Antigen-binding molecules capable of binding interferon gamma

Abstract

The present disclosure relates to bispecific antibodies against interferon gamma (IFNγ) and a tumour associated antigen to deliver IFNγ to the tumour, and to medical uses of such antibodies.

Description

[0001] ANTIGEN-BINDING MOLECULES CAPABLE OF BINDING INTERFERON GAMMA

[0002] Field of the Invention

[0003] The present disclosure relates to antigen binding molecules against interferon gamma (I FNy), and to medical uses of such antigen-binding molecules.

[0004] Background

[0005] Non-immunogenic or “cold” tumours” are more challenging to treat, because a lack of immune cells in the tumour region means that they respond less well to immunotherapy.

[0006] A range of targeted therapeutics have been used for treating cancers, giving rise to various degrees of immune engagement, including radioconjugates, ADC-antibody drug conjugates, and T-cell engagers.

[0007] IFNy is a cytokine with an established role in modulating immune and inflammatory processes. The interferon family has three main members, now designated interferon-alpha (IFNa), interferon-beta (IFNp), and interferon-gamma (IFNy). IFNy is generally recognised as pro-inflammatory.

[0008] Studies have shown that IFNy is vital to tumor surveillance by the immune system and a high correlation between IFNy production and tumor regression has been seen in immunotherapy. IFNy also has direct antitumor effects, as it is anti-angiogenic, inhibits proliferation, sensitizes tumor cells to apoptosis, upregulates MHC class I and II expression, and stimulates antitumor immune activity (Clinical uses of Interferon-y, Ann N Y Acad. Sci. 2009 December; 1182:69-79).

[0009] Clinical trials have been carried out with Actimmune®, a recombinant form of IFNy. Due to its pleiotropic effects, administration of IFNy is associated with systemic and local toxicity, which relate to the range of adverse events reported during execution of the clinical trials and collected in the prescribing information for Actimmune® (see HIGHLIGHTS OF PRESCRIBING INFORMATION. ACTIMMUNE® (interferon gamma-1 b) injection, for subcutaneous use. Initial U.S. Approval:

[0010] 1990. https: / / www.accessdata.fda.gov / drugsatfda_docs / label / 2015 / 103836s5182lbl.pdf). The most common adverse events were “flu-like,” such as fever, headache, chills, myalgia, or fatigue. However, a range of warnings and precautions are listed in the prescribing information, which include hepatic, renal and bone marrow toxicity, cardiovascular and neurological disorders, as well as other adverse immunological reactions that are dose limiting for the approved clinical indications. Local toxicity reactions result in administration site erythema or tenderness, which were reported in more than 20% of patients participating in the clinical trials, including adverse events of hemorrhage and pain at the injection site. Moreover, local cutaneous necrotizing lesions has been described with clinical use of IFNy (see Krainick U, Kantarjian H, Broussard S, Talpaz M. Local cutaneous necrotizing lesions associated with interferon injections. J Interferon Cytokine Res. 1998 Oct; 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

[0011] Dermatol. 2016 Sep-Oct;91 (5 suppl 1):76-78).

[0012] Summary of the Invention

[0013] The present disclosure provides an antigen-binding molecule, optionally an isolated antigen-binding molecule, which is capable of binding to interferon gamma (IFNy). The antigen-binding molecule may be an antibody.

[0014] The present inventors have recognised that it is possible to make use of the proinflammatory / anti- tumorigenic properties of IFNy at the same time as binding an antigen binding molecule thereto, in order to obtain various advantages, such as increasing the plasma concentration of endogenous IFNy, and / or increasing the half-life of exogenous administered IFNy, and / or targeting the IFNy to a target cell or tissue, such as a tumour or cancer cell or tissue.

[0015] Thus, in a first aspect, the present invention relates to a multispecific, e.g., bispecific, antigen-binding molecule comprising an antigen binding domain that binds to interferon gamma (IFNy) and an antigen binding domain that binds to a target antigen other than IFNy. In some embodiments, the antigen-binding molecule may be a multispecific or bispecific antibody.

[0016] The use of a targeted antibody / antigen-binding molecule according to the present invention has a number of advantages as compared to other approaches used in the art. For example, it engages the immune system by directly inflaming the tumour; it makes use of endogenous IFNy for reduced side-effects as compared to administration of an exogenous agent; and it is capable of capturing and prolonging local IFNy bursts produced by the tumour, thus enhancing efficacy. The targeted antibody / antigen-binding molecule has a unique three-stage mode of action, comprising binding the body’s own IFNy; targeted delivery to cells (e.g., tumour cells); and immune stimulation including e.g. attraction of immune cells and upregulation of MHC-1 to generate an immune response against the tumour.

[0017] In some embodiments, binding of said molecule to IFNy modulates cell signalling downstream of the IFNy receptor.

[0018] In one embodiment, binding of the molecule to IFNy partially inhibits the pro-inflammatory activity of IFNy. In some situations, partial reduction of proinflammatory / anti-tumorigenic activity in combination with targeting to a target cell or tissue (e.g., using a bispecific antibody) may be beneficial to reduce side effects such as systemic inflammation while accumulating proinflammatory / anti-tumorigenic activity at the target (and thus benefitting from higher concentrations and / or avidity effects at the target). The present inventors have further unexpectedly discovered that antigen-binding molecules binding to IFNy can bias the pro-and anti-inflammatory activities of IFNy in favour of the pro-inflammatory activities. This may offer improved treatment for diseases and conditions where the proinflammatory and / or anti- tumorigenic activities of IFNy are beneficial, such as in the treatment of cancer. Although IFNy is generally recognised as pro-inflammatory, there is also evidence which supports a dual role of IFNy in inflammation, including activation of some anti-inflammatory molecules, modulation of pro-inflammatory cytokine production, activation of apoptosis, and interference with the signal transduction machinery by induction of suppressors of cytokine signalling (SOCS) (Muhl and Pfeilshifter, International Immunopharmacology Vol 3, Issue, 9, September 2003, pages 1247-1255). (As used here, the term “proinflammatory” may be used interchangeably with “anti-tumorigenic”; and the term “anti-inflammatory” may be used interchangeably with the term “pro-tumorigenic”.)

[0019] In an embodiment, binding of said molecule to IFNy biases the pro-and anti-inflammatory activities of IFNy in favour of the pro-inflammatory activities. For example, in some embodiments, an antigen-binding molecule may preferentially inhibit the anti-inflammatory activities (i.e., may inhibit the anti-inflammatory activities while inhibiting the pro-inflammatory properties to a lesser degree or while not inhibiting the proinflammatory activities). In some embodiments, the antigen-binding molecule may partially inhibit the pro-inflammatory activity of IFNy, while inhibiting the anti-inflammatory activities of IFNy more strongly.

[0020] In some embodiments, the pro-inflammatory activity of the cytokine may be measured by the ability of the cytokine to induce expression of major histocompatibility complex class I (MHC-1) on the surface of a cell, e.g., a cell in vitro. In some embodiments, the anti-inflammatory activity of the cytokine may be measured by the ability of the cytokine to induce expression of programmed Cell Death Ligand 1 (PD-L1) on the surface of a cell, e.g., a cell in vitro.

[0021] Accordingly, in an embodiment, the molecule is capable of partially inhibiting the induction of MHC-1 expression (e.g., cell surface expression) and / or activity by IFNy. Additionally or alternatively, in some embodiments binding of said molecule to IFNy modulates cell signalling downstream of IFNy, 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 modified in favour of MHC-1 expression and / or activity.

[0022] In a second aspect, the present disclosure relates to an antigen-binding molecule as described in the following. The antigen binding molecules described in the following may bind to IFNy alone, or may bind to IFNy in addition to binding another antigen such as a TAA. In some embodiments, any of the following antigen-binding molecules may be present in a multispecific antigen binding molecule which is also capable of binding (independently) to an antigen other than interferon gamma (a target antigen), e.g., a multispecific or bispecific antibody. That is, in a multispecific antigen-binding molecule comprising an antigen binding domain that binds to interferon gamma (IFNy) and an antigen binding site that binds to a target antigen other than I FNy, as described in the first aspect and according to any of the embodiments further described herein, the antigen binding domain that binds to interferon gamma (IFNy) may be an antigen binding domain as follows.

[0023] In an embodiment, the present disclosure relates to an antigen binding molecule comprising an antigen binding domain for IFNy, wherein said antigen binding site comprises a heavy chain variable region comprising HC-CDR1 , HC-CDR2 and HC-CDR3, wherein: a) heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 4 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR1 are substituted with another amino acid; b) heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 5 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR2 are substituted with another amino acid; c) heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 6 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR3 are substituted with another amino acid; and / or wherein the antigen binding site comprises a light chain variable region comprising LC-CDR1 , LC- CDR2 and LC-CDR3, wherein: d) light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 7 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR1 are substituted with another amino acid; e) light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 8 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR2 are substituted with another amino acid; f) light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 9 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR3 are substituted with another amino acid.

[0024] In some embodiments, heavy chain CDR1 comprises a variant of SEQ ID NO: 4 with a substitution at position 30, numbered according to the IMGT scheme (i.e., GYTXTNYY (SEQ ID NO: 84), where X can be any amino acid), wherein the residue at this position is preferably D, E, N or Q, most preferably E. For instance, the heavy chain CDR1 may be GYTETNYY (SEQ ID NO: 85). IMGT position 30 corresponds to Kabat position 29.

[0025] In another embodiment, heavy chain CDR2 comprises a variant of SEQ ID NO: 5 with a substitution at position 63, numbered according to the IMGT scheme (i.e., INPSNXGT (SEQ ID NO: 86), where X can be any amino acid and is preferably other than D), wherein the residue at this position is preferably selected from E, H and Q, and most preferably is H. Removing D at this position removes a potential isomerisation site, since the residue pair DG forms a potential isomerisation sequence liability. For instance, the heavy chain CDR2 may 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 table, as the “D55H” variant. In another embodiment, light chain CDR1 comprises a variant of SEQ ID NO: 7 with substitutions at one or both of positions 29 and 35, numbered according to the IMGT scheme (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 is most preferably V and / or the residue at position 35 is preferably selected from D, E, N or Q and is most preferably N. For instance, the LC-CDR1 may be QSVLYSSNNKNY (SEQ ID NO: 89). IMFT positions 29 and 35 correspond to Kabat positions 27B and 29 respectively.

[0026] In some embodiments, the antigen-binding molecule comprises a VH region having at least at least 70% sequence identity, optionally at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:10 and / or a VL region having at least at least 70% sequence identity, optionally at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO: 11 .

[0027] An exemplary antigen-binding molecule may have a 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 thereto and a 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 thereto.

[0028] In one embodiment, a humanised antibody / antigen binding molecule may have a VH region having 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 thereto and a VL region having 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 thereto. In another embodiment, a humanised antibody / antigen binding molecule may have a VH region having 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 thereto and a VL region having 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 thereto. In another embodiment, a humanised antibody / antigen binding molecule may have a VH region having 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 thereto and a VL region having 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 thereto. In another embodiment, a humanised antibody / antigen binding molecule may have a VH region having 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 thereto and a VL region having 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 thereto. In another embodiment, a humanised antibody / antigen binding molecule may have a VH region having 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 thereto and a VL region having 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 thereto. In another embodiment, a humanised antibody / antigen binding molecule may have a VH region having 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 thereto and a VL region having 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 thereto. In another embodiment, a humanised antibody / antigen binding molecule may have a VH region having 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 thereto and a VL region having 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 thereto. In another embodiment, a humanised antibody / antigen binding molecule may have a VH region having 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 thereto and a VL region having 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 thereto. In another embodiment, a humanised antibody / antigen binding molecule may have a VH region having 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 thereto and a VL region having 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 thereto.

[0029] In another embodiment, the present disclosure relates to an antigen binding molecule comprising an antigen binding domain for I FNy, wherein said antigen binding site comprises a heavy chain variable region comprising HC-CDR1 , HC-CDR2 and HC-CDR3, wherein: a) heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 12 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR1 are substituted with another amino acid; b) heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 13 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR2 are substituted with another amino acid; c) heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 14 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR3 are substituted with another amino acid; and / or wherein the antigen binding site comprises a light chain variable region comprising LC-CDR1 , LC- CDR2 and LC-CDR3, wherein: d) light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 15 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR1 are substituted with another amino acid; e) light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 16 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR2 are substituted with another amino acid; f) light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 17 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR3 are substituted with another amino acid.

[0030] In some embodiments, the antibody / antigen binding molecule comprises a VH region having at least at least 70% sequence identity, optionally at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:18 and / or a VL region having at least at least 70% sequence identity, optionally at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:19.

[0031] In another embodiment, the present disclosure relates to an antigen binding molecule comprising an antigen binding domain for I FNy, wherein said antigen binding site comprises a heavy chain variable region comprising HC-CDR1 , HC-CDR2 and HC-CDR3, wherein: a) heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 20 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR1 are substituted with another amino acid; b) heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 21 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR2 are substituted with another amino acid; c) heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 22 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR3 are substituted with another amino acid; and / or wherein the antigen binding site comprises a light chain variable region comprising LC-CDR1 , LC- CDR2 and LC-CDR3, wherein: d) light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 23 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR1 are substituted with another amino acid; e) light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 24 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR2 are substituted with another amino acid; f) light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 25 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR3 are substituted with another amino acid.

[0032] In some embodiments, the antibody / antigen binding molecule comprises a VH region having at least at least 70% sequence identity, optionally at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:26 and / or a VL region having at least at least 70% sequence identity, optionally at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:27.

[0033] In another embodiment, the present disclosure relates to an antigen binding molecule comprising an antigen binding domain for I FNy, wherein said antigen binding site comprises a heavy chain variable region comprising HC-CDR1 , HC-CDR2 and HC-CDR3, wherein: a) heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 28 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR1 are substituted with another amino acid; b) heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 29 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR2 are substituted with another amino acid; c) heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 30 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR3 are substituted with another amino acid; and / or wherein the antigen binding site comprises a light chain variable region comprising LC-CDR1 , LC- CDR2 and LC-CDR3, wherein: d) light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 31 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR1 are substituted with another amino acid; e) light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 32 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR2 are substituted with another amino acid; f) light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 33 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR3 are substituted with another amino acid.

[0034] In some embodiments, the antibody / antigen binding molecule comprises a VH region having at least at least 70% sequence identity, optionally at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:34 and / or a VL region having at least at least 70% sequence identity, optionally at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:35.

[0035] In an embodiment, the present disclosure relates to an antigen binding molecule comprising an antigen binding domain for I FNy, wherein said antigen binding site comprises a heavy chain variable region comprising HC-CDR1 , HC-CDR2 and HC-CDR3, wherein: a) heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 36 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR1 are substituted with another amino acid; b) heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 37 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR2 are substituted with another amino acid; c) heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 38 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR3 are substituted with another amino acid; and / or wherein the antigen binding site comprises a light chain variable region comprising LC-CDR1 , LC- CDR2 and LC-CDR3, wherein: d) light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 39 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR1 are substituted with another amino acid; e) light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 40 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR2 are substituted with another amino acid; f) light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 41 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR3 are substituted with another amino acid.

[0036] In some embodiments, the antibody / antigen binding molecule comprises a VH region having at least at least 70% sequence identity, optionally at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:42 and / or a VL region having at least at least 70% sequence identity, optionally at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:43.

[0037] The antigen binding molecule may have any of the functional properties described above. For instance, it may be capable of binding IFNy and biasing the pro-and anti-inflammatory activities of IFNy in favour of the pro-inflammatory activities, and / or it may be non-inhibitory or partially inhibitory for the pro- inflammatory activity of IFNy. It may be capable of binding to interferon gamma (IFNy) and modulating cell signalling downstream of IFNy, such that the ratio of PD-L1 expression (e.g., cell surface expression) and / or activity to MHC-1 expression (e.g., cell surface expression) and / or activity which is induced by IFNy is modified in favour of MHC-1 expression and / or activity and / or it may be non-inhibitory or partially inhibitory for the induction of MHC-1 expression (e.g., cell surface expression) or activity by IFNy.

[0038] In a third aspect, the present invention relates to an antigen binding molecule comprising an antigen binding domain for IFNy binding to the same IFNy epitope as one of the antibodies described above, e.g., binding the same epitope as an antibody having a VH and VL sequence as described above. In some embodiments, the antigen binding molecule may be a multispecific or bispecific antigen binding molecule according to any of the embodiments described above and according to any of the embodiments further described herein.

[0039] In a fourth aspect, the invention provides an antigen-binding molecule that binds to interferon gamma (IFNy), wherein binding of said molecule to IFNy biases the pro-and anti-inflammatory activities of IFNy in favour of the pro-inflammatory activities. For example, in some embodiments, an antigen-binding molecule may preferentially inhibit the anti-inflammatory activities (i.e., may inhibit the anti-inflammatory activities while inhibiting the pro-inflammatory properties to a lesser degree or while not inhibiting the proinflammatory activities). In some embodiments, the antigen-binding molecule may partially inhibit the pro-inflammatory activity of IFNy, while inhibiting the anti-inflammatory activities of IFNy more strongly. Optionally the antibody is human or humanised.

[0040] As described above, the pro-inflammatory activity of the cytokine may be measured by the ability of the cytokine to induce expression of major histocompatibility complex class I (MHC-1) on the surface of a cell, e.g., a cell in vitro. In some embodiments, the anti-inflammatory activity of the cytokine may be measured by the ability of the cytokine to induce expression of programmed Cell Death Ligand 1 (PD-L1) on the surface of a cell, e.g., a cell in vitro. Accordingly, the present invention may provide an antigen-binding molecule, optionally an antibody, which is capable of binding to interferon gamma (IFNy) and which is capable of modulating cell signalling downstream of IFNy, such that the ratio of PD-L1 expression (e.g., cell surface expression) and / or activity to MHC-1 expression and / or activity which is induced by IFNy is modified in favour of MHC-1 expression (e.g., cell surface expression) and / or activity.

[0041] In any aspect or embodiment described herein, the antigen-binding molecule capable of binding to interferon gamma (alone, or in addition to binding to another antigen such as a TAA) may comprise an antibody Fc region and / or may be modified by conjugation to another half-life extending moiety. This may result in the antigen-binding molecule having a significantly extended half-life, in turn extending the halflife of an antigen bound by the molecule.

[0042] In any of the above aspects and embodiments, the antibody may be human or humanised.

[0043] In another aspect, the present invention relates to a pharmaceutical composition comprising any of the antigen binding molecules as described herein.

[0044] In another aspect, the disclosure relates to a method of treating a disease or disorder in which the pro- inflammatory and / or anti-tumorigenic activities of IFNy are beneficial, comprising administering to the subject the antigen binding molecule or the pharmaceutical composition of any one of the foregoing aspects or embodiments. In another aspect, the disclosure relates to methods of treating cancer in a subject in need thereof, comprising administering to the subject the antigen binding molecule or the pharmaceutical composition of any one of the foregoing aspects or embodiments.

[0045] The treatment may be in combination with another one or more additional anti-cancer therapies, as further discussed herein. In some embodiments the treatment may be combined with one or more checkpoint inhibitors as known in the art. Also disclosed are methods of sensitizing a subject with a cancer to treatment with a checkpoint inhibitor, comprising administering to the subject a composition comprising an antigen binding molecule or pharmaceutical composition disclosed herein.

[0046] The present disclosure further relates to an antigen binding molecule or a pharmaceutical composition of any one of the foregoing aspects or embodiments for use in a method of treatment, e.g., any method of treatment as described herein. For example, the present invention relates in one aspect to an antigenbinding molecule as described above for use in the treatment of a disorder in which the proinflammatory and / or anti-tumorigenic activities of IFNy are beneficial. More particularly, there is provided an antigen binding molecule or a pharmaceutical composition of any one of the foregoing aspects or embodiments for use in a method of treating cancer. Also disclosed is an antigen binding molecule or a pharmaceutical composition of any one of the foregoing aspects or embodiments for use in a method of sensitizing a subject with a cancer to treatment with a checkpoint inhibitor. Also disclosed is an antigen binding molecule or a pharmaceutical composition of any one of the foregoing aspects or embodiments for use in a method of treating cancer, said method further comprising administering one or more additional anticancer therapies. In some embodiments the one or more additional anti-cancer therapies may comprise a check point inhibitor. Also disclosed is an antigen binding molecule or a pharmaceutical composition of any one of the foregoing aspects or embodiments and one or more additional anti-cancer therapies for use in a method of treating cancer. In one embodiment the one or more additional anti-cancer therapies may comprise a checkpoint inhibitor.

[0047] Further provided is use of an antigen binding molecule or a pharmaceutical composition of any one of the foregoing aspects or embodiments in the manufacture of a medicament for the treatment of a disease or disorder as described herein, e.g., cancer. The treatment of cancer may be in combination with another one or more additional anti-cancer therapies, as further discussed herein.

[0048] The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

[0049] Summary of the Figures

[0050] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

[0051] FIGS. 1A-1E are plots showing binding of exemplary bivalent anti-IFNy antibodies of the disclosure against human recombinant IFNy (hrlFNy). Biacore sensorgrams and fitted curves are used to determine binding affinities for exemplary antibodies 4F4 (FIG. 1A), 3G1 (FIG. 1 B), 16D2 (FIG. 1 C), 9F1 (FIG. 1 D) and 2C1 (FIG. 1 E) to hrlFNy. The sensorgrams show binding curves of hrlFNy to mAb at two different dilution series of hrlFNy (lower curve: 1 .11 to 10nM series, top curve: 10 to 90 nM series) which together are fitted (black lines) to calculate the affinity. Affinity values in nM are shown on the right side of the plot.

[0052] Figure 1 F shows the ability of the anti-IFNy antibodies to inhibit the IFNy mediated heterotetramerization of the IFNyRI and IFNyR2 subunits, and thereby neutralizing the IFNy response. Commercial neutralizing and non-neutralizing antibodies are show as reference. Figure 1G shows the ability of a truncated monovalent version of antibody 4F4 prepared by Duobody technology (4F4-VH(K409R) (SEQ ID NO: 75):Truncated Fc - monovalent mAbs lgG1 .1 Fc(H-CH2-CH3)(F405L) (SEQ ID NO: 83) to increase endogenous IFNy plasma concentration over time, following a single intravenous administration to cynomolgus monkeys. Mean values incl. SEM from two cynomolgus monkeys per time point are shown.

[0053] FIGS. 2A-2E show normalized surface expression of Major Histocompatibility Complex Class I (MHC-1) proteins and Programmed Death-Ligand 1 (PD-L1) proteins on PC-3 prostate cancer cells treated with an IFNy antibody of the disclosure (4F4 (FIG. 2A), 3G1 (FIG. 2B), 16D2 (FIG. 2C), 9F1 (FIG. 2D) and 2C1 (FIG. 2E)) pre-complexed with 1 ng / mL (0.06 nM) recombinant IFNy. Surface expression levels of MHC-1 and PD-L1 are normalized against those of corresponding control cells treated with 1 ng / mL (0.06 nM) IFNy only. FIG. 2F, similarly to Figs. 2A-2E, show normalized surface expression of MHC-1 and PD-L1 proteins on A549 lung cancer cells treated with selected IFNy antibodies of the disclosure (4F4 and 9F1) pre-complexed with 1 ng / mL (0.06 nM) recombinant IFNy. Surface expression levels of MHC-1 and PD- L1 are normalized against those of corresponding control cells treated with 1 ng / mL (0.06 nM) IFNy only. FIG 2G left is a bar graph showing dependence of MHC-1 surface expression on IFNy concentration in the presence or absence of 50 nM (7,300 ng / mL) 4F4 antibody (FIG. 2G left). Fig 2G right is a bar graph showing dependence of PD-L1 surface expression on IFNy concentration in the presence or absence of 50 nM (7,300 ng / mL) 4F4 antibody (FIG. 2G right).

[0054] Figure 3 shows binding saturation curves of LDC-1 to PSMA-positive LNCaP cells and LDC-2, LDC-3, LDC-4 and LDC-5 to PSMA-transfected PC-3 cells obtained by flow cytometry. LDC-1 , LDC-2, LDC-3, LDC-4 and LDC-5 are bispecific versions of certain antibodies as disclosed herein, also having a binding domain for PSMA, as described in more detail in example 7. Binding affinities in nM of each LDC are stated in the lower right corner of each plot.

[0055] Figure 4A shows flow cytometry results of IFNy binding to PC-3 cells (PSMA negative) and LNCaP (PSMA positive) pre-incubated with or without LDC-1 . The binding of IFNy to cells was detected using an anti-IFNy antibody. Figure 4B shows binding of fluorescently labelled IFNy to PC-3 cells, either positive or negative for GFP-PSMA, that were pre-incubated with increasing concentrations of LDC-2. Figure 4C shows MHC-1 and PD-L1 expression levels (Mean Fluoresence Intensity, MFI) in PC-3 cells either positive or negative for GFP-PSMA, which were stimulated with 0.025 nM IFNy alone or with combinations of 0.025 nM IFNy and 0.1 , 1 , 10 or 100 nM LDC-4 or LDC-6 for 48 hr. The expression level of MHC-1 and PD-L1 on GFP-PSMA-expressing and PSMA-negative PC-3 cells was measured using an Attune flow cytometry setup.

[0056] Figure 5 shows that IFNy bound to KiH-3 and KiH-6 retains proinflammatory signalling across different cell lines. Normalized surface expression of Major Histocompatibility Complex Class I (MHC-1) proteins and Programmed Death-Ligand 1 (PD-L1) proteins assessed on three different cancer cell lines by flow cytometry is shown. HT29 colorectal cancer cells (FIG. 5A), PC3-FLU prostate cancer cells (FIG. 5B) and A549 lung cancer cells (FIG. 5C) were treated with a bispecific antibody of the disclosure (KiH-3 or KiH-6) pre-complexed with 1 ng / mL (0.06 nM) recombinant IFNy. Surface expression levels of MHC-1 and PD- L1 are normalized against those of corresponding control cells treated with 1 ng / mL (0.06 nM) IFNy only. A549 cells (FIG. 5C) were additionally treated with an IFNy neutralizing control antibody (MT111 W) precomplexed with 1 ng / mL (0.06 nM) recombinant IFNy. The IC50 range of MT111 W is indicated (grey bar).

[0057] Figure 6 shows MHC-1 and PD-L1 expression levels (Mean Fluorescence Intensity, MFI) in PC3 PIP cells expressing PSMA (FIG. 6A-B) or A549 cells not expressing PSMA (FIG. 6C-D) determined by flow cytometry at 6h, 24h, 48h and 72h. Cells were treated with a bispecific antibody of the disclosure (KiH-3 or KiH-6) alone, human IFNy (0.1 nM) alone, or a bispecific antibody of the disclosure (KiH-3 or KiH-6) pre-complexed with human IFNy (0.1 nM). IFNy neutralising antibody MT111W pre-complexed with human IFNy (0.1 nM) was used as a positive control.

[0058] Figure 7 shows flow cytometry results of IFNy stimulation (0.025 nM or 5.95 nM) of PC3-PIP cells (expressing PSMA), and stimulation with bispecific antibody KiH-3 at either 1 nM or 10 nM concentration with or without IFNy stimulation. MHC-1 (FIG. 7A) and PD-L1 (FIG. 7C) expression levels (Mean Fluorescence Intensity, MFI) are shown, as well as the percentage of cells which were MHC-I+ (FIG. 7B) and PD-L1 + (FIG 7D).

[0059] Figure 8 shows the PSMA targeted delivery of IFNy to a tumor with KIH-3. Figure 8A is a schematic depiction of a PSMA-expressing PC-3 prostate cancer xenograft tumor model. A tumor comprising PC-3 prostate cancer cells was established in mice by injecting the cells subcutaneously at day 0. On day 21 , mice were i.v. injected with bispecific antibody of the disclosure (KiH-3) with or without hlFNy. On day 24, tumor resection was carried out. Figure 8B shows the immunohistochemical staining of paraffin embedded sections obtained using the method shown in FIG 8A stained with an anti-IFNy antibody.

[0060] Figure 8C shows the results of an in vivo imaging experiment in which mice comprising a prostate cancer xenograft tumor were i.v. injected with Alexa680-labelled KiH-3 antibody and imaged using a far-red camera.

[0061] Figure 9 shows that KIH-3 activates the IFNy receptor and upregulates MHC-I in cold prostate cancer. Figure 9A is a schematic depiction of a PSMA-expressing PC-3 prostate cancer xenograft tumor model. A tumor comprising PC-3 prostate cancer cells was established in mice by injecting the cells subcutaneously at day 0. On day 21 , mice were i.v. injected with bispecific antibody of the disclosure (KiH-3) with or without hlFNy (or vehicle control). On day 24, tumor resection was carried out. Figure 9B shows the immunohistochemical staining of paraffin embedded sections obtained using the method shown in FIG 9A stained using an anti-MHC-l antibody.

[0062] Figures 10 and 11 show the results of a pharmacokinetics study of the KiH-16 antibody of the disclosure. Mice were dosed with 1 , 3 or 10 mg / kg KiH-16 (i.v.) or alternatively 10 mg / kg KiH-16 (i.p.), blood samples were taken at a series of time-points post-injection and the concentrations of KiH-16 and mlFN-y in the blood samples were measured by ELISA. Figure 10 shows the KiH-16 concentration in ug / mL (FIG. 10A) and uM (FIG. 10B) measured in the blood serum through binding to immobilized human PSMA protein and detection with HRP Goat anti-mouse lgG2a heavy chain (Abeam, Cat.ab97245). Figure 11 shows the accumulation of mlFN-y in serum samples following KiH-16 administration. mlFN-y was detected using the detection antibody #ab14-Biotin (1 pg / mL, Biocytogen) (FIG. 11 A). Figure 11 B shows the molar ratio of KiH-16 detected in the serum (FIG. 11A) versus mlFN-y detected in the serum (FIG. 11A).

[0063] Figure 12 shows the results of a test assay to determine the optimal conditions for mlFN-y stimulation of B-hPSMA MC38 cells. Cells were stimulated with 0.01 , 0.05, 0.1 , 0.5, 1 , 5, 10, 50, 100, 250, 500 ng / mL. mlFN-y for 24 or 48 h and surface expression levels of MHC-I and PD-L1 were measured by flow cytometry. 5 ng / mL mlFN-y is marked (black vertical line).

[0064] Figure 13 shows that KIH-16 does not neutralize the effect of IFNy. B-hPSMA MC38 cells were stimulated with 5 ng / mL mlFNy pre-mixed with either a bispecific antibody of the disclosure (KIH-16), nonneutralizing anti-mlFN-y antibody AN-18 or neutralizing anti-mlFN-y antibody XMG1.2 at a range of concentrations (0.9, 9. 90, 900 nM) for 48 h. After stimulation, cells were analysed by flow cytometry.

[0065] Figure 13A-B shows the percentage of cells which were mCD274+(FIG. 13A) and mMHC-l+(FIG 13.B) . Figure 13C-D shows PD-L1 (FIG. 13C) and MHC-1 (FIG. 13D) surface expression levels (Mean Fluorescence Intensity, MFI).

[0066] Figure 14 shows the mlFNy accumulation and immune infiltration potential of the KiH-16 antibody in a syngeneic mouse model. A tumor comprising MC38 cells (not expressing hPSMA) was established in mice by injecting the cells subcutaneously. Mice were dosed with anti-mouse PD-1 (1 mg / kg), with or without KiH-16 (1 mg / kg), by i.p. injection twice weekly and blood was collected 24 h post-injection following the 1st, 3rd, 5th and 7th (final) dose. mlFN-y levels in the blood were detected by ELISA using an anti mlFN-y antibody (FIG. 14A). Figure 14B-C shows the results of flow cytometry analysis of tumor tissues taken from the study endpoint. Surface expression of MHC-I (FIG 14B) and the percentage of mCD45+ cells (FIG 14C) in the tumour samples are shown.

[0067] Definitions

[0068] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. Generally, nomenclatures utilized in connection with, and techniques of, immunology, oncology, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those known and commonly used in the art. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0069] As used herein, singular forms “a,” “and,” and “the” include plurals unless the context clearly indicates otherwise.

[0070] As used herein, all numerical values or numerical ranges include whole integers within or encompassing such ranges and fractions of the values or the integers within or encompassing ranges unless the context clearly indicates otherwise. Thus, e.g., reference to a range of 90-100%, includes 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so forth.

[0071] “About” a number, as used herein, refers to a range including the number and ranging from 10% below that number to 10% above that number. “About” a range refers to 10% below the lower limit of the range, spanning to 10% above the upper limit of the range.

[0072] An ‘antigen-binding molecule’ refers to a molecule that binds to a given target antigen. Antigen-binding molecules include antibodies ( / .e. immunoglobulins (Igs)) and antigen-binding fragments thereof. An antigen-binding molecule that is capable of binding to a given target antigen may also be described as an antigen-binding molecule that binds to the given target antigen.

[0073] Antigen-binding molecules according to the present disclosure also include antibody-derived molecules, e.g. molecules comprising an antigen-binding region / domain derived from an antibody. Antibody-derived antigen-binding molecules may comprise an antigen-binding region / domain that comprises, or consists of, the 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 an antibody-derived antigen-binding molecule may be or comprise the Fv (e.g. provided as an scFv) or the Fab region of an antibody, or the whole antibody. Antigen-binding molecules according to the present disclosure also include multispecific antigen-binding molecules.

[0074] The antigen-binding molecule of the present disclosure comprises a moiety or moieties capable of binding to a target antigen(s). In some embodiments, the moiety capable of binding to a target antigen comprises an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) of an antibody capable of specific binding to the target antigen. In some embodiments, the moiety capable of binding to a target antigen comprises or consists of an aptamer capable of binding to the target antigen, e.g. a nucleic acid aptamer (reviewed, for example, in Zhou and Rossi Nat Rev Drug Discov. 2017 16(3): 181 -202). In some embodiments, the moiety capable of binding to a target antigen comprises or consists of an antigen-binding peptide / polypeptide, e.g. a peptide aptamer, thioredoxin, monobody, anticalin, Kunitz domain, avimer, knottin, fynomer, atrimer, DARPin, affibody, nanobody ( / .e. a singledomain antibody (sdAb), which may for example include a “camelised” human VH, or a human VH domain modified to be stable as a single-domain antibody), affilin, armadillo repeat protein (ArmRP), Obody or fibronectin - reviewed e.g. in Reverdatto et al., Curr Top Med Chem. 2015; 15(12): 1082-1101 , which is hereby incorporated by reference in its entirety (see also e.g. Boersma et al., J Biol Chem (2011) 286:41273-85 and Emanuel et al., Mabs (2011) 3:38-48).

[0075] As used herein “antibody” is used in the broadest sense and encompasses various antibody structures so long as 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, mutated antibodies, grafted antibodies, antibody fragments, and in vitro- generated antibodies having the antigen-binding activity. The term also includes heavy-chain only antibodies such as those comprising VHH fragments or nanobodies, including human VH domains modified to be stable as a single-domain antibody. The term also includes antibody conjugates having advantageous properties as compared to an unconjugated antibody (e.g., antibodies conjugated to a halflife extending moiety, e.g., a fatty acid). In some embodiments, an antibody may have a variable domain and a constant domain in each of a heavy chain and a light chain. Accordingly, antibodies may have a heavy chain variable domain (VH) and a light chain variable domain (VL) that together form the portion of the antibody that binds to the antigen, sometimes referred to as the “antigen receptor.” Within each variable domain are three complementarity-determining regions (CDR), which form loops in the VH and VL and contact the surface of the antigen.

[0076] The terms “antigen-binding portion of an antibody,” “antigen-binding fragment,” “antigen-binding domain,” “antibody fragment” are used interchangeably herein to refer to one or more fragments of an antibody that retain the ability to specifically bind to the antigen. Examples of antibody fragments include but are not limited to Fv, Fab, cross-Fab, Fab’, Fab’-SH, F(ab’)2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv, and scFab); single domain antibodies (dAbs) (also known as nanobodies); and multispecific antibodies formed from antibody fragments. Also included 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 domain of the heavy chain and the VL and CL domain of the light chain of an immunoglobulin. As used herein, a reference to a “Fab fragment” is intended to include a cross-Fab fragment or a scFab as well as a conventional Fab fragment (i.e., one comprising a light chain comprising a VL domain and a CL domain, and a heavy chain fragment comprising a VH domain and a CH1 domain). “Fab’ fragments” differ from Fab fragments by the addition of residues at the carboxy terminus of the CH1 domain including one or more cysteines from the antibody hinge region.

[0077] The term “cross-Fab fragment” or “xFab fragment” or “crossover Fab fragment” refers to a Fab fragment, wherein either the variable regions or the constant regions of the heavy and light chain are exchanged. A cross-Fab fragment comprises a polypeptide chain composed of the light chain variable region (VL) and the heavy chain constant region 1 (CH1), and a polypeptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL). For clarity, in a crossover Fab molecule wherein the variable regions of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain constant region is referred to herein as the "heavy chain" of the crossover Fab molecule. Conversely, in a crossover Fab molecule wherein the constant regions of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain variable region is referred to herein as the "heavy chain" of the crossover Fab molecule.

[0078] “Affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein “binding affinity” refers to intrinsic binding affinity which reflects a 1 :1 interaction between members of a binding pair (e.g., paratope and epitope). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD), and unless otherwise indicated, KD values as used herein refer to the intrinsic affinity. Binding affinity of antibodies or antigen-binding fragments is, e.g., measured by surface plasmon resonance (SPR) measurements, ELISA, AlphaLisa assays, flow cytometry, and other conventional methods. In one embodiment, binding affinity is measured using a BIACORE® surface plasmon resonance assay, such as a BIACORE surface plasmon resonance assay with captured antibody, at e.g. 25°C, e.g., at pH 7.4, optionally as described in detail in example 2 for single or multi-kinetic analysis. In another embodiment, the conditions for the BIACORE® analysis may be as described in example 19.

[0079] As used herein “complementarity-determining regions” or “CDRs” or “hypervariable regions” refer to the parts of the variable domains in antibodies that determine the binding specificities of the antibodies to their specific antigen. As noted, a single variable region of an antibody polypeptide will typically comprise three CDRs, usually designated CDR1 , CDR2, and CDR3. More particularly, a heavy chain variable region may contain CDRs designated H1 , H2, and H3; likewise, a light chain variable region may contain CDRs designated L1 , L2, and L3. Multiple methods may be used to define a CDR. The current art utilizes various numbering schemes with different definitions of CDR lengths and positions. For example, the Kabat numbering scheme is based on sequence alignment and uses "variability parameter" of a given amino acid position (the number of different amino acids at a given position divided by the frequency of the most occurring amino acid at that position) to predict CDRs. The Chothia numbering scheme, on the other hand, is a structure-based numbering scheme where antibody crystal structures are aligned as define the loop structures as CDRs. The Martin numbering scheme focuses on the structure alignment of different framework regions of unconventional lengths. IMGT numbering scheme is a standardized numbering system based on alignments of sequences from a complete reference gene database including the whole immunoglobulin superfamily. Honneger’s numbering scheme (AHo) is based on structural alignments of the 3D structure of the variable regions and uses structurally conserved Ca positions to deduce framework and CDR lengths. One skilled in the art will note that the definition of a CDR will vary based on the method used. Any method of defining a CDR is contemplated with the sequences disclosed herein. The CDRs as described herein have been determined using the IMGT scheme, but in other embodiments CDRs as determined by another scheme as described above may be substituted.

[0080] The “epitope” of an antibody is the specific site on the antigen to which the antibody binds. The epitope may be a linear epitope, consisting of a contiguous sequence of amino acids (i.e. an amino acid primary sequence). In some embodiments, an antigen-binding molecule may bind to a conformational epitope of I FNy, consisting of a discontinuous sequence of amino acids in the antigen. The region of a given target molecule to which an antigen-binding molecule binds can be determined by the skilled person using various methods well known in the art, including X-ray co-crystallography analysis of antibody-antigen complexes, peptide scanning, mutagenesis mapping, hydrogen-deuterium exchange analysis by mass spectrometry, phage display, competition ELISA and proteolysis-based ‘protection’ methods. Such methods are described, for example, in Gershoni et al., BioDrugs, 2007, 21 (3):145-156, which is hereby incorporated by reference in its entirety. An antibody may be considered to bind the “same epitope” as a reference antibody if they cross-block - e.g., if the test antibody inhibits binding of the reference antibody to the antigen, and the reference antibody inhibits binding of the test antibody to the antigen. The blocking may be by at least 50% in each case. In some embodiments, in the case of a linear epitope, an antibody may bind the “same epitope” as a reference antibody it binds to the same fragment of the antigen, said fragment consisting of 20 amino acids or less, optionally 15 amino acids or less or 14, 13, 12, 11 or 10 residues or less, and does not specifically bind a different fragment having no overlap therewith; alternatively, using peptide scanning, the epitope may be identified by the set of peptide fragments of the antigen to which the antibody specifically binds, and two antibodies may be considered to bind the same epitope if they bind to a set of peptide fragments consisting of fragments which fully overlap or which include no more than 4, 3, 2 or 1 non-overlapping residues when comparing the two sets. In some embodiments, peptides of various lengths may be screened to identify the smallest fragment that can specifically bind to the reference antibody, and it can be determined if another antibody binds to the same fragment. Such a fragment can also be used, if required, as an immunogen to obtain antibodies which bind the same epitope. In the case of a non-linear epitope, in some embodiments, the antibody may be considered to bind the “same epitope” as a reference antibody if it shares the same set of key contact residues in the antigen, wherein a key contact residue is defined as a residue having a distance of 4.5A or less between an antibody residue and antigen residue in a crystal structure, or as a residue having a distance of 4.5A or less between an antibody residue and antigen residue in a crystal structure and as being a residue for which mutation (e.g, to alanine) abolishes binding. In some embodiments, two epitopes are considered the same when they have an epitope similarity of 0.1 or above, 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. 2021 Jan-Dec;13(1):1873478. doi: 10.1080 / 19420862.2021 .1873478. PMID: 33448242; PMCID: PMC7833755, which is fully incorporated by reference.

[0081] The antibodies (including but not limited to bispecific antibodies) or antigen-binding fragments thereof disclosed herein are identified using any suitable techniques. For example, a computational approach can be used to design epitope-specific antibodies. Another approach can be used to identify antibodies that bind to specific epitopes from a library of antibodies that bind to the antigen, in certain embodiments, such as the following: first incorporate noncanonical amino acids (ncAAs) p-benzoyl-L-phenylalanine (pBpa) and p-azido-L-phenylalanine (pAzF) into the target epitope and then select the antibodies that cross-link with the ncAA incorporated epitope after UV irradiation. Because cross-linking only occurs when the distance between the antibody and the epitope is close enough, this method efficiently selects antibodies that specifically bind to the target epitope.

[0082] The term “Fc domain” herein is used to define a C-terminal region of an immunoglobulin that contains the constant regions of two heavy chains, optionally including part or all of the hinge region, but excluding the first constant region. Thus, Fc domain refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM. The constant regions from the two polypeptides together form the Fc region. The term includes native sequence Fc regions and variant Fc regions. In one aspect, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Therefore, an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include the full- length heavy chain, or it may include a cleaved variant of the full-length heavy chain. This may be the case where the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to EU index). Therefore, the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (Lys447), of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. A "subunit" of an Fc domain or “Fc subunit” as used herein refers to one of the two polypeptides forming the dimeric Fc domain, i.e. a polypeptide comprising C-terminal constant regions of an immunoglobulin heavy chain, capable of stable association with the other of the two polypeptides forming the dimeric Fc domain. For example, a subunit of an IgG Fc domain comprises an IgG CH2 and an IgG CH3 constant domain.

[0083] As used herein, “sequence identity” refers to the percent of nucleotides / amino acid residues in a subject sequence that are identical to nucleotides / amino acid residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum percent sequence identity between the sequences. Pairwise and multiple sequence alignment for the purposes of determining percent sequence identity between two or more amino acid or nucleic acid sequences can be achieved in various ways known to a person of skill in the art, for instance, using publicly available computer software such as ClustalOmega (Soding, J. 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) software. Other exemplary programs that compare and align pairs of sequences, include ALIGN (Myers and Miller, Comput Appl Biosci. 1988 Mar; 4(1 ):11-7), FASTA (Pearson and Lipman, Proc Natl Acad Sci USA. 1988 Apr; 85(8):2444-8; Pearson, Methods Enzymol. 1990; 183:63-98), gapped BLAST (Altschul et al., Nucleic Acids Res. 1997 Sep 1 ; 25(17): 3389-40), BLASTP, BLASTN, or GCG (Devereux et al., Nucleic Acids Res. 1984 Jan 11 ; 12(1 Pt 1):387-95). When using such software, the default parameters, e.g. for gap penalty and extension penalty, are preferably used.

[0084] The terms “polypeptide,” “peptide,” “oligopeptide,” and “protein” are used interchangeably herein and refer to a chain of amino acids. The polypeptides, etc. are not limited to a specific chain length of amino acids. This term also encompasses chains of peptides with post-expression modifications, e.g., glycosylation, acetylation, phosphorylation, and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. In certain embodiments, a polypeptide is an entire protein, or a fragment thereof.

[0085] The terms “preferentially binds” or “specifically binds” mean that the antibodies or fragments thereof bind to an epitope with greater affinity than it binds unrelated amino acid sequences. Binding of the antigenbinding molecules described herein to their antigen may be “specific” in this sense. In certain embodiments, such affinity is at least 1 .5-fold greater, 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1000-fold greater than the affinity of the antibody or fragment thereof for unrelated amino acid sequences. The term is also applicable where e.g., an antigen-binding domain is specific for a particular epitope which is carried by a number of antigens, in which case the antibody or antigen-binding fragment thereof carrying the antigen-binding domain will be able to bind to the various antigens carrying the epitope. Any of the antigen-binding molecules disclosed herein may specifically bind to their antigen.

[0086] The terms “recipient,” “individual,” “subject,” “host,” and “patient,” are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans. “Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and laboratory, zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc. In certain embodiments, the mammal is a human.

[0087] The term “therapeutically effective amount” generally refers to an amount of a disclosed antibody or a drug effective to “treat” a disease or disorder in a subject or mammal. In certain embodiments, a composition described herein is administered to a subject in an amount that is effective for producing some desired therapeutic effect by inhibiting a disease or disorder as described herein at a reasonable benefit / risk ratio applicable to any medical treatment. A therapeutically effective amount is an amount that achieves at least partially a desired therapeutic or prophylactic effect in an organ or tissue. The amount of an antibody necessary to bring about prevention and / or therapeutic treatment of a disease or disorder is not fixed per se. In certain embodiments, the amount of the antibody administered varies with the type of disease, extensiveness of the disease, and size of the mammal suffering from the disease or disorder. When used in conjunction with therapeutic methods involving administration of a therapeutic agent after the subject presents symptoms of a disease or disorder, the term “therapeutically effective” means that, after treatment, one or more signs or symptoms of the disease or disorder is ameliorated or eliminated.

[0088] In certain embodiments, administration of a therapeutic agent in a prophylactic method occurs prior to the manifestation of symptoms of an undesired disease or disorder, such that the disease or disorder is prevented or, alternatively, delayed in its progression. Thus, when used in conjunction with prophylactic methods, the term “therapeutically effective” means that, after treatment, a smaller number of subjects (on average) develop the undesired disease or disorder or progress in severity of symptoms.

[0089] As used herein, the terms “treatment,” “treating,” and the like, in some cases, refer to administering an agent, or carrying out a procedure, for the purposes of obtaining an effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or is therapeutic in terms of effecting a partial or complete cure for a disease and / or symptoms of the disease. “Treatment,” as used herein, includes treatment of a disease or disorder in a mammal, particularly in a human, and includes: (a) preventing the disease or a symptom of a disease from occurring in a subject which is predisposed to the disease but has not yet been diagnosed as having it (e.g., including diseases that is associated with or caused by a primary disease; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease. The term treating includes to any indicia of success in the treatment or amelioration or prevention of a disease or disorder, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating. The treatment or amelioration of symptoms is based on one or more objective or subjective parameters, including the results of an examination by a physician. Accordingly, the term “treating” includes the administration of the agents of the present disclosure to prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated with diseases. The term “therapeutic effect” refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject. A subject is “treated” for a disease or disorder if, after receiving a therapeutic amount of an antibody of the present disclosure, the patient shows observable and / or measurable change in a parameter or symptom of the disease or disorder.

[0090] Detailed Description of the Invention

[0091] Aspects and embodiments of the present invention will now be discussed. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0092] Interferon gamma (IFNy)

[0093] IFNy is a 20-25 kDa glycoprotein that exists as a homodimer in solution. In certain embodiments, the IFNy protein is a human IFNy protein. In certain embodiments, the human IFNy protein is a wild-type human IFNy protein, e.g., an endogenous IFNy. In certain embodiments, the human IFNy protein is a recombinant human IFNy protein (rlFNy). In certain embodiments, the human IFNy protein has an amino acid sequence having 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%) sequence identity to SEQ ID NO: 1 . In certain embodiments, the human IFNy protein has an amino acid sequence having at least 90% (e.g., at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more e.g., 100%) sequence identity to SEQ ID NO: 1 . In certain embodiments, the human IFNy protein has an amino acid sequence having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 1 . In certain embodiments, the human IFNy protein has an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 1 . In certain embodiments, the human IFNy protein has an amino acid sequence of SEQ ID NO: 1 (UniProt No.: P01579). In certain embodiments, the IFNy protein is an IFNy protein of a non-human primate (NHP; e.g., Macaca fascicularis or Macaca mulata). In certain embodiments, the NHP IFNy protein has an amino acid sequence having 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 to SEQ ID NO: 2. In certain embodiments, the NHP IFNy protein has an amino acid sequence having at least 90% (e.g., at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 2. In certain embodiments, the NHP IFNy protein has an amino acid sequence having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 2. In certain embodiments, the NHP IFNy protein has an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 2. In certain embodiments, the NHP IFNy protein has an amino acid sequence of SEQ ID NO: 2 (UniProt No.: P63309).

[0094] In certain embodiments, it may be preferred that the antigen binding molecule is cross reactive against SEQ ID NO: 1 and SEQ ID NO:2, e.g., to allow the antibody to be subject to tests in primate model animal. In certain embodiments, the binding agent exhibits an affinity to the IFNy protein of Macaca fascicularis that is at most 10-fold lower than an affinity of the binding agent to a human IFNy protein.

[0095] In some embodiments, an antigen binding molecule / antibody according to the invention may bind to IFNy, e.g., human and / or primate IFNy, preferably human IFNy, with an affinity in the micromolar range, i.e. KD = 9.9 x 10-4to 1 x 10-6M. In some embodiments, the antigen-binding molecule described herein binds to IFNy with sub-micromolar affinity, i.e. KD < 1 x 10-6M. In some embodiments, the antigen-binding molecule described herein binds to IFNy with an affinity in the nanomolar range, i.e. KD = 9.9 x 10-7to 1 x 10-9M. In some embodiments, the antigen-binding molecule described herein binds to IFNy with sub- nanomolar affinity, i.e. KD < 1 x 10-9M. In some embodiments, the antigen-binding molecule described herein binds to IFNy with an affinity in the picomolar range, i.e. KD = 9.9 x 10-1° to 1 x 10-12M. In some embodiments, the antigen-binding molecule described herein binds to IFNy with sub-picomolar affinity, i.e. KD < 1 x 10-12M. In certain embodiments, the binding agent binds to the IFNy 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 bind to human IFNy with a KD of less than 10 nM or 5nM.

[0096] Tumour associated antigens

[0097] In some embodiments, the antigen binding molecule (e.g., antibody) is a multispecific antigen binding molecule (e.g., antibody) which has an antigen-binding domain capable of binding to IFNy and which also has an antigen binding site capable of binding to another target antigen such as a tumour-associated antigen (TAA). In some embodiments, the antigen binding molecule / antibody may be bispecific. For example, it may bind to IFNy and a single target antigen other than IFNy.

[0098] In other embodiments, the antigen binding molecule / antibody may bind more than one target antigen / TAA or may bind more than one distinct epitope on a target antigen / TAA. For instance, in some embodiments, the antigen binding molecule may bind to two different target antigens / TAAs, such as two different TAAs associated with the same tumour / cancer type. Dual TAA-targeting antibodies may offer advantages such as increased tumour selectivity.

[0099] The term “tumour-associated antigen” or “tumour specific antigen” as used herein refers to any molecule (e.g., protein, peptide, lipid, carbohydrate, etc.) solely or predominantly expressed or over-expressed by tumour cells and / or cancer cells, or by other cells of the stroma of the tumour such as cancer-associated fibroblasts, such that the antigen is associated with the tumour(s) and / or cancer(s). The tumour- associated antigen is expressed on the cell surface. The tumour-associated antigen can additionally be expressed by normal, non-tumour, or non-cancerous cells. However, in such cases, the expression of the tumour-associated antigen by normal, non-tumour, or non-cancerous cells is not as robust as the expression by tumour or cancer cells. In this regard, the tumour or cancer cells can over-express the antigen or express the antigen at a significantly higher level, as compared to the expression of the antigen by normal, non-tumour, or non-cancerous cells. Also, the tumour-associated antigen can additionally be expressed by cells of a different state of development or maturation. For instance, the tumour-associated antigen can be additionally expressed by cells of the embryonic or foetal stage, which cells are not normally found in an adult host. Alternatively, the tumour-associated antigen can be additionally expressed by stem cells or precursor cells, which cells are not normally found in an adult host.

[0100] The tumour-associated antigen can be an antigen expressed by any cell of any cancer or tumour, including the cancers and tumours described herein. The tumour-associated antigen may be a tumour- associated antigen of only one type of cancer or tumour, such that the tumour-associated antigen is associated with or characteristic of only one type of cancer or tumour. Alternatively, the tumour- associated antigen may be a tumour-associated antigen (e.g., may be characteristic) of more than one type of cancer or tumour. By way of example, the tumour-associated antigen may be expressed by both breast and prostate cancer cells and not expressed at all by normal, non-tumour, or non-cancer cells.

[0101] Exemplary tumour-associated antigens to which the antibodies of the invention may bind include, but are not limited to, avp3 integrin (e.g., in adenocarcinomas), Bombesin R (e.g., in prostate cancer), CAiX (e.g., in colon cancer), CD13 (e.g., in myeloid malignancies and adenocarcinomas), CD44 v6 (e.g., in adenocarcinomas such as head and neck), CXCR4 (e.g., in breast cancer), EGFR (e.g., in head / neck and breast cancer), emmprin (e.g., in pancreatic cancer), endoglin (e.g., gastrointestinal tumors, malignant melanomas, and tumors of the central nervous system), EphA2 (e.g., in ovarian cancer and melanoma), folate R (e.g., in ovarian cancer), GRP78 (e.g., in breast cancer and glioblastoma), IGF-1 R (e.g., in colon cancer), matriptase (e.g., in colon cancer), cMET / HGFR (e.g., in lung cancer), MT1-MMP (e.g., in lung cancer), MT6-MMP (e.g., in head and neck cancer, breast cancer, and colon cancer), PSMA (e.g., in prostate cancer), Tn and STn antigen (e.g., adenocarcinomas), uPAR (e.g., in colon cancer), GD2 (e.g., in neuroblastoma, melanoma, sarcoma, solid childhood tumours), TncA1 (e.g., in breast cancer, AML, solid tumours, MCC), EBD (e.g., in melanoma, RCC, NSCLC, solid tumours, pancreatic carcinoma, colorectal carcinoma, DLBCL, glioblastoma, sarcoma, glioma), PD1 (e.g., in solid tumours), EDA, MHCII, EDB, A33, alphaFR, EGR, PS (phosphatidyl serine), Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP), Mucin 1 (MUC1 ; tumour-associated epithelial mucin) (e.g., adenocarcinomas), preferentially expressed antigen of melanoma (PRAME) (e.g., in melanoma), carcinoembryonic antigen (CEA) (e.g., colon cancer), PSCA (e.g., in prostate cancer), EpCAM (e.g., in SCLC, prostate carcinoma, ovarian carcinoma, breast carcinoma, bladder carcinoma, kidney carcinoma, lung carcinoma, in colon cancer, solid tumours), 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 related protein (TRP) I, and TRP2. The tumour antigen may also be selected from the group consisting of cluster of differentiation (CD) 19, CD20 (e.g., in B cell lymphoma), CD21 , CD22, CD25, CD30, CD33 (sialic acid binding Ig-like lectin 3, myeloid cell surface antigen), CD79b, CD123 (interleukin 3 receptor alpha), transferrin receptor, EGF receptor, mesothelin (e.g., in mesothelioma), cadherin, Lewis Y, Glypican-3, FAP (fibroblast activation protein alpha) (e.g., in cancer-associated fibroblasts, solid tumours, RCC, melanoma, pancreatic adenocarcinoma, breast carcinoma, HNC, oesophageal carcinoma, cervical carcinoma), GPRC5D (G Protein-Coupled Receptor Class C Group 5 Member D), CA9 = CAIX (carbonic anhydrase IX), LI CAM (neural cell adhesion molecule L 1 ), endosialin, 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 (six-transmembrane epithelial antigen of prostate 1), LIV-1 (SLC39A6), B7-H3 (B7 Homolog 3, also known as CD276), and CD46. For example, the tumour- associated antigen may be selected from PSMA, EGFR, CEA, HER2, Tn antigen, sTn antigen, CD44v6, B7-H3, and Trop2.

[0102] In certain embodiments, the tumour associated antigen may be prostate specific membrane antigen (PSMA). Prostate-Specific Membrane Antigen (PSMA), also known as glutamate carboxypeptidase II, N- acetyl-a-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 prostate cancer cells and in non-prostatic solid tumor neovasculature and expressed at lower levels in other tissues, including healthy prostate, kidney, liver, small intestine, small bowel, salivary gland, duodenal mucosa, proximal renal tubules, and brain. PSMA is a member of a superfamily of zinc-dependent exopeptidases which include carboxypeptidases with a mononuclear zinc active site.

[0103] In certain embodiments, the human PSMA protein has an amino acid sequence having 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 to SEQ ID NO: 3. In certain embodiments, the human PSMA protein has an amino acid sequence of SEQ ID NO: 3 (UniProt No.: Q04609).

[0104] The expression of PSMA is increased in prostate cancer, especially in poorly differentiated, metastatic, and / or hormone-refractory carcinomas. Typically, PSMA expression is found to increase with prostate cancer progression and metastasis. PSMA is also expressed in endothelial cells of capillary vessels in peritumoral and intratumoral areas of certain malignancies, including renal cell carcinomas, and colon carcinomas, but not in blood vessels from normal tissues. In addition, PSMA is reported to be related to tumor angiogenesis. PSMA has been demonstrated to be expressed in endothelial cells of tumor- associated neovasculature in carcinomas of the colon, breast, bladder, pancreas, kidney, and melanoma.

[0105] In other embodiments the tumour associated antigen may be B7-H3 (also known as CD276). B7-H3 protein is overexpressed in many human tumours compared to healthy tissues and therefore provides a tumour-associated antigen with broad applicability e.g., to a range of solid tumours.

[0106] In certain embodiments, the human B7-H3 may be of the 2lg-B7-H3 or 4lg-B7-H3 isoform (formed respectively of a single pair or two identical pairs of immunoglobulin variable (lgV)-like and immunoglobulin constant (IgC)-like domains). In certain embodiments, the human B7-H3 protein has an amino acid sequence of SEQ ID NO: 131 (UniProt No.: Q5ZPR3).

[0107] In some embodiments, antibodies according to the invention may bind to a TAA (e.g., one or more TAAs selected from those as described herein) with an affinity an affinity in the micromolar range, i.e. KD = 9.9 x 10-4to 1 x 10-6M. In some embodiments, the antigen-binding molecule described herein binds to the TAA with sub-micromolar affinity, i.e. KD < 1 x 10-6M. In some embodiments, the antigen-binding molecule described herein binds to the TAA with an affinity in the nanomolar range, i.e. KD = 9.9 x 10-7to 1 x 10-9M. In some embodiments, the antigen-binding molecule described herein binds to the TAA with sub- nanomolar affinity, i.e. KD < 1 x 10-9M. In some embodiments, the antigen-binding molecule described herein binds to the TAA with an affinity in the picomolar range, i.e. KD = 9.9 x 10-1° to 1 x 10-12M. In some embodiments, the antigen-binding molecule described herein binds to the TAA with sub-picomolar affinity, i.e. KD < 1 x 10-12M. In certain embodiments, the binding agent binds to the PSMA 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, an antigen binding molecule / antibody may bind to the TAA with a KD of less than 20 nM.

[0108] Properties of the Antigen Binding Molecules

[0109] The antigen binding molecule capable of binding to IFNy, e.g., binding to IFNy alone or a multispecific antigen binding molecule as described herein, may have the following properties.

[0110] Partially and / or preferentially inhibiting antigen binding molecule

[0111] The present inventors have determined that it is possible to obtain antibodies which are capable of binding IFNy while still maintaining at least partial proinflammatory activity or anti-tumorigenic activity of the cytokine. (As used here, the term “proinflammatory” may be used interchangeably with “anti- tumorigenic”; and the term “anti-inflammatory” may be used interchangeably with the term “pro- tumorigenic”.) The inventors have further recognised that it is possible to make use of the proinflammatory / anti-tumorigenic properties of IFNy at the same time as binding an antigen binding molecule thereto, in order to obtain various advantages, such as increasing the plasma concentration of the endogenous IFNy, and / or increasing the half-life of exogenous administered IFNy, and / or targeting the IFNy to a target cell or tissue, such as a tumour or cancer cell or tissue. The antibody / antigen binding molecule is capable of binding IFNy while maintaining at least partial proinflammatory activity of IFNy. (As used herein, reference to “partial reduction” or “partial inhibition” means that at least some proinflammatory activity is retained.)

[0112] The inventors have unexpectedly determined that an antibody can be used to obtain accumulation of IFNy in the blood at the same time as retaining IFNy activity, even though it may have been expected that in the absence of IFNy inhibition / blocking of receptor binding, receptor mediated clearance may still occur.

[0113] In some situations, partial reduction of proinflammatory / anti-tumorigenic activity in combination with targeting to a target cell or tissue (e.g., using a multispecific antibody) may be beneficial to reduce side effects such as systemic inflammation while accumulating proinflammatory / anti-tumorigenic activity at the target (and thus benefitting from higher concentrations and / or avidity effects at the target).

[0114] Inflammatory and / or anti-tumour effects of IFNy may include effects on immune cells and / or direct effects on cancer cells. For example, IFNy may induce proinflammatory / anti-tumorigenic effects in one or more of the following: T-cells including but not limited to CD-4 or CD-8 cells, regulatory T-cells (Tregs), and gamma delta 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 (DC), macrophages, premacrophage monocytes and monocytes; Thy-1 .2+ cells, and HLA-DR+ cells. For example, proinflammatory activity of IFNy may be measured as one or more of: increased recruitment of immune cells via induction of chemokines CXCL9, CXCL10 and CXCL11 and their cognate receptor CXCR3 on immune cells, increased phagocytic ability of phagocytes; increased microbial and / or tumor cell-killing ability of macrophages; upregulating the expression of MHC I complex on immune cells or cancer cells; activation and enhanced cytotoxic effect of CD8+ T-cells e.g. via FAS-FASL and upregulation of granzyme B and TNF-related apoptosis-inducing ligand (TRAIL); enhanced survival of CD4+ T-helper 1 cells and prevention of polarization to T-helper 17 regulatory T-cells and T-helper 2 cells; maturation of DCs and / or enhanced expression in DCs of costimulatory molecules such as CD40, CD54, CD80, CD86, and CCR7; secretion from DCs of IL-12 family cytokines and / or IL-1 p; inhibiting Treg proliferation and function; and activation of one or more members of the signal transducer and activator of transcription (STAT) family, e.g., 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. 2022 Mar;22(3): 158-172. doi: 10.1038 / S41577-021 -00566-3. Epub 2021 Jun 21.PMID: 34155388).

[0115] In some embodiments proinflammatory activity is measured by cell surface expression of major histocompatibility complex class I (MHC-1), as explained further below.

[0116] For example, in one embodiment, the effect of the antigen binding molecule is assessed in vitro when coadministered to cells with 1 ng / mL IFNy. The assessment may be made 48 hours after the coadministration. In one embodiment, it is assessed by contacting a plated test cell such as an A549 lung cancer cell or PC3 prostate cancer cell, e.g., a PC3-FLU cell, with IFNy at a concentration of 1 ng / mL in the absence of the test molecule and in the presence of the test molecule and assessing MHC-1 expression on the cell surface after 48 hours. Optionally the cell may be an A549 lung cancer cell. In one embodiment, an antibody / antigen binding molecule when tested in this way is non-inhibitory on proinflammatory activity (e.g., MHC-1 expression) when tested at a concentration of 0.1 nM, i.e., it does not reduce the expression of MHC-1 as compared to the same concentration of IFNy in the absence of the test molecule. Additionally or alternatively, the antibody / antigen binding molecule may be inhibitory when tested at a concentration of 100nM, i.e., at this concentration it does reduce the expression of MHC-1 as compared to the same concentration of IFNy in the absence of the antibody / antigen binding molecule. In some embodiments, the IC50 for IFNy-induced expression of MHC-1 may be in the nanomolar range, i.e., less than 1 mM and at least 1 nM, optionally at least 10nM. In other embodiments the IC50 may be greater than 1 nM, or greater than 10nM. In some embodiments, the antibody / antigen binding molecule has an IC50 in this assay which is at least 100 fold higher (100 fold less inhibitory) than the reference anti-IFNy antibody MT111 W (Mabtech). In some embodiments, the antibody / antigen binding molecule has an IC50 in this assay which is between 100 and 10,000 times higher than MT 111 W. In another embodiment, the antigen binding molecule is non-inhibitory on the ability of IFNyRI and IFNyR2 expressed on a cell to dimerise (i.e., on IFNy mediated hetero tetramerization on the cell surface of the IFNyRI and IFNyR2 subunits). For instance, the antigen binding molecules may be assessed at concentrations of 200ng / mL in the presence of IFNy at a concentration of 5ng / m. In some embodiments, the antigen binding molecule may be non-inhibitory when assessed using the assay of example 3.

[0117] As well as displaying proinflammatory or anti-tumorigenic effects, it has been shown that IFNy can exert certain anti-inflammatory or pro-tumorigenic effects under certain circumstances, including promoting the expression of PD-L1 , an immune inhibitory receptor ligand that leads to T-cell dysfunction and apoptosis.

[0118] The present inventors have further unexpectedly determined that it is possible to obtain anti-IFNy antibodies which can bias the pro-and anti-inflammatory activities of IFNy in favour of the pro- inflammatory or anti-tumorigenic activities. Thus, in one aspect, the present invention relates to an antigen binding molecule which is capable of binding IFNy and biasing the pro-and anti-inflammatory activities of IFNy in favour of the pro-inflammatory activities. For example, in some embodiments, such molecules may preferentially inhibit the anti-inflammatory activities (i.e., may inhibit the anti-inflammatory activities while inhibiting the pro-inflammatory properties to a lesser degree or while not inhibiting the proinflammatory properties). The molecules may be partially inhibitory for proinflammatory activity as described above (while being more strongly inhibitory for anti-inflammatory activity). Such molecules may be of therapeutic utility e.g., in the treatment of cancer.

[0119] In some embodiments, the pro-inflammatory activity of the cytokine may be measured by the ability of the cytokine to induce expression of major histocompatibility complex class I (MHC-1) on the surface of a cell, e.g., a cell in vitro. In some embodiments, the anti-inflammatory activity of the cytokine may be measured by the ability of the cytokine to induce expression of programmed Cell Death Ligand 1 (PD-L1) on the surface of a cell, e.g., a cell in vitro.

[0120] Accordingly, the present invention may provide an antigen-binding molecule, optionally an antibody, which is capable of binding to interferon gamma (IFNy) and which is capable of modulating cell signalling downstream of IFNy, such that the ratio of PD-L1 expression and / or activity to MHC-1 expression and / or activity which is induced by IFNy is modified in favour of MHC-1 expression and / or activity.

[0121] As an example, the MHC-1 and PD-1 expression may be measured in vitro on a model cell such as a PC3 prostate cancer cell or an A549 lung cancer cell. In one embodiment, a PC3 cell is used for the test assay, e.g, a PC3-FLU cell. In another embodiment, a A549 lung cancer cell is used.

[0122] MHC-1 expression on a cell can be detected using anti-MHC1 antibodies, labelled directly or indirectly.

[0123] 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 may be measured by flow cytometry. PD-L1 expression on a cell may be determined using commercially available antibodies, labelled directly or indirectly, such as directly or indirectly labelled anti-PD-L1 (e.g. clone 29E.2A3, Biolegend, conjugated with PE-Dazzle). It may be measured by flow cytometry. The MHC-1 and PD-1 expression may be cell surface expression, e.g., when measured with flow cytometry.

[0124] In one embodiment, the effect of the antigen binding molecule is assessed in vitro when co-administered to cells with 1 ng / mL IFNy. The assessment may be made 48 hours after the co-administration In one embodiment, it is assessed by contacting a plated test cell as described above, such as an A549 lung cancer cell or PC3 prostate cancer cell, with IFNy at a concentration of 1 ng / mL in the absence of the test molecule and in the presence of the test molecule at a concentration of 1 nM, 10nM or 100nM, preferably 10nM, and assessing MHC-1 expression and / or PD-L1 on the cell surface after 48 hours. In some embodiments, the assessment may be carried out using the method of example 5. (Based on our observations, it does not appear that the cell density is important to the assay, but in some embodiments the cells may be cultured to about 90% or more coverage before the assay is performed).

[0125] In some embodiments, the antigen binding molecule when co-administered with IFNy may be capable of increasing the ratio of MHC-1 to PD-L1 expression as compared to the ratio that is obtained by administering the same amount of IFNy in the absence of the antibody / antigen binding molecule. That is, the ratio of MHC-1 expression to PD-L1 expression may be greater in the presence of the antibody / antigen binding molecule than in its absence, in the presence of the same amount of IFNy.

[0126] (Both expression levels may be reduced in absolute terms, but MHC-1 expression may be reduced less than PD-L1 expression). In some embodiments, when expression values are normalised to those seen in the presence of the same amount of IFNy but in the absence of antibody / test molecule, such that the ratio of MHC-1 to PD-L1 expression in the absence of antibody is 1 (1 :1), an antibody / antigen binding molecule may be capable of increasing the 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, the normalisation may be carried out as described in example 5 (value of the test sample minus the value when no antibody / antigen binding molecule and no IFNy is added (baseline value), divided by the value when only IFNy is added minus the baseline value, i.e., (sample value - baseline value) / (IFNy only value - baseline value) *100.

[0127] In some embodiments, the antigen-binding molecule may be capable of reducing PD-L1 expression in the presence of IFNy (as compared to PD-L1 expression in presence of the same amount of IFNy but the absence of the antigen-binding molecule) by a greater percentage than it reduces MHC-1 expression in the presence of IFNy (as compared to MHC-1 expression in the presence of the same amount of IFNy but in the absence of the antigen-binding molecule). In some embodiments the IFNy-induced expression of MHC-1 is partially inhibited by the antibody / antigen binding molecule at a test concentration of 1 nM, 10nM, or 100mM, i.e., is less than 100% of the amount seen in the absence of antibody / antigen binding molecule. For example, the level of MHC-1 expression may be e.g., greater than 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15% or 20% of that which is seen in the presence of the same amount of IFNy but in the absence of the antigen-binding molecule but less than 100%. It can be envisaged that even low amounts of residual proinflammatory activity may be therapeutic relevant, particularly when combined with targeting and / or accumulation. In some embodiments, the level of MHC-1 expression may be less than 90% or less than 80% or less than 70% or less than 60% or less than 50% of that which is seen in the presence of the same amount of IFNy but in the absence of the antigen-binding molecule.

[0128] In certain embodiments, an antigen-binding molecule of the invention may have functional properties of strongly inhibiting PD-L1 expression to less than 25% of the level seen in the absence of the antibody / antigen binding molecule but in the presence of the same amount of IFNy (e.g., at 1 mM and / or 10mM and / or 100nM of the antigen binding molecule as described above) while maintaining a level of MHC-1 expression that is e.g., at least 50% of that which is seen in the presence of the same amount of IFNy but in the absence of the antigen-binding molecule. Additionally or alternatively, the properties may include maintaining a level of MHC-1 expression that is e.g., at least 50% of that which is seen in the presence of the same amount of IFNy but in the absence of the antigen-binding molecule, e.g., at 1 mM and / or 10mM of the antigen binding molecule as described above and / or even at a 100nM concentration of the antigen-binding molecule.

[0129] Extends the half-life of endogenously occurring IFNy in vivo and / or results in accumulation of endogenously occurring IFNy in vivo

[0130] Exogenously administered recombinant IFNy has an elimination half-life in the bloodstream after intravenous administration of around 38 min in healthy male subjects. The subcutaneous route generally results in a somewhat apparent extended half-life of 5-6 hours due to slow absorption from subcutaneous depot into the blood stream (https: / / www.hzndocs.com / ACTIMMUNE-Prescribing-lnformation.pdf).

[0131] The present inventors have determined that binding of an antigen-binding molecule can increase the halflife of endogenous or exogenously administered (e.g., recombinant) IFNy. This provides a novel approach to increasing the half-life of IFNy in the circulation. The antigen binding molecule may be any antigen binding molecule as disclosed herein, including one which binds to IFNy alone, or one which binds to IFNy and to at least one other antigen, e.g., a bispecific antibody. In such embodiments, it may be preferred that the antigen binding molecule comprises an Fc domain and / or is itself conjugated to another moiety which extends half-life. A variety of moieties which extend half-life are known in the art. For example, the moiety may be selected from a fatty acid, albumin and a hydrophilic polymer such as PEG, polysicalic acid (PSA), N-(2-hydroxypropyl)methacrylamide (HPMA) or dextran

[0132] The antibodies described herein may provide an elevated steady-state concentration of IFNy. In some embodiments, the antibodies may provide improved pharmacokinetics as compared to administration of recombinant IFNy, by providing an elevated steady-state concentration while reducing or avoiding peaks which may be associated with adverse effects and toxicity.

[0133] It may be preferred that the antigen binding molecule has an affinity for IFNy of 500pM or below. Modelling has shown that such affinities can result in accumulation of IFNy in the blood and at the tumour in the case of a tumour targeted antibody.

[0134] The antigen-binding molecule may bind to endogenous IFNy as it is synthesised and secreted into the blood stream. It may protect the IFNy from being degraded and eliminated or otherwise cleared from the circulation. Consequently, INFy may accumulate in the blood. Accumulation at the relevant site may be further assisted in the case of the novel multispecific molecules / bispecific antibodies by the targeting mechanism to reach pharmacological threshold concentration at target cells, e.g. cancer tissue (as discussed further below). Moreover, the antigen-binding molecule may assist in capturing local IFNy release e.g., following administration of a checkpoint inhibitor. For instance, anti-PD1 causes an intratumoral IFN burst. Such an IFNy burst may be prolonged by use of the antigen-binding molecules described herein, particularly in the case of tumour-targeted antibodies, providing an enhanced local IFNy concentration of endogenous IFNy.

[0135] In certain embodiments, an antigen binding molecule as disclosed herein increases the half-life of the IFNy protein by at least 1 .5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11 -fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold as compared to the half-life of the IFNy protein in the absence of the antibody / antigen binding molecule. In certain embodiments, an antibody / antigen binding molecule disclosed herein (e.g., a bispecific antibody) increases the half-life of the IFNy protein by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 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% as compared to the half-life of the IFNy protein in the absence of the antibody / antigen binding molecule. In certain embodiments, the IFNy is endogenous IFNy. The ability of an antibody / antigen binding molecule to increase 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 may be assessed by analysis of plasma concentrations of IFNy in the presence or absence of the antibody in a subject e.g., mice, rats or monkeys. In some embodiments, the ability of an antibody to increase half-life and / or accumulate IFNy may be determined by analysis of plasma levels of IFNy as described in example 4. If the antibody crossreacts to endogenous mouse, rat, or macaque IFNy, the ability of the antibody to accumulate mouse, rat, or monkey (e.g. macaque) IFNy may be assessed. In other embodiments, the ability of an antibody to increase half-life and / or accumulate IFNy may be assessed in the presence of exogenously administered IFNy to which the antibody binds, e.g., exogenously administered human IFNy. In still other embodiments, the test subject may be a human. In some embodiments, the test subject may be a subject not expressing the target antigen for the multispecific antibody. The subject may be a healthy subject, e.g., one not having a cancer expressing the TAA.

[0136] Accordingly, disclosed herein, in certain embodiments, are methods of extending the half-life of endogenous or exogenously administered (e.g., recombinant) IFNy protein in a subject, comprising administering to the subject an antigen binding molecule or a pharmaceutical composition disclosed herein. Preferably the method is a method of extending the half-life of endogenous IFNy.

[0137] Targeting

[0138] The presence of an antigen-binding domain that binds to a target antigen such as a tumour associated antigen (TAA) may be used to target the antigen binding molecule (and hence the IFNy associated with the antigen binding molecule) to a cell or tissue expressing the target antigen, such as a tumour or cancer cell / tissue in which the TAA is expressed.

[0139] This may assist in harnessing the proinflammatory and / or anti-tumour effects of IFNy at the target tissue, by achieving relatively high concentrations at the target tissue, while having a lower IFNy concentration in the blood stream to control toxicity. In the case where the antigen binding molecule partially inhibits the proinflammatory effects of IFNy, the accumulation at the target site can also serve to increase activity at the target site by virtue of higher concentration and / or avidity effects, while again controlling systemic inflammatory side effects.

[0140] Accordingly, the antigen-binding molecule described herein may be part of a multispecific or bispecific antibody also comprising an antigen binding site for a target antigen such as a TAA, as discussed further herein. Also disclosed herein, in certain embodiments, are methods of increasing localization (e.g., targeting) of an IFNy protein to cancer cells / tissues in a subject (e.g., a human), 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.

[0141] Valency for IFNy

[0142] It may be preferred that the antigen binding molecule is monovalent for IFNy. For example, an antibody may have a single binding site for IFNy present on one arm of an IgG. The present inventors have unexpectedly determined that the affinity for IFNy is significantly increased when the antibody is monovalent for IFNy. This has been observed for distinct IFNy binding sequences, i.e., it is not dependent on the sequence of the IFNy binder.

[0143] This format may be useful for both monospecific and multispecific (e.g., bispecific) molecules. In the context of multispecific antigen binding molecules, e.g., bispecific antibodies, the fact that monovalency for IFNy allows for unexpectedly good affinity means that formats with only two binding sites can be used, e.g., IgG formats in which each arm of the antibody binds a different antigen. In the context of monovalent antibodies, “one-armed” IgG formats can be used. One armed formats typically comprise one full length heavy chain and one heavy chain which consists of an Fc subunit (e.g., consists of Hinge- CH2-CH3 regions).

[0144] Exemplary Antigen Binding Molecules - IFNy binding domains

[0145] Exemplary antigen binding molecules comprising an IFNy binding domain according to the present disclosure are provided below. In some embodiments, the antigen binding molecules may be multispecific (e.g., bispecific), e.g., multispecific or bispecific antibodies comprising an IFNy binding domain as described herein. In other embodiments the antigen binding molecules may bind to IFNy alone, i.e., may be monospecific.

[0146] The antigen-binding molecules of the present disclosure may comprise an IFNy-binding domain comprising a VH and a VL of an antibody capable of specific binding to IFNy. The antigen-binding domain formed by a VH and a VL may also be referred to herein as an Fv region.

[0147] In some embodiments, the antigen binding molecules may comprise a single IFNy binding domain, as described above. Embodiments comprising a single IFNy binding domain may be preferred. In other embodiments, the antigen binding molecules may comprise more than one IFNy binding domain. For instance, the antigen binding molecule may comprise two such domains, as is the case for example for a conventional IgG. In some embodiments, the antigen-binding molecule comprises the CDRs, FRs and / or the VH and / or VL regions of a IFNy-binding antibody clone described herein, or CDRs, FRs and / or VH and / or VL regions which are derived from those of a IFNy-binding antibody clone described herein. In some embodiments, a IFNy-binding antibody clone is selected from: 4F4; 16D2; 3G1 ; 9F1 ; or 2C1.

[0148] 4F4 and antibodies derived therefrom

[0149] In an embodiment, the present disclosure relates to an antigen binding molecule comprising an IFNy binding domain, wherein said IFNy binding domain comprises a heavy chain variable region comprising HC-CDR1 , HC-CDR2 and HC-CDR3, wherein: a) heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 4 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR1 are substituted with another amino acid; b) heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 5 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR2 are substituted with another amino acid; c) heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 6 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR3 are substituted with another amino acid; and / or wherein the IFNy binding domain comprises a light chain variable region comprising LC-CDR1 , LC-CDR2 and LC-CDR3, wherein: d) light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 7 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR1 are substituted with another amino acid; e) light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 8 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR2 are substituted with another amino acid; f) light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 9 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR3 are substituted with another amino acid.

[0150] In some embodiments, the antigen binding molecule comprises an IFNy binding domain, wherein said IFNy binding domain comprises a heavy chain variable region comprising HC-CDR1 , HC-CDR2 and HC- CDR3, wherein: a) heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO:4 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR1 are substituted with another amino acid; b) heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO:5 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR2 are substituted with another amino acid; c) heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 6; and / or wherein the IFNy binding domain comprises a light chain variable region comprising LC-CDR1 , LC-CDR2 and LC-CDR3, wherein: d) light chain CDR1 comprises the amino acid sequence of SEQ ID NO:7 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR1 are substituted with another amino acid; e) light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 8 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR2 are substituted with another amino acid; f) light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 9.

[0151] In some embodiments, heavy chain CDR1 comprises a variant of SEQ ID NO: 4 with a substitution at position 30 numbered according to the IMGT scheme (i.e., GYTXTNYY (SEQ ID NO: 84), where X can be any amino acid), wherein the residue at this position is preferably D, E, N or Q, most preferably E. For instance, the heavy chain CDR1 may be selected from GYTFTNYY (SEQ ID NO: 4) and GYTETNYY (SEQ ID NO: 85).

[0152] In another embodiment, heavy chain CDR2 comprises a variant of SEQ ID NO: 5 with a substitution at position 63 numbered according to the IMGT scheme (i.e., INPSNXGT (SEQ ID NO: 86), where X can be any amino acid and is preferably other than D), wherein the residue at this position is preferably E, H or Q, most preferably H. Removing D at this position removes a potential isomerisation site, since the residue pair DG forms a potential isomerisation sequence liability. For instance, the heavy chain CDR2 may be selected from INPSNDGT (SEQ ID NO: 5) and INPSNHGT (SEQ ID NO: 87).

[0153] In another embodiment, light chain CDR1 comprises a variant of SEQ ID NO: SEQ ID NO: 7 with substitutions at one or both of positions 29 and 35, numbered according to the IMGT scheme (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 is most preferably V and / or the residue at position 35 is preferably selected from D, E, N or Q and is most preferably N. For instance, the LC-CDR1 may be selected from QSLLYSSNQKNY (SEQ ID NO: 7) and QSVLYSSNNKNY (SEQ ID NO: 89).

[0154] In one exemplary embodiment, the IFNy binding domain may comprise a heavy chain variable region comprising HC-CDR1 , HC-CDR2 and HC-CDR3, wherein: a) heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 4; b) heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 87; c) heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 6; and comprises a light chain variable region comprising LC-CDR1 , LC-CDR2 and LC-CDR3, wherein: d) light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 7; e) light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 8; f) light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 9. In one exemplary embodiment, the IFNy binding domain may comprise a heavy chain variable region comprising HC-CDR1 , HC-CDR2 and HC-CDR3, wherein: a) heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 85; b) heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 87; c) heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 6; and comprises a light chain variable region comprising LC-CDR1 , LC-CDR2 and LC-CDR3, wherein: d) light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 7; e) light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 8; f) light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 9.

[0155] In one exemplary embodiment, the IFNy binding domain may comprise a heavy chain variable region comprising HC-CDR1 , HC-CDR2 and HC-CDR3, wherein: a) heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 4; b) heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 87; c) heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 6; and comprises a light chain variable region comprising LC-CDR1 , LC-CDR2 and LC-CDR3, wherein: d) light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 89; e) light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 8; f) light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 9.

[0156] In another exemplary embodiment, the IFNy binding domain may comprise a heavy chain variable region comprising HC-CDR1 , HC-CDR2 and HC-CDR3, wherein: a) heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 85; b) heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 87; c) heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 6; and comprises a light chain variable region comprising LC-CDR1 , LC-CDR2 and LC-CDR3, wherein: d) light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 89; e) light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 8; f) light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 9.

[0157] In some embodiments, the antibody / antigen binding molecule comprises a VH region having at least at least 70% sequence identity, optionally at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:10 and / or a VL region having at least at least 70% sequence identity, optionally at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO: 11. In some embodiments, said antibody / antigen binding molecule comprises the CDRs as set out above (i.e. , any substitutions are in the framework regions). Optionally the substitutions in VH and / or VL (e.g., in the framework region) may be with the residue found in the corresponding position of a human germline antibody sequence.

[0158] In some embodiments, the antibody is a humanised antibody, for instance comprising the CDRs as set out above and human framework regions, i.e., the 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 in which 1-5 amino acids in HC-FR1 , and / or in which 1-5 amino acids in HC-FR2, and / or in which 1-5 amino acids in HC-FR3, and / or in which 1-5 amino acids in HC-FR4 and / or in which 1-5 amino acids in LC-FR1 , and / or in which 1-5 amino acids in LC-FR2, and / or in which 1-5 amino acids in LC-FR3, and / or in which 1-5 amino acids in LC-FR4 are substituted with another amino acid. In some embodiments, the substitution may be a back- mutation to the residue in the corresponding position of 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 embodiments, the total number of substitutions made to the human framework regions may be 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6 or 1 to 5; e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0159] Exemplary humanised antibodies comprise a heavy chain variable region comprising HC-FR1 , HC-FR2, HC-FR3 and HC-FR4, wherein:

[0160] HC-FR1 has a sequence selected from SEQ ID NO: 90, SEQ ID NO: 98 and SEQ ID NO: 102; HC-FR2 has a sequence set forth in SEQ ID NO: 91 ;

[0161] HC-FR3 has a sequence selected from SEQ ID NO: 92, SEQ ID NO: 99, SEQ ID NO: 103 and SEQ ID NO: 105; and

[0162] HC-FR4 has a sequence selected from SEQ ID NO: 93 and SEQ ID NO: 100; and / or comprise a light chain variable region comprising LC-FR1 , LC-FR2 and LC-FR3 and LC-FR4, wherein: LC-FR1 has a sequence selected from SEQ ID NO: 94 and SEQ ID NO: 101 ;

[0163] LC-FR2 has a sequence selected from SEQ ID NO: 95 and SEQ ID NO: 106;

[0164] LC-FR3 has a sequence selected from SEQ ID NO: 96 and SEQ ID NO: 104; and

[0165] LC-FR4 has a sequence set forth in SEQ ID NO: 97.

[0166] These framework regions may be combined with CDRs as described above.

[0167] An exemplary antibody / antigen binding molecule may have a 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 thereto and a 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 thereto. In one embodiment, an 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, an antigenbinding 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, an 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, an 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, an 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, an 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, an 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, an 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, an antigenbinding 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.

[0168] Any an antigen-binding molecule derived from 4F4 (i.e. , any of the antibodies / antigen-binding molecules set out above) may have any of the properties as set out above, e.g., may be partially or preferentially inhibiting as set out above, may extend the half-life of endogenously occurring IFNy in vivo and / or result in accumulation of endogenously occurring IFNy in vivo and / or may be provided as part of a multispecific antibody also comprising a binding site for a target molecule such as a TAA.

[0169] Any antigen-binding molecule derived from 4F4 (i.e., any of the antibodies / antigen-binding molecules set out above) may retain affinity for IFNy as described herein, e.g., may bind to IFNy, e.g, human and / or primate IFNy, with sub-micromolar affinity, i.e. KD < 1 x 10-6M. In some embodiments, the antigen-binding molecule described herein binds to IFNy with an affinity in the nanomolar range, i.e. KD = 9.9 x 10-7to 1 x 10-9M. In some embodiments, the antigen-binding molecule described herein binds to IFNy with sub- nanomolar affinity, i.e. KD < 1 x 10-9M. In some embodiments, the antigen-binding molecule described herein binds to IFNy with an affinity in the picomolar range, i.e. KD = 9.9 x 10-10to 1 x 10-12M. In some embodiments, the antigen-binding molecule described herein binds to IFNy with an affinity in the range KD = 9.9 x 10-1° to 1 x 10-13M. In some embodiments, the antigen-binding molecule described herein binds to IFNy with sub-picomolar affinity, i.e. KD < 1 x 10-12M. In some embodiments, the antibodies bind to human IFNy with a KD of less than 20nM, less than 10nM or less than 5nM. In a further embodiment, the present disclosure provides an antibody / antigen-binding molecule which 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 as set out above, e.g., may be partially or preferentially inhibiting as set out above. Any such antigen-binding molecule may have an affinity for IFNy as set out herein. Any such antigen-binding molecule may extend the half-life of endogenously occurring IFNy in vivo and / or result in accumulation of endogenously occurring IFNy in vivo and / or may be provided as part of a multispecific antibody also comprising a binding site for a target molecule such as a TAA.

[0170] 2C1 and antibodies derived therefrom

[0171] In an embodiment, the present disclosure relates to an IFNy binding domain comprising an antigen binding site specific for IFNy, wherein said an IFNy binding domain comprises a heavy chain variable region comprising HC-CDR1 , HC-CDR2 and HC-CDR3, wherein: a) heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 12 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR1 are substituted with another amino acid; b) heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 13 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR2 are substituted with another amino acid; c) heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 14 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR3 are substituted with another amino acid; and / or wherein the IFNy binding domain comprises a light chain variable region comprising LC- CDR1 , LC-CDR2 and LC-CDR3, wherein: d) light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 15 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR1 are substituted with another amino acid; e) light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 16 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR2 are substituted with another amino acid; f) light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 17 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR3 are substituted with another amino acid.

[0172] In some embodiments, the antigen binding molecule comprises an IFNy binding domain, wherein said antigen binding site comprises a heavy chain variable region comprising HC-CDR1 , HC-CDR2 and HC- CDR3, wherein: a) heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO:12 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR1 are substituted with another amino acid; b) heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO:13 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR2 are substituted with another amino acid; c) heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 14; and / or wherein the antigen binding site comprises a light chain variable region comprising LC-CDR1 , LC- CDR2 and LC-CDR3, wherein: d) light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 15 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR1 are substituted with another amino acid; e) light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 16 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR2 are substituted with another amino acid; f) light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 17.

[0173] In some embodiments, the antibody / antigen-binding molecule comprises a VH region having at least at least 70% sequence identity, optionally at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:18 and / or a VL region having at least at least 70% sequence identity, optionally at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:19. In some embodiments, said antibody / antigen-binding molecule comprises the CDRs as set out above (i.e., any substitutions are in the framework regions). Optionally the substitutions in VH and / or VL (e.g., in the framework region) may be with the residue found in the corresponding position of a human germline antibody sequence.

[0174] In some embodiments, the antibody / antigen-binding molecule is a humanised antibody, for instance comprising the CDRs as set out above and human framework regions, i.e., the 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 in which 1-5 amino acids in HC-FR1 , and / or in which 1-5 amino acids in HC-FR2, and / or in which 1 -5 amino acids in HC-FR3, and / or in which 1 -5 amino acids in HC-FR4 and / or in which 1-5 amino acids in LC-FR1 , and / or in which 1-5 amino acids in LC-FR2, and / or in which 1-5 amino acids in LC-FR3, and / or in which 1-5 amino acids in LC-FR4 are substituted with another amino acid. In some embodiments, the substitution may be a back-mutation to the residue in the corresponding position of SEQ ID NO 18 or 19. In some embodiments, the number of substitutions in each FR region may be 1 , 2, or 3. In some embodiments, the total number of substitutions made to the human framework regions may be 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6 or 1 to 5; e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0175] Any antibody / antigen-binding molecule derived from 2C1 (i.e., any of the antibodies set out above) , may have any of the properties as set out above, e.g., may be partially or preferentially inhibiting as set out above, may extend the half-life of endogenously occurring IFNy in vivo and / or result in accumulation of endogenously occurring IFNy in vivo and / or may be provided as part of a multispecific antibody / antigen- binding molecule also comprising a binding site for a target molecule such as a TAA.. Any antibody / antigen-binding molecule derived from 2C1 (i.e., any of the antibodies set out above) may retain affinity for IFNy as described herein, e.g., may binds to IFNy, e.g, human and / or primate I FNy, with an affinity in the micromolar range, i.e. KD = 9.9 x 10-4to 1 x 10-6M. In some embodiments, the antigenbinding molecule described herein binds to IFNy with sub-micromolar affinity, i.e. KD < 1 x 10-6M. In some embodiments, the antigen-binding molecule described herein binds to IFNy with an affinity in the nanomolar range, i.e. KD = 9.9 x 10-7to 1 x 10-9M. In some embodiments, the antigen-binding molecule described herein binds to IFNy with sub-nanomolar affinity, i.e. KD < 1 x 10-9M. In some embodiments, the antigen-binding molecule described herein binds to IFNy with an affinity in the picomolar range, i.e. KD = 9.9 x 10-1° to 1 x 10-12M. In some embodiments, the antigen-binding molecule described herein binds to IFNy with sub-picomolar affinity, i.e. KD < 1 x 10-12M. In some embodiments, the antibodies bind to human IFNy with a KD of less than 20nM, less than 10nM or less than 5nM.

[0176] In a further embodiment, the present disclosure provides an antibody / antigen-binding molecule which 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 as set out above, e.g., may be partially or preferentially inhibiting as set out above. Any such antibody / antigen- binding molecule may have affinity for IFNy as set out herein. Any such antibody may extend the half-life of endogenously occurring IFNy in vivo and / or result in accumulation of endogenously occurring IFNy in vivo and / or may be provided as part of a multispecific antibody / antigen-binding molecule also comprising a binding site for a target molecule such as a TAA.

[0177] 3G1 and antibodies derived therefrom

[0178] In an embodiment, the present disclosure relates to an antigen binding molecule comprising an IFNy binding domain, wherein said an IFNy binding domain comprises a heavy chain variable region comprising HC-CDR1 , HC-CDR2 and HC-CDR3, wherein: a) heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 20 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR1 are substituted with another amino acid; b) heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 21 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR2 are substituted with another amino acid; c) heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 22 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR3 are substituted with another amino acid; and / or wherein the IFNy binding domain comprises a light chain variable region comprising LC- CDR1 , LC-CDR2 and LC-CDR3, wherein: d) light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 23 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR1 are substituted with another amino acid; e) light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 24 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR2 are substituted with another amino acid; f) light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 25 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR3 are substituted with another amino acid.

[0179] In some embodiments, the antigen binding molecule comprises an IFNy binding domain, wherein said an IFNy binding domain comprises a heavy chain variable region comprising HC-CDR1 , HC-CDR2 and HC- CDR3, wherein: a) heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO:20 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR1 are substituted with another amino acid; b) heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO:21 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR2 are substituted with another amino acid; c) heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 22; and / or wherein the antigen binding site comprises a light chain variable region comprising LC-CDR1 , LC- CDR2 and LC-CDR3, wherein: d) light chain CDR1 comprises the amino acid sequence of SEQ ID NO:23 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR1 are substituted with another amino acid; e) light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 24 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR2 are substituted with another amino acid; f) light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 25.

[0180] In some embodiments, the antibody / antigen-binding molecule comprises a VH region having at least at least 70% sequence identity, optionally at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:26 and / or a VL region having at least at least 70% sequence identity, optionally at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:27. In some embodiments, said antibody / antigen-binding molecule comprises the CDRs as set out above (i.e., any substitutions are in the framework regions). Optionally the substitutions in VH and / or VL (e.g., in the framework region) may be with the residue found in the corresponding position of a human germline antibody sequence.

[0181] In some embodiments, the antibody / antigen-binding molecule is a humanised antibody / antigen-binding molecule, for instance comprising the CDRs as set out above and human framework regions, i.e., the 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 in which 1-5 amino acids in HC-FR1 , and / or in which 1-5 amino acids in HC-FR2, and / or in which 1 -5 amino acids in HC-FR3, and / or in which 1 -5 amino acids in HC-FR4 and / or in which 1-5 amino acids in LC-FR1 , and / or in which 1-5 amino acids in LC-FR2, and / or in which 1-5 amino acids in LC-FR3, and / or in which 1-5 amino acids in LC-FR4 are substituted with another amino acid. In some embodiments, the substitution may be a back-mutation to the residue in the corresponding position of SEQ ID NO 26 or 27. In some embodiments, the number of substitutions in each FR region may be 1 , 2, or 3. In some embodiments, the total number of substitutions made to the human framework regions may be 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6 or 1 to 5; e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0182] Any antibody / antigen-binding molecule derived from 3G1 (i.e., any of the antibodies set out above) may have any of the properties as set out above, e.g., may be partially or preferentially inhibiting as set out above, may extend the half-life of endogenously occurring IFNy in vivo and / or result in accumulation of endogenously occurring IFNy in vivo and / or may be provided as part of a multispecific antibody / antigen- binding molecule also comprising a binding site for a target molecule such as a TAA..

[0183] Any antibody / antigen-binding molecule derived from 3G1 (i.e., any of the antibodies set out above) may retain affinity for IFNy as described herein, e.g., may bind to IFNy, e.g, human and / or primate IFNy, with an affinity in the micromolar range, i.e. KD = 9.9 x 10-4to 1 x 10-6M. In some embodiments, the antigenbinding molecule described herein binds to IFNy with sub-micromolar affinity, i.e. KD < 1 x 10-6M. In some embodiments, the antigen-binding molecule described herein binds to IFNy with an affinity in the nanomolar range, i.e. KD = 9.9 x 10-7to 1 x 10-9M. In some embodiments, the antigen-binding molecule described herein binds to IFNy with sub-nanomolar affinity, i.e. KD < 1 x 10-9M. In some embodiments, the antigen-binding molecule described herein binds to IFNy with an affinity in the picomolar range, i.e. KD = 9.9 x 10-1° to 1 x 10-12M. In some embodiments, the antigen-binding molecule described herein binds to IFNy with sub-picomolar affinity, i.e. KD < 1 x 10-12M. In some embodiments, the antibodies bind to human IFNy with a KD of less than 20nM, less than 10nM or less than 5nM.

[0184] In a further embodiment, the present disclosure provides an antibody / antigen-binding molecule which 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 as set out above, e.g., may be partially or preferentially inhibiting as set out above. Any such antibody / antigen- binding molecule may have affinity for IFNy as set out herein. Any such antibody / antigen-binding molecule may extend the half-life of endogenously occurring IFNy in vivo and / or result in accumulation of endogenously occurring IFNy in vivo and / or may be provided as part of a multispecific antibody / antigen- binding molecule also comprising a binding site for a target molecule such as a TAA.

[0185] 9F1 and antibodies derived therefrom In an embodiment, the present disclosure relates to an antigen binding molecule comprising an IFNy binding domain, wherein said antigen binding site comprises a heavy chain variable region comprising HC-CDR1 , HC-CDR2 and HC-CDR3, wherein: a) heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 28 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR1 are substituted with another amino acid; b) heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 29 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR2 are substituted with another amino acid; c) heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 30 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR3 are substituted with another amino acid; and / or wherein the IFNy binding domain comprises a light chain variable region comprising LC- CDR1 , LC-CDR2 and LC-CDR3, wherein: d) light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 31 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR1 are substituted with another amino acid; e) light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 32 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR2 are substituted with another amino acid; f) light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 33 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR3 are substituted with another amino acid.

[0186] In some embodiments, the antigen binding molecule comprises an IFNy binding domain, wherein said antigen binding site comprises a heavy chain variable region comprising HC-CDR1 , HC-CDR2 and HC- CDR3, wherein: a) heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO:28 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR1 are substituted with another amino acid; b) heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO:29 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR2 are substituted with another amino acid; c) heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 30; and / or wherein the IFNy binding domain comprises a light chain variable region comprising LC-CDR1 , LC-CDR2 and LC-CDR3, wherein: d) light chain CDR1 comprises the amino acid sequence of SEQ ID NO:31 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR1 are substituted with another amino acid; e) light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 32 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR2 are substituted with another amino acid; f) light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 33.

[0187] In some embodiments, the antibody / antigen-binding molecule comprises a VH region having at least at least 70% sequence identity, optionally at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:34 and / or a VL region having at least at least 70% sequence identity, optionally at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:35. In some embodiments, said antibody / antigen-binding molecule comprises the CDRs as set out above (i.e., any substitutions are in the framework regions). Optionally the substitutions in VH and / or VL (e.g., in the framework region) may be with the residue found in the corresponding position of a human germline antibody sequence.

