected with the bispecific antibody (KiH-3) disclosed herein, with or without injection of hIFNγ (or a mediator control). On day 24, tumor resection was performed. Figure 9 B illustrates the use of staining with anti-MHC-I antibody. Figure 9 Immunohistochemical staining of paraffin-embedded sections obtained by the method shown in Figure A.

[0054] Figure 10 and Figure 11 The results of pharmacokinetic studies of the KiH-16 antibody disclosed herein are shown. Mice were administered 1, 3, or 10 mg / kg KiH-16 (intravenous) or 10 mg / kg KiH-16 (intraperitoneal), and blood samples were collected at a series of time points after injection. The concentrations of KiH-16 and mIFN-γ in the blood samples were measured by ELISA. Figure 10 This demonstrates the detection of serum levels measured in μg / mL by binding to immobilized human PSMA protein and using HRP goat anti-mouse IgG2a heavy chain (Abcam, catalog number ab97245). Figure 10 A) and uM ( Figure 10 B) The KiH-16 concentration. Figure 11The accumulation of mIFN-γ in serum samples after KiH-16 administration was shown. mIFN-γ was detected using the detection antibody #ab14-Biotin (1 μg / mL, Biocytogen). Figure 11 A). Figure 11 B shows the KiH-16 detected in serum ( Figure 11 A) and mIFN-γ detected in serum ( Figure 11 A) molar ratio.

[0055] Figure 12 The results of assays determining the optimal conditions for mIFN-γ stimulation of B-hPSMA MC38 cells are shown. Cells were stimulated with mIFN-γ at concentrations of 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 250, and 500 ng / mL for 24 or 48 hours, and the surface expression levels of MHC-I and PD-L1 were measured by flow cytometry. 5 ng / mL mIFN-γ is labeled (black vertical line).

[0056] Figure 13 The effect of KIH-16 in not neutralizing IFNγ was demonstrated. B-hPSMA MC38 cells were stimulated for 48 hours with 5 ng / mL mIFNγ premixed with a range of concentrations (0.9, 9, 90, 900 nM) of the bispecific antibody (KIH-16) of this disclosure, the non-neutralizing anti-mIFN-γ antibody AN-18, or the neutralizing anti-mIFN-γ antibody XMG1.2. Cells were analyzed by flow cytometry after stimulation. Figure 13 AB shows mCD274 + ( Figure 13 A) and mMHC-I + ( Figure 13 The percentage of cells in B). Figure 13 CD shows PD-L1 ( Figure 13 C) and MHC-1 ( Figure 13 D) Surface expression level (mean fluorescence intensity, MFI).

[0057] Figure 14 This study demonstrates the potential for mIFN-γ accumulation and immune infiltration of the KiH-16 antibody in a syngeneic mouse model. Tumors comprising MC38 cells (non-hPSMA-expressing) were established in mice via subcutaneous cell injection. Mice were administered anti-mouse PD-1 (1 mg / kg) via intraperitoneal injection twice weekly, with or without KiH-16 (1 mg / kg), and blood was collected 24 hours post-injection following the 1st, 3rd, 5th, and 7th (final) administrations. Blood mIFN-γ levels were detected by ELISA using the anti-mIFN-γ antibody. Figure 14A). Figure 14 BC shows the results of flow cytometry analysis of tumor tissue taken from the study endpoint. It shows the surface expression of MHC-I in the tumor samples. Figure 14 B) and the percentage of mCD45+ cells ( Figure 14 C).

[0058] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. Generally, the nomenclature and techniques used in conjunction with the immunology, oncology, cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization described herein are those known and commonly used in the art. It should be understood that the foregoing general description and the following detailed description are exemplary and interpretive only and do not limit any of the claimed subject matter. The section headings used herein are for organizational purposes only and are not construed as limiting the subject matter described.

[0059] As used herein, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include the plural.

[0060] As used herein, unless the context clearly indicates otherwise, all numerical values ​​or ranges include integers that fall within or contain such ranges, as well as fractions of values ​​or integers that fall within or contain such ranges. Thus, for example, references to the range 90%–100% include 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., and 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so on.

[0061] As used in this article, “approximately” means a range that includes the value and extends from 10% below the value to 10% above the value. “Approximately” range refers to a range that extends from 10% below the lower limit of the range to 10% above the upper limit of the range.

[0062] An 'antigen-binding molecule' is a molecule that binds to a given target antigen. Antigen-binding molecules include antibodies (i.e., immunoglobulins (Ig)) and their antigen-binding fragments. An antigen-binding molecule capable of binding to a given target antigen can also be described as an antigen-binding molecule that binds to a given target antigen.

[0063] The antigen-binding molecules according to this disclosure also include antibody-derived molecules, such as molecules comprising an antigen-binding region / domain derived from an antibody. Antibody-derived antigen-binding molecules may comprise an antigen-binding region / domain comprising or consisting of an antigen-binding region / domain of an antibody (e.g., an antigen-binding fragment of an antibody). In some embodiments, the antigen-binding region / domain of the antibody-derived antigen-binding molecule may be an Fv (e.g., provided in scFv form) or Fab region of the antibody, or the entire antibody, or comprising an Fv or Fab region of the antibody, or the entire antibody. The antigen-binding molecules according to this disclosure also include multispecific antigen-binding molecules.

[0064] The antigen-binding molecules disclosed herein comprise one or more portions capable of binding to a target antigen. In some embodiments, the portion capable of binding to the target antigen comprises an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) of an antibody capable of specifically binding to the target antigen. In some embodiments, the portion capable of binding to the target antigen comprises or is composed of an aptamer capable of binding to the target antigen, such as a nucleic acid aptamer (e.g., reviewed in Zhou and Rossi Nat Rev DrugDiscov. 2017 16(3):181-202). In some implementations, molecules capable of binding to the target antigen comprise or are composed of antigen-binding peptides / peptides, such as aptamers, thioredoxins, monomers, anticarrier proteins, Kunitz domains, avimers, knottins, fynomers, atrimers, DARPins, affinity molecules, nanobodies (i.e., single-domain antibodies (sdAbs), which may include, for example, “camelized” human VH, or modified to serve as a single-domain antibody-stabilized human VH domain), affilins, armadillo repeat proteins (ArmRPs), type O antibodies, or fibronectins – as reviewed, for example, in Reverdatto et al., Curr TopMed Chem. 2015; 15(12): 1082–1101, which is hereby incorporated in its entirety by reference (see also, for example, Boersma et al., J Biol Chem (2011) 286:41273-85 and Emanuel et al., Mabs (2011) 3:38-48).

[0065] As used herein, “antibody” is used in the broadest sense and encompasses a wide variety of antibody structures, provided they exhibit the desired antigen-binding activity. The term “antibody” includes, but is not limited to, monoclonal antibodies, monospecific antibodies, monovalent antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, chimeric antibodies, synthetic antibodies, recombinant antibodies, hybrid antibodies, mutant antibodies, transplanted antibodies, antibody fragments, and in vitro generated antibodies with antigen-binding activity. The term also includes heavy-chain-only antibodies, such as those containing VHH fragments or nanobodies, including human VH domains modified to act as single-domain antibody stabilizers. The term also includes antibody conjugates (e.g., antibodies conjugated to a half-life-extending portion, such as a fatty acid) that have advantageous properties compared to unconjugated antibodies. In some embodiments, antibodies may have variable and constant domains in each of the heavy and light chains. Thus, an antibody may have a heavy-chain variable domain (VH) and a light-chain variable domain (VL), which together form the antibody moiety that binds to the antigen, sometimes referred to as an “antigen receptor.” Within each variable domain are three complementarity-determining regions (CDRs), which form loops in the VH and VL and contact the surface of the antigen.

[0066] The terms “antigen-binding portion of an antibody,” “antigen-binding fragment,” “antigen-binding domain,” and “antibody fragment” are used interchangeably herein to refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of antibody fragments include, but are not limited to, Fv, Fab, cross-Fab, Fab', Fab'-SH, F(ab')2; biantibodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single-domain antibodies (dAbs) (also known as nanobodies); and multispecific antibodies formed from antibody fragments. Also included are antibodies comprising one or more (e.g., 1, 2, or more) single-chain variable fragments (scFvs). The term “Fab fragment” refers to a protein consisting of the VH and CH1 domains of the heavy chain of an immunoglobulin and the VL and CL domains of the light chain. As used herein, references to “Fab fragment” are intended to include cross-Fab fragments or scFab as well as conventional Fab fragments (i.e., Fab fragments comprising a light chain containing the VL and CL domains and a heavy chain containing the VH and CH1 domains). The “Fab” fragment differs from the Fab fragment in that it has a residue added to the carboxyl terminus of the CH1 domain, including one or more cysteine ​​residues from the antibody hinge region.

[0067] The terms "cross-Fab fragment," "xFab fragment," or "cross-type Fab fragment" refer to Fab fragments in which the variable or constant regions of the heavy and light chains are exchanged. Cross-Fab fragments comprise polypeptide chains consisting of a light chain variable region (VL) and a heavy chain constant region 1 (CH1), and polypeptide chains consisting of a heavy chain variable region (VH) and a light chain constant region (CL). For clarity, in cross-type Fab molecules in which the variable regions of the Fab light and Fab heavy chains are exchanged, the peptide chain containing the heavy chain constant region is referred to herein as the "heavy chain" of the cross-Fab molecule. Conversely, in cross-type Fab molecules in which the constant regions of the Fab light and Fab heavy chains are exchanged, the peptide chain containing the heavy chain variable region is referred to herein as the "heavy chain" of the cross-Fab molecule.

[0068] “Affinity” refers to the strength of the sum 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 stated, as used herein, “binding affinity” refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., complementary sites and epitopes). The affinity of molecule X for its partner Y can generally be determined by the dissociation constant (K0). D K represents, and unless otherwise stated, K as used herein. D The value refers to intrinsic affinity. The binding affinity of an antibody or antigen-binding fragment is measured, for example, by surface plasmon resonance (SPR) measurement, ELISA, AlphaLisa assay, flow cytometry, and other conventional methods. In one embodiment, binding affinity is measured using the BIACORE® surface plasmon resonance assay, such as the BIACORE surface plasmon resonance assay with captured antibodies, at, for example, 25°C and, for example, at pH 7.4, optionally as described in detail in Example 2 for single-kinetic or multi-kinetic analysis. In another embodiment, the conditions for the BIACORE® assay may be as described in Example 19.

[0069] As used herein, a "complementarity-determining region" or "CDR" or "hypervariant region" refers to a portion of an antibody's variable domain that determines the antibody's binding specificity to its specific antigen. As mentioned above, a single variable region of an antibody peptide typically contains three CDRs, usually named CDR1, CDR2, and CDR3. More specifically, heavy chain variable regions may contain CDRs named H1, H2, and H3; similarly, light chain variable regions may contain CDRs named L1, L2, and L3. Various methods can be used to define CDRs. Existing techniques utilize various numbering schemes with different definitions of CDR length and position. For example, the Kabat numbering scheme is based on sequence alignment and uses a "variability parameter" (the number of different amino acids at a given position divided by the frequency of the most frequent amino acid at that position) to predict CDRs. On the other hand, the Chothia numbering scheme is a structure-based numbering scheme in which antibody crystal structures are aligned to define loop structures as CDRs. The Martin numbering scheme focuses on the structural alignment of different frame regions of unconventional lengths. The IMGT numbering scheme is a standardized numbering system based on sequence alignments from a complete reference gene database encompassing the entire immunoglobulin superfamily. Honneger's numbering scheme (AHo) is based on structural alignments of the 3D structure of variable regions and uses structurally conserved Cα positions to infer frame and CDR length. Those skilled in the art will note that the definition of a CDR will vary depending on the method used. The sequences disclosed herein consider any method for defining a CDR. CDRs as described herein have been determined using the IMGT scheme, but in other embodiments, CDRs determined by another scheme as described above may be substituted.

[0070] An antibody's "epitope" is a specific site on an antigen that the antibody binds to. An epitope can be a linear epitope consisting of a continuous sequence of amino acids (i.e., a primary amino acid sequence). In some embodiments, the antigen-binding molecule can bind a conformational epitope of IFNγ, which consists of a discontinuous sequence of amino acids in the antigen. The region of a given target molecule that the antigen-binding molecule binds to can be determined by those skilled in the art using a variety of methods well known in the art, including X-ray cocrystallography of antibody-antigen complexes, peptide scanning, mutagenesis mapping, hydrogen-deuterium exchange analysis by mass spectrometry, phage display, competitive ELISA, and proteolysis-based "protection" methods. Such methods are described, for example, in Gershoni et al., BioDrugs, 2007, 21(3):145-156, which are hereby incorporated herein by reference in their entirety. If an antibody cross-blocks with a reference antibody, for example, if the test antibody inhibits the binding of the reference antibody to the antigen, and the reference antibody inhibits the binding of the test antibody to the antigen, then the antibody and the reference antibody can be considered to bind to the "same epitope". In each case, the blockade can be at least 50%. In some embodiments, in the case of linear epitopes, an antibody may bind to the “same epitope” as a reference antibody, which binds to the same fragment of the antigen, said fragment consisting of 20 or fewer amino acids, optionally 15 or fewer amino acids, or 14, 13, 12, 11, or 10 residues or fewer, and does not specifically bind to different fragments that do not overlap with it; alternatively, using peptide scanning, an epitope can be identified by a set of peptide fragments of the antigen that the antibody specifically binds to, and if two antibodies bind to a set of peptide fragments consisting of such fragments that completely overlap when the two sets are compared or include no more than 4, 3, 2, or 1 non-overlapping residue, then the two antibodies can be considered to bind to the same epitope. In some embodiments, peptides of various lengths can be screened to identify the smallest fragment that can specifically bind to a reference antibody, and it can be determined whether another antibody binds to the same fragment. If desired, such fragments can also be used as immunogens to obtain antibodies that bind to the same epitope. In the case of nonlinear epitopes, in some embodiments, if the antibody and the reference antibody share the same set of key contact residues in the antigen, the antibody and the reference antibody can be considered to bind to the "same epitope," wherein key contact residues are defined as residues in the crystal structure that are 4.5 Å or less apart from the antigen residues, or are defined as residues in the crystal structure that are 4.5 Å or less apart from the antigen residues. andMutations (e.g., mutations to alanine) eliminate the binding residues. In some embodiments, two epitopes are considered identical when they have 0.1 or higher epitope similarity, as calculated by the ab-Ligity method described in Wong WK, Robinson SA, Bujotzek A, Georges G, Lewis AP, Shi J, Snowden J, Taddese B, Deane CM. Ab-Ligity: identifying sequence-dissimilar antibodies that bind to the same epitope. MAbs. Jan-Dec 2021;13(1):1873478. doi:10.1080 / 19420862.2021.1873478. PMID: 33448242; PMCID: PMC7833755, which is fully incorporated by reference.

[0071] The antibodies disclosed herein (including, but not limited to, bispecific antibodies) or their antigen-binding fragments can be identified using any suitable technique. For example, computational methods can be used to design epitope-specific antibodies. In some embodiments, an alternative method can be used to identify antibodies binding to specific epitopes from an antigen-binding antibody library, such as the following approach: first, incorporating the non-classical amino acids (ncAA) p-benzoyl-L-phenylalanine (pBpa) and p-azido-L-phenylalanine (pAzF) into the target epitope, and then selecting antibodies that crosslink with the ncAA-incorporated epitope after UV irradiation. Because crosslinking only occurs when the distance between the antibody and the epitope is sufficiently close, this method effectively selects antibodies that specifically bind to the target epitope.

[0072] The term "Fc domain" is used herein to define the C-terminal region of an immunoglobulin, which comprises the constant regions of the two heavy chains, optionally including some or all of the hinge regions, but excluding the first constant region. Thus, the Fc domain refers to the last two constant regions of the immunoglobulin domains of IgA, IgD, and IgG, and the last three constant regions of the immunoglobulin domains of IgE and IgM. The constant regions from both polypeptides together form the Fc region. The term includes native sequence Fc regions and variant Fc regions. In one aspect, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the C-terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Therefore, antibodies produced by host cells by expressing a specific nucleic acid molecule encoding the full-length heavy chain may include the full-length heavy chain, or it may include cleaved variants of the full-length heavy chain. This could be the case where the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, according to EU index number). Therefore, the C-terminal lysine (Lys447) or C-terminal glycine (Gly446) and lysine (Lys447) in the Fc region may or may not be present. Unless otherwise stated herein, the amino acid residues in the Fc region or constant region are numbered 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 edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0073] As used herein, a “subunit” or “Fc subunit” of an Fc domain refers to one of the two polypeptides that form a dimer Fc domain, namely, a polypeptide containing the C-terminal constant region of the immunoglobulin heavy chain that can stably associate with the other of the two polypeptides that form the dimer Fc domain. For example, the subunit of the IgG Fc domain contains the IgG CH2 and IgG CH3 constant domains.

[0074] As used herein, “sequence identity” refers to the percentage of nucleotide / amino acid residues in the subject sequence that are identical to those in the reference sequence after alignment and, where necessary, the introduction of gaps to achieve maximum percentage sequence identity between sequences. For the purpose of determining the percentage of sequence identity between two or more amino acid or nucleic acid sequences, pairwise and multiple sequence alignments can be performed in various ways known to those skilled in the art, for example, using publicly available computer software such as ClustalOmega (Söding, J. 2005, Bioinformatics 21, 951-960), T-coffee (Notredame et al. 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6(298)) and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30(4) 772–780). Other exemplary programs for comparing and aligning sequence pairs include ALIGN (Myers and Miller, Comput Appl Biosci. March 1988; 4(1):11-7), FASTA (Pearson and Lipman, Proc Natl Acad Sci USA. April 1988; 85(8):2444-8; Pearson, Methods Enzymol. 1990; 183:63-98), BLAST (Altschul et al., Nucleic Acids Res. September 1, 1997; 25(17): 3389-40), BLASTP, BLASTN, or GCG (Devereux et al., Nucleic Acids Res. January 11, 1984; 12(1 Pt 1):387-95). When using such software, it is preferable to use the default parameters (e.g., for vacancy penalty and expansion penalty).

[0075] The terms “polypeptide,” “peptide,” “oligopeptide,” and “protein” are used interchangeably herein and refer to an amino acid chain. Polypeptides, etc., are not limited to amino acids of a specific chain length. The term also includes peptide chains with post-expression modifications, such as glycosylation, acetylation, phosphorylation, etc., and other modifications known in the art, including both naturally occurring and non-naturally occurring ones. In some embodiments, a polypeptide is a complete protein or a fragment thereof.

[0076] The terms "preferential binding" or "specific binding" mean that an antibody or fragment thereof binds to an epitope with a greater affinity than it binds to an irrelevant amino acid sequence. In this sense, the binding of an antigen-binding molecule described herein to its antigen can be "specific." In some embodiments, this affinity is at least 1.5 times, 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 100 times, or at least 1000 times greater than the affinity of the antibody or fragment thereof for an irrelevant amino acid sequence. This term also applies, for example, to cases where the antigen-binding domain is specific to a particular epitope carried by a number of antigens, in which case an antibody carrying the antigen-binding domain or an antigen-binding fragment thereof will be able to bind to various antigens carrying that epitope. Any antigen-binding molecule disclosed herein can specifically bind to its antigen.

[0077] The terms “recipient,” “individual,” “subject,” “host,” and “patient” are used interchangeably herein and refer to any mammalian subject, particularly a human, who requires diagnosis, treatment, or therapy. For therapeutic purposes, “mammal” means any animal classified as a mammal, including humans, livestock, and farm animals, as well as laboratory animals, zoo animals, sporting animals, or pet animals, such as dogs, horses, cats, cattle, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc. In some embodiments, the mammal is a human.

[0078] The term "therapeuticly effective amount" generally refers to the amount of a disclosed antibody or drug that effectively "treats" a subject or mammal's disease or condition. In some embodiments, the composition described herein is administered to a subject in an effective amount that produces some of the desired therapeutic effect by inhibiting the disease or condition described herein with a reasonable benefit / risk ratio suitable for any medical treatment. A therapeutically effective amount is the amount that at least partially achieves the desired therapeutic or preventive effect in an organ or tissue. The amount of antibody necessary for the prevention and / or therapeutic treatment of a disease or condition is not fixed in itself. In some embodiments, the amount of antibody administered varies depending on the type of disease, the extent of the disease, and the size of the mammal suffering from the disease or condition. When used in conjunction with a therapeutic method involving the administration of a therapeutic agent after a subject presents with symptoms of the disease or condition, the term "therapeuticly effective" means that one or more signs or symptoms of the disease or condition are improved or eliminated after treatment.

[0079] In some implementations, the administration of a therapeutic agent in a preventative approach occurs before the symptoms of an undesirable disease or condition appear, thereby preventing or delaying its progression. Therefore, when used in conjunction with a preventative approach, the term "therapeuticly effective" means that, after treatment, a smaller number of subjects (on average) develop the undesirable disease or condition or the severity of their symptoms.

[0080] As used herein, in some contexts, the terms “treatment,” “treating,” etc., refer to the administration of a drug or the performance of a procedure to achieve an effect. An effect may be preventative in relation to the complete or partial prevention of a disease or its symptoms, and / or therapeutic in relation to the partial or complete cure of a disease and / or its symptoms. As used herein, “treatment” includes treating a disease or condition in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or its symptoms in a subject who is susceptible to the disease but has not yet been diagnosed with it (e.g., including diseases associated with or caused by a primary disease); (b) suppressing a disease, i.e., halting its development; and (c) alleviating a disease, i.e., causing its remission. The term treatment includes any indication of success in treating or improving or preventing a disease or condition, including any objective or subjective parameters such as reduction; relief; decrease in symptoms or make the disease symptoms more tolerable to the patient; slowing deterioration or The rate of decline; or making the endpoint of decline less weak. Treatment or improvement of symptoms is based on one or more objective or subjective parameters, including the results of a physician's examination. Therefore, the term "treatment" includes the administration of the agents of this disclosure to prevent or delay, alleviate or stop or inhibit the development of symptoms or conditions associated with the disease. The term "treatment effect" refers to the reduction, elimination or prevention of the disease, disease symptoms or disease side effects in a subject. If a patient shows observable and / or measurable changes in parameters or symptoms of the disease or condition after receiving a therapeutic dose of the antibody of this disclosure, the subject is "treated" for the disease or condition. Detailed Implementation

[0081] Various aspects and embodiments of the invention will now be discussed. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned herein are incorporated by reference.

[0082] Interferon-γ (IFNγ) IFNγ is a 20-25 kDa glycoprotein that exists as a homodimer in solution. In some embodiments, the IFNγ protein is human IFNγ protein. In some embodiments, the human IFNγ protein is wild-type human IFNγ protein, such as endogenous IFNγ. In some embodiments, the human IFNγ protein is recombinant human IFNγ protein (rIFNγ). In some embodiments, the human IFNγ protein has an amino acid sequence that is at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, e.g., 100%) identical to the sequence in SEQ ID NO: 1. In some embodiments, the human IFNγ protein has an amino acid sequence that is at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, e.g., 100%) identical to the sequence in SEQ ID NO: 1. In some embodiments, the human IFNγ protein has an amino acid sequence that is at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99% or more) sequence identical to that of SEQ ID NO: 1. In some embodiments, the human IFNγ protein has an amino acid sequence that is at least 99% sequence identical to that of SEQ ID NO: 1. In some embodiments, the human IFNγ protein has the amino acid sequence of SEQ ID NO: 1 (UniProt number: P01579).

[0083] In some embodiments, the IFNγ protein is a non-human primate (NHP; e.g., cynomolgus monkey or rhesus monkey) IFNγ protein. In some embodiments, the NHP IFNγ protein has an amino acid sequence with at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with SEQ ID NO: 2. In some embodiments, the NHP IFNγ protein has an amino acid sequence with at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with SEQ ID NO: 2. In some embodiments, the NHP IFNγ protein has an amino acid sequence with at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99% or more) sequence identity with SEQ ID NO: 2. In some embodiments, the NHP IFNγ protein has an amino acid sequence that is at least 99% sequence identical to SEQ ID NO: 2. In some embodiments, the NHP IFNγ protein has the amino acid sequence of SEQ ID NO: 2 (UniProt No.: P63309).

[0084] In some embodiments, it may be preferred that the antigen-binding molecule is cross-reactive against SEQ ID NO: 1 and SEQ ID NO: 2, for example, to allow the antibody to be tested in primate models. In some embodiments, the binder exhibits an affinity for cynomolgus monkey IFNγ protein that is up to 10 times lower than the affinity of the binder for human IFNγ protein.

[0085] In some embodiments, the antigen-binding molecule / antibody according to the invention can bind IFNγ with an affinity in the micromolar range, such as human and / or primate IFNγ, preferably human IFNγ, i.e., K. D = 9.9 x 10 -4 Up to 1 x 10 -6 M. In some embodiments, the antigen-binding molecule described herein binds IFNγ, i.e., K, with sub-micromolar affinity. D <1 x 10 - 6 M. In some embodiments, the antigen-binding molecule described herein binds IFNγ, i.e., K, with an affinity in the nanomolar range. D = 9.9 x 10 -7 Up to 1 x 10 -9 M. In some embodiments, the antigen-binding molecule described herein binds IFNγ, i.e., K, with sub-nanomolar affinity.D <1 x 10 -9 M. In some embodiments, the antigen-binding molecule described herein binds IFNγ, i.e., K, with an affinity in the picomolar range. D = 9.9 x 10 -10 Up to 1 x 10 -12 M. In some embodiments, the antigen-binding molecule described herein binds IFNγ, i.e., K, with subpimolar affinity. D <1 x 10 -12 M. In some embodiments, the binder binds to the IFNγ protein with a dissociation constant (Kd) of less than 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 7 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.25 nM, or less. In some embodiments, the antigen-binding molecule / antibody binds to the IFNγ protein with a Kd of less than 10 nM or 5 nM. D Combined with human IFNγ.

[0086] Tumor-associated antigens In some embodiments, the antigen-binding molecule (e.g., antibody) is a multispecific antigen-binding molecule (e.g., antibody) having an antigen-binding domain capable of binding IFNγ and also having an antigen-binding site capable of binding another target antigen such as a tumor-associated antigen (TAA).

[0087] In some implementations, the antigen-binding molecule / antibody can be bispecific. For example, it can bind to IFNγ and a single target antigen other than IFNγ.

[0088] In other embodiments, the antigen-binding molecule / antibody may bind to more than one target antigen / TAA or to more than one different epitope on the target antigen / TAA. For example, in some embodiments, the antigen-binding molecule may bind to two different target antigens / TAAs, such as two different TAAs associated with the same tumor / cancer type. Dual TAA-targeting antibodies can provide advantages such as increased tumor selectivity.

[0089] As used herein, the term "tumor-associated antigen" or "tumor-specific antigen" refers to any molecule (e.g., protein, peptide, lipid, carbohydrate, etc.) that is expressed, primarily or overexpressed, by tumor cells and / or cancer cells, or by other cells of the tumor stroma (such as cancer-associated fibroblasts), such that the antigen is associated with a tumor and / or cancer. Tumor-associated antigens are expressed on cell surfaces. Tumor-associated antigens can also be expressed by normal, non-tumor, or non-cancer cells. However, in this case, the expression of tumor-associated antigens by normal, non-tumor, or non-cancer cells is less robust than that by tumor or cancer cells. In this respect, tumor or cancer cells can overexpress antigens or express antigens at significantly higher levels compared to the antigen expression by normal, non-tumor, or non-cancer cells. Furthermore, tumor-associated antigens can also be expressed by cells at different developmental or maturation stages. For example, tumor-associated antigens can also be expressed by cells at the embryonic or fetal stage, which are typically not present in adult hosts. Alternatively, tumor-associated antigens can also be expressed by stem cells or progenitor cells, which are typically not present in adult hosts.

[0090] Tumor-associated antigens (TAs) can be antigens expressed by any cells of any cancer or tumor (including the cancers and tumors described herein). A TA can be a TA associated with only one type of cancer or tumor, such that it is associated with or characterized by only one type of cancer or tumor. Alternatively, a TA can be a TA associated with more than one type of cancer or tumor (e.g., it can be characterized by it). For example, a TA can be expressed by breast cancer and prostate cancer cells and not at all by normal non-tumor or non-cancerous cells.

[0091] The antibodies of this invention can bind to exemplary tumor-associated antigens, including but not limited to avβ3 integrin (e.g., in adenocarcinoma), bufotoxin R (e.g., in prostate cancer), CAiX (e.g., in colon cancer), CD13 (e.g., in myeloma and adenocarcinoma), CD44 v6 (e.g., in adenocarcinomas such as head and neck cancer), CXCR4 (e.g., in breast cancer), EGFR (e.g., in head / neck cancer and breast cancer), emmprin (e.g., in pancreatic cancer), endothelial glycoproteins (e.g., in gastrointestinal tumors, malignant melanoma, and central nervous system tumors), EphA2 (e.g., in ovarian cancer and melanoma), folate R (e.g., in ovarian cancer), GRP78 (e.g., in breast cancer and glioblastoma), IGF-1R (e.g., in colon cancer), matrix proteases (e.g., in colon cancer), cMET / HGFR (e.g., in lung cancer), MT1-MMP (e.g., in lung cancer), and MT6-MMP. (e.g., in head and neck cancer, breast cancer, and colon cancer), PSMA (e.g., in prostate cancer), Tn and STn antigens (e.g., adenocarcinoma), uPAR (e.g., in colon cancer), GD2 (e.g., in neuroblastoma, melanoma, sarcoma, and pediatric solid tumors), TncA1 (e.g., in breast cancer, AML, solid tumors, and MCC), EBD (e.g., in melanoma, RCC, NSCLC, solid tumors, pancreatic cancer, colorectal cancer, DLBCL, glioblastoma, sarcoma, and glioma), PD1 (e.g., in solid tumors), EDA, MHCII, EDB, A33, αFR, EGR, PS (phosphatidylserine), melanoma-associated chondroitin sulfate proteoglycan (MCSP), mucin 1 (MUC1; tumor-associated epithelial mucin) (e.g., in adenocarcinoma), melanoma-preferred antigen (PRAME) (e.g., in melanoma), carcinoembryonic antigen (CEA) (e.g., in colon cancer), PSCA (e.g., in prostate cancer), EpCAM (e.g., in SCLC, prostate cancer, ovarian cancer, breast cancer, bladder cancer, kidney cancer, lung cancer, colon cancer, solid tumors), Trop2 (trophoblast-2, also known as EGP-1), granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR), CD56, human epidermal growth factor receptor 2 (HER2 / neu) (also known as erbB-2) (e.g., in breast cancer), CDS, CD7, tyrosinase-associated protein (TRP) I, and TRP2.Tumor antigens can also be selected from groups consisting of the following: differentiation clusters (CD) 19, CD20 (e.g., in B-cell lymphoma), CD21, CD22, CD25, CD30, CD33 (sialic acid-binding Ig-like lectin 3, bone marrow cell surface antigen), CD79b, CD123 (interleukin-3 receptor α), transferrin receptor, EGF receptor, mesothelin (e.g., in mesothelioma), cadherin, Lewis Y, phosphatidylinositol proteoglycan-3, FAP (fibroblast activation protein α) (e.g., in cancer-associated fibroblasts, solid tumors, RCC, melanoma, pancreatic adenocarcinoma, breast cancer, HNC, esophageal cancer, cervical cancer), GPRC5D (G protein-coupled receptor class C5 member D), CA9=CAIX (carbonic anhydrase IX), L1 CAM (neural cell adhesion molecule L1), endothelial sialic acid protein, HER3 (Activated conformation of epidermal growth factor receptor family member 3), Alkl / BMP9 complex (anaplastic lymphoma kinase 1 / bone morphogenetic protein 9), TPBG=5T4 (trophoblast glycoprotein), ROR1 (receptor tyrosine kinase-like surface antigen), HER1 (activated conformation of epidermal growth factor receptor), CLL1 (C-type lectin domain family 12, member A), STEAP1 (prostatic six-transmembrane epithelial antigen 1), LIV-1 (SLC39A6), B7-H3 (B7 homolog 3, also known as CD276), and CD46. For example, tumor-associated antigens can be selected from PSMA, EGFR, CEA, HER2, Tn antigen, sTn antigen, CD44v6, B7-H3, and Trop2.

[0092] In some implementations, the tumor-associated antigen may be prostate-specific membrane antigen (PSMA). Prostate-specific membrane antigen (PSMA), also known as glutamate carboxypeptidase II, N-acetyl-α-linked acidic dipeptidase I (Naaladase (NLD) I), or folate hydrolase, is a 750-residue type II transmembrane glycoprotein that has been found to be highly expressed in angiogenesis in prostate cancer cells and non-prostate solid tumors, and expressed at lower levels in other tissues, including the healthy prostate, kidney, liver, small intestine, salivary glands, duodenal mucosa, proximal renal tubules, and brain. PSMA is a member of the zinc-dependent exopeptidase superfamily, which includes carboxypeptidases with a single nuclear zinc active site.

[0093] In some embodiments, the human PSMA protein has an amino acid sequence that is at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the sequence of SEQ ID NO: 3. In some embodiments, the human PSMA protein has the amino acid sequence of SEQ ID NO: 3 (UniProt number: Q04609).

[0094] PSMA expression is increased in prostate cancer, particularly in poorly differentiated, metastatic, and / or hormone-refractory cancers. Typically, PSMA expression is found to increase with prostate cancer progression and metastasis. PSMA is also expressed in the endothelial cells of capillaries in the peritumoral and intratumoral regions of certain malignancies, including renal cell carcinoma and colon cancer, but not in blood vessels originating from normal tissue. Furthermore, PSMA has been reported to be involved in tumor angiogenesis. PSMA expression has been demonstrated in the endothelial cells of tumor-associated neovascularization systems in colon cancer, breast cancer, bladder cancer, pancreatic cancer, renal cell carcinoma, and melanoma.

[0095] In other embodiments, the tumor-associated antigen may be B7-H3 (also known as CD276). The B7-H3 protein is overexpressed in many tumors compared to healthy tissue, thus providing a tumor-associated antigen with broad applicability, for example, for a range of solid tumors.

[0096] In some embodiments, human B7-H3 can be a 2Ig-B7-H3 or 4Ig-B7-H3 isotype (formed by one or two identical pairs of immunoglobulin variable (IgV)-like domains and immunoglobulin constant (IgC)-like domains, respectively). In some embodiments, the human B7-H3 protein has the amino acid sequence of SEQ ID NO: 131 (UniProt number: Q5ZPR3).

[0097] In some embodiments, the antibody according to the invention can bind a TAA (e.g., one or more TAAs selected from those described herein) with an affinity in the micromolar range, i.e., K D = 9.9 x 10 -4 Up to 1 x 10 -6 M. In some embodiments, the antigen-binding molecule described herein binds to the TAA with submicromolar affinity, i.e., K. D <1 x 10 -6 M. In some embodiments, the antigen-binding molecule described herein binds to the TAA with an affinity in the nanomolar range, i.e., K. D = 9.9 x 10 -7 Up to 1 x 10-9 M. In some embodiments, the antigen-binding molecule described herein binds to the TAA with sub-nanomolar affinity, i.e., K. D <1 x 10 -9 M. In some embodiments, the antigen-binding molecule described herein binds to the TAA with an affinity in the picomolar range, i.e., K. D = 9.9 x 10 -10 Up to 1 x 10 -12 M. In some embodiments, the antigen-binding molecule described herein binds to the TAA with subpimolar affinity, i.e., K. D <1 x 10 -12 M. In some embodiments, the binder has a dissociation constant (K) of less than 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 7 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.25 nM or smaller. D The antigen-binding molecule / antibody binds to the PSMA protein. In some implementations, the antigen-binding molecule / antibody can be at a K+ level of less than 20 nM. D Combine with TAA.

[0098] Properties of antigen-binding molecules Antigen-binding molecules capable of binding IFNγ (e.g., binding IFNγ alone) or multispecific antigen-binding molecules as described herein may have the following properties.

