Conditional TNFR1 agonists

Abstract

The present invention relates to bi-, tri-, or oligospecific anti-TNFR1 fusion proteins, which are monovalent for TNFR1 and exhibit excellent productivity and strong conditional TNFR1 agonism. The invention also relates to bi-, tri-, or oligospecific anti-TNFR1 nanobody fusion proteins which display strong conditional TNFR1 agonism upon binding to cell surface or extracellular matrix antigens such as PD-L1 or CD70. The invention also relates to methods of producing such bi-, tri-, or oligospecific anti-TNFR1 nanobody fusion protein constructs, pharmaceutical compositions comprising the same, as well as their uses for treating cancer.

Description

[0001] Conditional TNFR1 agonists

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to bi-, tri-, or oligospecific anti-TNFR1 fusion proteins, which are monovalent for TNFR1 and exhibit excellent productivity and which display strong conditional TNFR1 agonism. The invention also relates to bi-, tri-, or oligospecific anti-TNFR1 nanobody fusion proteins which display strong conditional TNFR1 agonism upon binding to cell surface or extracellular matrix antigens such as PD-L1 or CD70. The invention also relates to methods of producing such bi-, tri-, or oligospecific anti-TN F R 1 nanobody fusion protein constructs, pharmaceutical compositions comprising the same, as well as their uses for treating cancer.

[0004] BACKGROUND

[0005] The cytokine tumor necrosis factor-alpha (TNF) has proinflammatory and cytotoxic activities but also elicits tissue protecting and immune suppressive effects (1 ,2). TNF acts via two receptors, TNF receptor-1 (TNFR1 ) and TNFR2 (3). TNFR1 is an extremely potent activator of proinflammatory signaling pathways but can also induce apoptotic and necroptotic cell death (3,4). Indeed, primarily the activities of TNFR1 make TNF a driver of autoimmune diseases. The therapeutic effect of TNF blockers in rheumatoid diseases, psoriasis and Crohn's disease therefore primarily result from preventing the activation of TNFR1 (1 ,2). TNFR2 has also inflammatory activities but above all mediates the anti-inflammatory and tissue protective activities of TNF (1 ,5).

[0006] Tumor necrosis factor (TNF) binds and activates two structurally related types of receptors, TNF receptor-1 (TNFR1) and TNFR2. TNFR1 is expressed by virtually all kinds of cells in the human body. Activation of TNFR1 leads to a strong release of immune stimulating factors as well as induction of programmed cell death program (3,4). TNF receptor-2 (TNFR2) is only expressed by a subset of cell types and, in addition to some pro-inflammatory functions, mainly mediates the cell-protective and immunosuppressive effects of TNF (1 ,2). As already indicated by its name, the combination of inflammatory cell activation and cell death induction of TNF via the TNFR1 axis leads in particular to the destruction of tumors (6). For this reason, already in the late 1990s a number of clinical trials were conducted using recombinantly produced soluble TNF to treat cancer (7). However, these studies were stopped after several years, not due to the lack of antitumor effect of TNF, but due to the serious systemic side effects of TNF on healthy cells and organs, which prevented effective tumor treatment (7). Nevertheless, recombinant TNF received clinical approval as early as 1999 for the treatment of soft tissue sarcomas as part of "isolated limb perfusion", in which the effect of TNF is locally limited by cutting off the blood supply to the treated limb (8). Interestingly, both the antitumor effects and the dose limiting "side effects" of TNF on healthy cells, tissues and organs play an important role in cancer therapy with the newly and extremely successfully used immune checkpoint inhibitors (I Cis) (9).

[0007] Cancers, among other things, exploit immune suppressive proteins, such as PD-L1 and CTLA-4, which are otherwise used by the body to restrict immune responses to evade tumor surveillance by the immune system. This so-called immune checkpoint can be quite successfully targeted with clinically approved inhibitory antibodies or soluble decoy receptors. In line with the central role of TNF and its receptors in immune regulation, it has been found that immune checkpoint blockade (ICB) comes along with increased TNF production. Indeed, expression of PD-L1 , a major ICI target on tumor cells has been implicated in the inhibition of signaling pathways resulting in TNF production (10). Now, the ICI-related stimulation of TNF production comes along with complex, partly opposing effects on ICB therapies including without claim on completeness:

[0008] 1. Negative feedback on ICB

[0009] Upregulation of PD-L1 expression on tumor infiltrating lymphocytes (TILs) and monocytes and macrophages by the ability of TNR1 to potently stimulate the classical NFkB pathway resulting in particular in induction of Cop9 signalosome 5 (CSN5) which inhibits ubiquitination and proteasomal degradation of PD-L1 (11-14). TNFR2 stimulation by membrane TNF can furthermore boost the proliferation, metabolic fitness and suppressive activity of MDSCs and Tregs (1 ,2,5).

[0010] 2. Costimulation of CD8+T-cells

[0011] Especially in the early phase of CD8+ T-cell stimulation, TNF via TNF2 can act as a costimulatory molecule enhancing T-cell activity and promoting the expression of anti-apoptotic molecules (1 ,2,5).

[0012] 3. Activation induced cell death of CD8+T-cells

[0013] TNFR1-induced cell death is one of several mechanisms involved in the downregulation of CD8+ T-cells responses including CD8+ TILs after repeated / prolonged stimulation. Worth mentioning, there is evidence from studies with TNF- and TNFR1 -deficient mice that this mechanism limits the efficacy of anti-PD1 ICB (12).

[0014] 4. Killing of tumor cells by CD8+T-cell and / or NK cell derived TNF. Crispr / Cas9 screens results in the identification of the cytotoxic TNF-TNFR1 signaling axis as a major factor for the success of ICB. TNFR1 and its cell death signaling intermediates caspase-8, FADD, RIPK1 as well as the TNFR1 killing-suppressing factors LUBAC, TRAF2 and cl AP 1 have all been identified in genome-wide Crispr / Cas9 screens as important factors involved in the control of tumor development by CD8+ T-cells and NK cells (15,16). Intriguingly, TRAF2 and clAP1 were furthermore found to be major sgRNA targets enhancing tumor cell killing under ICB (17). Moreover, TRAF2-deficiency and IAP antagonists sensitize for tumor cell killing by CD8+ T-cell / NK cell-derived TNF (10,17). Furthermore, comprehensive meta-analysis of genomic / transcriptional data of > 1000 ICI treated patients identified chromosomal loss of 9q34, the chromosomal region where the TRAF2 gene is located, as positively associated with the clinical response (18).

[0015] 5. Induction of of-tumor autoimmune pathologies

[0016] Similar as in patients suffering from autoimmune diseases, I Cis can trigger therapy limiting immune-related adverse events including the induction of colitis, cardiotoxicity or ICI-associated arthritis (19-21). These ICB- associated autoimmune syndromes are accordingly currently often treated with TNF blockers with complex results. Initial clinical data and preclinical animal model data give evidence for both inhibition of the ICB- associated TNF-driven autoimmune effects but also of the antitumoral activity but also for the inhibition of the ICB-associated TNF-driven autoimmune effects along with an improved anti-tumor activity.

[0017] It is tempting to speculate that the contrasting effects of a TNF blockade on the anti-tumor activity of ICIs reflect the varying relative importance of the TNF-related pro- (enhanced CD8+T-cell AICD, PD-L1 , MDSC / Treg stimulation) and antitumoral (tumor cell killing, CD8+T-cell costimulation) activities. This consideration prompt interest on the development of drugs specifically promoting the antitumoral effects of the combined blockade of immune checkpoints and TNF, e.g. drugs / biologicals sensitizing cells for TNFRI- induced cell death or TNFR1 agonists with tumor-localized, thus conditional activity. Of course, conditional TNFR1 agonists also promise to be useful in ICB-independent tumor therapy regimes.

[0018] Conditional TNFR1 agonists: State of the Art

[0019] Beside isolated limb perfusion, two concepts are under investigation aiming on the local tumor-associated in vivo activation with systemically applicable drugs. First, the development of TNF immunocytokines, thus TNF fusion proteins (typically antibody fusion proteins) that bind to tumor-associated structures and eventually have a broader therapeutic window due to the accumulation of the TNF immunocytokine in the tumor area. Although such constructs show an increased TNF effect in the tumor, they are still systemically active (22). The therapeutic window of such constructs might be widened by using TNF mutants with reduced bioactivity as basis (23). Second, tumor antigen-binding TNF prodrugs which connect eventually a TNF immunocytokine module via a tumor-associated protease sensitive linker with an intramolecular TNF-inhibitory domain. These constructs have a very high tumor cell- / tumor protease-associated TNF activity (24) but are challenging to develop biotech nologically due to their very complex domain architecture. Both of these TNF-based concepts of TNFR1 activation have not yet made it into the clinic and suffer from the effect that TNFR2 is also activated.

[0020] Bivalent anti-TNFR1 antibodies typically trigger significant TNFR1 signaling (25). Indeed, even a TNF- blocking anti-TNFR1 antibody, such as H398 which is used as experimental TNFR1 antagonist as it strongly reduces TNFR1 activation in response to TNF, elicit in the absence of TNF significant residual TNFR1 activation. The latter are in vivo relevant enough to prevent the clinical development of a bivalent H398- derived TNFR1 -blocking antibody. Only after conversion into a monovalent variant devoid of any agonistic activity H398 has been considered for further clinical development (2). The strong agonistic responsiveness of TNFR1 to anti-TNFR1 IgGs is in contrast to the majority of other receptors of the TNFRSF II which are typically not activated by “free” bivalent antibodies and only become efficiently stimulated by such antibodies when bound to FcyRs thus when presented in membrane-associated form.

[0021] DESCRIPTION OF THE INVENTION

[0022] The inventors have identified fusion proteins, comprising i) a TNFR1 -binding protein, wherein the TNFR1- binding protein is monovalent for TNFR1 , ii) a domain capable of binding to a cell surface antigen or an extracellular matrix antigen (also referred to as “anchoring domain”), and optionally further domains capable of binding to a different cell surface antigen or extracellular matrix antigen than the domain according to ii).These fusion proteins display strong targeting-dependent TNFR1 agonism. In contrast to TNF immunocytokines and conventional anti-TNFR1 antibodies, this novel type of conditional TNFR1 agonists is inactive or largely inactive in the absence of its anchoring target or even acts as a TNFR1 inhibitor. Advantageously, by selecting a domain (or domains) capable of binding to (a) cell surface antigen(s) or (an) extracellular matrix antigen(s) which is / are specific to a particular cell type(s) or tissue(s), the fusion proteins of the invention can be used as cell type-specific or tissue-specific TNFR1 agonists. For example, the cell surface antigen(s) or extracellular matrix antigen(s) may be a cancer antigen and / or an antigen of the tumor microenvironment. Thus, a fusion protein of the invention can be used as a tumor-specific TNFR1 agonist, or as a TNFR1 agonist preferentially targeting tumor(s), in the treatment of cancer.

[0023] Moreover, the fusion proteins of the invention do not stimulate TNFR2, irrespective of whether the molecules are anchored or not to the plasma membrane. The molecules are furthermore efficiently produced. Thus, the fusion proteins of the invention, which are a type of conditional TNFR1 agonists, overcome the limitations of TNF immunocytokines, TNF prodrugs and conventional anti-TNFR1 antibodies and therefore promise systemic applicability for tumor therapy. Accordingly, the invention relates to the following preferred embodiments:

[0024] 1 . A fusion protein, comprising: i) a TNFR1 -binding protein, wherein the TNFR1 -binding protein is monovalent for TNFR1, and ii) a domain capable of binding to a cell surface antigen or an extracellular matrix antigen.

[0025] 2. The fusion protein of item 1 , wherein: the TNFR1 -binding protein according to i) is an anti-TNFR1 antibody or a TNFR1 -binding portion thereof, wherein the anti-TNFR1 antibody or TNFR1 -binding portion thereof is monovalent for TNFR1.

[0026] 3. The fusion protein of item 1 , wherein:

[0027] The TNFR1 -binding protein according to i) is a mutant single-chain TNF variant, wherein the mutant single-chain TNF variant thereof is monovalent for TNFR1 .

[0028] 4. The fusion protein according to any one of the preceding items, wherein the fusion protein is monovalent for TNFR1.

[0029] 5. The fusion protein according to any one of items 1-2 and 4, wherein the TNFR1-binding protein according to i) is an anti-hTNFR1 antibody or hTNFRI -binding portion thereof.

[0030] 6. The fusion protein according to any one of items 1-2 and 4-5, wherein the TNFR1 -binding protein according to i) is a VHH, a Fab domain, or an scFv fragment.

[0031] 7. The fusion protein according to any one of items 1-2 and 4-6, wherein the TNFR1 -binding protein according to i) is a VHH.

[0032] 8. The fusion protein according to any one of items 1-2 and 4-6, wherein the TNFR1 -binding protein according to i) is a Fab domain.

[0033] 9. The fusion protein according to any one of items 1-2 and 4-6, wherein the TNFR1 -binding protein according to i) is an scFv fragment.

[0034] 10. The fusion protein according to any one of the preceding items, wherein said fusion protein is an agonist of TNFR1 when bound to TNFR1 and bound to the cell surface antigen or extracellular matrix antigen, and is not an agonist of TNFR1 when bound to TNFR1 but not bound to the cell surface antigen or extracellular matrix antigen. The fusion protein according to any one of the preceding items, wherein said fusion protein is an agonist of TNFR1 when bound to TNFR1 and bound to the cell surface antigen or extracellular matrix antigen, and is an antagonist of TNFR1 when bound to TNFR1 but not bound to the cell surface antigen or extracellular matrix antigen. The fusion protein according to any one of items 3-4 and 10-11 , wherein the mutant single-chain TNF variant comprises three peptide linker-connected TNF protomers covering the receptor binding domain, whereby two of the three protomers do not bind to or show reduced binding to TNFR1. The fusion protein according to any one of items 3-4 and 10-12, wherein the mutant single-chain TNF variant comprises two LTalpha protomers and one LTbeta protomer covering the receptor binding domains of these ligands. The fusion protein according to any one of the preceding items, wherein the domain according to ii) comprises an antibody or an antigen-binding portion, said antibody or antigen-binding portion being capable of binding to said cell surface antigen or extracellular matrix antigen. The fusion protein according to item 14, wherein said antibody or antigen-binding portion capable of binding to said cell surface antigen or extracellular matrix antigen comprises at least one Fab domain, VHH, or scFv fragment or a combination of at least one Fab domain, at least one VHH, and / or at least one scFv fragment. The fusion protein according to item 15, wherein said antibody or antigen-binding portion capable of binding to said cell surface antigen or extracellular matrix antigen comprises at least one Fab domain. The fusion protein according to item 15, wherein said antibody or antigen-binding portion capable of binding to said cell surface antigen or extracellular matrix antigen comprises at least one scFv fragment. The fusion protein according to item 15, wherein said antibody or antigen-binding portion capable of binding to said cell surface antigen or extracellular matrix antigen comprises at least one VHH. The fusion protein according to any one of the preceding items, wherein the domain according to ii) does not comprise an anti-TNFR1 -antibody or antigen-binding portion thereof. The fusion protein according to any one of the preceding items, wherein said cell surface antigen or extracellular matrix antigen is a protein. The fusion protein according to any one of the preceding items, wherein said cell surface antigen or extracellular matrix antigen is a cell surface antigen. The fusion protein according to any one of the preceding items, wherein said cell surface antigen or extracellular matrix antigen is an extracellular matrix antigen. The fusion protein according to any one of the preceding items, wherein said cell surface antigen is an antigen of an immune cell, preferably wherein the immune cell is a human immune cell, further preferably wherein the human immune cell is a human T lymphocyte. The fusion protein according to any one of the preceding items, wherein said cell surface antigen is a cancer antigen and / or an antigen of the tumor microenvironment. The fusion protein according to any one of the preceding items, wherein said antigen is PD-L1, CD70, CD40, CXCR4, BCMA, CTLA4, FAP or PD1. The fusion protein according to any one of the preceding items, wherein said antigen is PD-L1. The fusion protein according to any one of the items 1 -25, wherein said antigen is CD70. The fusion protein according to any one of the preceding items, wherein said antigen is a human antigen. The fusion protein according to any one of the preceding items, wherein said antibody or antigenbinding portion capable of binding to said cell surface antigen or extracellular matrix antigen is an immune checkpoint inhibitor. The fusion protein according to any one of items 1 to 29, wherein the domain according to ii) comprises an anti-CD70 Fab and the TNFR1 -binding protein according to i) is an anti-TNFR1 VHH, and the fusion protein is selected from the group consisting of: a1 ) a fusion protein comprising a first polypeptide chain having an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 1 , at least 90% identical to the amino acid sequence of SEQ ID NO: 1 , at least 95% identical to the amino acid sequence of SEQ ID NO: 1 , at least 98% identical to the amino acid sequence of SEQ ID NO: 1 , or at least 99% identical to the amino acid sequence of SEQ ID NO: 1 , and a second polypeptide chain having an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 2, at least 90% identical to the amino acid sequence of SEQ ID NO: 2, at least 95% identical to the amino acid sequence of SEQ ID NO: 2, at least 98% identical to the amino acid sequence of SEQ ID NO: 2, or at least 99% identical to the amino acid sequence of SEQ ID NO: 2; a2) a fusion protein comprising a first polypeptide chain having the amino acid sequence of SEQ ID NO: 1 , and a second polypeptide chain having the amino acid sequence of SEQ ID NO: 2; a3) a fusion protein competing with the fusion protein of a2) for specific binding to CD70 and for specific binding to TNFR1 ; a4) a fusion protein wherein the domain according to ii) comprises an anti-CD70 Fab having the same CDRs as the anti-CD70 Fab of the fusion protein of a2), and wherein the TNFR1 -binding protein according to i) is an anti-TNFR1 VHH having the same CDRs as the anti-TNFR1 VHH of the fusion protein of a2); b1 ) a fusion protein comprising a first polypeptide chain having an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 3, at least 90% identical to the amino acid sequence of SEQ ID NO: 3, at least 95% identical to the amino acid sequence of SEQ ID NO: 3, at least 98% identical to the amino acid sequence of SEQ ID NO: 3, or at least 99% identical to the amino acid sequence of SEQ ID NO: 3, and a second polypeptide chain having an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 5, at least 90% identical to the amino acid sequence of SEQ ID NO: 5, at least 95% identical to the amino acid sequence of SEQ ID NO: 5, at least 98% identical to the amino acid sequence of SEQ ID NO: 5, or at least 99% identical to the amino acid sequence of SEQ ID NO: 5; b2) a fusion protein comprising a first polypeptide chain having the amino acid sequence of SEQ ID NO: 3, and a second polypeptide chain having the amino acid sequence of SEQ ID NO: 5; b3) a fusion protein competing with the fusion protein of b2) for specific binding to CD70 and for specific binding to TNFR1 ; b4) a fusion protein wherein the domain according to ii) comprises an anti-CD70 Fab having the same CDRs as the anti-CD70 Fab of the fusion protein of b2), and wherein the TNFR1 -binding protein according to i) is an anti-TNFR1 VHH having the same CDRs as the anti-TNFR1 VHH of the fusion protein of b2); c1 ) a fusion protein comprising a first polypeptide chain having an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 4, at least 90% identical to the amino acid sequence of SEQ ID NO: 4, at least 95% identical to the amino acid sequence of SEQ ID NO: 4, at least 98% identical to the amino acid sequence of SEQ ID NO: 4, or at least 99% identical to the amino acid sequence of SEQ ID NO: 4, and a second polypeptide chain having an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 5, at least 90% identical to the amino acid sequence of SEQ ID NO: 5, at least 95% identical to the amino acid sequence of SEQ ID NO: 5, at least 98% identical to the amino acid sequence of SEQ ID NO: 5, or at least 99% identical to the amino acid sequence of SEQ ID NO: 5; c2) a fusion protein comprising a first polypeptide chain having the amino acid sequence of SEQ ID NO: 4, and a second polypeptide chain having the amino acid sequence of SEQ ID NO: 5; c3) a fusion protein competing with the fusion protein of c2) for specific binding to CD70 and for specific binding to TNFR1 ; c4) a fusion protein wherein the domain according to ii) comprises an anti-CD70 Fab having the same CDRs as the anti-CD70 Fab of the fusion protein of c2), and wherein the TNFR1 -binding protein according to i) is an anti-TNFR1 VHH having the same CDRs as the anti-TNFR1 VHH of the fusion protein of c2); d1 ) a fusion protein comprising a first polypeptide chain having an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 1 , at least 90% identical to the amino acid sequence of SEQ ID NO: 1 , at least 95% identical to the amino acid sequence of SEQ ID NO: 1 , at least 98% identical to the amino acid sequence of SEQ ID NO: 1 , or at least 99% identical to the amino acid sequence of SEQ ID NO: 1 , and a second polypeptide chain having an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 8, at least 90% identical to the amino acid sequence of SEQ ID NO: 8, at least 95% identical to the amino acid sequence of SEQ ID NO: 8, at least 98% identical to the amino acid sequence of SEQ ID NO: 8, or at least 99% identical to the amino acid sequence of SEQ ID NO: 8; d2) a fusion protein comprising a first polypeptide chain having the amino acid sequence of SEQ ID NO: 1 , and a second polypeptide chain having the amino acid sequence of SEQ ID NO: 8; d3) a fusion protein competing with the fusion protein of d2) for specific binding to CD70 and for specific binding to TNFR1 ; and d4) a fusion protein wherein the domain according to ii) comprises an anti-CD70 Fab having the same CDRs as the anti-CD70 Fab of the fusion protein of d 2), and wherein the TNFR1 -binding protein according to i) is an anti-TNFR1 VHH having the same CDRs as the anti-TNFR1 VHH of the fusion protein of d2). The fusion protein according to any one of items 1 to 29, wherein the domain according to ii) comprises an anti-PD-L1 Fab and the TNFR1 -binding protein according to i) is an anti-TNFR1 VHH, and the fusion protein is selected from the group consisting of: e1 ) a fusion protein comprising a first polypeptide chain having an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 6, at least 90% identical to the amino acid sequence of SEQ ID NO: 6, at least 95% identical to the amino acid sequence of SEQ ID NO: 6, at least 98% identical to the amino acid sequence of SEQ ID NO: 6, or at least 99% identical to the amino acid sequence of SEQ ID NO: 6, and a second polypeptide chain having an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 7, at least 90% identical to the amino acid sequence of SEQ ID NO: 7, at least 95% identical to the amino acid sequence of SEQ ID NO: 7, at least 98% identical to the amino acid sequence of SEQ ID NO: 7, or at least 99% identical to the amino acid sequence of SEQ ID NO: 7; e2) a fusion protein comprising a first polypeptide chain having the amino acid sequence of SEQ ID NO: 6, and a second polypeptide chain having the amino acid sequence of SEQ ID NO: 7; e3) a fusion protein competing with the fusion protein of e2) for specific binding to PD-L1 and for specific binding to TNFR1 ; e4) a fusion protein wherein the domain according to ii) comprises an anti-PD-L 1 Fab having the same CDRs as the anti-PD-L1 Fab of the fusion protein of e2), and wherein the TNFR1 -binding protein according to i) is an anti-TNFR1 VHH having the same CDRs as the anti-TNFR1 VHH of the fusion protein of e2); f1) a fusion protein comprising a first polypeptide chain having an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 6, at least 90% identical to the amino acid sequence of SEQ ID NO: 6, at least 95% identical to the amino acid sequence of SEQ ID NO: 6, at least 98% identical to the amino acid sequence of SEQ ID NO: 6, or at least 99% identical to the amino acid sequence of SEQ ID NO: 6, and a second polypeptide chain having an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 9, at least 90% identical to the amino acid sequence of SEQ ID NO: 9, at least 95% identical to the amino acid sequence of SEQ ID NO: 9, at least 98% identical to the amino acid sequence of SEQ ID NO: 9, or at least 99% identical to the amino acid sequence of SEQ ID NO: 9; f2) a fusion protein comprising a first polypeptide chain having the amino acid sequence of SEQ ID NO: 6, and a second polypeptide chain having the amino acid sequence of SEQ ID NO: 9; f3) a fusion protein competing with the fusion protein of f2) for specific binding to PD-L1 and for specific binding to TNFR1 ; and f4) a fusion protein wherein the domain according to ii) comprises an anti-PD-L1 Fab having the same CDRs as the anti-PD-L1 Fab of the fusion protein of f2), and wherein the TNFR1 -binding protein according to i) is an anti-TNFR1 VHH having the same CDRs as the anti-TNFR1 VHH of the fusion protein of f2). The fusion protein according to any one of the preceding items, wherein the fusion protein further comprises iii) an additional protein domain. The fusion protein according to item 32, wherein the additional protein domain according to iii) is serum albumin, transferrin or a Fab domain, VHH or scFv capable of binding to serum albumin or a transferrin receptor. The fusion protein according to item 32, wherein the additional protein domain according to iii) is serum albumin. The fusion protein according to any one of the preceding items, wherein the fusion protein further comprises iv) at least one domain capable of binding to a different cell surface antigen or extracellular matrix antigen than the domain according to ii). The fusion protein according to item 35, wherein the at least one domain capable of binding to a different cell surface antigen or extracellular matrix antigen is a domain capable of binding to a cell surface antigen or extracellular matrix antigen as defined in any one of items 14-31 . 37. A pharmaceutical composition comprising the fusion protein according to any one of the preceding items.