[0188] In some embodiments, the antibody / antigen-binding molecule is a humanised antibody / antigen-binding molecule, for instance comprising the CDRs as set out above and human framework regions, i.e., the 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 in which 1-5 amino acids in HC-FR1 , and / or in which 1-5 amino acids in HC-FR2, and / or in which 1 -5 amino acids in HC-FR3, and / or in which 1 -5 amino acids in HC-FR4 and / or in which 1-5 amino acids in LC-FR1 , and / or in which 1-5 amino acids in LC-FR2, and / or in which 1-5 amino acids in LC-FR3, and / or in which 1-5 amino acids in LC-FR4 are substituted with another amino acid. In some embodiments, the substitution may be a back-mutation to the residue in the corresponding position of SEQ ID NO 34 or 35. In some embodiments, the number of substitutions in each FR region may be 1 , 2, or 3. In some embodiments, the total number of substitutions made to the human framework regions may be 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6 or 1 to 5; e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0189] Any antibody / antigen-binding molecule derived from 9F1 (i.e., any of the antibodies set out above) may have any of the properties as set out above, e.g., may be partially or preferentially inhibiting as set out above, may extend the half-life of endogenously occurring IFNy in vivo and / or result in accumulation of endogenously occurring IFNy in vivo and / or may be provided as part of a multispecific antibody / antigen- binding molecule also comprising a binding site for a target molecule such as a TAA.

[0190] Any antibody / antigen-binding molecule derived from 9F1 (i.e., any of the antibodies set out above) may retain affinity for IFNy as described herein, e.g., may bind to IFNy, e.g, human and / or primate IFNy, with an affinity in the micromolar range, i.e. KD = 9.9 x 10-4to 1 x 10-6M. In some embodiments, the antigenbinding molecule described herein binds to IFNy with sub-micromolar affinity, i.e. KD < 1 x 10-6M. In some embodiments, the antigen-binding molecule described herein binds to IFNy with an affinity in the nanomolar range, i.e. KD = 9.9 x 10-7to 1 x 10-9M. In some embodiments, the antigen-binding molecule described herein binds to IFNy with sub-nanomolar affinity, i.e. KD < 1 x 10-9M. In some embodiments, the antigen-binding molecule described herein binds to IFNy with an affinity in the picomolar range, i.e. KD = 9.9 x 10-1° to 1 x 10-12M. In some embodiments, the antigen-binding molecule described herein binds to IFNy with sub-picomolar affinity, i.e. KD < 1 x 10-12M. In some embodiments, the antibodies bind to human IFNy with a KD of less than 20nM, less than 10mM, less than 5nM or less than 1 nM. In a further embodiment, the present disclosure provides an antibody / antigen-binding molecule which 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 as set out above e.g., may be partially or preferentially inhibiting as set out above. Any such antibody may have affinity for IFNy as set out herein. Any such antibody / antigen-binding molecule may extend the half-life of endogenously occurring IFNy in vivo and / or result in accumulation of endogenously occurring IFNy in vivo and / or may be provided as part of a multispecific antibody / antigen-binding molecule also comprising a binding site for a target molecule such as a TAA.

[0191] In some embodiments, an antibody / antigen-binding molecule derived from 9F1 or binding to the same epitope as 9F1 may have one or more of the following properties: i) strongly inhibiting PD-L1 expression to less than 25% of the level seen in the absence of the antibody but in the presence of the same amount of IFNy (e.g., at 1 mM and / or 10mM and / or 100nM of the antigen binding molecule as described above) while maintaining a level of MHC-1 expression that is e.g., at least 50% of that which is seen in the presence of the same amount of IFNy but in the absence of the antigen-binding molecule, e.g., in the assay of example 5; ii) maintaining a level of MHC-1 expression that is e.g., at least 50% of that which is seen in the presence of the same amount of IFNy but in the absence of the antigen-binding molecule, e.g., at 1 mM and / or 10mM of the antigen binding molecule as described above and / or even at a 100nM concentration of the antigen-binding molecule, e.g., in the assay of example 5; and / or iii) inhibiting the IFNy mediated hetero tetramerization of the IFNyRI and IFNyR2 subunits, e.g., at a concentration of 1 nM, e.g., in the assay described in example 3.

[0192] 16D2 and antibodies derived therefrom

[0193] In an embodiment, the present disclosure relates to an antigen binding molecule comprising an IFNy binding domain, wherein said an IFNy binding domain comprises a heavy chain variable region comprising HC-CDR1 , HC-CDR2 and HC-CDR3, wherein: a) heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 36 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR1 are substituted with another amino acid; b) heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 37 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR2 are substituted with another amino acid; c) heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 38 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR3 are substituted with another amino acid; and / or wherein the IFNy binding domain comprises a light chain variable region comprising LC- CDR1 , LC-CDR2 and LC-CDR3, wherein: d) light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 39 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR1 are substituted with another amino acid; e) light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 40 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR2 are substituted with another amino acid; f) light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 41 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR3 are substituted with another amino acid.

[0194] In some embodiments, the antigen binding molecule comprises an IFNy binding domain, wherein said an IFNy binding domain comprises a heavy chain variable region comprising HC-CDR1 , HC-CDR2 and HC- CDR3, wherein: a) heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO:36 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR1 are substituted with another amino acid; b) heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO:37 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR2 are substituted with another amino acid; c) heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 38; and / or wherein the IFNy binding domain comprises a light chain variable region comprising LC-CDR1 , LC-CDR2 and LC-CDR3, wherein: d) light chain CDR1 comprises the amino acid sequence of SEQ ID NO:39 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR1 are substituted with another amino acid; e) light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 40 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR2 are substituted with another amino acid; f) light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 41 .

[0195] In some embodiments, the antibody / antigen-binding molecule comprises a VH region having at least at least 70% sequence identity, optionally at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:42 and / or a VL region having at least at least 70% sequence identity, optionally at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:43. In some embodiments, said antibody / antigen-binding molecule comprises the CDRs as set out above (i.e., any substitutions are in the framework regions). Optionally the substitutions in VH and / or VL (e.g., in the framework region) may be with the residue found in the corresponding position of a human germline antibody / antigen-binding molecule sequence. In some embodiments, the antibody / antigen-binding molecule is a humanised antibody / antigen-binding molecule, for instance comprising the CDRs as set out above and human framework regions, i.e., the 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 in which 1-5 amino acids in HC-FR1 , and / or in which 1-5 amino acids in HC-FR2, and / or in which 1 -5 amino acids in HC-FR3, and / or in which 1 -5 amino acids in HC-FR4 and / or in which 1-5 amino acids in LC-FR1 , and / or in which 1-5 amino acids in LC-FR2, and / or in which 1-5 amino acids in LC-FR3, and / or in which 1-5 amino acids in LC-FR4 are substituted with another amino acid. In some embodiments, the substitution may be a back-mutation to the residue in the corresponding position of SEQ ID NO: 42 or 43. In some embodiments, the number of substitutions in each FR region may be 1 , 2, or 3. In some embodiments, the total number of substitutions made to the human framework regions may be 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6 or 1 to 5; e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0196] Any antibody / antigen-binding molecule derived from 16D2 (i.e., any of the antibodies set out above) may have the properties as set out above, e.g may be partially or preferentially inhibiting as set out above, may extend the half-life of endogenously occurring IFNy in vivo and / or result in accumulation of endogenously occurring IFNy in vivo and / or may be provided as part of a multispecific antibody / antigen- binding molecule also comprising a binding site for a target molecule such as a TAA.

[0197] Any antibody / antigen-binding molecule derived from 16D2 (i.e., any of the antibodies set out above) may retain affinity for IFNy as described herein, e.g., may bind to IFNy, e.g, human and / or primate IFNy, with an affinity in the micromolar range, i.e. KD = 9.9 x 10-4to 1 x 10-6M. In some embodiments, the antigenbinding molecule described herein binds to IFNy with sub-micromolar affinity, i.e. KD < 1 x 10-6M. In some embodiments, the antigen-binding molecule described herein binds to IFNy with an affinity in the nanomolar range, i.e. KD = 9.9 x 10-7to 1 x 10-9M. In some embodiments, the antigen-binding molecule described herein binds to IFNy with sub-nanomolar affinity, i.e. KD < 1 x 10-9M. In some embodiments, the antigen-binding molecule described herein binds to IFNy with an affinity in the picomolar range, i.e. KD = 9.9 x 10-10to 1 x 10-12M. In some embodiments, the antigen-binding molecule described herein binds to IFNy with sub-picomolar affinity, i.e. KD < 1 x 10-12M. In some embodiments, the antibodies bind to human IFNy with a KD of less than 20nM, less than 10mM or less than 5nM.

[0198] In a further embodiment, the present disclosure provides an antibody / antigen-binding molecule which 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 properties as set out above, e.g., may be partially or preferentially inhibiting as set out above. Any such antibody / antigen-binding molecule may have affinity for IFNy as set out herein. Any such antibody / antigen-binding molecule may extend the half-life of endogenously occurring IFNy in vivo and / or result in accumulation of endogenously occurring IFNy in vivo and / or may be provided as part of a multispecific antibody / antigen-binding molecule also comprising a binding site for a target molecule such as a TAA.

[0199] Exemplary Antigen Binding Molecules - PSMA binding domain

[0200] Any of the IFNy binding molecules as discussed herein may be present as part of a multispecific (e.g., bispecific) molecule which also binds to a TAA. Numerous TAAs are known in the art and antigenbinding molecules for these are widely known and are available to the skilled person.

[0201] In one specific embodiment, the TAA may be prostate-specific membrane antigen (PSMA). Exemplary known antibodies capable of binding PSMA include mAb 7E11-C5.3, capromab, ANT4044 (VH and VL shown as SEQ ID NO: 59 and 60; heavy chain and light chain shown as 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 , 3 / E6. In some embodiments, a multispecific (bispecific) antigen binding molecule which is capable of binding PSMA may comprise the VH and VL of any said antibody, or a variant thereof comprise the CDRs of said antibody and a VH or VL domain with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto. For instance, antibody J591 , is described in W02004 / 098535 and WO2017212250 (the VH and VL domain of a deimmunised antibody is 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 .

[0202] In some embodiments, the multispecific molecule as described herein may comprise an ScFv derived from J591 or ANT4044, by fusing said VH and VL domains or variants thereof with a linker.

[0203] Another exemplary known binder for PSMA is a nanobody as described in WO2021 / 038571 A1 , e.g., nanobody Nb7, Nb8, Nb13 or Nb37. In some embodiments, the PSMA binder may be VHH comprising CDRs 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 RRDYSLSSSSDDFDY (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).

[0204] In one embodiment, CDR-H2 may comprise the sequence GISSTMSGIIFAESKAGQFTISQDNA; SEQ ID NO:53.

[0205] In some embodiments, the PSMA-binding polypeptide may comprise one of the following sequences: QVQLQESGGG SVQAGGSLRL SCTAPGYTDS NYYMSWFRQA PGKEREWVAG

[0206] VNTGRGSTSY ADSVKGRFTI SQDNAKNTMF LQMNSLKPED TAIYYCAVAA

[0207] CHFCDSLPKT QDEYILWGQG TQVTVSSAAA YPYDVPDYGS (Nb7; SEQ ID NO: 54)

[0208] QVQLQESGGG SVQAGGSLRL SCARSGWPYS TYSMNWFRQA PGKEREAVAG ISSTMSGIIF AESKAGQFTI SQDNAKNTVY LQMNNLKPED TAIYYCAARR DYSLSSSSDD FDYWGQGTQV TVSSAAAYPY DVPDYGS (SEQ ID NO: 55)

[0209] QVQLQESGGG SVQTGGSLRL SCAASGYTAS FSWIGYFRQA PGKEREGVAV INVGVGSTYY ADSVKGRFTI SRDNTENTIS LEMNSLKPED TGLYYCAGSL

[0210] RWSRPPNPIS EDAYNYWGQG TQVTVSSAAA YPYDVPDYGS (SEQ ID NO: 56)

[0211] QVQLQESGGG SVEAGGSLRL SCARSGWPYS TYSMNWFRQA PGKEREAVAG ISSTMSGIIF AESKAGQFTI SQDNAKNTVY LQMNNLKPED TAIYYCAARR DYSLSSSSDD FDYWGQGTQV TVSSAAAYPY DVPDYGS (SEQ ID NO: 57)

[0212] Nb7 has picomolar binding affinity to PSMA and may be preferred in some embodiments.

[0213] In some embodiments, the PSMA-binding antibody may comprise a sequence with at least 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 54, 55 or 57, optionally while retaining the CDRs as set out above. In some embodiments, the sequence may be humanised. In some embodiments, the humanised antibody may bind to PSMA with a KD no more than 10 fold greater than the KD of the parent antibody. In some embodiments, the affinity for PSMA may be in the picomolar range, e.g., KD= 9.9X1 O-10to 1x10-12M.

[0214] Exemplary Antigen Binding Molecules - B7-H3

[0215] In another specific embodiment, the TAA may be B7-H3 (also known as CD276).

[0216] In one embodiment, the antigen binding moiety for B7-H3 may be an scFv. Exemplary scFvs capable of binding B7-H3 are described in WQ2012 / 081052 A1 and in Li et al Mature Communications (2023) 14:5920.

[0217] In one embodiment, the B7-H3 binding domain, e.g., scFv, comprises CDRs of the following sequences: i) GFTYNSYS (HC-CDR1 ; SEQ ID NO: 119) ii) INSGGSST (HC-CDR2; SEQ ID NO: 120) iii) AARSPSPLTFQTRTLREDSYNYW (HC-CDR3; SEQ ID NO: 121). In one embodiment, the B7-H3 binding domain is a scFv of the following sequence:

[0218] QVQLVESGGGSVQVGGSLRLSCAASGFTYNSYSVGWFRQAPGKEREGVAAINSGGSSTYYAASVKGRF TISRDNAKNTVYLQMNSLKPEDTAMYYCAARSPSPLTFQTRTLREDSYNYWGQGTQVTVSS (SEQ ID NO: 122; “B12” VHH as described in Li et al 2023).

[0219] In one embodiment, the B7-H3 binding domain may comprise a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 122, optionally while retaining the CDRs of SEQ ID NOs: 119, 120 and 121 as set out above. In some embodiments, the sequence may be humanised. In some embodiments, the humanised antibody may bind to B7-H3 with a KD no more than 10 fold greater than the KD of the parent antibody. In some embodiments, the affinity for B7-H3 may be in the nanomolar or range, e.g., KD= 9.9X10-7to 1x10-9M or in the picomolar range, e.g., KD= 9.9x10-10to 1x10-12M.

[0220] In another embodiment, the B7-H3 binding domain, e.g., scFv, comprises CDRs of the following sequences: i) EDSTSAMC (HC-CDR1 ; SEQ ID NO: 123) ii) INPTGEVT (HC-CDR2; SEQ ID NO: 124) iii) AARVTYGGDWSTDTDYEYW (HC-CDR3; SEQ ID NO: 125).

[0221] In one embodiment, the B7-H3 binding domain is a scFv of the following sequence:

[0222] EVQLVESGGGSVQAGGSLRLSCVASEDSTSAMCMGWFRQAPGKEREGVACINPTGEVTWYGDSVKGR FTISRDTVKKIVYLQMNSLKPEDTAMYYCAARVTYGGDWSTDTDYEYWGQGTQVTVSS (SEQ ID NO: 126; “C4” VHH as described in Li et al 2023).

[0223] In one embodiment, the B7-H3 binding domain may comprise a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 126, optionally while retaining the CDRs of SEQ ID NOs: 123, 124 and 125 as set out above. In some embodiments, the sequence may be humanised. In some embodiments, the humanised antibody may bind to B7-H3 with a KD no more than 10 fold greater than the KD of the parent antibody. In some embodiments, the affinity for B7-H3 may be in the nanomolar or range, e.g., KD= 9.9X10-7to 1x10-9M or in the picomolar range, e.g., KD= 9.9x10-10to 1x10-12M.

[0224] In another embodiment, the B7-H3 binding domain, e.g., scFv, comprises CDRs of the following sequences: i) GFTFSRYW (HC-CDR1 ; SEQ ID NO: 127) ii) INSGGGST (HC-CDR2; SEQ ID NO: 128)

[0225] Hi) AKEQWRTGSR (HC-CDR3; SEQ ID NO: 129).

[0226] In one embodiment, the B7-H3 binding domain is a scFv of the following sequence:

[0227] DVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMGWFRQAPGKGVEVWSTINSGGGSTYYADSVKGR FTISRDNAKNTLYLQLNNLKTEDTAMYYCAKEQWRTGSRGQGTQVTVSS (SEQ ID NO: 130; “G8” VHH as described in Li et al 2023).

[0228] In another embodiment, the B7-H3 binding domain may comprise a sequence with at least 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 130, optionally while retaining the CDRs of SEQ ID NOs: 127, 128 and 129 as set out above. In some embodiments, the sequence may be humanised. In some embodiments, the humanised antibody may bind to B7-H3 with a KD no more than 10 fold greater than the KD of the parent antibody. In some embodiments, the affinity for B7-H3 may be in the nanomolar or range, e.g., KD= 9.9X10-7to 1x10-9M or in the picomolar range, e.g., KD= 9.9X10'10to 1x1012M.

[0229] Other exemplary antibodies for 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 instance, the B7-H3 binding domain may comprise a VH of SEQ ID: NO: 132 and a VL of SEQ ID NO: 133.

[0230] In another embodiment, the B7-H3 binding domain may comprise: a humanised VH of SEQ ID: NO 134, optionally having one, two, three or four of the affinity enhancing mutations HC, A24T; HC, E42G; HC, G56D; and HC, A102G (linear numbering); and a humanised VL of SEQ ID NO: 135, optionally having one or both of the affinity-enhancing mutations LC, S20T and LC, H34Y (linear numbering).

[0231] In one embodiment, the VH may comprise these four affinity enhancing mutations and the VL may comprise these two affinity-enhancing mutations.

[0232] In one embodiment the VH and VL as described above are present in an scFv domain. In another embodiment, the VH and VL are present in a Fab domain. Fc regions

[0233] In some embodiments, the antigen-binding molecules of the present disclosure comprise an Fc region. The presence of the Fc region significantly enhances the half-life of the antibody and is expected also to significantly enhance the half-life of the IFNy molecule to which the antibody binds.

[0234] Fc-mediated functions include Fc receptor binding, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), formation of the membrane attack complex (MAC), cell degranulation, cytokine and / or chemokine production, and antigen processing and presentation. Modifications to antibody Fc regions that influence Fc-mediated functions are known in the art, such as those described e.g. in Wang et al., Protein Cell (2018) 9(1):63-73, which is hereby incorporated by reference in its entirety. Exemplary Fc region modifications known to influence antibody effector function are summarised in Table 1 of Wang et al., Protein Cell (2018) 9(1):63-73. In some embodiments, the antigen-binding molecule of the present disclosure comprises an Fc region comprising modification to increase or reduce an Fc-mediated function as compared to an antigen-binding molecule comprising the corresponding unmodified Fc region.

[0235] Where an Fc region / CH2 / CH3 is described as comprising modifications) ‘corresponding to’ reference substitutions), equivalent substitutions) in the homologous Fc / CH2 / CH3 are contemplated. By way of illustration, L234A / L235A substitutions in human lgG1 (numbered according to the EU numbering system as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991) correspond to L to A substitutions at positions 117 and 118 of the mouse Ig gamma-2A chain C region (UniProtKB: P01863-1 , v1).

[0236] Where an Fc region is described as comprising a modification, the modification may be present in one or both of the polypeptide chains which together form the Fc region.

[0237] In some embodiments, the antigen-binding molecule of the present disclosure comprises an Fc region comprising modification. In some embodiments, the antigen-binding molecule of the present disclosure comprises an Fc region comprising modification in one or more of the CH2 and / or CH3 regions.

[0238] In certain embodiments, the modified Fc region comprises one or more mutations selected from the group consisting of a R435H mutation, N434A mutation, T252L / T253S / T254F mutation, E294delta / T307P / N434Y mutation, T256N / A378V / S383N / N434Y mutation, E294 delta 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, T256D / H286D / T307R / Q311V / A378V mutation, H285N / T307Q / N315D mutation, T307Q / Q311V / A378V mutation, H285D / T307Q / A378V mutation, L234F / L235E / P331 S mutation, L234F / L235Q / K322Q mutation, L234F / L235Q / P331 G mutation, L234F / L235A / K322Q mutation, S228P / F234A / L235A / G237A / P238S mutation, L234A / L235E / G237A / A330S / P331S mutation, F243A / V264A mutation, S228P / L235E / P329G mutation, M252Y / M428L mutation, D259IA / 308F mutation, T307Q / N434S mutation, M428LA / 308F mutation, Q311V / N434S mutation, H433K / N434F mutation, E258FA / 427T 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, S354C / T366W / K409A mutation, L234S / L235T / G236R mutation, F243L / R292P / Y300LA / 305I / P396L mutation, S239D / I332E mutation, S239D / I332E / A330L mutation, S298A / E333A / K334A mutation, G236A / S239D / I332E mutation, K326W / E333S mutation, S267E / H268F / S324T mutation, mutation of L234Y / L235Q / G236W / S239M / H268D / D270E / S298A in one heavy chain and D270E / K326D / A330M / K334E in the other heavy chain, E345R / E430G / S440Y mutation, P329G mutation, S228P mutation, delta-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.

[0239] In certain embodiments, the modified Fc region comprises a half-life enhancing mutation selected from the group consisting of a 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, E294delta / T307P / N434Y mutation, T256N / A378V / S383N / N434Y mutation, M428L / N434S mutation, T307A / E380A / N434A mutation, T307Q / N434S mutation, Q311V / N434S mutation, H433K / N434F mutation, E294delta / 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 / M428F mutation, T250E / M428F mutation, T250E / M428L mutation, M252Y / M428L mutation, M428LA / 308F mutation, D259IA / 308F mutation, E258FA / 427T mutation, T256D / Q311V / A378V mutation, T256D / H286D / T307R / Q311V / A378V mutation, and K288E / H435K mutation.

[0240] In certain embodiments, an Fc region of an antibody disclosed herein (e.g., a bispecific antibody) is modified to reduce or silence effector functions of the antibody (e.g., antibody-dependent cellular cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), opsonization, phagocytosis, transcytosis, neutralization of infectivity, inflammation, mucosal immunity, and neonatal immunity). Modifications are, e.g., determined using conventional means in the art. In certain embodiments, the modified Fc region comprises one or more mutations selected from the group consisting of a R435H mutation, N434A mutation, T252L / T253S / T254F mutation, E294delta / T307P / N434Y mutation, T256N / A378V / S383N / N434Y mutation, E294 delta 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, T256D / H286D / T307R / Q311V / A378V mutation, H285N / T307Q / N315D mutation, T307Q / Q311 V / A378V mutation, H285D / T307Q / A378V mutation, L234F / L235E / P331 S mutation, L234F / L235Q / K322Q mutation, L234F / L235Q / P331G mutation, L234F / L235A / K322Q mutation, S228P / F234A / L235A / G237A / P238S mutation, L234A / L235E / G237A / A330S / P331 S mutation, F243AA / 264A mutation, S228P / L235E / P329G mutation, M252Y / M428L mutation, D259IA / 308F mutation, T307Q / N434S mutation, M428LA / 308F mutation, Q311V / N434S mutation, H433K / N434F mutation, E258FA / 427T 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, S354C / T366W / K409A mutation, L234S / L235T / G236R mutation, P329G mutation, S228P mutation, delta-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, P331 S 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.

[0241] In certain embodiments, an Fc region of an antibody disclosed herein (e.g., a bispecific antibody) is modified to enhance effector functions of the antibody (e.g., antibody-dependent cellular cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), or antibody-dependent cellular phagocytosis (ADCP). In certain embodiments, the modified Fc region comprises one or more mutations selected from the group consisting of a F243L / R292P / Y300LA / 305I / P396L mutation, a S239D / I332E mutation, a S239D / I332E / A330L mutation, a S298A / E333A / K334A mutation, a G236A / S239D / I332E mutation, a K326W / E333S mutation, a S267E / H268F / S324T mutation, a mutation of L234Y / L235Q / G236W / S239M / H268D / D270E / S298A in one heavy chain and D270E / K326D / A330M / K334E in the other heavy chain, a E345R / E430G / S440Y mutation (giving rise to hexamerization and enhanced CDC) and an IgG 1 / lgG3 cross subclass mutation. In certain embodiments, the modified Fc region comprises a mutation that improves stability or downstream processing of the antibody and is selected from the group consisting of a 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 / del / K447del mutation, K370Q mutation, K370E mutation, R409A mutation, R409E mutation, R409W mutation, C131 S mutation, K370 deletion or substitution, delta-K447 mutation, F405L mutation, K409R mutation, and F405L / R409K mutation.

[0242] In some embodiments - particularly embodiments in which the antigen-binding molecule is a multispecific (e.g. bispecific) antigen-binding molecule - the antigen-binding molecule comprises an Fc region comprising modification in one or more of the CH2 and CH3 regions promoting association of the Fc region. Recombinant co-expression of constituent polypeptides of an antigen-binding molecule and subsequent association leads to several possible combinations. To improve the yield of the desired combinations of polypeptides in antigen-binding molecules in recombinant production, it is advantageous to introduce in the Fc regions modifications) promoting association of the desired combination of heavy chain polypeptides. Modifications may promote e.g. hydrophobic and / or electrostatic interaction between CH2 and / or CH3 regions of different polypeptide chains. Suitable modifications are described e.g. in Ha et al., Front. Immnol (2016) 7:394, which is hereby incorporated by reference in its entirety.

[0243] In certain embodiments, each heavy chain may comprise a single point mutation in the CH3 domain which allows for a stronger interaction between the CH3 domains in the bispecific. In certain embodiments, the single point mutation in each CH3 domain is at amino acid residue 366, 368, 370, 399, 405, 407, or 409, numbered according to the EU numbering system. In certain embodiments, the single point mutation is located at a different residue in CH3 domain as compared to the other CH3 domain. For example, one can comprise the mutation F405L (i.e., a mutation from phenylalanine to leucine at residue 405), while the other can comprise the mutation K409R (i.e., a mutation from lysine to arginine at residue 409), numbered according to the EU numbering system. In certain embodiments, heavy chain constant regions of the monospecific antibodies are selected from an lgG1 , lgG2, lgG3, or lgG4 isotype (e.g., a human lgG1 isotype).

[0244] In certain embodiments, the antibody is an lgG4 and both heavy chain constant regions have the sequence as shown in SEQ ID NO: 141 . In another embodiment, the antibody is a one-armed version of lgG4, in which one heavy chain comprises the constant regions of SEQ ID NO: 141 (fused to the VH) and the other heavy chain comprises or consists of SEQ ID NO: 142. In certain embodiments, the sequences of SEQ ID NO: 141 and / or 142 may comprise a F234A / L235A mutation (i.e., F234A and L235A). Additionally or alternatively, the sequence of SEQ ID NO: 141 and / or SEQ ID NO: 142 may be modified by S228P for enhanced in vivo stability. In certain embodiments the antibody is an lgG4 and one heavy chain constant regions has the sequence as shown in SEQ ID NO: 141 modified by T366S, L368A and Y407V (and optionally further modified by F234A / L235A and / or S228P) and the other heavy chain has the sequence shown in SEQ ID NO; 141 modified by T366W (and optionally further modified by F234A / L235A and / or S228P). In another embodiment, the antibody is a one-armed version of lgG4, in which the first heavy chain comprises the constant regions of SEQ ID NO: 141 (fused to the VH), optionally comprising a F234A / L235A mutation and / or S228P, and the second heavy chain comprises or consists of SEQ ID NO: 142, optionally comprising a F234A / L235A mutation and / or S228P, and wherein one of said heavy chains comprise the mutation T366W and the other comprises the mutations T366S, L368A and Y407V,

[0245] In certain embodiments, the antibody is an lgG4 comprising a first heavy chain having a F405L / R409K mutation and a second heavy chain having no mutations. In certain embodiments, the antibody is an lgG1 comprising a first heavy chain having a K409R mutation and a second heavy chain having a F405L mutation.

[0246] In certain embodiments, an antibody disclosed herein (e.g., a bispecific antibody) is a human lgG4 antibody comprising a first heavy chain and a second heavy chain. In certain embodiments, the first heavy chain comprises a S228P / F405L / R409K / delta-K447 mutation and the second heavy chain comprises a S228P / delta-K447 mutation. In certain embodiments, the first heavy chain comprises a S228P / F405L / R409K / F234A / L235A / delta-K447 mutation and the second heavy chain comprises a S228P / F234A / L235A / delta-K447 mutation. In certain embodiments, the first heavy chain and the second heavy chain further comprise a M252Y / S254T / T256E mutation. In certain embodiments, the first heavy chain and the second heavy chain further comprise a F234A / L235A mutation.

[0247] In certain embodiments, the first heavy chain and the second heavy chain further comprise a F234A / L235A mutation. In certain embodiments, the antibody comprises a heavy chain constant region with a single point mutation in the CH3 domain at an amino acid position selected from the group consisting of 366, 368, 370, 399, 405, 407, and 409. In certain embodiments, the single point mutation is a F405L mutation or a K409R mutation. In certain embodiments, the antibody is an lgG4 comprising a first heavy chain having a F405L / R409K mutation and a second heavy chain having no mutations. In certain embodiments, the antibody is an lgG1 comprising a first heavy chain having a K409R mutation and a second heavy chain having a F405L mutation.

[0248] The antibodies may comprise combinations of mutations as described above, e.g., to enhance half-life and / or to reduce or silence effector function and / or to improve stability or downstream processing. Formats of Bispecific antigen binding molecules

[0249] As described herein, aspects of the present disclosure relate to antigen-binding molecules which have more than one binding domain, wherein each binding domain binds to a different antigen - i.e., multispecific antigen binding molecules. The term “multispecific” is used herein to include “bispecific”.

[0250] Multispecific antigen-binding molecules according to the present disclosure may be provided in any suitable format, such as those formats described in described in Brinkmann and Kontermann, MAbs (2017) 9(2): 182-212, which is hereby incorporated by reference in its entirety. Suitable formats include those shown in Figure 2 of Brinkmann and Kontermann, MAbs (2017) 9(2): 182-212: antibody conjugates, e.g. lgG2, F(ab’)2 or CovX-Body; IgG or IgG-like molecules, e.g. IgG, chimeric IgG, KA-body common HC; CH1 / CL fusion proteins, e.g. scFv2-CH1 / CL, VHH2-CH1 / CL; ‘variable domain only’ bispecific antigenbinding molecules, e.g. tandem scFv (taFV), triplebodies, diabodies (Db), dsDb, Db(kih), DART, scDB, dsFv-dsFv, tandAbs, triple heads, tandem dAbA / HH, tertravalent dAb.VHH; Non-lg fusion proteins, e.g. scFv2-albumin, scDb-albumin, taFv-albumin, taFv-toxin, miniantibody, DNL-Fab2, DNL-Fab2-scFv, DNL- Fab2-lgG-cytokine2, ImmTAC (TCR-scFv); modified Fc and CH3 fusion proteins, e.g. scFv-Fc(kih), scFv- Fc(CH3 charge pairs), 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), TriFabs; Fc fusions, e.g. Di-diabody, scDb-Fc, taFv-Fc, scFv-Fc-scFv, HCAb-VHH, Fab-scFv-Fc, scFv4-lg, scFv2-Fcab; CH3 fusions, e.g. Dia-diabody, scDb-CH3; IgE / IgM CH2 fusions, e.g. scFv-EHD2-scFv, scFvMHD2-scFv; Fab fusion proteins, e.g. Fab-scFv (bibody), Fab-scFv2 (tribody), Fab- Fv, Fab-dsFv, Fab-VHH, orthogonal Fab-Fab; non-lg fusion proteins, e.g. DNL-Fabs, DNL-Fab2-scFv, DNL-Fab2-lgG-cytokine2; asymmetric IgG or IgG-like molecules, e.g. IgG(kih), IgG(kih) common LC, ZW1 IgG common LC, Biclonics common LC, CrossMab, CrossMab(kih), scFab-lgG(kih), Fab-scFab-lgG(kih), orthogonal Fab IgG(kih), DuetMab, CH3 charge pairs + CH1 / CL charge pairs, hinge / CH3 charge pairs, SEED-body, Duobody, four-in-one-CrossMab(kih), LUZ-Y common LC; LUZ-Y scFab-IgG, FcFc*; appended and Fc-modified IgGs, e.g. lgG(kih)-Fv, IgG HA-TF-Fv, lgG(kih)scFab, scFab-Fc(kih)-scFv2, scFab-Fc(kih)-scFv, half DVD-lg, DVI-lg (four-in-one), CrossMab-Fab; modified Fc and CH3 fusion proteins, e.g. Fab-Fc(kih)-scFv, Fab-scFv-Fc(kih), Fab-scFv-Fc(BEAT), Fab-scFv-Fc-SEEDbody, TriFab; appended IgGs - HC fusions, e.g. IgG-HC, scFv, IgG-dAb, IgG-taFV, IgG-CrossFab, IgG-orthogonal Fab, IgG-(CaCp) Fab, scFv-HC-IgG, tandem Fab-IgG (orthogonal Fab), Fab-lgG(CaCp Fab), Fab-lgG(CR3), Fab-hinge-lgG(CR3); appended IgGs - LC fusions, e.g. IgG-scFv(LC), scFv(LC)-lgG, dAb-IgG; appended IgGs - HC and LC fusions, e.g. DVD-lg, TVD-lg, CODV-lg, scFv4-lgG, Zybody; Fc fusions, e.g. Fab-scFv- Fc, scFv4-lg; F(ab’)2 fusions, e.g. F(ab’)2-scFv2; CH1 / CL fusion proteins e.g. scFv2-CH1-hinge / CL; modified IgGs, e.g. DAF (two-in one-IgG), DutaMab, Mab2; and non-lg fusions, e.g. DNL-Fab4-lgG.

[0251] The skilled person is able to design and prepare bispecific antigen-binding molecules. Methods for producing bispecific antigen-binding molecules include chemically crosslinking antigen-binding molecules or antibody fragments, e.g. with reducible disulphide or non-reducible thioether bonds, for example as described in Segal and Bast, 2001. Production of Bispecific Antigen-binding molecules. 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, A / -succinimidyl-3-(-2-pyridyldithio)-propionate (SPDP) can be used to chemically crosslink e.g. Fab fragments via hinge region SH- groups, to create disulfide-linked bispecific F(ab)2 heterodimers.

[0252] Other methods for producing bispecific antigen-binding molecules include fusing antibody-producing hybridomas e.g. with polyethylene glycol, to produce a quadroma cell capable of secreting bispecific antibody, for example as described in D. M. and Bast, B. J. 2001 . Production of Bispecific Antigen-binding molecules. Current Protocols in Immunology. 14:IV:2.13:2.13.1-2.13.16.

[0253] Bispecific antigen-binding molecules according to the present disclosure can also be produced recombinantly, by expression from e.g. a nucleic acid construct encoding polypeptides for the antigenbinding molecules, for example as described in Antibody Engineering: Methods and Protocols, Second Edition (Humana Press, 2012), at Chapter 40: Production of Bispecific Antigen-binding molecules: Diabodies and Tandem scFv (Hornig and Farber-Schwarz), or French, How to make bispecific antigenbinding molecules, Methods Mol. Med. 2000; 40:333-339, the entire contents of both of which are hereby incorporated by reference.

[0254] For example, a DNA construct encoding the light and heavy chain variable domains for the two antigenbinding fragments ( / .e., binding IFNy and another target such as a TAA), and including sequences encoding a suitable linker or dimerization domain between the antigen-binding fragments can be prepared by molecular cloning techniques. Recombinant bispecific antibody can thereafter be produced by expression (e.g. in vitro) of the construct in a suitable host cell (e.g. a mammalian host cell), and expressed recombinant bispecific antibody can then optionally be purified.

[0255] Preferred formats according to the present invention include an Fc domain. The presence of an Fc domain significantly extends the half-life of the antibody. (In the absence of an Fc region, it may in some embodiments be preferred that the antigen binding molecule is conjugated to a half-life extending moiety such as fatty acid, albumin or a hydrophilic polymer as discussed further herein).

[0256] The present inventors have unexpectedly determined that the affinity for IFNy is significantly increased when the antibody is monovalent for IFNy. For example, the antibody may have two arms, wherein one arm comprises a single binding site for IFNy and the other arm comprises one or more binding sites for the TAA (e.g., a single binding site for the TAA). In some embodiments, multispecific (e.g., bispecific) antibodies according to the present invention may comprise a heterodimeric IgG antibody, in which one arm of the antibody (i.e. , one Fab region) binds to IFNy and the other arm (i.e., the other Fab) binds to the other target, e.g., the TAA. Thus, the bispecific antibody comprises a full-length antibody (e.g., an IgG) comprising a first and second antibody heavy chain and a first and second antibody light chain, wherein the first heavy chain and the first light chain assemble to form an antigen binding site for a first antigen, and wherein the second heavy chain and second light chain assemble to form an antigen binding site for a second antigen. Correct assembly of the heterodimeric heavy chains can be assisted e.g. by the use of knob into hole mutations and / or other modifications as discussed further below. Correct assembly of the light chains with their respective heavy chain can be assisted by using cross-mab technology and / or charge modification.

[0257] One exemplary embodiment of such a format is formed from the 4 chains as set out in SEQ ID Nos 75-78 (2 light chains and two heavy chains). The antibody formed therefrom is referred to herein as LDC-1 .

[0258] In some embodiments of the above format, the format may be bivalent, having a single binding site for IFNy and a single binding site for the TAA. In another possible embodiment, further antigen binding moieties may be fused e.g., to the first and / or second heavy chain to increase the valency for one or both antigens, although as discussed above, it may be preferred that the antibody is monovalent for IFNy.

[0259] In other embodiments, the multispecific (e.g., bispecific) antibodies may comprise a heterodimer formed of an Fc domain comprising a first and a second Fc subunit, wherein each submit is fused (directly or indirectly, via a linker) to an antigen-binding moiety, and wherein the first Fc subunit is fused, e.g., at its N-terminus, to an antigen-binding moiety capable of binding IFNy and the second Fc subunit is fused, e.g., at its N-terminus, to an antigen-binding moiety capable of binding a TAA. The antigen-binding moiety may be any antibody fragment as disclosed herein, e.g., a Fab, Fab’, F(ab’), F(ab’)2, Fd, scFv, minibody, a variable heavy domain, a variable light domain, a variable NAR domain, a single chain binding polypeptide, a dAb fragment, a nanobody, a VHH, and others also referred to as antigen-binding fragments. For instance, in some embodiments, the antigen binding moiety may be a Fab, ScFv, or a single domain antibody / nanobody. In certain embodiments, the first polypeptide chain and second polypeptide chain of the Fc comprises a hinge or linker covalently linked to a polypeptide comprising the CH2 and CH3 domains of the Fc domain. Where the antigen-binding moiety is a Fab, the heavy chain of the Fab may be fused to the Fc subunit via the hinge region - i.e., the antibody may comprise one complete IgG heavy chain, comprising CH3-CH2-hinge-CH1-VH, as well as a light chain comprising CL1- VL which associates with the heavy chain to form the Fab (optionally with exchange of the VL / VH or CL1 / CH1 regions as in a cross-mab). In one embodiment, the antibody comprises one arm comprising Fab and one arm comprising an ScFv or a nanobody / single domain antibody. That is, in certain embodiments, the antibody comprises: (1) a Fab fragment linked 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 linked 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 linked 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 linked at its C terminus to the N-terminus of the second polypeptide chain of the Fc domain. In one embodiment, the Fab is capable of binding IFNy and the scFv or nanobody is capable of binding the TAA.

[0260] One exemplary embodiment of such a format is formed from the light chain of SEQ ID NO: 65 and the heavy chains of SEQ ID NO: 66 and 67. The antibody formed therefrom is referred to herein as LDC-4.

[0261] Another exemplary embodiment of such a format is formed from the light chain of SEQ ID NO: 79 and the heavy chains of SEQ ID NO: 80 and 81 . The antibody formed therefrom is referred to herein as LDC-6.

[0262] Another exemplary embodiment of such a format is formed from the light chain of SEQ ID NO: 72 and the heavy chains of SEQ ID NO: 73 and 74. The antibody formed therefrom is referred to herein as LDC-2.

[0263] In another possible embodiment, further antigen binding moieties may be fused e.g., to the first and / or second heavy chain to increase the valency for one or both antigens, although as discussed above, it may be preferred that the antibody is monovalent for IFNy.

[0264] Another exemplary format comprises a full-length antibody such as an IgG, which may be an IgG 1 , lgG2, lgG3 or lgG4, optionally IgG 1 , or optionally lgG4, comprising an antigen binding site for the first antigen (e.g., which may be bivalent for the first antigen), fused (directly or indirectly, via a linker) to an antigen binding moiety for the second antigen. In some embodiments, the first antigen is IFNy and the second antigen in a TAA, in which case the antibody will be (at least) bivalent for IFNy. In other embodiments, the first antigen is the TAA and the second antigen in a IFNy. The antigen binding moiety for the second antigen may be any of the antibody fragments as set out herein, e.g., a Fab, Fab’, F(ab’), F(ab’)2, Fd, scFv, minibody, a variable heavy domain, a variable light domain, a variable NAR domain, a single chain binding polypeptide, a dAb fragment, a nanobody, a VHH, and others 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 an scFv or nanobody / single domain antibody, in some embodiments, a nanobody / single domain antibody or scFv may be fused to the C-terminus of one of both Fc subunits of the IgG. In the case of a Fab, one chain of the Fab can in some embodiments be fused via a linker to the N-terminus of one or both IgG heavy chains 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 the IgG. In still further embodiments, one chain of the Fab may be fused to the C-terminus of one Fc-subunit of the IgG, and the other chain of the Fab may be fused to the C-terminus of the other Fc subunit of the IgG. In all embodiments, fusion may be via a linker.

[0265] For instance, in certain embodiments, the antibody comprises: (1) a first Fab fragment linked at the C- terminus of the CH1 domain to the N-terminus of the first polypeptide chain of the Fc domain; (2) a first scFv linked at its N-terminus to the C-terminus of the first polypeptide chain of the Fc domain; (3) a second Fab fragment linked at the C-terminus of the CH1 domain to the N-terminus of the second polypeptide chain of the Fc domain; (2) a second scFv linked at its N-terminus to the C-terminus of the second polypeptide chain of the Fc domain.

[0266] One exemplary embodiment of such a format is formed from the light chain of SEQ ID NO: 70 and the heavy chain of SEQ ID NO: 71 . The antibody formed therefrom is referred to herein as LDC-3.

[0267] For instance, in other embodiments, the antibody comprises: (1) a first Fab fragment linked at the C- terminus of the CH1 domain to the N-terminus of the first polypeptide chain of the Fc domain; (2) a first nanobody linked at its N-terminus to the C-terminus of the first polypeptide chain of the Fc domain; (3) a second Fab fragment linked at the C-terminus of the CH1 domain to the N-terminus of the second polypeptide chain of the Fc domain; (2) a second nanobody linked at its N-terminus to the C-terminus of the second polypeptide chain of the Fc domain.

[0268] One exemplary embodiment of such a format is formed from the light chain of SEQ ID NO: 63 and the heavy chain of SEQ ID NO: 64. The antibody formed therefrom is referred to herein as LDC-5.

[0269] In any of the above embodiments, when the molecule comprises Fabs binding to different antigens, the Fabs for one of the antigens may be cross-fabs.

[0270] The valency of the molecule for either of the antigens may be varied using the knowledge of the skilled person. In certain embodiments, an antibody described herein has two or more (e.g., 2, 3, 4, or more) antigen-binding sites for binding to the TAA. In some embodiments, the multi-specific antigen binding molecule may have antigen-binding sites for more than one TAA, e.g., 2 different TAAs, or for more than one distinct (e.g., non-overlapping) epitopes in a TAA. In certain embodiments, an antibody described herein has two or more (e.g., 2, 3, 4, or more) antigen-binding sites for binding to I FNy, although as described above, antibodies which are monovalent for IFNy may be preferred due to the observed higher affinity.

[0271] In some embodiments of any of the above formants, the multispecific (e.g., bispecific) antibodies are at least bivalent for IFNy. Formats of monospecific antigen binding molecules

[0272] In some aspects, the antigen-binding molecules describes herein are monospecific, i.e., they comprise a single antigen binding site or they comprise more than one antigen binding site wherein the sites have the same specificity. Thus, they bind to IFNy without binding to another antigen.

[0273] In some embodiments the monospecific antigen binding molecules are also monovalent for IFNy, i.e, they comprise a single antigen binding site.

[0274] In some embodiments the antigen binding molecule may be an antibody. In some embodiments, it may be a one-armed IgG. A one-armed IgG may comprise an Fc domain comprising a first and a second Fc subunit, and a single Fab domain coupled to one of the Fc subunits (e.g., with the C-terminus of the VH domain of the Fab linked to the N-terminus of the Fc subunit via the hinge in the normal manner).

[0275] Linkers and additional seguences

[0276] In some embodiments, the antigen-binding molecules and polypeptides of the present disclosure comprise one or more linker sequences between amino acid sequences. A linker sequence may be provided at one or both ends of one or more of a VH, VL, CH1-CH2 hinge region, CH2 region and a CH3 region of the antigen-binding molecule / polypeptide.

[0277] Linker sequences are known to the skilled person, and are described, for example in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369, which is hereby incorporated by reference in its entirety. In some embodiments, a linker sequence may be a flexible linker sequence. Flexible linker sequences allow for relative movement of the amino acid sequences which are linked by the linker sequence. Flexible linkers are known to the skilled person, and several are identified in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369. Flexible linker sequences often comprise high proportions of glycine and / or serine residues.

[0278] In some embodiments, the linker sequence comprises at least one glycine residue and / or at least one serine residue. In some embodiments, the linker sequence consists of glycine and serine residues. In some embodiments, the linker sequence comprises one or more copies (e.g. in tandem) of the sequence motif G4S. In some embodiments, the linker sequence has a length of 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25, or 1-30 amino acids. For example, the linker may have a sequence (G4S)n(SEQ ID NO: 136), wherein n is 1-30, e.g., 1-10, preferably 2 or more or 3 or more, e.g., n=3 or n=6.

[0279] The antigen-binding molecules and polypeptides of the present disclosure may additionally comprise further amino acids or sequences of amino acids. For example, the antigen-binding molecules and polypeptides may comprise amino acid sequence(s) to facilitate expression, folding, trafficking, processing, purification or detection of the antigen-binding molecule / polypeptide. For example, the antigen-binding molecule / polypeptide may comprise a sequence encoding a His, (e.g. 6XHis), Myc, GST, MBP, FLAG, HA, E, or Biotin tag, optionally at the N- or C- terminus of the antigen-binding molecule / polypeptide. In some embodiments, the antigen-binding molecule / polypeptide comprises a detectable moiety, e.g. a fluorescent, luminescent, immuno-detectable, radio, chemical, nucleic acid or enzymatic label.

[0280] The antigen-binding molecules and polypeptides of the present disclosure may additionally comprise a signal peptide (also known as a leader sequence or signal sequence). Signal peptides normally consist of a sequence of 5-30 hydrophobic amino acids, which form a single alpha helix. Secreted proteins and proteins expressed at the cell surface often comprise signal peptides.

[0281] The signal peptide may be present at the N-terminus of the antigen-binding molecule / polypeptide and may be present in the newly synthesized antigen-binding molecule / polypeptide. The signal peptide provides for efficient trafficking and secretion of the antigen-binding molecule / polypeptide. Signal peptides are often removed by cleavage, and thus are not comprised in the mature antigen-binding molecule / polypeptide secreted from the cell expressing the antigen-binding molecule / polypeptide.

[0282] Signal peptides are known for many proteins, and are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro, and / or can be identified / predicted e.g. using amino acid sequence analysis tools such as SignalP (Petersen et al. , 2011 Nature Methods 8: 785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24: 2172- 2176).

[0283] Modifications and Conjugates

[0284] In certain embodiments, an antibody of the disclosure (e.g., a bispecific antibody) is modified, e.g., by addition of a moiety which increases the circulation time of the antibody in the circulation. Many such moieties are known in the prior art, including a fatty acid, albumin and a hydrophilic polymer such as PEG, polysicalic acid (PSA), N-(2-hydroxypropyl)methacrylamide (HPMA) or dextran.

[0285] In certain embodiments, an antibody of the disclosure (e.g., a bispecific antibody) is modified, e.g., by addition of polyethylene glycol (PEG). In certain embodiments, PEG modification (PEGylation) leads to one or more of improved circulation time, improved solubility, improved resistance to proteolysis, reduced antigenicity and immunogenicity, improved bioavailability, reduced toxicity, improved stability, and easier formulation. Glycosylation of immunoglobulins has been shown to have significant effects on their effector functions, structural stability, and rate of secretion from antibody-producing cells. The carbohydrate groups responsible for these properties are generally attached to the constant (C) regions of the antibodies. For example, glycosylation of IgG at asparagine 297 in the CH2 domain is required for full capacity of IgG to activate the classical pathway of complement-dependent cytolysis. Glycosylation of IgM at asparagine 402 in the CH3 domain is necessary for proper assembly and cytolytic activity of the antibody. Removal of glycosylation sites as positions 162 and 419 in the CH1 and CH3 domains of an IgA antibody led to intracellular degradation and at least 90% inhibition of secretion.

[0286] Glycosylation of immunoglobulins in the variable (V) region has also been observed. About 20% of human antibodies are glycosylated in the V region. Glycosylation of the V domain is believed to arise from fortuitous occurrences of the N-linked glycosylation signal Asn-Xaa-Ser / Thr in the V region sequence and has not been recognized in the art as playing a role in immunoglobulin function.

[0287] Glycosylation at a variable domain framework residue, in some cases, alters the binding interaction of the antibody with antigen. The present disclosure includes criteria by which a limited number of amino acids in the framework or CDRs of a humanized immunoglobulin chain are chosen to be mutated (e.g., by substitution, deletion, or addition of residues) to increase the affinity of an antibody. In certain embodiments, cysteine residue(s) are removed or introduced in the Fc region of an antibody or Fc- containing polypeptide, thereby eliminating or increasing interchain disulfide bond formation in this region.

[0288] It has been shown that sequences within the CDR can sometimes cause an antibody to bind to MHC Class II and trigger a helper T-cell response, which may be unwanted in some cases. In certain embodiments, a conservative substitution allows an antibody to retain binding activity yet reduce its ability to trigger an unwanted T-cell response. In one embodiment, one or more of the N-terminal 20 amino acids of the heavy or light chain is removed.

[0289] Covalent modifications of an antibody disclosed herein (e.g., a bispecific antibody) are also included herein. In certain embodiments, they are made by chemical synthesis or by enzymatic or chemical cleavage of the antibody, if applicable. In certain embodiments, other types of covalent modifications are introduced by reacting targeted amino acid residues with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues.

[0290] Cysteinyl residues are most commonly reacted with alpha-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, alpha-bromo-beta-(5- imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2- pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa- 1 ,3-diazole.

[0291] In certain embodiments, histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5- 7.0 because this agent is relatively specific for the histidyl side chain. In certain embodiments, parabromophenacyl bromide also is useful; the reaction, in certain embodiments, is performed in 0.1 M sodium cacodylate at pH 6.0.

[0292] In certain embodiments, lysinyl and amino-terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing alpha-amino-containing residues include imidoesters such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reaction with glyoxylate.

[0293] In certain embodiments, arginyl residues are modified by reaction with one or several conventional reagents, such as phenylglyoxal, 2,3-butanedione, 1 ,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents, in certain embodiments, react with the groups of lysine as well as the arginine epsilon-amino group.

[0294] In certain embodiments, the specific modification of tyrosyl residues is made, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidazole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively, in certain embodiments. Tyrosyl residues are iodinated using125l or1311 to prepare labeled proteins for use in radioimmunoassay.

[0295] Carboxyl side groups (aspartyl or glutamyl) are specifically modified by reaction with carbodiimides (R- N=C=N-R’), where R and R’ are different alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

[0296] In certain embodiments, glutaminyl and asparaginyl residues are deamidated to the corresponding glutamyl and aspartyl residues, respectively. These residues are deamidated under neutral or basic conditions.

[0297] Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains, acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group. Another type of covalent modification involves chemically or enzymatically coupling glycosides to the specific binding agent or antibody. These procedures do not require production of the polypeptide or antibody in a host cell that has glycosylation capabilities for N- or O-linked glycosylation. Depending on the coupling mode used, in certain embodiments, the sugar(s) are attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine.

[0298] Removal of any carbohydrate moieties present on the polypeptide or antibody can be accomplished chemically or enzymatically. Chemical deglycosylation involves exposure of the antibody to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the antibody intact. Enzymatic cleavage of carbohydrate moieties on an antibody can be achieved using a variety of endo- and exo-glycosidases.

[0299] Another type of covalent modification comprises linking an antibody of the disclosure (e.g., a bispecific antibody) to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, polyoxyethylated polyols, polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol, polyoxyalkylenes, or polysaccharide polymers such as dextran. Such methods are known in the art.

[0300] Affinity for binding a pre-determined polypeptide antigen, generally, is modulated by introducing one or more mutations into the V region framework, typically in areas adjacent to one or more CDRs and / or in one or more framework regions. Typically, such mutations involve the introduction of conservative amino acid substitutions that either destroy or create the glycosylation site sequences but do not substantially affect the hydropathic structural properties of the polypeptide. Typically, mutations that introduce a proline residue are avoided.

[0301] In certain embodiments, an antibody (e.g., a bispecific antibody) or antigen-binding fragment disclosed herein is conjugated or linked to a therapeutic moiety, an imaging or detectable moiety, or an affinity tag. Methods for conjugating or linking polypeptides are known in the art. Associations (binding) between compounds and labels include any means known in the art including, but not limited to, covalent and non- covalent interactions, chemical conjugation, as well as recombinant techniques. An antibody (e.g., a bispecific antibody) or antigen-binding fragment of the disclosure is conjugated to, or recombinantly engineered with, an affinity tag (e.g., a purification tag), in certain embodiments. Affinity tags such as poly-histidine tags (e.g., His6) are conventional in the art. In certain embodiments, an antibody (e.g., a bispecific antibody) or antigen-binding fragment further comprises a detectable moiety. Detections accomplished, e.g., in vitro, in vivo or ex vivo. In vitro assays for the detection and / or determination (quantification, qualification, etc.) of, e.g., IFNy protein levels using an antibody (e.g., a bispecific antibody) or antigen-binding fragment disclosed herein include but are not limited to, e.g., ELISAs, RIAs, and western blots. In certain embodiments, in vitro detection, diagnosis, or monitoring of the antigen of the antibodies occurs by obtaining a sample (e.g., a blood sample) from a subject and testing the sample in, e.g., a standard ELISA assay.

[0302] Nucleic Acids and Vectors

[0303] The present disclosure provides a nucleic acid, or a plurality of nucleic acids, encoding an antigen-binding molecule according to the present disclosure. In some embodiments, the nucleic acid(s) comprise or consist of DNA and / or RNA.

[0304] The present disclosure also provides a vector, or plurality of vectors, comprising the nucleic acid or plurality of nucleic acids according to the present disclosure.

[0305] Nucleic acids and vectors according to the present disclosure may be provided in purified or isolated form, i.e. from other nucleic acid, or naturally-occurring biological material.

[0306] The nucleotide sequence may be contained in a vector, e.g. an expression vector. A ‘vector’ as used herein is a nucleic acid molecule used as a vehicle to transfer exogenous nucleic acid into a cell. The vector may be a vector for expression of the nucleic acid in the cell. Such vectors may include a promoter sequence operably linked to the nucleotide sequence encoding the sequence to be expressed. A vector may also include a termination codon and expression enhancers. Any suitable vectors, promoters, enhancers and termination codons known in the art may be used to express a peptide or polypeptide from a vector according to the present disclosure.

[0307] The term ‘operably linked’ may include the situation where a selected nucleic acid sequence and regulatory nucleic acid sequence (e.g. promoter and / or enhancer) are covalently linked in such a way as to place the expression of nucleic acid sequence under the influence or control of the regulatory sequence (thereby forming an expression cassette). Thus, a regulatory sequence is operably linked to the selected nucleic acid sequence if the regulatory sequence is capable of effecting transcription of the nucleic acid sequence. The resulting transcripts) may then be translated into a desired peptide(s) / polypeptide(s). Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g. gammaretroviral vectors e.g. murine Leukemia virus (MLV)-derived vectors), lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors and herpesvirus vectors), transposon-based vectors, and artificial chromosomes (e.g. yeast artificial chromosomes).

[0308] In some embodiments, the vector may be a eukaryotic vector, e.g. a vector comprising the elements necessary for expression of protein from the vector in a eukaryotic cell. In some embodiments, the vector may be a mammalian vector, e.g. comprising a cytomegalovirus (CMV) or SV40 promoter to drive protein expression.

[0309] Constituent polypeptides of an antigen-binding molecule according to the present disclosure may be encoded by different nucleic acids of the plurality of nucleic acids, or by different vectors of the plurality of vectors.

[0310] Cells and methods of production

[0311] The present disclosure also provides a cell comprising or expressing an antigen-binding molecule according to the present disclosure. Also provided is a cell comprising or expressing a nucleic acid, a plurality of nucleic acids, a vector or a plurality of vectors according to the present disclosure.

[0312] The cell may be a eukaryotic cell, e.g. a mammalian cell. The mammal may be a primate (rhesus monkey, cynomolgous monkey, non-human primate or human) or a non-human mammal (e.g. rabbit, guinea pig, rat, mouse or other rodent (including any animal in the order Rodentia), cat, dog, pig, sheep, goat, cattle (including cows, e.g. dairy cows, or any animal in the order Bos), horse (including any animal in the order Equidae), donkey, and non-human primate).

[0313] In some embodiments, the cell is, or is derived from, a cell type commonly used for the expression of polypeptides for use in therapy in humans. Exemplary cells are described e.g. in Kunert and Reinhart, Appl Microbiol Biotechnol. (2016) 100:3451-3461 (hereby incorporated by reference in its entirety), and include e.g. CHO, HEK 293, PER.C6, NSO and BHK cells. In preferred embodiments, the cell is, or is derived from, a CHO cell.

[0314] The present disclosure also provides a method for producing a cell comprising a nucleic acid(s) or vector(s) according to the present disclosure, comprising introducing a nucleic acid, a plurality of nucleic acids, a vector or a plurality of vectors according to the present disclosure into a cell. In some embodiments, introducing an isolated nucleic acid(s) or vector(s) according to the present disclosure into a cell comprises transformation, transfection, electroporation or transduction (e.g. retroviral transduction). The present disclosure also provides a method for producing a cell expressing / comprising an antigenbinding molecule according to the present disclosure, comprising introducing a nucleic acid, a plurality of nucleic acids, a vector or a plurality of vectors according to the present disclosure in a cell. In some embodiments, the methods additionally comprise culturing the cell under conditions suitable for expression of the nucleic acid(s) or vector(s) by the cell. In some embodiments, the methods are performed in vitro.

[0315] The present disclosure also provides cells obtained or obtainable by the methods according to the present disclosure.

[0316] Compositions

[0317] The present disclosure also provides compositions comprising the antigen-binding molecules, nucleic acids, expression vectors and cells described herein.

[0318] The antigen-binding molecules, nucleic acids, expression vectors and cells described herein may be formulated as pharmaceutical compositions or medicaments for clinical use and may comprise a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. The composition may be formulated for topical, parenteral, systemic, intracavitary, intravenous, intra-arterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, intrathecal, oral or transdermal routes of administration which may include injection or infusion. In some embodiments, the composition may be formulated for administration into a tissue, e.g., by injection. In some embodiments, the composition may be formulated for subcutaneous injection.

[0319] Suitable formulations may comprise the antigen-binding molecule in a sterile or isotonic medium. Medicaments and pharmaceutical compositions may be formulated in fluid, including gel, form. Fluid formulations may be formulated for administration by injection or infusion (e.g. via catheter) to a selected region of the human or animal body.

[0320] In some embodiments, the composition is formulated for injection or infusion, e.g. into a blood vessel, tissue / organ of interest or tumor.

[0321] The present disclosure also provides methods for the production of pharmaceutically useful compositions, such methods of production may comprise one or more steps selected from: producing an antigenbinding molecule, nucleic acid (or plurality thereof), expression vector (or plurality thereof) or cell described herein; isolating an antigen-binding molecule, nucleic acid (or plurality thereof), expression vector (or plurality thereof) or cell described herein; and / or mixing an antigen-binding molecule, nucleic acid (or plurality thereof), expression vector (or plurality thereof) or cell described herein with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.

[0322] For example, a further aspect the present disclosure relates to a method of formulating or producing a medicament or pharmaceutical composition for use in the treatment of a disease / condition (e.g. a cancer), the method comprising formulating a pharmaceutical composition or medicament by mixing an antigen-binding molecule, nucleic acid (or plurality thereof), expression vector (or plurality thereof) or cell described herein with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.

[0323] Administration and therapeutic applications

[0324] Disclosed herein, in certain embodiments, are methods of treating a disease in a subject in need thereof, comprising administering to the subject the antigen binding molecule or the pharmaceutical composition of any one of the foregoing embodiments.

[0325] The disease or disorder to be treated may be any disease or disorder which is beneficially treated by the proinflammatory and / or anti-tumorigenic activities of IFNy.

[0326] In certain embodiments, the disease treated could be 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

[0327] Clinical Infectious Diseases, Volume 39, Issue 5, 1 September 2004, Pages 692-699), osteopetrosis (L L Key Jr 1 , R M Rodriguiz, S M Willi, N M Wright, H C Hatcher, D R Eyre, J K Cure, P P Griffin, W L Ries N Engl J Med. 1995 Jun 15;332(24):1594-9) , atopic dermatitis, and treatment of bacterial and fungal infections (Netea MG, Kullberg BJ, Van der Meer JW. Proinflammatory cytokines in the treatment of bacterial and fungal infections. BioDrugs. 2004;18(1):9-22). This may be through enhanced effect of IFNy on neutrophils, osteoclasts, and / or the innate immune system.

[0328] In other embodiments the disease or disorder is a tumor or cancer. Optionally the tumor may be a non- immunogenic tumor, such as a check-point inhibitor resistant tumor.

[0329] The treatment may be in combination with another one or more additional anti-cancer therapies, as further discussed herein.

[0330] Also disclosed are methods of sensitizing a subject with a cancer to treatment with a checkpoint inhibitor, comprising administering to the subject a composition comprising an antibody (including but not limited to a bispecific antibody) or pharmaceutical composition disclosed herein. In certain embodiments, the sensitizing results in an increased sensitivity of the cancer to treatment with a checkpoint inhibition and / or a reduced dosage of a checkpoint inhibitor necessary to reach a therapeutically desirable treatment benchmark (e.g., amelioration of symptoms, prolongation of progression, cure, remission, prolongation of survival, or other objective responses).

[0331] Also disclosed is an antigen binding molecule or a pharmaceutical composition of any one of the foregoing embodiments for use in a method of treatment. More particularly, there is provided an antigen binding molecule or a pharmaceutical composition of any one of the foregoing embodiments for use in a method of treating cancer.

[0332] Also disclosed is an antigen binding molecule or a pharmaceutical composition of any one of the foregoing embodiments for use in a method of sensitizing a subject with a cancer to treatment with a checkpoint inhibitor. Also provided is an antigen binding molecule or a pharmaceutical composition of any one of the foregoing embodiments for use in a method of treating cancer, said method further comprising administering one or more additional anti-cancer therapies. Also provided is an antigen binding molecule or a pharmaceutical composition of any one of the foregoing embodiments and one or more additional anti-cancer therapies for use in a method of treating cancer. In one embodiment the one or more additional anti-cancer therapies may comprise a checkpoint inhibitor.

[0333] Further provided is use of an antigen binding molecule or a pharmaceutical composition of any one of the foregoing embodiments in the manufacture of a medicament for the treatment of cancer. The treatment may be in combination with another one or more additional anti-cancer therapies, as further discussed herein.

[0334] In some embodiments, the disease / condition to be treated / prevented is a cancer. The cancer may be any unwanted cell proliferation (or any disease manifesting itself by unwanted cell proliferation), neoplasm or tumor. The cancer may be benign or malignant and may be primary or secondary (metastatic). A neoplasm or tumor may be any abnormal growth or proliferation of cells and may be located in any tissue. The cancer may be of tissues / cells derived from e.g. the adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain) cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g. renal epithelia), gallbladder, oesophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal glad, larynx, liver, lung, lymph, lymph node, lymphoblast, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissues, spleen, stomach, testis, thymus, thyroid gland, tongue, tonsil, trachea, uterus, vulva, white blood cells.

[0335] Tumors to be treated may be nervous or non-nervous system tumors. Nervous system tumors may originate either in the central or peripheral nervous system, e.g. glioma, medulloblastoma, meningioma, neurofibroma, ependymoma, Schwannoma, neurofibrosarcoma, astrocytoma and oligodendroglioma. Non-nervous system cancers / tumors may originate in any other non-nervous tissue, examples include melanoma, mesothelioma, lymphoma, myeloma, leukemia, Non-Hodgkin’s lymphoma (NHL), Hodgkin’s lymphoma, chronic myelogenous leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma (CTCL), chronic lymphocytic leukemia (CLL), hepatoma, epidermoid carcinoma, prostate carcinoma, breast cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, thymic carcinoma, NSCLC, hematologic cancer and sarcoma.

[0336] In some embodiments, the TAA may be PSMA and the cancer may be prostate cancer.

[0337] In certain embodiments, the antigen binding molecule (including but not limited to the bispecific antibody) or the pharmaceutical composition are administered in combination with an additional therapeutic modality. “In combination” as used herein refers to the use of two or more therapeutic modalities to achieve the intended use, indication or effect, where such modalities may be administered to the patient at the same time or sequentially.

[0338] In certain embodiments, the additional therapeutic modality is an anti-cancer therapy. In certain embodiments, the anti-cancer therapy is an immunotherapy such as a CAR-T cell therapy, T-cell engager, dendrite cell vaccine, radioimmunoconjugate, antibody drug conjugate, cytokine therapy, PARP inhibitor, AKT inhibitor, Tyr-kinase inhibitor, checkpoint inhibitor, chemotherapy, hormone therapy, radiation therapy, cryotherapy, alternating electric field therapy, hyperthermia, vaccine and / or surgical intervention such as surgical removal of tumor tissue.

[0339] In certain embodiments, the immunotherapy is a cytokine therapy. In certain embodiments, the cytokine therapy is recombinant IFNy such as recombinant IFNy-1 b (Actimmune®). In certain embodiments, recombinant IFNy-1 b is administered at a dose of between 5 and 50 pg / m2(e.g., 5, 10, 20, 30, 40, or 50, pg / m2). In certain embodiments, recombinant IFNy-1 b is administered at a dose of 50 pg / m2. In certain embodiments, the cytokine therapy is selected from the group consisting of NL-201 (IL-2 / IL-15), BNZ-1 (inhibitor of IL-2, IL-9, and IL-15), nemvaleukin alfa (I L-2R agonist), SON-3015 (anti-IL6-FHAB-Anti- TGFp), SON-1410 (IL-18-FHAB-IL-12), SON-1210 (IL-12-FHAB-IL-15), SON-1010 (IL-12-FHAB), PF- 07209960 (PDCD PD-1 targeting region fused to IL-15 mutein), XmAb306 (IL-15 / IL15Ra), Fibromun (L19 antibody and TNF), ProscaVax (PSA, IL-2, and GM-CSF), Bempegaldesleukin (IL-2), and INO-5151 (PSA, PSMA, and IL-12-encoding vector). In certain embodiments, the antigen binding molecules of the present invention may serve to increase half-life and / or plasma concentration of exogenously administered I FNy, and / or decrease side effects associated with exogenously administered IFNy (e.g., in the case of antigen binding molecules which bias the pro-and anti-inflammatory effects of IFNy in favour of the pro-inf I mmatory activities) and / or target exogenously administered IFNy to a target cell or tissue (e.g., in the case of multispecific, e.g., bispecific, antigen binding molecules).

[0340] In certain 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 certain embodiments, the checkpoint inhibitor is a PD-L1 inhibitory antibody selected from the group consisting of atezolizumab, avelumab, garivulimab, durvalumab, LY3300054 and BGB-A333.

[0341] In certain embodiments, the checkpoint inhibitor is a CTLA-4 inhibitor. In certain embodiments, the CTLA- 4 inhibitor is selected from the group consisting of ipilimumab, tremelimumab, pembralizumab, atezolizumab, and avelumab.

[0342] In certain embodiments, the checkpoint inhibitor is a LAG-3 inhibitor. In certain embodiments, 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 Biologies), SRF388 (Surface Oncology), IMP321 (Immutep), MK-4280 (Merck), REGN3767 (Regeneron), TSR-033 (GSK), and IBI110 (Innovent Biologies).

[0343] In certain embodiments, the checkpoint inhibitor is a TIGIT inhibitor. In certain embodiments, 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).

[0344] In certain embodiments, the checkpoint inhibitor is a TIM-3 inhibitor. In certain embodiments, 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).

[0345] In certain embodiments, the checkpoint inhibitor is a GITR inhibitor. In certain embodiments, 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), MK-4166 (Merck).

[0346] In certain embodiments, the checkpoint inhibitor is a CD40 / CD40L inhibitor. In certain embodiments, the CD40 / CD40L inhibitor is selected from the group consisting of dacetuzumab, selicrelumab, lucatumumab, mitazalimab, sotigalimab, CP-870893 (Pfizer), CDX-1140 (CellDex Therapeutics), NG-350A (PsiOxus Therapeutics), APX005M (Apexigen) and JNJ-64457107 (Alligator Bioscience / Janssen Biotech Inc.). In certain embodiments, the checkpoint inhibitor is an 0X40 inhibitor. In certain embodiments, the 0X40 inhibitor is selected from the group consisting of PF-04518600 (Pfizer), MEDI6469 (Medlmmune), GSK3174998 (GSK), MEDI0562 (Medlmmune), BMS986178 (Bristol-Meyer Squibb), INCAGN01949 (Agenus), MOXR0916 (Roche), INBRX-106 (Inhibrx), IBI101 (Innovent Biologies), MEDI6383 (Medlmmune), and BGB-A445 (BeiGene).

[0347] In certain embodiments, the checkpoint inhibitor is a 4-1 BB / CD137 inhibitor. In certain embodiments, the 4-1 BB / CD137 inhibitor is selected from the group consisting of urelumab, ADG106 (Adagene), MEDI1873 (Medlmmune), LVGN6051 (Lyvgen), and AGEN2373 (Agenus).

[0348] In certain embodiments, the checkpoint inhibitor is an ICOS inhibitor. In certain embodiments, the ICOS inhibitor is selected from the group consisting of GSK3359609 (GSK) and MEDI570 (Medlmmune). In certain embodiments, the checkpoint inhibitor is a NKG2A inhibitor. In certain embodiments, the NKG2A inhibitor is monalizumab. In certain embodiments, the checkpoint inhibitor is a CD47 inhibitor. In certain embodiments, the CD47 inhibitor is selected from the group consisting of megrolimab, IBI188 (Innovent Biologies), 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 certain embodiments, the checkpoint inhibitor is a SIRPa inhibitor. In certain embodiments, the SIRPa inhibitor is BI765063 (Behringer-Ingelheim) or CC-95251 (Celgene). In certain embodiments, the checkpoint inhibitor is an A2A inhibitor, such as Ciforadenant.

[0349] In certain embodiments, the immunotherapy is a T cell engager. In certain embodiments, the T cell engager 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.

[0350] In certain embodiments, the checkpoint inhibitor is a bispecific checkpoint inhibitor. In certain embodiments, the bispecific checkpoint inhibitor is specific for PD-1 and LAG-3. In certain embodiments, the PD-1 / LAG-3 bispecific checkpoint inhibitor is MGD013 (MacroGenics). In certain embodiments, the bispecific checkpoint inhibitor is specific for PD-1 and TIM-3. In certain embodiments, the PD-1 / TIM-3 bispecific checkpoint inhibitor is RO7121661 (Eli Lilly). In certain embodiments, the bispecific checkpoint inhibitor is specific for PD-1 and CD47. In certain embodiments, the PD-1 / CD47 bispecific checkpoint inhibitor is HX009 (HanxBio). In certain embodiments, the bispecific checkpoint inhibitor is specific for PD- L1 and CD47. In certain embodiments, the PD-L1 / CD47 bispecific checkpoint inhibitor is IBI322 (Innovent Biologies). In certain embodiments, the bispecific checkpoint inhibitor is specific for PD-L1 and 4-1 BB. In certain embodiments, the PD-L1 / 4-1 BB bispecific checkpoint inhibitor is MCLA-145 (Merus). In certain embodiments, the chemotherapy is selected from the group consisting of one or more (e.g., 1 , 2, 3, 4, 5, or more) of Docetaxel (Taxotere), Cabazitaxel (Jevtana), Mitoxantrone (Novantrone), Olaparib / Lynparza, Rucaparib / Rubraca (mCRPC), Carboplatin, Talazoparib (Talzenna), Niraparib / Zejula, lpatasertib / RG7440, Cabometyx / Cometriq, Lutrate (leuprolide acetate; anti-hormone), PNT2002 (radioligand), 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 that targets B7- H3), and FOR46 (CD46-ADC).

[0351] In certain embodiments, 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.

[0352] In certain embodiments, the radiotherapy is selected from the group consisting of radium-223 chloride injection, lutetium (Lu)-177 vipivotide tetraxetan, and BAY2315497.

[0353] In certain embodiments, the vaccine is a dendritic cell (DC)-targeting vaccine selected from Sipuleucel-T and Stapuldencel T. In certain embodiments the vaccine is rilimogene galvacirepvec / glafolivec or CAN- 2409.

[0354] In certain embodiments, the one or more additional therapeutic modalities is surgery (e.g., tumor resection, orchiectomy, cryosurgery, and radiosurgery).

[0355] In one embodiment the one or more additional therapeutic modalities includes a virus-based therapy such as Prostvac and CAN-2409.

[0356] In certain embodiments, the additional therapeutic modality is administered to the subject prior to, concurrently with, or following administration of the antigen binding molecule or the pharmaceutical composition.

[0357] When two or more compositions are administered, the compositions are, e.g., administered in combination (either sequentially or simultaneously). In certain embodiments, the one or more additional therapeutic agents or modalities are administered immediately before or after the composition, or 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, one week, two weeks, 1 month, or more before or after administration of the composition. In certain embodiments, a composition is administered in a single dose or multiple doses. It is not required that the compositions are formulated or packaged together or administered at the same time.

[0358] The antigen-binding moieties of the invention may be administered using any route of administration known in the art, e.g., topical, parenteral, systemic, intracavitary, intravenous, intra-arterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, intrathecal, oral or transdermal routes of administration which may include injection or infusion. In some embodiments, the antigen-binding moieties of the present application are administered into tissue, e.g., intradermally, intramuscularly or subcutaneously. The administration may be by injection. For instance, administration may be by subcutaneous injection. The molecules of the present invention may have the advantage of avoiding local side effects in tissue and / or at the injection site as compared to equivalent methods of administering IFNy itself.

[0359] Subjects

[0360] The subject in accordance with aspects of the present disclosure may be any animal or human. The subject is preferably mammalian, more preferably human. The subject may be male or female. The subject may be a patient. A subject may have been diagnosed with a disease or condition requiring treatment (e.g. a cancer), may be suspected of having such a disease / condition, or may be at risk of developing / contracting such a disease / condition.

[0361] In embodiments according to the present disclosure the subject is preferably a human subject. In some embodiments, the subject to be treated according to a therapeutic or prophylactic method of the present disclosure is a subject having, or at risk of developing, a disease described herein (e.g. a cancer). In embodiments according to the present disclosure, a subject may be selected for treatment according to the methods based on characterisation for certain markers of such disease / condition. The subject may have (e.g. may have been determined to have) a cancer described herein.

[0362] Sequences

[0363]

[0364] The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.

[0365] 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 given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention.

[0366] For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations.

[0367] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0368] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0369] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example + / - 10%.

[0370] EXAMPLES

[0371] For standard molecular biology techniques, see Sambrook, J., Russel, D.W. Molecular Cloning, A Laboratory Manual. 3 ed. 2001 , Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press. The following examples are put forth to provide those of ordinary skill in the art with a description of how the compositions and methods described herein may be used, made, and evaluated, and are intended to be purely exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their invention.

[0372] Example 1 : Anti-IFNy antibody clone generation, selection, and expansion

[0373] Methods

[0374] Two mice immunized with recombinant human IFNy 8 months prior were intraperitoneally (i.p.) injected with 90 pg recombinant IFNy (Cat. No. IFNG-1164R; Creative BioMart, New York, USA), and the spleen and lymph nodes (where visible) were excised three days later under sterile conditions. All subsequent work was carried out in a Class II microbiological safety hood with aseptic technique. All excised tissues were combined, placed in a 40 pm filter, mechanically disrupted using a 10 mL syringe plunger, and washed with 50 mL Opti-MEM (Thermofisher Scientific, Loughborough, UK) to create a single cell suspension. Cells were pelleted, washed twice with 50 mL Opti-MEM, and resuspended in 0.5 mL MACS buffer (Opti-MEM, 20% FCS; GE Healthcare, Little Chalfont, UK), 1 % BM Condimed H1 (Sigma-Aldrich, Poole, UK)). Live cell counting using trypan-blue viability stain was carried out using a Luna-Il instrument (Logos Biosystems, Annandale, USA). For negative MACS selection, 100 pL of Pan B Cell Biotin- Antibody Cocktail (Miltenyi, Woking, UK) were added to 400 pL of MACS buffer containing 1 *108cells, incubated at 4 °C for 5 minutes. To remove lgM+ naive B cells, 200 pL anti-mouse IgM Microbeads (Miltenyi, Woking, UK), 100 pL MACS buffer and 200 pL anti-biotin microbeads (Miltenyi, Woking, UK) were added and the mixture incubated at 4 °C for 15 minutes. 20 mL of MACS buffer were added, cells pelleted and resuspended in 500 pL of MACS buffer and allowed to pass through a pre-equilibrated LS Column (Miltenyi, Woking, UK) under gravity following which cells were washed out with 3x3 mL of MACS buffer. An equal number of logarithmically growing SP2 / 0 myeloma cells were added to the negatively selected B cells, pelleted, washed twice with 20 mL BTXpress Cytofusion Medium C (VWR, Lutterworth, UK) and resuspended in 2 mL of Cytofusion Medium C. Washed cells were transferred to a 2 mL electrofusion chamber (VWR, Lutterworth, UK) and electrofused in a ECM2001 + electroporator under conditions recommended by the manufacturer for electrofusion of fusion partners spleen (B cell) and SP2 / 0 myeloma cells (Harvard Apparatus U.K., Cambridge, UK). Cells were 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 following day, cells were pelleted, resuspended in 1 mL Opti-MEM and added to 199 mL of semisolid ClonaCell-HY Medium D (Stem Cell Technologies, Waterbeach, UK) containing 3 pg / mL FITC conjugated rlNFy protein (labeled using the Lightning-Link Fluorescein Labelling Kit; Expedeon Ltd, Cambridge, UK) according to the manufacturer’s instructions), 3.75 pg / mL AF647-conjugated F(ab’)2 fragment goat anti-mouse IgG (Stratech Scientific, Ely, UK) and 1 x Hypoxanthine / Azaserine selection agent (Sigma-Aldrich, Poole, UK). 9 mL of semisolid medium were added to each of 20 single-well PetriWell plates that were subsequently incubated at 37 °C 1 5% CO2 for 11 days. Growth plates were imaged by bright light, FITC and AF647 fluorescent detection channels in the ClonePix 2 colony picker (Molecular Devices, Wokingham, UK), and hybridoma colonies picked into 200 pL of ClonaCell-HY Medium E recovery medium (Stem Cell Technologies, Waterbeach, UK) based on colony size, morphology and EMI. Two hybridoma colonies that were identified by ClonePix 2 as positive but were too close to the side wall of growth well to be picked were aspirated manually using a P20 pipette. After 7 days at 37 °C / 5% CO2, culture supernatants were tested for the presence of anti-rhlFNy (recombinant human IFNy) and anti-rrlNFy (recombinant rhesus IFNy, the amino acid sequence of which is identical to the amino acid sequence of cynomolgus IFNy) antibodies by ELISA. Selected cultures were expanded up to 10 mL volume with hybridoma growth media (DMEM; Thermofisher Scientific, Loughborough, UK), 20% FCS, 1% BM Condimed H1 , 1 % Penicillin / streptomycin), cells frozen down in cryopreservative (90% FCS, 10% DMSO) and stored in the vapor phase of a liquid nitrogen dewar. Culture supernatants were tested initially for the presence of anti- rhlFNy and anti-rrlFNy antibodies by ELISA. 50 pL / well of 2 pg / mL rhlFNy or rrlFNy protein in PBS buffer pH 7.4 was used to coat a MICROLON medium binding ELISA plate (Greiner Bio One Ltd, Stonehouse, UK) and incubated at 4 °C overnight. The coating solution was aspirated, washed twice with PBS and blocked for 1 hour in blocking buffer (PBS / 0.8% BSA) at room temperature. Culture samples were applied neat at 50 pL / well and incubated for 60 min at 37 °C. The reagent blank consisted of 50 pL culture medium only. Samples were aspirated and wells washed four times in PBST (PBS / 0.05% Tween-20) and blotted 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 pL / well and incubated for 60 min at 37 °C. Antibody solution was subsequently removed, and wells washed three times with PBST and three times with water. A volume of 100 pL / well of HRP substrate was added and color development stopped after 4 min at room temperature with the addition of 100 pL / well 1 M hydrochloric acid. Absorbance was determined at OD450nm on a Dynex Technologies MRX TC II plate reader. All sample values were corrected by subtracting against the reagent blank value that was the average of 2 readings on every plate. Mouse IgG was quantified in supernatants by ELISA using purified mouse lgG1 protein (Sigma, Dorset, UK) as standard. 50 pL / well of 2 pg / mL goat anti-mouse IgG (Jackson ImmunoResearch Europe, Ely, UK) in PBS buffer pH 7.4 was used to coat a MICROLON medium binding ELISA plate (Greiner Bio One Ltd, Stonehouse, UK) and incubated at 4 °C overnight. The coating solution was aspirated, washed twice with PBS, and blocked for 1 hr in blocking buffer (PBS / 0.8% BSA) at room temperature. Supernatant samples were diluted serially from 1 :100 to 1 :1000 in blocking solution at 50 pL / well and incubated for 60 min at 37 °C. Sample blanks consisted of unused hybridoma growth medium diluted as samples. The mouse IgG 1 standard was added in duplicate from 1000 to 0.5 ng / mL while the reagent blank consisted of 50 pL culture medium only. Samples were aspirated and wells washed four times in PBST (PBS / 0.05% Tween-20) and blotted 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 pL / well and incubated for 60 min at 37 °C and processed as above.

[0375] Results

[0376] A negative MACS selection protocol removed non-lgG+ cells (e.g., T cells, monocytes, macrophages, neutrophils, platelets, fibroblasts, granulocytes, and erythrocytes) and lgM+ B cells from spleen and lymph node cell suspensions harvested from two mice and yielded 0.21 *107cells upon thawing. After combining with an equal number of SP2 / 0 cells (1 :1 ratio), electrofusion was carried out at a density of 0.21 X107cells / mL. After overnight culture, recovered fused cells were pelleted, resuspended in Opti-MEM and seeded in semisolid growth medium at 1 .2x104cells per mL. From 20 growth plates, 179 hybridoma colonies were selected and picked by the ClonePix 2 instrument based on size, morphology, and AF647 and FITC EMI fluorescence due to formation of immune complexes between secreted IgG immunoglobulins and immunogen specific secreted IgG immunoglobulins respectively.

[0377] Example 2: Kinetic analysis of antibodies binding to IFNy

[0378] Methods

[0379] To investigate the binding kinetics, Biacore single-cycle kinetic analysis was performed on supernatants in which the monoclonal antibody was captured on the Fc surface as the ligand and IFNy homodimer flowed over as the analyte. In the examples, both bivalent and monovalent antibody formats have been analyzed, as set out 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 run at 25 °C with either 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. Supernatants containing the antibody were diluted in running buffer and at the start of each cycle were loaded onto Fc2, Fc3, and Fc4 of the CM5 chip previously coupled with an anti-mouse capture antibody using standard amine chemistry (GE Healthcare, Little Chalfont, UK). Antibodies were captured at a flow rate of 10 pL / min to give an immobilization level (RL) of -150 RU. The surface was then allowed to stabilize. Single-cycle kinetic data was obtained with recombinant human (rh)IFNy (Aero Biosystems, Newark, USA) as the analyte at a flow rate of 40 pL / min to minimize any potential mass transport limitations. The signal from the reference channel Fc1 (no antibody) was subtracted from that of Fc2, Fc3, and Fc4 to correct for differences in non-specific binding to a reference surface. For initial screening, the affinities of the antibodies were unknown. In order generate data for all antibodies tested irrespective of their affinities, two different three-point, three-fold dilution ranges (from 1.11 nM to 10 nM or 10 nM to 90 nM) of IFNy without regeneration between each concentration was used. When recombinant rhesus (rr)IFNy produced in HEK cells from Creative BioMart (Shirley, USA) was used for screening a single three-point, three-fold dilution range from 1.11 nM to 10 nM was used.

[0380] The association phase for the three injections of increasing concentrations of IFNy was monitored for 180 seconds each time and a single dissociation phase was measured for 500 seconds following the last injection of IFNy. Regeneration of the anti-mouse capture surface was conducted with 10 mM glycine- HCL pH 1 .7. The signal from the reference channel Fc1 was subtracted from that of Fc2, Fc3, and Fc4 to correct for differences in non-specific binding to a reference surface, and a global Rmax parameter was used in the 1 -to-1 binding model.

[0381] Biacore multi-cycle kinetic analysis was also performed on selected antibodies (4F4 and 2C1) using a Biacore T200 instrument running Biacore T200 Evaluation Software V3.0.1 . In this particular example, bivalent antibody formats were used, but the same methods may also be applied to a monovalent version of the antibodies. All multi cycle kinetic experiments were run at 25°C with HBS-P+ running buffer (pH 7.4) (GE Healthcare, Little Chalfont, UK) containing 1 mg / mL BSA with 2.5 mM CaCh. Antibody supernatants were diluted in running buffer and at the start of each cycle loaded onto Fc2, Fc3 and Fc4 of the Anti-mouse capture kit (GE Healthcare, Little Chalfont, UK). Antibodies were captured at a flow rate of 10 pL / min to give an immobilization level (RL) of ~ 150 RU. The surface was then allowed to stabilize. Multi-cycle kinetic data was obtained with human (Aero Biosystems, Cat. No. IFG-H4211) or rhesus IFNy (Creative Biomart, Cat. No. IFNG-1164R) diluted in running buffer as analyte and using a flow rate of 40 pL / min to minimize any potential mass transfer effects. Multiple repeats of a blank and a repeat of a single concentration of the analyte were programmed into the kinetic run in order to check the stability of both the surface and analyte over the kinetic cycles. For kinetic analysis a six point, two-fold dilution range was selected from 100 nM to 3.125 nM IFNy. The association phase of IFNy was monitored for 200 seconds and the dissociation phase was monitored for 1200 seconds. Regeneration of the anti-mouse capture surface was conducted using injections of 10 mM glycine-HCL pH 1 .7. The signal from the reference channel Fc1 was subtracted from that of Fc2, Fc3 and Fc4 to correct for differences in nonspecific binding to a reference surface, and a global Rmax parameter was used in the 1-to-1 binding model.

[0382] Results

[0383] The tested clones displayed affinities ranging from no apparent binding to picomolar affinities. Biacore single-cycle kinetic sensorgrams and affinities for the antibody clones selected for further study (4F4, 3G1 , 16D2, 9F1 and 2C1) are shown in FIGS. 1A-1 E and affinities determined by single- or multi-cycle kinetic analysis are summarized in the table below. The 4F4 clone was also tested against rhesus IFNy in both bivalent and monovalent format (data in 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 form, and the results are shown below.

[0384] *Note: The amino acid sequence of rhesus IFNy is identical to the sequence of cynomolgus IFNy

[0385] Example 3: Screening for IFNy Neutralizing Activity Using the PathHunter® Cellular Assay Methods

[0386] To screen clones for neutralizing activity, the PathHunter® express IFNGR1 / IFNGR2 Dimerization Assay (Eurofins, Brussels, Belgium) was performed. This assay detects ligand induced dimerization of two subunits of a receptor-dimer pair. The cells have been engineered to co-express one receptor subunit fused to Enzyme Donor, and a second dimer partner fused to Enzyme Acceptor. Binding of an agonist to one receptor subunit induces it to interact with its dimer partner, forcing complementation of the two enzyme fragments. This results in the formation of a functional enzyme that hydrolyzes a substrate to generate a chemiluminescent signal.

[0387] The assay was performed according to manufacturer’s instructions. Briefly, one vial of cells were thawed, resuspended in 10 mL of pre-warmed Cell Plating Reagent, seeded at 20 pL / well in 384-well, white, clear flat-bottom tissue culture plates (Eurofins, Brussels, Belgium), and incubated overnight in a 37°C 5% CO2 humidified incubator. The following morning, 10x serial dilutions of rhlFNy (Aero Biosystems, Newark, USA) and test or control antibodies were prepared using AssayComplete™ Protein Dilution Buffer (Eurofins, Brussels, Belgium) as diluent. Dilutions of IFNy and test or control antibodies were co-incubated for 30 min at room temperature prior to addition to cells, then 5 pL of these pre-mixes were transferred to the cells, and plates were incubated in a 37 °C 5% CO2 humidified incubator for 6 hr. Detection Reagent was prepared by mixing 1 part Flash Cell Assay Buffer and 4 parts Flash Substrate Reagent. 30 pL Detection Reagent was added per well, and the plate was incubated at room temperature for 1 hr in the dark. Luminescence was measured on a SpectraMax i3x plate reader (Molecular Devices, Wokingham, UK). GraphPad Prism 8 was used for data analysis and data was fitted using a four-parameter non-linear regression. In the initial screen, supernatants from the 30 ELISA positive clones, selected based on performance in the rhlFNy binding ELISA, were assessed at 200, 66.6, 22.2 and 7.4 ng / mL final concentrations. In cases where samples were not concentrated enough, the titration was started at the highest possible concentration. Results were compared to reference neutralising (Mabtech MT111W, Sweden) and non-neutralising (Mabtech 11 i, Sweden) antibodies (prepared as a seven point, two-fold dilution series starting at 100 ng / mL). IFNy was diluted to a 5 ng / mL final concentration for all screening experiments.

[0388] In the second screen, the 22 expanded clones were quantified and tested as serial dilutions to obtain dose-responses and IC50 values. Test antibodies were prepared at 200 ng / mL starting concentration, and a seven point, two-fold serial dilution was performed. IFNy was diluted to a 5 ng / mL final concentration. Only selected data from second screen is shown, Fig. 1 F.

[0389] Results

[0390] The effect of the antibodies ranged from no effect to very potent inhibition at all concentrations in the case of 9F1.

[0391] The five selected antibody clones 4F4, 3G1 , 16D2, 9F1 and 2C1 are shown in FIG. 1 F. Two of the clones 3G1 and 4F4 showed no apparent inhibition of receptor tetramerization in the tested concentration range. Two of the clones 2C1 and 16D2 displayed a dose dependent inhibition of the receptor tetramerization and the final mAb 9F1 induced an almost complete inhibition even at the lowest dose.

[0392] Example 4: Antibody-mediated IFNy accumulation in cynomolgus monkeys

[0393] Methods

[0394] To probe the ability of the disclosed IFNy antibodies to stabilize and or accumulate endogenous IFNy in vivo, experiments were performed in cynomolgus monkeys (Macaca fascicularis).

[0395] For these experiments, truncated monovalent versions of IFNy antibody clone 2C4, 8D1 , 4F4 and 19F10 were prepared in the following way: Monovalent lgG1 .1 / Fc1 .1 (K409R / F405L) 2C4, 8D1 , 4F4 (SEQ ID NO: 75 and 76) or 19F10 antibody was produced through the redox reaction between lgG1 .1 (K409R) 2C4, 8D1 , 4F4 or 19F10 chimeric antibody and lgG1 .1 Fc(H-CH2-CH3)(F405L) (SEQ ID NO: 83) both produced in CHO cells. For example, the monovalent version of antibody 4F4 was composed 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 4.7mL column (GE Healthcare) using 20 mM Sodium Acetate, pH 5.0 with a 0-100% gradient of 300 mM Sodium Chloride as the mobile phase. Peak fractions, eluting around 150 mM Sodium Chloride were analyzed and those containing the desired monovalent species were pooled and filter sterilised before quantification by A280nm using an extinction coefficient (Ec(0.1 %)) based on the predicted amino acid sequence.

[0396] Antibody clones (in the truncated monovalent versions) 2C4, 8D1 , 4F4 and 19F10 were tested in single bolus injections of two dosages (1 mg / kg and 3 mg / kg) in cynomolgus monkeys. One peptide variant (Actimmune®) was tested in one dosage every fourth day (1 .5 pg / kg). The results were compared to naive animals injected with a saline single bolus (control group). Each group included two animals. The animals were sedated and dosed with antibodies / Actimmune® through intravenous injection. The subjects of the control group were sedated and dosed with saline through intravenous injection. Subsequently, blood samples were collected on day 0; 24 hrs; 48 hrs; 96 hrs; 192 hrs; 288 hrs; and 360 hrs. Blood samples were analyzed to determine the circulating concentrations of the antibodies and IFNy using ELISA kits. The study was terminated after two weeks, at which point the antibodies were expected to have been eliminated and IFNy levels to have normalized.

[0397] Results

[0398] Four antibodies capable of binding rhesus ( / W. Mulata) IFNy and human IFNy were tested for their ability to accumulate IFNy in cynomolgus monkeys ( / W. fascicularis) . As the amino acid sequence of rhesus monkey IFNy is identical to the amino acid sequence of cynomolgus monkey IFNy, the binding characteristics of the antibodies to IFNy from these two species are expected to be similar. The animals were injected with either 1 mg / kg or 3 mg / kg of antibody, and the resulting increase in IFNy plasma concentration over time was recorded using ELISA. All four antibodies produced an elevated concentration of IFNy in cynomolgus monkeys. The 4F4 antibody, having an affinity of 0.1 nM towards rhesus IFNy, elevated the IFNy concentration from 10-100 pg / mL to about 5000 pg / mL, corresponding to a 50-500 fold increase, at its peak at four days post-injection when applied at 3 mg / kg (FIG. 1G).

[0399] Example 5: IFNy / IFNyR-biasing properties of antibodies

[0400] To test the effects of the antibodies on the cellular cascades downstream of the IFNgR we chose to use two cellular outputs of the IFNyR signaling cascade as a proxy. We sought to identify antibodies that maintain or boost IFNy mediated MHC-1 expression on cancer cells and at the same time reduce PD-L1 expression as this bias is understood to be pro inflammatory and thus beneficial for cancer therapy. To do so PC-3 prostate cancer cells or A549 lung cancer cells were stimulated for 48 hours with a fixed dose of IFNg at 1 ng / ml (0.6 nM) which gives a full activation of the receptor but still is within the dynamic concentration range of the stimulation. To test the effects of the antibodies these were added on top at a concentration range going from 0.1 nM to 100 nM for PC-3 cells and 100 to 10,000 ng / mL for A549 cells.

[0401] Methods

[0402] For PC-3 cells:

[0403] PC-3 cells (not expressing PSMA) were seeded in 25 cm2flasks with 4 ml medium with supplements and 0.1-0.2 million cells per flask on day 1 (a 10x dilution).

[0404] On day 3 cells were stimulated with addition of IFNy / mAbs prepared as follows:

[0405] IFNy and antibody were sterile filtered and added to a cell culture medium (400pl) such that when subsequently added to the medium in a cell culture flask (4ml) the final concentration of IFNy would be 1 ng / ml and the final concentration of antibody would be 0.1 , 1 , 10 or 100 nM. The cell culture medium comprising the mix of IFNy and antibody was incubated at room temp for 30 min to allow pre-complexing between IFNy and antibody before adding to the cell culture flask.

[0406] Cells were then incubated for an additional 48h at 37°C before flow cytometry was performed on day 5. 48h post stimulation cells growing in 25 cm2flasks were harvested by removing medium by decanting and adding 1 ml Trypsin solution (Thermofisher: 25200056) at RT. After about 10 min 1 ml cell culture medium with FBS was added and the total of 2 ml was transferred to a 2 ml Eppendorf tube and the cells were pelleted by centrifugation at 100 g for 5 min at 4C. Cells were resuspended in 1 ml medium with FBS and 450 ul was transferred into 2 x 1 .5 ml Eppendorf tubes and pelleted again.

[0407] After removing medium one 1 .5 ml tube had 500 ul medium containing 0.5 ul mouse anti MHC-1 (Merck: MABN1783) and 0.5 ul rabbit anti PD-L1 (Abeam: ab205921) mixed in. Cells were shaken and incubated on ice for 40 min. Cells were then pelleted again by centrifugation at 100 g for 5 min, washed in 500 ul medium + FBS, pelleted at 100 g and then incubated with 500 ul medium containing 0.25 ul Goat anti mouse Alexa 488 (ThermoFisher: A-11001) and 0.25 ul Goat anti rabbit Alexa 647 (ThermoFisher: A- 21244) for 20 min.

[0408] Then the cells were washed again in 500 ul medium and resuspended into 200 ul medium and transferred to a 96 well plate (Thermo scientific: 249570). Cells were immediately thereafter analyzed by flow cytometry using the Invitrogen Attune Next 4 laser system.

[0409] The Attune Next 4 laser system lasers were calibrated using standard beads according to the guidelines of the system. Lasers used were FSC, SSC, BL1 (GFP) and RL1 (Alexa 647). A gate was defined around the event population representing cells and laser intensities were adjusted such that the signal for unstained cells were about 10A3 units in the RL1 and BL1 channels respectively.

[0410] Each sample (100 ul of each of the loaded samples) was injected into the system at 200 ul / min and the signal in BL1 and RL1 were recorded. The BL-1 channel was used to detect Alexa 488 as a measure of MHC-1 and the RL-1 was used to detect Alexa 647 as a measure of PD-L1 expression.

[0411] The increase in MHC-1 and PDL-I expression when stimulating cells with IFNy vs untreated cells was apparent, especially for PD-L1 , by an increase in the RL-1 signal. Also note that the morphology of the PC- 3 cells changed upon IFNy treatment such that the cluster of cells would move slightly upwards and to the left with the gate defined in the SSC / FSC plot.

[0412] Data treatment was done using the FlowJo software package. As in the attune software a gate was defined around cells in the first sample.

[0413] Next the mean BL-1 A (Alexa488) and RL-1 A signal (Alexa 647) was plotted by activating the statistics menu by right clicking on the line for each gate. The gate setting was then copied onto all subsequent samples in the data set by drag and drop. The statistics value for each population was then manually copied to a prism data sheet and plotted.

[0414] For A549 cells:

[0415] A549 cells were cultured in RPMI 1640 medium containing 10% FBS and 1 % PenStrep at 37 °C in 5% CO2.

[0416] Cells were plated into 96-well plates and stimulated with 1 ng / mL human IFNy pre-complexed with selected IFNy antibodies at three concentrations (10,000, 1000, 100 ng / mL or ~ 67, 6.7 and 0.7 nM) for 48h in duplicate. MT111 W neutralising antibody was used as internal control (1000 - 0.1 ng / mL) also in duplicate. The assay window was defined by A549 cells + / - 1 ng / mL human IFNy.

[0417] For 4F4, the concentration was too low to achieve 10,000 ng / mL. Therefore, the max concentration was adjusted to 5000 ng / mL or the highest possible.

[0418] Cells were detached with PBS + 12.5 mM EDTA, fixed and analysed by flow cytometry using Attune NxT.

[0419] Dead cells were discriminated using the Live / Dead near IR (Invitrogen), non-specific antibody binding was minimized using Human FC Block (BD) and surface staining was performed with PE-Dazzle conjugated anti-PD-L1 (clone 29E.2A3, Biolegend) and v450-conjugated anti-HLA-ABC (clone G46-2.6, BD)

[0420] For both PC-3 and A549 cells:

[0421] MFI values representing MHC-1 and PD-L1 surface expression were normalized by subtracting from the MFI value of the sample (“sample MFI”) the MFI value when no antibody and no IFNy was added (“baseline MFI”) and dividing this by the MFI value when only IFNy was added (IFNy only MFI) with baseline MFI subtracted ((sample MFI - baseline MFI) / (IFNy only MFI - baseline MFI))*100.

[0422] Results

[0423] Tested antibodies showed a range of properties. The five tested IFNy antibody clones 4F4, 3G1 , 16D2, 9F1 and 2C1 were shown to inhibit PD-L1 surface expression more strongly than MHC-1 on PC-3 cells as seen by a “left shift” of the PD-L1 inhibition curves relative to their respective MHC-1 curves - see FIG. 2A- E. The same properties were seen for 4F4 and 9F1 tested on A549 cells - see FIG. 2F.

[0424] Thus, it is concluded that all these antibodies are “preferentially inhibitory of PD-L1 over MHC-1”. 9F1 showed particularly high inhibition of PD-L1 , while maintaining a level of MHC-1 expression at 50%-80% on PC-3 cells.