[0099] Partial and / or preferential inhibition of antigen-binding molecules The inventors have determined that it is possible to obtain antibodies capable of binding IFNγ while still maintaining at least a portion of the cytokine's pro-inflammatory or antitumor activity. (As used herein, the term "pro-inflammatory" is used interchangeably with "antitumor"; and the term "anti-inflammatory" is used interchangeably with "protumor".) The inventors further recognize that it is possible to utilize the pro-inflammatory / antitumor properties of IFNγ while it binds to antigen-binding molecules to obtain various advantages, such as increasing plasma concentrations of endogenous IFNγ, and / or increasing the half-life of exogenously administered IFNγ, and / or targeting IFNγ to target cells or tissues, such as tumors or cancer cells or tissues. The antibody / antigen-binding molecule is capable of binding IFNγ while maintaining at least a portion of the IFNγ's pro-inflammatory activity. (As used herein, references to "partially reduce" or "partially inhibit" mean retaining at least some pro-inflammatory activity.) The inventors unexpectedly determined that antibodies can be used to achieve IFNγ accumulation in the blood while preserving IFNγ activity, even though receptor-mediated clearance may still occur in the absence of IFNγ inhibition / blocking of receptor binding.

[0100] In some cases, a partial reduction in pro-inflammatory / anti-tumor activity in combination with targeting target cells or tissues (e.g., using multispecific antibodies) can be beneficial in reducing side effects such as systemic inflammation, while accumulating pro-inflammatory / anti-tumor activity at the target site (and thus benefiting from higher concentrations and / or affinity effects at the target site).

[0101] The inflammatory and / or antitumor effects of IFNγ can include its effects on immune cells and / or its direct effects on cancer cells. For example, IFNγ can induce pro-inflammatory / antitumor effects in one or more of the following cell types: T cells, including but not limited to CD4 or CD8 cells, regulatory T cells (Tregs), and γδ T cells; B cells, including but not limited to ICAM-1+ B cells; natural killer (NK) cells; phagocytes, including but not limited to dendritic cells (DCs), macrophages, pre-macrophage monocytes, and monocytes; Thy-1.2+ cells and HLA-DR+ cells. For example, the pro-inflammatory activity of IFNγ can be measured as one or more of the following: increased recruitment of immune cells via induction of chemokines CXCL9, CXCL10, and CXCL11 and their homologous receptor CXCR3 on immune cells; increased phagocytic capacity of phagocytes; increased microbial and / or tumor cell killing capacity of macrophages; upregulation of MHC I complex expression on immune cells or cancer cells; activation and enhanced cytotoxicity of CD8+ T cells via FAS-FASL, and upregulation of granzyme B and TNF-associated apoptosis-inducing ligand (TRAIL); CD4+ Enhanced survival of T helper type 1 cells and prevention of polarization to T helper type 17 regulatory T cells and T helper type 2 cells; enhanced expression of DC maturation and / or co-stimulatory molecules such as CD40, CD54, CD80, CD86 and CCR7 in DCs; secretion of IL-12 family cytokines and / or IL-1β from DCs; inhibition of Treg proliferation and function; and activation of one or more members of the signal transduction and transcription activator (STAT) family, such as STAT4, T-box transcription factor (T-bet), activator protein 1 (AP1) and / or Eomes (Jorgovanovic et al. Biomarker Research 2020 8:49, Gocher et al. Nat Rev Immunol. Mar 2022;22(3):158-172. doi: 10.1038 / s41577-021-00566-3. E-published on 21 June 2021. PMID: 34155388).

[0102] In some implementations, pro-inflammatory activity is measured by cell surface expression of major histocompatibility complex class I (MHC-1), as explained further below.

[0103] For example, in one embodiment, the effect of the antigen-binding molecule is evaluated in vitro when co-administered with 1 ng / mL IFNγ to cells. Evaluation can be performed 48 hours after co-administration. In one embodiment, evaluation is performed by contacting plated test cells (such as A549 lung cancer cells or PC3 prostate cancer cells, e.g., PC3-FLU cells) with 1 ng / mL IFNγ in the absence and presence of the test molecule, and then evaluating MHC-1 expression on the cell surface after 48 hours. Optionally, the cells can be A549 lung cancer cells. In one embodiment, when tested at a concentration of 0.1 nM, the antibody / antigen-binding molecule tested in this manner is non-inhibitory to pro-inflammatory activity (e.g., MHC-1 expression), i.e., it does not reduce MHC-1 expression compared to the same concentration of IFNγ in the absence of the test molecule. Alternatively, when tested at a concentration of 100 nM, the antibody / antigen binding molecule can be inhibitory; that is, at this concentration, it does indeed reduce MHC-1 expression compared to the same concentration of IFNγ in the absence of the antibody / antigen binding molecule. In some embodiments, the IC50 of IFNγ-induced MHC-1 expression is... 50 It can be in the nanomolar range, i.e., less than 1 mM and at least 1 nM, optionally at least 10 nM. In other embodiments, IC 50 It can be greater than 1 nM or greater than 10 nM. In some embodiments, the IC50 of the antibody / antigen binding molecule in this assay is... 50 It is at least 100-fold more potent than the reference anti-IFNγ antibody MT111W (Mabtech) (but 100-fold less inhibitory). In some embodiments, the IC50 of the antibody / antigen binding molecule in this assay is... 50 It is 100 to 10,000 times more powerful than the MT111W.

[0104] In another embodiment, the ability of the antigen-binding molecule to dimerize IFNγR1 and IFNγR2 expressed on cells (i.e., IFNγ-mediated heterotetramerization of IFNγR1 and IFNγR2 subunits on the cell surface) is non-inhibitory. For example, the antigen-binding molecule can be evaluated at a concentration of 200 ng / mL in the presence of 5 ng / mL IFNγ. In some embodiments, the antigen-binding molecule may be non-inhibitory when evaluated using the assay of Example 3.

[0105] In addition to demonstrating pro-inflammatory or anti-tumor effects, IFNγ has been shown to exert certain anti-inflammatory or pro-tumor effects under certain circumstances, including promoting the expression of PD-L1 (an immunosuppressive receptor ligand that leads to T cell dysfunction and apoptosis).

[0106] The inventors have also unexpectedly determined that it is possible to obtain anti-IFNγ antibodies that can bias the pro-inflammatory and anti-inflammatory activities of IFNγ towards pro-inflammatory or anti-tumor activity. Therefore, in one aspect, the present invention relates to an antigen-binding molecule capable of binding IFNγ and biasing its pro-inflammatory and anti-inflammatory activities towards pro-inflammatory activity. For example, in some embodiments, such a molecule can preferentially inhibit anti-inflammatory activity (i.e., it can inhibit anti-inflammatory activity while simultaneously inhibiting pro-inflammatory properties to a lower degree or not inhibiting pro-inflammatory properties at all). As described above, these molecules can be partially inhibitory of pro-inflammatory activity (while being more inhibitory of anti-inflammatory activity). Such molecules can have therapeutic efficacy, for example, in the treatment of cancer.

[0107] In some embodiments, the pro-inflammatory activity of cytokines can be measured by their ability to induce the expression of major histocompatibility complex class I (MHC-1) on the surface of cells (e.g., in vitro cells). In some embodiments, the anti-inflammatory activity of cytokines can be measured by their ability to induce the expression of programmed cell death ligand 1 (PD-L1) on the surface of cells (e.g., in vitro cells).

[0108] Therefore, the present invention can provide an antigen-binding molecule, optionally an antibody, which is capable of binding interferon-γ (IFNγ) and regulating cellular signaling downstream of IFNγ, such that the ratio of IFNγ-induced PD-L1 expression and / or activity to MHC-1 expression and / or activity is altered to favor MHC-1 expression and / or activity.

[0109] As an example, MHC-1 and PD-1 expression can be measured in vitro in model cells such as PC3 prostate cancer cells or A549 lung cancer cells. In one embodiment, PC3 cells, such as PC3-FLU cells, are used for the assay. In another embodiment, A549 lung cancer cells are used.

[0110] MHC-1 expression on cells can be detected using directly or indirectly labeled anti-MHC1 antibodies. Various anti-MHC-1 antibodies are commercially available, such as anti-HLA-ABC (e.g., clone G46.2.6, BD, e.g., conjugated with v450). It can be measured by flow cytometry. Similarly, PD-L1 expression on cells can be determined using directly or indirectly labeled commercially available antibodies, such as directly or indirectly labeled anti-PD-L1 (e.g., clone 29E.2A3, Biolegend, conjugated with PE-Ddazzle). It can be measured by flow cytometry. MHC-1 and PD-1 expression can be cell surface expression, for example, when measured by flow cytometry.

[0111] In one embodiment, the effect of the antigen-binding molecule is evaluated in vitro when co-administered with 1 ng / mL IFNγ to cells. Evaluation can be performed 48 hours after co-administration. In one embodiment, evaluation is performed by contacting test cells (such as A549 lung cancer cells or PC3 prostate cancer cells) plated as described above with 1 ng / mL IFNγ in the absence of the test molecule and in the presence of the test molecule at concentrations of 1 nM, 10 nM, or 100 nM, preferably 10 nM, and evaluating MHC-1 expression and / or PD-L1 on the cell surface after 48 hours. In some embodiments, the method of Example 5 can be used for evaluation. (Based on our observations, it appears that cell density is not important for the assay, but in some embodiments, cells can be cultured to approximately 90% or higher coverage before the assay.)

[0112] In some implementations, when co-administered with IFNγ, the antigen-binding molecule may increase the ratio of MHC-1 to PD-L1 expression compared to the ratio obtained by administering the same amount of IFNγ in the absence of an antibody / antigen-binding molecule. That is, in the presence of the same amount of IFNγ, the ratio of MHC-1 to PD-L1 expression may be greater in the presence of the antibody / antigen-binding molecule than in its absence. (In absolute terms, both expression levels may decrease, but the decrease in MHC-1 expression may be less than that in PD-L1 expression). In some embodiments, when expression values ​​are normalized to those observed in the presence of the same amount of IFNγ but in the absence of antibody / test molecules, such that the MHC-1 to PD-L1 expression ratio is 1 (1:1) in the absence of antibody, the antibody / antigen binding molecule can be able to increase this ratio to at least 1.11 (1:0.9), 1.25 (1:0.8), 1.33 (1:0.75), 1.43 (1:0.7), 1.67 (1:0.6), or 2 (1:0.5) or higher. In some embodiments, normalization can be performed as described in Example 5 (the value of the test sample minus the value without the addition of antibody / antigen binding molecules and IFNγ (baseline value), divided by the value with only IFNγ added minus the baseline value, i.e., (sample value - baseline value) / (IFNγ value only - baseline value) * 100.

[0113] In some embodiments, the antigen-binding molecule may be able to reduce PD-L1 expression by a greater percentage in the presence of IFNγ (compared to PD-L1 expression in the presence of the same amount of IFNγ but without the antigen-binding molecule) than it reduces MHC-1 expression in the presence of IFNγ (compared to MHC-1 expression in the presence of the same amount of IFNγ but without the antigen-binding molecule).

[0114] In some embodiments, IFNγ-induced MHC-1 expression is partially inhibited by antibody / antigen-binding molecules at test concentrations of 1 nM, 10 nM, or 100 mM, i.e., less than 100% of the level observed in the absence of antibody / antigen-binding molecules. For example, the level of MHC-1 expression may be, for instance, greater than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, or 20% of the level observed in the presence of the same amount of IFNγ but in the absence of antigen-binding molecules, but less than 100%. It is conceivable that even small amounts of residual pro-inflammatory activity may be treatment-relevant, particularly when combined with targeting and / or accumulation. In some embodiments, the level of MHC-1 expression may be less than 90%, less than 80%, less than 70%, less than 60%, or less than 50% of the level observed in the presence of the same amount of IFNγ but in the absence of antigen-binding molecules.

[0115] In some embodiments, the antigen-binding molecule of the present invention may have the following functional properties: strongly inhibiting PD-L1 expression to less than 25% of the level observed in the absence of antibody / antigen-binding molecules but in the presence of the same amount of IFNγ (e.g., at 1 mM and / or 10 mM and / or 100 nM of the antigen-binding molecule as described above), while maintaining a level of MHC-1 expression that is, for example, at least 50% of the level observed in the presence of the same amount of IFNγ but in the absence of antigen-binding molecules. Alternatively or additionally, these properties may include maintaining a level of MHC-1 expression that is, for example, at at least 50% of the level observed in the presence of the same amount of IFNγ but in the absence of antigen-binding molecules, for example, at 1 mM and / or 10 mM of the antigen-binding molecule as described above and / or even at a concentration of 100 nM of antigen-binding molecules.

[0116] Prolonging the half-life of endogenous IFNγ in vivo and / or causing endogenous IFNγ to accumulate in vivo tired The elimination half-life of exogenously administered recombinant IFNγ in the bloodstream in healthy male subjects after intravenous administration was approximately 38 minutes. The subcutaneous route typically results in a somewhat significantly prolonged half-life of 5–6 hours due to the slow absorption from the subcutaneous reservoir into the bloodstream (https: / / www.hzndocs.com / ACTIMMUNE-Prescribing-Information.pdf).

[0117] The inventors have determined that the binding of an antigen-binding molecule can increase the half-life of endogenously or exogenously administered (e.g., recombinant) IFNγ. This provides a novel method for increasing the half-life of IFNγ in circulation. The antigen-binding molecule can be any antigen-binding molecule disclosed herein, including antigen-binding molecules that bind IFNγ alone, or antigen-binding molecules that bind IFNγ and at least one other antigen, such as bispecific antibodies.

[0118] In such embodiments, it is preferable that the antigen-binding molecule includes an Fc domain and / or is itself conjugated to another portion that extends the half-life. Various portions that extend the half-life are known in the art. For example, this portion may be selected from fatty acids, albumins, and hydrophilic polymers such as PEG, polysialic acid (PSA), N-(2-hydroxypropyl)methacrylamide (HPMA), or dextran. The antibodies described herein can provide elevated steady-state IFNγ concentrations. In some embodiments, compared to administration of recombinant IFNγ, antibodies can provide improved pharmacokinetics by providing elevated steady-state concentrations while reducing or avoiding peaks that may be associated with adverse effects and toxicity.

[0119] Preferably, the antigen-binding molecule has an affinity of 500 pM or lower for IFNγ. Modeling shows that, in the case of tumor-targeting antibodies, this affinity can lead to the accumulation of IFNγ in the blood and at the tumor site.

[0120] Antigen-binding molecules can bind to endogenous IFNγ during its synthesis and secretion into the bloodstream. This protects IFNγ from degradation and elimination or other forms of clearance from circulation. Therefore, IFNγ can accumulate in the blood. In the case of novel multispecific molecules / bispecific antibodies, accumulation at relevant sites can be further facilitated through targeting mechanisms to reach pharmacologically threshold concentrations at target cells (e.g., cancerous tissue) (as discussed further below). Furthermore, antigen-binding molecules can help capture local IFNγ release, for example, after administration of checkpoint inhibitors. For instance, anti-PD1 induces an intratumoral IFN burst. This IFNγ burst can be prolonged by using the antigen-binding molecules described herein, particularly in the case of tumor-targeting antibodies, thereby providing enhanced local IFNγ concentrations of endogenous IFNγ.

[0121] In some embodiments, the antigen-binding molecules disclosed herein increase the half-life of the IFNγ protein by at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times compared to the half-life of the IFNγ protein in the absence of an antibody / antigen-binding molecule. In some embodiments, the antibody / antigen-binding molecules disclosed herein (e.g., bispecific antibodies) increase the half-life of the IFNγ protein by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, or 600% compared to the half-life of the IFNγ protein in the absence of an antibody / antigen-binding molecule. %, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 1,000%, 1,500%, 2,000%, 2,500%, 3,000%, 3,500%, 4,000%, 4,500%, 5,000%, 5,500%, 6,000%, 6,500%, 7,000%, 7,500%, 8,000%, 8,500%, 9,000%, 9,500%, or 10,000%. In some embodiments, IFNγ is endogenous IFNγ.

[0122] The ability of an antibody / antigen binding molecule to increase its half-life can be determined by well-known methods, such as those described in Pharmacokinetics and Pharmacodynamic Data Analysis: concepts and applications (Gabrielsson and Weiner). For example, it can be assessed by analyzing plasma concentrations of IFNγ in the presence or absence of the antibody in a subject (e.g., mouse, rat, or monkey). In some embodiments, the ability of an antibody to increase its half-life and / or accumulate IFNγ can be determined by analyzing plasma levels of IFNγ as described in Example 4. The ability of the antibody to accumulate mouse, rat, or monkey (e.g., rhesus monkey) IFNγ can be assessed if the antibody cross-reacts with endogenous mouse, rat, or macaque IFNγ. In other embodiments, the ability of an antibody to increase its half-life and / or accumulate IFNγ can be assessed in the presence of exogenously administered IFNγ (e.g., exogenously administered human IFNγ) bound to the antibody. In other embodiments, the test subject can be a human. In some embodiments, the test subject can be a subject who does not express a target antigen of a multispecific antibody. The subject can be a healthy subject, such as a subject who does not have cancer expressing a TAA.

[0123] Therefore, in some embodiments, methods for prolonging the half-life of endogenous or exogenously administered (e.g., recombinant) IFNγ protein in a subject are disclosed herein, comprising administering to the subject the antigen-binding molecule or pharmaceutical composition disclosed herein. Preferably, the method is a method for prolonging the half-life of endogenous IFNγ.

[0124] Targeted The presence of an antigen-binding domain that binds to target antigens such as tumor-associated antigens (TAAs) can be used to target antigen-binding molecules (and thus IFNγ associated with the antigen-binding molecules) to cells or tissues that express the target antigen, such as tumors or cancer cells / tissues that express TAAs.

[0125] Achieving relatively high concentrations at target tissues while maintaining low IFNγ concentrations in the bloodstream to control toxicity can facilitate the utilization of the pro-inflammatory and / or anti-tumor effects of IFNγ at target tissues. In cases where antigen-binding molecules partially inhibit the pro-inflammatory effects of IFNγ, accumulation at the target site can also be used to increase activity at the target site through higher concentrations and / or affinity effects, while again controlling systemic inflammatory side effects.

[0126] Therefore, the antigen-binding molecules described herein may be part of a multispecific or bispecific antibody that also contains an antigen-binding site against a target antigen such as a TAA, as discussed further herein. In some embodiments, methods for increasing the localization (e.g., targeting) of IFNγ protein to cancer cells / tissues in a subject (e.g., a human) are also disclosed herein, comprising administering to the subject a composition comprising a multispecific antigen-binding molecule (e.g., a bispecific antigen-binding molecule / antibody) or a pharmaceutical composition disclosed herein.

[0127] valence of IFNγ Preferably, the antigen-binding molecule is monovalent to IFNγ. For example, an antibody may have a single binding site against IFNγ present on one arm of IgG. The inventors unexpectedly determined that when the antibody is monovalent to IFNγ, the affinity for IFNγ is significantly increased. This has been observed for different IFNγ binding sequences, i.e., it is independent of the sequence of the IFNγ conjugate.

[0128] This form is useful for both monospecific and multispecific (e.g., bispecific) molecules. In the case of multispecific antigen-binding molecules (e.g., bispecific antibodies), the fact that the monovalent nature of IFNγ allows for surprisingly good affinity means that a form with only two binding sites, such as the IgG form, can be used, where each arm of the antibody binds a different antigen. In the case of monovalent antibodies, a “single-arm” IgG form can be used. The single-arm form typically consists of a full-length heavy chain and a heavy chain composed of Fc subunits (e.g., a hinge-CH2-CH3 region).

[0129] Exemplary antigen-binding molecule - IFNγ binding domain The following provides exemplary antigen-binding molecules comprising an IFNγ binding domain according to this disclosure. In some embodiments, the antigen-binding molecule may be multispecific (e.g., bispecific), such as a multispecific or bispecific antibody comprising an IFNγ binding domain as described herein. In other embodiments, the antigen-binding molecule may bind only IFNγ, i.e., it may be monospecific.

[0130] The antigen-binding molecule disclosed herein may include IFNγ-binding domains of VH and VL containing antibodies capable of specifically binding IFNγ. The antigen-binding domain formed by VH and VL may also be referred to herein as the Fv region.

[0131] In some embodiments, the antigen-binding molecule may contain a single IFNγ-binding domain, as described above. Embodiments containing a single IFNγ-binding domain may be preferred. In other embodiments, the antigen-binding molecule may contain more than one IFNγ-binding domain. For example, the antigen-binding molecule may contain two such domains, as in the case of, for example, conventional IgG.

[0132] In some embodiments, the antigen-binding molecule comprises the CDR, FR, and / or VH and / or VL regions of the IFNγ-binding antibody clone described herein, or is derived from the CDR, FR, and / or VH and / or VL regions of the IFNγ-binding antibody clone described herein. In some embodiments, the IFNγ-binding antibody clone is selected from: 4F4, 16D2, 3G1, 9F1, or 2C1.

[0133] 4F4 and antibodies derived from it In one embodiment, this disclosure relates to an antigen-binding molecule comprising an IFNγ-binding domain, wherein the IFNγ-binding domain comprises a heavy chain variable region containing HC-CDR1, HC-CDR2, and HC-CDR3, wherein: a) The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 4 or a variant thereof in which one, two or three amino acids in HC-CDR1 are replaced by another amino acid; b) The heavy chain CDR2 contains the amino acid sequence of SEQ ID NO: 5 or a variant thereof in which one, two or three amino acids in HC-CDR2 are replaced by another amino acid; c) The heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 6 or a variant thereof in which one, two or three amino acids in HC-CDR3 are replaced by another amino acid; And / or the IFNγ-binding domain contains a light chain variable region comprising LC-CDR1, LC-CDR2, and LC-CDR3, wherein: d) The light chain CDR1 contains the amino acid sequence of SEQ ID NO: 7 or a variant thereof in which one, two, or three amino acids in LC-CDR1 are replaced by another amino acid; e) The light chain CDR2 contains the amino acid sequence of SEQ ID NO: 8 or a variant thereof in which one, two or three amino acids in LC-CDR2 are replaced by another amino acid; f) The light chain CDR3 contains the amino acid sequence of SEQ ID NO: 9 or a variant thereof in which one, two or three amino acids in LC-CDR3 are replaced by another amino acid.

[0134] In some embodiments, the antigen-binding molecule includes an IFNγ-binding domain, wherein the IFNγ-binding domain includes a heavy chain variable region containing HC-CDR1, HC-CDR2, and HC-CDR3, wherein: a) The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO:4 or a variant thereof in which one, two or three amino acids in HC-CDR1 are replaced by another amino acid. b) The heavy chain CDR2 contains the amino acid sequence of SEQ ID NO:5 or a variant thereof in which one, two or three amino acids in HC-CDR2 are replaced by another amino acid. c) The heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 6; And / or the IFNγ-binding domain contains a light chain variable region comprising LC-CDR1, LC-CDR2, and LC-CDR3, wherein: d) The light chain CDR1 contains the amino acid sequence of SEQ ID NO:7 or a variant thereof in which one, two, or three amino acids in LC-CDR1 are replaced by another amino acid; e) The light chain CDR2 contains the amino acid sequence of SEQ ID NO: 8 or a variant thereof in which one, two or three amino acids in LC-CDR2 are replaced by another amino acid; f) The light chain CDR3 contains the amino acid sequence of SEQ ID NO: 9.

[0135] In some embodiments, the heavy chain CDR1 comprises a variant of SEQ ID NO: 4 with a substitution at position 30 according to the IMGT scheme number (i.e., GYTXTNYY (SEQ ID NO: 84), where X can be any amino acid), wherein the residue at that position is preferably D, E, N, or Q, most preferably E. For example, the heavy chain CDR1 may be selected from GYTFTNYY (SEQ ID NO: 4) and GYTETNYY (SEQ ID NO: 85).

[0136] In another embodiment, the heavy chain CDR2 comprises a variant of SEQ ID NO: 5 with a substitution at position 63 according to the IMGT scheme number (i.e., INPSNXGT (SEQ ID NO: 86), where X can be any amino acid and preferably not D), wherein the residue at that position is preferably E, H, or Q, most preferably H. Removing D at this position eliminates a potential isomerization site because the residue forms a potential isomerization sequence defect for DG. For example, the heavy chain CDR2 may be selected from INPSNDGT (SEQ ID NO: 5) and INPSNHGT (SEQ ID NO: 87).

[0137] In another embodiment, the light chain CDR1 comprises a variant of SEQ ID NO: SEQ ID NO: 7 with substitutions at one or both positions 29 and 35 according to the IMGT scheme number (i.e., QSXLYSSNXKNY (SEQ ID NO: 88), where X can be any amino acid), wherein the residue at position 29 is preferably selected from G, A, V, L, or I, and most preferably V, and / or the residue at position 35 is preferably selected from D, E, N, or Q, and most preferably N. For example, LC-CDR1 can be selected from QSLLYSSNQKNY (SEQ ID NO: 7) and QSVLYSSNNKNY (SEQ ID NO: 89).

[0138] In one exemplary embodiment, the IFNγ binding domain may include a heavy chain variable region containing HC-CDR1, HC-CDR2, and HC-CDR3, wherein: a) The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 4; b) The heavy chain CDR2 contains the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 87; c) The heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 6; It also includes light chain variable regions containing LC-CDR1, LC-CDR2, and LC-CDR3, wherein: d) The light chain CDR1 contains the amino acid sequence of SEQ ID NO: 7; e) The light chain CDR2 contains the amino acid sequence of SEQ ID NO: 8; f) The light chain CDR3 contains the amino acid sequence of SEQ ID NO: 9.

[0139] In one exemplary embodiment, the IFNγ binding domain may include a heavy chain variable region containing HC-CDR1, HC-CDR2, and HC-CDR3, wherein: a) The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 85; b) The heavy chain CDR2 contains the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 87; c) The heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 6; It also includes light chain variable regions containing LC-CDR1, LC-CDR2, and LC-CDR3, wherein: d) The light chain CDR1 contains the amino acid sequence of SEQ ID NO: 7; e) The light chain CDR2 contains the amino acid sequence of SEQ ID NO: 8; f) The light chain CDR3 contains the amino acid sequence of SEQ ID NO: 9.

[0140] In one exemplary embodiment, the IFNγ binding domain may include a heavy chain variable region containing HC-CDR1, HC-CDR2, and HC-CDR3, wherein: a) The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 4; b) The heavy chain CDR2 contains the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 87; c) The heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 6; It also includes light chain variable regions containing LC-CDR1, LC-CDR2, and LC-CDR3, wherein: d) The light chain CDR1 contains the amino acid sequence of SEQ ID NO: 89; e) The light chain CDR2 contains the amino acid sequence of SEQ ID NO: 8; f) The light chain CDR3 contains the amino acid sequence of SEQ ID NO: 9.

[0141] In another exemplary embodiment, the IFNγ binding domain may include a heavy chain variable region containing HC-CDR1, HC-CDR2, and HC-CDR3, wherein: a) The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 85; b) The heavy chain CDR2 contains the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 87; c) The heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 6; It also includes light chain variable regions containing LC-CDR1, LC-CDR2, and LC-CDR3, wherein: d) The light chain CDR1 contains the amino acid sequence of SEQ ID NO: 89; e) The light chain CDR2 contains the amino acid sequence of SEQ ID NO: 8; f) The light chain CDR3 contains the amino acid sequence of SEQ ID NO: 9.

[0142] In some embodiments, the antibody / antigen binding molecule comprises a VH region having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 10, optionally at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or a VL region having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 11, optionally at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the antibody / antigen binding molecule comprises a CDR (i.e., any substitutions are within the frame region) as listed above. Optionally, the substitutions in VH and / or VL (e.g., in the frame region) may be residues found at the corresponding positions in the human lineage antibody sequence.

[0143] In some embodiments, the antibody is a humanized antibody, such as comprising the CDR and human frame regions as listed above, i.e., HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LCFR3, and LC-FR4 of a human germline antibody; or variants thereof wherein 1-5 amino acids in HC-FR1, and / or 1-5 amino acids in HC-FR2, and / or 1-5 amino acids in HC-FR3, and / or 1-5 amino acids in HC-FR4, and / or 1-5 amino acids in LC-FR1, and / or 1-5 amino acids in LC-FR2, and / or 1-5 amino acids in LC-FR3, and / or 1-5 amino acids in LC-FR4 are substituted with another amino acid. In some embodiments, the substitution may be a reversion mutation of the residues at the corresponding positions in SEQ ID NO 10 or 11. In some embodiments, the number of substitutions in each FR region may be 1, 2, or 3. In some implementations, the total number of replacements made to the human frame region can be 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, or 1 to 5; for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0144] An exemplary humanized antibody comprises a heavy chain variable region containing HC-FR1, HC-FR2, HC-FR3, and HC-FR4, wherein: HC-FR1 has sequences selected from SEQ ID NO: 90, SEQ ID NO: 98 and SEQ ID NO: 102; HC-FR2 has the sequence shown in SEQ ID NO: 91; HC-FR3 has sequences selected from SEQ ID NO: 92, SEQ ID NO: 99, SEQ ID NO: 103, and SEQ ID NO: 105; and HC-FR4 has sequences selected from SEQ ID NO: 93 and SEQ ID NO: 100; and / or It includes light chain variable regions containing LC-FR1, LC-FR2, LC-FR3, and LC-FR4, wherein: LC-FR1 has sequences selected from SEQ ID NO: 94 and SEQ ID NO: 101; LC-FR2 has sequences selected from SEQ ID NO: 95 and SEQ ID NO: 106; LC-FR3 has sequences selected from SEQ ID NO: 96 and SEQ ID NO: 104; and LC-FR4 has the sequence shown in SEQ ID NO: 97.

[0145] These frame regions can be combined with CDRs as described above.

[0146] An exemplary antibody / antigen binding molecule may have a VH region and a VL region, wherein the VH region has a sequence selected from SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112 and SEQ ID NO:113 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with such sequences, and the VL region has a sequence selected from SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117 and SEQ ID NO:118 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with such sequences.

[0147] In one embodiment, the antigen-binding molecule may have: a VH region having the sequence of SEQ ID NO: 109 and a VL region having the sequence of SEQ ID NO: 116. In another embodiment, the antigen-binding molecule may have: a VH region having the sequence of SEQ ID NO: 110 and a VL region having the sequence of SEQ ID NO: 117. In another embodiment, the antigen-binding molecule may have: a VH region having the sequence of SEQ ID NO: 111 and a VL region having the sequence of SEQ ID NO: 118. In another embodiment, the antigen-binding molecule may have: a VH region having the sequence of SEQ ID NO: 110 and a VL region having the sequence of SEQ ID NO: 118. In another embodiment, the antigen-binding molecule may have: a VH region having the sequence of SEQ ID NO: 111 and a VL region having the sequence of SEQ ID NO: 117. In another embodiment, the antigen-binding molecule may have: a VH region having the sequence of SEQ ID NO: 112 and a VL region having the sequence of SEQ ID NO: 117. In another embodiment, the antigen-binding molecule may have: a VH region having the sequence of SEQ ID NO: 112 and a VL region having the sequence of SEQ ID NO: 118. In another embodiment, the antigen-binding molecule may have: a VH region having the sequence of SEQ ID NO: 113 and a VL region having the sequence of SEQ ID NO: 117. In another embodiment, the antigen-binding molecule may have: a VH region having the sequence of SEQ ID NO: 113 and a VL region having the sequence of SEQ ID NO: 118.

[0148] Any antigen-binding molecule derived from 4F4 (i.e., any of the antibody / antigen-binding molecules listed above) may have any of the properties listed above, such as being able to partially or preferentially inhibit, prolong the half-life of endogenously present IFNγ in vivo, and / or cause the accumulation of endogenously present IFNγ in vivo, and / or be provided as part of a multispecific antibody that also contains a binding site against a target molecule such as TAA.

[0149] Any antigen-binding molecule derived from 4F4 (i.e., any of the antibody / antigen-binding molecules listed above) can retain the affinity for IFNγ as described herein, for example, it can bind IFNγ with submicromolar affinity, such as human and / or primate IFNγ, i.e., K D <1 x 10 -6 M. In some embodiments, the antigen-binding molecule described herein binds IFNγ, i.e., K, with an affinity in the nanomolar range.D = 9.9 x 10 -7 Up to 1 x 10 -9 M. In some embodiments, the antigen-binding molecule described herein binds IFNγ, i.e., K, with sub-nanomolar affinity. D <1 x 10 -9 M. In some embodiments, the antigen-binding molecule described herein binds IFNγ, i.e., K, with an affinity in the picomolar range. D = 9.9 x 10 -10 Up to 1 x 10 -12 M. In some embodiments, the antigen-binding molecule described herein has a KD of 9.9 x 10⁻⁶. -10 Up to 1 x 10 -13 The antigen-binding molecule described herein binds to IFNγ with an affinity in the M range. In some embodiments, the antigen-binding molecule binds to IFNγ with a subpicomolar affinity, i.e., K... D <1 x 10 -12 M. In some embodiments, the antibody is in K0 of less than 20 nM, less than 10 nM, or less than 5 nM. D Combined with human IFNγ.

[0150] In another embodiment, this disclosure provides an antibody / antigen binding molecule that binds to the same epitope as an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 10 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 11. Any such antigen binding molecule may have the properties listed above, for example, it may partially or preferentially inhibit as listed above. Any such antigen binding molecule may have the affinity for IFNγ as listed herein. Any such antigen binding molecule may prolong the half-life of endogenously present IFNγ in vivo and / or lead to the accumulation of endogenously present IFNγ in vivo and / or may be provided as part of a multispecific antibody that also comprises a binding site against a target molecule such as a TAA.

[0151] 2C1 and antibodies derived from it In one embodiment, this disclosure relates to an IFNγ-binding domain comprising an antigen-binding site specific for IFNγ, wherein the IFNγ-binding domain comprises a heavy chain variable region containing HC-CDR1, HC-CDR2, and HC-CDR3, wherein: a) The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 12 or a variant thereof in which one, two or three amino acids in HC-CDR1 are replaced by another amino acid; b) The heavy chain CDR2 contains the amino acid sequence of SEQ ID NO: 13 or a variant thereof in which one, two or three amino acids in HC-CDR2 are replaced by another amino acid; c) The heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 14 or its variants in which one, two or three amino acids in HC-CDR3 are replaced by another amino acid; And / or the IFNγ-binding domain contains a light chain variable region comprising LC-CDR1, LC-CDR2, and LC-CDR3, wherein: d) The light chain CDR1 contains the amino acid sequence of SEQ ID NO: 15 or a variant thereof in which one, two, or three amino acids in LC-CDR1 are replaced by another amino acid; e) The light chain CDR2 contains the amino acid sequence of SEQ ID NO: 16 or a variant thereof in which one, two or three amino acids in LC-CDR2 are replaced by another amino acid; f) The light chain CDR3 contains the amino acid sequence of SEQ ID NO: 17 or a variant thereof in which one, two or three amino acids in LC-CDR3 are replaced by another amino acid.

[0152] In some embodiments, the antigen-binding molecule includes an IFNγ-binding domain, wherein the antigen-binding site includes a heavy chain variable region containing HC-CDR1, HC-CDR2, and HC-CDR3, wherein: a) The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO:12 or a variant thereof in which one, two or three amino acids in HC-CDR1 are replaced by another amino acid; b) The heavy chain CDR2 contains the amino acid sequence of SEQ ID NO:13 or a variant thereof in which one, two or three amino acids in HC-CDR2 are replaced by another amino acid; c) The heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 14; And / or wherein the antigen binding site comprises a light chain variable region containing LC-CDR1, LC-CDR2 and LC-CDR3, wherein: d) The light chain CDR1 contains the amino acid sequence of SEQ ID NO:15 or a variant thereof in which one, two, or three amino acids in LC-CDR1 are replaced by another amino acid; e) The light chain CDR2 contains the amino acid sequence of SEQ ID NO: 16 or a variant thereof in which one, two or three amino acids in LC-CDR2 are replaced by another amino acid; f) The light chain CDR3 contains the amino acid sequence of SEQ ID NO: 17.

[0153] In some embodiments, the antibody / antigen binding molecule comprises a VH region having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 18, optionally at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or a VL region having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 19, optionally at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the antibody / antigen binding molecule comprises a CDR (i.e., any substitution is within the frame region) as listed above. Optionally, the substitutions in VH and / or VL (e.g., in the frame region) may be residues found at the corresponding positions in the human lineage antibody sequence.