[0035] 38. A pharmaceutical composition comprising a fusion protein according to any one of items 1-36, for use as a medicament.

[0036] 39. A pharmaceutical composition according to item 38 or a fusion protein according to any one of items 1-36, for use in the treatment of cancer.

[0037] 40. The pharmaceutical composition or fusion protein for use according to item 39, wherein the cancer is a hematologic malignancy.

[0038] 41. The pharmaceutical composition or fusion protein for use according to item 39, wherein the cancer is a solid cancer.

[0039] 42. The pharmaceutical composition or fusion protein for use according to any one of items 39-41 , wherein the use is a use in combination with an immune checkpoint inhibitor.

[0040] 43. A nucleic acid, or a set of nucleic acids, encoding the fusion protein according to any one of items 1-36.

[0041] 44. A recombinant cell containing a nucleic acid, or a set of nucleic acids, according to item 43 and expressing the fusion protein of any one of items 1-36.

[0042] 45. A method for producing a fusion protein according to any one of items 1 to 36, the method comprising expressing the nucleic acid or set of nucleic acids according to item 43 in a cell according to item 44, and harvesting the fusion protein.

[0043] BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 : Scheme of domain architecture of Fab(muCD70)-HC:VHH(muTNFR1) and Fab(mu / hPDL1)- HC:VHH(muTNFR1).

[0045] Figure 2: Biochemical characterization of purified Fab(muCD70)-HC:VHH(muTNFR1). (A,B) Fab(muCD70)-HC:VHH(muTNFR1) was purified by affinity chromatography on anti-Flag M2 agarose. Silver stained SDS-PAGE gel (A). Chromatogram of gel filtration on a MabPac SEC-1 (B).

[0046] Figure 3: Fab(muCD70)-HC:VHH(muTNFR1) binds murine CD70 and murine TNFR1. Binding of GpL-

[0047] TNC-muCD70 and muTNFR1-GpL to plastic immobilized Fab(muCD70)-HC:VHH(muTNFR1). Figure 4: Fab(muCD70)-HC:VHH(muTNFR1) inhibits muCD70-interaction. Specific binding of 25 ng / ml GpL-TNC-muCD70 to human HT1080-CD27 cells in the presence and absence of 10 g / ml Fab(muCD70)- HC:VHH(muTNFR1).

[0048] Figure 5: Membrane muCD70-dependent activation of murine TNFR1. PancO2 murine cells, which produce MCP-1 in response to TNFR1 activation, were co-cultivated with human HEK293 cells transiently transfected with empty vector or an expression vector encoding full-length, thus transmembrane murine CD70. Cocultures were stimulated with the indicated concentrations of Fab(muCD70)-HC:VHH(muTNFR1) and the next day muMCP-1 production was measured by ELISA.

[0049] Figure 6: Biochemical characterization of purified Fab(hCD70)-HC:VHH(muTNFR1). A) Binding of GpL- TNC-huCD70 and muTNFR1-GpL to plastic immobilized Fab(hCD70)-HC:VHH(muTNFR1). (B, C) Fab(hCD70)-HC:VHH(muTNFR1) was purified by affinity chromatography on anti-Flag M2 agarose. Silver stained SDS-PAGE gel (B). Chromatogram of gel filtration on a MabPac SEC-1 (C).

[0050] Figure 7: Membrane hCD70-dependent activation of murine TNFR1. Murine PancO2 cells were cocultivated with human BJAB cells which endogenously express membrane hCD70 or human Jurkat cells not expressing CD70. Cocultures were stimulated with the indicated concentrations of Fab(hCD70)- HC:VHH(muTNFR1) and the next day muMCP-1 production was measured by ELISA.

[0051] Figure 8: Membrane hCD70-dependent activation of human TNFR1. (A) U2OS cells expressing medium levels of endogenous transmembrane CD70 were supplemented with Hek293 cells transiently transfected with empty vector or a transmembrane CD70 expression plasmid and stimulated overnight with the indicated concentrations of Fab(CD70)-HC:VHH(TNFR1) in the absence and presence of 10 pg / ml of the parental anti-CD70 antibody 9G2. Finally, IL8 production, as an indicator of TNFR1 activation, was measured by ELISA. (B) HT1080 cells were supplemented with BJAB cells expressing endogenous CD70 (Silence et al., 2014) or Jurkat cells not expressing CD70 and stimulated then overnight with the indicated concentrations of Fab(CD70)-HC:VHH(TNFR1). IL8 production was again measured as an indicator of TNFR1 activation. Jurkat and BJAB express TNFR1 but does not produce IL8.

[0052] Figure 9: Biochemical characterization of purified Fab(mu / hPDL1)-HC:VHH(muTNFR1). (A) Binding of GpL-TNC-muPDL1 and muTNFR1-GpL to plastic immobilized Fab(h / muPDL1)-HC:VHH(muTNFR1). (B, C) Fab(h / muPDL1)-HC:VHH(muTNFR1) was purified by affinity chromatography on anti-Flag M2 agarose. Silver stained SDS-PAGE gel (B). Chromatogram of gel filtration on a MabPac SEC-1 (C). Figure 10: Membrane hPDL1-dependent activation of murine TNFR1. (A) PancO2 murine cells, which produce MCP-1 in response to TNFR1 activation, were co-cultivated with human HEK293 transfectants expressing human PDL1 or as a negative control empty vector. (B) In another approach, PancO2 cells cocultures were co-cultivated with human ES-2 cells which express PDL1 endogenously and a PDL1 knockout variant derived thereof. (A,B) Cocultures were stimulated with the indicated concentrations of Fab(mu / hPDL1)-HC:VHH(muTNFR1). Next day, muMCP-1 production was measured by ELISA. (C) PancO2 murine cells were co-cultivated with human HEK293 transfectants expressing murine PDL1 or as a negative control empty vector.

[0053] Figure 11 : Scheme of domain architecture of serum albumin fusion proteins of Fab(muCD70)- HC:VHH(muTNFR1) and Fab(mu / hPDL1)-HC:VHH(muTNFR1).

[0054] Figure 12: Serum albumin fusion proteins of Fab(muCD70)-HC:VHH(muTNFR1) and Fab(mu / hPDL1)- HC:VHH(muTNFR1) retain their conditional TNFR1 agonistic activity. (A) Western blot analysis of constructs. (B,C) PancO2 cells were co-cultivated with human Hek293 transfectants expressing murine CD70 (B), human PDL1 (C) or empty vector (B,C). Cocultures were stimulated with the indicated concentrations of Fab(muCD70)-LC:MSA-HC:VHH(muTNFR1) or Fab(mu / hPDL1)-LC:MSA- HC:VHH(muTNFR1) and the next day muMCPI production was evaluated by ELISA.

[0055] Figure 13: Biochemical characterization of purified Fab(muCD70)-LC:MSA-HC:VHH(muTNFR1) and Fab(mu / hPDL1)-LC:MSA-HC:VHH(muTNFR1). (A,B) Fab(muCD70)-LC:MSA-HC:VHH(muTNFR1) and Fab(mu / hPDL1)-LC:MSA-HC:VHH(muTNFR1) were purified by affinity chromatography on anti-Flag M2 agarose. Silver stained SDS-PAGE gel (A). Chromatogram of gel filtration on a MabPac SEC-1 (B).

[0056] Figure 14: (A) Scheme of domain architecture of Fab(h / muFAP)-C1 H:VHH(muTNFR1). (B,C) Fab(h / muFAP)-C1 / H:VHH(muTNFR1) was purified by affinity chromatography on anti-Flag M2 agarose. Silver stained SDS-PAGE gel (B). Chromatogram of gel filtration on a MabPac SEC-1 (C). (D) PancO2 murine cells, which produce MCP-1 in response to TNFR1 activation, were co-cultivated with murine B16 cells stably transfected with human FAP (B16-FAP) as a negative control with B16 cells. Cocultures were stimulated with the indicated concentrations of Fab(h / muFAP)-C1 H:VHH(muTNFR1). Next day, muMCP-1 production was measured by ELISA.

[0057] DETAILED DESCRIPTION OF THE INVENTION

[0058] Definitions and General Techniques Unless otherwise defined below, the terms used in the present invention shall be understood in accordance with their common meaning known to the person skilled in the art. All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. Publications referred to herein may be cited by specifying the full literature reference in the text, by naming the author and the publication year (e.g., “Wajant et al. 2003”) and by specifying the corresponding full literature reference in the “references” section, or by naming a number (e.g., “(2)”) and by specifying the corresponding full literature reference in the “references” section.

[0059] All proteins in accordance with the invention, including the fusion proteins of the invention, can be obtained by methods known in the art. Such methods include methods for the production of recombinant proteins. The fusion proteins of the invention can be expressed in recombinant host cells according to the invention. Recombinant host cells of the invention are preferably mammalian cells such as HEK or PancO2 cells. It will be understood that the fusion proteins of the invention are meant to optionally include a secretion signal peptide sequence. Similarly, the fusion proteins of the invention are meant to also optionally include affinity tags, e.g., in order to facilitate purification, and optional protease cleavage sites between the tag and the fusion proteins, e.g., in order to facilitate removal of the tags by protease cleavage. Likewise, it will be understood that the fusion proteins of the invention are meant to optionally include the respective propeptides.

[0060] It is also understood that any reference to amino acid sequences referred to herein is meant to encompass not only the unmodified amino acid sequence but also typical posttranslational modifications of these amino acid sequences (e.g., glycosylation or deamidation of amino acids, the clipping of particular amino acids or other posttranslational modifications) occurring in cellular expression systems known in the art, including mammalian cells such as HEK or PancO2 cells.

[0061] The term “antibody” as used herein refers to any functional antibody that is capable of specific binding to the antigen of interest, as generally outlined in chapter 7 of Paul, W.E. (Ed.).: Fundamental Immunology 2nd Ed. Raven Press, Ltd., New York 1989, which is incorporated herein by reference. Without particular limitation, the term “antibody” encompasses antibodies from any appropriate source species, including chicken and mammalian such as mouse, goat, non-human primate and human. Preferably, the antibody is a humanized or human antibody. The antibody is preferably a monoclonal antibody which can be prepared by methods well-known in the art. The term “antibody” encompasses an lgG-1 , -2, -3, or -4, IgE, IgA, IgM, or IgD isotype antibody. The term “antibody” encompasses monomeric antibodies (such as IgD, IgE, IgG) or oligomeric antibodies (such as IgA or IgM). The term “antibody” also encompasses - without particular limitations - isolated antibodies and modified antibodies such as genetically engineered antibodies, e.g., chimeric, humanized or human antibodies. For example, the terms “lgG1”, “lgG2”, “lgG3” or “lgG4” as used in accordance with the invention encompass chimeric, humanized and human lgG1 , lgG2, lgG3 and lgG4, respectively. Known types of modified antibodies also include bispecific antibodies, bispecific antibodies, knob into hole antibodies and CrossMAb antibodies. For example, an “anti-TNFR1 antibody” as a “TNFR1- binding protein” as part of the fusion proteins of the invention can be a bispecific antibody which has one valency for TNFR1 and one valency for an antigen other than TNFR1 . Such a bispecific antibody may be a knob into hole antibody or a CrossMAb antibody but is not limited to such antibodies.

[0062] As used herein, an “IgG” can be a naturally occurring IgG or a mutated IgG, as is known in the art.

[0063] The nomenclature of the antibodies, antibody fragments and mutations thereof follow the terms as known in the art. It will be understood by a person skilled in the art that in accordance with the invention, this known nomenclature is to be applied also to the fusion proteins of the invention. For example, the known nomenclature is to be applied also to an antibody which forms part of a fusion protein of the invention.

[0064] Each monomer of an antibody comprises two heavy chains and two light chains, as generally known in the art. Of these, each heavy and light chain comprises a variable domain (termed VH for the heavy chain and VL for the light chain) which is important for antigen binding. These heavy and light chain variable domains comprise (in an N-terminal to C-terminal order) the regions FR1 , CDR1, FR2, CDR2, FR3, CDR3, and FR4 (FR, framework region; CDR, complementarity determining region which is also known as hypervariable region). The identification and assignment of the above-mentioned antibody regions within the antibody sequence is generally in accordance with Kabat et al. (Sequences of proteins of immunological interest, U.S. Dept, of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda, Md. 1983), or Chothia et al. (Conformations of immunoglobulin hypervariable regions. Nature. 1989 Dec 21- 28;342(6252): 877-83.), or may be performed by using the IMGTA / -QUEST software described in Giudicelli et al. (IMGTA / -QUEST, an integrated software program for immunoglobulin and T cell receptor V-J and V-D-J rearrangement analysis. Nucleic Acids Res. 2004 Jul 1 ;32(Web Server issue): W435-40.), which is incorporated herein by reference. Preferably, the antibody regions indicated above are identified and assigned by using the IMGTA / -QUEST software.

[0065] A “monoclonal antibody” is an antibody from an essentially homogenous population of antibodies, wherein the antibodies are substantially identical in sequence (i.e., identical except for minor fraction of antibodies containing naturally occurring sequence modifications such as amino acid modifications at their N- and C- termini). Unlike polyclonal antibodies which contain a mixture of different antibodies directed to either a single epitope or to numerous different epitopes, monoclonal antibodies are directed to the same epitope and are therefore highly specific. The term “monoclonal antibody” includes (but is not limited to) antibodies which are obtained from a monoclonal cell population derived from a single cell clone, as for instance the antibodies generated by the hybridoma method described in Kohler and Milstein (Nature, 1975 Aug 7;256(5517):495-

[0066] 7) or Harlow and Lane (“Antibodies: A Laboratory Manual” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York 1988). A monoclonal antibody may also be obtained from other suitable methods, including phage display techniques such as those described in Clackson et al. (Nature. 1991 Aug 15;352(6336):624-

[0067] 8) or Marks et al. (J Mol Biol. 1991 Dec 5;222(3):581 -97). A monoclonal antibody may be an antibody that has been optimized for antigen-binding properties such as decreased Kd values, optimized association and dissociation kinetics by methods known in the art. For instance, Kd values may be optimized by display methods including phage display, resulting in affinity-matured monoclonal antibodies. The term “monoclonal antibody” is not limited to antibody sequences from particular species of origin or from one single species of origin. Thus, the meaning of the term “monoclonal antibody” encompasses chimeric monoclonal antibodies such as humanized monoclonal antibodies and human antibodies.

[0068] The term “monovalent antibody” is well known in the art and relates to an antibody that has specificity for the same antigen or epitope. Typically, monoclonal antibodies are produced by a single clone of plasma B cells, i.e., that the same clone of plasma B cells.

[0069] “Humanized antibodies” are antibodies which contain human sequences and a minor portion of non-human sequences which confer binding specificity to an antigen of interest. Typically, humanized antibodies are generated by replacing hypervariable region sequences from a human acceptor antibody by hypervariable region sequences from a non-human donor antibody (e.g., a mouse, rabbit, rat donor antibody) that binds to an antigen of interest. In some cases, framework region sequences of the acceptor antibody may also be replaced by the corresponding sequences of the donor antibody. In addition to the sequences derived from the donor and acceptor antibodies, a “humanized antibody” may either contain other (additional or substitute) residues or sequences or not. Such other residues or sequences may serve to further improve antibody properties such as binding properties (e.g., to decrease Kd values) and / or immunogenic properties (e.g., to decrease antigenicity in humans). Non-limiting examples for methods to generate humanized antibodies are known in the art, e.g., from Riechmann et al. (Nature. 1988 Mar 24; 332(6162):323-7) or Jones et al. (Nature. 1986 May 29-Jun 4; 321 (6069): 522-5).

[0070] The term “human antibody” relates to an antibody containing human variable and constant domain sequences. This definition encompasses antibodies having human sequences bearing single amino acid substitutions or modifications which may serve to further improve antibody properties such as binding properties (e.g., to decrease Kd values) and / or immunogenic properties (e.g., to decrease antigenicity in humans). The term “human antibody” excludes humanized antibodies where a portion of non-human sequences confers binding specificity to an antigen of interest.

[0071] An “antigen-binding portion” of an antibody as used herein refers to a portion of an antibody that retains the capability of the antibody to specifically bind to the antigen. This capability can, for instance, be determined by determining the capability of the antigen-binding portion to compete with the antibody for specific binding to the antigen by methods known in the art. The antigen-binding portion may contain one or more fragments of the antibody. Without particular limitation, the antigen-binding portion can be produced by any suitable method known in the art, including recombinant DNA methods and preparation by chemical or enzymatic fragmentation of antibodies. Antigen-binding portions may be Fab fragments, F(ab’) fragments, Fab2 fragments, single-chain variable fragments (scFv), VHH (also known as single-domain antibodies or nanobodies), diabodies or any other portion(s) of the antibody that retain the capability of the antibody to specifically bind to the antigen. It will be understood that in accordance with the meaning of the term “Fab2” as known in the art, the term “Fab2” is synonymous with the terms “Fab2”, “Fab2”, and “FAB2”. Thus, for the purposes of the present application, the term “Fab2” is used interchangeably with the terms “Fab2”, “Fab2”, and “FAB2”.

[0072] The term “VHH” is to be understood in accordance with its known meaning in the art. VHH are oftentimes also referred to as “nanobodies”. VHH can, for instance, be engineered from heavy-chain antibodies such as those found in camelids. VHH engineered from heavy-chain antibodies are oftentimes referred to as “singledomain antibody (sdAb)”. Thus, in one preferred embodiment, one, more, or all occurrences of a “VHH” in connection with the invention can optionally be replaced by “single-domain antibody (sdAb)”.

[0073] An “fusion protein” according to the invention contains one antigen binding site for TNFRI , as defined herein, and a domain capable of binding to a cell surface antigen or an extracellular matrix antigen, which is also referred to as “anchoring domain”. A fusion protein is capable of binding to its specific target antigen, i.e., TNFR1. It is understood that a fusion protein according to the invention may be a protein consisting of a single polypeptide chain, or it may be a protein wherein two or more polypeptide chains are linked together. They can be linked together covalently, e.g., by covalent linkages. Such covalent linkages may be one or more disulfide linkages. Alternatively, the covalent linkages may be obtained by chemical conjugation (preferably by chemical conjugation using click chemistry), and / or they may be any other covalent linkage which is known in the art as a suitable link for proteins. Alternatively, a fusion protein according to the invention may be a protein wherein two or more polypeptide chains are linked together non-covalently (e.g., by non-covalent interactions). Generally, in connection with all fusion proteins of the invention, it will be understood that the parts of the fusion proteins may be fused using linker sequences. In that case, the fusion protein of the invention will comprise such linker sequences. Suitable linker sequences are known in the art and comprise, for example, peptide linkers, without being limited thereto.

[0074] A fusion protein according to the invention may a fusion protein which is derivatized or linked to a different molecule. For example, molecules that may be linked to the fusion protein are a molecular label (e.g., a fluorescent, luminescent, colored, or radioactive molecule) and / or a pharmaceutical agent.

[0075] The meaning of the terms “antigen binding site” and “antigen binding sites” as used herein is known in the art. Typically, an antigen binding site comprises six complementarity-determining regions (CDRs). The six complementarity-determining regions (CDRs) are typically located in the VH and VL domains, i.e., a CDR1, a CDR2 and a CDR3 in the VH (i.e., in the variable domain of the heavy chain) and a CDR1 , a CDR2 and a CDR3 in the VL (i.e., in the variable domain of the light chain). Various forms of antigen binding sites are known in the art and include, without limitation, antigen binding sites contained in the VH and VH of antibodies, or antigen binding sites contained in their antibody fragments such as scFvs.

[0076] The term “TNFR1” is known in the art. Similarly, the term “hTNFRI” is also known in the art and refers to human TNFR1 (which is also known as TNFRSF1 B, CD120a, TNFR60, TNF-R55, orTNF-R1). In connection with the fusion proteins of the invention, TNFR1 is preferably human TNFR1 (hTNFRI).

[0077] Terms such as “TNFR1 agonist” or “agonist of TNFR1” refer to a molecule that is able to trigger TNFR1 signal transduction. TNFR1 signal transduction can be triggered, for example, by tumor necrosis factor (TNF) superfamily ligands such as TNFalpha or LTalpha but also bi- or oligovalent TNFR1 -specific antibodies or antibody formulations. Activation of TNFR1 leads to a strong release of immune stimulating factors as well as induction of programmed cell death program. TNFR1 -induced cell death is one of several mechanisms involved in the downregulation of CD8+T-cells responses including CD8+ TILs after repeated / prolonged stimulation. Hence, whether a fusion protein is an TNFR1 agonist can be determined by methods known in the art, including, without limitation, methods for measuring muMCP-1 and IL8 secretion in cells expressing TNFR1 (e.g. by ELISA), determining activation of the classical NFkB pathway in cells expressing TNFR1 (and methods for measuring TNFR1 -induced cell death in cells expressing TNFR1 . Cells expressing TNFR1 are known in the art.

[0078] A ’’cell surface antigen or an extracellular matrix antigen” as referred to herein in relation to the invention is not particularly limited. It is understood that the “cell surface antigen or an extracellular matrix antigen” is an antigen that can be bound by the fusion proteins of the invention, i.e., that it is exposed on the cell surface or in the extracellular matrix, respectively. Preferred cell surface or extracellular matrix antigens include checkpoint molecules. Examples of preferred cell surface or extracellular matrix antigens are PD-L1 and CD70.

[0079] It is understood that the term ’’wherein the mutant single-chain TNF variant comprises three peptide linker- connected TNF protomers covering the receptor binding domain, whereby two of the three protomers do not bind to or show reduced binding to TNFR1” preferably comprises a polypeptide of the following structure

[0080] TNF protomer-linker-TNF protomer- linker-TNF protomer, wherein the polypeptide is a single-chain (sc) TNF domain wherein two of the protomer domains are mutated such that they do not bind to TNFR1 / 2. This (intramolecularly assembled) trimeric TNF domain can only bind to one TNFR molecule but not to three TNFR molecules.