[0425] Example 6: IFNy / IFNy-R interaction modulatory properties of antibody 4F4 on PC-3 cells

[0426] To gain further insight to the difference in inhibition of MHC-1 vs PD-L1 by our IFNy antibodies as shown in example 5, saturation stimulation curves of IFNy on PC-3 cells were made with and without the presence of 50 nM antibody 4F4.

[0427] Methods

[0428] PC-3 cells (not expressing PSMA) were seeded in 25 cm2flasks 48 hr prior to stimulation. A fixed amount of 4F4 antibody giving a final concentration of 50 nM after being applied to the cells was mixed with ActiveMax IFNy to give a final dose 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) IFNy in the cell culture. The 4F4 / IFNy mix (400 pL) was incubated at room temperature for 30 min to allow pre-complexing between the proteins before being added to the cell culture flasks giving a final volume of 4.4 mL as described in example 5. After an additional 48 hr, the 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 trafficking. The effect of the combination treatment of 4F4 / IFNy was then probed using a co-staining with 1 :500 mouse anti-MHC-1 (Sigma MABN1783) and 1 :500 rabbit anti-PD-L1 (Abeam ab205921) on ice for 45 min, followed by a wash and treatment with 1 :2000 goat anti-mouse Alexa 488 (ThermoFisher A-11001) and goat anti-rabbit Alexa 647 (ThermoFisher A-21244) for 30 min. After washing, the labeling intensity was determined by flow cytometry using the Attune Nxt system and Blue-1 and Red- 1 lasers. Data was analysed using the FlowJo software package as described in example 5.

[0429] Results

[0430] The surface expression level of MHC-1 and PD-L1 upon antibody / IFNy stimulation is shown in Figure 2G. Data show that both the expression of MHC-1 and PD-L1 were inhibited by 4F4 to a similar relative level as it was seen on A549 lung cancer cells (Fig. 2F). This confirms that the recombinant 4F4 retains the properties of the original clone and that the effect of 4F4 can be transferred between cancer types and cell lines.

[0431] Furthermore, the results suggest that the modulating / biasing effect of 4F4 may involve a mechanism that lowers the effective affinity between IFNy and IFNyR as demonstrated by Mendoza et al [Nature, Vol 567, 2019] using variants of IFNy with different affinities to IFNyR. An effect which is more pronounced when probing of PD-L1 versus MHC-1 signaling. This is apparent from a rightward shift of the dose response curves (Fig. 2G) for both MHC-1 and PD-L1 but at a more pronounced shift for PD-L1 over MHC-1 .

[0432] Example 7: Bispecific antibody design

[0433] Methods

[0434] DuoBody recombination (LDC-1)

[0435] The VH region of antibody 4F4 was fused to a human IgG 1 constant region (amino acid residues 120-449) with mutations L234A / L235E / G237A / A330S / P331 S and the DuoBody mutation K409R (SEQ ID NO: 75) and the VL region of 4F4 (SEQ ID NO: 76) was grafted onto a human IgKappa. A variant of the PSMA- binding antibody J591 ANT4044 was fused onto a L234A / L235E / G237A / A330S / P331S and F405L lgG1 constant region (SEQ ID NO: 77 and 78). The antibodies were custom expressed by Genscript and a Fab arm exchange was conducted by mixing a 1 molar 4044 F405L to 1 .2 molar 4F4 K409R ratio at a total of 1 mg / mL in PBS. Reduction of disulfide bonds was achieved by added 75 mM 2-MEA for 4 hours at 31 °C after which 2-MEA was removed using dialysis into PBS in 3 mL 10 kDa dialysis cassettes. This protein construct constitutes LDC-1. The sequences of this antibody are shown as SEQ ID NOs: 75-78 (two light chains and two heavy chains).

[0436] Knob-into-hole design (LDC-2, LDC-4 and LDC-6)

[0437] A single chain variable fragment (scFv) domain variant of the PSMA-binding antibody ANT4044 derived from antibody J591 was designed by fusing Vnwith VL via an 18 residue -GSTSGGGSGGGSGGGGSS linker (SEQ ID NO: 82). The scFv was fused at the hinge region (before Glu216) of a human IgG C220A + hole mutations Y349C / T366S / L368A / Y407V / F405K. The VH region of antibody 4F4 was fused to a human IgG 1 constant region with knob mutations S354C / T366W / K409A. These were co-expressed and purified as described below in a 1 :1 :1 molar ratio with 4F4 light chain. This combination constitutes LDC- 2. The sequences of this antibody are shown as SEQ ID NO: 72-74 (two heavy chains and one light chain).

[0438] In a similar way LDC-4 was generated using a construct in which the ANT4044 ScFv was replaced by the nanobody number 7 (Nb7) reported by Rosenfeld et al 2020 (J. Med. Chem. 2020, 63, 14, 7601-7615). The sequences of LDC-4 are shown in SEQ ID Nos: 65-67 (two heavy chains and one light chain).

[0439] LDC-6 is similar to LDC-4 except that the heavy and light chains of antibody 4F4 are replaced by those of 9F1. Thus, LCD-6 comprises nanobody Nb7 fused at the hinge region (before Glu216) of a human IgG C220A + hole mutations Y349C / T366S / L368A / Y407V / F405K. The VH region of antibody 9F1 was fused to a human IgG 1 constant region with knob mutations S354C / T366W / K409A. These were co-expressed and purified as described below in a 1 :1 :1 molar ratio with 9F1 light chain. The sequences of LDC-6 are shown in SEQ ID NO: 79-81 (two heavy chains and one light chain). lgG(scFv / Nb)2 design (LDC-3 and LDC-5)

[0440] The above ANT4044 scFv was fused to the C-terminal of 4F4 lgG1 L234A / L235E / G237A / A330S / P331 S This construct constituting LDC-3 was co-expressed with 4F4 light chain in a 1 :1 ration and purified as described below. The sequences of LDC-3 are shown as SEQ ID NO: 70 and 71 .

[0441] 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.

[0442] The features of the various bispecific antibodies are summarized below.

[0443]

[0444] Example 8: Bispecific antibody expression and purification

[0445] Bispecific antibodies as described above were fused with signal peptides for secretion (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) and the 5-day expression protocol according to the manufacturer. Secreted antibodies were purified from the cell culture medium using centrifugation to remove cells and loading onto 1 ml HiTrap® MabSelect™ PrismA (Cytiva 17549852) at 0.5 mL / min. Columns were washed in 20 mM Hepes pH 7.4, 100 mM NaCI and eluted using 100 mM Na-citrate after which collected samples were partially neutralized using Tris pH 9.0 and put into dialysis ON in PBS buffer.

[0446] Example 9: Anti-PSMA antibody binding affinity determination

[0447] Methods

[0448] The affinities of the disclosed bispecific antibody constructs against PSMA were determined by making saturation curves using either LNCaP cells naturally expressing PSMA or PC-3 cells transiently transfected with eGFP-PSMA, as described below.

[0449] Cell maintenance and transfection

[0450] LNCaP (Merck: 89110211-1 VL) cells were maintained 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) in 75 cm2cell culture flasks with about 11-12 ml medium in each at 37 °C and 5% CO2.

[0451] PC-3 cells were maintained in RPMI 1640 + Glutamax (ThermoFisher: 72400-021) containing 10 % FBS (Merck: F2442-100ML) and 1% Penstrep (ThermoFisher: 15140122) in 75 cm2cell culture flasks with about 11 -12 ml medium in each at 37 °C and 5% CO2.

[0452] Cells were split Monday and Friday at 10x and 20x dilution each time for PC-3 and 4x and 6x for LNCaP as these grew slower. Cells were split using 3 ml Trypsin-EDTA (ThermoFisher 25200056) per 75 cm2 and then diluted with 3 ml medium incl. FBS to quench the Trypsin. Cells were then pelleted at 100 g for 5 min. before seeding into 10 ml fresh medium.

[0453] PC-3 cells were transiently transfected with eGFP-M1 A / L5A-PSMA in pcDNA3.1 . The M1 A / L5A mutation set was introduced to reduce internalization. Transfections were made using Lipofectamine 2000 (Invitrogen: 11668030). 1.5 ml RMPI-1640 without supplements had 50 pl Lipofectamine added, and 1.5 ml had 20 pl 1 mg / ml DNA added. After 5-10 min at room temp, the two tubes were mixed and incubated at room temp, for an additional 20-30 min after which the mix was added to cells. Cells were split 2x Monday from about 80% (about 6 mio cells pr 75 cm2) confluent cell culture flask into a 75 cm2flask, such that that this was about 70% confluent Tuesday. Medium was then replaced with 8 ml serum and Penstrep-free medium and the 3 ml DNA / Lipofectamine mix was added on top, and cells were returned to 37 °C for 4 hours after which cells were split into normal serum-containing medium in 25 cm2flasks with about 0.4-0.5 million cells per flask and 4 ml medium in total.

[0454] Determining Affinity

[0455] At the appropriate time (typically after 48h in the case of transfected cells) cells were harvested from the culture flasks using trypsin, washed and resuspended in cell culture medium and kept on ice to stop membrane protein trafficking. Cells were then resuspended in either 1.5 mL Eppendorf tubes (about 0.6 million cells / tube) or 96-well plates in cell medium in which a dilution curve of the bispecific antibody to be tested was made (500 pL if using Eppendorf tubes). After 45 min of incubation on ice, cells were centrifuged for 5 min at 100-150 g to pellet cells, and the cells were washed once in cell medium before being incubated for 30 min in medium containing 1 :2000 goat anti-human Alexa 488 (Invitrogen A-11013) or goat anti-human Alexa 647 (Invitrogen A-21445), if using GFP-PSMA transfected cells. Cells were centrifuged for 5 min at 100-150 g to pellet cells, and the cells were washed once in cell medium after which the labeling intensity was determined using the Attune Nxt flow cytometry system and Blue-1 and Red-1 lasers. Data was analyzed using the FlowJo software package.

[0456] Results

[0457] To test the bispecific antibodies for their affinity towards PSMA, saturation curves using PSMA-expressing prostate cancer cells were generated (FIG. 3). With a Kd of 4 nM, LDC-1 largely retained the affinity of the parent ANT4044 antibody. The KiH-scFv (LDC-2) and the Nb7 based LDC-4 and LDC-5 displayed affinities from 1 .5 to 7 nM, respectively. lgG(scFv)2 (LDC-3) had a weaker affinity (about 120 nM), however this antibody was not purified from the cell culture medium by SEC before testing affinity, which may affect the results.

[0458] Example 10: LDC-mediated IFNy recruitment to PSMA expressing prostate cancer cells

[0459] To confirm that 4F4 retained its IFNy-binding properties after being converted into a bispecific format and that this format can bind PSMA to recruit IFNy specifically to PSMA-expressing cells, binding experiments were performed on cells expressing PSMA using purified IFNy protein.

[0460] Methods

[0461] The ability of our 4F4-based bispecific antibodies to recruit IFNy to PSMA-expressing PC cells was shown using two different setups. LDC-1 (a DuoBody format based on 4F4 and ANT4044) was tested using a binding assay on LNCaP (PSMA-positive) versus PC-3 (PSMA-negative) cells using an anti-IFNy antibody to detect the presence of IFNy. To test LDC-1 , LNCaP and PC-3 cells were cultured in parallel, harvested using trypsin, and washed in culture medium on ice. Cells were incubated with or without 20 nM LDC-1 for 45 min, pelleted at 100 g for 5 min and then washed in culture medium. Then cells were incubated with 100 nM IFNy (Acrobiosystems cat. Nr. IFG-H4211) for 30 min and then washed. The bound IFNy was then detected using 1 :500 rat anti-IFNy (Mabtech MT111W) for 30 min followed by 1 :1000 anti-rat Alexa 488 secondary antibody (ThermoFisher cat. Nr. A-11006) for 15 min. After washing, the labeling intensity was determined by flow cytometry using the Attune Nxt system and Blue-1 laser.

[0462] To test the IFNy recruiting properties of LDC-2 (a KiH format based on 4F4 and ANT4044 ScFv), human IFNy was labeled with Alexa 647 fluorophore. Human IFNy (Acrobiosystems cat. Nr. IFG-H4211) was dissolved in water at 1 mg / mL and buffer exchanged into PBS buffer adjusted to pH 6.5 with 20 mM Na- Citrate (pH 3.0) buffer using Micro Bio-Spin™ P-6 Gel Columns (BioRad cat. Nr. 7326221). The IFNy was then labeled with either 5- or 20-fold molar excess of NHS-Alexa 647 (ThermoFisher cat. Nr. A37573) ON at 4C. Excess dye was removed using Micro Bio-spin column exchange into PBS buffer. PC-3 cells transfected with GFP-PSMA were cultured in 75 cm2flasks in RPMI 1640 containing 10% FBS and 1% PenStrep at 37 °C and 5% CO2 and then harvested using trypsin and washed in culture medium by centrifugation at 150 g for 5 min. Approx. 0.6 million cells were distributed into 1 .5 mL Eppendorf tubes and incubated on ice for 45 min with increasing concentrations of LDC-2. Cells were centrifuged for 5 min at 100-150 g to pellet cells and the cells were washed once in cell medium after which the cells were incubated with 50 nM Alexa 647 IFNy for 45 min. Cells were washed and the labeling intensity was determined using the Attune Nxt system Blue-1 laser to distinguish between GFP-PSMA expressing and non-expressing cells and its Red-1 laser to quantify the amount of bound IFNy. Data was analysed using the FlowJo software package.

[0463] Results

[0464] LDC-1 was tested for its ability to recruit IFNy to PSMA-expressing LNCaP cells (FIG. 4A, black bars). PSMA-negative PC-3 cells were tested (FIG. 4A, gray bars).

[0465] PC-3 cells were shown to have higher background binding than LNCaP cells, as seen by a stronger binding of IFNy to the cells regardless of the presence of LDC-1. As expected, the presence of LDC-1 did not increase the recruitment of IFNy to PSMA-negative PC-3 cells.

[0466] On the other hand, PSMA-expressing LNCaP cells exhibited approximately a 4- to 5-fold increase of IFNy binding in the presence of LDC-1 , showing that the combination of LDC-1 and PSMA-expression results in a robust recruitment of IFNy to the PSMA-expressing cells.

[0467] LDC-2 was tested for IFNy recruitment using PC-3 cells transfected with GFP-PSMA. IFNy was detected via direct labeling with Alexa 647. Cells which did not uptake any GFP-PSMA DNA, and, therefore, did not express PSMA, served as an internal control. The saturation curve of AF647-IFNy binding (FIG. 4B) shows an increase of AF647-IFNy binding to the cells with increasing amounts of bound LDC-2. This effect was only observed on PSMA-positive cells, confirming that the binding was specific.

[0468] Example 11 : Bispecific antibody modulated IFNy stimulation of prostate cancer cells

[0469] Objectives

[0470] The objective of this experiment was to confirm that the preferential inhibition of PD-L1 is also obtained in the bispecific format.

[0471] Methods

[0472] PC-3 IFNy sensitive cells were transfected with GFP-PSMA on day 1 as described above. The following bispecific antibodies were tested: LDC-4 and LDC-6

[0473] On day 2, 400 ul IFNy / mAbs mix was prepared to achieve a final concentration in the cell culture flasks (4 mL) of 0.025 nM (0.4 ng / ml) IFNy and 0.1 , 1 , 10 or 100 nM of antibody. This mix was incubated at room temp for 30 min to allow prebinding between IFNg and antibody before being added to the cell culture flasks containing PC-3 cells transiently transfected with GFP-PSMA.

[0474] After additional 48 hr (day 4), the effect of the combination treatment of IFNy and the LDC-4 or LDC-6 antibodies was then probed using 1 :500 mouse anti-human MHC-1 (Sigma MABN1783) or 1 :500 rabbit anti-human PD-L1 (Abeam ab205921) on ice for 45 min, followed by washing, and treatment with 1 :2000 goat anti-mouse Alexa 647 (Invitrogen A-21235) or goat anti-rabbit Alexa 647 (Invitrogen A-21244), respectively for 30 min. After washing the labeling intensity as well as GFP-PSMA expression was determined using the Attune Nxt flow cytometer system and Blue-1 and Red-1 lasers. Data was analyzed using the Flow Jo software package. As the PC-3 cells were transiently transfected (Lipofectamine) with GFP-PSMA, only a subset of these cells expressed GFP-PSMA. Therefore, by separating PC-3 cells into PSMA-expressing and non-expressing cells based on their GFP fluorescence, the cell samples effectively contained an internal control. Only the cells expressing PSMA would be expected to bind LDCs.

[0475] Results

[0476] Cells stimulated with only IFNy as expected showed a significant increase in both MHC-1 and PD-L1 surface expression over non-stimulated cells, regardless of the GFP-PSMA expression level (FIG. 4C). For both LDC-4 and -6 a dose dependent inhibition of both MHC-1 and PD-L1 surface expression regardless of PSMA expression was seen. This observation was expected based on previous experiments.

[0477] In un-transfected cells, LDC-6 inhibited MHC-1 expression to a slightly larger extent than LDC-4. This inhibitory effect was expected to partially mask the effect of any increase in IFNy-mediated upregulation of MHC-1 expression due to recruitment by LDC to PSMA-expressing cells.

[0478] Both LDC-4 and -6 inhibited PD-L1 surface expression to a greater degree than MHC-1 expression. LDC- 6 strongly decreased PD-L1 expression in both PSMA positive and PSMA negative cells.

[0479] Example 12: Bispecific antibody retains proinflammatory signaling across different cell lines

[0480] The objective of this experiment was to confirm that IFNy bound to the bispecific antibodies retains proinflammatory signaling across different cell lines.

[0481] Methods

[0482] The constructs used in this experiment are KiH-3 and KiH-6 discussed above. KiH-3 comprises the 4F4 IFNy binder and Nb7 as the TAA binder, and KiH-6 comprises 9F1 as the IFNy binder and Nb7 as the TAA binder.

[0483] The IFNy / IFNyR-biasing properties of antibodies KiH-3 and KiH-6 were tested using surface expression of MHC-I and PD-L1 on cancer cells as a proxy for IFNyR signaling as described in Example 5. PC3-FLU prostate cancer cells, A549 lung cancer cells or HT29 colorectal cancer cells, all of which are PSMA nonexpressing were stimulated for 48 hours with a fixed dose of IFNy at 1 ng / ml (0.6 nM) which gives activation of the receptor within the dynamic range of stimulation. To test the effects of the bispecific antibodies, these were additionally added at a range of concentrations. The methods were as in example 5, except that an additional concentration of antibody was tested for the A549 cells (1 Opg / mL; 87nM). HT29 cells were tested in the same way as PC3-FLU cells.

[0484] A time-course study was also performed to evaluate the effect of the bispecific antibodies KiH-3, 10 nM or KiH-6, 10nM on surface expression of MHC-I and PD-L1 on A549 cells (not expressing PSMA) and PC3- PIP cells (expressing PSMA) at 6h, 24h, 48h and 72h, with and without the presence of human IFNy (0.1 nM).

[0485] To gain further insight into the difference in inhibition of MHC-1 versus PD-L1 by the IFNy bispecific antibodies, IFNy stimulation (0.025 nM or 5.95 nM) of PC3-PIP cells (expressing PSMA) was performed, as well as stimulation with bispecific antibody KiH-3 at either 1 nM or 10 nM concentration, with or without 0.025 nM IFNy.

[0486] Results

[0487] The results of the IFNy / IFNyR-biasing experiment are shown in Figure 5. MHC-1 expression for IFNy was maintained across the different cell lines with both KiH-3 and KiH-6. MHC-1 expression was fully maintained in all cell lines at concentrations of 0.1 nM antibody. In HT29 and AT549 cells, MHC-1 expression was fully maintained at antibody concentrations of 10nM. The antibodies were partially inhibitory, since some inhibition was seen at higher concentrations, but the ICsowas greater than 0.1 nM in all cell lines. Both KiH-3 and KiH-6 were also less inhibitory than an IFNy neutralizing control antibody in A549 cells as shown in Figure 5c. The IC50 was around 1000-fold higher.

[0488] The IFNy / IFNyR-biasing properties of antibodies KiH-3 and KiH-6 are also shown in Figure 5. Both antibodies were shown to inhibit surface expression of PD-L1 more strongly than MHC-1 , particularly on A549 cells, as seen by a “left shift” of the PD-L1 inhibition curves relative to their respective MHC-1 curves (FIG. 5).

[0489] The results of the time-course study are shown in Figure 6. KiH-3 and KiH-6 did not stimulate surface expression of MHC-I or PD-L1 on any of the cells in the absence of IFNy. For the A549 cells, no inhibition of MHC-I expression was observed for either of the bispecific antibodies, whereas PD-L1 expression was inhibited by about 20% (KiH-3) and about 50% (KiH-6), respectively, relative to IFNy alone (FIG. 6C). For the PC3-PIP cells, KiH-3 inhibited MHC-I expression by 50% and PD-L1 expression by 80% relative to IFNy alone (FIG. 6A-B). Similar results were seen for KiH-6.

[0490] From this, it was concluded that both KiH-3 and KiH-6 maintain pro-inflammatory activity of IFNy and that the percentage inhibition of PD-L1 cell-surface expression was greater than that of MHC-I cell-surface expression.

[0491] This was further investigated with IFNy stimulation experiments performed with and without the presence of KiH-3, the results of which are shown in Figure 7. KiH-3 suppressed surface expression of both MHC-I and PD-L1 on PC3-PIP cells as compared to IFNy alone Further, when surface expression of MHC-I and PD-L1 in the presence of KiH-3 was normalized against IFNy alone, KiH3 suppressed MHC-I expression by 40% (1 nM) or 52% (10 nM), respectively, and suppressed PD-L1 expression by 55% (1 nM) or 61 % (10 nM) respectively. Example 13: PSMA targeted delivery of IFNy to a tumor with KiH-3

[0492] The aim of this experiment was to evaluate the ability of the KiH-3 bispecific antibody to target delivery of IFNy to a tumor. To do so, a PSMA-expressing PC-3 prostate cancer xenograft tumor model was used (FIG. 8A).

[0493] Methods

[0494] Prostate cancer xenograft tumor model

[0495] ATHYMIC Nude (BALB / cAnNRj-Foxn1 nu / nu) immunodeficient mice (male, 6 weeks of age) were purchased from Janvier, Europe. To prepare the xenograft tumor model, 1 x106PC3-PIP cells in 50 pl Matrigel were subcutaneously inoculated into the flank of each mouse. By approximately 3 weeks after inoculation, tumors had reached a size of 250 mm3.

[0496] IHC staining

[0497] When the resulting tumors reached a size of 250 mm3, mice were inoculated by intravenous administration by tail vein under isoflurane anaesthesia using a syringe. For the test group, 10 mg / kg of KiH-3 was pre-mixed with 1 .5 mg / kg IFNy (1 :1 molar ratio) and incubated for 30 min at room temperature to allow pre-complexing of the proteins prior to inoculation. For the control groups, the inoculum comprised IFNy alone (1 .5 mg / kg). After 72 h, tumors were excised from each mouse, fixed in 10% neutral-buffered formalin for 24 h and thereafter moved to 70% EtOH. The tissue specimens were then paraffin embedded within 2-3 days. The samples were trimmed, and sections were obtained for H&E staining to ensure the presence of tumor area. Slides were stained using anti-IFNy (D3H2, Cell Sign. Techn.). Digital slides were obtained from all stained slides using a bright field scanner (Zeiss AxioScan, 20x).

[0498] In Vivo Imaging

[0499] KiH3 was labelled with Alexa 680 NHS ester dye (Invitrogen: A37574) following a specific protocol designed to minimize perturbation of the antibody's function. KiH3 was first purified and dialyzed overnight. Alexa 680-NHS was dissolved in DMSO (Sigma: D2650-100ml) at a concentration of 100 pg / 10 pl. For labelling, a reaction mixture was prepared by mixing 30 pl of Alexa 680-NHS solution with 696 pl of KiH3 at a concentration of 61 ,775 nM (7.1 mg / ml) in PBS (~2 mL total volume). In this way, a molar ratio of 5 dyes per antibody was achieved. The reaction mixture was incubated overnight at 4°C. The following day, excess dye was removed using PD-10 desalting columns (Cytiva: 17085101). The columns were prepared and equilibrated with PBS buffer, and the reaction mixture was processed to collect the clean, labelled antibody. The labelled antibody was then further purified via size exclusion chromatography to remove aggregates. Three mice comprising a PC-3 prostate cancer xenograft tumor prepared as described were each inoculated with 1 dose (1-1 .5 nmol) of the Alexa680-labeled KiH-3 by intravenous administration by tail vein under isoflurane anaesthesia using a syringe. The mice were imaged using a far-red camera 3 days post-injection.

[0500] Results

[0501] The hlFNy staining observed in tumor samples from mice who received hlFNy in combination with KiH3 was stronger as compared to hlFNy alone (FIG. 8B). In particular, the presence of hlFNy was detected around the blood vessels. Thus, the levels of hlFNy in the tumor were increased in the presence of KiH3. It was therefore concluded that KiH-3 successfully targeted hlFNy to the tumor.

[0502] The results of the in vivo imaging are shown in Figure 8C. The fluorescently-labelled KiH3 antibody was detected in the tumor by in vivo imaging 3 days post-injection.

[0503] Example 14: Upregulation of MHC-I in xenograft tumors in mice

[0504] The aim of this study was to examine MHC-I expression in PSMA positive PC3-PIP prostate cancer xenograft tumors in mice dosed with KiH-3:IFNy complex by immunohistochemistry staining. MHC-I staining in this context can be seen as a signal of immune stimulation in the tumor.

[0505] Methods

[0506] The prostate cancer xenograft tumor model preparation and IHC staining were both performed as described in Example 13, except that an additional vehicle control inoculation was used and the slides were stained using anti-MHC-l (EP1395Y, #ab52922).

[0507] Results

[0508] The results are shown in Figure 9. MHC-I staining was observed in tumors of mice injected with either IFNy alone or the KiH3:IFNy complex, as compared to the vehicle control group. Notably, MHC-I was upregulated in tumors from mice which were inoculated with the KiH3:IFNy complex as compared to IFNy alone, as shown by the increase in MHC-I staining intensity.

[0509] Example 15: in vivo syngeneic model

[0510] The objective of this study was to produce a syngeneic model suitable for performing experiments in mice without inducing unwanted immunogenicity. To this end, bispecific antibody KiH-16 was prepared based on the KiH-3 construct described in Example 7, but comprising a mouse lgG2a isotype with effector silencing in place of the human constant domain region. The pharmacokinetic properties of KiH-16 were measured in this study, including tolerability and accumulation of mlFN-y in serum following KiH-16 administration.

[0511] Methods

[0512] Pharmacokinetics of KiH-16

[0513] Six C57BL / 6 mice were included per group, with each group further divided into two subgroups, designated as subgroups -a and -b, each consisting of three mice. Treatment was started on the day of grouping (Day 0). The Dosing regimen used was as follows:

[0514] Approximately 150 pL of blood was collected from each mouse at 24 h (subgroup-a) or 72 h (subgroup-b) after dosing. Approximately 500 pL termination blood was collected from each mouse either immediately (pre-dose), 144 h after dosing (subgroup-a) or 192 h after dosing (subgroup-b). Blood samples were maintained at room temperature for 30-60 minutes and then centrifuged at approximately 2000* g for 15 minutes at RT to obtain serum. The serum samples were stored at -80 °C and then used for detection of (i) mouse IFN-y and (ii) KiH-16 by ELISA. mlFN-y ELISA: mlFN-y monoclonal antibody XMG1.2 (Thermo Fisher Scientific, Cat#16-7311-85) was diluted to a final concentration of 2 pg / ml. 100 ml of diluted antibody solution was then applied onto a 96-well flat microplate (American Burton Instruments Co., Ltd., Bio Tek H1MF). The plate was incubated overnight at 4°C. The following day, the coated plate was washed 4 times with PBST and blocked with 2% BSA, then incubated at 37°C for 1 hour with shaking at 400 rpm. The plate was washed a further 4 times with PBST, followed by the addition of 100 pL of diluted standard mouse IFN-y or serum samples as above and incubated at 37°C for 1 hour with shaking at 400 rpm. After washing 4 times with PBST, 100 pL of detection antibody #ab14-Biotin (1 pg / mL, Biocytogen) was added to each well and incubated at 37°C for 1 hour with shaking at 400 rpm. The plate was washed 4 times with PBST and bound antibodies were detected using Pierce™ Streptavidin Poly-HRP (Thermo Scientific, Cat: 21140). After 4 washes with PBST, 100 pL of TMB substrate solution was added to each well the reaction was allowed to proceed for up to 20 minutes to allow the color development, before being stopped with 0.1 N HCI. Finally, the OD value was then measured at 450 nm and 570 nm using a microplate reader (American Burton Instruments Co., Ltd.), and the concentration of mlFN-y was calculated.

[0515] KiH-16 ELISA:

[0516] The ELISA was developed to detect the presence of KiH-16 by binding to immobilized human PSMA protein. The assay plate was prepared by adding 200 pL of 2% BSA to each well of a streptavidin coated 96 well plate (Thermo Scientific™, Cat.15120) and incubating the plate for 1 hour at 25°C. Following this, the plate was washed 4 times with PBST buffer, and then 100 pl of diluted biotinylated human PSMA protein was added to each well at a concentration of 0.5 pg / mL (Aero, Cat. PSA-H82Qb) and incubated for 1 hour at 25°C. The plate was then washed 4 times with PBST. Diluted serum samples were added to the wells at a volume of 100 pL per well and incubated for 1 .5 hours at 25°C. The plate was washed four times with PBST buffer to remove any unbound samples. The secondary antibody, HRP Goat anti-mouse lgG2a heavy chain (Abeam, Cat.ab97245), was then diluted 1 :100,000 in 1% BSA and 100 pL of this diluted solution was added to each well. The plate was incubated for one hour at 25°C, and washed four times with PBST buffer. 100 pL of TMB substrate was added to each well and the reaction was allowed to proceed for 5-20 minutes at room temperature. The reaction was stopped by adding 100 pL of stop solution (Beyotime, Cat.P0215-100mL) to each well. Finally, the OD value of the wells was measured at 450 nm and 630 nm using an ELISA microplate reader (BioTek, EPOCH2).

[0517] Results

[0518] All of the tested doses of KiH-16 and both routes of injection tested were well-tolerated by the mice.

[0519] The results of the KiH-16 exposure experiments are shown in Figure 10. Dose-linearity was observed for the increasing concentrations of KiH-16 tested by i.v. injection. High bioavailability was also observed using i.p. injection, which resulted in a KiH-16 plasma concentration which was 80% of that obtained with the equivalent i.v. injection. The KiH-16 antibody also had a long half-life in vivo, which was consistent with expectation for the selected mouse constant region.

[0520] A mlFN-y accumulation in serum was observed in all mice following a single dose of KiH-16 as compared to the baseline concentration in pre-dose control serum (FIG. 11 A). The accumulation over time was most notable for the 10 mg / kg dose (i.p. and i.v.). The molar ratio of KiH-16 versus mlFN-y detected in the serum at each time point decreased over time (FIG.11 B). Example 16: in vitro syngeneic model

[0521] The aim of this study was to evaluate the effect of KiH-16 in the presence of mlFN-y on B-hPSMA MC38 cells compared to a neutralizing anti-mlFN-y antibody XMG1.2 (Invitrogen, cat.16-7311-81) and a nonneutralizing anti-mlFN-y antibody AN-18 (Invitrogen, cat.16-7313-85).

[0522] Methods

[0523] In vitro functional assessment of KiH-16 in B-hPSMA MC38 cells

[0524] The MC38 murine colon carcinoma cell line was purchased from Shunran Shanghai Biological Technology Co., Ltd. The MC38 cells were genetically modified to express human PSMA and the resultant cells were named B-hPSMA MC38 by Biocytogen Pharmaceuticals (Beijing) Co., Ltd. The cells were maintained in vitro as a monolayer culture in DMEM supplemented with 10% heat inactivated FBS at 37°C in a humidified atmosphere of 5% CO2.

[0525] Determination of the stimulation concentration of mlFN-y:

[0526] B-hPSMA MC38 cells were seeded at a density of 3x104per well in 48 well plates. Following overnight attachment, cells were stimulated with a range of concentrations of mlFN-y (Sino Biological, cat.50709- MNAH) in duplicates for 24 or 48 h at 37°C in 5% CO2. The concentrations tested were 0.01 , 0.05, 0.1 , 0.5, 1 , 5, 10, 50, 100, 250, 500 ng / mL. After stimulation, dead cells were discriminated using the Live / Dead near IR (Invitrogen), and surface staining was performed with anti-PD-L1 (Biolegend, cat.124312) and anti-MHC-l (Biolegend, cat.114606) and analyzed by flow cytometry.

[0527] Evaluation of KiH-16 function in the presence of mlFN-y:

[0528] B-hPSMA MC38 cells were seeded at a density of 3x104per well in 48 well plates. A range of concentrations of KIH-16, AN-18 or XMG1 .2 (0.9, 9. 90, 900 nM) were pre-mixed with 5 ng / mL mlFN-y for 30 min at room temperature. A further highest concentration of 9000 nM was additionally tested for KIH- 16. Following attachment, cells were stimulated with the pre-mixed solutions for 48 h at 37°C in 5% CO2. After stimulation, dead cells were discriminated using the Live / Dead near IR (Invitrogen), and surface staining was performed with anti-PD-L1 (Biolegend, cat.124312) and anti-MHC-l (Biolegend, cat.114606) and analyzed by flow cytometry.

[0529] Results

[0530] The results of the mlFN-y stimulation determination assay are shown in Figure 12. The sensitivity of MC38 cells to mlFN-y was almost as high as for human cells to hlFNy. A 5 ng / mL concentration of mlFN- y and 48 h stimulation duration were selected as the optimal conditions for the assay going forward. Under these conditions, both the MHC-I and PD-L1 surface expression levels had plateaued. The results of the in vitro functional assay are shown in Figure 13. For both KiH-16 and AN-18 (nonneutralizing control), no change was observed in the surface expression level of both MHC-I and PD-L1 on B-hPSMA MC38 cells at increasing concentrations of antibody. In contrast, a clear decrease was observed for the neutralizing control antibody XMG1.2. From this, it was concluded that KiH-16 did not neutralize the effect of IFNy.

[0531] Example 17: Syngeneic mouse model

[0532] The aim of this study was to evaluate the mlFNy accumulation and immune infiltration potential of both innate and adaptive immune system of the KiH-16 antibody. The MC38 cells used in this example do not express hPSMA and so KiH16 does not act as a targeted antibody in this model.

[0533] Methods

[0534] The MC38 murine colon carcinoma cell line was purchased from Shunran Shanghai Biological Technology Co., Ltd. The cells were maintained as a monolayer culture in DMEM supplemented with 10% heat inactivated FBS at 37°C in a humidified atmosphere of 5% CO2. C57BL / 6 mice were subcutaneously injected with MC38 tumor cells (5x105cells per mouse) in 0.1 mL PBS in the right front flank for tumor development. 8 mice were randomly enrolled into each group when the mean tumor size reached 80-120 mm3. Treatment started on the day of grouping (Day 0). Anti-mouse PD-1 (1 mg / kg) with or without KiH16 (3 mg / kg) was administered intraperitoneally twice a week for a total of 7 times, starting on day 0. Blood was collected from each mouse 24 h post-injection following the 1st, 3rd, 5thand 7th(final) dose. The blood samples were maintained at room temperature for 30-60 minutes and then centrifuged at approximately 2000x g for 15 minutes at RT to obtain serum. The serum samples were stored at -80 °C for mlFN-y detection by ELISA. At the study endpoint, the animals were euthanized with CO2, and 4 mice from each group were selected from which the tumors were collected. The tumors were stained with the Live / Dead near IR (Invitrogen), and antibodies against mCD45, mCD3, mF4 / 80, mCD66a, mPD-L1 and mMHC-l, and analyzed by flow cytometry. mlFN-y ELISA:

[0535] IFN-y monoclonal antibody XMG1 .2 (Thermo Fisher Scientific, Cat#16-7311-85) was diluted to a final concentration of 2 ug / ml and 100 ml of the diluted antibody was applied onto 96-well flat microplate (American Burton Instruments Co., Ltd., Bio Tek H1 MF). The plate was incubated overnight at 4°C. The following day, the coated plate was washed 4 times with PBST and blocked with 2% BSA, then incubated at 37°C for 1 hour with shaking at 400 rpm. The plate was washed 4 times with PBST, followed by the addition of 100 uL of diluted standard mouse IFN-y or serum samples and incubated at 37°C for 1 hour with shaking at 400 rpm. After washing 4 times with PBST, 100 uL of detection Ab #ab14-Biotin (1 pg / mL, Biocytogen) was added to each well and incubated at 37°C for 1 hour with shaking at 400 rpm, after which the plate was washed 4 times with PBST and bound antibodies were detected using Pierce™ Streptavidin Poly-HRP (Thermo Scientific, Cat: 21140). After 4 washes with PBST, 100 uL of TMB substrate solution was added to each well the reaction was allowed to proceed for up to 20 minutes to allow the color development before being stopped with 0.1 N HCI. Finally, the OD value was then measured at 450 nm and 570 nm using a microplate reader (American Burton Instruments Co., Ltd.), and the concentration of mlFN-y was calculated.

[0536] FACS staining:

[0537] Tumor tissues were mechanically chopped into small pieces 2-4 mm3in size, transferred into the tube containing an enzyme mix and placed on the gentleMACS Octo Dissociator. The samples were then ground with a sterile syringe tail with 10 mL of RPMI 1640 at 2-8°C. The cell suspension was centrifuged at 300*g for 7 minutes at 4°C and the supernatant was aspirated completely. The cells were resuspended in PBS to the required volume for further applications. Single cells from tumor tissue were incubated with fixable viability dye staining and surface marker staining followed by acquiring via the cell flow cytometry. Data were analyzed using Flowjo software.

[0538] Results

[0539] The results are shown in Figure 14. Accumulation of mlFN-y over time was observed in mice who received doses of KiH-16 in combination with anti-mouse PD-1 (FIG. 14A). In fact, this accumulation was more the 1000 fold greater than that observed when mice were treated with anti-mouse PD-1 alone.

[0540] Further, an increase in MHC-I expression (FIG. 14B) and infiltra...

Claims

Claims:1 . A multispecific antigen-binding molecule comprising an antigen binding domain that binds to interferon gamma (IFNy) and an antigen binding domain that binds to a target antigen other than IFNy.

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

3. The multispecific antigen-binding molecule of any one of the preceding claims, wherein the molecule binds to IFNy with 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, as assessed with a BIACORE® surface plasmon resonance assay.

4. The multispecific antigen-binding molecule of any one of the preceding claims, wherein the molecule binds to IFNy with a KD of less than 200 nM, or less than 20 nM or less than 10 nM or less than 5 nM.

5. The multispecific antigen-binding molecule of any one of the preceding claims, wherein the molecule binds to the target antigen with 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, as assessed with a BIACORE® surface plasmon resonance assay.

6. The multispecific antigen-binding molecule of any one of the preceding claims, wherein the molecule binds to the target antigen with a KD of less than 20 nM, as assessed with a 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-related cell adhesion molecule), HER2 (Receptor tyrosine-protein kinase erbB-2), TN antigen (Tenascin), 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 of any one of the preceding claims, wherein binding of said molecule to IFNy modulates cell signalling downstream of IFNy.

9. The multispecific antigen-binding molecule of claim 1 or claim 8, wherein binding of said molecule to IFNy biases the pro- and anti-inflammatory activities of IFNy in favour of the pro-inflammatory activities.

10. The multispecific antigen-binding molecule of claim 9, wherein binding of said molecule to IFNy modulates cell signalling downstream of IFNy, such that the ratio of MHC-1 expression and / or activity to PD-L1 expression and / or activity which is induced by IFNy is modified in favour of MHC-1 expression and / or activity.11 . The multispecific antigen-binding molecule of any one of the preceding claims, wherein binding of said molecule to IFNy partially inhibits the pro-inflammatory activity of IFNy.

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

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

14. The multispecific antigen-binding molecule of any one of the preceding claims which is a multispecific antibody.

15. The multispecific antigen-binding molecule of any one of the preceding claims, which comprises an Fc region and / or is conjugated to a half-life extending moiety.

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 mutations: i) which further enhance the half-life of the molecule, selected from the group consisting of a 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, E294delta / T307P / N434Y mutation, T256N / A378V / S383N / N434Y mutation, M428L / N434S mutation, T307A / E380A / N434A mutation, T307Q / N434S mutation, Q311V / N434S mutation, H433K / N434F mutation, E294delta / 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 / M428F 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; and / or ii) which reduce or silence effector functions of the antibody, selected from the group consisting of a R435H mutation, N434A mutation, T252L / T253S / T254F mutation, E294delta / T307P / N434Y mutation, T256N / A378V / S383N / N434Y mutation, E294 delta 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, T256D / H286D / T307R / Q311V / A378V mutation, H285N / T307Q / N315D mutation, T307Q / Q311V / A378V mutation, H285D / T307Q / A378V mutation, L234F / L235E / P331 S mutation, L234F / L235Q / K322Q mutation, L234F / L235Q / P331 G mutation, L234F / L235A / K322Q mutation, S228P / F234A / L235A / G237A / P238S mutation, L234A / L235E / G237A / A330S / P331S mutation, F243A / V264A mutation, S228P / L235E / P329G mutation, M252Y / M428L mutation, D259IA / 308F mutation, T307Q / N434S mutation, M428LA / 308F mutation, Q311V / N434S mutation, H433K / N434Fmutation, 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, S354C / T366W / K409A mutation, L234S / L235T / G236R mutation, P329G mutation, S228P mutation, delta-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, P331 S 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) which enhance the effector functions of the antibody, selected from the group consisting of a F243L / R292P / Y300L / V305I / P396L mutation, a S239D / I332E mutation, a S239D / I332E / A330L mutation, a S298A / E333A / K334A mutation, a G236A / S239D / I332E mutation, a K326W / E333S mutation, a S267E / H268F / S324T mutation, a mutation of L234Y / L235Q / G236W / S239M / H268D / D270E / S298A in one heavy chain and D270E / K326D / A330M / K334E in the other heavy chain, a E345R / E430G / S440Y mutation and an lgG1 / lgG3 cross subclass mutation; and / or iv) which improve stability or downstream processing of the antibody, selected from the group consisting of a 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 / del / K447del mutation, K370Q mutation, K370E mutation, R409A mutation, R409E mutation, R409W mutation, C131 S mutation, K370 deletion or substitution, delta-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, which comprises a heterodimeric IgG comprising a first and second antibody heavy chain and a first and second antibody light chain, wherein the first heavy chain and the first light chain assemble to form an antigen binding domain for I FNy, and wherein the second heavy chain and second light chain assemble to form an antigen binding domain for the TAA.

18. The multispecific antigen-binding molecule of any one of claims 2 to 16, which comprises an Fc domain comprising a first and a second Fc subunit, wherein each subunit is fused directly or indirectly to an antigen-binding moiety, and wherein the first Fc subunit is fused to an antigen-binding moiety capableof binding IFNy 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 moiety is an antibody fragment selected from an Fv, Fab, cross-Fab, Fab', Fab’-SH, F(ab')2; a diabody; a linear antibody; a single-chain antibody molecule and a single domain antibody.

20. The multispecific antigen-binding molecule of claim 18 or claim 19, wherein the first and / or second subunit are fused via their N-terminus to the antigen-binding moiety.21 . The multispecific antigen-binding molecule of any one of the preceding claims, wherein the molecule is monovalent for IFNy.

22. The multispecific antigen-binding molecule of any one of the preceding claims, which comprises a binding site for IFNy and binding sites for more than one TAA.

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

24. The multispecific antigen-binding molecule of any one of the preceding claims, wherein said IFNy binding domain comprises a heavy chain variable region comprising HC-CDR1 , HC-CDR2 and HC- CDR3, wherein: a) heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 4 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR1 are substituted with another amino acid; b) heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 5 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR2 are substituted with another amino acid; and c) heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 6 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR3 are substituted with another amino acid; and / or wherein the IFNy binding domain comprises a light chain variable region comprising LC- CDR1 , LC-CDR2 and LC-CDR3, wherein: d) light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 7 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR1 are substituted with another amino acid; e) light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 8 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR2 are substituted with another amino acid; and f) light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 9 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR3 are substituted with another amino acid.

25. The multispecific antigen-binding molecule of claim 24, wherein heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 6 and / or 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 said heavy chain CDR1 comprises the sequence GYTXTNYY, wherein X can be any amino acid.

27. The multispecific antigen-binding molecule of claim 26, wherein said 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 said heavy chain CDR2 comprises the sequence INPSNXGT wherein X can be any amino acid.

29. The multispecific antigen-binding molecule of claim 28, wherein said 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 said light chain CDR1 comprises the sequence QSXLYSSNXKNY where X can be any amino acid.31 . The multispecific antigen-binding molecule of claim 30, wherein said 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 said IFNy binding domain comprises a heavy chain variable region comprising HC-CDR1 , HC-CDR2 and HC- CDR3, wherein: a) heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 4; b) heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 87; and c) heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 6; and comprises a light chain variable region comprising LC-CDR1 , LC-CDR2 and LC-CDR3, wherein: d) light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 7; e) light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 8; and f) light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 9.

33. The multispecific antigen-binding molecule of any one of claims 24 to 31 , wherein said IFNy binding domain comprises a heavy chain variable region comprising HC-CDR1 , HC-CDR2 and HC- CDR3, wherein: a) heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 85; b) heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 87; and c) heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 6; and comprises a light chain variable region comprising LC-CDR1 , LC-CDR2 and LC-CDR3, wherein: d) light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 7; e) light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 8; and f) light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 9.

34. The multispecific antigen-binding molecule of any one of claims 24 to 31 , wherein said IFNy binding domain comprises a heavy chain variable region comprising HC-CDR1 , HC-CDR2 and HC- CDR3, wherein: a) heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 4; b) heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 87; and c) heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 6; and comprises a light chain variable region comprising LC-CDR1 , LC-CDR2 and LC-CDR3, wherein: d) light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 89;e) light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 8; and f) light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 9.

35. The multispecific antigen-binding molecule of any one of claims 24 to 31 , wherein said IFNy binding domain comprises a heavy chain variable region comprising HC-CDR1 , HC-CDR2 and HC- CDR3, wherein: a) heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 85; b) heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 87; and c) heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 6; and comprises a light chain variable region comprising LC-CDR1 , LC-CDR2 and LC-CDR3, wherein: d) light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 89; e) light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 8; and f) light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 9.

36. The multispecific antigen-binding molecule of any one of claims 24 to 35, which is humanised.

37. The multispecific antigen-binding molecule of claim 36, wherein said IFNy binding domain comprises a heavy chain variable region comprising HC-FR1 , HC-FR2, HC-FR3 and HC-FR4, wherein:HC-FR1 has a sequence selected from SEQ ID NO: 90, SEQ ID NO: 98 and SEQ ID NO: 102;HC-FR2 has a sequence set forth in SEQ ID NO: 91 ;HC-FR3 has a sequence selected from SEQ ID NO: 92, SEQ ID NO: 99, SEQ ID NO: 103 and SEQ ID NO: 105; andHC-FR4 has a sequence selected from SEQ ID NO: 93 and SEQ ID NO: 100; and / or a light chain variable region comprising LC-FR1 , LC-FR2, LC-FR3 and LC-FR4, wherein: LC-FR1 has a sequence selected from SEQ ID NO: 94 and SEQ ID NO: 101 ;LC-FR2 has a sequence selected from SEQ ID NO: 95 and SEQ ID NO: 106;LC-FR3 has a sequence selected from SEQ ID NO: 96 and SEQ ID NO: 104; andLC-FR4 has a sequence set forth in SEQ ID NO: 97.

38. The multispecific antigen-binding molecule of any one of claims 24 to 37, wherein the IFNy binding domain comprises a 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 thereto and a 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 thereto.

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

40. An antigen-binding molecule comprising an IFNy binding domain, wherein said IFNy binding domain comprises a heavy chain variable region comprising HC-CDR1 , HC-CDR2 and HC-CDR3, wherein: a) heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 4 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR1 are substituted with another amino acid; b) heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 5 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR2 are substituted with another amino acid; and c) heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 6 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR3 are substituted with another amino acid; and / or wherein the IFNy binding domain comprises a light chain variable region comprising LC- CDR1 , LC-CDR2 and LC-CDR3, wherein: d) light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 7 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR1 are substituted with another amino acid; e) light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 8 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR2 are substituted with another amino acid; and f) light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 9 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR3 are substituted with another amino acid.41 . The antigen-binding molecule of claim 40, wherein heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 6 and / or 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 said heavy chain CDR1 comprises the sequence GYTXTNYY, wherein X can be any amino acid.

43. The antigen-binding molecule of claim 42, wherein said 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 said heavy chain CDR2 comprises the sequence INPSNXGT, wherein X can be any amino acid.

45. The antigen-binding molecule of claim 44, wherein said 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 said light chain CDR1 comprises the sequence QSXLYSSNXKNY, where X can be any amino acid.

47. The antigen-binding molecule of claim 46, wherein said 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 said IFNy binding domain comprises a heavy chain variable region comprising HC-CDR1 , HC-CDR2 and HC-CDR3, wherein: a) heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 4; b) heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 5 or 87; and c) heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 6; and comprises a light chain variable region comprising LC-CDR1 , LC-CDR2 and LC-CDR3, wherein: d) light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 7; e) light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 8; and f) light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 9.

49. The antigen-binding molecule of any one of claims 40 to 47, wherein said IFNy binding domain comprises a heavy chain variable region comprising HC-CDR1 , HC-CDR2 and HC-CDR3, wherein: a) heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 85; b) heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 87; and c) heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 6; and comprises a light chain variable region comprising LC-CDR1 , LC-CDR2 and LC-CDR3, wherein: d) light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 7; e) light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 8; and f) light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 9.

50. The antigen-binding molecule of any one of claims 40 to 47, wherein said IFNy binding domain comprises a heavy chain variable region comprising HC-CDR1 , HC-CDR2 and HC-CDR3, wherein: a) heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 4;b) heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 87; and c) heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 6; and comprises a light chain variable region comprising LC-CDR1 , LC-CDR2 and LC-CDR3, wherein: d) light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 89; e) light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 8; and f) light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 9.

51. The antigen-binding molecule of any one of claims 40 to 47, wherein said IFNy binding domain comprises a heavy chain variable region comprising HC-CDR1 , HC-CDR2 and HC-CDR3, wherein: a) heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 85; b) heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 87; and c) heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 6; and comprises a light chain variable region comprising LC-CDR1 , LC-CDR2 and LC-CDR3, wherein: d) light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 89; e) light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 8; and f) light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 9.

52. The antigen-binding molecule of any one of claims 40 to 51 , which is a humanised antibody.

53. The humanised antibody of claim 52, wherein said IFNy binding domain comprises a heavy chain variable region comprising HC-FR1 , HC-FR2, HC-FR3 and HC-FR4, wherein:HC-FR1 has a sequence selected from SEQ ID NO: 90, SEQ ID NO: 98 and SEQ ID NO: 102;HC-FR2 has a sequence set forth in SEQ ID NO: 91 ;HC-FR3 has a sequence selected from SEQ ID NO: 92, SEQ ID NO: 99, SEQ ID NO: 103 and SEQ ID NO: 105; andHC-FR4 has a sequence selected from SEQ ID NO: 93 and SEQ ID NO: 100; and / or a light chain variable region comprising LC-FR1 , LC-FR2, LC-FR3 and LC-FR4, wherein: LC-FR1 has a sequence selected from SEQ ID NO: 94 and SEQ ID NO: 101 ;LC-FR2 has a sequence selected from SEQ ID NO: 95 and SEQ ID NO: 106;LC-FR3 has a sequence selected from SEQ ID NO: 96 and SEQ ID NO: 104; andLC-FR4 has a sequence set forth in SEQ ID NO: 97.

54. The antigen-binding molecule of any one of claims 40 to 53, wherein the IFNy binding domain comprises a 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 thereto and a 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 thereto.

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

56. An antigen-binding molecule comprising an IFNy binding domain, wherein said IFNy binding domain comprises a heavy chain variable region comprising HC-CDR1 , HC-CDR2 and HC-CDR3, wherein: a) heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 12 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR1 are substituted with another amino acid; b) heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 13 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR2 are substituted with another amino acid; and c) heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 14 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR3 are substituted with another amino acid; and / or wherein the IFNy binding domain comprises a light chain variable region comprising LC- CDR1 , LC-CDR2 and LC-CDR3, wherein: d) light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 15 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR1 are substituted with another amino acid; e) light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 16 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR2 are substituted with another amino acid; and f) light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 17 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR3 are substituted with another amino acid.

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

59. The antigen-binding molecule of any one of claims 56 to 58, which is a humanised antibody.

60. An antigen-binding molecule comprising an IFNy binding domain, wherein said IFNy binding domain comprises a heavy chain variable region comprising HC-CDR1 , HC-CDR2 and HC-CDR3, wherein: a) heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 20 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR1 are substituted with another amino acid; b) heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 21 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR2 are substituted with another amino acid; and c) heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 22 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR3 are substituted with another amino acid; and / or wherein the IFNy binding domain comprises a light chain variable region comprising LC- CDR1 , LC-CDR2 and LC-CDR3, wherein: d) light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 23 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR1 are substituted with another amino acid; e) light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 24 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR2 are substituted with another amino acid; and f) light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 25 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR3 are substituted with another amino acid.61 . The antigen-binding molecule of claim 60, wherein heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 22 and / or 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 IFNy binding domain which binds to the same epitope of IFNy 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.

63. The antigen-binding molecule of any one of claims 60 to 62, which is a humanised antibody.

64. An antigen-binding molecule comprising an IFNy binding domain, wherein said IFNy binding domain comprises a heavy chain variable region comprising HC-CDR1 , HC-CDR2 and HC-CDR3, wherein: a) heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 28 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR1 are substituted with another amino acid;b) heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 29 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR2 are substituted with another amino acid; and c) heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 30 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR3 are substituted with another amino acid; and / or wherein the IFNy binding domain comprises a light chain variable region comprising LC- CDR1 , LC-CDR2 and LC-CDR3, wherein: d) light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 31 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR1 are substituted with another amino acid; e) light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 32 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR2 are substituted with another amino acid; and f) light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 33 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR3 are substituted with another amino acid.

65. The antigen-binding molecule of claim 64, wherein heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 30 and / or 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 IFNy binding domain which binds to the same epitope of IFNy 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.

67. The antigen-binding molecule of any one of claims 64 to 66, which is a humanised antibody.

68. An antigen-binding molecule comprising an IFNy binding domain, wherein said IFNy binding domain comprises a heavy chain variable region comprising HC-CDR1 , HC-CDR2 and HC-CDR3, wherein: a) heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 36 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR1 are substituted with another amino acid; b) heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 37 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR2 are substituted with another amino acid; and c) heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 38 or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR3 are substituted with another amino acid; and / or wherein the IFNy binding domain comprises a light chain variable region comprising LC- CDR1 , LC-CDR2 and LC-CDR3, wherein: d) light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 39 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR1 are substituted with another amino acid; e) light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 40 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR2 are substituted with another amino acid; and f) light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 41 or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR3 are substituted with another amino acid.

69. The antigen-binding molecule of claim 68, wherein heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 38 and / or 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 IFNy binding domain, which binds to the same epitope of IFNy 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: 43.71 . The antigen-binding molecule of any one of claims 68 to 70, which is a humanised antibody.

72. The antigen-binding molecule of any one of claims 40 to 71 , wherein the molecule binds to IFNy with 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.

73. The antigen-binding molecule of claim 72, wherein the molecule binds to IFNy with a KD of less than 20 nM or less than 10 nM or less than 5 nM.

74. The antigen-binding molecule of any one of claims 40 to 73, wherein binding of the antigenbinding molecule to IFNy modulates cell signalling downstream of IFNy.

75. The antigen-binding molecule of any one of claims 40 to 74, wherein binding of the antigenbinding molecule to IFNy biases the pro- and anti-inflammatory activities of IFNy in favour of the pro- inflammatory activities.

76. The antigen-binding molecule of any one of claims 40 to 75, wherein the ratio of MHC-1 expression and / or activity to PD-L1 expression and / or activity which is induced by IFNy is modified in favour of MHC-1 expression and / or activity.

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

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

79. The antigen-binding molecule of any one of claims 40 to 78, which comprises an Fc region and / or is conjugated to a half-life extending moiety.

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 mutations: i) which further enhance the half-life of the molecule, selected from the group consisting of a 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, E294delta / T307P / N434Y mutation, T256N / A378V / S383N / N434Y mutation, M428L / N434S mutation, T307A / E380A / N434A mutation, T307Q / N434S mutation, Q311V / N434S mutation, H433K / N434F mutation, E294delta / 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 / M428F 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; and / or ii) which reduce or silence effector functions of the antibody, selected from the group consisting of a R435H mutation, N434A mutation, T252L / T253S / T254F mutation, E294delta / T307P / N434Y mutation, T256N / A378V / S383N / N434Y mutation, E294 delta 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, T256D / H286D / T307R / Q311V / A378V mutation, H285N / T307Q / N315D mutation, T307Q / Q311V / A378V mutation, H285D / T307Q / A378V mutation, L234F / L235E / P331 S mutation, L234F / L235Q / K322Q mutation, L234F / L235Q / P331G mutation, L234F / L235A / K322Q mutation, S228P / F234A / L235A / G237A / P238S mutation, L234A / L235E / G237A / A330S / P331 S mutation, F243AA / 264A mutation, S228P / L235E / P329G mutation, M252Y / M428L mutation, D259I / V308F mutation, T307Q / N434S mutation, M428L / V308F mutation, Q311V / N434S mutation, H433K / N434F mutation, E258FA / 427T 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, S354C / T366W / K409A mutation, L234S / L235T / G236R mutation, P329G mutation, S228P mutation, delta- 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, P331 S 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) which enhance the effector functions of the antibody, selected from the group consisting of a F243L / R292P / Y300L / V305I / P396L mutation, a S239D / I332E mutation, a S239D / I332E / A330L mutation, a S298A / E333A / K334A mutation, a G236A / S239D / I332E mutation, a K326W / E333S mutation, a S267E / H268F / S324T mutation, a mutation of L234Y / L235Q / G236W / S239M / H268D / D270E / S298A in one heavy chain and D270E / K326D / A330M / K334E in the other heavy chain, a E345R / E430G / S440Y mutation and an lgG1 / lgG3 cross subclass mutation; and / or iv) which improve stability or downstream processing of the antibody, selected from the group consisting of a 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 / del / K447del mutation, K370Q mutation, K370E mutation, R409A mutation, R409E mutation, R409W mutation, C131S mutation, K370 deletion or substitution, delta-K447 mutation, F405L mutation, K409R mutation, and F405L / R409K mutation; or any combination thereof.81 . The antigen-binding molecule of any one of claims 40 to 80, which is a multispecific antigenbinding molecule further comprising an antigen-binding domain for a target antigen other than IFNy.

82. The antigen-binding molecule of claim 81 , which is the multispecific antigen-binding molecule of any one of claims 1 to 39.

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

84. A combination therapeutic comprising the antigen-binding molecule of any one 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 of claim 84 for use in a method of treatment.

86. The antigen-binding molecule of any one of claims 1 to 82, the pharmaceutical composition of claim 83 or the combination therapeutic of claim 84, for use in the treatment of a tumour 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 treatment of a tumour or cancer, wherein the use is in combination with a checkpoint inhibitor and / or the treatment sensitises a tumour or cancer to a checkpoint inhibitor.

88. An antigen-binding molecule that binds to interferon gamma (IFNy), wherein said binding biases the pro- and anti-inflammatory activities of IFNy in favour of the pro-inflammatory activities, for use in a method of treatment.

89. The antigen-binding molecule for use of claim 88, wherein binding of said molecule to IFNy modulates cell signalling downstream of IFNy, such that the ratio of MHC-1 expression and / or activity to PD-L1 expression and / or activity which is induced by IFNy is modified in favour of MHC-1 expression.

90. The antigen-binding molecule for use of claim 88 or 89, wherein binding of said molecule to IFNy partially inhibits the pro-inflammatory activity of IFNy.91 . The antigen-binding molecule for use of claim 90, wherein binding of said molecule to IFNy partially inhibits the induction of MHC-1 expression and / or activity by IFNy.

92. The antigen-binding molecule for use of any one of claims 88 to 91 , which is a humanised antibody.

93. The antigen-binding molecule for use of any one of claims 88 to 92, which binds to IFNy with 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.

94. The antigen-binding molecule for use of any one of claims 88 to 93, which binds to IFNy with a KD of less than 20 nM or less than 10 nM or less than 5 nM.

95. The antigen-binding molecule for use of any one of claims 88 to 94, which comprises an Fc region or is conjugated to a half-life extending moiety.

96. The antigen-binding molecule for use of claim 95, wherein the molecule comprises an Fc region and wherein the Fc region comprises one or more mutations selected from mutations: i) which enhance the half-life of the molecule, selected from the group consisting of a 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, E294delta / T307P / N434Y mutation, T256N / A378V / S383N / N434Y mutation, M428L / N434S mutation, T307A / E380A / N434A mutation, T307Q / N434S mutation, Q311V / N434S mutation, H433K / N434F mutation, E294delta / 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 / M428F mutation, T250E / M428F mutation, T250E / M428L mutation, M252Y / M428L mutation, M428LA / 308F mutation, D259IA / 308F mutation, E258FA / 427T mutation, T256D / Q311V / A378V mutation, T256D / H286D / T307R / Q311V / A378V mutation, and K288E / H435K mutation; and / or ii) which reduce or silence effector functions of the antibody, selected from the group consisting of a R435H mutation, N434A mutation, T252L / T253S / T254F mutation, E294delta / T307P / N434Y mutation, T256N / A378V / S383N / N434Y mutation, E294 delta 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, T256D / H286D / T307R / Q311V / A378V mutation, H285N / T307Q / N315D mutation, T307Q / Q311V / A378V mutation, H285D / T307Q / A378V mutation, L234F / L235E / P331 S mutation, L234F / L235Q / K322Q mutation, L234F / L235Q / P331G mutation, L234F / L235A / K322Q mutation, S228P / F234A / L235A / G237A / P238S mutation, L234A / L235E / G237A / A330S / P331 S mutation, F243AA / 264A mutation, S228P / L235E / P329G mutation, M252Y / M428L mutation, D259I / V308F mutation, T307Q / N434S mutation, M428L / V308F mutation, Q311V / N434S mutation, H433K / N434F mutation, E258FA / 427T 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, S354C / T366W / K409A mutation, L234S / L235T / G236R mutation, P329G mutation, S228P mutation, delta-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, P331 S mutation, T299A mutation, F234A mutation, T366W mutation, T366Smutation, 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) which enhance the effector functions of the antibody, selected from the group consisting of a F243L / R292P / Y300L / V305I / P396L mutation, a S239D / I332E mutation, a S239D / I332E / A330L mutation, a S298A / E333A / K334A mutation, a G236A / S239D / I332E mutation, a K326W / E333S mutation, a S267E / H268F / S324T mutation, a mutation of L234Y / L235Q / G236W / S239M / H268D / D270E / S298A in one heavy chain and D270E / K326D / A330M / K334E in the other heavy chain, a E345R / E430G / S440Y mutation and an lgG1 / lgG3 cross subclass mutation; and / or iv) which improve stability or downstream processing of the antibody, selected from the group consisting of a 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 / del / K447del mutation, K370Q mutation, K370E mutation, R409A mutation, R409E mutation, R409W mutation, C131 S mutation, K370 deletion or substitution, delta-K447 mutation, F405L mutation, K409R mutation, and F405L / R409K mutation; or any combination thereof.

97. The antigen-binding molecule for use of any one of claims 88 to 96, wherein the treatment is treatment of a tumour or cancer.

98. A pharmaceutical composition comprising an antigen-binding molecule that binds to interferon gamma (IFNy), wherein said binding biases the pro-and anti-inflammatory activities of IFNy in favour of the pro-inflammatory activities.

99. The pharmaceutical composition of claim 98, wherein binding of said molecule to IFNy modulates cell signalling downstream of IFNy, such that the ratio of MHC-1 expression and / or activity to PD-L1 expression and / or activity which is induced by IFNy is modified in favour of MHC-1 expression and / or activity.

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

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

102. The pharmaceutical composition of any one of claims 98 to 101 , wherein the antigenbinding molecule is a humanised antibody.

103. The pharmaceutical composition of any one of claims 98 to 102, wherein the antigenbinding molecule binds to IFNy with 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.

104. The pharmaceutical composition of claim 103, wherein the antigen-binding molecule binds to IFNy with a KD of less than 20 nM or less thanl 0 nM or less than 5 nM.

105. The pharmaceutical composition of any one of claims 98 to 104, wherein the antigenbinding molecule comprises an Fc region or is conjugated to a half-life extending moiety.

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 mutations: i) which enhance the half-life of the molecule, selected from the group consisting of a 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, E294delta / T307P / N434Y mutation, T256N / A378V / S383N / N434Y mutation, M428L / N434S mutation, T307A / E380A / N434A mutation, T307Q / N434S mutation, Q311V / N434S mutation, H433K / N434F mutation, E294delta / 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 / M428F mutation, T250E / M428F mutation, T250E / M428L mutation, M252Y / M428L mutation, M428LA / 308F mutation, D259IA / 308F mutation, E258FA / 427T mutation, T256D / Q311V / A378V mutation, T256D / H286D / T307R / Q311V / A378V mutation, and K288E / H435K mutation; and / or ii) which reduce or silence effector functions of the antibody, selected from the group consisting of a R435H mutation, N434A mutation, T252L / T253S / T254F mutation, E294delta / T307P / N434Y mutation, T256N / A378V / S383N / N434Y mutation, E294 delta 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, T256D / H286D / T307R / Q311V / A378V mutation, H285N / T307Q / N315D mutation, T307Q / Q311V / A378V mutation, H285D / T307Q / A378V mutation, L234F / L235E / P331 S mutation, L234F / L235Q / K322Q mutation, L234F / L235Q / P331G mutation, L234F / L235A / K322Q mutation, S228P / F234A / L235A / G237A / P238S mutation, L234A / L235E / G237A / A330S / P331 S mutation, F243AA / 264A mutation, S228P / L235E / P329G mutation, M252Y / M428L mutation, D259I / V308F mutation, T307Q / N434S mutation, M428L / V308F mutation, Q311V / N434S mutation, H433K / N434F mutation, E258FA / 427T 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, S354C / T366W / K409A mutation, L234S / L235T / G236R mutation, P329G mutation, S228P mutation, delta-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, P331 S mutation, T299A mutation, F234A mutation, T366W mutation, T366Smutation, 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) which enhance the effector functions of the antibody, selected from the group consisting of a F243L / R292P / Y300L / V305I / P396L mutation, a S239D / I332E mutation, a S239D / I332E / A330L mutation, a S298A / E333A / K334A mutation, a G236A / S239D / I332E mutation, a K326W / E333S mutation, a S267E / H268F / S324T mutation, a mutation of L234Y / L235Q / G236W / S239M / H268D / D270E / S298A in one heavy chain and D270E / K326D / A330M / K334E in the other heavy chain, a E345R / E430G / S440Y mutation and an IgG 1 ZlgG3 cross subclass mutation; and / or iv) which improve stability or downstream processing of the antibody, selected from the group consisting of a 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 / del / K447del mutation, K370Q mutation, K370E mutation, R409A mutation, R409E mutation, R409W mutation, C131 S mutation, K370 deletion or substitution, delta-K447 mutation, F405L mutation, K409R mutation, and F405L / R409K mutation; or any combination thereof.

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