[0154] In some embodiments, the antibody / antigen binding molecule is a humanized antibody, such as comprising the CDR and human framework region as listed above, i.e., HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LCFR3, and LC-FR4 of a human germline antibody; or a variant thereof wherein 1-5 amino acids in HC-FR1, and / or 1-5 amino acids in HC-FR2, and / or 1-5 amino acids in HC-FR3, and / or 1-5 amino acids in HC-FR4, and / or 1-5 amino acids in LC-FR1, and / or 1-5 amino acids in LC-FR2, and / or 1-5 amino acids in LC-FR3, and / or 1-5 amino acids in LC-FR4 are substituted with another amino acid. In some embodiments, the substitution may be a reversion mutation of the residues at the corresponding positions in SEQ ID NO 18 or 19. In some implementations, the number of substitutions in each FR region can be 1, 2, or 3. In some implementations, the total number of substitutions in the human frame region can be 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, or 1 to 5; for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0155] Any antibody / antigen binding molecule derived from 2C1 (i.e., any of the antibodies listed above) may have any of the properties listed above, such as being able to partially or preferentially inhibit, prolong the half-life of endogenously present IFNγ in vivo, and / or cause the accumulation of endogenously present IFNγ in vivo, and / or may be provided as part of a multispecific antibody / antigen binding molecule that also contains a binding site against a target molecule such as TAA.

[0156] Any antibody / antigen binding molecule derived from 2C1 (i.e., any of the antibodies listed above) can retain the affinity for IFNγ as described herein, for example, it can bind IFNγ with an affinity in the micromolar range, such as human and / or primate IFNγ, i.e., K D = 9.9 x 10 -4 Up to 1 x 10 -6 M. In some embodiments, the antigen-binding molecule described herein binds IFNγ, i.e., K, with sub-micromolar affinity. D <1 x 10 -6 M. In some embodiments, the antigen-binding molecule described herein binds IFNγ, i.e., K, with an affinity in the nanomolar range. D = 9.9 x 10 -7 Up to 1 x 10 -9 M. In some embodiments, the antigen-binding molecule described herein binds IFNγ, i.e., K, with sub-nanomolar affinity. D <1 x 10 -9 M. In some embodiments, the antigen-binding molecule described herein binds IFNγ, i.e., K, with an affinity in the picomolar range. D = 9.9 x 10 -10 Up to 1 x 10 -12 M. In some embodiments, the antigen-binding molecule described herein binds IFNγ, i.e., K, with subpimolar affinity. D <1 x 10 -12 M. In some embodiments, the antibody is in K0 of less than 20 nM, less than 10 nM, or less than 5 nM. D Combined with human IFNγ.

[0157] In another embodiment, this disclosure provides an antibody / antigen binding molecule that binds to the same epitope as an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 18 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 19. Any such antibody / antigen binding molecule may have the properties listed above, for example, it may partially or preferentially inhibit as listed above. Any such antibody / antigen binding molecule may have the affinity for IFNγ as listed herein. Any such antibody may prolong the half-life of endogenously present IFNγ in vivo and / or cause the accumulation of endogenously present IFNγ in vivo and / or may be provided as part of a multispecific antibody / antigen binding molecule that also includes a binding site against a target molecule such as a TAA.

[0158] 3G1 and antibodies derived from it In one embodiment, this disclosure relates to an antigen-binding molecule comprising an IFNγ-binding domain, wherein the IFNγ-binding domain comprises a heavy chain variable region containing HC-CDR1, HC-CDR2, and HC-CDR3, wherein: a) The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 20 or a variant thereof in which one, two or three amino acids in HC-CDR1 are replaced by another amino acid; b) The heavy chain CDR2 contains the amino acid sequence of SEQ ID NO: 21 or a variant thereof in which one, two or three amino acids in HC-CDR2 are replaced by another amino acid; c) The heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 22 or a variant thereof in which one, two or three amino acids in HC-CDR3 are replaced by another amino acid; And / or the IFNγ-binding domain contains a light chain variable region comprising LC-CDR1, LC-CDR2, and LC-CDR3, wherein: d) The light chain CDR1 contains the amino acid sequence of SEQ ID NO: 23 or a variant thereof in which one, two, or three amino acids in LC-CDR1 are replaced by another amino acid; e) The light chain CDR2 contains the amino acid sequence of SEQ ID NO: 24 or a variant thereof in which one, two or three amino acids in LC-CDR2 are replaced by another amino acid; f) The light chain CDR3 contains the amino acid sequence of SEQ ID NO: 25 or a variant thereof in which one, two or three amino acids in LC-CDR3 are replaced by another amino acid.

[0159] In some embodiments, the antigen-binding molecule includes an IFNγ-binding domain, wherein the IFNγ-binding domain includes a heavy chain variable region containing HC-CDR1, HC-CDR2, and HC-CDR3, wherein: a) The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO:20 or a variant thereof in which one, two or three amino acids in HC-CDR1 are replaced by another amino acid; b) The heavy chain CDR2 contains the amino acid sequence of SEQ ID NO:21 or a variant thereof in which one, two or three amino acids in HC-CDR2 are replaced by another amino acid. c) The heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 22; And / or wherein the antigen binding site comprises a light chain variable region containing LC-CDR1, LC-CDR2 and LC-CDR3, wherein: d) The light chain CDR1 contains the amino acid sequence of SEQ ID NO:23 or a variant thereof in which one, two, or three amino acids in LC-CDR1 are replaced by another amino acid; e) The light chain CDR2 contains the amino acid sequence of SEQ ID NO: 24 or a variant thereof in which one, two or three amino acids in LC-CDR2 are replaced by another amino acid; f) The light chain CDR3 contains the amino acid sequence of SEQ ID NO: 25.

[0160] In some embodiments, the antibody / antigen binding molecule comprises a VH region having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 26, optionally at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or a VL region having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 27, optionally at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the antibody / antigen binding molecule comprises a CDR (i.e., any substitutions are within the frame region) as listed above. Optionally, the substitutions in VH and / or VL (e.g., in the frame region) may be residues found at the corresponding positions in the human lineage antibody sequence.

[0161] In some embodiments, the antibody / antigen binding molecule is a humanized antibody / antigen binding molecule, for example comprising the CDR and human framework region as listed above, i.e., HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LCFR3, and LC-FR4 of human germline antibodies; or variants thereof wherein 1-5 amino acids in HC-FR1, and / or 1-5 amino acids in HC-FR2, and / or 1-5 amino acids in HC-FR3, and / or 1-5 amino acids in HC-FR4, and / or 1-5 amino acids in LC-FR1, and / or 1-5 amino acids in LC-FR2, and / or 1-5 amino acids in LC-FR3, and / or 1-5 amino acids in LC-FR4 are substituted with another amino acid. In some embodiments, the substitution may be a reversion mutation of the residues at the corresponding positions of SEQ ID NO 26 or 27. In some implementations, the number of substitutions in each FR region can be 1, 2, or 3. In some implementations, the total number of substitutions in the human frame region can be 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, or 1 to 5; for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0162] Any antibody / antigen binding molecule derived from 3G1 (i.e., any of the antibodies listed above) may have any of the properties listed above, such as being able to partially or preferentially inhibit, prolong the half-life of endogenously present IFNγ in vivo, and / or cause the accumulation of endogenously present IFNγ in vivo, and / or may be provided as part of a multispecific antibody / antigen binding molecule that also contains a binding site against a target molecule such as TAA.

[0163] Any antibody / antigen binding molecule derived from 3G1 (i.e., any of the antibodies listed above) can retain the affinity for IFNγ as described herein, for example, it can bind IFNγ with an affinity in the micromolar range, such as human and / or primate IFNγ, i.e., K D = 9.9 x 10 -4 Up to 1 x 10 -6 M. In some embodiments, the antigen-binding molecule described herein binds IFNγ, i.e., K, with sub-micromolar affinity. D <1 x 10 -6 M. In some embodiments, the antigen-binding molecule described herein binds IFNγ, i.e., K, with an affinity in the nanomolar range. D = 9.9 x 10 -7 Up to 1 x 10 -9M. In some embodiments, the antigen-binding molecule described herein binds IFNγ, i.e., K, with sub-nanomolar affinity. D <1 x 10 -9 M. In some embodiments, the antigen-binding molecule described herein binds IFNγ, i.e., K, with an affinity in the picomolar range. D = 9.9 x 10 -10 Up to 1 x 10 -12 M. In some embodiments, the antigen-binding molecule described herein binds IFNγ, i.e., K, with subpimolar affinity. D <1 x 10 -12 M. In some embodiments, the antibody is in K0 of less than 20 nM, less than 10 nM, or less than 5 nM. D Combined with human IFNγ.

[0164] In another embodiment, this disclosure provides an antibody / antigen binding molecule that binds to the same epitope as an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 26 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 27. Any such antibody / antigen binding molecule may have the properties listed above, for example, it may partially or preferentially inhibit as listed above. Any such antibody / antigen binding molecule may have the affinity for IFNγ as listed herein. Any such antibody / antigen binding molecule may prolong the half-life of endogenously present IFNγ in vivo and / or lead to the accumulation of endogenously present IFNγ in vivo and / or may be provided as part of a multispecific antibody / antigen binding molecule that also includes a binding site against a target molecule such as TAA.

[0165] 9F1 and antibodies derived from it In one embodiment, this disclosure relates to an antigen-binding molecule comprising an IFNγ-binding domain, wherein the antigen-binding site comprises a heavy chain variable region containing HC-CDR1, HC-CDR2, and HC-CDR3, wherein: a) The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 28 or a variant thereof in which one, two or three amino acids in HC-CDR1 are replaced by another amino acid; b) The heavy chain CDR2 contains the amino acid sequence of SEQ ID NO: 29 or a variant thereof in which one, two or three amino acids in HC-CDR2 are replaced by another amino acid; c) The heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 30 or a variant thereof in which one, two or three amino acids in HC-CDR3 are replaced by another amino acid; And / or the IFNγ-binding domain contains a light chain variable region comprising LC-CDR1, LC-CDR2, and LC-CDR3, wherein: d) The light chain CDR1 contains the amino acid sequence of SEQ ID NO: 31 or a variant thereof in which one, two or three amino acids in LC-CDR1 are replaced by another amino acid; e) The light chain CDR2 contains the amino acid sequence of SEQ ID NO: 32 or a variant thereof in which one, two or three amino acids in LC-CDR2 are replaced by another amino acid; f) The light chain CDR3 contains the amino acid sequence of SEQ ID NO: 33 or its variants in which one, two or three amino acids in LC-CDR3 are replaced by another amino acid.

[0166] In some embodiments, the antigen-binding molecule includes an IFNγ-binding domain, wherein the antigen-binding site includes a heavy chain variable region containing HC-CDR1, HC-CDR2, and HC-CDR3, wherein: a) The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO:28 or a variant thereof in which one, two, or three amino acids in HC-CDR1 are replaced by another amino acid; b) The heavy chain CDR2 contains the amino acid sequence of SEQ ID NO:29 or a variant thereof in which one, two or three amino acids in HC-CDR2 are replaced by another amino acid; c) The heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 30; And / or the IFNγ-binding domain contains a light chain variable region comprising LC-CDR1, LC-CDR2, and LC-CDR3, wherein: d) The light chain CDR1 contains the amino acid sequence of SEQ ID NO:31 or a variant thereof in which one, two, or three amino acids in LC-CDR1 are replaced by another amino acid; e) The light chain CDR2 contains the amino acid sequence of SEQ ID NO: 32 or a variant thereof in which one, two or three amino acids in LC-CDR2 are replaced by another amino acid; f) The light chain CDR3 contains the amino acid sequence of SEQ ID NO: 33.

[0167] In some embodiments, the antibody / antigen binding molecule comprises a VH region having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 34, optionally at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or a VL region having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 35, optionally at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the antibody / antigen binding molecule comprises a CDR (i.e., any substitutions are within the frame region) as listed above. Optionally, the substitutions in VH and / or VL (e.g., in the frame region) may be residues found at the corresponding positions in the human lineage antibody sequence.

[0168] In some embodiments, the antibody / antigen binding molecule is a humanized antibody / antigen binding molecule, for example comprising the CDR and human framework region as listed above, i.e., HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LCFR3, and LC-FR4 of human germline antibodies; or variants thereof wherein 1-5 amino acids in HC-FR1, and / or 1-5 amino acids in HC-FR2, and / or 1-5 amino acids in HC-FR3, and / or 1-5 amino acids in HC-FR4, and / or 1-5 amino acids in LC-FR1, and / or 1-5 amino acids in LC-FR2, and / or 1-5 amino acids in LC-FR3, and / or 1-5 amino acids in LC-FR4 are substituted with another amino acid. In some embodiments, the substitution may be a reversion mutation of the residues at the corresponding positions of SEQ ID NO 34 or 35. In some implementations, the number of substitutions in each FR region can be 1, 2, or 3. In some implementations, the total number of substitutions in the human frame region can be 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, or 1 to 5; for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0169] Any antibody / antigen binding molecule derived from 9F1 (i.e., any of the antibodies listed above) may have any of the properties listed above, such as being able to partially or preferentially inhibit, prolong the half-life of endogenously present IFNγ in vivo, and / or cause the accumulation of endogenously present IFNγ in vivo, and / or may be provided as part of a multispecific antibody / antigen binding molecule that also contains a binding site against a target molecule such as TAA.

[0170] Any antibody / antigen-binding molecule derived from 9F1 (i.e., any of the antibodies listed above) can retain the affinity for IFNγ as described herein, for example, it can bind IFNγ with an affinity in the micromolar range, such as human and / or primate IFNγ, i.e., K. D = 9.9 x 10 -4 Up to 1 x 10 -6 M. In some embodiments, the antigen-binding molecule described herein binds IFNγ, i.e., K, with sub-micromolar affinity. D <1 x 10 -6 M. In some embodiments, the antigen-binding molecule described herein binds IFNγ, i.e., K, with an affinity in the nanomolar range. D = 9.9 x 10 -7 Up to 1 x 10 -9 M. In some embodiments, the antigen-binding molecule described herein binds IFNγ, i.e., K, with sub-nanomolar affinity. D <1 x 10 -9 M. In some embodiments, the antigen-binding molecule described herein binds IFNγ, i.e., K, with an affinity in the picomolar range. D = 9.9 x 10 -10 Up to 1 x 10 -12 M. In some embodiments, the antigen-binding molecule described herein binds IFNγ, i.e., K, with subpimolar affinity. D <1 x 10 -12 M. In some embodiments, the antibody is in K0 of less than 20 nM, less than 10 mM, less than 5 nM, or less than 1 nM. D Combined with human IFNγ.

[0171] In another embodiment, this disclosure provides an antibody / antigen binding molecule that binds to the same epitope as an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 34 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 35. Any such antibody / antigen binding molecule may have the properties listed above, for example, it may partially or preferentially inhibit as listed above. Any such antibody may have the affinity for IFNγ as listed above. Any such antibody / antigen binding molecule may prolong the half-life of endogenously present IFNγ in vivo and / or lead to the accumulation of endogenously present IFNγ in vivo and / or may be provided as part of a multispecific antibody / antigen binding molecule that also includes a binding site against a target molecule such as a TAA.

[0172] In some implementations, antibody / antigen binding molecules derived from or binding to the same epitope as 9F1 may have one or more of the following properties: i) PD-L1 expression is strongly suppressed to less than 25% of the level observed in the absence of antibody but in the presence of the same amount of IFNγ (e.g., at 1 mM and / or 10 mM and / or 100 nM of antigen-binding molecules as described above), while maintaining the level of MHC-1 expression, which is at least 50% of the level observed, for example, in the presence of the same amount of IFNγ but in the absence of antigen-binding molecules, as in the assay of Example 5. ii) Maintain MHC-1 expression levels that are at least 50% of the levels observed, for example, in the presence of the same amount of IFNγ but in the absence of antigen-binding molecules, for example at 1 mM and / or 10 mM of antigen-binding molecules as described above and / or even at a concentration of 100 nM of antigen-binding molecules, as in the assay of Example 5; and / or iii) Inhibit the heterotetramerization of IFNγR1 and IFNγR2 subunits mediated by IFNγ, for example at a concentration of 1 nM, as described in the assay in Example 3.

[0173] 16D2 and antibodies derived from it In one embodiment, this disclosure relates to an antigen-binding molecule comprising an IFNγ-binding domain, wherein the IFNγ-binding domain comprises a heavy chain variable region containing HC-CDR1, HC-CDR2, and HC-CDR3, wherein: a) The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 36 or a variant thereof in which one, two or three amino acids in HC-CDR1 are replaced by another amino acid; b) The heavy chain CDR2 contains the amino acid sequence of SEQ ID NO: 37 or a variant thereof in which one, two or three amino acids in HC-CDR2 are replaced by another amino acid; c) The heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 38 or a variant thereof in which one, two or three amino acids in HC-CDR3 are replaced by another amino acid; And / or the IFNγ-binding domain contains a light chain variable region comprising LC-CDR1, LC-CDR2, and LC-CDR3, wherein: d) The light chain CDR1 contains the amino acid sequence of SEQ ID NO: 39 or a variant thereof in which one, two, or three amino acids in LC-CDR1 are replaced by another amino acid; e) The light chain CDR2 contains the amino acid sequence of SEQ ID NO: 40 or a variant thereof in which one, two or three amino acids in LC-CDR2 are replaced by another amino acid; f) The light chain CDR3 contains the amino acid sequence of SEQ ID NO: 41 or a variant thereof in which one, two or three amino acids in LC-CDR3 are replaced by another amino acid.

[0174] In some embodiments, the antigen-binding molecule includes an IFNγ-binding domain, wherein the IFNγ-binding domain includes a heavy chain variable region containing HC-CDR1, HC-CDR2, and HC-CDR3, wherein: a) The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO:36 or a variant thereof in which one, two or three amino acids in HC-CDR1 are replaced by another amino acid. b) The heavy chain CDR2 contains the amino acid sequence of SEQ ID NO:37 or a variant thereof in which one, two or three amino acids in HC-CDR2 are replaced by another amino acid; c) The heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 38; And / or the IFNγ-binding domain contains a light chain variable region comprising LC-CDR1, LC-CDR2, and LC-CDR3, wherein: d) The light chain CDR1 contains the amino acid sequence of SEQ ID NO:39 or a variant thereof in which one, two, or three amino acids in LC-CDR1 are replaced by another amino acid; e) The light chain CDR2 contains the amino acid sequence of SEQ ID NO: 40 or a variant thereof in which one, two or three amino acids in LC-CDR2 are replaced by another amino acid; f) The light chain CDR3 contains the amino acid sequence of SEQ ID NO: 41.

[0175] In some embodiments, the antibody / antigen binding molecule comprises a VH region having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 42, optionally at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or a VL region having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 43, optionally at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the antibody / antigen binding molecule comprises a CDR (i.e., any substitutions are within the frame region) as listed above. Optionally, the substitutions in VH and / or VL (e.g., in the frame region) may be residues found at corresponding positions in the sequence of human antibody / antigen binding molecules.

[0176] In some embodiments, the antibody / antigen binding molecule is a humanized antibody / antigen binding molecule, for example comprising the CDR and human framework region as listed above, i.e., HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LCFR3, and LC-FR4 of human germline antibodies; or variants thereof wherein 1-5 amino acids in HC-FR1, and / or 1-5 amino acids in HC-FR2, and / or 1-5 amino acids in HC-FR3, and / or 1-5 amino acids in HC-FR4, and / or 1-5 amino acids in LC-FR1, and / or 1-5 amino acids in LC-FR2, and / or 1-5 amino acids in LC-FR3, and / or 1-5 amino acids in LC-FR4 are substituted with another amino acid. In some embodiments, the substitution may be a reversion mutation of the residues at the corresponding positions of SEQ ID NO 42 or 43. In some implementations, the number of substitutions in each FR region can be 1, 2, or 3. In some implementations, the total number of substitutions in the human frame region can be 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, or 1 to 5; for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0177] Any antibody / antigen binding molecule derived from 16D2 (i.e., any of the antibodies listed above) may have the properties listed above, such as being able to partially or preferentially inhibit, as listed above, prolong the half-life of endogenously present IFNγ in vivo and / or cause the accumulation of endogenously present IFNγ in vivo and / or be provided as part of a multispecific antibody / antigen binding molecule that also contains a binding site against a target molecule such as TAA.

[0178] Any antibody / antigen binding molecule derived from 16D2 (i.e., any of the antibodies listed above) can retain the affinity for IFNγ as described herein, for example, it can bind IFNγ with an affinity in the micromolar range, such as human and / or primate IFNγ, i.e., K D = 9.9 x 10 -4 Up to 1 x 10 -6 M. In some embodiments, the antigen-binding molecule described herein binds IFNγ, i.e., K, with sub-micromolar affinity. D <1 x 10 -6 M. In some embodiments, the antigen-binding molecule described herein binds IFNγ, i.e., K, with an affinity in the nanomolar range. D = 9.9 x 10 -7 Up to 1 x 10 -9 M. In some embodiments, the antigen-binding molecule described herein binds IFNγ, i.e., K, with sub-nanomolar affinity. D <1 x 10 -9 M. In some embodiments, the antigen-binding molecule described herein binds IFNγ, i.e., K, with an affinity in the picomolar range. D = 9.9 x 10 -10 Up to 1 x 10 -12 M. In some embodiments, the antigen-binding molecule described herein binds IFNγ, i.e., K, with subpimolar affinity. D <1 x 10 -12 M. In some implementations, the antibody is in K0 of less than 20 nM, less than 10 mM, or less than 5 nM. D Combined with human IFNγ.

[0179] In another embodiment, this disclosure provides an antibody / antigen binding molecule that binds to the same epitope as an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 42 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 43. Any such antibody / antigen binding molecule may have the properties listed above, for example, it may partially or preferentially inhibit as listed above. Any such antibody / antigen binding molecule may have the affinity for IFNγ as listed herein. Any such antibody / antigen binding molecule may prolong the half-life of endogenously present IFNγ in vivo and / or lead to the accumulation of endogenously present IFNγ in vivo and / or may be provided as part of a multispecific antibody / antigen binding molecule that also includes a binding site against a target molecule such as TAA.

[0180] Exemplary antigen-binding molecule - PSMA binding domain Any IFNγ-binding molecule discussed herein may exist as part of a multispecific (e.g., bispecific) molecule that also binds to a TAA. Many TAAs are known in the art, and the antigen-binding molecules of these TAAs are widely known and available to those skilled in the art.

[0181] In one specific implementation, the TAA may be prostate-specific membrane antigen (PSMA). Exemplary known antibodies capable of binding to PSMA include mAb 7E11-C5.3, capromab, ANT4044 (VH and VL as shown in SEQ ID NO: 59 and 60; heavy chain and light chain as shown in SEQ ID NO: 61 and 62), huJ591, J951, J415, J533, E99, 5D3, D2B, 107-1A4, YPSMA-1, YPSMA-2, 3E6, 2G7, 24.4E6, GCP-02, GCP-04, GCP-05, 3 / A12, 3 / E7, 3 / F11, and 3 / E6. In some embodiments, a multispecific (bispecific) antigen-binding molecule capable of binding PSMA may comprise the VH and VL domains of any of the antibodies, or variants thereof may comprise the CDR of the antibody and a VH or VL domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it. For example, antibody J591 is described in WO2004 / 098535 and WO2017212250 (the VH and VL domains of the deimmunized antibody are given as SEQ ID NO 25 and 26 of WO2017212250, respectively). ANT4044 is derived from J591 and is described as AB-03 in WO2017 / 212250 A1.

[0182] In some embodiments, the multispecific molecule as described herein may include ScFv derived from J591 or ANT4044 by fusing the VH and VL domains or variants thereof with a linker.

[0183] Another exemplary known conjugate of PSMA is a nanobody as described in WO2021 / 038571A1, such as nanobodies Nb7, Nb8, Nb13, or Nb37. In some embodiments, the PSMA conjugate may be a VHH comprising a CDR selected from the group consisting of: i) GYTDSNYYMS (CDRH1; SEQ ID NO: 44); GVNTGRGSTSYADSVKG (CDR-H2; SEQ ID NO: 45); and AACHFCDSLPKTQDEYIL (CDR-H3; SEQ ID NO: 46) ii) GWPYSTYSMN (CDR-H1; SEQ ID NO:47); GISSTMSGIIFAES (CDR-H2; SEQ ID NO:48); and RRDYSLSSSSSDDFDY (CDRH3; SEQ ID NO:49); and iii) GYTASFS (CDR-H1; SEQ ID NO:50); GVAVINVGVGSTYYADSV (CDR-H2; SEQ ID NO:51) and SLRWSRPPNPISEDAYNY (CDR-H3; SEQ ID NO:52).

[0184] In one implementation, CDR-H2 may contain the sequence GISSTMSGIIFAESKAGQFTISQDNA; SEQ ID NO: 53.

[0185] In some implementations, the PSMA-binding polypeptide may comprise one of the following sequences: QVQLQESGGG SVQAGGSLRL SCTAPGYTDS NYYMSWFRQA PGKEREWVAG VNTGRGSTSYADSVKGRFTI SQDNAKNTMF LQMNSLKPED TAIYYCAVAA CHFCDSLPKT QDEYILWGQG TQVTVSSAAAYPYDVPDYGS (Nb7; SEQ ID NO: 54) QVQLQESGGG SVQAGGSLRL SCARSGWPYS TYSMNWFRQA PGKEREAVAG ISSTMSGIIFAESKAGQFTI SQDNAKNTVY LQMNNLKPED TAIYYCAARR DYSLSSSSDD FDYWGQGTQV TVSSAAAYPYDVPDYGS (SEQ ID NO: 55) QVQLQESGGG SVQTGGSLRL SCAASGYTAS FSWIGYFRQA PGKEREGVAV INVGVGSTYYADSVKGRFTI SRDNTENTIS LEMNSLKPED TGLYYCAGSL RWSRPPNPIS EDAYNYWGQG TQVTVSSAAAYPYDVPDYGS (SEQ ID NO: 56) QVQLQESGGG SCARSGWPYS TYSMNWFRQA PGKEREAVAG ISSTMSGIIFAESKAGQFTI SQDNAKNTVY LQMNNLKPED TAIYYCAARR DYSLSSSSDD FDYWGQGTQV TVSSAAAYPYDVPDYGS (SEQ ID NO: 57) Nb7 has a picomolar binding affinity for PSMA and may be preferred in some embodiments.

[0186] In some embodiments, the PSMA-binding antibody may comprise a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with SEQ ID NO: 54, 55 or 57, optionally while retaining the CDRs listed above. In some embodiments, the sequence may be humanized. In some embodiments, the humanized antibody may contain a Ki of no more than that of the parent antibody. D 10 times K D Combined with PSMA. In some implementations, the affinity for PSMA can be in the picomolar range, for example, K. D = 9.9x10 -10 Up to 1x10 -12 M.

[0187] Exemplary antigen-binding molecule – B7-H3 In another specific implementation, the TAA can be B7-H3 (also known as CD276).

[0188] In one implementation, the antigen-binding portion of B7-H3 may be an scFv. Exemplary scFvs capable of binding B7-H3 are described in WO2012 / 081052 A1 and Li et al. Mature Communications (2023) 14:5920.

[0189] In one implementation, the B7-H3 binding domain (e.g., scFv) contains CDRs of the following sequence: i) GFTYNSYS (HC-CDR1; SEQ ID NO: 119) ii) INSGGSST (HC-CDR2; SEQ ID NO: 120) iii) AARSPSPLTFQTRTLREDSYNYW (HC-CDR3; SEQ ID NO: 121).

[0190] In one implementation, the B7-H3 binding domain is the scFv of the following sequence: QVQLVESGGGSVQVGGSLRLSCAASGFTYNSYSVGWFRQAPGKEREGVAAINSGGSSTYYAASVKGRFTISRDNAKNTVYLQMNSLKPEDTAMYYCAARSPSPLTFQTRTLREDSYNYWGQGTQVTVSS (SEQ ID NO:122; as described by Li et al. 2023, “B12” V) H H).

[0191] In one embodiment, the B7-H3 binding domain may comprise a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with SEQ ID NO: 122, optionally while retaining the CDRs of SEQ ID NO: 119, 120 and 121 as listed above. In some embodiments, the sequence may be humanized. In some embodiments, the humanized antibody may have a KD not exceeding 10 times that of the parent antibody. D Binding to B7-H3. In some implementations, the affinity for B7-H3 can be in the nanomolar range or, for example, K. D = 9.9x10 -7 Up to 1x10 -9 M, or within the picomolar range, for example, KD = 9.9 x 10⁻⁶. -10 Up to 1x10 -12 M.

[0192] In another implementation, the B7-H3 binding domain (e.g., scFv) contains CDRs of the following sequence: i) EDSTSAMC (HC-CDR1; SEQ ID NO: 123) ii) INPTGEVT (HC-CDR2; SEQ ID NO: 124) iii) AARVTYGGDWSTDTDYEYW (HC-CDR3; SEQ ID NO: 125).

[0193] In one implementation, the B7-H3 binding domain is the scFv of the following sequence: V H H).

[0194] In one embodiment, the B7-H3 binding domain may comprise a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with SEQ ID NO: 126, optionally while retaining the CDRs of SEQ ID NO: 123, 124 and 125 as listed above. In some embodiments, the sequence may be humanized. In some embodiments, the humanized antibody may contain a K0 of no more than the parent antibody. D 10 times K D Binding to B7-H3. In some implementations, the affinity for B7-H3 can be in the nanomolar range or, for example, K. D = 9.9x10 -7 Up to 1x10 -9 M, or within the picomolar range, for example, KD = 9.9 x 10⁻⁶. -10 Up to 1x10 -12 M.

[0195] In another implementation, the B7-H3 binding domain (e.g., scFv) contains CDRs of the following sequence: i) GFTFSRYW (HC-CDR1; SEQ ID NO: 127) ii) INSGGGST (HC-CDR2; SEQ ID NO: 128) iii) AKEQWRTGSR (HC-CDR3; SEQ ID NO: 129).

[0196] In one implementation, the B7-H3 binding domain is the scFv of the following sequence: DVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMGWFRQAPGKGVEWVSTINSGGGSTYYADSVKGRFTISRDNAKNTLYLQLNNLKTEDTAMYYCAKEQWRTGSRGQGTQVTVSS (SEQ ID NO: 130; as described by Li et al. 2023, “G8” V) H H).

[0197] In another embodiment, the B7-H3 binding domain may comprise a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with SEQ ID NO: 130, optionally while retaining the CDRs of SEQ ID NO: 127, 128 and 129 as listed above. In some embodiments, the sequence may be humanized. In some embodiments, the humanized antibody may have a KD not exceeding 10 times that of the parent antibody. D Binding to B7-H3. In some implementations, the affinity for B7-H3 can be in the nanomolar range or, for example, K. D = 9.9x10 -7 Up to 1x10 -9 M, or in the picomoles range, such as K D = 9.9x10 -10 Up to 1x10 -12 M.

[0198] Other exemplary antibodies against B7-H3 are described in Ahmed M et al., Humanized Affinity-matured Monoclonal Antibody 8H9 Has Potent Antitumor Activity and Binds to FG Loop of Tumor Antigen B7-H3. J Biol Chem. 2015 Dec 11;290(50):30018-29. For example, the B7-H3 binding domain may include VH of SEQ ID NO: 132 and VL of SEQ ID NO: 133.

[0199] In another implementation, the B7-H3 binding domain may include: Humanized VH of SEQ ID: NO 134, optionally having one, two, three, or four of the following affinity-enhancing mutations: HC, A24T; HC, E42G; HC, G56D; and HC, A102G (linearly numbered); and Humanized VL of SEQ ID NO: 135, which optionally has one or both of the affinity-enhancing mutations LC, S20T and LC, H34Y (linear numbering).

[0200] In one implementation, VH may contain all four affinity-enhancing mutations, and VL may contain both affinity-enhancing mutations.

[0201] In one implementation, VH and VL, as described above, reside in the scFv domain. In another implementation, VH and VL reside in the Fab domain.

[0202] Fc area In some embodiments, the antigen-binding molecule of this disclosure includes an Fc region. The presence of the Fc region significantly prolongs the half-life of the antibody, and is also expected to significantly prolong the half-life of the antibody-bound IFNγ molecule.

[0203] Fc-mediated functions include Fc receptor binding, antibody-dependent cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), formation of the membrane attack complex (MAC), cell degranulation, production of cytokines and / or chemokines, and antigen processing and presentation. Modifications of the antibody Fc region affecting Fc-mediated functions are known in the art, such as those described, for example, in Wang et al., Protein Cell (2018) 9(1):63-73, which are hereby incorporated in their entirety by reference. Exemplary Fc region modifications known to affect antibody effector function are summarized in Table 1 of Wang et al., Protein Cell (2018) 9(1):63-73. In some embodiments, the antigen-binding molecules of this disclosure contain an Fc region modified to increase or decrease Fc-mediated function, compared to antigen-binding molecules containing a corresponding unmodified Fc region.

[0204] In cases where the Fc region / CH2 / CH3 is described as containing modifications “corresponding” to a reference substitution, consider equivalent substitutions in the homologous Fc / CH2 / CH3. For example, the L234A / L235A substitution in human IgG1 (according to the EU numbering system described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition. Public Health Service, National Institutes of Health, Bethesda, MD, 1991) corresponds to the L-to-A substitution at positions 117 and 118 in the C region of the mouse Ig γ-2A chain (UniProtKB: P01863-1, v1).

[0205] When the Fc region is described as containing a modification, that modification can be present in one or two polypeptide chains that together form the Fc region.

[0206] In some embodiments, the antigen-binding molecule of this disclosure includes a modified Fc region. In some embodiments, the antigen-binding molecule of this disclosure includes a modified Fc region in one or more CH2 and / or CH3 regions.

[0207] In some implementations, the modified Fc region contains one or more mutations selected from the group consisting of: R435H mutation, N434A mutation, T252L / T253S / T254F mutation, E294 deletion / T307P / N434Y mutation, T256N / A378V / S383N / N434Y mutation, E294 deletion mutation, M252Y / S254T / T256E mutation, M428L / N434S mutation, T307A / E380A / N434A mutation, T250Q / M428L mutation, T250Q / M428F mutation, T250E / M428F mutation, T250E / M428F mutation, T250E / M4 28L mutation, T256D / Q311V / A378V mutation, T256D / H286D / T307R / Q311V / A378V mutation, H285N / T307Q / N315D mutation, T307Q / Q311V / A378V mutation, H285D / T307Q / A378V mutation, L234F / L235E / P331S mutation, L234F / L235Q / K322Q mutation, L234F / L235Q / P331G mutation, L234F / L235A / K322Q mutation, S228P / F234A / L235A / G237A / P238S mutation, L23 4A / L235E / G237A / A330S / P331S mutation, F243A / V264A mutation, S228P / L235E / P329G mutation, M252Y / M428L mutation, D259I / V308F mutation, T307Q / N434S mutation, M428L / V308F mutation, Q311V / N434S mutation, H433K / N434F mutation, E258F / V427T mutation, K288E / H435K mutation, F234A / L235A mutation, F234A / L235A / S228P mutation, L234A / L235A / P329G mutation, S228 P / F234A / L235A / P329G mutation, Y349C / T366S / L368A / Y407V / F405K mutation, S354C / T366W / K409A mutation, L234S / L235T / G236R mutation, F243L / R292P / Y300L / V305I / P396L mutation, S239D / I332E mutation, S239D / I332E / A330L mutation, S298A / E333A / K334A mutation, G236A / S239D / I332E mutation, K326W / E333S mutation, S267E / H268F / S324T mutation.Mutations in L234Y / L235Q / G236W / S239M / H268D / D270E / S298A in one heavy chain and D270E / K326D / A330M / K334E in another heavy chain, E345R / E430G / S440Y mutation, P329G mutation, S228P mutation, deletion-K447 mutation, L234A mutation, L23 5A mutation, L235E mutation, G237A mutation, V308P mutation, V308W mutation, V308Y mutation, V308F mutation, N434S mutation, T307Q mutation, T307P mutation, T307A mutation, T307R mutation, Q312A mutation, Q318A mutation, Q233P mutation, K322A mutation, P329A mutation, P331 A mutation, P238A mutation, P238S mutation, F241A mutation, D265A mutation, D269A mutation, D270A mutation, N297A mutation, A327Q mutation, P329A mutation, S239A mutation, E294A mutation, Q295A mutation, V303A mutation, V264A mutation, A330R mutation, A330L mutation, A330S mutation, P331S mutation, T299A mutation, F234A mutation, T366W mutation, T366S mutation, L368A mutation, Y407V mutation, Y349C mutation, F405L mutation, F405K mutation, S354C mutation, T366W mutation, K409R mutation, K409A mutation, F405L / R409K mutation, and any combination thereof.

