WO2023088889A1 - CD137 ligands - Google Patents

Abstract

The invention relates to CD137 ligand moieties with improved protein properties. Ligands of the invention exhibit increased solubility and pH stability, as well as improved folding kinetics, leading to higher expression titers during production in eukaryotic cells. CD137 ligands of the invention are well suited for the construction of therapeutic multivalent CD137L fusion proteins employing homotrimeric or single-chain based assembly of the CD137L protomers. The invention further relates to nucleic acids and transfected host cells for the production of said improved CD137L fusion proteins.

Description

CD137 ligands

Field of the invention

The present invention provides specific CD137 ligand proteins comprising selected mutations improving their solubility and stability during secretory pathway based expression in mammalian cells. It also provides nucleic acid molecules encoding the specific CD137 ligand proteins, and uses thereof. The specific CD137 ligand proteins are suitable for therapeutic, diagnostic and/or research applications.

Background of the invention

The diverse functions of the immune system are orchestrated by a complex and delicately balanced interplay of stimulatory and inhibitory signals. Many key regulators of immune cell function belong to the so-called tumor necrosis factor superfamily (TNFSF) and their cognate receptors, the so-called TNF receptor superfamily (TNFRSF). The TNFSF consists 19 structurally related ligands; type-ll transmembrane proteins comprising the receptor-binding-domain at its C-terminus. Said ligands assert their biological function as self-assembling, noncovalent trimers capable of binding and activating one or more members of the 29 members of the TNFSF receptor family.

TNFSF receptors are of great importance in the anti-tumor immune response and the regulation of inflammatory processes. They are expressed by a wide variety of immune cells including T cells and antigen-presenting cell populations, such as dendritic cells and macrophages, as well as by tumor cells themselves. This diverse expression pattern highlights the critical role that TNFSF receptors play in many parts of the body and in the various phases of the anti-tumor immune response (Dostert et al, Physiol Rev. 2019 Jan 1 ;99(1): 115-160. doi: 10.1152/physrev.00045.2017).

CD137 is a prominent member of the TNFSF receptors. Its expression on a variety of different cell types such as T- and B-lymphocytes, NK-cells, monocytes, neutrophils, mast cells and dendritic, epithelial cells and cancer cells is mostly inducible and triggered by T- cell or B-cell receptor dependent stimulatory signals. Expression of CD137L is more restricted, mainly to antigen presenting cells (APC) such as B-cells, dendritic cells (DCs) and macrophages. Inducible expression of CD137L is characteristic for T-cells, including both ap and y6 T-cell subsets, and endothelial cells (Shao and Schwarz, 2011)

PCT/EP2016/075543 discloses Fc-fused trivalent CD137L protein moieties in a single single-chain configuration. Said fusion proteins comprise three soluble, stalk depleted and C-terminal shortened CD137L receptor binding domains connected by short (3-8) amino acids linkers and fused by a stabilizing hinge linker to an IGG1-Fc-mutein. These fusion proteins avoid Fc-receptor-dependent pathways and, as pure agonists, exert their biological activity via CD137/CD137L signalling only.

PCT/EP2021/063005 discloses various multi-specific and bi-specific TNF superfamily fusion protein assemblies comprising at least (i) one protein moiety which comprises a single-chain TNF superfamily receptor binding domain and (ii) a protein moiety capable of specific binding to a cell surface antigen or an immune modulating protein. Fusion proteins of PCT/EP2021/063005 allow for locally enhanced or locally enriched TNF receptor superfamily (TNFRSF) agonistic activity.

CD137L fusion proteins of PCT/EP2021/063005 and PCT/EP2016/075543 (contents of both aforementioned patent applications incorporated by reference herein in their entirety) are well suited for therapeutic use.

However, it is striking that the CD137L domains, which have a high content of hydrophobic amino acids exposed at their surface, show limited expression rates in specific trivalent configurations, e.g. with very short linkers, when produced in mammalian cells. Most likely, this reduced expression rates are the result of extensive formation of insoluble multimers driven by the hydrophobic surface of the CD137L domains. This multimer formation of recombinant CD137L proteins within the secretory pathway may lead to their aggregation inside the cells, induction of rapid internal protein decay and subsequent low yields. There is a need in the art for novel CD137L proteins that exhibit improved solubility, high stability and allow for more efficient recombinant manufacturing.

Summary of the invention

One aim of the present invention was therefore to provide CD137L muteins that allow higher expression levels in eukaryotic cells when used as fusion partners in different protein formats. Another aim of the present invention was to potentially identify CD137L muteins with enhanced stability to enable sufficient productivities of CD137L fusion proteins in general.

The invention further relates to nucleic acid molecules encoding CD137L mutein as described herein and to a cell or a non-human organism transformed or transfected with a nucleic acid molecule as described herein.

The invention also relates to a pharmaceutical or diagnostic composition comprising as an active agent a CD137L mutein, a nucleic acid molecule, or a cell as described herein. The invention also relates to a CD137L mutein, a nucleic acid molecule, or a cell as described herein for use in therapy, e.g., the use of a CD137L mutein, a nucleic acid molecule, or a cell as described herein for the preparation of a pharmaceutical composition in the prophylaxis and/or treatment of disorders caused by, associated with and/or accompanied by dysfunction of CD137/CD137L signaling, particularly proliferative disorders, such as tumours, e.g. solid or lymphatic tumours; infectious diseases; inflammatory diseases; metabolic diseases; autoimmune disorders, e.g. rheumatoid and/or arthritic diseases; degenerative diseases, e.g. neurodegenerative diseases such as multiple sclerosis; apoptosis-associated diseases or transplant rejections.

Description of the Figures

Figure 1 Schematic illustration of hexavalent single-chain CD137L-Fc fusion protein (A) and homotrimeric (ht) CD137L (B). A typical multispecific immune- Modulator of the invention can be achieved by combining a CD137L-Fcfusion protein of the invention with a second non-CD137L scTNFSF-Fc fusion protein (A).

Figure 2 Schematic illustration of bispecific trivalent scCD137L-Fab-Fc-fusion protein (=Single-arm-bispecific, SAB) (A) and bispecific trivalent scCD137L-Fab- fusion protein (B).

Figure 3 Surface glycosylation of homotrimeric (ht) CD137L-Variants. Introduction of surface glycosylation sites at different positions in CD137L-receptor binding domain (RBD). (A) Comparison of affinity chromatography product titers after transient gene expression (A) or stable cell expression (B) in CHO cells. The titers of surface glycosylation variants were compared to aglycosylated wildtype sequence. The expressed proteins contain the CD137L-RBD htCD137L-Var2 to htCD137L-Var7 fused to a trimerization domain (SEQ-ID: 51). (C) SDS-PAGE of AFC eluates indicates the molecular weight shift and uniformity of surface glycosylation. Only E128N variant demonstrates a complete and uniform glycosylation pattern in addition to highest expression titers observed.

Figure 4 Intraprotomer cysteine bridge incorporation in homotrimeric CD137L- Variants. Introduction of intraprotomeric cysteine bridge mutations at different positions in CD137L-receptor binding domain (RBD). