WO2019185792A1 - Cancer treatment using immunoconjugates and immune check-point inhibitors - Google Patents

Abstract

This invention relates to therapeutic combinations comprising a check-point inhibitor, such as an anti-PD1 or anti-PD-L1 antibody and either (i) an immunoconjugate comprising tumour necrosis factor (TNF) and an anti- fibronectin antibody and/or an immunoconjugate comprising interleukin 2 (IL2) and an anti-fibronectin antibody; or (ii) a dual immunoconjugate comprising IL2, TNF and an anti-fibronectin antibody. Therapeutic combinations and methods of use in the treatment of cancer are provided.

Description

Cancer Treatment using Immunoconiuaates and Immune check-point inhibitors

Field

The present invention relates to the use of immunocytokines in combination with immune check-point inhibition to treat cancer.

Background

Immune check-point inhibitors are rapidly changing the clinical management of patients with cancer [1 ,2]. Ipilimumab (blocking CTLA-4), Nivolumab or Pembrolizumab (blocking PD-1 ) and Avelumab (Blocking PD- L1 ) [3-6] have gained marketing authorization for the treatment of different types of malignancies, on the basis of an impressive clinical benefit offered to a subset of patients. Unfortunately, not all cancer types and not all patients respond equally well to immune oncology drugs and many combination strategies are currently being investigated, with the aim to improve therapeutic activity with acceptable toxicity [3-7].

The therapeutic activity of immune check-point inhibitors often correlates with the quantity and quality of lymphocyte infiltrate into the solid tumor mass [2]. On one hand, the nature of tumor rejection antigens presented by the tumor influences the anti-cancer activity of specific cytotoxic T cells [8,9]. A growing body of experimental evidence indicates that both mutational load and HLA class I genotype potently influence response to immunotherapy in patients [10] Moreover, various experimental strategies are under development, with the aim to turn“cold” tumors“hot”, by increasing the density of lymphocytes in the neoplastic lesions and by tilting the cytokine balance towards a more inflammatory phenotype [11].

Recombinant cytokines (e.g., IL2, TNF, IFNy) have been used for many years, with the aim to boost the patient’s anticancer activity, with some encouraging results. Treatment with recombinant IL2 mediates a long-term survival for a relatively small proportion of patients with metastatic melanoma and renal cell carcinoma [12]. TNF has received marketing authorization in Europe for the treatment of soft-tissue sarcoma with isolated limb perfusion procedures [13], while recombinant IFNy has been used for decades to treat various types of cancer [14]. However, the clinical use of anti-cancer cytokines is often limited to substantial toxicity (sometimes even at sub-milligram dose levels), preventing escalation to therapeutically active regimens [12-15].

In order to improve the therapeutic index of pro-inflammatory cytokines for oncological applications, the fusion of these immunomodulatory payloads with tumor-targeting monoclonal antibodies has been proposed [16-18]. Both intact immunoglobulins and antibody fragments have been used to generate fusion proteins with cytokines (“immunocytokines”). Some of these products have moved to clinical trials [19], on the basis of promising preclinical results.

Two antibody-cytokine fusion proteins (L19-IL2 and L19-TNF) are currently being investigated by the present applicant in Phase III clinical trials [EudraCT number 2015-002549-72], after having shown encouraging activity in Phase II clinical studies [20-22] These products recognize the alternatively-spliced EDB domain of fibronectin, a marker of tumor angiogenesis [23]. The simultaneous delivery of two cytokine payloads to the tumor environment may exhibit a synergistic anticancer effect. For example, the combination of IL2- and TNF-based immunocytokine products was able to eradicate lesions in immunocompetent mouse models [24] and to induce complete responses in patients with stage IIIB/C melanoma [22]. In an attempt to combine the therapeutic activity of IL2 and of TNF into a single molecular entity, a class of biopharmaceuticals, termed“potency-matched dual cytokine fusions” was recently described [25]. Specifically, a novel fusion protein (termed IL2-F8-TNF^mut), featuring the scFv (F8) antibody fragment (specific to the alternatively-spliced EDA domain of fibronectin) fused to murine interleukin-2 (IL2) and to a single-amino acid TNF mutant was generated [25]. The R111 W mutation in the TNF moiety was introduced in order to match the potency of the IL2 and TNF payloads [25]. The EDA domain of fibronectin is expressed in the majority of tumors both in mouse and in man, while being virtually undetectable in normal adult tissues [26]. The F8 antibody recognizes murine and human EDA with identical affinity [27].

L19-TNF, also known as "FIBROMUN", is an immunocytokine developed by the present applicant, consisting of three polypeptides each of which is composed of TNFalpha (TNFa) fused at its N-terminus, via a linker, to the C-terminus of recombinant monoclonal antibody L19. In this fusion, L19 is an antibody molecule in the scFV format, which recognizes the alternatively spliced extra-domain B (EDB) of fibronectin (FN), a marker of tumor angiogenesis. The soluble TNFa domain homotrimerises, hence L19-TNFu itself is a trimer comprising three L19 scFV domains. The construction of L19-TNF is disclosed in W001/062298. Certain formulations of L19-TNF are disclosed in WO2018/011404.

As briefly indicated above L19-TNF has been studied in a number of pre-clinicai models of cancer, in particular:

Borsi et al. [46] report in 2003 the therapeutic activity of L19-TNF in F9 teratocarcinoma bearing mice and in WEH!-164 fibrosarcoma. The results show that L19-TNF alone or in combination with L19-IL2 can delay tumor growth in F9 teratocarcinoma. The results also show that L19-TNF in combination with elphalan can delay tumor growth in WEHI-164 fibrosarcoma.

Halin et al. [42] report in 2003 the therapeutic activity of L19-TNF in F9 teratocarcinoma bearing mice. The results show that when mice grafted with tumor volumes of -300 mm³ were treated with a single injection of 2 mg L19-TNF alone or in combination with L19-IL12, the combination induced a tumor growth regression for some days but not eradication. In a second experiment, mice bearing small F9 teratocarcinoma, were treated 5 days after tumor implantation with 2 mg L19-TNF. In the combination group four out five mice showed complete tumor regression.

Balza et al. [47] report in 2006 the therapeutic activity of L19-TNF in C51 colon carcinoma and in WEHI-164 fibrosarcoma. The results show that L19-TNF in combination with melphalan induced complete and irreversible tumor eradication in 83% of Balc-C mice bearing WEHI-164 and in 33% of mice bearing C51. In i m m u no-com prom ised SCID mice, resulted in tumor growth retardation but in no eradication. Mortara et al. [48] report in 2007 that the therapeutic activity of the combination between L19-TNF and melphalan described by Balza et al, is due to the action of CD4+ and CD4+ cells but not NK cells.

Schwager et al. [24] and WO2013/045125 (filed by the present applicant) report that mice bearing F9 murine teratocarcinoma (70mg) who received an intratumoral injection of 7 mg L19-TNF in combination with 30 mg L19-IL2, exhibited a complete cure in five out of five mice. When bigger tumors were studied (140 mm³) two out of five mice could be cured after first intratumoral injection and five mice out of six could be cured with a second intratumoral injection.

In light of such promising pre-clinical results, L19-TNF has been further studied into clinical trials.

Papadia et al. [21] report in 2012 the therapeutic activity of L19-TNF in combination with melphalan and mild hyperthermia in 17 patients with histologically or cytologically confirmed extremity melanoma of the lower limb. The patients received in an isolated limb perfusion setting, two preset L19-TNF doses (325 or 650 mg TNF activity). At 10 weeks after L19-TNF ILP, objective tumor responses were found in 86% (6/7) and 89% (9/10) of the previously progressing extremity melanoma patients at a L19-TNF dose of 325 mg and 650 pg, respectively. Interestingly, at the 650 pg dose of L19-TNF, complete remissions in the ILP area were achieved in 5 of 10 patients at the 10-week assessment and 4 of 10 patients were still alive 12 months after treatment with one patient still alive 24 months after treatment. All patients who achieved objective tumor response had also a significant relief of symptoms.

Spitaleri et al. [20] report in 2013 that 32 patients with metastatic solid cancer were treated with L19-TNF monotherapy in a Phase I/ll clinical trial with doses ranging between 1 ,3 and 13 pg/kg. Tumor response was evaluated in 31 patients and 14 patients achieved stable disease.

Danielli et al. [22] report in 2015 that a combination of L19-IL2 and L19-TNF was administered intralesionally in 20 efficacy-evaluable patients with stage MIC / IVM1 a metastatic melanoma who were not candidate for surgery. Thirty-two melanoma lesions exhibited complete responses upon intralesional administration of the two products. Complete responses were recorded in 7/13 of non-injected lesions suggesting a bystander effect of the combination. Nineteen patients were evaluable for overall survival. One patient died at 292 days after the date of first treatment. The other 18 patients had survived at the time point of last follow-up for periods ranging from 189 to >365 days.

Despite this promising body of evidence, a better therapeutic modality for L19-TNF remains to be found. Summary

The present inventors have unexpectedly recognised that immune checkpoint inhibition potentiates the anticancer properties of IL2 and TNF targeted to fibronectin as immunocytokines.

In particular, the present inventors have found that when L19-TNF is administered in combination with an anti-PD-1 antibody, complete eradication of large tumors (volumes up to 250 mm³) in mouse models was achieved. This result is surprising because previous combinations with L19-TNF at the same dose in preclinical models did not achieve completeeradication in 100% of the mice treated.

A first aspect of the invention provides a therapeutic combination comprising an immune check-point inhibitor and fibronectin-targeted IL2 and/or fibronectin-targeted TNF.

A second aspect of the invention provides a therapeutic combination comprising an immune check-point inhibitor and an im mu noconjugate comprising tumor necrosis factor (TNF) and an anti-fibronectin antibody.

A third aspect of the invention provides a therapeutic combination comprising an anti-PD1 antibody and an immunoconjugate comprising tumor necrosis factor (TNF) and an anti-fibronectin antibody.

A fourth aspect of the invention provides a therapeutic combination comprising an anti-PD1 antibody and an immunoconjugate comprising tumor necrosis factor (TNF) and an anti-ED-B antibody.

A fifth aspect of the invention provides a therapeutic combination comprising an anti-PD1 antibody and an immunoconjugate comprising tumor necrosis factor (TNF) and the L19 antibody.

A sixth aspect of the invention provides a therapeutic combination comprising an immune check-point inhibitor and an immunoconjugate comprising interleukin 2 (IL2) and an anti-fibronectin antibody.

A seventh aspect of the invention provides a therapeutic combination comprising an immune check-point inhibitor; a first immunoconjugate comprising tumor necrosis factor (TNF) and an anti-fibronectin antibody and a second immunoconjugate comprising interleukin 2 (IL2) and an anti-fibronectin antibody.

An eight aspect of the invention provides a therapeutic combination comprising an immune check-point inhibitor and a dual immunoconjugate comprising IL2, TNF and an anti-fibronectin antibody.

A ninth aspect of the invention provides a therapeutic combination comprising a PD-L1 inhibitor and a dual immunoconjugate comprising IL2, TNF and an anti-fibronectin antibody.

A tenth aspect of the invention provides a therapeutic combination comprising a PD-L1 inhibitor and a dual immunoconjugate comprising IL2, TNF and an anti-ED-A antibody.

An eleventh aspect of the invention provides a therapeutic combination comprising a PD-L1 inhibitor and a dual immunoconjugate comprising IL2, TNF and an F8 antibody.

A twelfth aspect of the invention provides a method of treating cancer comprising administering to an individual in need thereof a therapeutic combination according to any of the first to eleventh aspects. A thirteenth aspect of the invention provides a therapeutic combination any of the first to eleventh aspects for use in a method of treating cancer, for example a method of the twelfth aspect.

A fourteenth aspect of the invention provides an immunoconjugate comprising TNF and an anti-fibronectin antibody for use in a method of treating cancer comprising administering said immunoconjugate in combination with a check-point inhibitor to an individual in need thereof, for example a method of the twelfth aspect.

A fifteenth aspect of the invention provides an immunoconjugate comprising IL2 and an anti-fibronectin antibody for use in a method of treating cancer comprising administering said immunoconjugate in combination with a check-point inhibitor to an individual in need thereof, for example a method of the twelfth aspect.

A sixteenth aspect of the invention provides a first immunoconjugate comprising TNF and an anti-fibronectin antibody and a second immunoconjugate comprising IL2 and an anti-fibronectin antibody for use in a method of treating cancer comprising administering said first and second immunoconjugates and a check-point inhibitor to an individual in need thereof, for example a method of the twelfth aspect.

A seventeenth aspect of the invention provides a dual immunoconjugate comprising TNF, IL2 and an anti- fibronectin antibody for use in a method of treating cancer comprising administering said dual

immunoconjugate and a check-point inhibitor to an individual in need thereof, for example a method of the twelfth aspect.

Other aspects and embodiments of the invention are described in more detail below.

Brief Description of the Figures

Figure 1 shows reagents and tumor models characterization (a) Schematic representation of the domain assembly of IL2-F8-TNF^mut. (b) Microscopic fluorescence analysis of EDA expression on CT26, WEHI-164, LLC and F9 tumor sections detected with IL2-F8-TNF^mut or IL2-KSF-TNF^mut (green for anti-murine IL2, Alexa Fluor 488) and anti-CD31 (red, Alexa Fluor 594), 20x magnification, scale bars = 100pm.

Figure 2 shows therapeutic performance of ll2-F8-TNF^mut in combination with anti-PD-L1 treatment. Data represent mean tumor volume ± SEM. For all therapies mice were injected three times intravenously (black arrows) every 48 hours with either PBS, IL2-F8-TNFmut, 200pg anti-mouse PD-L1 or a combination of the two (IL2-F8-TNF^mut six hours before anti-PD-L1 or the opposite) n = 5 mice per group (unless stated elsewhere), CR = complete response (a) Therapy in Balb/c mice bearing CT26 colon carcinoma lesions. Treatment started when tumors reached a volume of 80 mm³, IL2-F8-TNF^mut was dosed at 50pg. n = 5 mice per group, CR = complete response (b) Tumor re-challenge study. After 37 days, mice with complete responses were injected subcutaneously with 4 x 10^® CT26 cells in the flank. n= 5 for PBS group (n = 4 from day 12), n= 4 for re-challenge group (c) Therapy in Balb/c mice bearing WEHI-164 tumors. Treatment started when tumors reached a volume of 70 mm³, IL2-F8-TNF^mut was dosed at 20pg. (d) Therapy in 129/SvEv mice bearing F9 teratocarci nomas. Treatment started when tumors reached a volume of 80 mm³, IL2-F8-TNF^mut was dosed at 40pg. (e) Therapy in C57BL/6 mice bearing LLC tumors. Treatment started when tumors reached a volume of 70 mm³, IL2-F8-TNF^mut was dosed at 40pg. n=4 from day 15 for groups IL2-F8-TNF^mut + anti-PD-L1 and PBS, from day 16 for group anti-PD-L1 and from day 20 for groups anti-PD- L1 + IL2-F8-TNF^mut and IL2-F8-TNF^mut.

Figure 3 shows the body weight profiles. Weight change was monitoring during the therapy of subcutaneous CT26 (a), WEHI-164 (b), F9 (c) and LLC (d). Data represent mean % weight (± SEM).

Figure 4 shows microscopic analysis of therapeutic performance of N2-F8-TNF^mut in combination with anti- PD-L1 treatment (a) Immunofluorescence analysis of tumor targeting properties of an anti-PD-L1 antibody 24 hours after IL2-F8-TNF^mut + anti-PD-L1 treatment in mice bearing CT26 or LLC lesions, cryosections were stained with anti-rat IgG (green, Alexa Fluor 488) and anti-CD31 (red, Alexa Fluor 594), 20x magnification, scale bars = 100pm. Immunofluorescence analysis of tumor infiltrating cells on CT26 (b) or LLC (c) tumor sections 24 hours after treatment with PBS or IL2-F8-TNF^mut + anti-PD-L1 , marker specific for NK cells (NCR1 ), CD4+ T cells (CD4), CD8+ T cells (CD8) and T regs (FoxP3) were stained in green (Alexa Fluor 488), anti-CD31 (red, Alexa Fluor 594), 20x magnification, scale bars = 100pm

Figure 5 shows the combination experiment of L19-TNF + anti-PD-1 in WEHI-164 tumor bearing mice. Three mice were injected three times intravenously every 48 hours with L19-TNF and anti-PD-1. CR = complete response.

Figure 6 shows the body weight profiles. Weight change was monitoring during the therapy of subcutaneous WEHI-164 tumors.

Detailed Description

This invention relates to the treatment of cancer using a combination of an immune check-point inhibitor and fibronectin-targeted immunocytokines IL2 and/or TNF. The combination may comprise an immune checkpoint inhibitor and one or more fibronectin-targeted immunoconjugates. The one or more fibronectin-targeted immunoconjugates may comprise (i) a im mu noconjugate comprising IL2 and an anti-fibronectin antibody (ii) an immunoconjugate comprising TNF and an anti-fibronectin antibody; (iii) a first immunoconjugate comprising IL2 and an anti-fibronectin antibody and a second immunoconjugate comprising TNF and an anti- fibronectin antibody; or (iv) a single dual immunoconjugate comprising IL2, TNF and an anti-fibronectin antibody.

In some embodiments, the therapeutic combination may comprise an immune check-point inhibitor and one or more fibronectin-targeted immunoconjugates, the one or more fibronectin-targeted immunoconjugates comprising an immunoconjugate comprising TNF and an anti-fibronectin antibody. The one and more fibronectin-targeted immunoconjugates may comprise an immunoconjugate comprising TNF and an anti ED- B antibody. Preferably, the one and more fibronectin-targeted immunoconjugates may comprise an immunoconjugate comprising TNF and L19 antibody.

In some preferred embodiments, the therapeutic combination may comprise an immune check-point PD-1 inhibitor and one fibronectin-targeted immunoconjugate comprising an immunoconjugate comprising TNF and L19 antibody.

In other embodiments, the therapeutic combination may comprise an immune check-point inhibitor and one and more fibronectin-targeted immunoconjugates, the one and more fibronectin-targeted immunoconjugates comprising a single dual immunoconjugate comprising IL2, TNF and an anti-fibronectin antibody. The one and more fibronectin-targeted immunoconjugates may comprise a single dual immunoconjugate comprising IL2, TNF and an anti-EDA antibody. Preferably, the one and more fibronectin-targeted immunoconjugates comprise a single dual immunoconjugate comprising IL2, TNF and a F8 antibody. In some preferred embodiments, the therapeutic combination may comprise an immune check-point PD-L1 inhibitor and one fibronectin-targeted immunoconjugate, the fibronectin-targeted immunoconjugate comprising a single dual immunoconjugate comprising IL2, TNF and a F8 antibody.

Immune checkpoint proteins negatively regulate the activation or function of T-cells. Immune checkpoint inhibition increases or promote T-cell activation or function by totally or partially inhibiting or reducing the expression or activity of an immune checkpoint protein. An immune checkpoint inhibitor may for example inhibit or block the interaction of an immune checkpoint protein with one of its ligands or receptors.

Numerous immune checkpoint proteins are known, including CTLA-4 (Cytotoxic T-Lymphocyte Associated protein 4) and its ligands CD80 and CD86; PD-1 (Programmed Death 1 ) with its ligands PD-L1 and PD-L2 (Pardall, Nature Reviews Cancer 12: 252-264, 2012); TIM-3 (T-cell Immunoglobulin domain and Mucin domain 3); LAG-3 (Lymphocyte Activation Gene-3); BTLA (CD272 or B and T Lymphocyte Attenuator), KIR (Killer-cell Immunoglobulin-like Receptor); VISTA (V-domain immunoglobulin suppressor of T-cell activation); and A2aR (Adenosine A2A receptor). These proteins are responsible for down-regulating T-cell responses. Immune checkpoint proteins regulate and maintain self-tolerance and the duration and amplitude of physiological immune responses.

Cytotoxic T-lymphocyte associated antigen 4 (CTLA-4) is an immune checkpoint protein that down-regulates pathways of T-cell activation (Fong et al., Cancer Res. 69(2):609-5 615, 2009; Weber Cancer Immunol. Immunother, 58:823-830, 2009). CTLA-4 is a negative regulator of T-cell activation. Blockade of CTLA-4 has augment T-cell activation and proliferations. Inhibitors of CTLA-4 include anti-CTLA-4 antibodies. Anti-CTLA- 4 antibodies bind to CTLA-4 and block the interaction of CTLA-4 with its ligands CD80/CD86 expressed on antigen presenting cells and thereby blocking the negative down regulation of the immune responses elicited by the interaction of these molecules. Examples of anti-CTLA-4 antibodies are described in US Patent Nos: 5,811 ,097; 5,811 ,097; 5,855,887; 6,051 ,227; 6,207, 157; 6,682,736; 6,984,720; and 7,605,238. Anti-CTLA-4 antibodies include tremelimumab, (ticilimumab, CP-675,206), ipilimumab (also known as IODI, MDX-0010; marketed under the name Yervoy™ and) a fully human monoclonal IgG antibody that binds to CTLA-4 approved for the treatment of unresectable or metastatic melanoma.

Programmed cell death 1 (PD-1 ; also called CD279) is a type I membrane protein that mediates T cell exhaustion. It has two ligands, PD-L 1 and PD-L2. The PD-1 pathway is a key immune-inhibitory mediator Blockade of this pathway leads to T-cell activation, expansion, and enhanced effector functions. As such, PD-1 negatively regulates T cell responses. PD-1 has been identified as a marker of exhausted T cells in chronic disease states, and blockade of PD-1 :PD-L1 interactions has been shown to partially restore T cell function. (Sakuishi et al., JEMVol. 207, September 27, 2010, pp2187-2194). PD-1 limits the activity of T cells in peripheral tissues at the time of an inflammatory response to infection and to limit autoimmunity. PD-1 blockade in vitro enhances T-cell proliferation and cytokine production in response to a challenge by specific antigen targets or by allogeneic cells in mixed lymphocyte reactions. A strong correlation between PD-1 expression and response was shown with blockade of PD-1 (Pardall, Nature Reviews Cancer, 12: 252-264,

2012). PD-1 blockade can be accomplished by a variety of mechanisms including antibodies that bind PD-1 or its ligand, PD-L1 , or soluble PD-1 decoy receptors (e.g. sPD-1 , see Pan et al., Oncology Letters 5: 90-96,

2013). Examples of PD-1 and PD-L1 blockers are described in US7488802; US7943743; US8008449; US8168757; US8217149, W02003042402, WO2008156712, W02010089411 , W02010036959,

WO2011066342, WO2011159877, WO201 1082400, and WO2011161699.

PD-1 blockers include anti-PD-L 1 antibodies and proteinaceous binding agents. Nivolumab (BMS-936558) is an anti-PD-1 antibody that was approved for the treatment of melanoma in Japan in July 2014. It is a fully human lgG4 antibody that binds to and blocks the activation of PD-1 by its ligands PD-L 1 and PD-L2. Other anti-PD-1 antibodies include lambrolizumab (pembrolizumab; MK-3475 or SCH 900475), a humanized monoclonal lgG4 antibody against PD-1 ; CT-01 1 a humanized antibody that binds PD-1. AMP-224 is a fusion protein of 87-DC; an antibody Fe portion; BMS-936559 (MDX-1105-01 ) for PD-L 1 (87-HI) blockade. Other anti-PD-1 antibodies are described in WO 2010/077634, WO 2006/121168, WO 2008/156712 and WO 2012/135408. AUNP-12 (Aurigene) is a branched 29 amino acid peptide antagonist of the interaction of PD-1 with PD-L 1 or PD-L2 and has been shown to inhibit tumor growth and metastasis in preclinical models of cancer.

T cell immunoglobulin mucin 3 (TIM-3) is an immune regulator identified as being upregulated on exhausted coa+ T cells (Sakuishi et al., JEM (2010) 207 2187-2194 and Fourcade et al J. Exp. Med. (2010) 207:2175- 86). TIM-3 was originally identified as being selectively expressed on IFN-y-secreting Th1 and Tc1 cells. Interaction of TIM-3 with its ligand, galectin-9, triggers cell death in TIM-3+ T cells. Anti-TIM-3 antibodies are described for example in Ngiow et al (Cancer Res. 2011 May 15; 71 (10):3540-51 ) and in US8552156.

Other immune-checkpoint inhibitors include lymphocyte activation gene-3 (LAG-3) inhibitors, such as IMP321 , a soluble Ig fusion protein (Brignone et al., 2007, J. Immunol.179:4202-4211 ). Other immune- checkpoint inhibitors include B7 inhibitors, such as B7-H3 and B7-H4 inhibitors. In particular, the anti-B7-H3 antibody MGA271 (Loo et al. (2012) 5 Clin. Cancer Res. July 15 (18) 3834). Preferred immune check-point inhibitors for use as described herein include anti-CTLA4 antibodies, such as ipilimumab; anti-PD-1 antibodies, such as nivolumab and pembrolizumab; and an anti-PD-L1 antibody, such as is atezolizumab, avelumab or durvalumab. Preferably, the immune check-point inhibitor is an anti-PD-L1 antibody. Most preferably, the immune check-point inhibitor is an anti-PD-1 antibody.

Immune checkpoint inhibitors are administered as described herein in combination with the fibronectin- targeted immunocytokines IL2 and TNF. The fibronectin-targeted immunocytokines may be contained in one or more immunoconjugates. In a preferred embodiment, the fibronectin-targeted immunocytokines may be contained in one immu noconjugate.

A fibronectin-targeted immunoconjugate specifically binds to fibronectin. Fibronectin is subject to alternative splicing, and a number of alternative isoforms of fibronectin are known, including alternatively spliced isoforms A-FN and B-FN, comprising domains ED-A or ED-B respectively, which are known markers of angiogenesis and are selectively expressed in the neovasculature. A fibronectin-targeted immunoconjugate may selectively bind to one or more isoforms of fibronectin that are selectively expressed in the neovasculature. For example, an immunoconjugate may specifically bind to fibronectin isoform A-FN, e.g. it may bind the extra domain A (ED-A); or fibronectin isoform B-FN, e.g. it may bind extra domain B (e.g. ED- B).

In some embodiments, the one or more fibronectin-targeted immunoconjugates may comprise an immunoconjugate comprising IL2 and an anti-fibronectin antibody or an immunoconjugate comprising TNF and an anti-fibronectin antibody. In some preferred embodiments, the fibronectin-targeted

immunoconjugates may comprise an immunoconjugate comprising TNF and an anti-fibronectin antibody. Preferably, the fibronectin-targeted immunoconjugates may comprise an immunoconjugate comprising TNF and an anti ED-B antibody. More preferably, the one and more fibronectin-targeted immunoconjugates may comprise an immunoconjugate comprising TNF and L19 antibody.

In other embodiments, the one or more fibronectin-targeted immunoconjugates may comprise a combination of a first immunoconjugate comprising IL2 and an anti-fibronectin antibody and a second immunoconjugate comprising TNF and an anti-fibronectin antibody.

In other embodiments, the one or more fibronectin-targeted immunoconjugates may comprise a dual immunoconjugate comprising IL2, TNF and an anti-fibronectin antibody. Preferably, the one and more fibronectin-targeted immunoconjugates may comprise a single dual immunoconjugate comprising IL2, TNF and an anti-EDA antibody. More preferably, the one and more fibronectin-targeted immunoconjugates may comprise a single dual immunoconjugate comprising IL2, TNF and a F8 antibody.

An immunoconjugate as described herein may be targeted to fibronectin through the presence of an anti- fibronectin antibody. The term“antibody” describes an immunoglobulin whether natural or partly or wholly synthetically produced. The term also relates to any polypeptide or protein comprising an antibody antigen-binding site. Antibodies may have been isolated or obtained by purification from natural sources, or else obtained by genetic recombination, or by chemical synthesis, and that they may contain unnatural amino acids.

An antigen binding site is the part of a molecule that recognises and binds to all or part of a target antigen.

In an antibody molecule, it is referred to as the antibody antigen-binding site or paratope, and comprises the part of the antibody that recognises and binds to all or part of the target antigen. Where an antigen is large, an antibody may only bind to a particular part of the antigen, which part is termed an epitope. An antibody antigen-binding site may be provided by one or more antibody variable domains. An antibody antigenbinding site preferably comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

An antigen binding site may be provided by means of arrangement of complementarity determining regions (CDRs). The structure for carrying a CDR or a set of CDRs will generally be an antibody heavy or light chain sequence or substantial portion thereof in which the CDR or set of CDRs is located at a location corresponding to the CDR or set of CDRs of naturally occurring VH and VL antibody variable domains encoded by rearranged immunoglobulin genes. The structures and locations of immunoglobulin variable domains may be determined by reference to Kabat et al. (1987) (Sequences of Proteins of Immunological Interest. 4^th Edition. US Department of Health and Human Services.), and updates thereof, now available on the Internet (at immuno.bme.nwu.edu or find“Kabat” using any search engine).

By CDR region or CDR, it is intended to indicate the hypervariable regions of the heavy and light chains of the immunoglobulin as defined by Kabat et al. (1987) Sequences of Proteins of Immunological Interest, 4^th Edition, US Department of Health and Human Services (Kabat et al., (1991a), Sequences of Proteins of Immunological Interest, 5^th Edition, US Department of Health and Human Services, Public Service, NIH, Washington, and later editions). An antibody typically contains 3 heavy chain CDRs and 3 light chain CDRs. The term“CDR” or“CDRs” may indicate, according to the case, one of these regions or several, or even the whole, of these regions which contain the majority of the amino acid residues responsible for the binding by affinity of the antibody for the antigen or the epitope which it recognizes.

Among the six short CDR sequences, the third CDR of the heavy chain (HCDR3) has a greater size variability (greater diversity essentially due to the mechanisms of arrangement of the genes which give rise to it). It can be as short as 2 amino acids although the longest size known is 26. Functionally, HCDR3 plays a role in part in the determination of the specificity of the antibody (Segal et al., (1974), PNAS, 71 :4298- 4302; Amit et al., (1986), Science, 233:747-753; Chothia et al., (1987), J. Mol. Biol., 196:901-917; Chothia et al., (1989), Nature, 342:877-883; Caton et al., (1990), J. Immunol., 144:1965-1968; Sharon et al., (1990a), PNAS, 87:4814-4817; Sharon et al., (1990b), J. Immunol., 144:4863-4869; Kabat et al., (1991 b), J.

Immunol., 147:1709-1719). As antibodies can be modified in a number of ways, the term "antibody” should be construed as covering any specific binding member or substance having an antibody antigen-binding site with the required specificity and/or binding, for example to fibronectin or an immune checkpoint inhibitor. Thus, this term covers antibody fragments, in particular antigen-binding fragments, and derivatives, including any polypeptide comprising an antibody antigen-binding site, whether natural or wholly or partially synthetic. Chimeric molecules comprising an antibody antigen-binding site, or equivalent, fused to another polypeptide (e.g. belonging to another antibody class or subclass) are therefore included. Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023, and a large body of subsequent literature.

As mentioned above, fragments of a whole antibody can perform the function of binding an antigen.

Examples of binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward et al. (1989) Nature 341 , 544-546; McCafferty et a!., (1990) Nature, 348, 552-554; Holt et al. (2003) Trends in Biotechnology 21 , 484-490), which consists of a VH or a VL domain; (v) isolated CDR regions; (vi) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al. (1988) Science, 242, 423-426; Huston et al. (1988) PNAS USA, 85, 5879-5883); (viii) bispecific single chain Fv dimers (PCT/US92/09965); (ix) "diabodies", multivalent or multispecific fragments constructed by gene fusion (WO94/13804; Holliger et al. (1993a), Proc. Natl. Acad. Sci. USA 90 6444-6448) and (x) a single chain diabody format wherein each of the VH and VL domains within a set is connected by a short or 'non- flexible^' peptide linker. Fv, scFv or diabody molecules may be stabilized by the incorporation of disulphide bridges linking the VH and VL domains (Reiter et al. (1996), Nature Biotech, 14, 1239-1245). A single chain Fv (scFv) may be comprised within a mini-immunoglobulin or small immunoprotein (SIP), e.g. as described in (Li et al., (1997), Protein Engineering, 10: 731-736). A SIP may comprise an scFv molecule fused to the CH4 domain of the human IgE secretory isoform lgE-S2 (E_S2-CH4; Batista et al., (1996), J. Exp. Med., 184: 2197- 205) forming a homo-dimeric mini-immunoglobulin antibody molecule. Minibodies comprising a scFv joined to a CH3 domain may also be made (Hu et al. (1996), Cancer Res., 56(13):3055-61 ). Other examples of binding fragments are Fab’, which differs from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region, and Fab’-SH, which is a Fab’ fragment in which the cysteine residue(s) of the constant domains bear a free thiol group.

An immunoconjugate is a molecule comprising an anti-fibronectin antibody and one or more cytokines selected from IL2 and TNF (e.g. IL2, TNF or IL2 and TNF). The cytokines present in an immunoconjugate may be referred to as immunocytokines.

A fibronectin targeted immunoconjugate as described herein, may comprise an antibody that specifically binds to fibronectin (i.e. an anti-fibronectin antibody), preferably a single chain Fv (scFv), diabody or single chain diabody that binds fibronectin. Diabodies and scFvs do not comprise an antibody Fc region, thus potentially reducing the effects of anti-idiotypic reaction. Preferably, the anti-fibronectin antibody for use in the immunoconjugates described herein is a scFv.

Where the anti-fibronectin antibody is a scFv, the VH and VL domains of the antibody are preferably linked by a 10 to 20 amino acid linker, by a 14 to 20 amino acid linker, preferably by a 10 to 14 amino acid linker. Suitable linkers are known in the art and available to the skilled person.

Where the anti-fibronectin antibody is a diabody, the VH and VL domains may be linked by a 5 to 12 amino acid linker. A diabody comprises two VH-VL molecules which associate to form a dimer. The VH and VL domains of each VH-VL molecule may be linked by a 5 to 12 amino acid linker.

An immunoconjugate described herein may specifically bind to the ED-A of fibronectin, and thus also A-FN. Suitable anti-ED-A antibodies are known in the art (see for example Villa et al Int J Cancer. (2008)

1 2(11 ):2405-13, Rybak et al.(2007) Cancer Res. 67, 10948-10957, W02008/120101 , W02009/013619, WO2010/078950, WO2011/015333) and include antibody F8. An anti-fibronectin antibody as described herein may comprise the complementarity determining regions (CDRs) of antibody F8 set forth in SEQ ID NOs 1 to 6. More preferably, an antibody for use as described herein may comprise the VH and/or VL domains of antibody F8 set forth in SEQ ID NOs 7 and 8, respectively or variants thereof. Yet more preferably, an antibody for use as described herein comprises the VH and VL domains of antibody F8 set forth in SEQ ID NOs 7 and 8, respectively or variants thereof. The F8 antibody is preferably in scFv or diabody format, most preferably in scFv format. Where the F8 antibody is in scFv format, the antibody molecule for use as described herein preferably has the amino acid sequence set forth in SEQ ID NO: 9 or a variant thereof.

An anti-fibronectin antibody for use as described herein may bind the A-FN and/or the ED-A of fibronectin, with the same affinity as anti-ED-A antibody F8 e.g. in scFv format, or with a higher affinity.

An anti-fibronectin antibody for use as described herein may bind to the same epitope on A-FN and/or the ED-A of fibronectin as anti-ED-A antibody F8.

An immunoconjugate described herein may specifically bind to the ED-B of fibronectin, and thus also B-FN. Suitable anti-ED-B antibodies and conjugates comprising anti-EDB antibodies such as the L19 antibody are known in the art (see for example W01999/058570, W02001/062298, WO2007/128563, WO2013/045125, and W02018/011404). An anti-fibronectin antibody for use as described herein may comprise the CDRs of antibody L19 set forth in SEQ ID NOs 10-15. More preferably, an antibody for use as described herein may comprise the VH and/or VL domains of antibody L19 set forth in SEQ ID NOs 16 and 17 or variants thereof. Yet more preferably, an antibody for use as described herein comprises the VH and VL domains of antibody L19 set forth in SEQ ID Nos 16 and 17 or variants thereof. The L19 antibody is preferably in scFv or diabody format, most preferably in scFv format. Where the L19 antibody is in scFv format, the antibody molecule for use as described herein preferably has the amino acid sequence set forth in SEQ ID NO: 18 or a variant thereof. An anti-fibronectin antibody for use as described herein may bind the B-FN and/or the ED-B of fibronectin, with the same affinity as anti-ED-B antibody L19 e.g. in scFv format, or with a higher affinity.

An anti-fibronectin antibody for use as described herein may bind to the same epitope on B-FN and/or the ED-B of fibronectin as anti-ED-B antibody L19 as described by Fattorusso et al., Structure 1999, 7, 381-390.

Variants of an antibody disclosed herein may be produced and used as an anti-fibronectin antibody as described herein. The techniques required to make substitutions within amino acid sequences of CDRs, antibody VH or VL domains, in particular the framework regions of the VH and VL domains, and antibody molecules generally are available in the art. Variant sequences may be made, with substitutions that may or may not be predicted to have a minimal or beneficial effect on activity, and tested for ability to bind antigen, such as A-FN and/or the ED-A of fibronectin, B-FN and/or the ED-B of fibronectin, and/or for any other desired property.

It is contemplated that from 1 to 5, e.g. from 1 to 4, including 1 to 3, or 1 or 2, or 3 or 4, amino acid alterations (addition, deletion, substitution and/or insertion of an amino acid residue) may be made in one or more of the CDRs and/or the VH and/or the VL domain of an antibody molecule as described herein. For example, anti-fibronectin antibody may comprise the CDRs and/or the VH and/or the VL domain of antibody F8 or L19 described herein with 5 or fewer, for example, 5, 4, 3, 2 or 1 amino acid alterations within the CDRs and/or the VH and/or the VL domain. For example, an antibody which binds the FN-A or FN-B, may comprise the VH and/or the VL domain of antibody F8 or L19 described herein with 5 or fewer, for example, 5, 4, 3, 2 or 1 amino acid alterations within the framework region of the VH and/or VL domain. An antibody molecule that binds the FN-A or ED-A of fibronectin, as referred to herein, thus may comprise the VH domain shown in SEQ ID NO: 7 and/or the VL domain shown in SEQ ID NO: 8 with 5 or fewer, for example, 5, 4, 3, 2 or 1 amino acid alterations within the framework region of the VH and/or VL domain. Such an antibody molecule may bind the ED-A isoform or ED-A of fibronectin with the same or substantially the same, affinity as an antibody molecule comprising the VH domain shown in SEQ ID NO: 7 and the VL domain shown in SEQ ID NO: 8 or may bind the ED-A isoform or ED-A of fibronectin with a higher affinity than an antibody molecule comprising the VH domain shown in SEQ ID NO: 7 and the VL domain shown in SEQ ID NO: 8.

An antibody that binds the FN-B or ED-B of fibronectin, as referred to herein, thus may comprise the VH domain shown in SEQ ID NO: 16 and/or the VL domain shown in SEQ ID NO: 17 with 5 or fewer, for example, 5, 4, 3, 2 or 1 amino acid alterations within the framework region of the VH and/or VL domain.

Such an antibody molecule may bind the ED-B isoform or ED-B of fibronectin with the same or substantially the same, affinity as an antibody molecule comprising the VH domain shown in SEQ ID NO: 16 and the VL domain shown in SEQ ID NO: 17 or may bind the ED-B isoform or ED-B of fibronectin with a higher affinity than an antibody molecule comprising the VH domain shown in SEQ ID NO: 16 and the VL domain shown in SEQ ID NO: 17. An anti-fibronectin antibody as described herein may comprise a VH and/or VL domain that has at least 70%, more preferably one of at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to the F8 antibody VH and/or VL domains set forth in SEQ ID NOs 7 and 8, respectively, or the L19 antibody VH and/or VL domains set forth in SEQ ID NOs 16 and 17, respectively. An anti-fibronectin antibody as described herein may have at least 70%, more preferably one of at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to the VH or VL amino acid sequence of the F8 or L19 antibodies set forth in SEQ ID NOs 7, 8, 16 and 17, respectively. In some embodiments, the anti- fibronectin antibody may comprise the VH and VL CDR sequences of the F8 or L19 antibodies set forth in SEQ ID NOs 1-6 and 10-15, respectively i.e. the variation may be in the framework regions.

An immunoconjugate, second immunoconjugate or a dual im mu noconjugate described herein may comprise TNF.

TNF is preferably human TNF. Where the tumour necrosis factor is TNFa, the TNFa is preferably human TNFa.

Human TNFa consists of a 35 amino acid cytoplasmic domain, a 20 amino acid transmembrane domain and a 177 amino acid extracellular domain. The 177 amino acid extracellular domain is cleaved to produce a 157 amino acid soluble form, which is biologically active, and which forms a non-covalently linked trimer in solution. Human TNFa is preferably the soluble form of the extracellular domain of human TNFa, or the extracellular domain of human TNFa. The sequence of the soluble form of the extracellular domain of human TNFa is shown in SEQ ID NO: 19. The TNFa thus preferably comprises or consist of the sequence set forth in SEQ ID NO: 19. Typically, TNFa has at least 70%, more preferably one of at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 19. The sequence of the extracellular domain of human TNFa is shown in SEQ ID NO: 20. Thus, alternatively the TNFa may comprise or consist of the sequence set forth in SEQ ID NO: 20. In this case, the TNFa may have at least 70%, more preferably one of at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 20. TNFa in conjugates of the invention retains a biological activity of human TNFa, e.g. the ability to inhibit cell proliferation.

TNF may be a wild type human TNF or a TNF mutant which retains biological function of human TNF, e.g. the ability to inhibit cell proliferation but has a reduced activity relative to the wild-type human TNF.

The TNF mutant may comprise one or more mutations which reduce activity relative to the wild-type TNF which lacks the one or more mutations i.e. the TNF mutant is less potent than wild-type TNF. For example, the TNF mutant may comprise a mutation at the position corresponding to position 32 in SEQ ID NO: 19 or position 52 of SEQ ID NO: 20. In some embodiments, the R at said position may be substituted for a different amino acid, preferably an amino acid other than G, for example a non-polar amino acid, preferably A, F, or V, most preferably A. For example, a mutant TNFa may comprise or consist of the sequence shown in SEQ ID NO: 19 or 20, except that the residue at position 32 of SEQ ID NO: 19 or at position 52 of SEQ ID NO: 20 is an alanine residue rather than an arginine residue. This sequence is shown in SEQ ID NO: 21 or 22. The mutant of TNFa thus preferably comprises or consist of the sequence set forth in SEQ ID NO: 21. Typically, the mutant of TNFa has at least 70%, more preferably one of at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 21 with an A at the position corresponding to position 32 in SEQ ID NO: 21. Thus, alternatively the TNFa may comprise or consist of the sequence set forth in SEQ ID NO: 22. In this case, the TNFa may have at least 70%, more preferably one of at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 22 with an A at the position corresponding to position 52 in SEQ ID NO: 22. In some preferred embodiments, a dual immunoconjugate as described herein may comprise a TNF mutant.

An immunoconjugate, first immunoconjugate or a dual immunoconjugate described herein may comprise IL2.

The IL2 may be human IL2.

The IL2 preferably comprises or consist of the sequence set forth in SEQ ID NO: 38 or a variant thereof. Typically, IL2 has at least 70%, more preferably one of at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 38. IL2 in an immunoconjugate described herein may retain a biological activity of human IL2, e.g. the ability to inhibit cell proliferation.

Preferably, the anti-fibronectin antibody may be connected to IL2 and/or TNF in an immunoconjugate through linkers, for example peptide linkers. Alternatively, the anti-fibronectin antibody and IL2 and/or TNF may be connected directly, e.g. through a chemical bond. The chemical bond may be, for example, a covalent or ionic bond. Examples of covalent bonds include peptide bonds (amide bonds) and disulphide bonds. The anti-fibronectin antibody and IL2 and/or TNF may be covalently linked, for example by peptide bonds (amide bonds). Thus, the antibody molecule, in particular a scFv portion of an antibody molecule, and IL2 and/or TNF may be produced as a fusion protein. By“fusion protein’’ is meant a polypeptide that is a translation product resulting from the fusion of two or more genes or nucleic acid coding sequences into one open reading frame (ORF).

Where the anti-fibronectin antibody of the immunoconjugate is a two-chain or multi-chain molecule (e.g. a diabody), IL2 and/or TNF may be conjugated as a fusion protein with one or more polypeptide chains in the anti-fibronectin antibody.

The peptide linkers connecting the anti-fibronectin antibody and IL2 and/or TNF in an immunoconjugate described herein may be a flexible peptide linker. Suitable examples of peptide linker sequences are known in the art. The linker may be 10-20 amino acids, preferably 10-15 or 15-18 amino acids in length. Most preferably, the linker is 1 1-15 or 16-18 amino acids in length, for example 17 amino acids in length. The linker may have the sequence set forth in SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 41. In a dual immunoconjugate in which the anti-fibronectin antibody is, or comprises, an scFv, the IL2 may be linked to the N-terminus of the VH domain of the scFv via a peptide linker and TNF may be linked to the C- terminus of the VL domain of the scFv via a peptide linker. Alternatively, the TNF may be linked to the N- terminus of the VH domain of the scFv via a peptide linker and the IL2 may be linked to the C-terminus of the VL domain of the scFv via a peptide linker. Alternatively, the IL2 and TNF may be linked to the C-terminus of the VL domain or the N-terminus of the VH domain of the antibody, e.g. in scFv format, via a peptide linker. The IL2 and TNF may be in any order and/or may optionally be linked to one another via a peptide linker. Suitable peptide linkers are described herein. In some preferred dual immunoconjugates, the IL2 and the TNF may be linked to the anti-fibronectin antibody by the linkers set forth in SEQ ID NO: 23 and SEQ ID NO: 24, respectively; or the linkers set forth in SEQ ID NO: 24 and SEQ ID NO: 25, respectively.

An immunoconjugate or first immunoconjugate as described herein may comprise or consist of the sequence shown in SEQ ID NO: 26 or 32 or may be a variant thereof. An immunoconjugate or second

immunoconjugate as described herein may comprise or consist of the sequence shown in SEQ ID NO: 27 or 33 or may be a variant thereof. Suitable first and second immunoconjugates are described, for example in W02001/062298, WO2013/045125, Hemmerle et al. (2013) Br. J. Cancer 109, 1206-1213; Frey et al. (2008) J. Urol. 184, 2540-2548. In some preferred embodiments, the immunoconjugate or second immunoconjugate comprises or consists of the sequence shown in SEQ ID NO: 33.

A dual immunoconjugate as described herein may comprise the amino acid sequence of any one of SEQ ID NOs: 28 to 31 and 34 to 37 or a variant of any one of these sequences. In some preferred embodiments, a dual cytokine may be a potency matched immunoconjugate, for example an immunoconjugate comprising the sequence of any one of SEQ ID NOs: 30 to 31 and 34 to 35 or a variant of any one of these sequences. Suitable dual immunoconjugates are described in W02016/180715 and PCT/EP2017/078652, De Luca R, et al (2017), Molecular Cancer Therapeutics 16 (1 1 ):2442

A variant of a reference sequence, such as an antibody or immunoconjugate sequence set out herein may have an amino acid sequence having at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the reference amino acid sequence. Suitable reference amino acid sequences for anti-fibronectin antibodies, IL2, TNF, linkers and immunoconjugates are provided herein.

Amino acid sequence identity is generally defined with reference to the algorithm GAP (GCG Wisconsin Package™, Accelrys, San Diego CA). GAP uses the Needleman & Wunsch algorithm (J. Mol. Biol. (48): 444-453 (1970)) to align two complete sequences that maximizes the number of matches and minimizes the number of gaps. Generally, the default parameters are used, with a gap creation penalty = 12 and gap extension penalty = 4. Use of GAP may be preferred but other algorithms may be used, e.g. BLAST, psiBLAST or TBLASTN (which use the method of Altschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448), or the Smith-Waterman algorithm (Smith and Waterman (1981 ) J. Mol Biol. 147: 195-197), generally employing default parameters. Particular amino acid sequence variants may differ from a reference sequence by insertion, addition, substitution or deletion of 1 amino acid, 2, 3, 4, 5-10, 10-20 or 20-30 amino acids. In some embodiments, a variant sequence may comprise the reference sequence with 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more residues inserted, deleted or substituted. For example, up to 15, up to 20, up to 30 or up to 40 residues may be inserted, deleted or substituted.

In some preferred embodiments, a variant may differ from a reference sequence by 1 , 2, 3, 4, 5, 6, 7, 8, 9,

10 or more substitutions, preferably conservative substitutions. Conservative substitutions involve the replacement of an amino acid with a different amino acid having similar properties. For example, an aliphatic residue may be replaced by another aliphatic residue, a non-polar residue may be replaced by another nonpolar residue, an acidic residue may be replaced by another acidic residue, a basic residue may be replaced by another basic residue, a polar residue may be replaced by another polar residue or an aromatic residue may be replaced by another aromatic residue. Conservative substitutions may, for example, be between amino acids within the following groups:

(i) alanine and glycine;

(ii) glutamic acid, aspartic acid, glutamine, and asparagine

(iii) arginine and lysine;

(iv) asparagine, glutamine, glutamic acid and aspartic acid

(v) isoleucine, leucine and valine;

(vi) phenylalanine, tyrosine and tryptophan

(vii) serine, threonine, and cysteine.

Fibronectin-targeted immunoconjugates and immune check-point inhibitors will usually be administered in the form of pharmaceutical compositions, which may comprise at least one component in addition to the active agent.

Fibronectin-targeted immunoconjugates and immune check-point inhibitors may be formulated into a single combined composition or any formulated into separate compositions. Separate compositions preparations may be useful, for example, to facilitate separate and sequential or simultaneous administration, and allow administration of the components by different routes.

Pharmaceutical compositions may comprise, in addition to an active ingredient described herein, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be by injection, e.g. intravenous or subcutaneous. Preferably, the therapeutic combination of the present invention is administered intravenously.

Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. For intravenous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be employed, as required. Many methods for the preparation of pharmaceutical formulations are known to those skilled in the art. See e.g. Robinson ed., Sustained and Controlled Release Drug Delivery Systems, Marcel Dekker, Inc., New York, 1978.

A therapeutic combination as described herein may be for use in a method of treatment of the human or animal body, for example a method of treating cancer. A method of treating cancer as described herein may comprise administering a first immunoconjugate comprising IL2 and an anti-fibronectin antibody and/or a second immunoconjugate comprising TNF and an anti-fibronectin antibody and an immune check-point inhibitor to an individual in need thereof. Some preferred methods of treating cancer as described herein may comprise administering an immunoconjugate comprising TNF and an anti-fibronectin antibody and an immune check-point inhibitor to an individual in need thereof. Alternatively, a method of treating cancer may comprise administering antibody to an individual in need thereof an immune checkpoint inhibitor and a dual immunoconjugate that comprises IL2, TNF and an anti-fibronectin. Other aspects of the invention provide a therapeutic combination; a first immunoconjugate; a second immunoconjugate; a first immunoconjugate and a second immunoconjugate; or a dual immunoconjugate for use in the above methods of treating cancer; and the use of a therapeutic combination; a first immunoconjugate; a second immunoconjugate; a first immunoconjugate and a second immunoconjugate; or a dual immunoconjugate in the manufacture of a medicament for use in the above methods of treating cancer.

Cancer, as referred to herein, may be a cancer which expresses, or has been shown to express, an antigen, such as an extracellular matrix component, that is associated with neoplastic growth and/or angiogenesis. Preferably, the cancer is a cancer which expresses, or has been shown to express, the ED-A or ED-B isoform of fibronectin. More preferably, the cancer expresses the ED-B isoform of fibronectin.

The cancer may be any type of solid or non-solid cancer or malignant lymphoma. For example, the cancer may be selected from the group consisting of skin cancer (in particular melanoma), head and neck cancer, kidney cancer, sarcoma, germ cell cancer (such as teratocarcinoma), liver cancer, lymphoma (such as Hodgkin's or non-Hodgkin's lymphoma), leukaemia (e.g. acute myeloid leukaemia), skin cancer, bladder cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer, colorectal cancer, cervical cancer, oesophageal cancer, pancreatic cancer, stomach cancer, and cerebral cancer. Cancers may be familial or sporadic. Cancers may be metastatic or non-metastatic. Preferably, the cancer is a cancer selected from the group consisting of a melanoma, head and neck cancer, kidney cancer, and a sarcoma. The reference to a cancer as mentioned above normally refers to a malignant transformation of the cells in question. Thus, kidney cancer, for example, refers to a malignant transformation of cells in the kidney. The cancer may be located at its primary location, such as the kidney in the case of kidney cancer, or at a distant location in the case of metastases. A tumour as referred to herein may be the result of any of the cancers mentioned above. Preferably, a tumour is the result of a melanoma, head and neck cancer, kidney cancer, or a sarcoma. A tumour which is the result of a particular cancer includes both a primary tumour and tumour metastases of said cancer. Thus, a tumour which is the result of head and neck cancer, for example, includes both a primary tumour of head and neck and cancer and metastases of head and neck cancer found in other parts of a patient’s body.

Administration of the immune check-point inhibitor and the immunoconjugate(s) can include coadministration, either in a single pharmaceutical formulation or using separate formulations, or consecutive administration in either order but generally within a time period such that all active agents can exert their biological activities simultaneously. Examples of sequential administration of IL2-based immunocytokines and anti-CTLA-4 checkpoint inhibitors include W02013/010749.

Compositions provided may be administered to mammals, preferably humans. Administration may be in a "therapeutically effective amount", this being sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom. Thus“treatment” of a specified disease refers to amelioration of at least one symptom. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated, the particular patient being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the composition, the type of conjugate, the method of administration, the scheduling of administration and other factors known to medical practitioners. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and may depend on the severity of the symptoms and/or progression of a disease being treated. Appropriate doses of antibody are well known in the art (Ledermann et al. (1991 ) Int. J. Cancer 47: 659-664; and Bagshawe et al. (1991 ) Antibody, Immunoconjugates and Radiopharmaceuticals 4: 915-922). Specific dosages indicated herein, or in the Physician's Desk Reference (2003) as appropriate for the type of medicament being administered, may be used. A therapeutically effective amount or suitable dose of a conjugate for use in the invention can be determined by comparing its in vitro activity and in vivo activity in an animal model. Methods for extrapolation of effective dosages in mice and other test animals to humans are known. The precise dose will depend upon a number of factors, including whether the antibody is for diagnosis, prevention or for treatment, the size and location of the area to be treated, the precise nature of the conjugate. A typical conjugate dose will be in the range 100 pg to 1 g for systemic applications. An initial higher loading dose, followed by one or more lower doses, may be administered. This is a dose for a single treatment of an adult patient, which may be proportionally adjusted for children and infants, and also adjusted according to conjugate format in proportion to molecular weight. Treatments may be repeated at daily, twice-weekly, weekly or monthly intervals, at the discretion of the physician. Treatments may be every two to four weeks for subcutaneous administration and every four to eight weeks for intravenous administration. Treatment may be periodic, and the period between administrations is about two weeks or more, e.g. about three weeks or more, about four weeks or more, or about once a month. In some embodiments, treatment may be given before, and/or after surgery, and may be administered or applied directly at the anatomical site of surgical treatment. A therapeutic combination as described herein may be administered alone or in combination with other cancer treatments, concurrently or sequentially or as a combined preparation with another therapeutic agent or agents, for the treatment of cancer. For example, a therapeutic combination of the invention may be used in combination with an existing therapeutic agent for cancer.

Other aspects and embodiments of the invention provide the aspects and embodiments described above with the term“comprising” replaced by the term“consisting of and the aspects and embodiments described above with the term“comprising” replaced by the term“consisting essentially of.

It is to be understood that the application discloses all combinations of any of the above aspects and embodiments described above with each other, unless the context demands otherwise. Similarly, the application discloses all combinations of the preferred and/or optional features either singly or together with any of the other aspects, unless the context demands otherwise.

Modifications of the above embodiments, further embodiments and modifications thereof will be apparent to the skilled person on reading this disclosure, and as such, these are within the scope of the present invention.

All documents and sequence database entries mentioned in this specification are incorporated herein by reference in their entirety for all purposes.

“and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example“A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

Experimental

Example 1- Therapeutic activity of IL2-F8-TNF^mut in combination with an anti-PD-L1 antibody

1 _ Materials and Methods

Tumor cell lines and reagents

WEHI-164 fibrosarcoma cells, F9 teratocarcinoma cells, CT26 colon carcinoma cells and LLC Lewis lung carcinoma cells were grown according to the supplier’s protocol and kept in culture for no longer than 14 passages. Authentication of the cell lines also including check of post-freeze viability, growth properties and morphology, test for mycoplasma contamination, isoenzyme assay and sterility test were performed by the cell bank before shipment.

The production and purification of IL2-F8-TNF^mut was performed as described before [25] and IL2-F8-TNF^mut has the amino acid sequence set forth in SEQ ID NO 39. The commercial anti-PD-L1 was purchased at BioXCell (clone 10F.9G2; BE0101 ). Tumor models and therapy studies

Tumor cells were implanted subcutaneously in the flank of BALB/c mice using 4 x 10^® cells (CT26), 5 x 10^® cells (WEHI-164). 15 x 10^® cells (F9) in 129/ScEv mice and 2 x 10^® cells (LLC) in C57BL/6 mice.

Experiments were performed under a project license granted by the Veterinaramt des Kantons Zurich, Switzerland (27/2015).

Mice were monitored daily and tumor volume was measured with a calliper (volume = length x width² x 0.5). When tumors reached a suitable volume (approx. 70-80 mm³), mice were injected three times into the lateral tail vein with the pharmacological agents. IL2-F8-TNF^mut and the commercial anti-PD-L1 antibody (clone 10F.9G2, BioXCell) were dissolved in phosphate buffered saline (PBS), also used as negative control, and administered every 48 hours. Weight of the mice was also recorded during the therapy and expressed as percent of weight change.

IL2-F8-TNF^mut was administered at 50pg, 20pg, 40pg and 40pg for the therapy in CT26, WEHI-164, F9 and LLC, respectively. The commercial anti-PD-L1 antibody was administered at 200pg (Mosely et al. (2017) Cancer Immunol Res. 5: 29-41 ). In a combination group IL2-F8-TNF^mut was administered 6 hours before anti-PD-L1 , while in a second combination group anti-PD-L1 was administered 6 hours before IL2-F8-TNF^mut.

For the tumor re-challenge study, mice with complete responses were injected subcutaneously with 4 x 10^® CT26 cells in the flank.

Immunofluorescence studies

EDA expression was assessed on ice-cold acetone fixed 8- pm cryostat sections of WEHI-164, CT26, F9 and LLC stained with IL2-F8-TNF^mut (final concentration 5pg/mL), as negative control IL2-KSF-TNF^mut (specific for an irrelevant antigen) was used. Both antibodies were detected with rat anti-IL2 and anti-rat

AlexaFluor488. For vascular staining goat anti-CD31 and anti-goat AlexaFluor594 antibodies were used.

For ex-vivo immunofluorescence analysis, mice bearing CT26 or LLC lesions were injected once with IL2- F8-TNF^muf + anti-PD-L1 or saline according to the therapy schedule and sacrificed 24h after injection.

Tumors were excised and embedded in cryoembedding medium and cryostat sections (8pm) were stained using the following antibodies: rabbit anti-CD4, rabbit anti-CD8, rabbit anti-FoxP3, rabbit anti-NCR1 , goat anti-CD31 and detected with anti-rat AlexaFluor488, anti-rabbit AlexaFluor488, anti-goat AlexaFluor594. Slides were mounted with fluorescent mounting medium and analysed with Axioskop2 mot plus microscope.

2. _ Results

Therapy experiments in immunocompetent mouse models of cancer

Fig. 1 describes the reagents used for therapy experiments and provides information about the murine tumor models used in the study. IL2-F8-TNF^mut, assembled into a stable non-covalent homotrimeric structure [Fig. 1a], was produced and purified as previously described [25], while a commercial antibody specific to murine PD-L1 was used as a surrogate for clinically-approved anti-PD-L1 products [11]. The IL2-F8-TNF^mut product strongly stained the neo-vasculature of F9 teratocarcinomas [Fig. 1 b], while the three other models used in our study (CT26, WEHI-164 and LLC) displayed a more diffuse stromal staining pattern. When IL2-KSF- TNF^mut (a fusion protein with identical format but specific to hen egg lysozyme) was used as negative control in immunofluorescence procedures, no detectable staining was observed in addition to the CD31 signal.

The therapeutic activity of IL2-F8-TNF^mut (administered at a dose of 50pg) was compared with the one of an anti-mouse PD-L1 antibody (used at 200pg) in immunocompetent BALB/c mice bearing CT26 carcinomas [Fig. 2a]. Treatment with the anti-PD-L1 antibody resulted in a tumor growth profile similar to the one obtained in the saline control group (PBS). A tumor growth inhibition was observed in the IL2-F8-TNF^mut group, as well as in the combination group where the anti-PD-L1 antibody had been administered six hours before IL2-F8-TNF^mut. By contrast, the combination IL2-F8-TNF^mut followed by anti-PD-L1 six hours later induced complete responses in 80% of the treated mice. Animals which had been cured by the combined administration of IL2-F8-TNF^mut + anti-PD-L1 were re-challenged with CT26 cells and were surprisingly found to have acquired a protective immunity against a second tumor implantation [Fig. 2b]

In a second therapy experiment, immunocompetent mice bearing WEHI-164 sarcomas were treated [Fig.

2c]. In this model, which is very sensitive to the action of TNF, the dose of IL2-F8-TNF^mut was reduced to 20pg, in order to assess whether a synergistic effect with anti-PD-L1 treatment could be observed [25]. An extremely potent anti-tumor activity was observed for IL2-F8-TNF^mut used as single agent and its combination with PD-L1 blockade led to cancer cures in 4/5 mice, irrespective of the order of administration of the two biopharmaceuticals.

A potent inhibition of tumor cell growth was observed in mice bearing F9 and LLC lesions [Fig. 2d and 2e], but complete cancer eradication was difficult to achieve in these models. All regimens were tolerated as evidenced by the comparison of body weight profiles [Fig. 3]. In particular, in the experiments performed in mice bearing the F9 tumors, treatments had to be stopped at day 1 1 due to ethical reasons (ulceration of lesions). Three out of five mice in the group treated with IL2-F8-TNF^mut + anti-PD-L1 could complete the course of therapy.

Microscopic analysis of immune cell infiltrate

The tumor-targeting properties of the commercial anti-PD-L1 antibody were investigated by analyzing sections of CT26 (a tumor that could be cured) or LLC (a tumor that could not be cured in our experimental setting) 24 hours after intravenous administration of 200 pg of this product. An ex vivo immunofluorescence staining, performed using an anti-rat secondary antibody reagent, revealed a patchy uptake of the anti-PD- L1 antibody at the tumor site in both models [Fig. 4a].

A microscopic analysis of leukocyte infiltrate in sections of CT26 tumors [Fig. 4b], obtained 24 hours after a single injection of IL2-F8-TNF^mut + anti-PD-L1 or saline, revealed a substantial increase in the intratumoral density of NK cells and CD4+ T cells after combination therapy. Tumor-resident CD8+ T cells were detectable also prior to treatment. By contrast, the density of FoxP3-positive lymphocytes decreased, as a result of pharmacological intervention, indicating product activity against regulatory T cells. A similar analysis, performed in sections of LLC tumors [Fig. 4c], revealed a distinct increase in the intratumoral density of NK cells, CD4+ T cells and CD8+ T cells after combination therapy. Only a small reduction in the density of FoxP3-positive lymphocytes was observed.

The targeted delivery of cytokines to the tumor environment increases the therapeutic index of those immune modulators [28,29,19,30-36] and various antibody-cytokine fusions are currently being investigated in clinical trials for oncological applications. As the combination of cytokines is often required in order to control immunological processes [37], it is attractive to use mixtures of antibody-cytokine fusions in therapy experiments [38-40,24,41] or to develop antibody fusions, featuring multiple cytokine payloads [40,42,25]. Here, we investigated the therapeutic activity of a novel antibody-cytokine fusion protein (IL2-F8-TNF^mut) in four immunocompetent mouse models of cancer (CT26, WEHI-164, F9 and LLC). The product was used alone or in combination with an anti-PD-L1 antibody (a surrogate for the human-specific PD-L1 blockers Avelumab, Durvalumab and Atezolizumab, which have gained marketing authorization for cancer therapy) [6,43,44]

A potent tumor growth inhibition was observed for IL2-F8-TNF™' used as single agents in CT26 tumors, while PD-L1 blockade had a minimal anti-cancer activity in all experimental models, in keeping with previous reports [1 1] The combination of the two products led to long-lasting complete responses, when the immune check-point inhibitor was given after immunocytokine treatment. This observation and studies of the immune infiltrate into the tumor mass suggests that the targeted delivery of pro-inflammatory cytokines (IL2 and TNF) increase the density and activity of lymphocytes in the neoplastic mass, whose activity can be further modulated by PD-L1 blockade.

A potentiation of the therapeutic activity of IL2-F8-TNF™' in combination with the anti-PD-L1 antibody was observed in WEHI-164, leading to cure in most treated animals. Sarcomas are extremely sensitive to the targeted delivery of TNF, which rapidly kills the majority of tumor cells. The anti-cancer role of lymphocytes is mainly confined to the eradication of the few tumor cells, which survive the induction of hemorrhagic necrosis into the neoplastic mass [45,25]

A potent tumor growth inhibition was observed when F9 tumors were treated with IL2-F8-TNF^mut, alone or in combination with anti-PD-L1. However, only one mouse could be cured in the combination group. For LLC tumors, no cures were observed and the potency-matched immunocytokine product could mediate only a partial tumor growth inhibition. No apparent benefit was observed in the combination therapy group.

Example 2- Therapeutic activity of L19-TNF in combination with an anti-PD-1 antibody

T _ Materials and Methods

Therapy experiments in immunocompetent mouse models of cancer

Tumor cells were implanted subcutaneously in the flank of eight week old female Balb/c mice using 5 x 10^® cells (WEHI-164). Mice were monitored daily and tumor volume was measured with a caliper (volume = length x width² x 0.5). When tumors reached a suitable volume (approx. 70 mm³), pharmacological treatment was performed. The production and purification of L19-mTNF was performed as described before [46] and L19-mTNF has the amino acid sequence set forth in SEQ ID NO 40. L19-mTNF was dissolved in phosphate buffered saline (PBS), and administered at 2pg/mouse into the lateral tail vein three times every 48h. The commercial anti- PD-1 antibody (clone 29F.1A12, BioXCell, Catalog # BE0273) was administered at 200pg/mouse into the lateral tail vein three times every 48h, together with L19-mTNF. Weight of the mice was also recorded during the therapy and expressed as percent of weight change.

2. _ Results

The combined administration of L19-TNF + anti-PD-1 was able to completely eradicate tumor lesions [Fig. 5]. The treatment was tolerated as evidenced by the comparison of body weight profiles [Fig. 6].

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Sequences

1. Amino acid

of the F8 CDR’s

F8 CDR1 VH - LFT (SEQ ID NO: 1 )

F8 CDR2 VH - SGSGGS (SEQ ID NO: 2)

F8 CDR3 VH - STHLYL (SEQ ID NO: 3)

F8 CDR1 VL - MPF (SEQ ID NO: 4)

F8 CDR2 VL -GASSRAT (SEQ ID NO: 5)

F8 CDR3 VL -MRGRPP (SEQ ID NO: 6)

2. Amino acid sequence of the F8 VH domain (SEQ ID NO: 7)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTI SRDNSKNTLYLQMNSLRAE DTAVYYCAKSTHLYLFDYWGQGTLVTVSS

4. Amino acid

of the F8 VL domain

EIVLTQSPGTLSLSPGERATLSCRASQSVSMPFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTI SRLEPEDFAVYYC QQMRGRPPTFGQGTKVEIK

5. Amino acid sequence of the F8 scFv (SEQ ID NO: 9)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTI SRDNSKNTLYLQMNSLRAE DTAVYYCAKSTHLYLFDYWGQGTLVTVSSGGGGSGGGGSGGGGEIVLTQSPGTLSLSPGERATLSCRASQSVSMPFLAWYQQKPGQAPR LLIYGASSRATGI PDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK

6. Amino acid sequence of L19 CDR’s

Ll 9 CDR1 VH -SFSMS (SEQ ID NO: 10)

LI 9 CDR2 VH -SISGSSGTTYYADSVKG (SEQ ID NO: 11)

Ll 9 CDR3 VH -PFPYFDY (SEQ ID NO: 12)

Ll 9 CDR1 VL -RASQSVSSSFLA (SEQ ID NO: 13)

Ll 9 CDR2 VL -YASSRAT (SEQ ID NO: 14)

Ll 9 CDR3 VL -QQTGRIPPT (SEQ ID NO: 15)

7. Amino acid sequence of the L19 VH domain (SEQ ID NO: 16)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTI SRDNSKNTLYLQMNSLRAE DTAVYYCAKPFPYFDYWGQGTLVTVSS

8. Amino acid sequence of the L19 VL domain (SEQ ID NO: 17)

EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGI PDRFSGSGSGTDFTLTI SRLEPEDFAVYYC QQTGRIPPTFGQGTKVEIK

9. Amino acid

of the L19 scFv

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAE DTAVYYCAKPFPYFDYWGQGTLVTVSSGDGSSGGSGGASEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIY

YASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRI PPTFGQGTKVEIK 10. Amino acid sequence of the soluble form of the extracellular domain of human TNFa (huTNFcO (SEQ ID

NO: 19).

VRSSSRTPSDKPVAHWANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQT KVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGI IAL

11. Amino acid sequence of the extracellular domain of human TNFa (huTNFa) [extracellular domainl (SEQ

ID NO: 20).

GPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLWPSEGLYLIYSQVLFKGQGC PSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGI IAL

12. Amino acid sequence of the soluble form of the extracellular domain of human TNFa (R32A) mutant

(huTNFa R32A) (SEQ ID NO: 21 ). The R32A is underlined in bold.

VRSSSRTPSDKPVAHWANPQAEGQLQWLNRAANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQT KVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGI IAL

13. Amino acid sequence of the extracellular domain of human TNFa (R52A1 mutant (huTNFa R52A)

[extracellular domainl (SEQ ID NO:22). R52A is underlined in bold.

GPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRAANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGC PSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGI IAL

14. Amino acid sequence of linker (SEQ ID NO: 23)

GDGSSGGSGGAS

15. Amino acid sequence of linker (SEQ ID NO: 241

SSSSGSSSSGSSSSG

16. Amino acid sequence of linker (SEQ ID NO: 251

GGGGSGGGGSGGGG

17. Amino acid sequence of the F8-hulL2 conjugate (SEQ ID NO: 26)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTI SRDNSKNTLYLQMNSLRAE DTAVYYCAKSTHLYLFDYWGQGTLVTVSSGGSGGEIVLTQSPGTLSLSPGERATLSCRASQSVSMPFLAWYQQKPGQAPRLLIYGASSR ATGIPDRFSGSGSGTDFTLTI SRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIKSSSSGSSSSGSSSSGAPTSSSTKKTQLQLEHLLLDL QMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADE TATIVEFLNRWITFCQSIISTLT

18. Amino acid sequence of the F8-huTNF conjugate (SEQ ID NO: 27)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTI SRDNSKNTLYLQMNSLRAE DTAVYYCAKSTHLYLFDYWGQGTLVTVSSGGGGSGGGGSGGGGEIVLTQSPGTLSLSPGERATLSCRASQSVSMPFLAWYQQKPGQAPR LLIYGASSRATGIPDRFSGSGSGTDFTLTI SRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIKSSSSGSSSSGSSSSGVRSSSRTPSDKP VAHWANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTI SRIAVSYQTKVNLLSAIKSPC QRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGI IAL

19. Amino acid sequence of the hulL2-F8-huTNFa [soluble forml conjugate (SEQ ID NO: 28) The amino acid sequence of the hull_2-F8-huTNFa [soluble form] conjugate (human IL2 - linker - F8 VH - linker - F8 VL - linker - human TNFa [soluble form]) is shown below. The linker sequences are underlined. The human TNFa in this conjugate is the soluble form of the extracellular domain of TNFa. APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNI NVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSI ISTLTGDGSSGGSGGASEVQLLESGGGLVQPGGSLRLSCAASGFTFSLFT MSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSSGGGG SGGGGSGGGGEIVLTQSPGTLSLSPGERATLSCRASQSVSMPFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTI SRL EPEDFAVYYCQQMRGRPPTFGQGTKVEIKSSSSGSSSSGSSSSGVRSSSRTPSDKPVAHWANPQAEGQLQWLNRRANALLANGVELRD NQLWPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSA

EINRPDYLDFAESGQVYFGI IAL

20. Amino acid sequence of the hulL2-F8-huTNFa [extracellular domain] conjugate (SEQ ID NO: 29]

The amino acid sequence of the hull_2-F8-huTNFa [extracellular domain] conjugate (human IL2 - linker - F8 VH - linker - F8 VL - linker - human TNFa [extracellular domain]) is shown below. The linker sequences are underlined. The human TNFa in this conjugate is the extracellular domain of TNFa.

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNI NVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSI I STLTGDGSSGGSGGASEVQLLESGGGLVQPGGSLRLSCAASGFTFSLFT MSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSSGGGG SGGGGSGGGGEIVLTQSPGTLSLSPGERATLSCRASQSVSMPFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTI SRL

EPEDFAVYYCQQMRGRPPTFGQGTKVEIKSSSSGSSSSGSSSSGGPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQ LQWLNRRANALLANGVELRDNQLWPSEGLYLIYSQVLFKGQGCPSTHVLLTHTI SRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPW YEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGI IAL 21. Amino acid sequence of the hulL2-F8-huTNFa (R32A1 mutant [soluble forml conjugate (SEQ ID NO: 30)

The amino acid sequence of the hulL2-F8-huTNFa (R32A) mutant [soluble form] conjugate (human IL2 - linker - F8 VH - linker - F8 VL - linker - human TNFa (R32A) mutant [soluble form]) is shown below. The linker sequences are underlined and the R32A is underlined in bold. The mutant of human TNFa (R32A) in this conjugate is the soluble form of the extracellular domain of TNFa.

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNI NVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSI I STLTGDGSSGGSGGASEVQLLESGGGLVQPGGSLRLSCAASGFTFSLFT MSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSSGGGG SGGGGSGGGGEIVLTQSPGTLSLSPGERATLSCRASQSVSMPFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTI SRL EPEDFAVYYCQQMRGRPPTFGQGTKVEIKSSSSGSSSSGSSSSGVRSSSRTPSDKPVAHWANPQAEGQLQWLNRAANALLANGVELRD NQLWPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSA

EINRPDYLDFAESGQVYFGI IAL

22. Amino acid sequence of the hulL2-F8-huTNFa (R52A) mutant (huTNFa R52A) [extracellular domain] conjugate (SEQ ID NO: 311

The amino acid sequence of the hulL2-F8-huTNFa (R52A) mutant [extracellular domain] conjugate (human IL2 - linker - F8 VH - linker - F8 VL - linker - human TNFa (R52A1 mutant [extracellular domain]) is shown below. The linker sequences are underlined and the R52A is in underlined in bold. The human TNFa (R52A) mutant in this conjugate is the extracellular domain of TNFa.

APTSSSTKKTQLQLEHLLLDLQMILNGIN YKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLI SNI NVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSI ISTLTGDGSSGGSGGASEVQLLESGGGLVQPGGSLRLSCAASGFTFSLFT MSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSSGGGG SGGGGSGGGGEIVLTQSPGTLSLSPGERATLSCRASQSVSMPFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTI SRL EPEDFAVYYCQQMRGRPPTFGQGTKVEIKSSSSGSSSSGSSSSGGPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQ LQWLNRAANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPW YEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGI IAL

23. Amino acid sequence of the L19- hulL2 conjugate (SEQ ID NO: 32) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTI SRDNSKNTLYLQMNSLRAE PTAVYYCAKPFPYFPYWGQGTLVTVSSGPGSSGGSGGASEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIY YASSRATGIPPRFSGSGSGTPFTLTISRLEPEPFAVYYCQQTGRI PPTFGQGTKVEIKEFSSSSGSSSSGSSSSGAPTSSSTKKTQLQL EHLLLPLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRPLISNINVIVLELKGSETTF MCEYAPETATIVEFLNR ITFCQSI ISTLT

24. Amino acid sequence of the L19-huTNF conjugate (SEQ ID NO: 33)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYAPSVKGRFTI SRPNSKNTLYLQMNSLRAE PTAVYYCAKPFPYFPYWGQGTLVTVSSGPGSSGGSGGASEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIY YASSRATGIPPRFSGSGSGTPFTLTISRLEPEPFAVYYCQQTGRI PPTFGQGTKVEIKEFSSSSGSSSSGSSSSGVRSSSRTPSPKPVA HWANPQAEGQLQWLNRRANALLANGVELRPNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQR ETPEGAEAKPWYEPIYLGGVFQLEKGPRLSAEINRPPYLPFAESGQVYFGI IAL

25. Amino acid sequence of the hulL2-L19-huTNFa (R32A) mutant [soluble forml conjugate (SEQ ID NO:

)

The amino acid sequence of the hull_2-L19-huTNFa (R32A1 mutant [soluble form] conjugate (human IL2 - linker - L19 VH - linker - L19 VL - linker - human TNFa (R32A) mutant [soluble form]) is shown below. The linker sequences are underlined and the R32A is underlined in bold. The human TNFa mutant in this conjugate is the soluble form of the extracellular domain of TNFa.

APTSSSTKKTQLQLEHLLLPLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRPLISNI NVIVLELKGSETTFMCEYAPETATIVEFLNRWITFCQSI ISTLTGGGGSGGGGSGGGGEVQLLESGGGLVQPGGSLRLSCAASGFTFSS FSMSWVRQAPGKGLEWVSSISGSSGTTYYAPSVKGRFTI SRPNSKNTLYLQMNSLRAEPTAVYYCAKPFPYFPYWGQGTLVTVSSGPGS SGGSGGASEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGI PPRFSGSGSGTPFTLTI SRLEP EPFAVYYCQQTGRIPPTFGQGTKVEIKSSSSGXSXSGSSXXGVRSSSRTPSPKPVAHVVANPQAEGQL·QWl·NRAANALl·ANGVELRPNQ LWPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGPRLSAEI NRPPYLPFAESGQVYFGI IAL

26. Amino acid sequence of the hulL2-L19-huTNFa (R52A1 mutant [extracellular domain] conjugate (SEQ ID NO: 351

The amino acid sequence of the hulL2-L19-huTNFa (R52A) mutant [extracellular domain] conjugate (human IL2 - linker - L19 VH - linker - L19 VL - linker - human TNFa (R52A) mutant) is shown below. The linker sequences are underlined and the R52A is underlined in bold. The human TNFa mutant in this conjugate is the extracellular domain of TNFa.

APTSSSTKKTQLQLEHLLLQLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRPLISNI NVIVLELKGSETTFMCEYAPETATIVEFLNRWITFCQSI ISTLTGGGGSGGGGSGGGGEVQLLESGGGLVQPGGSLRLSCAASGFTFSS FSMSWVRQAPGKGLEWVSSISGSSGTTYYAPSVKGRFTI SRPNSKNTLYLQMNSLRAEPTAVYYCAKPFPYFPYWGQGTLVTVSSGPGS SGGSGGASEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPPRFSGSGSGTPFTLTI SRLEP EPFAVYYCQQTGRIPPTFGQGTKVEIKSSSSGSSSSGSSSSGGPQREEFPRPLSLISPLAQAVRSSSRTPSPKPVAHVVANPQAEGQLQ WLNRAANALLANGVELRPNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYE PIYLGGVFQLEKGPRLSAEINRPPYLPFAESGQVYFGI IAL

27. Amino acid sequence of the hulL2-L19-huTNFa [soluble form] conjugate (SEQ ID NO: 36)

The amino acid sequence of the hull_2-L19-huTNFa [soluble form] conjugate (human IL2 - linker - L19 VH - linker - L19 VL - linker - human TNFa [soluble form]) is shown below. The linker sequences are underlined. The human TNFa in this conjugate is the soluble form of the extracellular domain of TNFa.

APTSSSTKKTQLQLEHLLLELQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRPLI SNI NVIVLELKGSETTFMCEYAPETATIVEFLNRWITFCQSI ISTLTGGGGSGGGGSGGGGEVQLLESGGGLVQPGGSLRLSCAASGFTFSS FSMSWVRQAPGKGLEWVSSISGSSGTTYYAPSVKGRFTI SRPNSKNTLYLQMNSLRAEETAVYYCAKPFPYFPYWGQGTLVTVSSGPGS SGGSGGASEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPPRFSGSGSGTPFTLTI SRLEP EPFAVYYCQQTGRIPPTFGQGTKVEIKSSSSGSSSSGSSSSGVRSSSRTPSPKPVAHVVANPQAEGQLQWLNRRANALLANGVELRPNQ LWPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGPRLSAEI NRPPYLPFAESGQVYFGI IAL

28. Amino acid sequence of the hulL2-L19-huTNFa [extracellular domain] conjugate (SEQ ID NO: 37) The amino acid sequence of the hull_2-L19-huTNFa [extracellular domain] conjugate (human IL2 - linker - L19 VH - linker - L19 VL - linker - human TNFa) is shown below. The linker sequences are underlined. The human TNFa in this conjugate is the extracellular domain of TNFa.

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNI NVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSI ISTLTGGGGSGGGGSGGGGEVQLLESGGGLVQPGGSLRLSCAASGFTFSS FSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSGDGS SGGSGGASEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTI SRLEP EDFAVYYCQQTGRIPPTFGQGTKVEIKSSSSGSSXSGSSXSGGPQREEFPRDLSl·ISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQ WLNRRANALLANGVELRDNQLWPSEGLYLIYSQVLFKGQGCPSTHVLLTHTI SRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYE PIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGI IAL

29. Amino acid sequence of human IL2 (hulL21 in the conjugates (SEQ ID NO: 381

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNI NVIVLELKGSETTFMCEYADETATIVEFLNR ITFCQSI ISTLT

30. Amino acid sequence of the mlL2-F8-mTNFa (R32W) mutant [soluble forml conjugate (SEQ ID NO: 39)

APTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQS KSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDESATWDFLRRWIAFCQSIISTSPQGDGSSGGSGGASEVQLLESGGGLVQPGGS LRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFD YWGQGTLVTVSSGGGGSGGGGSGGGGEIVLTQSPGTL·Sl·SPGERATl·SCRASQSVXMPFL·AWYQQKPGQAPRLLIYGASSRATGIPDRF SGSGSGTDFTLTI SRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIKSSSSGSSSSGSSSSGLRSSSQNSSDKPVAHVVANHQVEEQLEWL SQWANALLANGMDLKDNQLVVPADGLYLVYSQVLFKGQGCPDYVLLTHTVSRFAISYQEKVNLLSAVKSPCPKDTPEGAELKPWYEPIY LGGVFQLEKGDQLSAEVNLPKYLDFAESGQVYFGVIAL

31. Amino acid sequence of the L19-mTNFa [soluble forml conjugate (SEQ ID NO: 40)

The amino acid sequence of the L19-mTNFa [soluble form] conjugate_(L19_VH - linker - L19_.VL - linker - mTNFal is shown below. The linker sequences are underlined. The murine TNFa in this conjugate is the soluble form of the extracellular domain of TNFa.

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTI SRDNSKNTLYLQMNSLRAE DTAVYYCAKPFPYFDYWGQGTLVTVSSGDGSSGGSGGASEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLIIY YASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRI PPTFGQGTKVEIKSSSSGSSSSGSSSSGLRSSSQNSSDKPVAHV VANHQVEEQLEWLSQRANALLANGMDLKDNQLVVPADGLYLVYSQVLFKGQGCPDYVLLTHTVSRFAI SYQEKVNLLSAVKSPCPKDTP EGAELKPWYEPIYLGGVFQLEKGDQLSAEVNLPKYLDFAESGQVYFGVIAL

32. Amino acid sequence of linker (SEQ ID NO: 41 )

EFSSSSGSSSSGSSSSG

Claims

Claims

1. A therapeutic combination comprising an immune check-point inhibitor and one or more fibronectin- targeted immunocytokines, wherein the one or more fibronectin-targeted immunocytokines comprise IL2 and/or TNF.

2. A therapeutic combination according to claim 1 wherein the one or more fibronectin-targeted immunocytokines comprise an immunoconjugate comprising TNF and an anti-fibronectin antibody.

3. A therapeutic combination according to claim 1 wherein the one or more fibronectin-targeted immunocytokines comprise an immunoconjugate comprising IL2 and an anti-fibronectin antibody.

4. A therapeutic combination according to claim 1 wherein the one or more fibronectin-targeted immunocytokines comprise a first immunoconjugate comprising IL2 and an anti-fibronectin antibody and a second immunoconjugate comprising TNF and an anti-fibronectin antibody.

5. A therapeutic combination according to claim 1 wherein the one or more fibronectin-targeted immunocytokines comprise a dual immunoconjugate comprising IL2, TNF and an anti-fibronectin antibody.

6. A therapeutic combination according to any one of the preceding claims wherein the immune checkpoint inhibitor is an anti- PD-1 antibody.

7. A therapeutic combination according to claim 6 wherein the anti- PD-1 antibody is nivolumab or pembrolizumab.

8. A therapeutic combination according to any one of claims 1 to 5 wherein the immune check-point inhibitor is an anti- CTLA4 antibody.

9. A therapeutic combination according to claim 8 wherein the anti- CTLA4 antibody is Ipilimumab.

10. A therapeutic combination according to any one of claims 1 to 5 wherein the immune check-point inhibitor is an anti-PD-L1 antibody.

11. A therapeutic combination according to claim 10 wherein the anti-PD-L1 antibody is atezolizumab, avelumab or durvalumab.

12. A therapeutic combination according to any one of the preceding claims wherein the anti-fibronectin antibody comprises a single chain Fv (scFv).

13. A therapeutic combination according to any one of claims 1-1 1 wherein the anti-fibronectin antibody comprises a diabody.

14. A therapeutic combination according to any one of claims 1-13 wherein the anti-fibronectin antibody is specific for the EDB domain of Fibronectin.

15. A therapeutic combination according to claim 14 wherein the anti-fibronectin antibody comprises an antigen binding site having the complementarity determining regions (CDRs) of antibody L19 set forth in SEQ ID NOs 10-15.

16. A therapeutic combination according to claim 14 or claim 15 wherein the anti-fibronectin antibody comprises the L19 VH domain amino acid sequence of SEQ ID NO: 16 or a variant thereof.

17. A therapeutic combination according to any one of claims 14-16 wherein the anti-fibronectin antibody comprises the L19 VL domain amino acid sequence of SEQ ID NO: 17 or a variant thereof.

18. A therapeutic combination according to any one of claims 14-17 wherein the anti-fibronectin antibody comprises the VH and VL domains of antibody L19 set forth in SEQ ID NOs 16 and 17 or variants thereof.

19. A therapeutic combination according to any one of claims 14-18 wherein the anti-fibronectin antibody comprises the amino acid sequence of scFv L19 set forth in SEQ ID NO: 18 or a variant thereof.

20. A therapeutic combination according to any one of the preceding claims wherein the anti-fibronectin antibody is specific for the EDA domain of Fibronectin.

21. A therapeutic combination according to claim 20 wherein the anti-fibronectin antibody comprises an antigen binding site having the complementarity determining regions (CDRs) of antibody F8 set forth in SEQ ID NOs 1-6.

22. A therapeutic combination according to claim 20 or claim 21 wherein the anti-fibronectin antibody comprises the F8 VH domain amino acid sequence of SEQ ID NO: 7 or a variant thereof.

23. A therapeutic combination according to any one of claims 20-22 wherein the anti-fibronectin antibody comprises the F8 VL domain amino acid sequence of SEQ ID NO: 8 or a variant thereof.

24. A therapeutic combination according to any one of claims 20-23 wherein the anti-fibronectin antibody comprises the VH and VL domains of antibody F8 set forth in SEQ ID NOs 7 and 8 or variants thereof.

25. A therapeutic combination according to any one of claims 20-24 wherein the anti-fibronectin antibody comprises the amino acid sequence of scFv F8 set forth in SEQ ID NO: 9 or a variant thereof.

26. A therapeutic combination according to any one of the preceding claims wherein TNF comprises the amino acid sequence of SEQ ID NOs: 19 or 20 or a variant thereof.

27. A therapeutic combination according to any one claims 1 to 25 wherein TNF is a mutant TNF with reduced activity relative to wild-type TNF.

28. A therapeutic combination according to claim 27 wherein TNF comprises the amino acid sequence of SEQ ID NOs: 21 or 22 or a variant thereof.

29. A therapeutic combination according to any one of the preceding claims wherein IL2 comprises the amino acid sequence of SEQ ID NO: 38 or a variant thereof.

30. A therapeutic combination according to any one of the preceding claims wherein the one or more fibronectin-targeted immunoconjugates comprise an immunoconjugate comprising an amino acid sequence of SEQ ID NO: 26 or 32 or a variant of any one of these sequences.

31. A therapeutic combination according to any one of claims 1 to 29 wherein the one or more fibronectin-targeted immunoconjugates comprise an immunoconjugate comprising an amino acid sequence of SEQ ID NO: 27 or 33 or a variant of any one of these sequences.

32. A therapeutic combination according to any one of claims 1 to 29 wherein the one or more fibronectin-targeted immunoconjugates comprise a first immunoconjugate comprising an amino acid sequence of SEQ ID NO: 26 or 32 or a variant of any one of these sequences and a second

immunoconjugate comprising an amino acid sequence of SEQ ID NO: 27 or 33 or a variant of any one of these sequences.

33. A therapeutic combination according to any one of claims 1 to 29 wherein one or more fibronectin- targeted immunoconjugates comprise a dual immunoconjugate comprising an amino acid sequence of any one of SEQ ID NOs: 28-31 and 34-37 or a variant of any one of these sequences.

34. A therapeutic combination comprising an immune check-point inhibitor according to claim 6 or claim 7 and one fibronectin-targeted immunocytokine, wherein the fibronectin-targeted immunocytokine comprises an anti-fibronectin antibody according to any one of claims 14 to 19 and TNF according to any one of claims 26 to 28.

35. A therapeutic combination according to claim 34 comprising an immune check-point inhibitor according to claim 6 or claim 7 and a fibronectin-targeted immunoconjugate comprise an immunoconjugate comprising an amino acid sequence of SEQ ID NO: 33 or a variant of this sequence.

36. A therapeutic combination comprising an immune check-point inhibitor according to claim 10 or claim 11 and one fibronectin-targeted immunocytokine comprising a dual immunoconjugate comprising IL2 according to claim 29, TNF according to any one of claims 26 to 28 and an anti-fibronectin antibody according to any one of claims 20 to 25.

37. A therapeutic combination according to claim 36 comprising an immune check-point inhibitor according to claim 10 or claim 1 1 and one fibronectin-targeted immunocytokine comprising a dual immunoconjugate comprising an amino acid sequence of any one of SEQ ID NOs: 28-31 or a variant of any one of these sequences.

38. A therapeutic combination according to any one of the preceding claims wherein the one or more fibronectin-targeted immunoconjugates and the immune checkpoint inhibitor are formulated in a single composition.

39. A therapeutic combination according to any one of claims 1 to 37 wherein the one or more fibronectin-targeted immunoconjugates and the immune checkpoint inhibitor are formulated in separate compositions.

40. A therapeutic combination according to any one of the preceding claims for use in a method of treatment of the human or animal body.

41. A method of treatment of cancer comprising administering a therapeutic combination according to any one of claims 1 to 39 to an individual in need thereof.

42. A therapeutic combination according to any one of claims 1 to 39 for use in a method of treatment of cancer.

43. An immunoconjugate comprising IL2 and an anti-fibronectin antibody and/or an immunoconjugate comprising TNF and an anti-fibronectin antibody for use in a method of treating cancer according to claim 41.

44. A dual immunoconjugate comprising IL2, TNF and an anti-fibronectin antibody for use in a method of treating cancer according to claim 41.