[0208] In some embodiments, the modified Fc region contains half-life-extending mutations selected from the group consisting of: R435H mutation, T307Q mutation, T307P mutation, T307A mutation, T307R mutation, N265 mutation, D265A mutation, N434S mutation, T250Q mutation, T250E mutation, M428L mutation, M428F mutation, V308W mutation, V308Y mutation, and V308F mutation. E294 deletion / T307P / N434Y mutation, T256N / A378V / S383N / N434Y mutation, M428L / N434S mutation, T307A / E380A / N434A mutation, T307Q / N434S mutation, Q311V / N434S mutation, H433K / N434F mutation, E294 deletion / T307P / N434Y mutation, T307A / E380A / N434A mutation, T256D / H286D / T307R / Q311V / A378V mutation, H285N / T307Q / N315D mutation, T307Q / Q311V / A378V mutation, H285D / T307Q / A378V mutation, T307Q / N434S mutation, M252Y / S254T / T256E mutation, T250Q / M428L mutation, T250Q / M4 28F mutation, T250E / M428F mutation, T250E / M428L mutation, M252Y / M428L mutation, M428L / V308F mutation, D259I / V308F mutation, E258F / V427T mutation, T256D / Q311V / A378V mutation, T256D / H286D / T307R / Q311V / A378V mutation and K288E / H435K mutation.

[0209] In some embodiments, the Fc region of the antibodies disclosed herein (e.g., bispecific antibodies) is modified to reduce or silence the effector functions of the antibody (e.g., antibody-dependent cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), opsonization, phagocytosis, transcytosis, infection neutralization, inflammation, mucosal immunity, and neonatal immunity). The modification is determined, for example, using methods conventional in the art. In some implementations, the modified Fc region contains one or more mutations selected from the group consisting of: R435H mutation, N434A mutation, T252L / T253S / T254F mutation, E294 deletion / T307P / N434Y mutation, T256N / A378V / S383N / N434Y mutation, E294 deletion mutation, M252Y / S254T / T256E mutation, M428L / N434S mutation, T307A / E380A / N434A mutation, T250Q / M428L mutation, T250Q / M428F mutation, T25 0E / M428F mutation, T250E / M428L mutation, T256D / Q311V / A378V mutation, T256D / H286D / T307R / Q311V / A378V mutation, H285N / T307Q / N315D mutation, T307Q / Q311V / A378V mutation, H285D / T307Q / A378V mutation, L234F / L235E / P331S mutation, L234F / L235Q / K322Q mutation, L234F / L235Q / P331G mutation, L234F / L235A / K322Q mutation Mutations include: S228P / F234A / L235A / G237A / P238S mutation, L234A / L235E / G237A / A330S / P331S mutation, F243A / V264A mutation, S228P / L235E / P329G mutation, M252Y / M428L mutation, D259I / V308F mutation, T307Q / N434S mutation, M428L / V308F mutation, Q311V / N434S mutation, H433K / N434F mutation, E258F / V427T mutation, K288E / H435K mutation, and F23. 4A / L235A mutation, F234A / L235A / S228P mutation, L234A / L235A / P329G mutation, S228P / F234A / L235A / P329G mutation, Y349C / T366S / L368A / Y407V / F405K mutation, S354C / T366W / K409A mutation, L234S / L235T / G236R mutation, P329G mutation, S228P mutation, deletion-K447 mutation, L234A mutation, L235A mutation, L235E mutation, G237A mutation, V308P mutation,V308W mutation, V308Y mutation, V308F mutation, N434S mutation, T307Q mutation, T307P mutation, T307A mutation, T307R mutation, Q312A mutation, Q318A mutation, Q233P mutation, K322A mutation, P329A mutation, P331A mutation, P238A mutation, P238S mutation, F241A mutation, D265A mutation, D269A mutation, D270A mutation, N297A mutation, A327Q mutation, P329A mutation, S239A mutation The following mutations are considered: E294A, Q295A, V303A, V264A, A330R, A330L, A330S, P331S, T299A, F234A, T366W, T366S, L368A, Y407V, Y349C, F405L, F405K, S354C, T366W, K409R, K409A, F405L / R409K, and any combination thereof.

[0210] In some embodiments, the Fc region of the antibodies disclosed herein (e.g., bispecific antibodies) is modified to enhance the effector functions of the antibody (e.g., antibody-dependent cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), or antibody-dependent phagocytosis (ADCP). In some embodiments, the modified Fc region contains one or more mutations selected from the group consisting of: F243L / R292P / Y300L / V305I / P396L mutation, S239D / I332E mutation, S239D / I332E / A330L mutation, S... 298A / E333A / K334A mutation, G236A / S239D / I332E mutation, K326W / E333S mutation, S267E / H268F / S324T mutation, L234Y / L235Q / G236W / S239M / H268D / D270E / S298A in one heavy chain and D270E / K326D / A330M / K334E in another heavy chain, E345R / E430G / S440Y mutation (resulting in hexamerization and enhanced CDC), and IgG1 / IgG3 cross subclass mutation.

[0211] In some embodiments, the modified Fc region contains mutations that improve antibody stability or downstream processing and are selected from the group consisting of: S228P mutation, R409K mutation, R409T mutation, R409M mutation, R409L mutation, S228P / L235E / R409K mutation, S228P / L235E / R409T mutation, S228P / L235E / R409M mutation, S228P / L235E / R409L mutation, G446 / deletion / K447 deletion mutation, K370Q mutation, K370E mutation, R409A mutation, R409E mutation, R409W mutation, C131S mutation, K370 deletion or substitution, deletion-K447 mutation, F405L mutation, K409R mutation, and F405L / R409K mutation.

[0212] In some embodiments, particularly those in which the antigen-binding molecule is a multispecific (e.g., bispecific) antigen-binding molecule, the antigen-binding molecule comprises an Fc region containing modifications in one or more CH2 and CH3 regions that promote association of the Fc region. Recombinant co-expression and subsequent association of the constituent peptides of the antigen-binding molecule result in several possible combinations. To improve the yield of desired peptide combinations in the antigen-binding molecule during recombinant production, it is advantageous to introduce modifications in the Fc region that promote association of the desired heavy chain peptide combination. These modifications can promote, for example, hydrophobic and / or electrostatic interactions between the CH2 and / or CH3 regions of different peptide chains. Suitable modifications are described, for example, in Ha et al., Front. Immnol (2016) 7:394, which is hereby incorporated in its entirety by reference.

[0213] In some embodiments, each heavy chain may contain a single-point mutation in the CH3 domain that allows for stronger interactions between the CH3 domains in bispecific antibody. In some embodiments, the single-point mutation in each CH3 domain is located at amino acid residues 366, 368, 370, 399, 405, 407, or 409 according to the EU numbering system. In some embodiments, the single-point mutation is located at a different residue in the CH3 domain compared to other CH3 domains. For example, according to the EU numbering system, one may contain the mutation F405L (i.e., a mutation from phenylalanine to leucine at residue 405), while another may contain the mutation K409R (i.e., a mutation from lysine to arginine at residue 409). In some embodiments, the heavy chain constant region of the monospecific antibody is selected from IgG1, IgG2, IgG3, or IgG4 isotypes (e.g., human IgG1 isotype).

[0214] In some embodiments, the antibody is IgG4, and both heavy chain constant regions have the sequence shown in SEQ ID NO: 141. In another embodiment, the antibody is a single-arm form of IgG4, wherein one heavy chain contains the constant region of SEQ ID NO: 141 (fused with VH), and the other heavy chain contains or consists of SEQ ID NO: 142. In some embodiments, the sequences of SEQ ID NO: 141 and / or 142 may contain the F234A / L235A mutation (i.e., F234A and L235A). Alternatively or additionally, the sequences of SEQ ID NO: 141 and / or SEQ ID NO: 142 may be modified with S228P to enhance in vivo stability. In some embodiments, the antibody is IgG4, and one heavy chain constant region has a sequence modified with T366S, L368A and Y407V as shown in SEQ ID NO:141 (and optionally further modified with F234A / L235A and / or S228P), and the other heavy chain has a sequence modified with T366W as shown in SEQ ID NO:141 (and optionally further modified with F234A / L235A and / or S228P). In another embodiment, the antibody is a single-arm form of IgG4, wherein the first heavy chain contains the constant region (fused with VH) of SEQ ID NO: 141, optionally containing the F234A / L235A mutation and / or S228P, and the second heavy chain contains or consists of SEQ ID NO: 142, optionally containing the F234A / L235A mutation and / or S228P, and wherein one of the heavy chains contains the mutation T366W and the other heavy chain contains the mutations T366S, L368A, and Y407V. In some embodiments, the antibody is IgG4, which comprises a first heavy chain with an F405L / R409K mutation and a second heavy chain without the mutation. In some embodiments, the antibody is IgG1, which comprises a first heavy chain with a K409R mutation and a second heavy chain with an F405L mutation.

[0215] In some embodiments, the antibodies disclosed herein (e.g., bispecific antibodies) are human IgG4 antibodies comprising a first heavy chain and a second heavy chain. In some embodiments, the first heavy chain comprises S228P / F405L / R409K / deletion-K447 and the second heavy chain comprises the S228P / deletion-K447 mutation. In some embodiments, the first heavy chain comprises S228P / F405L / R409K / F234A / L235A / deletion-K447 and the second heavy chain comprises the S228P / F234A / L235A / deletion-K447 mutation. In some embodiments, the first and second heavy chains also comprise the M252Y / S254T / T256E mutation. In some embodiments, the first and second heavy chains also comprise the F234A / L235A mutation.

[0216] In some embodiments, the first and second heavy chains further comprise the F234A / L235A mutation. In some embodiments, the antibody comprises a heavy chain constant region within the CH3 domain having a single-point mutation at an amino acid position selected from the group consisting of 366, 368, 370, 399, 405, 407, and 409. In some embodiments, the single-point mutation is either the F405L mutation or the K409R mutation. In some embodiments, the antibody is IgG4, comprising a first heavy chain with the F405L / R409K mutation and a second heavy chain without the mutation. In some embodiments, the antibody is IgG1, comprising a first heavy chain with the K409R mutation and a second heavy chain with the F405L mutation.

[0217] Antibodies may contain combinations of mutations as described above, for example, to prolong half-life and / or reduce or silence effector function and / or improve stability or downstream processing.

[0218] Form of bispecific antigen-binding molecules As described herein, aspects of this disclosure relate to antigen-binding molecules having more than one binding domain, wherein each binding domain binds a different antigen – i.e., multispecific antigen-binding molecules. The term “multispecific” is used herein to include “bispecific”.

[0219] The multispecific antigen-binding molecules according to this disclosure may be provided in any suitable form, such as those described in Brinkmann and Kontermann, MAbs (2017) 9(2): 182-212, which are hereby incorporated in their entirety by reference. Suitable forms include those described in Brinkmann and Kontermann, MAbs (2017) 9(2): 182-212: antibody conjugates Figure 2The forms shown include, for example, IgG2, F(ab')2, or CovX-Body; IgG or IgG-like molecules, such as IgG, chimeric IgG, κλ-common HC; CH1 / CL fusion proteins, such as scFv2-CH1 / CL, VHH2-CH1 / CL; 'variable domain only' bispecific antigen-binding molecules, such as tandem scFv (taFV), trisomy, dimer (Db), dsDb, Db(kih), DART, scDB, dsFv-dsFv, tandAb, trihead, tandem dAb / VHH, tetravalent dAb.VHH; non-Ig fusion proteins, such as scFv2-albumin, scDb-albumin, taFv-albumin, taFv-toxin, microantibodies, DNL-Fab2, DNL-Fab2-scFv, DNL-Fab2-IgG-cytokine 2, ImmTAC. (TCR-scFv); modified Fc and CH3 fusion proteins, such as scFv-Fc(kih), scFv-Fc(CH3 charge pair), scFv-Fc (EW-RVT), scFv-fc (HA-TF), scFv-Fc(SEEDbody), taFv-Fc(kih), scFv-Fc(kih)-Fv, Fab-Fc(kih)-scFv, Fab-scFv-Fc(kih), Fab-scFv-Fc(BEAT), Fab-scFv-Fc (SEEDbody), DART-Fc, scFv-CH3(kih), TriFab; Fc fusions, such as diadiposome, scDb-Fc, taFv-Fc, scFv-Fc-scFv, HCAb-VHH, Fab-scFv-Fc, scFv4-Ig, scFv2-Fcab; CH3 fusions, such as diadiposome, scDb-CH3; IgE / IgM CH2 fusions, such as scFv-EHD2-scFv, scFvMHD2-scFv; Fab fusion proteins, such as Fab-scFv (diadiposome), Fab-scFv2 (triadiposome), Fab-Fv, Fab-dsFv, Fab-VHH, orthogonal Fab-Fab; non-Ig fusion proteins, such as DNL-Fab3, DNL-Fab2-scFv, DNL-Fab2-IgG-cytokine 2;Asymmetric IgG or IgG-like molecules, such as IgG(kih), IgG(kih) co-LC, ZW1 IgG co-LC, Biclonics co-LC, CrossMab, CrossMab(kih), scFab-IgG(kih), Fab-scFab-IgG(kih), orthogonal Fab IgG(kih), DuetMab, CH3 charge pair + CH1 / CL charge pair, hinge / CH3 charge pair, SEED body, Duobody, tetra-CrossMab(kih), LUZ-Y co-LC; LUZ-Y scFab-IgG, FcFc*; IgG with appendage and Fc modification, such as IgG(kih)-Fv, IgG HA-TF-Fv, IgG(kih)scFab, scFab-Fc(kih)-scFv2, scFab-Fc(kih)-scFv, half-DVD-Ig, DVI-Ig. (Four-in-one), CrossMab-Fab; modified Fc and CH3 fusion proteins, such as Fab-Fc(kih)-scFv, Fab-scFv-Fc(kih), Fab-scFv-Fc(BEAT), Fab-scFv-Fc-SEEDbody, TriFab; IgG-HC fusions, such as IgG-HC, scFv, IgG-dAb, IgG-taFV, IgG-CrossFab, IgG-orthogonal Fab, IgG-(CαCβ) Fab, scFv-HC-IgG, tandem Fab-IgG (orthogonal Fab), Fab-IgG (CαCβ Fab), Fab-IgG (CR3), Fab-hinge-IgG (CR3); IgG-LC fusions, such as IgG-scFv(LC), scFv(LC)-IgG, dAb-IgG; IgG- HC and LC fusions, such as DVD-Ig, TVD-Ig, CODV-Ig, scFv4-IgG, Zybody; Fc fusions, such as Fab-scFv-Fc, scFv4-Ig; F(ab')2 fusions, such as F(ab')2-scFv2; CH1 / CL fusion proteins, such as scFv2-CH1-hinge / CL; modified IgGs, such as DAF (dual IgG), DutaMab, Mab; 2 ; and non-Ig fusion compounds, such as DNL-Fab4-IgG.

[0220] Technicians are capable of designing and preparing bispecific antigen-binding molecules. Methods for generating bispecific antigen-binding molecules include, for example, chemically cross-linking the antigen-binding molecule or antibody fragment with reducible disulfide bonds or non-reducible thioether bonds, as described in, for example, Segal and Bast, 2001. Production of Bispecific Antigen-binding molecules. Current Protocols in Immunology. 14:IV:2.13:2.13.1–2.13.16, which is hereby incorporated by reference in its entirety. For example, N 3-(-2-pyridyl dithio)-propionate (SPDP) can be used, for example, to chemically crosslink Fab fragments via the SH- group in the hinge region to produce disulfide-linked bispecific F(ab)2 heterodimers.

[0221] Other methods for generating bispecific antigen-binding molecules include fusing antibody-producing hybridomas, for example, with polyethylene glycol, to generate tetrahybridoma cells capable of secreting bispecific antibodies, as described in, for example, DM and Bast, BJ2001. Production of Bispecific Antigen-binding molecules. Current Protocols in Immunology. 14:IV:2.13:2.13.1–2.13.16.

[0222] The bispecific antigen-binding molecules disclosed herein can also be recombinantly generated by expression from, for example, nucleic acid constructs encoding peptides of antigen-binding molecules, as described in, for example, Antibody Engineering: Methods and Protocols, 2nd ed. (Humana Press, 2012), Chapter 40: Production of Bispecific Antigen-binding molecules: Diabodies and Tandem scFv (Hornig and Färber-Schwarz), or French, How to make bispecific antigen-binding molecules, MethodsMol. Med. 2000; 40:333-339, the entire contents of which are hereby incorporated by reference.

[0223] For example, a DNA construct encoding variable domains of light and heavy chains for two antigen-binding fragments (i.e., binding IFNγ and another target such as TAA) and including sequences encoding suitable linker or dimerizing domains between the antigen-binding fragments can be prepared using molecular cloning techniques. Subsequently, a recombinant bispecific antibody can be generated by expressing (e.g., in vitro) the construct in a suitable host cell (e.g., mammalian host cells), and the expressed recombinant bispecific antibody can then optionally be purified.

[0224] A preferred embodiment of the invention includes an Fc domain. The presence of the Fc domain significantly prolongs the half-life of the antibody. (In the absence of an Fc region, in some embodiments, it may be preferred that the antigen-binding molecule is conjugated to a half-life-extending portion such as a fatty acid, albumin, or hydrophilic polymer, as further discussed herein.)

[0225] The inventors unexpectedly determined that when the antibody is monovalent to IFNγ, the affinity for IFNγ increases significantly. For example, the antibody may have two arms, one arm containing a single binding site for IFNγ, and the other arm containing one or more binding sites for TAA (e.g., a single binding site for TAA).

[0226] In some embodiments, the multispecific (e.g., bispecific) antibody according to the invention may comprise a heterodimeric IgG antibody, wherein one arm of the antibody (i.e., a Fab region) binds IFNγ, and the other arm (i.e., another Fab) binds another target (e.g., TAA). Thus, the bispecific antibody comprises a full-length antibody (e.g., IgG) containing a first antibody heavy chain and a second antibody heavy chain, as well as a first antibody light chain and a second antibody light chain, wherein the first heavy chain and the first light chain assemble to form an antigen-binding site against a first antigen, and wherein the second heavy chain and the second light chain assemble to form an antigen-binding site against a second antigen. Proper assembly of the heterodimeric heavy chain can be assisted, for example, by using a club-and-mortar mutation and / or other modifications discussed further below. Proper assembly of the light chain with its corresponding heavy chain can be assisted by using cross-mab technology and / or charge modifications.

[0227] An exemplary embodiment of this form is formed from four chains (two light chains and two heavy chains) as listed in SEQ ID No. 75-78. The antibody thus formed is referred to herein as LDC-1.

[0228] In some embodiments of the above-described form, the form may be bivalent, having a single binding site for IFNγ and a single binding site for TAA. In another possible embodiment, an additional antigen-binding moiety may be fused to, for example, a first heavy chain and / or a second heavy chain to increase the valence of one or both antigens, although, as discussed above, it is preferred that the antibody be monovalent for IFNγ.

[0229] In other embodiments, the multispecific (e.g., bispecific) antibody may comprise a heterodimer formed of an Fc domain comprising a first Fc subunit and a second Fc subunit, wherein each subunit is fused to an antigen-binding moiety (directly or indirectly, via a linker), and wherein the first Fc subunit is fused, for example, at its N-terminus to an antigen-binding moiety capable of binding IFNγ, and the second Fc subunit is fused, for example, at its N-terminus to an antigen-binding moiety capable of binding TAA. The antigen-binding moiety may be any antibody fragment as disclosed herein, such as Fab, Fab', F(ab'), F(ab')2, Fd, scFv, microantibodies, variable heavy domains, variable light domains, variable NAR domains, single-chain binding peptides, dAb fragments, nanobodies, VHH, and other antibody fragments also referred to as antigen-binding fragments. For example, in some embodiments, the antigen-binding moiety may be Fab, ScFv, or a single-domain antibody / nanobody. In some embodiments, the first and second polypeptide chains of the Fc contain hinges or linkers covalently linked to the polypeptide containing the CH2 and CH3 domains of the Fc domain. In cases where the antigen-binding portion is Fab, the heavy chain of Fab can be fused to the Fc subunit via a hinge region – i.e., the antibody can comprise a complete IgG heavy chain containing CH3-CH2-hinge-CH1-VH, and a light chain containing CL1-VL, which conjugates with the heavy chain to form Fab (optionally with VL / VH or CL1 / CH1 region exchange, as in cross-mab). In one embodiment, the antibody comprises an arm containing Fab and an arm containing ScFv or nanobody / single-domain antibody. That is, in some embodiments, the antibody comprises: (1) a Fab fragment attached at the C-terminus of the CH1 domain to the N-terminus of the first polypeptide chain of the Fc domain; and (2) an scFv attached at its C-terminus to the N-terminus of the second polypeptide chain of the Fc domain. In other embodiments, the antibody comprises: (1) a Fab fragment attached at the C-terminus of the CH1 domain to the N-terminus of the first polypeptide chain of the Fc domain; and (2) a nanobody / single-domain antibody attached at its C-terminus to the N-terminus of the second polypeptide chain of the Fc domain. In one implementation, Fab is capable of binding IFNγ, and scFv or nanobody is capable of binding TAA.

[0230] An exemplary embodiment of this form is formed from the light chain of SEQ ID NO: 65 and the heavy chains of SEQ ID NO: 66 and 67. The antibody thus formed is referred to herein as LDC-4.

[0231] Another exemplary embodiment of this form is formed from the light chain of SEQ ID NO: 79 and the heavy chains of SEQ ID NO: 80 and 81. The antibody thus formed is referred to herein as LDC-6.

[0232] Another exemplary embodiment of this form is formed from the light chain of SEQ ID NO: 72 and the heavy chains of SEQ ID NO: 73 and 74. The antibody thus formed is referred to herein as LDC-2.

[0233] In another possible implementation, additional antigen-binding portions may be fused to, for example, a first heavy chain and / or a second heavy chain to increase the valence of one or both antigens, although, as discussed above, it is preferred that the antibody be monovalent for IFNγ.

[0234] Another exemplary form includes a full-length antibody, such as IgG, which may be IgG1, IgG2, IgG3, or IgG4, optionally IgG1 or optionally IgG4, containing an antigen-binding site against a first antigen (e.g., which may be bivalent for the first antigen), fused (directly or indirectly, via a linker) to an antigen-binding moiety against a second antigen. In some embodiments, the first antigen is IFNγ and the second antigen is in a TAA, in which case the antibody will be (at least) bivalent for IFNγ. In other embodiments, the first antigen is a TAA and the second antigen is IFNγ. The antigen-binding moiety against the second antigen may be any of the antibody fragments listed herein, such as Fab, Fab', F(ab'), F(ab')2, Fd, scFv, microantibodies, variable heavy domains, variable light domains, variable NAR domains, single-chain binding peptides, dAb fragments, nanobodies, VHHs, and other antibody fragments also referred to as antigen-binding fragments. In some embodiments, it may be a Fab, ScFv, or nanobody / single-domain antibody. In the case of scFv or nanobody / single-domain antibody, in some embodiments, the nanobody / single-domain antibody or scFv may be fused to the C-terminus of one of the two Fc subunits of IgG. In the case of Fab, in some embodiments, one chain of the Fab may be fused to the N-terminus of one or both IgG heavy chains via a linker to provide a tandem Fab. In some cases, one of the Fabs in a tandem Fab may be a cross-Fab. In other embodiments, one chain of the Fab may be fused to the C-terminus of one or both of the Fc subunits of IgG. In yet another embodiment, one chain of the Fab may be fused to the C-terminus of one Fc subunit of IgG, and the other chain of the Fab may be fused to the C-terminus of the other Fc subunit of IgG. In all embodiments, fusion may be performed via a linker.

[0235] For example, in some embodiments, the antibody comprises: (1) a first Fab fragment attached to the N-terminus of a first polypeptide chain in the Fc domain at the C-terminus of the CH1 domain; (2) a first scFv attached to the C-terminus of the first polypeptide chain in the Fc domain at its N-terminus; (3) a second Fab fragment attached to the N-terminus of a second polypeptide chain in the Fc domain at the C-terminus of the CH1 domain; and (4) a second scFv attached to the C-terminus of the second polypeptide chain in the Fc domain at its N-terminus.

[0236] An exemplary embodiment of this form is formed from the light chain of SEQ ID NO: 70 and the heavy chain of SEQ ID NO: 71. The antibody thus formed is referred to herein as LDC-3.

[0237] For example, in other embodiments, the antibody comprises: (1) a first Fab fragment attached to the N-terminus of a first polypeptide chain in the Fc domain at the C-terminus of the CH1 domain; (2) a first nanobody attached to the C-terminus of the first polypeptide chain in the Fc domain at its N-terminus; (3) a second Fab fragment attached to the N-terminus of a second polypeptide chain in the Fc domain at the C-terminus of the CH1 domain; and (4) a second nanobody attached to the C-terminus of the second polypeptide chain in the Fc domain at its N-terminus.

[0238] An exemplary embodiment of this form is formed from the light chain of SEQ ID NO: 63 and the heavy chain of SEQ ID NO: 64. The antibody thus formed is referred to herein as LDC-5.

[0239] In any of the above embodiments, when the molecule contains a Fab that binds to different antigens, the Fab targeting one of the antigens can be a crossfab.

[0240] The valence of any antigen molecule can be altered using the knowledge of a person skilled in the art. In some embodiments, the antibodies described herein have two or more (e.g., 2, 3, 4 or more) antigen-binding sites for binding to TAAs. In some embodiments, multispecific antigen-binding molecules may have antigen-binding sites targeting more than one TAA (e.g., two different TAAs) or targeting more than one different (e.g., non-overlapping) epitopes among TAAs. In some embodiments, the antibodies described herein have two or more (e.g., 2, 3, 4 or more) antigen-binding sites for binding IFNγ, although, as stated above, monovalent antibodies for IFNγ may be preferred due to the observed higher affinity.

[0241] In some embodiments of any of the above forms, the multispecific (e.g., bispecific) antibody is at least bivalent for IFNγ.

[0242] The form of single-specific antigen-binding molecules In some respects, the antigen-binding molecules described herein are single-specific, meaning they contain a single antigen-binding site, or they contain more than one antigen-binding site, where these sites have the same specificity. Therefore, they bind IFNγ without binding to another antigen.

[0243] In some implementations, monospecific antigen-binding molecules are also monovalent for IFNγ, meaning they contain a single antigen-binding site.

[0244] In some embodiments, the antigen-binding molecule may be an antibody. In some embodiments, it may be a single-arm IgG. A single-arm IgG may comprise an Fc domain containing a first Fc subunit and a second Fc subunit, and a single Fab domain coupled to one of the Fc subunits (e.g., wherein the C-terminus of the VH domain of the Fab is normally connected to the N-terminus of the Fc subunit via a hinge).

[0245] Connectors and other sequences In some embodiments, the antigen-binding molecule and peptide of this disclosure include one or more linker sequences between amino acid sequences. The linker sequence may be provided at one or both ends of one or more of the VH, VL, CH1-CH2 hinge regions, CH2 region, and CH3 region of the antigen-binding molecule / peptide.

[0246] Linker sequences are known to those skilled in the art and described, for example, in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369, which are hereby incorporated in their entirety by reference. In some embodiments, the linker sequence may be a flexible linker sequence. A flexible linker sequence allows relative movement of the amino acid sequences linked by the linker sequence. Flexible linkers are known to those skilled in the art and several have been identified in Chen et al., Adv Drug Deliv Rev (2013) 65(10):1357-1369. Flexible linker sequences typically contain a high proportion of glycine and / or serine residues.

[0247] In some embodiments, the adapter sequence comprises at least one glycine residue and / or at least one serine residue. In some embodiments, the adapter sequence consists of glycine and serine residues. In some embodiments, the adapter sequence comprises one or more copies (e.g., tandem) of the sequence motif G4S. In some embodiments, the adapter sequence has a length of 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25, or 1-30 amino acids. For example, the adapter may have the sequence (G4S). n (SEQ ID NO: 136), where n is 1-30, for example 1-10, preferably 2 or greater or 3 or greater, for example n=3 or n=6.

[0248] The antigen-binding molecules and peptides disclosed herein may additionally comprise additional amino acids or amino acid sequences. For example, antigen-binding molecules and peptides may comprise amino acid sequences that facilitate the expression, folding, transport, processing, purification, or detection of the antigen-binding molecule / peptide. For example, the antigen-binding molecule / peptide may optionally comprise a sequence encoding His (e.g., 6XHis), Myc, GST, MBP, FLAG, HA, E, or a biotinylate tag at its N- or C-terminus. In some embodiments, the antigen-binding molecule / peptide comprises a detectable moiety, such as fluorescent, luminescent, immunoassay, radiometric, chemical, nucleic acid, or enzyme labeling.

[0249] The antigen-binding molecules and peptides disclosed herein may additionally include a signal peptide (also known as a leader sequence or signal sequence). A signal peptide typically consists of a sequence of 5-30 hydrophobic amino acids forming a single α-helix. Secreted proteins and proteins expressed at the cell surface often contain signal peptides.

[0250] Signal peptides can be present at the N-terminus of antigen-binding molecules / peptides and can be present in newly synthesized antigen-binding molecules / peptides. Signal peptides facilitate the efficient transport and secretion of antigen-binding molecules / peptides. Signal peptides are typically removed by cleavage and are therefore not included in mature antigen-binding molecules / peptides secreted from cells expressing them.

[0251] Signal peptides are known for many proteins and are documented in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resources, Protein Databases, Ensembl, and InterPro, and / or can be identified / predicted using amino acid sequence analysis tools such as SignalP (Petersen et al., 2011 Nature Methods 8: 785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24:2172-2176).

[0252] Modifications and conjugates In some embodiments, the antibodies of this disclosure (e.g., bispecific antibodies) are modified, for example, by adding a portion that increases the cycle time of the antibody in circulation. Many such portions are known in the art, including fatty acids, albumins, and hydrophilic polymers such as PEG, polysialic acid (PSA), N-(2-hydroxypropyl)methacrylamide (HPMA), or dextran.

[0253] In some embodiments, the antibodies of this disclosure (e.g., bispecific antibodies) are modified, for example, by adding polyethylene glycol (PEG). In some embodiments, PEG modification (PEGylation) results in one or more of the following: improved cycle time, improved solubility, improved resistance to proteolysis, reduced antigenicity and immunogenicity, improved bioavailability, reduced toxicity, improved stability, and easier formulation.

[0254] It has been shown that glycosylation of immunoglobulins has a significant impact on their effector function, structural stability, and the rate of secretion from antibody-producing cells. The carbohydrate groups responsible for these properties are typically attached to the constant (C) region of the antibody. For example, IgG is located at C... H Glycosylation at asparagine 297 in the 2 domain is essential for the complete ability of IgG to activate the classical complement-dependent cytolysis pathway. IgM at C H Glycosylation at asparagine 402 in the 3-domain is essential for proper antibody assembly and cytolytic activity. The C3 domain of IgA antibodies... H 1 and C H Removal of glycosylation sites at positions 162 and 419 in the 3 domain leads to intracellular degradation and at least 90% inhibition of secretion.

[0255] Glycosylation of immunoglobulins in the variable (V) region was also observed. Approximately 20% of human antibodies were glycosylated in the V region. Glycosylation of the V domain is thought to be caused by the incidental N-linked glycosylation signal Asn-Xaa-Ser / Thr in the V region sequence and has not been previously recognized as playing a role in immunoglobulin function.

[0256] In some cases, glycosylation at framework residues of variable domains alters the binding interaction between the antibody and the antigen. This disclosure includes criteria for selecting a limited number of amino acids in the framework or CDR of a humanized immunoglobulin chain to mutate (e.g., by substitution, deletion, or addition of residues) to increase antibody affinity. In some embodiments, cysteine ​​residues are removed or introduced into the Fc region of the antibody or Fc-containing polypeptide, thereby eliminating or increasing interchain disulfide bond formation in that region.

[0257] It has been shown that sequences within the CDR can sometimes induce antibody binding to MHC class II and trigger helper T cell responses, which may be undesirable in some cases. In some embodiments, conserved substitution allows the antibody to retain its binding activity but reduces its ability to trigger unwanted T cell responses. In one embodiment, one or more amino acids from the N-terminal 20 amino acids of the heavy or light chain are removed.

[0258] Covalent modifications of antibodies (e.g., bispecific antibodies) disclosed herein are also included herein. In some embodiments, they are prepared by chemical synthesis or by enzymatic or chemical cleavage of the antibody, if applicable. In some embodiments, other types of covalent modifications are introduced by reacting the target amino acid residues with an organic derivatizer capable of reacting with selected side chains, or N-terminal or C-terminal residues.

[0259] Cysteine ​​residues most commonly react with α-haloacetic esters (and their corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxamide-methyl derivatives. Cysteine ​​residues are also derivatized by reactions with bromotrifluoroacetone, α-bromo-β-(5-imidozoyl))propionic acid, chloroacetyl phosphate, N-alkylmaleimide, 3-nitro-2-pyridyl disulfide, methyl-2-pyridyl disulfide, p-chloromercuric benzoate, 2-chloromercuric-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0260] In some embodiments, the histidine residues are derivatized by reacting with diethyl pyrocarbonate at pH 5.5–7.0, as this reagent is relatively specific to the histidine side chain. In some embodiments, p-bromobenzoylmethyl bromide is also useful; in some embodiments, the reaction is carried out in 0.1 M sodium dimethylarsinate at pH 6.0.

[0261] In some embodiments, the lysyl and amino-terminal residues react with succinic anhydride or other carboxylic anhydrides. Derivatization with these reagents has the effect of reversing the charge of the lysyl residues. Other suitable reagents for derivatizing α-amino residues include imine esters (such as methyl pyridinium imide), pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reactions with glyoxylic acid.

[0262] In some embodiments, the arginyl residue is modified by reacting with one or more conventional reagents such as benzoylcarbaldehyde, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Due to the high pKa of the guanidine functional group, the derivatization of the arginine residue requires the reaction to be carried out under basic conditions. Furthermore, in some embodiments, these reagents react with the lysine group and the ε-amino group of arginine.

[0263] In some embodiments, specific modification of tyrosine residues is performed, with particular interest in introducing spectral labeling into the tyrosine residues through reaction with aromatic diazo compounds or tetranitromethane. Most commonly, in some embodiments, N-acetylimidazolium and tetranitromethane are used to form O-acetyltyrosine substances and 3-nitro derivatives, respectively. 125 I or 131I iodide tyrosine residues to prepare labeled proteins for use in radioimmunoassays.

[0264] The carboxyl side group (aspartic or glutamine) is specifically modified by reacting with a carbodiimide (RN=C=N-R'), where R and R' are different alkyl groups, such as 1-cyclohexyl-3-(2-morpholino-4-ethyl)carbodiimide or 1-ethyl-3-(4-azainium-4,4-dimethylpentyl)carbodiimide. Furthermore, aspartic and glutamine residues are converted to asparagine and glutamine residues by reacting with ammonium ions.

[0265] In some embodiments, the glutamine and asparagine residues are deamidated to the corresponding glutamine and asparagine residues, respectively. These residues are deamidated under neutral or alkaline conditions.

[0266] Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of serine or threonine residues, methylation of the α-amino groups of the lysine, arginine, and histidine side chains, acetylation of N-terminal amines, and amidation of any C-terminal carboxyl groups.