[0081] Similarly, it is also understood that the term ’’wherein the mutant single-chain TNF variant comprises two LTalpha protomers and one LTbeta protomer covering the receptor binding domains of these ligands” preferably comprises a polypeptide according to one of the following structures:

[0082] LTalpha protomer- linker— LTalpha protomer- linker— LTbeta protomer;

[0083] LTbeta protomer-linker-LTalpha protomer— linker— LTalpha protomer; and LTalpha protomer-linker-LTbeta protomer-linker-LTalpha protomer.

[0084] These polypeptide are a single-chain (sc) LTalpha2beta domains, wherein only the LTalpha-alpha interface binds to TNFR1 / 2 but the LTalpha-beta and LTbeta-alpha interfaces do not bind to TNFR1 / 2.

[0085] The cancers to be treated according to the present invention are preferably hematologic malignancies or solid cancers preferentially multiple myeloma, PDAC, colon cancer, AML, lung cancer.

[0086] A “solid cancer” is a cancer which forms one or more solid tumors. Such solid cancers forming solid tumors are generally known in the art. The term “solid cancer” encompasses both a primary tumor formed by the cancer and possible secondary tumors, which are also known as metastases. Known solid cancers to be treated according to the invention include, but are not limited to melanoma, colorectal cancer, prostate cancer, head and neck cancer, urothelial cancer, stomach cancer, pancreatic cancer, liver cancer, testis cancer, ovarian cancer, endometrial cancer, cervical cancer, brain cancer, breast cancer, gastric cancer, renal cell carcinoma, Ewing’s sarcoma, non-small cell lung cancer and small cell lung cancer. Terms such as “treatment of cancer” or “treating cancer” according to the present invention refer to a therapeutic treatment. An assessment of whether or not a therapeutic treatment works can, for instance, be made by assessing whether the treatment inhibits cancer growth in the treated patient or patients. Preferably, the inhibition is statistically significant as assessed by appropriate statistical tests which are known in the art. Inhibition of cancer growth may be assessed by comparing cancer growth in a group of patients treated in accordance with the present invention to a control group of untreated patients, or by comparing a group of patients that receive a standard cancer treatment of the art plus a treatment according to the invention with a control group of patients that only receive a standard cancer treatment of the art. Such studies for assessing the inhibition of cancer growth are designed in accordance with accepted standards for clinical studies, e.g., double-blinded, randomized studies with sufficient statistical power. The term “treating cancer” includes an inhibition of cancer growth where the cancer growth is inhibited partially (i.e., where the cancer growth in the patient is delayed compared to the control group of patients), an inhibition where the cancer growth is inhibited completely (i.e., where the cancer growth in the patient is stopped), and an inhibition where cancer growth is reversed (i.e. the cancer shrinks). Preferably, an assessment of whether or not a therapeutic treatment works can be made based on a classification of responders and non-responders by using the response evaluation criteria in solid tumours, version 1.1 (RECIST v1 .1) (Eisenhauer et al.: New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). In: Eur. J. Cancer. 45, No. 2, January 2009, pp. 228-47). Alternatively, or additionally, an assessment of whether or not a therapeutic treatment works can be made based on known clinical indicators of cancer progression.

[0087] The treatment of cancer according to the invention can be a first-line therapy, a second-line therapy or a third-line therapy or a therapy that is beyond third-line therapy. The meaning of these terms is known in the art and in accordance with the terminology that is commonly used by the US National Cancer Institute.

[0088] Whether a cancer is a cancer expressing CD70 or PD-L1 can be determined by methods known in the art, including, without limitation, immunohistochemistry methods using anti-CD70 or anti-PD-L1 antibodies to analyze a biopsy sample of the cancer, qPCR.

[0089] In accordance with the present invention, each occurrence of the term “comprising” may optionally be substituted with the term “consisting of”.

[0090] Methods and Techniques

[0091] Generally, unless otherwise defined herein, the methods used in the present invention (e.g., cloning methods or methods relating to antibodies) are performed in accordance with procedures known in the art, e.g. the procedures described in Sambrook et al. (“Molecular Cloning: A Laboratory Manual.”, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York 1989), Ausubel et al. (“Current Protocols in Molecular Biology.” Greene Publishing Associates and Wiley Interscience; New York 1992), and Harlow and Lane (“Antibodies: A Laboratory Manual” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York 1988), all of which are incorporated herein by reference.

[0092] Protein-protein binding, such as binding of antibodies to their respective target proteins, can be assessed by methods known in the art. Protein-protein binding is preferably assessed by surface plasmon resonance spectroscopy measurements.

[0093] Sequence Alignments of sequences according to the invention are performed by using the BLAST algorithm (see Altschul et al. (1990) “Basic local alignment search tool.” Journal of Molecular Biology 215. p. 403-410.; Altschul et al.: (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402.). Appropriate parameters for sequence alignments of short peptides by the BLAST algorithm, which are suitable for peptide antigens in accordance with the invention, are known in the art. Most software tools using the BLAST algorithm automatically adjust the parameters for sequence alignments for a short input sequence. In one embodiment, the following parameters are used: Max target sequences 10; Word size 3; BLOSUM 62 matrix; gap costs: existence 11 , extension 1 ; conditional compositional score matrix adjustment. Thus, when used in connection with sequences, terms such as “identity” or “identical” preferably refer to the identity value obtained by using the BLAST algorithm.

[0094] Preparation of pharmaceutical compositions of the Invention

[0095] Pharmaceutical compositions of the present invention are prepared in accordance with known standards for the preparation of pharmaceutical compositions. For instance, the pharmaceutical compositions are prepared in a way that they can be stored and administered appropriately. The pharmaceutical compositions of the invention may therefore comprise pharmaceutically acceptable components such as carriers, excipients and / or stabilizers. Such pharmaceutically acceptable components are not toxic in the amounts used when administering the pharmaceutical composition to a human patient. The pharmaceutical acceptable components added to the pharmaceutical compositions may depend on the chemical nature of the active ingredients present in the composition, the particular intended use of the pharmaceutical compositions and the route of administration. In general, the pharmaceutically acceptable components used in connection with the present invention are used in accordance with knowledge available in the art, e.g., from Remington's Pharmaceutical Sciences, Ed.AR Gennaro, 20th edition, 2000, Williams & Wilkins, PA, USA.

[0096] Sequences Preferred amino acid sequences referred to in the present application can be independently selected from the following sequences. The sequences are represented in an N-terminal to C-terminal order; and they are represented in the one-letter amino acid code.

[0097] The following non-limiting exemplary sequences were used in the experimental examples of the present application:

[0098] Table 1 : amino acid sequences of heavy and light chain variants of anti-TNFR1 agonists constructs.

[0099] Legend:

[0100] XXX linker or internal flag tag

[0101] XXX const, light or const, heavy chain

[0102] XXX VH (variable heavy) or VL (variable light) domain

[0103] XXX Tenascin C (TNC) trimerization domain

[0104] (DIACGCAAAPDIKDLLSRLEELEGLVSSLREQGTGGGSGGRG;

[0105] SEQ ID NO: 13)

[0106] For each of the plasmids No. 1 , 3, 4, 6, and 13, the encoded amino acid sequence is as follows: The indicated amino acid sequence of SEQ ID NOs 1 , 3, 4, 6, and 16, respectively, is preceded by an N-terminal amino acid sequence consisting of (in an N- to C-terminal order): a leader sequence (SED ID NO: 14: MNFGFSLIFLVLVLKGVQCEVKLVPR), a first linker consisting of the amino acids QL, a flag tag (SEQ ID: 15: DYKDDDDK), and a second linker consisting of the amino acids EF.

[0107] For each of the plasmids No. 2, 5, 7-9, and 14 the encoded amino acid sequence is as follows: The indicated amino acid sequence of SEQ ID NOs 2, 5, and 7-9, and 17 respectively, is preceded by an N-terminal amino acid sequence consisting of (in an N- to C-terminal order): a leader sequence (SED ID NO: 14: MNFGFSLIFLVLVLKGVQCEVKLVPR), a first linker consisting of the amino acids QL, a flag tag (SEQ ID: 15: DYKDDDDK), and a second linker consisting of the amino acids EL. For the plasmids No. 10 and 12, the encoded amino acid sequences are as follows: The indicated amino acid sequences of SEQ ID NO: 10 and 12 are preceded by an N-terminal amino acid sequence consisting of (in an N- to C-terminal order): a leader sequence (SEQ ID NO: 14: MNFGFSLIFLVLVLKGVQCEVKLVPR) and a linker consisting of the amino acids GS. The amino acid sequences of SEQ ID NO: 10 and 12 carry an internal first linker consisting of the amino acids GS, an internal flag tag (SEQ ID NO: 15; DYKDDDDK), a Tenascin C (TNG) trimerization domain (SEQ ID NO: 13; DIACGCAAAPDIKDLLSRLEELEGLVSSLREQGTGGGSGGRG), and a second internal linker consisting of the amino acids EF.

[0108] For the plasmid No. 11 , the encoded amino acid sequence is as follows: The indicated amino acid sequence of SEQ ID NO: 11 carries (in an N- to C-terminal order): an internal first linker consisting of the amino acids GS, a second internal first linker consisting of the amino acids RS, an first internal flag tag (SEQ ID NO: 15; DYKDDDDK), a third internal linker consisting of the amino acids EF, an second internal flag tag (SEQ ID NO: 15; DYKDDDDK), and a fourth internal linker consisting of the amino acids LE.

[0109] Note that in accordance with the invention, the above-indicated linker sequences QL, EF, EL, GS, RS, LE, and GT are cloning-related amino acid linker sequences and may be independently removed from the amino acid sequences of SEQ ID NOs 1-12 or they may be independently replaced by another linker sequence comprising 1-20 amino acids.

[0110] Table 2: Heavy and light chain composition of XX.

[0111] EXAMPLES

[0112] The present invention is further illustrated by the following non-limiting examples. Materials and Methods

[0113] Production and purification of fusion proteins

[0114] Fusion proteins were produced by transient transfection of HEK293 cells by the PEI method (PEI, polyethylenimine; Polyscience Inc., Warrington, USA) using 1 :1 mixtures of the expression plasmids encoding the antibody heavy and light chain variants of interest (for details see Kums et al., 2017 (29)). To control size and chain balance and to estimate the concentration of the various TNFR1 -targeting antibody fusion proteins, 10 pl of the supernatant of the cells producing the construct of interest were analysed by anti-Flag western blotting with the Flag tag-specific antibody M2 (Sigma-Aldrich, Saint Louis, USA) exploiting the fact that all antibody chain variants used were cloned with an N-terminal Flag tag. The concentrations of the fusion proteins were estimated by comparison with an in parallel processed in house produced Flag- tagged control antibody of known concentration. For purification by affinity chromatography on anti-Flag M2 agarose (Sigma-Aldrich, Steinheim, Germany), the supernatants containing the Flag-tagged fusion proteins and the anti-Flag agarose column(s) were prepared as described by the supplier and described in Fick et al., 2012 (30). After binding of the Flag-tagged constructs the anti-Flag M2 agarose gel, the columns were washed with TBS and the remaining proteins bound were eluted using a buffer with an excess of Flag® peptide (100 pig / ml). The eluted fusion proteins were dialyzed against PBS to reduce the Flag® peptide content.

[0115] Binding studies (Fig. 3)

[0116] Black 96 well high binding plates were coated with 1 g / ml of Fab(muCD70)-HC:VHH(muTNFR1) or an irrelevant construct in coating buffer at 4°C overnight. After blocking the immobilized constructs along with the irrelevant construct were incubated for 1 h at 37 °C with increasing concentrations of GpL-TNC-muCD70 and muTNFR1-GpL. After removal of unbound proteins (5 washes with ice-cold PBS), the remaining well- associated GpL activity was measured. Specific binding of GpL-TNC-muCD70 and muTNFR1-GpL was calculated by subtracting the unspecific binding values of wells coated with the irrelevant construct from the corresponding total binding values derived of the Fab(muCD70)-HC:VHH(muTNFR1) coated wells. KD values were calculated by fitting, using the “one site specific binding” function of the GraphPad Prism 5 software.

[0117] SDS-PAGE and silver staining (Figs. 2, 6, 9, 13)

[0118] Concentrations and purity of purified proteins were analysed by dissolving the proteins in Laemmli sample buffer containing 100 mM DTT and separation of the proteins in a 12.5 % polyacrylamid gel by SDS-PAGE and silver staining of the protein gel with the Pierce Silver Stain Kit (Thermo Fischer Scientific, USA) and 1 comparison with the protein standards of the LMW Calibration Kit for SDS Electrophoresis from Amersham (GE Healthcare).

[0119] Gel filtration (Figs. 2, 6, 9, 13)

[0120] The purity and integrity of purified recombinant antibodies were furthermore analysed using High Performance Liquid Chromatography (HPLC) (UltiMate 3000, Thermo Fischer Scientific, USA) using a MabPac SEC-1 column (Thermo Fisher, #088460). The column was pre-equilibrated with PBS at a flow rate of 0.76 mL / min until the column pressure stayed stable. Protein samples (100 pl, 50 - 1000 pg / ml) were manually injected into the injector and analyzed by UV at 280 nm.

[0121] Coculture assays to determine TNFR1 activation by analysing TNFR1 target gene production (Figs. 5, 7, 8, 10, 12)

[0122] To quantify TNFR1 activation by the various TNFR1 -targeting antibody fusion proteins their ability to stimulate the production of cytokines known to be strongly induced by TNFR1 via the classical NFKB signaling pathway in certain cell lines (“responder cells”), was determined. For this purpose, the murine cell line PancO2 and the human cell line U2OS were used which produce in response to TNFR1 stimulation muMCP- 1 and IL8, respectively. The responder cells were cultivated overnight in 96-well plates (2 X 104 cells / well) and the next day medium was replaced with fresh medium supplemented with HEK293 cells (2 X 104 cells / well) transfected with empty vector or expression vectors encoding murine or human PD-L1 or murine or human CD70 along with the TNFR1 -targeting antibody fusion proteins of interest. After an additional overnight cultivation, the amount of muMCP-1 or IL8 in the supernatant, as an indicator of TNFR1 activity was determined using commercially available ELISA kits for muMCP-1 and IL8 (BD Biosciences, San Diego, USA). OD values were measured with a PHOmo photometer (anthos Mikrosysteme GmbH, Friesoythe, Germany) and normalized by help of cytokines of known concentrations processed ion parallel.

[0123] Examples

[0124] Amongst others, the inventors developed bispecific anti-TNFR1 nanobody fusion proteins with excellent productivity, which display strong conditional TNFR1 agonism upon binding to PD-L1 or CD70 binding. PD- L1 and CD70 are promising anchoring targets for conditional TNFR1 agonists for the following reasons: PD- L1 is expressed by cells of the tumor microenvironment. Moreover, recent studies gave evidence that tumor cell killing by CD8+T cell-derived TNF contributes to the therapeutic success of immune checkpoint blockade (9,26,27). Since a PD-L1 blocking Fab domain as anchoring domain for the conditional TNFR1 agonist was used, the construct not only engages TNFR1 but also elicits constitutive checkpoint inhibitor activity. CD70 (CD27L) is a membrane-bound T cell costimulatory ligand of the TNF superfamily. CD70 can promote Treg development and T cell exhaustion. Accordingly, CD70 is frequently aberrantly expressed in hematologic malignancies and attracts considerable attention as a tumor target (28). To construct the conditional TNFR1 agonists with CD70-restricted activity, blocking Fab domains were used as anchoring domain. Thus, the constructs not only engage TNFR1 but also blocks CD70-CD27 interaction promising synergistic antitumoral activities.

[0125] According to the present invention, the same anchoring domain-restricted mode of TNFR1 agonism can also be achieved with scFv or nanobody or peptide / protein domains targeting other anchoring structures than PD- L1 or CD70. Likewise, other monovalent TNFR1 binding sites may similarly useful and can replace the murine TNFR1 -specific binding domain. Indeed, a construct of a murine TNFR1 -specific nanobody domain and a murine CD70-specific Fab domain (Fab(muCD70)-HC:VHH(muTNFR1)) as well as a similar construct recognizing hCD70 and hTNFRI shows both CD70-restricted TNFR1 agonism. The TNFR1 agonism empowering effect of membrane-associated presentation which converts per se non-agonistic monovalent TNFR1 binders to TNFR1 agonists also applies to bivalent anti-TNFR1 antibodies which have very poor or no agonistic activity. This suggests that attachment / anchoring to the plasma membrane via an anchoring domain is a general mean to empower non-agonistic or weakly agonistic TNFR1 binders into potent TNFR1 agonists. Importantly, the functionality of the construct type is not disturbed when additional functional domains are attached by genetic engineering as long as these domains do not affect the correct assembly of the two chains of the Fab / IgG scaffold. This is particular useful in the Fab based conditional TNFR1 agonists constructs as this allow introduction of domains improving serum retention of the molecules, e.g., a serum albumin domain.

[0126] Example 1 :

[0127] A Fab-VHH fusion protein (Fig. 1 and 2) targeting murine CD70 and murine TNFR1 (Fig. 3) was investigated with respect to engage muTNFRI signaling. The muCD70-specific Fab domain used inhibits muCD70 binding to CD27 (Fig. 4). The construct only induced increased production of MCP-1 by murine PancO2 cells in the presence of human HEK293 cells transfected with murine membrane CD70 but not in the presence of HEK293 cells transfected with empty vector (Fig. 5). This demonstrates muCD70-dependent muTNFRI activation.

[0128] Example 2:

[0129] A Fab-VHH fusion protein (Fig. 1) targeting human CD70 and murine TNFR1 (Fig. 6) was investigated with respect to engage muTNFRI signaling. The human CD70-specific Fab domain used was derived of the antibody 9G2 which inhibits CD70 binding to CD27 (Silence et al., 2014). The construct only induced increased production of MCP-1 by murine PancO2 cells in the presence of human BJAB cells expressing endogenously human CD70 but not in the presence of human Jurkat cells which do not express CD70 (Fig. 7). This demonstrates hCD70-dependent muTNFRI activation.

[0130] Example 3:

[0131] A Fab-VHH fusion protein (Fig. 1) targeting human CD70 and human TNFR1 was investigated with respect to engage human TNFR1 signaling. The human CD70-specific Fab domain used was derived again of the antibody 9G2 which inhibits CD70 binding to CD27 (Silence et al., 2014). In cocultures of human U2OS cells, which produce high amounts of IL8 in response to TNFR1 activation, and HEK293 cells transfected with empty vector (EV), the construct induced increased production of MCP-1 (Fig. 8) Moreover, when the U2OS cells were cocultured with human CD70-transfected HEK293, the IL8 induction by the construct was further strongly increased (Fig. 8). Worth mentioning, in the presence of an excess of the anti-CD70 antibody 9G2, IL8 production was strongly reduced in both scenarios. Please note, HEK293 cell are poor producers of IL8 and U2OS cells have moderate endogenous CD70 expression. In sum, these data thus demonstrate hCD70- dependent human TNFR1 activation.

[0132] Example 4:

[0133] A Fab-VHH fusion protein (Fig. 1) targeting murine TNFR1 and PDL1 of human or murine PDL1 was investigated with respect to engage muTNFRI signaling (Fig. 9). The human mouse cross-reactive PDL1- specific Fab domain was derived of the anti-PDL1 antibody Avelumab which inhibits PDL1-PD interaction (Fallon et al., 2017). The construct only induced increased production of MCP-1 by murine PancO2 cells in the presence of human or murine PDL1 expressing cells (Fig. 10). This demonstrates PDL1 -dependent muTNFRI activation.

[0134] Example 5:

[0135] A Fab-VHH fusion protein (Fig. 14A, B,C) targeting murine TNFR1 and fibroblast activation protein (FAP) of human or murine origin was investigated with respect to engage muTNFRI signaling (Fig. 14 D). The human mouse cross-reactive FAP-specific Fab domain was derived of the anti-PDL1 antibody Avelumab which inhibits PDL1 -PD interaction (Fallon et al., 2017). The construct only induced increased production of MCP- 1 by murine PancO2 cells in the presence of human FAP expressing stable transfectants derived of murine B16 cells (Fig. 14D). This demonstrates FAP-dependent muTNFRI activation. The Fab-VHH fusion protein (Fab(h / mu)FAP)-C1 H:Nb(mu)TNFR1 was purified by affinity chromatography using its internal Flag tags and was subjected to SDS-PAGE under reducing conditions to a 13.5 % polyacrylamide gel followed by silver staining of the proteins (Fig. 14B) or to gel filtration on a MabPac SEC-1 column (Fig. 14C) to control purity (Fig. 14B) and lack of high molecule weight aggregates (Fig. 14C). The B16- and B16-FAP cells have been previously described in:

[0136] Roos C, Wicovsky A, Muller N, Salzmann S, Rosenthal T, Kalthoff H, et al. Soluble and transmembrane TNF- like weak inducer of apoptosis differentially activate the classical and noncanonical NF-kappa B pathway. J Immunol 2010, 185(3): 1593-1605.

[0137] The human / murin FAP cross-reactive single-chain fragment sc36 has been described under the name “scFv MO36” in:

[0138] Brocks B, Garin-Chesa P, Behrle E, Park JE, Rettig WJ, Pfizenmaier K, Moosmayer D. Species- crossreactive scFv against the tumor stroma marker "fibroblast activation protein" selected by phage display from an immunized FAP- / - knock-out mouse. Mol Med. 2001 Jul;7(7):461-9.

[0139] The methods and materials which have been used to obtain Figure 14 are the same as those that have already been described above, including:

[0140] - Production and purification of fusion proteins

[0141] - SDS-PAGE and silver staining (Figs. 2, 6, 9, 13)

[0142] - Gel filtration (Figs. 2, 6, 9, 13)

[0143] - Coculture assays to determine TNFR1 activation by analysing TNFR1 target gene production (Figs. 5, 7, 8, 10, 12)

[0144] - The amino acids encoded by the plasmids #13 and #14 in Table 1.

[0145] INDUSTRIAL APPLICABILITY

[0146] The pharmaceutical compositions, polypeptides, nucleic acids, cells, and products for use in the invention are industrially applicable. For example, they can be used in the manufacture of, or as, pharmaceutical products.

[0147] REFERENCES

[0148] 1. Salomon BL. Insights into the biology and therapeutic implications of TNF and regulatory T cells. Nat Rev Rheumatol. 2021 Aug; 17(8):487-504.

[0149] 2. Siegmund D, Wajant H. TNF and TNF receptors as therapeutic targets for rheumatic diseases and beyond. Nat Rev Rheumatol. 2023 Sep;19(9):576-591.

[0150] 3. Wajant H, Pfizenmaier K, Scheurich P. Tumor necrosis factor signaling. Cell Death Differ. 2003 Jan;10(1):45-65.

[0151] 4. Wajant H, Siegmund D. TNFR1 and TNFR2 in the Control of the Life and Death Balance of Macrophages. Front Cell Dev Biol. 2019 May 29;7:91. 5. Medler J, Wajant H. Tumor necrosis factor receptor-2 (TNFR2): an overview of an emerging drug target. Expert Opin Ther Targets. 2019 Apr;23(4):295-307.

[0152] 6. Wajant H. The role of TNF in cancer. Results Probl Cell Differ. 2009;49:1-15.

[0153] 7. Hieber U, Heim ME. Tumor necrosis factor for the treatment of malignancies. Oncology. 1994 Mar- Apr; 51 (2): 142-53.

[0154] 8. Jakob J, Hohenberger P. Role of isolated limb perfusion with recombinant human tumor necrosis factor a and melphalan in locally advanced extremity soft tissue sarcoma. Cancer. 2016 Sep 1;122(17):2624- 32.

[0155] 9. Chen AY, Wolchok JD, Bass AR. TNF in the era of immune checkpoint inhibitors: friend or foe? Nat Rev Rheumatol. 2021 Apr; 17(4):213-223.

[0156] 10. Kearney CJ, Lalaoui N, Freeman AJ, Ramsbottom KM, Silke J, Oliaro J. PD-L1 and lAPs co-operate to protect tumors from cytotoxic lymphocyte-derived TNF. Cell Death Differ. 2017 Oct;24(10):1705-1716.

[0157] 11. Lim SO, Li CW, Xia W, Cha JH, Chan LC, Wu Y, Chang SS, Lin WC, Hsu JM, Hsu YH, Kim T, Chang WC, Hsu JL, Yamaguchi H, Ding Q, Wang Y, Yang Y, Chen CH, Sahin AA, Yu D, Hortobagyi GN, Hung MC. Deubiquitination and Stabilization of PD-L1 by CSN5. Cancer Cell. 2016 Dec 12;30(6):925-939.

[0158] 12. Bertrand F, Montfort A, Marcheteau E, Imbert C, Gilhodes J, Filleron T, Rochaix P, Andrieu-Abadie N, Levade T, Meyer N, Colacios C, Segui B. TNFa blockade overcomes resistance to anti-PD-1 in experimental melanoma. Nat Commun. 2017 Dec 22;8(1):2256.

[0159] 13. Hartley G, Regan D, Guth A, Dow S. Regulation of PD-L1 expression on murine tumor-associated monocytes and macrophages by locally produced TNF-a. Cancer Immunol Immunother. 2017 Apr; 66(4) : 523- 535.

[0160] 14. Wen Y, Wang X, Meng W, Guo W, Duan C, Cao J, Kang L, Guo N, Lin Q, Lv P, Zhang R, Xing L, Zhang X, Shen H. TNF-a-dependent lung inflammation upregulates PD-L1 in monocyte-derived macrophages to contribute to lung tumorigenesis. FASEB J. 2022 Nov;36(11 ):e22595.

[0161] 15. Kearney CJ, Vervoort SJ, Hogg SJ, Ramsbottom KM, Freeman AJ, Lalaoui N, Pijpers L, Michie J, Brown KK, Knight DA, Sutton V, Beavis PA, Voskoboinik I, Darcy PK, Silke J, Trapani JA, Johnstone RW, Oliaro J. Tumor immune evasion arises through loss of TNF sensitivity. Sci Immunol. 2018 May 18;3(23):eaar3451.

[0162] 16. Zhang Z, Kong X, Ligtenberg MA, van Hal-van Veen SE, Visser NL, de Bruijn B, Stecker K, van der Helm PW, Kuilman T, Hoefsmit EP, Vredevoogd DW, Apriamashvili G, Baars B, Voest EE, Klarenbeek S, Altelaar M, Peeper DS. RNF31 inhibition sensitizes tumors to bystander killing by innate and adaptive immune cells. Cell Rep Med. 2022 Jun 21 ;3(6): 100655.

[0163] 17. Vredevoogd DW, Kuilman T, Ligtenberg MA, Boshuizen J, Stecker KE, de Bruijn B, Krijgsman O, Huang X, Kenski JCN, Lacroix R, Mezzadra R, Gomez-Eerland R, Yildiz M, Dagidir I, Apriamashvili G, Zandhuis N, van der Noort V, Visser NL, Blank CU, Altelaar M, Schumacher TN, Peeper DS. Augmenting Immunotherapy Impact by Lowering Tumor TNF Cytotoxicity Threshold. Cell. 2020 Jan 23; 180(2):404-405.

[0164] 18. Litchfield K, Reading JL, Puttick C, Thakkar K, Abbosh C, Bentham R, Watkins TBK, Rosenthal R, Biswas D, Rowan A, Lim E, Al Bakir M, Turati V, Guerra-Assungao JA, Conde L, Furness AJS, Saini SK, Hadrup SR, Herrero J, Lee SH, Van Loo P, Enver T, Larkin J, Hellmann MD, Turajlic S, Quezada SA, McGranahan N, Swanton C. Meta-analysis of tumor- and T cell-intrinsic mechanisms of sensitization to checkpoint inhibition. Cell. 2021 Feb 4;184(3):596-614.e14.

[0165] 19. Perez-Ruiz E, Minute L, Otano I, Alvarez M, Ochoa MC, Belsue V, de Andrea C, Rodriguez-Ruiz ME, Perez-Gracia JL, Marquez-Rodas I, Llacer C, Alvarez M, de Luque V, Molina C, Teijeira A, Berraondo P, Melero I. Prophylactic TNF blockade uncouples efficacy and toxicity in dual CTLA-4 and PD-1 immunotherapy. Nature. 2019 May;569(7756):428-432.

[0166] 20. Chen Y, Liu Y, Wang Y, Chen X, Wang C, Chen X, Yuan X, Liu L, Yang J, Zhou X. Prevotellaceae produces butyrate to alleviate PD-1 / PD-L1 inhibitor-related cardiotoxicity via PPARa-CYP4X1 axis in colonic macrophages. J Exp Clin Cancer Res. 2022 Jan 3;41 (1 ):1 .

[0167] 21. Gao J, Miao J, Sun H, Fu X, Zhang P, Chen Z, Zhu P. TNF-a inhibitor ameliorates immune-related arthritis and pneumonitis in humanized mice. Front Immunol. 2022 Aug 9;13:955812.

[0168] 22. Neri D. Antibody-Cytokine Fusions: Versatile Products for the Modulation of Anticancer Immunity. Cancer Immunol Res. 2019 Mar;7(3):348-354.

[0169] 23. Huyghe L, Van Parys A, Cauwels A, Van Lint S, De Munter S, Bultinck J, Zabeau L, Hostens J, Goethals A, Vanderroost N, Verhee A, Uze G, Kley N, Peelman F, Vandekerckhove B, Brouckaert P, Tavernier J. Safe eradication of large established tumors using neovasculature-targeted tumor necrosis factor-based therapies. EMBO Mol Med. 2020 Feb 7;12(2):e11223.

[0170] 24. Gerspach J, Pfizenmaier K, Wajant H. Improving TNF as a cancer therapeutic: tailor-made TNF fusion proteins with conserved antitumor activity and reduced systemic side effects. Biofactors. 2009 Jul- Aug;35(4):364-72.

[0171] 25. Medler J, Nelke J, Weisenberger D, Steinfatt T, Rothaug M, Berr S, Hiinig T, Beilhack A, Wajant H. TNFRSF receptor-specific antibody fusion proteins with targeting controlled FcyR-independent agonistic activity. Cell Death Dis. 2019 Mar 4;10(3):224.

[0172] 26. Montfort A, Virazels M, Colacios C, Meyer N, Segui B. Combining TNF blockade with immune checkpoint inhibitors in patients with cancer. Nat Rev Rheumatol. 2021 Sep; 17(9):577.

[0173] 27. Vredevoogd DW, Apriamashvili G, Peeper DS. The (re)discovery of tumor-intrinsic determinants of immune sensitivity by functional genetic screens. Immunooncol Technol. 2021 Oct 28; 11 :100043.

[0174] 28. Wajant H. Therapeutic targeting of CD70 and CD27. Expert Opin Ther Targets. 2016 Aug;20(8):959- 73. 29. Kums J, Nelke J, Ruth B, Schafer V, Siegmund D, Wajant H. Quantitative analysis of cell surface antigenantibody interaction using Gaussia princeps luciferase antibody fusion proteins. MAbs. 2017;9:506-20

[0175] 30. Fick A, Wyzgol A, Wajant H. Production, purification, and characterization of scFv TNF ligand fusion proteins. Methods Mol Biol. 2012;907:597-609

[0176] Silence K, Dreier T, Moshir M, Ulrichts P, Gabriels SM, Saunders M, Wajant H, Brouckaert P, Huyghe L, Van Hauwermeiren T, Thibault A, De Haard HJ. ARGX-110, a highly potent antibody targeting CD70, eliminates tumors via both enhanced ADCC and immune checkpoint blockade. MAbs. 2014 Mar-Apr;6(2):523-32.

[0177] Fallon JK, Vandeveer AJ, Schlom J, Greiner JW. Enhanced antitumor effects by combining an IL-12 / anti- DNA fusion protein with avelumab, an anti-PD-L1 antibody. Oncotarget. 2017 Mar 28;8(13):20558-20571.

Claims

CLAIMS1 . A fusion protein, comprising: i) a TNFR1 -binding protein, wherein the TNFR1 -binding protein is monovalent for TNFR1, and ii) a domain capable of binding to a cell surface antigen or an extracellular matrix antigen.

2. The fusion protein of claim 1 , wherein: the TNFR1 -binding protein according to i) is an anti-TNFR1 antibody or a TNFR1 -binding portion thereof, wherein the anti-TNFR1 antibody or TNFR1 -binding portion thereof is monovalent for TNFR1.

3. The fusion protein of claim 1 , wherein:The TNFR1 -binding protein according to i) is a mutant single-chain TNF variant, wherein the mutant single-chain TNF variant thereof is monovalent for TNFR1 .

4. The fusion protein according to any one of the preceding claims, wherein the fusion protein is monovalent for TNFR1.

5. The fusion protein according to any one of claims 1-2 and 4, wherein the TNFR1 -binding protein according to i) is an anti-hTNFR1 antibody or hTNFRI -binding portion thereof.

6. The fusion protein according to any one of claims 1-2 and 4-5, wherein the TNFR1 -binding protein according to i) is a VHH, a Fab domain, or an scFv fragment.

7. The fusion protein according to any one of claims 1-2 and 4-6, wherein the TNFR1 -binding protein according to i) is a VHH.

8. The fusion protein according to any one of claims 1-2 and 4-6, wherein the TNFR1 -binding protein according to i) is a Fab domain.

9. The fusion protein according to any one of claims 1-2 and 4-6, wherein the TNFR1 -binding protein according to i) is an scFv fragment.

10. The fusion protein according to any one of the preceding claims, wherein said fusion protein is an agonist of TNFR1 when bound to TNFR1 and bound to the cell surface antigen or extracellular matrix antigen, and is not an agonist of TNFR1 when bound to TNFR1 but not bound to the cell surface antigen or extracellular matrix antigen.

11. The fusion protein according to any one of the preceding claims, wherein said fusion protein is an agonist of TNFR1 when bound to TNFR1 and bound to the cell surface antigen or extracellular matrixantigen, and is an antagonist of TNFR1 when bound to TNFR1 but not bound to the cell surface antigen or extracellular matrix antigen.

12. The fusion protein according to any one of claims 3-4 and 10-11 , wherein the mutant single-chain TNF variant comprises three peptide linker-connected TNF protomers covering the receptor binding domain, whereby two of the three protomers do not bind to or show reduced binding to TNFR1.

13. The fusion protein according to any one of claims 3-4 and 10-12, wherein the mutant single-chain TNF variant comprises two LTalpha protomers and one LTbeta protomer covering the receptor binding domains of these ligands.

14. The fusion protein according to any one of the preceding claims, wherein the domain according to ii) comprises an antibody or an antigen-binding portion, said antibody or antigen-binding portion being capable of binding to said cell surface antigen or extracellular matrix antigen.

15. The fusion protein according to claim 14, wherein said antibody or antigen-binding portion capable of binding to said cell surface antigen or extracellular matrix antigen comprises at least one Fab domain, VHH, or scFv fragment or a combination of at least one Fab domain, at least one VHH, and / or at least one scFv fragment.

16. The fusion protein according to claim 15, wherein said antibody or antigen-binding portion capable of binding to said cell surface antigen or extracellular matrix antigen comprises at least one Fab domain.

17. The fusion protein according to claim 15, wherein said antibody or antigen-binding portion capable of binding to said cell surface antigen or extracellular matrix antigen comprises at least one scFv fragment.

18. The fusion protein according to claim 15, wherein said antibody or antigen-binding portion capable of binding to said cell surface antigen or extracellular matrix antigen comprises at least one VHH.

19. The fusion protein according to any one of the preceding claims, wherein the domain according to ii) does not comprise an anti-TNFR1 -antibody or antigen-binding portion thereof.

20. The fusion protein according to any one of the preceding claims, wherein said cell surface antigen or extracellular matrix antigen is a protein.

21. The fusion protein according to any one of the preceding claims, wherein said cell surface antigen or extracellular matrix antigen is a cell surface antigen.

22. The fusion protein according to any one of the preceding claims, wherein said cell surface antigen or extracellular matrix antigen is an extracellular matrix antigen.

23. The fusion protein according to any one of the preceding claims, wherein said cell surface antigen is an antigen of an immune cell, preferably wherein the immune cell is a human immune cell, further preferably wherein the human immune cell is a human T lymphocyte.

24. The fusion protein according to any one of the preceding claims, wherein said cell surface antigen is a cancer antigen and / or an antigen of the tumor microenvironment.

25. The fusion protein according to any one of the preceding claims, wherein said antigen is PD-L1, CD70, CD40, CXCR4, BCMA, CTLA4, FAP or PD1.

26. The fusion protein according to any one of the preceding claims, wherein said antigen is PD-L1.

27. The fusion protein according to any one of the claims 1-25, wherein said antigen is CD70.

28. The fusion protein according to any one of the preceding claims, wherein said antigen is a human antigen.

29. The fusion protein according to any one of the preceding claims, wherein said antibody or antigenbinding portion capable of binding to said cell surface antigen or extracellular matrix antigen is an immune checkpoint inhibitor.

30. The fusion protein according to any one of claims 1 to 29, wherein the domain according to ii) comprises an anti-CD70 Fab and the TNFR1 -binding protein according to i) is an anti-TNFR1 VHH, and the fusion protein is selected from the group consisting of: a1 ) a fusion protein comprising a first polypeptide chain having an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 1 , at least 90% identical to the amino acid sequence of SEQ ID NO: 1 , at least 95% identical to the amino acid sequence of SEQ ID NO: 1 , at least 98% identical to the amino acid sequence of SEQ ID NO: 1 , or at least 99% identical to the amino acid sequence of SEQ ID NO: 1 , and a second polypeptide chain having an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 2, at least 90% identical to the amino acid sequence of SEQ ID NO: 2, at least 95% identical to the amino acid sequence of SEQ ID NO: 2, at least 98% identical to the amino acid sequence of SEQ ID NO: 2, or at least 99% identical to the amino acid sequence of SEQ ID NO: 2; a2) a fusion protein comprising a first polypeptide chain having the amino acid sequence of SEQ ID NO: 1 , and a second polypeptide chain having the amino acid sequence of SEQ ID NO:a3) a fusion protein competing with the fusion protein of a2) for specific binding to CD70 and for specific binding to TNFR1 ; a4) a fusion protein wherein the domain according to ii) comprises an anti-CD70 Fab having the same CDRs as the anti-CD70 Fab of the fusion protein of a2), and wherein the TNFR1 -binding protein according to i) is an anti-TNFR1 VHH having the same CDRs as the anti-TNFR1 VHH of the fusion protein of a2); b1 ) a fusion protein comprising a first polypeptide chain having an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 3, at least 90% identical to the amino acid sequence of SEQ ID NO: 3, at least 95% identical to the amino acid sequence of SEQ ID NO: 3, at least 98% identical to the amino acid sequence of SEQ ID NO: 3, or at least 99% identical to the amino acid sequence of SEQ ID NO: 3, and a second polypeptide chain having an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 5, at least 90% identical to the amino acid sequence of SEQ ID NO: 5, at least 95% identical to the amino acid sequence of SEQ ID NO: 5, at least 98% identical to the amino acid sequence of SEQ ID NO: 5, or at least 99% identical to the amino acid sequence of SEQ ID NO: 5; b2) a fusion protein comprising a first polypeptide chain having the amino acid sequence of SEQ ID NO: 3, and a second polypeptide chain having the amino acid sequence of SEQ ID NO: 5; b3) a fusion protein competing with the fusion protein of b2) for specific binding to CD70 and for specific binding to TNFR1 ; b4) a fusion protein wherein the domain according to ii) comprises an anti-CD70 Fab having the same CDRs as the anti-CD70 Fab of the fusion protein of b2), and wherein the TNFR1 -binding protein according to i) is an anti-TNFR1 VHH having the same CDRs as the anti-TNFR1 VHH of the fusion protein of b2); c1 ) a fusion protein comprising a first polypeptide chain having an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 4, at least 90% identical to the amino acid sequence of SEQ ID NO: 4, at least 95% identical to the amino acid sequence of SEQ ID NO: 4, at least 98% identical to the amino acid sequence of SEQ ID NO: 4, or at least 99% identical to the amino acid sequence of SEQ ID NO: 4, and a second polypeptide chain having an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 5, at least 90% identical to the amino acid sequence of SEQ ID NO: 5, at least 95% identical to the amino acid sequence of SEQ ID NO: 5, at least 98% identical to the amino acid sequence of SEQ ID NO: 5, or at least 99% identical to the amino acid sequence of SEQ ID NO: 5; c2) a fusion protein comprising a first polypeptide chain having the amino acid sequence of SEQ ID NO: 4, and a second polypeptide chain having the amino acid sequence of SEQ ID NO: 5; c3) a fusion protein competing with the fusion protein of c2) for specific binding to CD70 and for specific binding to TNFR1 ; c4) a fusion protein wherein the domain according to ii) comprises an anti-CD70 Fab having the same CDRs as the anti-CD70 Fab of the fusion protein of c2), and wherein the TNFR1 -bindingprotein according to i) is an anti-TNFR1 VHH having the same CDRs as the anti-TNFR1 VHH of the fusion protein of c2); d1 ) a fusion protein comprising a first polypeptide chain having an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 1 , at least 90% identical to the amino acid sequence of SEQ ID NO: 1 , at least 95% identical to the amino acid sequence of SEQ ID NO: 1 , at least 98% identical to the amino acid sequence of SEQ ID NO: 1 , or at least 99% identical to the amino acid sequence of SEQ ID NO: 1 , and a second polypeptide chain having an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 8, at least 90% identical to the amino acid sequence of SEQ ID NO: 8, at least 95% identical to the amino acid sequence of SEQ ID NO: 8, at least 98% identical to the amino acid sequence of SEQ ID NO: 8, or at least 99% identical to the amino acid sequence of SEQ ID NO: 8; d2) a fusion protein comprising a first polypeptide chain having the amino acid sequence of SEQ ID NO: 1 , and a second polypeptide chain having the amino acid sequence of SEQ ID NO: 8; d3) a fusion protein competing with the fusion protein of d2) for specific binding to CD70 and for specific binding to TNFR1 ; and d4) a fusion protein wherein the domain according to ii) comprises an anti-CD70 Fab having the same CDRs as the anti-CD70 Fab of the fusion protein of d 2), and wherein the TNFR1 -binding protein according to i) is an anti-TNFR1 VHH having the same CDRs as the anti-TNFR1 VHH of the fusion protein of d2).

31. The fusion protein according to any one of claims 1 to 29, wherein the domain according to ii) comprises an anti-PD-L1 Fab and the TNFR1 -binding protein according to i) is an anti-TNFR1 VHH, and the fusion protein is selected from the group consisting of: e1 ) a fusion protein comprising a first polypeptide chain having an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 6, at least 90% identical to the amino acid sequence of SEQ ID NO: 6, at least 95% identical to the amino acid sequence of SEQ ID NO: 6, at least 98% identical to the amino acid sequence of SEQ ID NO: 6, or at least 99% identical to the amino acid sequence of SEQ ID NO: 6, and a second polypeptide chain having an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 7, at least 90% identical to the amino acid sequence of SEQ ID NO: 7, at least 95% identical to the amino acid sequence of SEQ ID NO: 7, at least 98% identical to the amino acid sequence of SEQ ID NO: 7, or at least 99% identical to the amino acid sequence of SEQ ID NO: 7; e2) a fusion protein comprising a first polypeptide chain having the amino acid sequence of SEQ ID NO: 6, and a second polypeptide chain having the amino acid sequence of SEQ ID NO: 7; e3) a fusion protein competing with the fusion protein of e2) for specific binding to PD-L1 and for specific binding to TNFR1 ; e4) a fusion protein wherein the domain according to ii) comprises an anti-PD-L 1 Fab having the same CDRs as the anti-PD-L1 Fab of the fusion protein of e2), and wherein the TNFR1 -bindingprotein according to i) is an anti-TNFR1 VHH having the same CDRs as the anti-TNFR1 VHH of the fusion protein of e2); f1) a fusion protein comprising a first polypeptide chain having an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 6, at least 90% identical to the amino acid sequence of SEQ ID NO: 6, at least 95% identical to the amino acid sequence of SEQ ID NO: 6, at least 98% identical to the amino acid sequence of SEQ ID NO: 6, or at least 99% identical to the amino acid sequence of SEQ ID NO: 6, and a second polypeptide chain having an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 9, at least 90% identical to the amino acid sequence of SEQ ID NO: 9, at least 95% identical to the amino acid sequence of SEQ ID NO: 9, at least 98% identical to the amino acid sequence of SEQ ID NO: 9, or at least 99% identical to the amino acid sequence of SEQ ID NO: 9; f2) a fusion protein comprising a first polypeptide chain having the amino acid sequence of SEQ ID NO: 6, and a second polypeptide chain having the amino acid sequence of SEQ ID NO: 9; f3) a fusion protein competing with the fusion protein of f2) for specific binding to PD-L1 and for specific binding to TNFR1 ; and f4) a fusion protein wherein the domain according to ii) comprises an anti-PD-L1 Fab having the same CDRs as the anti-PD-L1 Fab of the fusion protein of f2), and wherein the TNFR1 -binding protein according to i) is an anti-TNFR1 VHH having the same CDRs as the anti-TNFR1 VHH of the fusion protein of f2).

32. The fusion protein according to any one of the preceding claims, wherein the fusion protein further comprises iii) an additional protein domain.

33. The fusion protein according to claim 32, wherein the additional protein domain according to iii) is serum albumin, transferrin or a Fab domain, VHH or scFv capable of binding to serum albumin or a transferrin receptor.

34. The fusion protein according to claim 32, wherein the additional protein domain according to iii) is serum albumin.

35. The fusion protein according to any one of the preceding claims, wherein the fusion protein further comprises iv) at least one domain capable of binding to a different cell surface antigen or extracellular matrix antigen than the domain according to ii).

36. The fusion protein according to claim 35, wherein the at least one domain capable of binding to a different cell surface antigen or extracellular matrix antigen is a domain capable of binding to a cell surface antigen or extracellular matrix antigen as defined in any one of claims 14-31 .

37. A pharmaceutical composition comprising the fusion protein according to any one of the preceding claims.

38. A pharmaceutical composition comprising a fusion protein according to any one of claims 1-36, for use as a medicament.

39. A pharmaceutical composition according to claim 38 or a fusion protein according to any one of claims 1-36, for use in the treatment of cancer.

40. The pharmaceutical composition or fusion protein for use according to claim 39, wherein the cancer is a hematologic malignancy.

41. The pharmaceutical composition or fusion protein for use according to claim 39, wherein the cancer is a solid cancer.

42. The pharmaceutical composition or fusion protein for use according to any one of claims 39-41 , wherein the use is a use in combination with an immune checkpoint inhibitor.

43. A nucleic acid, or a set of nucleic acids, encoding the fusion protein according to any one of claims 1-36.

44. A recombinant cell containing a nucleic acid, or a set of nucleic acids, according to claim 43 and expressing the fusion protein of any one of claims 1-36.

45. A method for producing a fusion protein according to any one of claims 1 to 36, the method comprising expressing the nucleic acid or set of nucleic acids according to claim 43 in a cell according to claim 44, and harvesting the fusion protein.

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