[0267] Another type of covalent modification involves chemically or enzymatically coupling a glycoside to a specific binder or antibody. These processes do not require the production of a polypeptide or antibody with N- or O-linked glycosylation capabilities in the host cell. Depending on the coupling mode used, in some embodiments, the sugar is attached to (a) arginine and histidine, (b) a free carboxyl group, (c) a free thiol group, such as those of cysteine, (d) a free hydroxyl group, such as those of serine, threonine, or hydroxyproline, (e) an aromatic residue, such as those of phenylalanine, tyrosine, or tryptophan, or (f) an amide group of glutamine.

[0268] The removal of any carbohydrate moieties present on a peptide or antibody can be accomplished by chemical or enzymatic methods. Chemical deglycosylation involves exposing the antibody to the compound trifluoromethanesulfonic acid or an equivalent. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylglucosamine), while keeping the antibody intact. Enzymatic cleavage of carbohydrate moieties on antibodies can be achieved using a variety of endoglucosidases and exoglucosidases.

[0269] Another type of covalent modification involves linking an antibody (e.g., a bispecific antibody) of this disclosure to one of a variety of non-protein polymers, such as polyethylene glycol, polypropylene glycol, polyoxyethylene polyols, polyoxyethylene sorbitol, polyoxyethylene glucose, polyoxyethylene glycerol, polyoxyethylene olefins, or polysaccharide polymers such as dextran. Such methods are known in the art.

[0270] Typically, the affinity for binding a predetermined peptide antigen is modulated by introducing one or more mutations into the V-region framework, usually in regions adjacent to one or more CDRs and / or in one or more framework regions. Such mutations typically involve introducing conserved amino acid substitutions that disrupt or create glycosylation sites, but do not substantially affect the hydrophilic structural properties of the peptide. Mutations involving proline residues are generally avoided.

[0271] In some embodiments, the antibodies (e.g., bispecific antibodies) or antigen-binding fragments disclosed herein are conjugated or linked to therapeutic, imaging, or detectable portions or affinity tags. Methods for conjugating or linking peptides are known in the art. Association (binding) between compounds and tags includes any means known in the art, including but not limited to covalent and non-covalent interactions, chemical conjugation, and recombinant techniques. In some embodiments, the antibodies (e.g., bispecific antibodies) or antigen-binding fragments disclosed herein are conjugated to or recombinantly engineered with affinity tags (e.g., purification tags). Affinity tags such as polyhistidine tags (e.g., His6) are conventional in the art.

[0272] In some embodiments, the antibody (e.g., a bispecific antibody) or antigen-binding fragment also includes a detectable portion. For example, the detection is performed in vitro, in vivo, or ex vivo. In vitro assays for detecting and / or determining (quantitative, qualitative, etc.) levels of, for example, IFNγ protein using the antibodies (e.g., bispecific antibodies) or antigen-binding fragments disclosed herein include, but are not limited to, ELISA, RIA, and Western blot. In some embodiments, the in vitro detection, diagnosis, or monitoring of the antigen of the antibody is performed by obtaining a sample (e.g., a blood sample) from a subject and testing that sample in, for example, a standard ELISA assay.

[0273] Nucleic acid and vector This disclosure provides one or more nucleic acids that encode an antigen-binding molecule according to this disclosure. In some embodiments, the nucleic acid comprises or is composed of DNA and / or RNA.

[0274] This disclosure also provides one or more vectors that contain one or more nucleic acids according to this disclosure.

[0275] The nucleic acids and vectors disclosed herein can be provided in purified or isolated form, i.e., derived from other nucleic acids or naturally occurring biological materials.

[0276] Nucleotide sequences can be contained in vectors, such as expression vectors. As used herein, a “vector” is a nucleic acid molecule used as a medium for transferring exogenous nucleic acids into cells. A vector can be a carrier for expressing nucleic acids in cells. Such vectors may include a promoter sequence operatively linked to a nucleotide sequence encoding the sequence to be expressed. Vectors may also include a stop codon and an expression enhancer. Any suitable vector, promoter, enhancer, and stop codon known in the art can be used to express peptides or polypeptides from the vectors disclosed herein.

[0277] The term "operably linked" can include cases where a selected nucleic acid sequence and a regulatory nucleic acid sequence (e.g., a promoter and / or enhancer) are covalently linked in such a way that the expression of the nucleic acid sequence is under the influence or control of the regulatory sequence (thus forming an expression cassette). Therefore, if the regulatory sequence can influence the transcription of the nucleic acid sequence, the regulatory sequence is operably linked to the selected nucleic acid sequence. The resulting transcript can then be translated into the desired peptide / polypeptide.

[0278] Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g., gamma retroviral vectors (e.g., vectors derived from murine leukemia virus (MLV)), lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors, and herpesvirus vectors), transposon-based vectors, and artificial chromosomes (e.g., yeast artificial chromosomes).

[0279] In some embodiments, the vector may be a eukaryotic vector, such as a vector containing elements necessary for protein expression from a protein in a eukaryotic cell. In some embodiments, the vector may be a mammalian vector, such as one containing a cytomegalovirus (CMV) or SV40 promoter to drive protein expression.

[0280] According to this disclosure, the constituent polypeptides of the antigen-binding molecule can be encoded by different nucleic acids from a variety of nucleic acids or different vectors from a variety of vectors.

[0281] Cells and production methods This disclosure also provides a cell that contains or expresses an antigen-binding molecule according to this disclosure. A cell that contains or expresses a nucleic acid, multiple nucleic acids, a vector, or multiple vectors according to this disclosure is also provided.

[0282] The cell can be a eukaryotic cell, such as a mammalian cell. Mammals can be primates (rhesus monkeys, cynomolgus monkeys, non-human primates or humans) or non-human mammals (such as rabbits, guinea pigs, rats, mice or other rodents (including any animal in the order Rodentia), cats, dogs, pigs, sheep, goats, cattle (including dairy cows, such as dairy cows, or any animal in the family Bovidae), horses (including any animal in the family Equidae), donkeys and non-human primates).

[0283] In some embodiments, the cells are or are derived from cell types commonly used to express peptides for use in human treatment. Exemplary cells are described, for example, in Kunert and Reinhart, Appl Microbiol Biotechnol. (2016) 100:3451–3461 (incorporated herein by reference in its entirety), and include, for example, CHO, HEK 293, PER.C6, NSO, and BHK cells. In a preferred embodiment, the cells are or are derived from CHO cells.

[0284] This disclosure also provides a method for generating cells comprising a nucleic acid or vector according to this disclosure, the method comprising introducing a nucleic acid, multiple nucleic acids, a vector, or multiple vectors according to this disclosure into the cells. In some embodiments, introducing the isolated nucleic acid or vector according to this disclosure into the cells includes transformation, transfection, electroporation, or transduction (e.g., retroviral transduction).

[0285] This disclosure also provides a method for generating cells expressing / containing an antigen-binding molecule according to this disclosure, comprising introducing a nucleic acid, multiple nucleic acids, a vector, or multiple vectors according to this disclosure into the cells. In some embodiments, the method further comprises culturing the cells under conditions suitable for cell expression of the nucleic acid or vector. In some embodiments, the method is performed in vitro.

[0286] This disclosure also provides cells that can be obtained or obtained by the methods according to this disclosure.

[0287] Composition This disclosure also provides compositions comprising the antigen-binding molecules, nucleic acids, expression vectors, and cells described herein.

[0288] The antigen-binding molecules, nucleic acids, expression vectors, and cells described herein can be formulated into pharmaceutical compositions or drugs for clinical use and may contain pharmaceutically acceptable carriers, diluents, excipients, or adjuvants. The compositions can be formulated for topical, parenteral, systemic, intracavitary, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, intrathecal, oral, or transdermal administration, and may include injection or infusion. In some embodiments, the compositions can be formulated for administration, for example, to tissues by injection. In some embodiments, the compositions can be formulated for subcutaneous injection.

[0289] Suitable formulations may contain antigen-binding molecules in a sterile or isotonic medium. Drugs and drug compositions may be formulated into fluid forms, including gel forms. Fluid formulations may be formulated for administration to selected areas of a human or animal body by injection or infusion (e.g., via a catheter).

[0290] In some embodiments, the composition is formulated for injection or infusion, such as into a blood vessel, tissue / organ of interest, or tumor.

[0291] This disclosure also provides methods for producing pharmaceutically useful compositions, such methods may include one or more steps selected from: producing the antigen-binding molecule, nucleic acid (or more thereof), expression vector (or more thereof) or cells described herein; isolating the antigen-binding molecule, nucleic acid (or more thereof), expression vector (or more thereof) or cells described herein; and / or mixing the antigen-binding molecule, nucleic acid (or more thereof), expression vector (or more thereof) or cells described herein with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.

[0292] For example, another aspect of this disclosure relates to a method of formulating or producing a medicament or pharmaceutical composition for use in treating a disease / condition (e.g., cancer), the method comprising formulating the pharmaceutical composition or medicament by mixing the antigen-binding molecule, nucleic acid (or a plurality thereof), expression vector (or a plurality thereof) or cell described herein with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.

[0293] Application and therapeutic use In some embodiments, this document discloses methods for treating a disease in a subject in need, which include administering to the subject an antigen-binding molecule or pharmaceutical composition of any of the foregoing embodiments.

[0294] The disease or condition to be treated can be any disease or condition that is beneficially treated by the pro-inflammatory and / or anti-tumor activity of IFNγ.

[0295] In some implementations, the disease being treated can be a non-cancerous disease, such as chronic granulomatous disease (Beatriz E. Marciano, Robert Wesley, Ellen S. De Carlo, Victoria L. Anderson, Lisa A. Barnhart, Dirk Darnell, Harry L. Malech, John I. Gallin, Steven M. Holland Clinical Infectious Diseases, Vol. 39, No. 5, September 1, 2004, pp. 692-699), osteosclerosis (LL Key Jr 1, RM Rodriguiz, SM Willi, NM Wright, HC Hatcher, D REyre, JK Cure, PP Griffin, WL Ries N Engl J Med. June 15, 1995; 332(24):1594-9), atopic dermatitis, and the treatment of bacterial and fungal infections (Netea MG, Kullberg BJ, Van derMeer JW. Proinflammatory cytokines in the treatment of bacterial and fungal infections. BioDrugs. 2004;18(1):9-22). This may be through the enhancing effects of IFNγ on neutrophils, osteoclasts and / or the innate immune system.

[0296] In other implementations, the disease or symptom is a tumor or cancer. Optionally, the tumor can be a non-immunogenic tumor, such as a checkpoint inhibitor-resistant tumor.

[0297] This treatment can be combined with one or more other anticancer therapies, as discussed further in this article.

[0298] Methods for sensitizing cancer subjects to checkpoint inhibitor therapy are also disclosed, comprising administering to the subject a composition containing antibodies (including, but not limited to, bispecific antibodies) or pharmaceutical compositions disclosed herein. In some embodiments, sensitization results in increased cancer sensitivity to checkpoint inhibitor therapy and / or a reduced dose of checkpoint inhibitor required to achieve therapeutically desired baseline outcomes (e.g., improvement of symptoms, prolongation of progression, cure, remission, prolongation of survival, or other objective response).

[0299] The invention also discloses an antigen-binding molecule or pharmaceutical composition of any of the foregoing embodiments for use in treatment methods. More specifically, an antigen-binding molecule or pharmaceutical composition of any of the foregoing embodiments is provided for use in methods of treating cancer.

[0300] The invention also discloses an antigen-binding molecule or pharmaceutical composition of any of the foregoing embodiments for use in methods of sensitizing a subject with cancer to checkpoint inhibitor therapy. It also provides an antigen-binding molecule or pharmaceutical composition of any of the foregoing embodiments for use in methods of treating cancer, said methods further comprising administering one or more additional anticancer therapies. It also provides an antigen-binding molecule or pharmaceutical composition of any of the foregoing embodiments and one or more additional anticancer therapies for use in methods of treating cancer. In one embodiment, the one or more additional anticancer therapies may include a checkpoint inhibitor.

[0301] The use of any of the antigen-binding molecules or pharmaceutical compositions described in the foregoing embodiments in the manufacture of a medicament for treating cancer is also provided. This treatment may be combined with one or more other anticancer therapies, as further discussed herein.

[0302] In some implementations, the disease / symptom to be treated / prevented is cancer. Cancer can be any unwanted cell proliferation (or any disease manifested by unwanted cell proliferation itself), growth, or tumor. Cancer can be benign or malignant, and can be primary or secondary (metastatic). A growth or tumor can be any abnormal growth or proliferation of cells and can be located in any tissue. Cancer can originate from the following tissues / cells: adrenal glands, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g., renal epithelium), gallbladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph nodes, lymphoblasts, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary glands, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testes, thymus, thyroid gland, tongue, tonsils, trachea, uterus, vulva, and leukocytes.

[0303] The tumor to be treated can be a nervous system tumor or a non-nervous system tumor. Nervous system tumors can originate from the central or peripheral nervous system, such as gliomas, medulloblastomas, meningiomas, neurofibromas, ependymomas, schwannomas, neurofibrosarcomas, astrocytomas, and oligodendrogliomas. Non-nervous system cancers / tumors can originate from any other non-nervous tissue, examples include melanomas, mesotheliomas, lymphomas, myelomas, leukemias, non-Hodgkin lymphomas (NHL), Hodgkin lymphomas, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), cutaneous T-cell lymphomas (CTCL), chronic lymphocytic leukemia (CLL), liver cancer, epidermoid carcinoma, prostate cancer, breast cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, thymic cancer, NSCLC, hematologic malignancies, and sarcomas.

[0304] In some implementations, the TAA can be PSMA, and the cancer can be prostate cancer.

[0305] In some implementations, antigen-binding molecules (including, but not limited to, bispecific antibodies) or pharmaceutical compositions are administered in combination with other modes of treatment. As used herein, “combination” means the use of two or more modes of treatment to achieve the intended use, indication, or effect, wherein such modes of treatment may be administered to the patient simultaneously or sequentially.

[0306] In some implementations, an additional treatment modality is anticancer therapy. In some implementations, anticancer therapy includes immunotherapy, such as CAR-T cell therapy, T-cell connectives, dendritic cell vaccines, radioimmunoassay conjugates, antibody-drug conjugates, cytokine therapy, PARP inhibitors, AKT inhibitors, Tyr-kinase inhibitors, checkpoint inhibitors, chemotherapy, hormone therapy, radiotherapy, cryotherapy, alternating electric field therapy, hyperthermia, vaccines, and / or surgical interventions, such as surgical removal of tumor tissue.

[0307] In some embodiments, the immunotherapy is a cytokine therapy. In some embodiments, the cytokine therapy is recombinant IFNγ, such as recombinant IFNγ-1b (Actimmune®). In some embodiments, recombinant IFNγ-1b is administered at a dose of 5 to 50 μg / m². 2 (For example, 5, 10, 20, 30, 40 or 50, μg / m 2 The dosage is as follows. In some embodiments, recombinant IFNγ-1b is administered at 50 μg / m². 2Dosage administration. In some implementations, the cytokine therapy is selected from the group consisting of: NL-201 (IL-2 / IL-15), BNZ-1 (an inhibitor of IL-2, IL-9, and IL-15), nemvaleukinalfa (an IL-2R agonist), SON-3015 (anti-IL6-FHAB-anti-TGFβ), SON-1410 (IL-18-FHAB-IL-12), SON-1210 (IL-12-FHAB-IL-15), SON-1010 (IL-12-FHAB), PF-07209960 (a PDCD PD-1 targeting region fused with a mutant IL-15 protein), XmAb306 (IL-15 / IL15Rα), Fibromun (an L19 antibody and TNF), ProscaVax (PSA, IL-2, and GM-CSF), and beempegaldesleukin. (IL-2) and INO-5151 (PSA, PSMA, and IL-12 encoding vectors). In some embodiments, the antigen-binding molecules of the present invention can be used to increase the half-life and / or plasma concentration of exogenously applied IFNγ, and / or reduce side effects associated with exogenously applied IFNγ (e.g., in cases where the pro-inflammatory and anti-inflammatory effects of IFNγ are biased towards antigen-binding molecules that favor pro-inflammatory activity) and / or to target exogenously applied IFNγ to target cells or tissues (e.g., in cases of multispecific, such as bispecific, antigen-binding molecules).

[0308] In some embodiments, the checkpoint inhibitor is a PD-1 inhibitory antibody selected from the group consisting of: pembrolizumab, nivolumab, cemiplimab, dostarlimab, tislelizumab, retifanlimab, pidilizumab, TSR-042, PDR-001, and Sym021. In some embodiments, the checkpoint inhibitor is a PD-L1 inhibitory antibody selected from the group consisting of: atezolizumab, avelumab, garivulimab, durvalumab, LY3300054, and BGB-A333.

[0309] In some implementations, the checkpoint inhibitor is a CTLA-4 inhibitor. In some implementations, the CTLA-4 inhibitor is selected from the group consisting of: ipilimumab, tremelimumab, pembralizumab, atezolizumab, and avelumumab.

[0310] In some implementations, the checkpoint inhibitor is a LAG-3 inhibitor. In some implementations, the LAG-3 inhibitor is selected from the group consisting of: relatlimab, leramilimab, eftilagimod alpha, favezelimab, fianlimab, encelimab, INCAGN2385 (Agenus), Sym022 (Symphogen), FS118 (F-Star Therapeutics), XmAb22841 (Xencor), RO7247669 (Roche), IBI110 (Innovent Biologics), SRF388 (Surface Oncology), IMP321 (Immutep), MK-4280 (Merck), REGN3767 (Regeneron), TSR-033 (GSK), and IBI110 (Innovent Biologics).

[0311] In some implementations, the checkpoint inhibitor is a TIGIT inhibitor. In some implementations, the TIGIT inhibitor is selected from the group consisting of: tiragolumab, 5MK-7684 (Merck), BMS-986207 (Bristol-Meyer Squibb), AB154 (Arcus Bio), BGB-A1217 (BeiGene), M6223 (Merck), and COM902 (Compugen).

[0312] In some implementations, the checkpoint inhibitor is a TIM-3 inhibitor. In some implementations, the TIM-3 inhibitor is selected from the group consisting of: sabatolimab, INCAGN02390 (Agenus), TSR-022 (Tesaro), Sym023 (Symphogen), LY3321367 (Eli Lilly), MBG453 (Novartis), and BGB-A425 (BeiGene).

[0313] In some implementations, the checkpoint inhibitor is a GITR inhibitor. In some implementations, the GITR inhibitor is selected from the group consisting of: INCAGN01876 (Agenus), TRX518 (Leap Therapeutics), BMS-986156 (Bristol-Meyer Squibb), ASP1951 (Astellas / Potenza Therapeutics), GWN323 (Novartis), and MK-4166 (Merck).

[0314] In some embodiments, the checkpoint inhibitor is a CD40 / CD40L inhibitor. In some embodiments, the CD40 / CD40L inhibitor is selected from the group consisting of: dacetuzumab, selicrelumab, lucarumumab, mitazalimab, sotigalimab, CP-870893 (Pfizer), CDX-1140 (CellDex Therapeutics), NG-350A (PsiOxus Therapeutics), APX005M (Apexigen), and JNJ-64457107 (AlligatorBioscience / Janssen Biotech Inc.).

[0315] In some embodiments, the checkpoint inhibitor is an OX40 inhibitor. In some embodiments, the OX40 inhibitor is selected from the group consisting of: PF-04518600 (Pfizer), MEDI6469 (MedImmune), GSK3174998 (GSK), MEDI0562 (MedImmune), BMS986178 (Bristol-Meyer Squibb), INCAGN01949 (Agenus), MOXR0916 (Roche), INBRX-106 (Inhibrx), IBI101 (Innovent Biologics), MEDI6383 (MedImmune), and BGB-A445 (BeiGene).

[0316] In some implementations, the checkpoint inhibitor is a 4-1BB / CD137 inhibitor. In some implementations, the 4-1BB / CD137 inhibitor is selected from the group consisting of: urelumab, ADG106 (Adagene), MEDI1873 (MedImmune), LVGN6051 (Lyvgen), and AGEN2373 (Agenus).

[0317] In some embodiments, the checkpoint inhibitor is an ICOS inhibitor. In some embodiments, the ICOS inhibitor is selected from the group consisting of GSK3359609 (GSK) and MEDI570 (MedImmune). In some embodiments, the checkpoint inhibitor is an NKG2A inhibitor. In some embodiments, the NKG2A inhibitor is monalizumab. In some embodiments, the checkpoint inhibitor is a CD47 inhibitor. In some embodiments, the CD47 inhibitor is selected from the group consisting of megrolimab, IBI188 (Innovent Biologics), TTI-621 / 622 (Trillium), AO-176 (Arch Oncology), ALX148 (ALX Oncology), CC-90002 (Celgene), ZL-1201 (Zai Lab), IMC-002 (ImmuneOncia), and SRF231 (Surface Oncology). In some embodiments, the checkpoint inhibitor is a SIRPα inhibitor. In some implementations, the SIRPα inhibitor is BI765063 (Behringer-Ingelheim) or CC-95251 (Celgene). In some implementations, the checkpoint inhibitor is an A2A inhibitor, such as Civoradenant.

[0318] In some implementations, the immunotherapy is a T-cell connective. In some implementations, the T-cell connective is selected from the group consisting of: JNJ-63898081, acaptamab, CCW-702, AMG-340, CC-1, REGN-5678, JNJ-63898081, TMB-585, HPN424, AMG-160, CCW702, AMG-509, XmAb808, solitomab, and tebentafusp.

[0319] In some embodiments, the checkpoint inhibitor is a bispecific checkpoint inhibitor. In some embodiments, the bispecific checkpoint inhibitor is specific for PD-1 and LAG-3. In some embodiments, the PD-1 / LAG-3 bispecific checkpoint inhibitor is MGD013 (MacroGenics). In some embodiments, the bispecific checkpoint inhibitor is specific for PD-1 and TIM-3. In some embodiments, the PD-1 / TIM-3 bispecific checkpoint inhibitor is RO7121661 (Eli Lilly). In some embodiments, the bispecific checkpoint inhibitor is specific for PD-1 and CD47. In some embodiments, the PD-1 / CD47 bispecific checkpoint inhibitor is HX009 (HanxBio). In some embodiments, the bispecific checkpoint inhibitor is specific for PD-L1 and CD47. In some embodiments, the PD-L1 / CD47 bispecific checkpoint inhibitor is IBI322 (Innovent Biologics). In some embodiments, the bispecific checkpoint inhibitor is specific for PD-L1 and 4-1BB. In some implementations, the PD-L1 / 4-1BB bispecific checkpoint inhibitor is MCLA-145 (Merus).

[0320] In some implementations, the chemotherapy therapy is selected from one or more of the following (e.g., 1, 2, 3, 4, 5 or more): docetaxel (Taxotere), cabazitaxel (Jevtana), mitoxantrone (Novantrone), olaparib / Lynparza, rucaparib / Rubraca (mCRPC), carboplatin, talazoparib (Talzenna), niraparib / Zejula, ipatasertib / RG7440, Cabometyx / Cometriq, lutrate (leuprorelin acetate; anti-steroid), PNT2002 (Radioligands), Sabizabulin, Talzenna, Masitinib, Capivasertib, Verzenio, ARX-517 (anti-PSMA antibody-drug conjugate), IMMU-132 (Sacituzumab Govitecan-hziy), SGN-LIV1A (LIV1-ADC), MGC018 (humanized B7-H3 monoclonal antibody-ADC), DS-7300 (exatecan derivative payload targeting B7-H3), and FOR46 (CD46-ADC).

[0321] In some implementations, the hormone therapy is selected from the group consisting of: enzalutamide, abiraterone (e.g., abiraterone acetate), darolutamide, Erleada, Nubeqa, Zytiga, Zoladex, Trelstar, Leuplin, and Orgovyx.

[0322] In some implementations, the radiotherapy is free from the group consisting of: radium chloride-223 injection, lutetium (Lu)-177 vipivotide tetraxetan, and BAY2315497.

[0323] In some implementations, the vaccine is a dendritic cell (DC) targeted vaccine selected from Sipuleucel-T and Stapuldencel T. In some implementations, the vaccine is rilimogene galvacirepvec / glafolivec or CAN-2409.

[0324] In some implementations, one or more additional treatment methods are surgery (e.g., tumor resection, orchiectomy, cryosurgery, and radiation surgery).

[0325] In one implementation, one or more additional treatments include virus-based therapies such as Prostvac and CAN-2409.

[0326] In some implementations, additional treatments are administered to the subject before, simultaneously with, or after the administration of the antigen-binding molecule or pharmaceutical composition.

[0327] When two or more compositions are applied, these compositions are applied, for example, in combination (sequentially or simultaneously). In some embodiments, one or more additional therapeutic agents or methods are applied immediately before or after the composition, or for a period of 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 1 week, 2 weeks, 1 month, or longer before or after the application of the composition. In some embodiments, the composition is applied in a single dose or multiple doses. It is not necessary to formulate or package the compositions together or apply them simultaneously.

[0328] The antigen-binding portion of the present invention can be administered using any route of administration known in the art, such as local, parenteral, systemic, intracavitary, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, intrathecal, oral, or transdermal administration, which may include injection or infusion. In some embodiments, the antigen-binding portion of the present application is administered, for example, intradermally, intramuscularly, or subcutaneously, to a tissue. Administration can be performed by injection. For example, administration can be performed by subcutaneous injection. Compared to equivalent methods of administering IFNγ itself, the molecules of the present invention may have the advantage of avoiding local side effects in tissues and / or at the injection site.

[0329] Subjects The subject according to this disclosure can be any animal or human. The subject is preferably a mammal, more preferably a human. The subject can be male or female. The subject can be a patient. The subject may have been diagnosed with a disease or condition requiring treatment (e.g., cancer), may be suspected of having such a disease / condition, or may be at risk of developing / contracting such a disease / condition.

[0330] In embodiments according to this disclosure, the subject is preferably a human subject. In some embodiments, the subject being treated according to the treatment or prevention methods of this disclosure is a subject who has a disease described herein (e.g., cancer) or is at risk of developing a disease described herein (e.g., cancer). In embodiments according to this disclosure, subjects may be selected for treatment according to these methods based on the characteristics of certain biomarkers of such disease / symptom. The subject may have (e.g., may have been diagnosed with) the cancer described herein.

[0331] sequence

[0332] The features disclosed in the foregoing description, the appended claims, or the drawings, expressed in their particular form or by means of performing the disclosed functions or by methods or processes for obtaining the disclosed results, may, where appropriate, be used alone or in any combination of such features to implement the invention in its various forms.

[0333] While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when this disclosure is given. Therefore, the exemplary embodiments set forth above are to be considered illustrative rather than restrictive. Various changes may be made to the described embodiments without departing from the spirit and scope of the invention.

[0334] To avoid any doubt, any theoretical explanations provided herein are intended to enhance the reader's understanding. The inventor does not wish to be bound by any of these theoretical explanations.

[0335] Any chapter headings used in this article are for organizational purposes only and should not be construed as limiting the subject matter described.

[0336] Throughout the specification, including the appended claims, unless the context otherwise requires, the words “comprising” and “including” and their variations shall be understood to implicitly include the indicated integer or step or group of steps, but not exclude any other integer or step or group of steps.

[0337] It must be noted that, unless the context clearly indicates otherwise, the singular forms “a” and “the” as used in this specification and the appended claims include a plural referent. A range may be expressed herein as “about” a particular value and / or “about” another particular value. When such a range is expressed, another embodiment includes a range from one particular value and / or to another particular value. Similarly, when a value is expressed as an approximation using the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” with respect to numerical values ​​is optional and means, for example, + / - 10%.

[0338] Example For standard molecular biology techniques, see Sambrook, J., Russell, DW Molecular Cloning, A Laboratory Manual, 3rd ed. 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.

[0339] The following examples are provided to provide those skilled in the art with a description of how the compositions and methods described herein can be used, prepared, and evaluated, and are intended to be purely illustrative and not to limit the scope of what the inventors consider to be their invention.

[0340] Example 1: Anti-IFNγ antibody cloning, selection, and amplification method Two mice, immunized 8 months prior with recombinant human IFNγ, were intraperitoneally (ip) injected with 90 μg of recombinant IFNγ (catalog number IFNG-1164R; Creative BioMart, New York, USA). Three days later, the spleen and visible lymph nodes were aseptically removed. All subsequent procedures were performed in a Class II microbial safety enclosure using aseptic techniques. All excised tissues were pooled, placed in a 40 μm filter, mechanically disrupted using a 10 mL syringe plunger, and washed with 50 mL Opti-MEM (Thermofisher Scientific, Loughborough, UK) to produce a single-cell suspension. The cell pellet was washed twice with 50 mL Opti-MEM and resuspended in 0.5 mL MACS buffer (Opti-MEM, 20% FCS; GE Healthcare, Little Chalfont, UK) and 1% BM Condimed H1 (Sigma-Aldrich, Poole, UK). Viable cell counts using trypan blue viability staining were performed using a Luna-II instrument (Logos Biosystems, Annandale, USA). For negative MACS selection, add 100 μL of pan-B cytokinin-antibody mixture (Miltenyi, Woking, UK) to 400 μL containing 1×10 8Cells were incubated in MACS buffer at 4°C for 5 minutes. To remove IgM+ naive B cells, 200 μL of anti-mouse IgM beads (Miltenyi, Woking, UK), 100 μL of MACS buffer, and 200 μL of avidin beads (Miltenyi, Woking, UK) were added, and the mixture was incubated at 4°C for 15 minutes. 20 mL of MACS buffer was added to pellet the cells, which were then resuspended in 500 μL of MACS buffer and passed under gravity through a pre-equilibrated LS column (Miltenyi, Woking, UK), followed by washing with 3 × 3 mL of MACS buffer. An equal volume of logarithmically grown SP2 / 0 myeloma cells was added to the negatively selected B cells, pelleted, washed twice with 20 mL of BTXpress cell fusion medium C (VWR, Lutterworth, UK), and resuspended in 2 mL of cell fusion medium C. Washed cells were transferred to a 2 mL electrofusion chamber (VWR, Lutterworth, UK) and electrofused in an ECM2001+ electroporator under the manufacturer's recommended conditions for fusion of mate body spleen (B cells) and SP2 / 0 myeloma cells (Harvard Apparatus UK, Cambridge, UK). Cells were then transferred to 45 mL of medium (Opti-MEM, 20% FCS, 2.5% BM Condimed H1, 1% penicillin / streptomycin; ThermoFisher Scientific, Loughborough, UK) and incubated overnight at 37°C / 5% CO2. The next day, the cell pellet was resuspended in 1 mL of Opti-MEM and added to 199 mL of semi-solid ClonaCell-HY medium D (Stem Cell Technologies, Waterbeach, UK), which contained 3 μg / mL FITC-conjugated rINFγ protein (labeled using a Lightning-Link fluorescent labeling kit according to the manufacturer's instructions; Expedeon Ltd, Cambridge, UK), 3.75 μg / mL AF647-conjugated F(ab')2 fragment goat anti-mouse IgG (Stratech Scientific, Ely, UK), and 1× hypoxanthine / diazoserine selector (Sigma-Aldrich, Poole, UK). 9 mL of the semi-solid medium was added to each of 20 wells in a Petri Well plate, which was then incubated at 37°C / 5% CO2 for 11 days.The growth plate was imaged using the bright, FITC, and AF647 fluorescence detection channels of a ClonePix 2 colony selector (Molecular Devices, Wokingham, UK), and hybridoma colonies were selected into 200 μL ClonaCell-HY recovery medium (Stem Cell Technologies, Waterbeach, UK) based on colony size, morphology, and EMI. Two hybridoma colonies identified as positive by the ClonePix 2 but too close to the sidewall of the growth well to be selected were manually aspirated using a P20 pipette. After 7 days at 37°C / 5% CO2, the presence of anti-rhIFNγ (recombinant human IFNγ) and anti-rrINFγ (recombinant rhesus macaque IFNγ with the same amino acid sequence as cynomolgus macaque IFNγ) antibodies in the culture supernatant was detected by ELISA. Selected cultures were expanded to 10 mL using hybridoma growth medium (DMEM; Thermofisher Scientific, Loughborough, UK), 20% FCS, 1% BM Condimed H1, and 1% penicillin / streptomycin. Cells were frozen in cryopreservative (90% FCS, 10% DMSO) and stored in the gas phase of a liquid nitrogen Dewar flask. The presence of anti-rhIFNγ and anti-rrIFNγ antibodies in the culture supernatant was initially tested by ELISA. 50 μL / well of 2 μg / mL rhIFNγ or rrIFNγ protein in pH 7.4 PBS buffer was used to coat MICROLON medium bound to ELISA plates (Greiner Bio One Ltd, Stonehouse, UK) and incubated overnight at 4°C. The coating solution was aspirated, washed twice with PBS, and blocked for 1 hour at room temperature in blocking buffer (PBS / 0.8% BSA). Culture samples were applied unchanged at 50 μL / well and incubated at 37°C for 60 minutes. The reagent blank consisted of only 50 μL of culture medium. Samples were aspirated and wells were washed four times with PBST (PBS / 0.05% Tween-20) and aspirated dry. Polyclonal goat anti-mouse IgG-HRP secondary antibody (Southern Biotech, Cambridge Bioscience, Cambridge, UK) was diluted 1 / 10,000 in blocking solution and applied at 50 μL / well, incubated at 37°C for 60 min. The antibody solution was then removed, and wells were washed three times with PBST and three times with water. 100 μL / well of HRP substrate was added, and color development was stopped after 4 min at room temperature by adding 100 μL / well of 1 M hydrochloric acid.The absorbance at OD 450 nm was determined using a Dynex Technologies MRX TC II plate reader. All sample values ​​were corrected by subtracting a reagent blank, which was the average of two readings per plate. Purified mouse IgG1 protein (Sigma, Dorset, UK) was used as a standard, and mouse IgG in the supernatant was quantified by ELISA. MICROLON medium-bound ELISA plates (Greiner Bio One Ltd, Stonehouse, UK) were coated with 50 μL / well of 2 μg / mL goat anti-mouse IgG (Jackson ImmunoResearch Europe, Ely, UK) in pH 7.4 PBS buffer and incubated overnight at 4°C. The coating solution was aspirated, washed twice with PBS, and blocked for 1 hour at room temperature in blocking buffer (PBS / 0.8% BSA). Supernatant samples were serially diluted 50 μL / well in blocking buffer from 1:100 to 1:1000 and incubated at 37°C for 60 minutes. The sample blank consisted of unused hybridoma growth medium diluted as a sample. Mouse IgG1 standards were added in duplicate at a concentration of 1000 to 0.5 ng / mL, while the reagent blank consisted of only 50 μL of medium. Samples were aspirated, and the wells were washed four times in PBST (PBS / 0.05% Tween-20) and aspirated dry. Polyclonal goat anti-mouse IgG-HRP secondary antibody (Southern Biotech, Cambridge Bioscience, Cambridge, UK) was diluted 1 / 10,000 in blocking solution, applied at 50 μL / well, and incubated at 37°C for 60 min, as described above.

[0341] result The negative MACS selection protocol removed non-IgG+ cells (e.g., T cells, monocytes, macrophages, neutrophils, platelets, fibroblasts, granulocytes, and erythrocytes) and IgM+ B cells from spleen and lymph node cell suspensions harvested from two mice, and produced 0.21 × 10⁻⁶ cells upon thawing. 7 Cells. After being combined with an equal amount of SP2 / 0 cells (1:1 ratio), at 0.21 × 10⁻⁶ cells. 7 Electrofusion was performed at a density of cells / mL. After overnight culture, the recovered fused cell pellet was resuspended in Opti-MEM and electrofused at 1.2 × 10⁻⁶ cells / mL. 4Cells / mL were seeded in semi-solid growth medium. Due to the formation of immune complexes between secreted IgG immunoglobulins and immunogen-specific IgG immunoglobulins, 179 hybridoma colonies were selected and picked from 20 growth plates using a ClonePix 2 instrument based on size, morphology, and AF647 and FITC EMI fluorescence.

[0342] Example 2: Kinetic analysis of antibody binding to IFNγ method To investigate binding kinetics, the supernatant was subjected to Biacore single-cycle kinetic analysis, in which monoclonal antibodies were captured on the Fc surface as ligands, and IFNγ homodimers were passed through as analytes. In this example, both divalent and monovalent antibody forms were analyzed, as listed in the table below. Kinetic experiments were performed on a Biacore T200 running Biacore T200 control software V2.0.1 and evaluation software V3.0 (GE Healthcare, Uppsala, Sweden). All single-cycle kinetic experiments were performed at 25°C with HBS-P+ running buffer (pH 7.4, GE Healthcare, Little Chalfont, UK) containing 1 mg / mL BSA or PBS (pH 6.0, GE Healthcare, Little Chalfont, UK) containing 1 mg / mL BSA. The antibody-containing supernatant was diluted in run buffer and loaded onto Fc2, Fc3, and Fc4 of a CM5 chip previously conjugated with an anti-mouse capture antibody using standard amine chemistry (GE Healthcare, Little Chalfont, UK) at the start of each cycle. The antibody was captured at a flow rate of 10 μL / min to obtain a fixed level (RL) of approximately 150 RU. The surface was then stabilized. Single-cycle kinetic data were obtained using recombinant human (rh)IFNγ (Acro Biosystems, Newark, USA) as the analyte at a flow rate of 40 μL / min to minimize any potential mass transfer limitations. The signal from the reference channel Fc1 (antibody-free) was subtracted from the signals from Fc2, Fc3, and Fc4 to correct for differences in nonspecific binding to the reference surface. For initial screening, the antibody affinity was unknown. To generate data for all tested antibodies regardless of their affinity, two different three-point, three-fold dilution ranges of IFNγ (from 1.11 nM to 10 nM or 10 nM to 90 nM) were used, with no regeneration between each concentration. When screening with recombinant rhesus monkey (rr) IFNγ generated from HEK cells from Creative BioMart (Shirley, USA), a single three-point, three-fold dilution range from 1.11 nM to 10 nM was used. The association phase was monitored for 180 seconds after each three injections of incremental concentrations of IFNγ, and a single dissociation phase was measured for 500 seconds after the last injection of IFNγ. Regeneration of the anti-mouse capture surface was performed using 10 mM glycine-HCl at pH 1.7. The signal from the reference channel Fc1 was subtracted from the signals of Fc2, Fc3, and Fc4 to correct for differences in nonspecific binding with the reference surface, and the global Rmax parameter was used in a 1-to-1 binding model.

[0343] The selected antibodies (4F4 and 2C1) were also subjected to Biacore multicycle kinetic analysis using a Biacore T200 instrument running Biacore T200 evaluation software V3.0.1. In this particular embodiment, the bivalent antibody form was used, but the same method can also be applied to the monovalent antibody form. All multicycle kinetic experiments were performed at 25°C using HBS-P+ running buffer (pH 7.4) containing 1 mg / mL BSA and 2.5 mM CaCl2. The antibody supernatant was diluted in the running buffer and loaded onto Fc2, Fc3, and Fc4 of the Anti-Mouse Capture Kit (GE Healthcare, Little Chalfont, UK) at the start of each cycle. The antibody was captured at a flow rate of 10 µL / min to obtain a fixed level (RL) of approximately 150 RU. The surface was then stabilized. Human IFNγ (Acro Biosystems, catalog IFG-H4211) or rhesus monkey IFNγ (Creative Biomart, catalog IFNG-1164R) diluted in run buffer was used as the analyte, and a flow rate of 40 μL / min was employed to minimize any potential mass transfer effects to obtain multi-cycle kinetic data. Multiple replicates of blank and single replicates of analyte at a single concentration were programmed into the kinetic run to examine the stability of both the surface and the analyte during kinetic cycling. For kinetic analysis, a six-point, 2-fold dilution range of IFNγ from 100 nM to 3.125 nM was selected. The associative phase of IFNγ was monitored for 200 seconds, and the dissociation phase for 1200 seconds. Regeneration of the anti-mouse capture surface was performed using an injection of 10 mM glycine-HCl at pH 1.7. The signal from the reference channel Fc1 was subtracted from the signals of Fc2, Fc3, and Fc4 to correct for differences in nonspecific binding with the reference surface, and the global Rmax parameter was used in a 1-to-1 binding model.

[0344] result The tested clones showed an affinity range from no obvious binding to picomolar affinity. Biacore single-cycle kinetics and affinities for the antibody clones selected for further research (4F4, 3G1, 16D2, 9F1, and 2C1) are shown in [the figure]. Figure 1 A to Figure 1The affinities determined by single-cycle or multi-cycle kinetic analysis in E are summarized in the table below. The 4F4 clone was also tested against rhesus monkey IFNγ in both bivalent and monovalent forms (data shown in the table below). Additional antibodies 8D1 (SEQ ID NO: 137 and 138) and 19F10 (SEQ ID NO: 139 and 140) were also tested in both bivalent and monovalent forms, and the results are shown below.

[0345] *Note: The amino acid sequence of rhesus monkey IFNγ is the same as that of cynomolgus monkey IFNγ. Example 3: Screening for IFNγ neutralizing activity using PathHunter® cell assay method To screen clones for neutralizing activity, a PathHunter® eXpress IFNGR1 / IFNGR2 dimerization assay was performed (Eurofins, Brussels, Belgium). This assay detects the dimerization of two subunits of a ligand-induced receptor-dimer pair. These cells have been engineered to co-express a receptor subunit fused to the enzyme donor and a second dimer conjugate fused to the enzyme receptor. The binding of an agonist to one receptor subunit induces its interaction with its dimer conjugate, forcing the two enzyme fragments to complement each other. This results in the formation of a functional enzyme that hydrolyzes the substrate to generate a chemiluminescent signal.

[0346] Perform the assay according to the manufacturer's instructions. In short, thaw a vial of cells, resuspend it in 10 mL of pre-warmed cell plating reagent, and seed 20 μL / well in 384-well white clear flat-bottomed tissue culture plates (Eurofins, Brussels, Belgium), and incubate overnight at 37°C in a humidified 5% CO2 incubator. The next morning, prepare 10x serial dilutions of rhIFNγ (Acro Biosystems, Newark, USA) and test or control antibodies using AssayComplete™ protein dilution buffer (Eurofins, Brussels, Belgium) as the diluent. Incubate the IFNγ and test or control antibody dilutions at room temperature for 30 minutes before adding them to the cells, then transfer 5 μL of these premixed solutions to the cells and incubate the plates at 37°C in a humidified 5% CO2 incubator for 6 hours. Assays are prepared by mixing 1 part Flash cell assay buffer with 4 parts Flash substrate reagent. Add 30 μL of detection reagent to each well and incubate the plate in the dark at room temperature for 1 hour. luminescence was measured using a SpectraMax i3x plate reader (Molecular Devices, Wokingham, UK). Data analysis was performed using a GraphPad Prism 8, and a four-parameter nonlinear regression was used to fit the data.

[0347] In the initial screening, supernatants from 30 ELISA-positive clones selected based on performance in rhIFNγ binding ELISA were evaluated at final concentrations of 200, 66.6, 22.2, and 7.4 ng / mL. In cases where sample concentrations were insufficient, titrations were initiated at the highest possible concentration. Results were compared to a reference neutralizing antibody (Mabtech MT111W, Sweden) and a non-neutralizing antibody (Mabtech 11i, Sweden) (prepared as a seven-point, 2-fold dilution series starting at 100 ng / mL). IFNγ was diluted to a final concentration of 5 ng / mL for all screening experiments.

[0348] In the second screening, 22 amplified clones were quantified and tested as serial dilutions to obtain dose-response and IC50 values. Test antibodies were prepared at a starting concentration of 200 ng / mL and subjected to seven-point, 2-fold serial dilutions. IFNγ was diluted to a final concentration of 5 ng / mL. Figure 1 F only shows the data from the selections in the second screening.

[0349] result In the case of 9F1, the antibody's effectiveness ranges from ineffective to highly effective inhibition at all concentrations.

[0350] The five selected antibody clones, 4F4, 3G1, 16D2, 9F1, and 2C1, are shown in... Figure 1 In F, clones 3G1 and 4F4 did not show significant inhibition of receptor tetramerization within the tested concentration range. Clones 2C1 and 16D2 exhibited dose-dependent inhibition of receptor tetramerization, and the final mAb 9F1 induced almost complete inhibition even at the lowest dose.

[0351] Example 4: Antibody-mediated IFNγ accumulation in cynomolgus monkeys method To investigate the ability of the disclosed IFNγ antibody to stabilize and / or accumulate endogenous IFNγ in vivo, experiments were conducted in cynomolgus monkeys (Macaca fascicularis).

[0352] For these experiments, truncated monovalent forms of IFNγ antibody clones 2C4, 8D1, 4F4, and 19F10 were prepared as follows: Monovalent IgG1.1 / Fc1.1 (K409R / F405L) 2C4, 8D1, 4F4 (SEQ ID NO: 75 and 76) or 19F10 antibodies were generated by a redox reaction between chimeric IgG1.1 (K409R) 2C4, 8D1, 4F4, or 19F10 antibodies, all produced in CHO cells, and IgG1.1 Fc(H-CH2-CH3)(F405L) (SEQ ID NO: 83). For example, the monovalent form of antibody 4F4 consists of (4F4-VH(K409R) (SEQ ID NO: 75) and Fc(H-CH2-CH3)(F405L) (SEQ ID NO: 83). The monovalent antibody was subsequently purified by cation exchange chromatography on a HiScreen SP HP 4.7 mL column (GE Healthcare) using 20 mM sodium acetate (pH 5.0) and a 0-100% gradient of 300 mM sodium chloride as the mobile phase. Peak fractions eluted at approximately 150 mM sodium chloride were analyzed, and those containing the desired monovalent substance were combined, filtered sterilized, and then quantified using the extinction coefficient (Ec(0.1%)) at A280 nm based on the predicted amino acid sequence.

[0353] Antibody clones (truncated monovalent forms) 2C4, 8D1, 4F4, and 19F10 were tested in cynomolgus monkeys via single bolus injection at two doses (1 mg / kg and 3 mg / kg). A peptide variant (Actimmune®) was tested at a dose of 1.5 μg / kg every four days. Results were compared with those of juvenile animals (control group) administered a single bolus injection of saline. Each group consisted of two animals. Animals were sedated and administered the antibody / Actimmune® via intravenous injection. Subjects in the control group were sedated and administered saline via intravenous injection. Blood samples were subsequently collected at day 0, 24 hours, 48 ​​hours, 96 hours, 192 hours, 288 hours, and 360 hours. Blood samples were analyzed using an ELISA kit to determine circulating concentrations of the antibody and IFNγ. The study was terminated after two weeks, at which point the antibody was expected to have been cleared and IFNγ levels to have normalized.

[0354] result Four types of antibodies that can bind to rhesus monkeys were tested. M. Mulata Antibodies against IFNγ and human IFNγ in cynomolgus monkeys ( M. fascicularis The ability of rhesus macaques to accumulate IFNγ. Since the amino acid sequence of rhesus macaque IFNγ is identical to that of cynomolgus macaque IFNγ, it is expected that the binding properties of antibodies to IFNγ in these two species will be similar. Animals were injected with antibodies at 1 mg / kg or 3 mg / kg, and the resulting plasma IFNγ concentrations were recorded over time using ELISA. All four antibodies produced elevated concentrations of IFNγ in cynomolgus macaques. The 4F4 antibody, with 0.1 nM affinity for rhesus macaque IFNγ, increased IFNγ concentrations from 10-100 pg / mL to approximately 5000 pg / mL, corresponding to a 50-500-fold increase, reaching its peak at 3 mg / kg four days post-injection. Figure 1 G).

[0355] Example 5: IFNγ / IFNγR bias of antibodies To test the effects of antibodies on the cellular cascade downstream of IFNγR, we selected two cellular outputs of the IFNγR signaling cascade as surrogates. We sought to identify antibodies that maintained or enhanced IFNγ-mediated MHC-1 expression on cancer cells while simultaneously reducing PD-L1 expression, as this bias is considered pro-inflammatory and therefore beneficial for cancer therapy. For this purpose, PC-3 prostate cancer cells or A549 lung cancer cells were stimulated for 48 hours with a fixed dose of 1 ng / ml (0.6 nM) of IFNγ, which fully activated the receptor but remained within the dynamic concentration range of stimulation. To test the efficacy of the antibodies, these antibodies were added to the top at concentrations ranging from 0.1 nM to 100 nM for PC-3 cells and from 100 to 10,000 ng / mL for A549 cells.

[0356] method For PC-3 cells: On day 1, PC-3 cells (not expressing PSMA) were seeded in 25 cm... 2 Each flask contains 4 ml of culture medium and supplements, as well as 100,000-200,000 cells (10x dilution).

[0357] On day 3, cells were stimulated by adding IFNγ / mAb prepared as follows: IFNγ and antibody were aseptically filtered and added to cell culture medium (400 µl) so that when subsequently added to the culture medium in a cell culture flask (4 ml), the final concentration of IFNγ would be 1 ng / ml, and the final concentration of antibody would be 0.1, 1, 10, or 100 nM. The cell culture medium containing the mixture of IFNγ and antibody was incubated at room temperature for 30 minutes to allow pre-complexation between IFNγ and antibody before being added to the cell culture flask.

[0358] Cells were then incubated at 37°C for another 48 hours, followed by flow cytometry on day 5. Forty-eight hours after stimulation, cells were harvested at 25 cm⁻¹ by decantation to remove the culture medium and adding 1 ml of trypsin solution (Thermofisher: 25200056) at RT. 2 Cells grown in the flask. After about 10 minutes, add 1 ml of cell culture medium containing FBS, and transfer a total of 2 ml to 2 ml Eppendorf tubes. Precipitate the cells by centrifugation at 100 g for 5 minutes at 4°C. Resuspend the cells in 1 ml of FBS-containing medium, and transfer 450 μL to two 1.5 ml Eppendorf tubes for further precipitation.

[0359] After removing the culture medium, a 1.5 ml tube was filled with 500 μL of medium containing 0.5 μL mouse anti-MHC-1 (Merck: MABN1783) and 0.5 μL rabbit anti-PD-L1 (Abcam: ab205921). The cells were shaken and incubated on ice for 40 min. The cells were then reprecipitated by centrifugation at 100 g for 5 min, washed in 500 μL of medium + FBS, precipitated at 100 g, and then incubated for 20 min with 500 μL of medium containing 0.25 μL goat anti-mouse Alexa 488 (ThermoFisher: A-11001) and 0.25 μL goat anti-rabbit Alexa 647 (ThermoFisher: A-21244).

[0360] Cells were then washed again in 500 μL of medium, resuspended in 200 μL of medium, and transferred to 96-well plates (Thermo Scientific: 249570). Cells were immediately analyzed by flow cytometry using an Invitrogen Attune Next 4 laser system.

[0361] The Attune Next 4 laser system lasers were calibrated using standard beads according to the system guidelines. The lasers used were FSC, SSC, BL1 (GFP), and RL1 (Alexa 647). Gating was defined around the event population representing the cells, and the laser intensity was adjusted so that the signal from unstained cells was approximately 10^3 units in the RL1 and BL1 channels, respectively.

[0362] Each sample (100 μL per loaded sample) was injected into the system at a rate of 200 μL / min, and signals in BL1 and RL1 were recorded. The BL-1 channel was used to detect Alexa 488 as a measure of MHC-1, and RL-1 was used to detect Alexa 647 as a measure of PD-L1 expression.

[0363] Compared to untreated cells, increased expression of MHC-1 and PDL-I, particularly PD-L1, was evident when cells were stimulated with IFNγ, via increased RL-1 signaling. It was also noted that the morphology of PC-3 cells changed after IFNγ treatment, causing cell clusters to shift slightly upwards and to the left, where gating is defined in the SSC / FSC plot.

[0364] Data processing was performed using the FlowJo software package. For example, in the Attune software, gating was defined around the cells in the first sample.

[0365] Next, activate the statistics menu by right-clicking the line for each gate and plot the mean BL-1A (Alexa 488) and RL-1A signal (Alexa 647). Then, copy the gate settings to all subsequent samples in the dataset by dragging and dropping. Finally, manually copy the statistics for each population to the Prism data table and plot them.

[0366] For A549 cells: A549 cells were cultured in RPMI 1640 medium containing 10% FBS and 1% PenStrep at 37°C in 5% CO2.

[0367] Cells were seeded into 96-well plates and stimulated for 48 hours with 1 ng / mL human IFNγ pre-conjugated with three concentrations (10,000, 1,000, 100 ng / mL or approximately 67, 6.7, and 0.7 nM) of the selected IFNγ antibody, in duplicate. MT111W neutralizing antibody was used as an internal control (1,000 – 0.1 ng / mL), also in duplicate. The assay window was defined by A549 cells + / - 1 ng / mL human IFNγ.

[0368] For 4F4, the concentration was too low to reach 10,000 ng / mL. Therefore, the maximum concentration was adjusted to 5,000 ng / mL or the highest possible concentration.

[0369] Cells were isolated using PBS + 12.5 mM EDTA, fixed, and analyzed by flow cytometry using Attune NxT.

[0370] Dead cells were distinguished using Live / Dead near-infrared staining (Invitrogen), non-specific antibody binding was minimized using human FC blocking agent (BD), and surface staining was performed using PE-Dazzle conjugated anti-PD-L1 (clone 29E.2A3, Biolegend) and v450 conjugated anti-HLA-ABC (clone G46-2.6, BD). For both PC-3 and A549 cells: The MFI values ​​representing MHC-1 and PD-L1 surface expression are normalized by subtracting the MFI value without antibody and IFNγ addition (“baseline MFI”) from the MFI value of the sample (“sample MFI”) and dividing it by the MFI value with only IFNγ addition (IFNγ-only MFI) minus the baseline MFI ((sample MFI - baseline MFI) / (IFNγ-only MFI - baseline MFI))*100.

[0371] result The tested antibodies exhibited a range of properties. Five IFNγ antibody clones tested on PC-3 cells—4F4, 3G1, 16D2, 9F1, and 2C1—showed stronger inhibition of PD-L1 surface expression than MHC-1, as seen by the “left shift” of the PD-L1 inhibition curves relative to their corresponding MHC-1 curves—see figure. Figure 2 AE. The same properties were observed for 4F4 and 9F1 cells tested on A549 cells - see [link to relevant documentation]. Figure 2 F.

[0372] Therefore, it was concluded that all these antibodies "preferentially inhibit PD-L1 relative to MHC-1." 9F1 showed particularly high inhibition of PD-L1 on PC-3 cells while maintaining MHC-1 expression levels at 50%-80%.

[0373] Example 6: Regulatory properties of antibody 4F4 on IFNγ / IFNγ-R interaction in PC-3 cells As shown in Example 5, in order to further understand the difference in the inhibition of MHC-1 and PD-L1 by our IFNγ antibody, saturation stimulation curves of PC-3 cells were generated in the presence and absence of 50 nM antibody 4F4.

[0374] method 48 hours before stimulation, PC-3 cells (not expressing PSMA) were seeded in 25 cm² incubators. 2In flasks, a fixed amount of 4F4 antibody, yielding a final concentration of 50 nM after cell administration, was mixed with ActiveMax IFNγ to produce IFNγ at final doses of 0, 0.002, 0.01, 0.05, 0.25, and 1 nM (0, 0.04, 0.2, 0.8, 4.2, and 17 ng / ml) in cell cultures. As described in Example 5, the 4F4 / IFNγ mixture (400 μL) was incubated at room temperature for 30 minutes to allow pre-complexation between proteins, and then added to cell culture flasks to obtain a final volume of 4.4 mL. After another 48 hours, cells were harvested from the culture flasks using trypsin, washed, and resuspended in cell culture medium, and kept on ice to stop further membrane protein transport. The 4F4 / IFNγ combination treatment was then co-stained on ice for 45 minutes with 1:500 mouse anti-MHC-1 (Sigma MABN1783) and 1:500 rabbit anti-PD-L1 (Abcam ab205921), followed by washing and treatment with 1:2000 goat anti-mouse Alexa 488 (ThermoFisher A-11001) and goat anti-rabbit Alexa 647 (ThermoFisher A-21244) for 30 minutes to probe the effect of the combined treatment. After washing, the labeling intensity was determined by flow cytometry using an Attune Nxt system and Blue-1 and Red-1 lasers. Data were analyzed using the FlowJo software package as described in Example 5.

[0375] result The surface expression levels of MHC-1 and PD-L1 after antibody / IFNγ stimulation were shown in Figure 2 In G, data showed that the expression of both MHC-1 and PD-L1 was suppressed by 4F4 to a relative level similar to that observed in A549 lung cancer cells. Figure 2 F). This confirms that recombinant 4F4 retains the characteristics of the original clone, and that the effects of 4F4 can be transferred between cancer types and cell lines.

[0376] Furthermore, the results indicate that the modulatory / biased effect of 4F4 may be involved in a mechanism that reduces the effective affinity between IFNγ and IFNγR, as demonstrated by Mendoza et al. [Nature, Vol. 567, 2019] using IFNγ variants with different affinities to IFNγR. This effect is more pronounced when probing PD-L1 and MHC-1 signaling. This is evident from the dose-response curves of both MHC-1 and PD-L1 ( Figure 2 The rightward shift of G) is obvious, but the shift of PD-L1 is more pronounced than that of MHC-1.

[0377] Example 7: Bispecific Antibody Design method DuoBody Recombinant (LDC-1) V of antibody 4F4 H The region is fused with the human IgG1 constant region (amino acid residues 120-449) containing mutants L234A / L235E / G237A / A330S / P331S and DuoBody mutant K409R (SEQ ID NO: 75) and the 4F4 V L The region (SEQ ID NO: 76) was transplanted onto human Igκ. A variant of the PSMA-binding antibody J591 ANT4044 was fused to the L234A / L235E / G237A / A330S / P331S and F405L IgG1 constant regions (SEQ ID NO: 77 and 78). The antibody was custom-expressed by Genscript and Fab arm exchanged by mixing 1 molar of 4044 F405L with 1.2 molar of 4F4 K409R in PBS at a total ratio of 1 mg / mL. Disulfide bond reduction was achieved by adding 75 mM 2-MEA at 31 °C for 4 h, followed by dialysis with PBS in a 3 mL 10 kDa dialysis box to remove 2-MEA. This protein construct constitutes LDC-1. The sequence of the antibody is shown in SEQ ID NO: 75-78 (two light chains and two heavy chains).

[0378] Pestle insertion design (LDC-2, LDC-4 and LDC-6) A PSMA-derived variant of antibody J591 binds to the single-chain variable fragment (scFv) domain of antibody ANT4044 via V H With V L The design was achieved via a fusion of an 18-residue GSTSGGGSGGGSGGGGSS linker (SEQ ID NO: 82). The scFv was fused to the hinge region (before Glu216) of the human IgG C220A+ acetamipel mutant Y349C / T366S / L368A / Y407V / F405K. The V of antibody 4F4 was then fused. H The region was fused with the constant region of human IgG1 with the cleavage mutation S354C / T366W / K409A. These were then co-expressed and purified with the 4F4 light chain at a 1:1:1 molar ratio, as described below. This combination constitutes LDC-2. The sequence of this antibody is shown in SEQ ID NO:72-74 (two heavy chains and one light chain).

[0379] In a similar manner, LDC-4 was generated using a construct in which ANT4044 ScFv was replaced with nanobody number 7 (Nb7) reported by Rosenfeld et al. 2020 (J. Med. Chem. 2020, 63, 14, 7601–7615). The sequence of LDC-4 is shown in SEQ ID No: 65-67 (two heavy chains and one light chain).

[0380] LDC-6 is similar to LDC-4, except that the heavy and light chains of antibody 4F4 are replaced by the heavy and light chains of 9F1. Therefore, LDC-6 comprises the nanobody Nb7 fused with the hinge region (before Glu216) of the human IgG C220A+ acetamipel mutation Y349C / T366S / L368A / Y407V / F405K. The VH region of antibody 9F1 was fused with the constant region of human IgG1 having the acetamipel mutation S354C / T366W / K409A. These were co-expressed and purified with the 9F1 light chain at a 1:1:1 molar ratio as described below. The sequence of LDC-6 is shown in SEQ ID NO: 79-81 (two heavy chains and one light chain).

[0381] IgG (scFv / Nb) 2 Design (LDC-3 and LDC-5) The ANT4044 scFv was fused to the C-terminus of 4F4 IgG1 L234A / L235E / G237A / A330S / P331S. This construct constituting LDC-3 was co-expressed with the 4F4 light chain at a 1:1 ratio and purified as described below. The sequence of LDC-3 is shown in SEQ ID NO: 70 and 71.

[0382] A similar LDC-5 was generated by introducing Nb7 instead of ANT4044 ScFv. The sequences of LDC-5 are shown as SEQ ID NO: 63 and 64.

[0383] The characteristics of various bispecific antibodies are summarized below.

[0384] Example 8: Expression and purification of bispecific antibodies The bispecific antibody described above was fused with a secretion signal peptide (heavy chain MGWSLILLFLVAVATRVHS (SEQ ID NO: 68) and light chain MRVPAQLLGLLLLWLPGARC (SEQ ID NO: 69)), cloned into the pcDNA3.4 vector, and expressed in ExpiCHO cells using the ExpiCHO™ Expression System Kit (ThermoFisher) according to the manufacturer's 5-day expression protocol. The secreted antibody was purified from the cell culture medium by centrifugation to remove cells and loaded onto 1 ml HiTrap® MabSelect™ PrismA (Cytiva 17549852) at 0.5 mL / min. The column was washed with 20 mM HEpes pH 7.4, 100 mM NaCl, and eluted with 100 mM sodium citrate. The collected sample was then partially neutralized with Tris pH 9.0 and dialyzed overnight in PBS buffer.

[0385] Example 9: Determination of anti-PSMA antibody binding affinity method As described below, the affinity of the disclosed bispecific antibody construct for PSMA was determined by using LNCaP cells naturally expressing PSMA or PC-3 cells transiently transfected with eGFP-PSMA to generate saturation curves.

[0386] Cell maintenance and transfection Each in a 75 cm culture medium containing approximately 11-12 ml of culture medium 2 In cell culture flasks, LNCaP (Merck: 89110211-1VL) cells were maintained at 37°C and 5% CO2 in RPMI-1640 (Merck: R8758-500ML) supplemented with 10% FBS (Merck: F2442-100ML), 1% Penstrep (ThermoFisher: 15140122), 2 mM glutamine (Merck: G7513-100ML), and 1 mM Na-Pyruvate (Merck: S8636-100ML).

[0387] Each in a 75 cm culture medium containing approximately 11-12 ml of culture medium 2PC-3 cells were maintained in cell culture flasks at 37°C and 5% CO2 in RPMI 1640 + Glutamax (ThermoFisher: 72400-021) containing 10% FBS (Merck: F2442-100ML) and 1% Penstrep (ThermoFisher: 15140122).

[0388] Cells were passaged on Mondays and Fridays, with PC-3 diluted at 10x and 20x each time, and LNCaP diluted at 4x and 6x each time, as they grow more slowly. Per 75 cm⁻¹ 2 Cells were passaged using 3 ml of trypsin-EDTA (ThermoFisher25200056), and then diluted with 3 ml of FBS-containing medium to quench the trypsin. Cells were then pelleted at 100 g for 5 minutes. They were then seeded into 10 ml of fresh medium.

[0389] PC-3 cells were transiently transfected with eGFP-M1A / L5A-PSMA in pcDNA3.1. The M1A / L5A mutant group was introduced to reduce internalization. Transfection was performed using Lipofectamine 2000 (Invitrogen: 11668030). 1.5 ml of supplement-free RMPI-1640 was supplemented with 50 µl of Lipofectamine, and 1.5 ml was supplemented with 20 µl of 1 mg / ml DNA. After 5–10 minutes at room temperature, the two tubes were mixed and incubated at room temperature for another 20–30 minutes before adding the mixture to the cells. On Monday, cells were collected from approximately 80% (per 75 cm⁻¹) 2 Approximately 6 million cells were passaged twice in a cell culture flask to a density of 75 cm. 2 In culture flasks, cells were cultured until confluence reached approximately 70%. The medium was then replaced with 8 ml of serum-free and Penstrep-containing medium, and 3 ml of DNA / Lipofectamine mixture was added on top. The cells were then returned to 37°C for 4 hours, after which they were passaged to 25 cm⁻¹ cells. 2 In a normal serum-containing culture medium, each flask contains approximately 400,000 to 500,000 cells and a total of 4 ml of culture medium.

[0390] Determine affinity At the appropriate time (usually 48 hours after transfection), harvest cells from the culture flask using trypsin, wash and resuspend in cell culture medium, and keep on ice to stop membrane protein transport. Then resuspend the cells in 1.5 mL Eppendorf tubes (approximately 600,000 cells / tube) or 96-well plates containing a dilution profile of the bispecific antibody to be tested (500 μL if using Eppendorf tubes). After incubating on ice for 45 minutes, if cells were transfected with GFP-PSMA, centrifuge at 100–150 g for 5 minutes to pellet the cells, wash once in cell culture medium, and then incubate for 30 minutes in medium containing 1:2000 goat anti-human Alexa 488 (Invitrogen A-11013) or goat anti-human Alexa 647 (Invitrogen A-21445). Cells were centrifuged at 100–150 g for 5 minutes to pellet the cells, and then washed once in cell culture medium. Labeling intensity was then determined using an Attune Nxt flow cytometer system and Blue-1 and Red-1 lasers. Data were analyzed using the FlowJo software package.

[0391] result To test the affinity of the bispecific antibody for PSMA, saturation curves were generated using prostate cancer cells expressing PSMA. Figure 3 At a Kd of 4 nM, LDC-1 largely retained the affinity of the parental ANT4044 antibody. KiH-scFv (LDC-2), as well as Nb7-based LDC-4 and LDC-5, showed affinities ranging from 1.5 to 7 nM. IgG(scFv)2 (LDC-3) exhibited a weaker affinity (approximately 120 nM); however, this antibody was not purified from cell culture medium by SEC prior to affinity testing, which may have affected the results.

[0392] Example 10: LDC-mediated recruitment of IFNγ to PSMA-expressing prostate cancer cells To confirm that 4F4 retains its IFNγ binding properties after conversion to a bispecific form, and that this form can bind to PSMA to specifically recruit IFNγ to PSMA-expressing cells, binding experiments were performed on PSMA-expressing cells using purified IFNγ protein.

[0393] method Two different settings were used to demonstrate the ability of our 4F4-based bispecific antibody to recruit IFNγ to PSMA-expressing PC cells. LDC-1 (a 4F4 and ANT4044-based DuoBody form) was tested using a binding assay against LNCaP (PSMA-positive) and PC-3 (PSMA-negative) cells, with the presence of IFNγ detected using an anti-IFNγ antibody. To test LDC-1, LNCaP and PC-3 cells were cultured in parallel, harvested using trypsin, and washed in medium on ice. Cells were incubated for 45 min with or without 20 nM LDC-1, precipitated with 100 g for 5 min, and then washed in medium. Cells were then incubated with 100 nM IFNγ (Acrobiosystems catalog number IFG-H4211) for 30 min, followed by washing. The binding of IFNγ was then detected for 30 minutes using 1:500 rat anti-IFNγ (Mabtech MT111W), followed by 15 minutes using 1:1000 anti-rat Alexa 488 secondary antibody (ThermoFisher catalog number A-11006). After washing, the labeling intensity was determined by flow cytometry using an Attune Nxt system and a Blue-1 laser.

[0394] To test the IFNγ recruitment properties of LDC-2 (in the KiH form based on 4F4 and ANT4044 ScFv), human IFNγ was labeled with the Alexa 647 fluorophore. Human IFNγ (Acrobiosystems catalog IFG-H4211) was dissolved in water at 1 mg / mL, and the buffer was exchanged to PBS buffer adjusted to pH 6.5 with 20 mM sodium citrate (pH 3.0) buffer using a Micro Bio-Spin™ P-6 gel column (BioRad catalog 7326221). IFNγ was then labeled overnight at 4C with a 5- or 20-fold molar excess of NHS-Alexa 647 (ThermoFisher catalog A37573). Excess dye was removed by exchanging the PBS buffer using a Micro Bio-Spin column. PC-3 cells transfected with GFP-PSMA were incubated in RPMI 1640 containing 10% FBS and 1% PenStrep at 37°C and 5% CO2 at 75 cm⁻¹. 2Cells were cultured in flasks, harvested using trypsin, and washed in culture medium by centrifugation at 150 g for 5 min. Approximately 600,000 cells were aliquoted into 1.5 mL Eppendorf tubes and incubated on ice with incremental concentrations of LDC-2 for 45 min. Cells were centrifuged at 100–150 g for 5 min to pellet the cells, washed once in cell culture medium, and then incubated with 50 nM Alexa 647 IFNγ for 45 min. Cells were washed, and labeling intensity was determined using the Attune Nxt system Blue-1 laser to distinguish between cells expressing GFP-PSMA and those not expressing GFP-PSMA, and the amount of bound IFNγ was quantified using its Red-1 laser. Data were analyzed using the FlowJo software package.

[0395] result The ability of LDC-1 to recruit IFNγ to LNCaP cells expressing PSMA was tested. Figure 4 A, black bar). PSMA-negative PC-3 cells were tested ( Figure 4 A, gray bar).

[0396] PC-3 cells showed higher background binding than LNCaP cells, with IFNγ binding to cells being stronger regardless of the presence of LDC-1. As expected, the presence of LDC-1 did not increase the recruitment of IFNγ to PSMA-negative PC-3 cells.

[0397] On the other hand, in the presence of LDC-1, LNCaP cells expressing PSMA showed an approximately 4 to 5-fold increase in IFNγ binding, indicating that the combination of LDC-1 and PSMA expression leads to robust recruitment of IFNγ to PSMA-expressing cells.

[0398] IFNγ recruitment of LDC-2 cells was tested using PC-3 cells transfected with GFP-PSMA. IFNγ was detected via direct labeling with Alexa 647. Cells that did not take up any GFP-PSMA DNA and therefore did not express PSMA served as an internal control. Saturation curve of AF647-IFNγ binding ( Figure 4 B) shows that the binding of AF647-IFNγ to cells increases with the amount of bound LDC-2. This effect was observed only in PSMA-positive cells, confirming that the binding is specific.

[0399] Example 11: IFNγ stimulation of bispecific antibody-regulated prostate cancer cells Target The purpose of this experiment is to confirm that preferential inhibition of PD-L1 is also achieved in the bispecific form.

[0400] method As described above, PC-3 IFNγ-sensitive cells were transfected with GFP-PSMA on day 1. The following bispecific antibodies were tested: LDC-4 and LDC-6. On day 2, a 400 μL IFNγ / mAb mixture was prepared to achieve a final concentration of 0.025 nM (0.4 ng / mL) IFNγ and 0.1, 1, 10, or 100 nM antibody in a cell culture flask (4 mL). The mixture was incubated at room temperature for 30 minutes to allow pre-binding between IFNγ and the antibody, and then added to a cell culture flask containing PC-3 cells transiently transfected with GFP-PSMA.

[0401] After an additional 48 hours (day 4), the combined treatment with IFNγ and LDC-4 or LDC-6 antibodies was probed by incubating on ice for 45 minutes with either 1:500 mouse anti-human MHC-1 (Sigma MABN1783) or 1:500 rabbit anti-human PD-L1 (Abcam ab205921) at 1:2000 goat anti-mouse Alexa 647 (Invitrogen A-21235) or goat anti-rabbit Alexa 647 (Invitrogen A-21244) at 1:2000 for 30 minutes. Following washing, the labeling intensity and GFP-PSMA expression were determined using a tune Nxt flow cytometry system and Blue-1 and Red-1 lasers. Data were analyzed using the FlowJo software package. When PC-3 cells were transiently transfected with GFP-PSMA (Lipofectamine), only a fraction of these cells expressed GFP-PSMA. Therefore, by separating PC-3 cells into PSMA-expressing and PSMA-non-PSMA-expressing cells based on their GFP fluorescence, the cell samples effectively contained an internal control. It was expected that only PSMA-expressing cells would bind LDC.

[0402] result As expected, cells stimulated with IFNγ alone showed significantly increased expression of both MHC-1 and PD-L1 on the surface compared to unstimulated cells, independent of GFP-PSMA expression levels. Figure 4 C).

[0403] For LDC-4 and -6, dose-dependent inhibition of MHC-1 and PD-L1 surface expression was observed, independent of PSMA expression. This observation was expected based on previous experiments.

[0404] In untransfected cells, LDC-6 suppressed MHC-1 expression to a slightly greater extent than LDC-4. This inhibition is expected to partially mask any increase in IFNγ-mediated MHC-1 expression upregulation resulting from LDC recruitment to PSMA-expressing cells.

[0405] Both LDC-4 and LDC-6 inhibited PD-L1 surface expression to a greater extent than MHC-1 expression. LDC-6 significantly reduced PD-L1 expression in both PSMA-positive and PSMA-negative cells.

[0406] Example 12: Bispecific antibodies preserve pro-inflammatory signal transduction across different cell lines The aim of this experiment is to demonstrate that IFNγ bound to bispecific antibodies retains pro-inflammatory signal transduction across different cell lines.

[0407] method The constructs used in this experiment are KiH-3 and KiH-6, which were discussed above. KiH-3 contains a 4F4 IFNγ conjugate and Nb7 as a TAA conjugate, while KiH-6 contains a 9F1 conjugate as an IFNγ conjugate and Nb7 as a TAA conjugate.

[0408] As described in Example 5, the IFNγ / IFNγR bias of antibodies KiH-3 and KiH-6 was tested using the surface expression of MHC-I and PD-L1 on cancer cells as a proxy for IFNγR signaling. PC3-FLU prostate cancer cells, A549 lung cancer cells, or HT29 colorectal cancer cells (all of which do not express PSMA) were stimulated with a fixed dose of 1 ng / ml (0.6 nM) of IFNγ for 48 hours, which produced receptor activation within the dynamic range of stimulation. To test the effects of the bispecific antibodies, a series of concentrations of these bispecific antibodies were additionally added. The method was the same as in Example 5, except that additional concentrations of antibody (10 µg / mL; 87 nM) were tested against A549 cells. HT29 cells were tested in the same manner as PC3-FLU cells.

[0409] Time-course studies were also conducted to evaluate the effects of the bispecific antibodies KiH-3 (10 nM) or KiH-6 (10 nM) on the surface expression of MHC-I and PD-L1 on A549 cells (non-PSMA-expressing) and PC3-PIP cells (PSMA-expressing) in the presence and absence of human IFNγ (0.1 nM) at 6, 24, 48, and 72 hours.

[0410] To further understand the difference in the inhibition of MHC-1 and PD-L1 by the bispecific IFNγ antibody, PC3-PIP cells (expressing PSMA) were stimulated with IFNγ (0.025 nM or 5.95 nM), and stimulated with the bispecific antibody KiH-3 at concentrations of 1 nM or 10 nM with or without 0.025 nM IFNγ.

[0411] result The results of the IFNγ / IFNγR bias experiment are shown in Figure 5 In this study, both KiH-3 and KiH-6 maintained MHC-1 expression of IFNγ across different cell lines. MHC-1 expression was completely maintained in all cell lines at a concentration of 0.1 nM antibody. In HT29 and AT549 cells, MHC-1 expression was completely maintained at a concentration of 10 nM antibody. The antibody was partially inhibitory, as some inhibitory effect was observed at higher concentrations, but the IC50 was low in all cell lines. 50 Greater than 0.1 nM. For example... Figure 5 As shown in c, in A549 cells, the inhibitory activity of both KiH-3 and KiH-6 was lower than that of the IFNγ neutralizing control antibody. IC 50 It is about 1,000 times higher.

[0412] The IFNγ / IFNγR bias characteristics of antibodies KiH-3 and KiH-6 are also shown in Figure 5 Both antibodies showed stronger inhibition of PD-L1 surface expression than MHC-1, particularly on A549 cells, as evidenced by the “left shift” of the PD-L1 inhibition curves relative to their corresponding MHC-1 curves. Figure 5 ).

[0413] The results of the time history study are shown in Figure 6 In the absence of IFNγ, KiH-3 and KiH-6 did not stimulate the surface expression of MHC-I or PD-L1 on any cells. For A549 cells, no inhibition of MHC-I expression was observed with either bispecific antibody compared to IFNγ alone, while PD-L1 expression was inhibited by approximately 20% (KiH-3) and approximately 50% (KiH-6), respectively. Figure 6 C). For PC3-PIP cells, KiH-3 inhibited MHC-I expression by 50% and PD-L1 expression by 80% compared to IFNγ alone. Figure 6 Similar results were observed for KiH-6.

[0414] Therefore, it can be concluded that both KiH-3 and KiH-6 maintain the pro-inflammatory activity of IFNγ, and the percentage of inhibition of PD-L1 cell surface expression is greater than that of MHC-I cell surface expression.

[0415] Further investigation was conducted using IFNγ stimulation experiments in the presence and absence of KiH-3, and the results are shown in... Figure 7 In comparison with IFNγ alone, KiH-3 inhibited the surface expression of both MHC-I and PD-L1 on PC3-PIP cells. Furthermore, when the surface expression of MHC-I and PD-L1 in the presence of KiH-3 was normalized against IFNγ alone, KiH3 inhibited MHC-I expression by 40% (1 nM) or 52% (10 nM), and PD-L1 expression by 55% (1 nM) or 61% (10 nM), respectively.

[0416] Example 13: Targeted delivery of IFNγ PSMA to tumors using KiH-3 The aim of this experiment was to evaluate the ability of the KiH-3 bispecific antibody to target and deliver IFNγ to tumors. For this purpose, a PC-3 prostate cancer xenograft tumor model expressing PSMA was used. Figure 8 A).

[0417] method Prostate cancer xenograft tumor model Athymic nude mice (BALB / cAnNRj-Foxn1 nu / nu) immunodeficient mice (male, 6 weeks old) were purchased from Janvier, Europe. To establish a xenograft tumor model, 1 x 10⁻⁶ mcg of thymic matrix gel was used. 6 PC3-PIP cells were subcutaneously injected into the flank of each mouse. Approximately 3 weeks post-inoculation, the tumor had reached 250 mm. 3 Size.

[0418] IHC staining When the tumor reaches 250 mm 3The inoculum was administered intravenously to mice via the tail vein under isoflurane anesthesia using a syringe. For the test group, 10 mg / kg KiH-3 was premixed with 1.5 mg / kg IFNγ (1:1 molar ratio) and incubated at room temperature for 30 minutes to allow the protein to pre-complex before inoculation. For the control group, the inoculum contained IFNγ alone (1.5 mg / kg). After 72 hours, tumors were excised from each mouse, fixed in 10% neutral buffered formalin for 24 hours, and then transferred to 70% EtOH. Tissue samples were then paraffin-embedded over 2–3 days. Samples were trimmed and sections were obtained for H&E staining to ensure the presence of tumor areas. Slides were stained using anti-IFNγ (D3H2, Cell Sign. Techn.). Digital slides were obtained from all stained slides using a brightfield scanner (Zeiss AxioScan, 20x).

[0419] In vivo imaging KiH3 was labeled with Alexa 680 NHS ester dye (Invitrogen: A37574) according to a specific protocol designed to minimize interference with antibody function. KiH3 was first purified and dialyzed overnight. Alexa 680-NHS was dissolved in DMSO (Sigma: D2650-100ml) at a concentration of 100 µg / 10 µl. For labeling, the reaction mixture was prepared by mixing 30 µl of Alexa 680-NHS solution with 696 µl of KiH3 at a concentration of 61,775 nM (7.1 mg / ml) in PBS (total volume approximately 2 mL). In this way, a molar ratio of 5 dyes per antibody was achieved. The reaction mixture was incubated overnight at 4°C. The next day, excess dye was removed using a PD-10 desalting column (Cytiva: 17085101). The column was prepared and equilibrated with PBS buffer, and the reaction mixture was treated to collect clean labeled antibodies. The labeled antibody was then further purified by size exclusion chromatography to remove aggregates.

[0420] Under isoflurane anesthesia, three mice containing PC-3 prostate cancer xenograft tumors prepared as described were each injected intravenously via the tail vein with one dose (1-1.5 nmol) of Alexa680-labeled KiH-3. The mice were imaged using a far-infrared camera three days post-injection.

[0421] result Compared to hIFNγ alone, stronger hIFNγ staining was observed in tumor samples from mice that received a combination of hIFNγ and KiH3. Figure 8(B) Specifically, the presence of hIFNγ was detected around blood vessels. Therefore, the level of hIFNγ in the tumor increased in the presence of KiH3. Thus, it is concluded that KiH-3 successfully targeted hIFNγ to the tumor.

[0422] The results of in vivo imaging are shown in Figure 8 In C, fluorescently labeled KiH3 antibodies were detected in the tumor via in vivo imaging 3 days after injection.

[0423] Example 14: Upregulation of MHC-I in xenograft tumors in mice The aim of this study was to detect MHC-I expression in PSMA-positive PC3-PIP prostate cancer xenografts in mice treated with the KiH-3:IFNγ complex via immunohistochemical staining. In this context, MHC-I staining can be considered a signal of immune stimulation within the tumor.

[0424] method The prostate cancer xenograft tumor model was prepared and IHC staining was performed as described in Example 13, except that an additional medium was used for control inoculation and anti-MHC-I (EP1395Y, #ab52922) was used to stain the slides.

[0425] result The results are shown in Figure 9 In contrast to the vector control group, MHC-I staining was observed in tumors of mice injected with either IFNγ alone or the KiH3:IFNγ complex. Notably, MHC-I was upregulated in tumors of mice inoculated with the KiH3:IFNγ complex compared to IFNγ alone, as indicated by the increased MHC-I staining intensity.

[0426] Example 15: In vivo homologous model The aim of this study was to generate a syngeneic model suitable for experimentation in mice without inducing unwanted immunogenicity. To this end, a bispecific antibody, KiH-16, was prepared based on the KiH-3 construct described in Example 7, but containing a mouse IgG2a isotype with effector silencing instead of a human constant domain region.

[0427] In this study, the pharmacokinetic properties of KiH-16 were measured, including tolerability and the accumulation of mIFN-γ in serum after KiH-16 administration.

[0428] method Pharmacokinetics of KiH-16 Each group consisted of six C57BL / 6 mice, and each group was further divided into two subgroups, referred to as subgroup-a and subgroup-b, with each subgroup consisting of three mice. Treatment began on the day of grouping (day 0). The dosing regimen used was as follows:

[0429] Approximately 150 μL of blood was collected from each mouse 24 h (subgroup-a) or 72 h (subgroup-b) after administration. Approximately 500 μL of stop blood was collected from each mouse immediately (before administration), 144 h (subgroup-a) after administration, or 192 h (subgroup-b) after administration. Blood samples were kept at room temperature for 30 to 60 minutes and then centrifuged at approximately 2000 × g for 15 minutes at RT to obtain serum. Serum samples were stored at -80°C and then used to detect (i) mouse IFN-γ and (ii) KiH-16 by ELISA.

[0430] mIFN-γ ELISA: The mIFN-γ monoclonal antibody XMG1.2 (Thermo Fisher Scientific, catalog number 16-7311-85) was diluted to a final concentration of 2 μg / mL. 100 mL of the diluted antibody solution was then applied to 96-well flat-bottom microplates (American Burton Instruments Co., Ltd., Bio Tek H1MF). The plates were incubated overnight at 4°C. The next day, the coated plates were washed four times with PBST, blocked with 2% BSA, and then incubated at 37°C with shaking at 400 rpm for 1 hour. The plates were washed four more times with PBST, followed by the addition of 100 μL of the above-diluted standard mouse IFN-γ or serum sample, and incubated at 37°C with shaking at 400 rpm for 1 hour. After washing four times with PBST, 100 μL of the detection antibody #ab14-Biotin (1 μg / mL, Biocytogen) was added to each well and incubated at 37°C with shaking at 400 rpm for 1 hour. The plate was washed four times with PBST, and the bound antibody was detected using Pierce™ streptavidin poly-HRP (Thermo Scientific, catalog number: 21140). After washing four times with PBST, 100 μL of TMB substrate solution was added to each well, and the reaction was allowed to proceed for up to 20 minutes to allow for color development, then terminated with 0.1 N HCl. Finally, the OD values ​​were measured at 450 nM and 570 nM using a microplate reader (American Burton Instruments Co., Ltd.), and the concentration of mIFN-γ was calculated.

[0431] KiH-16 ELISA: An ELISA was developed to detect the presence of KiH-16 by binding to immobilized human PSMA protein. Assay plates were prepared by adding 200 μL of 2% BSA to each well of a streptavidin-coated 96-well plate (Thermo Scientific™, catalog 15120) and incubating at 25°C for 1 hour. The plates were then washed four times with PBST buffer, and 100 μL of diluted biotinylated human PSMA protein (Acro, catalog PSA-H82Qb) at a concentration of 0.5 μg / mL was added to each well, and the plate was incubated at 25°C for 1 hour. The plate was then washed four times with PBST. Diluted serum samples were added to each well at a volume of 100 μL, and the plate was incubated at 25°C for 1.5 hours. The plate was then washed four times with PBST buffer to remove any unbound sample. The secondary antibody HRP (Hydrogen Pro-Goat Anti-Mouse IgG2a Heavy Chain) (Abcam, catalog number ab97245) was then diluted 1:100,000 in 1% BSA, and 100 μL of this diluted solution was added to each well. The plate was incubated at 25°C for one hour and washed four times with PBST buffer. 100 μL of TMB substrate was added to each well, and the reaction was allowed to proceed at room temperature for 5–20 minutes. The reaction was terminated by adding 100 μL of stop solution (Beyotime, catalog number P0215-100 mL) to each well. Finally, the OD values ​​of the wells were measured at 450 nM and 630 nM using an ELISA reader (BioTek, EPOCH2).

[0432] result Mice tolerated all tested doses of KiH-16 well and via both tested injection routes.

[0433] The results of the KiH-16 exposure experiment are shown in Figure 10 In vitro, dose-linearity was observed for escalating concentrations of KiH-16 as tested via intravenous injection. High bioavailability was also observed with intraperitoneal injection, resulting in KiH-16 plasma concentrations that were 80% of those obtained with equivalent intravenous injection. The KiH-16 antibody also exhibited a long half-life in vivo, consistent with expectations for the selected mouse constant region.

[0434] Compared to baseline concentrations in control serum before administration, mIFN-γ accumulation was observed in serum in all mice after a single dose of KiH-16. Figure 11 A). For a dose of 10 mg / kg (intraperitoneal and intravenous), accumulation over time was most significant. The molar ratio of KiH-16 to mIFN-γ detected in serum at each time point decreased over time. Figure 11 B).

[0435] Example 16: In vitro homologous model The aim of this study was to evaluate the effects of KiH-16 on B-hPSMA MC38 cells in the presence of mIFN-γ compared to neutralizing anti-mIFN-γ antibody XMG1.2 (Invitrogen, catalog number 16-7311-81) and non-neutralizing anti-mIFN-γ antibody AN-18 (Invitrogen, catalog number 16-7313-85).

[0436] method In vitro functional assessment of KiH-16 in B-hPSMA MC38 cells The MC38 mouse colon cancer cell line was purchased from Shunran Shanghai Biological Technology Co., Ltd. MC38 cells were genetically modified to express human PSMA, and the resulting cells were named B-hPSMA MC38 by Biocytogen Pharmaceuticals (Beijing) Co., Ltd. Cells were maintained in vitro as a monolayer culture in DMEM supplemented with 10% heat-inactivated FBS at 37°C under a humidified atmosphere of 5% CO2.

[0437] Determination of the stimulation concentration of mIFN-γ: B-hPSMA MC38 cells were loaded at 3×10⁻⁶ 4 Cells were seeded at a density of / wells in 48-well plates. After overnight adhesion, cells were stimulated in duplicate with a series of concentrations of mIFN-γ (Sino Biological, catalog number 50709-MNAH) in 5% CO2 at 37°C for 24 or 48 hours. The tested concentrations were 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 250, and 500 ng / mL. After stimulation, dead cells were distinguished using Live / Dead Near-Infrared Staining (Invitrogen), and surface staining was performed with anti-PD-L1 (Biolegend, catalog number 124312) and anti-MHC-I (Biolegend, catalog number 114606), followed by analysis by flow cytometry.

[0438] Evaluation of KiH-16 functionality in the presence of mIFN-γ: B-hPSMA MC38 cells were loaded at 3×10⁻⁶ 4Cells were seeded at a density of 1 / 2 well in 48-well plates. A series of concentrations of KIH-16, AN-18, or XMG1.2 (0.9, 9, 90, 900 nM) were premixed with 5 ng / mL mIFN-γ at room temperature for 30 min. For KIH-16, another maximum concentration of 9000 nM was also tested. After attachment, cells were stimulated with the premixed solution at 37°C in 5% CO2 for 48 h. After stimulation, dead cells were distinguished using Live / Dead Near-Infrared Staining Agent (Invitrogen), and surface staining was performed with anti-PD-L1 (Biolegend, catalog number 124312) and anti-MHC-I (Biolegend, catalog number 114606), followed by analysis by flow cytometry.

[0439] result The results of the mIFN-γ stimulation assay are shown in Figure 12 In this study, MC38 cells showed almost the same sensitivity to mIFN-γ as human cells to hIFN-γ. A concentration of 5 ng / mL of mIFN-γ and a stimulation duration of 48 hours were selected as the optimal conditions for the next assay. Under these conditions, the surface expression levels of both MHC-I and PD-L1 reached a plateau.

[0440] The results of the in vitro functional tests are shown in Figure 13 In B-hPSMA MC38 cells, no changes in the surface expression levels of MHC-I and PD-L1 were observed with increasing antibody concentrations for KiH-16 and AN-18 (non-neutralizing control). In contrast, a significant decrease was observed with the neutralizing control antibody XMG1.2. Therefore, it is concluded that KiH-16 does not neutralize IFNγ.

[0441] Example 17: Homologous Mouse Model The aim of this study was to evaluate the potential of KiH-16 antibody to accumulate mIFNγ and infiltrate the innate and adaptive immune systems. The MC38 cells used in this example do not express hPSMA, and therefore KiH16 does not act as a targeting antibody in this model.

[0442] method MC38 mouse colon cancer cell line was purchased from Shunran Shanghai Biological Technology Co., Ltd. Cells were maintained as a monolayer culture in DMEM supplemented with 10% heat-inactivated FBS at 37°C under a humidified atmosphere of 5% CO2. MC38 tumor cells (5 × 10⁶ cells per mouse) in 0.1 mL PBS were subcutaneously injected into the right anterior side of C57BL / 6 mice.5 (Each cell) is involved in tumor development. When the average tumor size reaches 80 to 120 mm... 3 Eight mice were randomly recruited for each group. Treatment began on the day of grouping (Day 0). Starting from Day 0, anti-mouse PD-1 (1 mg / kg) was administered intraperitoneally twice weekly for a total of seven times, with or without KiH16 (3 mg / kg). Blood was collected from each mouse 24 hours after the first, third, fifth, and seventh (final) administrations. Blood samples were kept at room temperature for 30 to 60 minutes and then centrifuged at approximately 2000 × g for 15 minutes at RT to obtain serum. Serum samples were stored at -80°C for the detection of mIFN-γ by ELISA. At the study endpoint, animals were euthanized with CO2, and four mice from each group were selected from which tumors were collected. Tumors were stained using Live / Dead near-infrared staining agent (Invitrogen) and antibodies against mCD45, mCD3, mF4 / 80, mCD66a, mPD-L1, and mMHC-I, and analyzed by flow cytometry.

[0443] mIFN-γ ELISA: The IFN-γ monoclonal antibody XMG1.2 (Thermo Fisher Scientific, catalog number 16-7311-85) was diluted to a final concentration of 2 μg / ml, and 100 ml of the diluted antibody was applied to 96-well flat-bottom microplates (American Burton Instruments Co., Ltd., Bio Tek H1MF). The plates were incubated overnight at 4°C. The next day, the coated plates were washed four times with PBST, blocked with 2% BSA, and then incubated at 37°C with shaking at 400 rpm for 1 hour. The plates were washed four times with PBST, and then 100 μL of diluted standard mouse IFN-γ or serum sample was added, and the plates were incubated at 37°C with shaking at 400 rpm for 1 hour. After washing four times with PBST, 100 μL of detection Ab #ab14-Biotin (1 μ...

Claims

1. A multispecific antigen-binding molecule comprising an antigen-binding domain for binding interferon-γ (IFNγ) and an antigen-binding domain for binding target antigens other than IFNγ.

2. The multispecific antigen-binding molecule of claim 1, wherein the target antigen is a tumor-associated antigen (TAA).

3. The multispecific antigen-binding molecule of any one of the preceding claims, wherein the molecule has a Kd of less than 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 7 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, or 0.25 nM. D Binding to IFNγ, as assessed with a BIACORE® surface plasmon resonance assay.

4. The multispecific antigen-binding molecule as described in any of the preceding claims, wherein the molecule is in a K+ concentration of less than 200 nM, or less than 20 nM, or less than 10 nM, or less than 5 nM. D Combined with IFNγ.

5. The multispecific antigen-binding molecule as described in any of the preceding claims, wherein the molecule is in the form of a K+ concentration of less than 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 7 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, or 0.25 nM. D Combined with the target antigen, as evaluated using BIACORE® surface plasmon resonance assay.

6. The multispecific antigen-binding molecule as described in any of the preceding claims, wherein the molecule is in a K+ concentration of less than 20 nM. D Combined with the target antigen, as evaluated using BIACORE® surface plasmon resonance assay.

7. The multispecific antigen-binding molecule of any one of claims 2 to 6, wherein the TAA is selected from the group consisting of: PSMA (prostate-specific membrane antigen), EGFR (epidermal growth factor receptor), CEA (carcinoembryonic antigen-associated cell adhesion molecule), HER2 (receptor tyrosine protein kinase erbB-2), TN antigen (tenosynovin), STN antigen (CST complex subunit STN), CD44 (CD44 antigen), Trop2 (tumor-associated calcium signal transducer 2), and B7-H3 (B7 homolog 3).

8. The multispecific antigen-binding molecule as described in any of the preceding claims, wherein the binding of the molecule to IFNγ regulates cellular signaling downstream of IFNγ.

9. The multispecific antigen-binding molecule of claim 1 or claim 8, wherein the binding of the molecule to IFNγ biases the pro-inflammatory and anti-inflammatory activities of IFNγ toward the pro-inflammatory activity.

10. The multispecific antigen-binding molecule of claim 9, wherein the binding of the molecule to IFNγ regulates cellular signaling downstream of IFNγ, such that the ratio of IFNγ-induced MHC-1 expression and / or activity to PD-L1 expression and / or activity is altered to favor MHC-1 expression and / or activity.

11. The multispecific antigen-binding molecule as claimed in any of the preceding claims, wherein the binding of the molecule to IFNγ partially inhibits the pro-inflammatory activity of IFNγ.

12. The multispecific antigen-binding molecule of claim 11, wherein the binding of the molecule to IFNγ partially inhibits the induction of MHC-1 expression and / or activity by IFNγ.

13. The multispecific antigen-binding molecule of claim 11 or claim 12, wherein the IC50 of the antibody against IFNγ activity is less than 1 mM and at least 1 nM, and the activity is assessed by contacting plated test A549 lung cancer cells with IFNγ at a concentration of 1 ng / mL in the absence and presence of the test antibody and evaluating the expression of MHC-1 on the cell surface as a readout of IFNγ activity after 48 hours.

14. The multispecific antigen-binding molecule as described in any of the preceding claims is a multispecific antibody.

15. The multispecific antigen-binding molecule as described in any of the preceding claims, comprising an Fc region and / or conjugated to a half-life extension portion.

16. The multispecific antigen-binding molecule of claim 15, wherein the molecule comprises an Fc region, and the Fc region comprises one or more mutations selected from the group consisting of: i) Further extending the half-life of the molecule by selecting from the group consisting of: R435H mutation, T307Q mutation, T307P mutation, T307A mutation, T307R mutation, N265 mutation, D265A mutation, N434S mutation, T250Q mutation, T250E mutation, M428L mutation, M428F mutation, V308W mutation, V308Y mutation, V308F mutation, E294 deletion / T3 07P / N434Y mutation, T256N / A378V / S383N / N434Y mutation, M428L / N434S mutation, T307A / E380A / N434A mutation, T307Q / N434S mutation, Q311V / N434S mutation, H433K / N434F mutation, E294 deletion / T307P / N434Y mutation, T307A / E380A / N434A mutation, The following mutations are associated with the following individuals: T256D / H286D / T307R / Q311V / A378V, H285N / T307Q / N315D, T307Q / Q311V / A378V, H285D / T307Q / A378V, T307Q / N434S, M252Y / S254T / T256E, T250Q / M428L, and T250Q / M428F. T250E / M428F mutation, T250E / M428L mutation, M252Y / M428L mutation, M428L / V308F mutation, D259I / V308F mutation, E258F / V427T mutation, T256D / Q311V / A378V mutation, T256D / H286D / T307R / Q311V / A378V mutation, and K288E / H435K mutation; and / or ii) The antibody reduces or silences the effector function of the antibody, selected from the group consisting of: R435H mutation, N434A mutation, T252L / T253S / T254F mutation, E294 deletion / T307P / N434Y mutation, T256N / A378V / S383N / N434Y mutation, E294 deletion mutation, M252Y / S254T / T256E mutation, M428L / N434S mutation, T307A / E380A / N434A mutation, T250Q / M428L mutation, T250Q / M428F mutation, T250E / M428F mutation, T250E / M428L mutation, T256D / Q311V / A378V mutation. Mutations include: T256D / H286D / T307R / Q311V / A378V, H285N / T307Q / N315D, T307Q / Q311V / A378V, H285D / T307Q / A378V, L234F / L235E / P331S, L234F / L235Q / K322Q, L234F / L235Q / P331G, L234F / L235A / K322Q, S228P / F234A / L235A / G237A / P238S, L234A / L235E / G237A / A330S / P331S, and F243A / V264A. Mutations, S228P / L235E / P329G mutation, M252Y / M428L mutation, D259I / V308F mutation, T307Q / N434S mutation, M428L / V308F mutation, Q311V / N434S mutation, H433K / N434F mutation, E258F / V427T mutation, K288E / H435K mutation, F234A / L235A mutation, F234A / L235A / S228P mutation, L234A / L235A / P329G mutation, S228P / F234A / L235A / P329G mutation, Y349C / T366S / L368A / Y407V / F405K mutation, S 354C / T366W / K409A mutation, L234S / L235T / G236R mutation, P329G mutation, S228P mutation, deletion-K447 mutation, L234A mutation, L235A mutation, L235E mutation, G237A mutation, V308P mutation, V308W mutation, V308Y mutation, V308F mutation, N434S mutation, T307Q mutation, T307P mutation, T307A mutation, T307R mutation, Q312A mutation, Q318A mutation, Q233P mutation, K322A mutation, P329A mutation, P331A mutation, P238A mutation, P238S mutation, F241A mutation, D265A mutation.D269A mutation, D270A mutation, N297A mutation, A327Q mutation, P329A mutation, S239A mutation, E294A mutation, Q295A mutation, V303A mutation, V264A mutation, A330R mutation, A330L mutation, A330S mutation, P331S mutation, T299A mutation, F234A mutation, T366W mutation, T366S mutation, L368A mutation, Y407V mutation, Y349C mutation, F405L mutation, F405K mutation, S354C mutation, T366W mutation, K409R mutation, K409A mutation, F405L / R409K mutation, and any combination thereof; and / or, iii) The effector function of the antibody is enhanced by selecting from the group consisting of: F243L / R292P / Y300L / V305I / P396L mutation, S239D / I332E mutation, S239D / I332E / A330L mutation, S298A / E333A / K334A mutation, G236A / S239D / I332E mutation, K326W / E3 33S mutation, S267E / H268F / S324T mutation, L234Y / L235Q / G236W / S239M / H268D / D270E / S298A in one heavy chain and D270E / K326D / A330M / K334E in another heavy chain, E345R / E430G / S440Y mutation and IgG1 / IgG3 cross subclass mutation; And / or iv) The antibody’s stability or downstream processing is improved by means of the group consisting of: S228P mutation, R409K mutation, R409T mutation, R409M mutation, R409L mutation, S228P / L235E / R409K mutation, S228P / L235E / R409T mutation, S228P / L235E / R409M mutation, S228P / L235E / R409L mutation, G446 / deletion / K447 deletion mutation, K370Q mutation, K370E mutation, R409A mutation, R409E mutation, R409W mutation, C131S mutation, K370 deletion or substitution, deletion-K447 mutation, F405L mutation, K409R mutation and F405L / R409K mutation; Or any combination thereof.

17. The multispecific antigen-binding molecule of any one of claims 2 to 16, comprising a heterodimeric IgG containing a first antibody heavy chain and a second antibody heavy chain, a first antibody light chain, and a second antibody light chain, wherein the first heavy chain and the first light chain are assembled to form an antigen-binding domain against IFNγ, and wherein the second heavy chain and the second light chain are assembled to form an antigen-binding domain against the TAA.

18. The multispecific antigen-binding molecule of any one of claims 2 to 16, comprising an Fc domain containing a first Fc subunit and a second Fc subunit, wherein each subunit is directly or indirectly fused to an antigen-binding moiety, and wherein the first Fc subunit is fused to an antigen-binding moiety capable of binding IFNγ, and the second Fc subunit is fused to an antigen-binding moiety capable of binding the TAA.

19. The multispecific antigen-binding molecule of claim 18, wherein the antigen-binding portion is an antibody fragment selected from the group consisting of: Fv, Fab, cross-Fab, Fab', Fab'-SH, F(ab')2; biantibody; linear antibody; single-chain antibody molecule and single-domain antibody.

20. The multispecific antigen-binding molecule of claim 18 or claim 19, wherein the first subunit and / or the second subunit are fused to the antigen-binding portion via their N-terminus.

21. The multispecific antigen-binding molecule as described in any of the preceding claims, wherein the molecule is monovalent for IFNγ.

22. The multispecific antigen-binding molecule as claimed in any of the preceding claims, comprising a binding site for IFNγ and a binding site for more than one TAA.

23. The multispecific antigen-binding molecule according to any one of claims 1 to 21, wherein it is a bispecific antigen-binding molecule, optionally a bispecific antibody.

24. The multispecific antigen-binding molecule as claimed in any of the preceding claims, wherein the IFNγ-binding domain comprises a heavy chain variable region containing HC-CDR1, HC-CDR2, and HC-CDR3, wherein: a) The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 4 or a variant thereof in which one, two or three amino acids in HC-CDR1 are replaced by another amino acid; b) The heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 5 or a variant thereof in which one, two, or three amino acids in HC-CDR2 are substituted by another amino acid; and c) The heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 6 or a variant thereof in which one, two or three amino acids in HC-CDR3 are replaced by another amino acid; And / or wherein the IFNγ-binding domain comprises a light chain variable region containing LC-CDR1, LC-CDR2 and LC-CDR3, wherein: d) The light chain CDR1 contains the amino acid sequence of SEQ ID NO: 7 or a variant thereof in which one, two, or three amino acids in LC-CDR1 are replaced by another amino acid; e) The light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 8 or a variant thereof in which one, two, or three amino acids in LC-CDR2 are substituted with another amino acid; and f) The light chain CDR3 contains the amino acid sequence of SEQ ID NO: 9 or a variant thereof in which one, two or three amino acids in LC-CDR3 are replaced by another amino acid.

25. The multispecific antigen-binding molecule of claim 24, wherein the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 6 and / or the light chain CDR3 comprises the amino acid sequence of SEQ ID NO:

9.

26. The multispecific antigen-binding molecule of claim 24 or claim 25, wherein the heavy chain CDR1 comprises the sequence GYTXTNYY, wherein X can be any amino acid.

27. The multispecific antigen-binding molecule of claim 26, wherein the heavy chain CDR1 comprises GYTFTNYY (SEQ ID NO: 4) or GYTETNYY (SEQ ID NO: 85).

28. The multispecific antigen-binding molecule of any one of claims 24 to 27, wherein the heavy chain CDR2 comprises the sequence INPSNXGT, wherein X can be any amino acid.

29. The multispecific antigen-binding molecule of claim 28, wherein the heavy chain CDR2 comprises INPSNDGT (SEQ ID NO: 5) or INPSNHGT (SEQ ID NO: 87).

30. The multispecific antigen-binding molecule of any one of claims 24 to 29, wherein the light chain CDR1 comprises the sequence QSXLYSSNXKNY, wherein X can be any amino acid.

31. The multispecific antigen-binding molecule of claim 30, wherein the light chain CDR1 comprises the sequence QSLLYSSNQKNY (SEQ ID NO: 7) or QSVLYSSNNKNY (SEQ ID NO: 89).

32. The multispecific antigen-binding molecule of any one of claims 24 to 31, wherein the IFNγ-binding domain comprises a heavy chain variable region containing HC-CDR1, HC-CDR2, and HC-CDR3, wherein: a) The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 4; b) The heavy chain CDR2 contains the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 87; and c) The heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 6; It also includes light chain variable regions containing LC-CDR1, LC-CDR2, and LC-CDR3, wherein: d) The light chain CDR1 contains the amino acid sequence of SEQ ID NO: 7; e) The light chain CDR2 contains the amino acid sequence of SEQ ID NO: 8; and f) The light chain CDR3 contains the amino acid sequence of SEQ ID NO:

9.

33. The multispecific antigen-binding molecule of any one of claims 24 to 31, wherein the IFNγ-binding domain comprises a heavy chain variable region containing HC-CDR1, HC-CDR2, and HC-CDR3, wherein: a) The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 85; b) The heavy chain CDR2 contains the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 87; and c) The heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 6; It also includes light chain variable regions containing LC-CDR1, LC-CDR2, and LC-CDR3, wherein: d) The light chain CDR1 contains the amino acid sequence of SEQ ID NO: 7; e) The light chain CDR2 contains the amino acid sequence of SEQ ID NO: 8; and f) The light chain CDR3 contains the amino acid sequence of SEQ ID NO:

9.

34. The multispecific antigen-binding molecule of any one of claims 24 to 31, wherein the IFNγ-binding domain comprises a heavy chain variable region containing HC-CDR1, HC-CDR2, and HC-CDR3, wherein: a) The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 4; b) The heavy chain CDR2 contains the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 87; and c) The heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 6; It also includes light chain variable regions containing LC-CDR1, LC-CDR2, and LC-CDR3, wherein: d) The light chain CDR1 contains the amino acid sequence of SEQ ID NO: 89; e) The light chain CDR2 contains the amino acid sequence of SEQ ID NO: 8; and f) The light chain CDR3 contains the amino acid sequence of SEQ ID NO:

9.

35. The multispecific antigen-binding molecule of any one of claims 24 to 31, wherein the IFNγ-binding domain comprises a heavy chain variable region containing HC-CDR1, HC-CDR2, and HC-CDR3, wherein: a) The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 85; b) The heavy chain CDR2 contains the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 87; and c) The heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 6; It also includes light chain variable regions containing LC-CDR1, LC-CDR2, and LC-CDR3, wherein: d) The light chain CDR1 contains the amino acid sequence of SEQ ID NO: 89; e) The light chain CDR2 contains the amino acid sequence of SEQ ID NO: 8; and f) The light chain CDR3 contains the amino acid sequence of SEQ ID NO:

9.

36. The multispecific antigen-binding molecule according to any one of claims 24 to 35, wherein it is humanized.

37. The multispecific antigen-binding molecule of claim 36, wherein the IFNγ-binding domain comprises Heavy chain variable regions containing HC-FR1, HC-FR2, HC-FR3, and HC-FR4, wherein: HC-FR1 has sequences selected from SEQ ID NO: 90, SEQ ID NO: 98 and SEQ ID NO: 102; HC-FR2 has the sequence shown in SEQ ID NO: 91; HC-FR3 has sequences selected from SEQ ID NO: 92, SEQ ID NO: 99, SEQ ID NO: 103, and SEQ ID NO: 105; and HC-FR4 has sequences selected from SEQ ID NO: 93 and SEQ ID NO: 100; and / or The light chain variable region contains LC-FR1, LC-FR2, LC-FR3 and LC-FR4, wherein: LC-FR1 has sequences selected from SEQ ID NO: 94 and SEQ ID NO: 101; LC-FR2 has sequences selected from SEQ ID NO: 95 and SEQ ID NO: 106; LC-FR3 has sequences selected from SEQ ID NO: 96 and SEQ ID NO: 104; and LC-FR4 has the sequence shown in SEQ ID NO:

97.

38. The multispecific antigen-binding molecule according to any one of claims 24 to 37, wherein the IFNγ-binding domain comprises a VH region and a VL region, the VH region having a sequence selected from SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112 and SEQ ID NO: 113 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity therewith, and the VL region having a sequence selected from SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 and SEQ ID NO: 118 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity therewith.

39. The multispecific antigen-binding molecule of any one of claims 24 to 38, wherein the IFNγ-binding domain comprises: a) The VH region of the sequence having SEQ ID NO: 109 and the VL region of the sequence having SEQ ID NO: 116; b) The VH region of the sequence having SEQ ID NO: 110 and the VL region of the sequence having SEQ ID NO: 117; c) The VH region of the sequence having SEQ ID NO: 111 and the VL region of the sequence having SEQ ID NO: 118; d) The VH region of the sequence having SEQ ID NO: 110 and the VL region of the sequence having SEQ ID NO: 118; e) The VH region of the sequence having SEQ ID NO: 111 and the VL region of the sequence having SEQ ID NO: 117; f) The VH region of the sequence having SEQ ID NO: 112 and the VL region of the sequence having SEQ ID NO: 117; g) The VH region having the sequence of SEQ ID NO: 112 and the VL region having the sequence of SEQ ID NO: 118; h) The VH region of the sequence having SEQ ID NO: 113 and the VL region of the sequence having SEQ ID NO: 117; or i) The VH region having the sequence of SEQ ID NO: 113 and the VL region having the sequence of SEQ ID NO:

118.

40. An antigen-binding molecule comprising an IFNγ-binding domain, wherein the IFNγ-binding domain comprises a heavy chain variable region containing HC-CDR1, HC-CDR2, and HC-CDR3, wherein: a) The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 4 or a variant thereof in which one, two or three amino acids in HC-CDR1 are replaced by another amino acid; b) The heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 5 or a variant thereof in which one, two, or three amino acids in HC-CDR2 are substituted by another amino acid; and c) The heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 6 or a variant thereof in which one, two or three amino acids in HC-CDR3 are replaced by another amino acid; And / or wherein the IFNγ-binding domain comprises a light chain variable region containing LC-CDR1, LC-CDR2 and LC-CDR3, wherein: d) The light chain CDR1 contains the amino acid sequence of SEQ ID NO: 7 or a variant thereof in which one, two, or three amino acids in LC-CDR1 are replaced by another amino acid; e) The light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 8 or a variant thereof in which one, two, or three amino acids in LC-CDR2 are substituted with another amino acid; and f) The light chain CDR3 contains the amino acid sequence of SEQ ID NO: 9 or a variant thereof in which one, two or three amino acids in LC-CDR3 are replaced by another amino acid.

41. The antigen-binding molecule of claim 40, wherein the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 6 and / or the light chain CDR3 comprises the amino acid sequence of SEQ ID NO:

9.

42. The antigen-binding molecule of claim 40 or claim 41, wherein the heavy chain CDR1 comprises the sequence GYTXTNYY, wherein X can be any amino acid.

43. The antigen-binding molecule of claim 42, wherein the heavy chain CDR1 comprises GYTFTNYY (SEQ ID NO: 4) or GYTETNYY (SEQ ID NO: 85).

44. The antigen-binding molecule of any one of claims 40 to 43, wherein the heavy chain CDR2 comprises the sequence INPSNXGT, wherein X can be any amino acid.

45. The antigen-binding molecule of claim 44, wherein the heavy chain CDR2 comprises INPSNDGT (SEQ ID NO: 5) or INPSNHGT (SEQ ID NO: 87).

46. ​​The antigen-binding molecule of any one of claims 40 to 45, wherein the light chain CDR1 comprises the sequence QSXLYSSNXKNY, wherein X can be any amino acid.

47. The antigen-binding molecule of claim 46, wherein the light chain CDR1 comprises the sequence QSLLYSSNQKNY (SEQ ID NO: 7) or QSVLYSSNNKNY (SEQ ID NO: 89).

48. The antigen-binding molecule of any one of claims 40 to 47, wherein the IFNγ-binding domain comprises a heavy chain variable region containing HC-CDR1, HC-CDR2, and HC-CDR3, wherein: a) The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 4; b) The heavy chain CDR2 contains the amino acid sequence of SEQ ID NO: 5 or 87; and c) The heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 6; It also includes light chain variable regions containing LC-CDR1, LC-CDR2, and LC-CDR3, wherein: d) The light chain CDR1 contains the amino acid sequence of SEQ ID NO: 7; e) The light chain CDR2 contains the amino acid sequence of SEQ ID NO: 8; and f) The light chain CDR3 contains the amino acid sequence of SEQ ID NO:

9.

49. The antigen-binding molecule of any one of claims 40 to 47, wherein the IFNγ-binding domain comprises a heavy chain variable region containing HC-CDR1, HC-CDR2, and HC-CDR3, wherein: a) The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 85; b) The heavy chain CDR2 contains the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 87; and c) The heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 6; It also includes light chain variable regions containing LC-CDR1, LC-CDR2, and LC-CDR3, wherein: d) The light chain CDR1 contains the amino acid sequence of SEQ ID NO: 7; e) The light chain CDR2 contains the amino acid sequence of SEQ ID NO: 8; and f) The light chain CDR3 contains the amino acid sequence of SEQ ID NO:

9.

50. The antigen-binding molecule of any one of claims 40 to 47, wherein the IFNγ-binding domain comprises a heavy chain variable region containing HC-CDR1, HC-CDR2, and HC-CDR3, wherein: a) The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 4; b) The heavy chain CDR2 contains the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 87; and c) The heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 6; It also includes light chain variable regions containing LC-CDR1, LC-CDR2, and LC-CDR3, wherein: d) The light chain CDR1 contains the amino acid sequence of SEQ ID NO: 89; e) The light chain CDR2 contains the amino acid sequence of SEQ ID NO: 8; and f) The light chain CDR3 contains the amino acid sequence of SEQ ID NO:

9.

51. The antigen-binding molecule of any one of claims 40 to 47, wherein the IFNγ-binding domain comprises a heavy chain variable region containing HC-CDR1, HC-CDR2, and HC-CDR3, wherein: a) The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 85; b) The heavy chain CDR2 contains the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 87; and c) The heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 6; It also includes light chain variable regions containing LC-CDR1, LC-CDR2, and LC-CDR3, wherein: d) The light chain CDR1 contains the amino acid sequence of SEQ ID NO: 89; e) The light chain CDR2 contains the amino acid sequence of SEQ ID NO: 8; and f) The light chain CDR3 contains the amino acid sequence of SEQ ID NO:

9.

52. The antigen-binding molecule according to any one of claims 40 to 51, wherein it is a humanized antibody.

53. The humanized antibody of claim 52, wherein the IFNγ-binding domain comprises a heavy chain variable region containing HC-FR1, HC-FR2, HC-FR3, and HC-FR4, wherein: HC-FR1 has sequences selected from SEQ ID NO: 90, SEQ ID NO: 98 and SEQ ID NO: 102; HC-FR2 has the sequence shown in SEQ ID NO: 91; HC-FR3 has sequences selected from SEQ ID NO: 92, SEQ ID NO: 99, SEQ ID NO: 103, and SEQ ID NO: 105; and HC-FR4 has sequences selected from SEQ ID NO: 93 and SEQ ID NO: 100; and / or The light chain variable region contains LC-FR1, LC-FR2, LC-FR3 and LC-FR4, wherein: LC-FR1 has sequences selected from SEQ ID NO: 94 and SEQ ID NO: 101; LC-FR2 has sequences selected from SEQ ID NO: 95 and SEQ ID NO: 106; LC-FR3 has sequences selected from SEQ ID NO: 96 and SEQ ID NO: 104; and LC-FR4 has the sequence shown in SEQ ID NO:

97.

54. The antigen-binding molecule according to any one of claims 40 to 53, wherein the IFNγ-binding domain comprises a VH region and a VL region, the VH region having a sequence selected from SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112 and SEQ ID NO: 113 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity therewith, and the VL region having a sequence selected from SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 and SEQ ID NO: 118 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity therewith.

55. The antigen-binding molecule of any one of claims 40 to 54, wherein the IFNγ-binding domain comprises: a) The VH region of the sequence having SEQ ID NO: 109 and the VL region of the sequence having SEQ ID NO: 116; b) The VH region of the sequence having SEQ ID NO: 110 and the VL region of the sequence having SEQ ID NO: 117; c) The VH region of the sequence having SEQ ID NO: 111 and the VL region of the sequence having SEQ ID NO: 118; d) The VH region of the sequence having SEQ ID NO: 110 and the VL region of the sequence having SEQ ID NO: 118; e) The VH region of the sequence having SEQ ID NO: 111 and the VL region of the sequence having SEQ ID NO: 117; f) The VH region of the sequence having SEQ ID NO: 112 and the VL region of the sequence having SEQ ID NO: 117; g) The VH region having the sequence of SEQ ID NO: 112 and the VL region having the sequence of SEQ ID NO: 118; h) The VH region of the sequence having SEQ ID NO: 113 and the VL region of the sequence having SEQ ID NO: 117; or i) The VH region having the sequence of SEQ ID NO: 113 and the VL region having the sequence of SEQ ID NO:

118.

56. An antigen-binding molecule comprising an IFNγ-binding domain, wherein the IFNγ-binding domain comprises a heavy chain variable region containing HC-CDR1, HC-CDR2, and HC-CDR3, wherein: a) The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 12 or a variant thereof in which one, two or three amino acids in HC-CDR1 are replaced by another amino acid; b) The heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 13 or a variant thereof in which one, two, or three amino acids in HC-CDR2 are substituted by another amino acid; and c) The heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 14 or its variants in which one, two or three amino acids in HC-CDR3 are replaced by another amino acid; And / or wherein the IFNγ-binding domain comprises a light chain variable region containing LC-CDR1, LC-CDR2 and LC-CDR3, wherein: d) The light chain CDR1 contains the amino acid sequence of SEQ ID NO: 15 or a variant thereof in which one, two, or three amino acids in LC-CDR1 are replaced by another amino acid; e) The light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 16 or a variant thereof in which one, two, or three amino acids in LC-CDR2 are substituted by another amino acid; and f) The light chain CDR3 contains the amino acid sequence of SEQ ID NO: 17 or a variant thereof in which one, two or three amino acids in LC-CDR3 are replaced by another amino acid.

57. The antigen-binding molecule of claim 56, wherein the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 14 and / or the light chain CDR3 comprises the amino acid sequence of SEQ ID NO:

17.

58. The antigen-binding molecule of claim 56 or claim 57, which binds to the same epitope of IFNγ to an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 18 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:

19.

59. The antigen-binding molecule according to any one of claims 56 to 58, wherein it is a humanized antibody.

60. An antigen-binding molecule comprising an IFNγ-binding domain, wherein the IFNγ-binding domain comprises a heavy chain variable region containing HC-CDR1, HC-CDR2, and HC-CDR3, wherein: a) The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 20 or a variant thereof in which one, two or three amino acids in HC-CDR1 are replaced by another amino acid; b) The heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 21 or a variant thereof in which one, two, or three amino acids in HC-CDR2 are substituted by another amino acid; and c) The heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 22 or a variant thereof in which one, two or three amino acids in HC-CDR3 are replaced by another amino acid; And / or wherein the IFNγ-binding domain comprises a light chain variable region containing LC-CDR1, LC-CDR2 and LC-CDR3, wherein: d) The light chain CDR1 contains the amino acid sequence of SEQ ID NO: 23 or a variant thereof in which one, two, or three amino acids in LC-CDR1 are replaced by another amino acid; e) The light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 24 or a variant thereof in which one, two, or three amino acids in LC-CDR2 are substituted with another amino acid; and f) The light chain CDR3 contains the amino acid sequence of SEQ ID NO: 25 or a variant thereof in which one, two or three amino acids in LC-CDR3 are replaced by another amino acid.

61. The antigen-binding molecule of claim 60, wherein the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 22 and / or the light chain CDR3 comprises the amino acid sequence of SEQ ID NO:

25.

62. The antigen-binding molecule of claim 60 or claim 61, comprising an IFNγ-binding domain having the same epitope as an antibody binding IFNγ having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 26 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:

27.

63. The antigen-binding molecule according to any one of claims 60 to 62, wherein it is a humanized antibody.

64. An antigen-binding molecule comprising an IFNγ-binding domain, wherein the IFNγ-binding domain comprises a heavy chain variable region containing HC-CDR1, HC-CDR2, and HC-CDR3, wherein: a) The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 28 or a variant thereof in which one, two or three amino acids in HC-CDR1 are replaced by another amino acid; b) The heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 29 or a variant thereof in which one, two, or three amino acids in HC-CDR2 are substituted by another amino acid; and c) The heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 30 or a variant thereof in which one, two or three amino acids in HC-CDR3 are replaced by another amino acid; And / or wherein the IFNγ-binding domain comprises a light chain variable region containing LC-CDR1, LC-CDR2 and LC-CDR3, wherein: d) The light chain CDR1 contains the amino acid sequence of SEQ ID NO: 31 or a variant thereof in which one, two or three amino acids in LC-CDR1 are replaced by another amino acid; e) The light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 32 or a variant thereof in which one, two, or three amino acids in LC-CDR2 are substituted by another amino acid; and f) The light chain CDR3 contains the amino acid sequence of SEQ ID NO: 33 or its variants in which one, two or three amino acids in LC-CDR3 are replaced by another amino acid.

65. The antigen-binding molecule of claim 64, wherein the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 30 and / or the light chain CDR3 comprises the amino acid sequence of SEQ ID NO:

33.

66. The antigen-binding molecule of claim 64 or claim 65, comprising an IFNγ-binding domain having the same epitope as an antibody binding IFNγ having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 34 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:

35.

67. The antigen-binding molecule according to any one of claims 64 to 66, wherein it is a humanized antibody.

68. An antigen-binding molecule comprising an IFNγ-binding domain, wherein the IFNγ-binding domain comprises a heavy chain variable region containing HC-CDR1, HC-CDR2, and HC-CDR3, wherein: a) The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 36 or a variant thereof in which one, two or three amino acids in HC-CDR1 are replaced by another amino acid; b) The heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 37 or a variant thereof in which one, two, or three amino acids in HC-CDR2 are substituted by another amino acid; and c) The heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 38 or a variant thereof in which one, two or three amino acids in HC-CDR3 are replaced by another amino acid; And / or wherein the IFNγ-binding domain comprises a light chain variable region containing LC-CDR1, LC-CDR2 and LC-CDR3, wherein: d) The light chain CDR1 contains the amino acid sequence of SEQ ID NO: 39 or a variant thereof in which one, two, or three amino acids in LC-CDR1 are replaced by another amino acid; e) The light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 40 or a variant thereof in which one, two, or three amino acids in LC-CDR2 are substituted with another amino acid; and f) The light chain CDR3 contains the amino acid sequence of SEQ ID NO: 41 or a variant thereof in which one, two or three amino acids in LC-CDR3 are replaced by another amino acid.

69. The antigen-binding molecule of claim 68, wherein the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 38 and / or the light chain CDR3 comprises the amino acid sequence of SEQ ID NO:

41.

70. The antigen-binding molecule of claim 68 or claim 69, comprising an IFNγ-binding domain having the same epitope as an antibody binding IFNγ having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 42 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:

43.

71. The antigen-binding molecule according to any one of claims 68 to 70, wherein it is a humanized antibody.

72. The antigen-binding molecule of any one of claims 40 to 71, wherein the molecule is in a K+ concentration of less than 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 7 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, or 0.25 nM. D Combined with IFNγ.

73. The antigen-binding molecule of claim 72, wherein the molecule is in a K+ concentration of less than 20 nM, less than 10 nM, or less than 5 nM. D Combined with IFNγ.

74. The antigen-binding molecule of any one of claims 40 to 73, wherein the binding of the antigen-binding molecule to IFNγ regulates cellular signaling downstream of IFNγ.

75. The antigen-binding molecule of any one of claims 40 to 74, wherein the binding of the antigen-binding molecule to IFNγ biases the pro-inflammatory and anti-inflammatory activities of IFNγ toward the pro-inflammatory activity.

76. The antigen-binding molecule of any one of claims 40 to 75, wherein the ratio of IFNγ-induced MHC-1 expression and / or activity to PD-L1 expression and / or activity is altered to favor MHC-1 expression and / or activity.

77. The antigen-binding molecule of any one of claims 40 to 76, wherein the binding of the molecule to IFNγ partially inhibits the pro-inflammatory activity of IFNγ.

78. The antigen-binding molecule of claim 77, wherein the binding of the molecule to IFNγ partially inhibits the induction of MHC-1 expression and / or activity by IFNγ.

79. The antigen-binding molecule of any one of claims 40 to 78, comprising an Fc region and / or conjugated to a half-life extension portion.

80. The antigen-binding molecule of claim 79, wherein the molecule comprises an Fc region, and the Fc region comprises one or more mutations selected from the group consisting of: i) Further extending the half-life of the molecule by selecting from the group consisting of: R435H mutation, T307Q mutation, T307P mutation, T307A mutation, T307R mutation, N265 mutation, D265A mutation, N434S mutation, T250Q mutation, T250E mutation, M428L mutation, M428F mutation, V308W mutation, V308Y mutation, V308F mutation, E294 deletion / T3 07P / N434Y mutation, T256N / A378V / S383N / N434Y mutation, M428L / N434S mutation, T307A / E380A / N434A mutation, T307Q / N434S mutation, Q311V / N434S mutation, H433K / N434F mutation, E294 deletion / T307P / N434Y mutation, T307A / E380A / N434A mutation, The following mutations are associated with the following individuals: T256D / H286D / T307R / Q311V / A378V, H285N / T307Q / N315D, T307Q / Q311V / A378V, H285D / T307Q / A378V, T307Q / N434S, M252Y / S254T / T256E, T250Q / M428L, and T250Q / M428F. T250E / M428F mutation, T250E / M428L mutation, M252Y / M428L mutation, M428L / V308F mutation, D259I / V308F mutation, E258F / V427T mutation, T256D / Q311V / A378V mutation, T256D / H286D / T307R / Q311V / A378V mutation, and K288E / H435K mutation; and / or ii) The antibody reduces or silences the effector function of the antibody, selected from the group consisting of: R435H mutation, N434A mutation, T252L / T253S / T254F mutation, E294 deletion / T307P / N434Y mutation, T256N / A378V / S383N / N434Y mutation, E294 deletion mutation, M252Y / S254T / T256E mutation, M428L / N434S mutation, T307A / E380A / N434A mutation, T250Q / M428L mutation, T250Q / M428F mutation, T250E / M428F mutation, T250E / M428L mutation, T256D / Q311V / A378V mutation. Mutations include: T256D / H286D / T307R / Q311V / A378V, H285N / T307Q / N315D, T307Q / Q311V / A378V, H285D / T307Q / A378V, L234F / L235E / P331S, L234F / L235Q / K322Q, L234F / L235Q / P331G, L234F / L235A / K322Q, S228P / F234A / L235A / G237A / P238S, L234A / L235E / G237A / A330S / P331S, and F243A / V264A. Mutations, S228P / L235E / P329G mutation, M252Y / M428L mutation, D259I / V308F mutation, T307Q / N434S mutation, M428L / V308F mutation, Q311V / N434S mutation, H433K / N434F mutation, E258F / V427T mutation, K288E / H435K mutation, F234A / L235A mutation, F234A / L235A / S228P mutation, L234A / L235A / P329G mutation, S228P / F234A / L235A / P329G mutation, Y349C / T366S / L368A / Y407V / F405K mutation, S 354C / T366W / K409A mutation, L234S / L235T / G236R mutation, P329G mutation, S228P mutation, deletion-K447 mutation, L234A mutation, L235A mutation, L235E mutation, G237A mutation, V308P mutation, V308W mutation, V308Y mutation, V308F mutation, N434S mutation, T307Q mutation, T307P mutation, T307A mutation, T307R mutation, Q312A mutation, Q318A mutation, Q233P mutation, K322A mutation, P329A mutation, P331A mutation, P238A mutation, P238S mutation, F241A mutation, D265A mutation.D269A mutation, D270A mutation, N297A mutation, A327Q mutation, P329A mutation, S239A mutation, E294A mutation, Q295A mutation, V303A mutation, V264A mutation, A330R mutation, A330L mutation, A330S mutation, P331S mutation, T299A mutation, F234A mutation, T366W mutation, T366S mutation, L368A mutation, Y407V mutation, Y349C mutation, F405L mutation, F405K mutation, S354C mutation, T366W mutation, K409R mutation, K409A mutation, F405L / R409K mutation, and any combination thereof; and / or, iii) The effector function of the antibody is enhanced by selecting from the group consisting of: F243L / R292P / Y300L / V305I / P396L mutation, S239D / I332E mutation, S239D / I332E / A330L mutation, S298A / E333A / K334A mutation, G236A / S239D / I332E mutation, K326W / E3 33S mutation, S267E / H268F / S324T mutation, L234Y / L235Q / G236W / S239M / H268D / D270E / S298A in one heavy chain and D270E / K326D / A330M / K334E in another heavy chain, E345R / E430G / S440Y mutation and IgG1 / IgG3 cross subclass mutation; And / or iv) The antibody’s stability or downstream processing is improved by means of the group consisting of: S228P mutation, R409K mutation, R409T mutation, R409M mutation, R409L mutation, S228P / L235E / R409K mutation, S228P / L235E / R409T mutation, S228P / L235E / R409M mutation, S228P / L235E / R409L mutation, G446 / deletion / K447 deletion mutation, K370Q mutation, K370E mutation, R409A mutation, R409E mutation, R409W mutation, C131S mutation, K370 deletion or substitution, deletion-K447 mutation, F405L mutation, K409R mutation and F405L / R409K mutation; Or any combination thereof.

81. The antigen-binding molecule according to any one of claims 40 to 80, wherein it is a multispecific antigen-binding molecule further comprising an antigen-binding domain for a target antigen other than IFNγ.

82. The antigen-binding molecule of claim 81, wherein it is a multispecific antigen-binding molecule as described in any one of claims 1 to 39.

83. A pharmaceutical formulation comprising an antigen-binding molecule as described in any one of the preceding claims and a pharmaceutically acceptable carrier.

84. A combination therapy comprising an antigen-binding molecule as described in any of the preceding claims and a checkpoint inhibitor.

85. The antigen-binding molecule of any one of claims 1 to 82, the pharmaceutical formulation of claim 83, or the combination therapeutic agent of claim 84, for use in a treatment method.

86. The antigen-binding molecule of any one of claims 1 to 82, the pharmaceutical composition of claim 83, or the combination therapeutic agent of claim 84, for use in the treatment of tumors or cancer.

87. The antigen-binding molecule of any one of claims 1 to 82 or the pharmaceutical formulation of claim 83, for use in a method of treating a tumor or cancer, wherein the use is in combination with a checkpoint inhibitor and / or the treatment sensitizes the tumor or cancer to the checkpoint inhibitor.

88. An antigen-binding molecule that binds to interferon-γ (IFNγ), wherein the binding biases the pro-inflammatory and anti-inflammatory activities of IFNγ toward the pro-inflammatory activity, for use in a therapeutic treatment.

89. The antigen-binding molecule used as claimed in claim 88, wherein the binding of said molecule to IFNγ regulates cellular signaling downstream of IFNγ such that the ratio of IFNγ-induced MHC-1 expression and / or activity to PD-L1 expression and / or activity is altered to favor MHC-1 expression.

90. The antigen-binding molecule used as described in claim 88 or 89, wherein the binding of the molecule to IFNγ partially inhibits the pro-inflammatory activity of IFNγ.

91. The antigen-binding molecule used as described in claim 90, wherein the binding of said molecule to IFNγ partially inhibits the induction of MHC-1 expression and / or activity by IFNγ.

92. The antigen-binding molecule used as described in any one of claims 88 to 91 is a humanized antibody.

93. The antigen-binding molecule used as described in any one of claims 88 to 92, wherein the K+ is less than 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 7 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, or 0.25 nM. D Combined with IFNγ.

94. The antigen-binding molecule used as described in any one of claims 88 to 93, wherein the K+ is less than 20 nM, less than 10 nM, or less than 5 nM. D Combined with IFNγ.

95. The antigen-binding molecule used as described in any one of claims 88 to 94, comprising an Fc region or conjugated with a half-life extension portion.

96. The antigen-binding molecule used as claimed in claim 95, wherein the molecule comprises an Fc region, and wherein the Fc region comprises one or more mutations selected from the group consisting of: i) The molecule's half-life is extended by selecting from the group consisting of: R435H mutation, T307Q mutation, T307P mutation, T307A mutation, T307R mutation, N265 mutation, D265A mutation, N434S mutation, T250Q mutation, T250E mutation, M428L mutation, M428F mutation, V308W mutation, V308Y mutation, V308F mutation, E294 deletion / T307 P / N434Y mutation, T256N / A378V / S383N / N434Y mutation, M428L / N434S mutation, T307A / E380A / N434A mutation, T307Q / N434S mutation, Q311V / N434S mutation, H433K / N434F mutation, E294 deletion / T307P / N434Y mutation, T307A / E380A / N434A mutation, T 256D / H286D / T307R / Q311V / A378V mutation, H285N / T307Q / N315D mutation, T307Q / Q311V / A378V mutation, H285D / T307Q / A378V mutation, T307Q / N434S mutation, M252Y / S254T / T256E mutation, T250Q / M428L mutation, T250Q / M428F mutation, T 250E / M428F mutation, T250E / M428L mutation, M252Y / M428L mutation, M428L / V308F mutation, D259I / V308F mutation, E258F / V427T mutation, T256D / Q311V / A378V mutation, T256D / H286D / T307R / Q311V / A378V mutation, and K288E / H435K mutation; and / or ii) The antibody reduces or silences the effector function of the antibody, selected from the group consisting of: R435H mutation, N434A mutation, T252L / T253S / T254F mutation, E294 deletion / T307P / N434Y mutation, T256N / A378V / S383N / N434Y mutation, E294 deletion mutation, M252Y / S254T / T256E mutation, M428L / N434S mutation, T307A / E380A / N434A mutation, T250Q / M428L mutation, T250Q / M428F mutation, T250E / M428F mutation, T250E / M428L mutation, T256D / Q311V / A378V mutation. Mutations include: T256D / H286D / T307R / Q311V / A378V, H285N / T307Q / N315D, T307Q / Q311V / A378V, H285D / T307Q / A378V, L234F / L235E / P331S, L234F / L235Q / K322Q, L234F / L235Q / P331G, L234F / L235A / K322Q, S228P / F234A / L235A / G237A / P238S, L234A / L235E / G237A / A330S / P331S, and F243A / V264A. Mutations, S228P / L235E / P329G mutation, M252Y / M428L mutation, D259I / V308F mutation, T307Q / N434S mutation, M428L / V308F mutation, Q311V / N434S mutation, H433K / N434F mutation, E258F / V427T mutation, K288E / H435K mutation, F234A / L235A mutation, F234A / L235A / S228P mutation, L234A / L235A / P329G mutation, S228P / F234A / L235A / P329G mutation, Y349C / T366S / L368A / Y407V / F405K mutation, S 354C / T366W / K409A mutation, L234S / L235T / G236R mutation, P329G mutation, S228P mutation, deletion-K447 mutation, L234A mutation, L235A mutation, L235E mutation, G237A mutation, V308P mutation, V308W mutation, V308Y mutation, V308F mutation, N434S mutation, T307Q mutation, T307P mutation, T307A mutation, T307R mutation, Q312A mutation, Q318A mutation, Q233P mutation, K322A mutation, P329A mutation, P331A mutation, P238A mutation, P238S mutation, F241A mutation, D265A mutation.D269A mutation, D270A mutation, N297A mutation, A327Q mutation, P329A mutation, S239A mutation, E294A mutation, Q295A mutation, V303A mutation, V264A mutation, A330R mutation, A330L mutation, A330S mutation, P331S mutation, T299A mutation, F234A mutation, T366W mutation, T366S mutation, L368A mutation, Y407V mutation, Y349C mutation, F405L mutation, F405K mutation, S354C mutation, T366W mutation, K409R mutation, K409A mutation, F405L / R409K mutation, and any combination thereof; and / or, iii) The effector function of the antibody is enhanced by selecting from the group consisting of: F243L / R292P / Y300L / V305I / P396L mutation, S239D / I332E mutation, S239D / I332E / A330L mutation, S298A / E333A / K334A mutation, G236A / S239D / I332E mutation, K326W / E3 33S mutation, S267E / H268F / S324T mutation, L234Y / L235Q / G236W / S239M / H268D / D270E / S298A in one heavy chain and D270E / K326D / A330M / K334E in another heavy chain, E345R / E430G / S440Y mutation and IgG1 / IgG3 cross subclass mutation; And / or iv) The antibody’s stability or downstream processing is improved by means of the group consisting of: S228P mutation, R409K mutation, R409T mutation, R409M mutation, R409L mutation, S228P / L235E / R409K mutation, S228P / L235E / R409T mutation, S228P / L235E / R409M mutation, S228P / L235E / R409L mutation, G446 / deletion / K447 deletion mutation, K370Q mutation, K370E mutation, R409A mutation, R409E mutation, R409W mutation, C131S mutation, K370 deletion or substitution, deletion-K447 mutation, F405L mutation, K409R mutation and F405L / R409K mutation; Or any combination thereof.

97. The antigen-binding molecule used as described in any one of claims 88 to 96, wherein the treatment is a treatment for tumors or cancer.

98. A pharmaceutical composition comprising an antigen-binding molecule that binds to interferon-γ (IFNγ), wherein the binding biases the pro-inflammatory and anti-inflammatory activities of IFNγ toward the pro-inflammatory activity.

99. The pharmaceutical composition of claim 98, wherein the binding of the molecule to IFNγ regulates cellular signaling downstream of IFNγ, such that the ratio of IFNγ-induced MHC-1 expression and / or activity to PD-L1 expression and / or activity is altered to favor MHC-1 expression and / or activity.

100. The pharmaceutical composition of claim 98 or claim 99, wherein the binding of the molecule to IFNγ partially inhibits the pro-inflammatory activity of IFNγ.

101. The pharmaceutical composition of claim 100, wherein the binding of the molecule to IFNγ partially inhibits the induction of MHC-1 expression and / or activity by IFNγ.

102. The pharmaceutical composition of any one of claims 98 to 101, wherein the antigen-binding molecule is a humanized antibody.

103. The pharmaceutical composition of any one of claims 98 to 102, wherein the antigen-binding molecule is in a Kc concentration of less than 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 7 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, or 0.25 nM. D Combined with IFNγ.

104. The pharmaceutical composition of claim 103, wherein the antigen-binding molecule is in a Kc concentration of less than 20 nM, less than 10 nM, or less than 5 nM. D Combined with IFNγ.

105. The pharmaceutical composition of any one of claims 98 to 104, wherein the antigen-binding molecule comprises an Fc region or is conjugated to a half-life extension portion.

106. The pharmaceutical composition of claim 105, wherein the molecule comprises an Fc region, and the Fc region comprises one or more mutations selected from the group consisting of: i) The molecule's half-life is extended by selecting from the group consisting of: R435H mutation, T307Q mutation, T307P mutation, T307A mutation, T307R mutation, N265 mutation, D265A mutation, N434S mutation, T250Q mutation, T250E mutation, M428L mutation, M428F mutation, V308W mutation, V308Y mutation, V308F mutation, E294 deletion / T307 P / N434Y mutation, T256N / A378V / S383N / N434Y mutation, M428L / N434S mutation, T307A / E380A / N434A mutation, T307Q / N434S mutation, Q311V / N434S mutation, H433K / N434F mutation, E294 deletion / T307P / N434Y mutation, T307A / E380A / N434A mutation, T 256D / H286D / T307R / Q311V / A378V mutation, H285N / T307Q / N315D mutation, T307Q / Q311V / A378V mutation, H285D / T307Q / A378V mutation, T307Q / N434S mutation, M252Y / S254T / T256E mutation, T250Q / M428L mutation, T250Q / M428F mutation, T 250E / M428F mutation, T250E / M428L mutation, M252Y / M428L mutation, M428L / V308F mutation, D259I / V308F mutation, E258F / V427T mutation, T256D / Q311V / A378V mutation, T256D / H286D / T307R / Q311V / A378V mutation, and K288E / H435K mutation; and / or ii) The antibody reduces or silences the effector function of the antibody, selected from the group consisting of: R435H mutation, N434A mutation, T252L / T253S / T254F mutation, E294 deletion / T307P / N434Y mutation, T256N / A378V / S383N / N434Y mutation, E294 deletion mutation, M252Y / S254T / T256E mutation, M428L / N434S mutation, T307A / E380A / N434A mutation, T250Q / M428L mutation, T250Q / M428F mutation, T250E / M428F mutation, T250E / M428L mutation, T256D / Q311V / A378V mutation. Mutations include: T256D / H286D / T307R / Q311V / A378V, H285N / T307Q / N315D, T307Q / Q311V / A378V, H285D / T307Q / A378V, L234F / L235E / P331S, L234F / L235Q / K322Q, L234F / L235Q / P331G, L234F / L235A / K322Q, S228P / F234A / L235A / G237A / P238S, L234A / L235E / G237A / A330S / P331S, and F243A / V264A. Mutations, S228P / L235E / P329G mutation, M252Y / M428L mutation, D259I / V308F mutation, T307Q / N434S mutation, M428L / V308F mutation, Q311V / N434S mutation, H433K / N434F mutation, E258F / V427T mutation, K288E / H435K mutation, F234A / L235A mutation, F234A / L235A / S228P mutation, L234A / L235A / P329G mutation, S228P / F234A / L235A / P329G mutation, Y349C / T366S / L368A / Y407V / F405K mutation, S 354C / T366W / K409A mutation, L234S / L235T / G236R mutation, P329G mutation, S228P mutation, deletion-K447 mutation, L234A mutation, L235A mutation, L235E mutation, G237A mutation, V308P mutation, V308W mutation, V308Y mutation, V308F mutation, N434S mutation, T307Q mutation, T307P mutation, T307A mutation, T307R mutation, Q312A mutation, Q318A mutation, Q233P mutation, K322A mutation, P329A mutation, P331A mutation, P238A mutation, P238S mutation, F241A mutation, D265A mutation.D269A mutation, D270A mutation, N297A mutation, A327Q mutation, P329A mutation, S239A mutation, E294A mutation, Q295A mutation, V303A mutation, V264A mutation, A330R mutation, A330L mutation, A330S mutation, P331S mutation, T299A mutation, F234A mutation, T366W mutation, T366S mutation, L368A mutation, Y407V mutation, Y349C mutation, F405L mutation, F405K mutation, S354C mutation, T366W mutation, K409R mutation, K409A mutation, F405L / R409K mutation, and any combination thereof; and / or, iii) The effector function of the antibody is enhanced by selecting from the group consisting of: F243L / R292P / Y300L / V305I / P396L mutation, S239D / I332E mutation, S239D / I332E / A330L mutation, S298A / E333A / K334A mutation, G236A / S239D / I332E mutation, K326W / E333S Mutations, S267E / H268F / S324T mutations, L234Y / L235Q / G236W / S239M / H268D / D270E / S298A in one heavy chain and D270E / K326D / A330M / K334E in another heavy chain, E345R / E430G / S440Y mutations, and IgG1 / IgG3 cross-subclass mutations; and / or iv) The antibody’s stability or downstream processing is improved by means of the group consisting of: S228P mutation, R409K mutation, R409T mutation, R409M mutation, R409L mutation, S228P / L235E / R409K mutation, S228P / L235E / R409T mutation, S228P / L235E / R409M mutation, S228P / L235E / R409L mutation, G446 / deletion / K447 deletion mutation, K370Q mutation, K370E mutation, R409A mutation, R409E mutation, R409W mutation, C131S mutation, K370 deletion or substitution, deletion-K447 mutation, F405L mutation, K409R mutation and F405L / R409K mutation; Or any combination thereof.

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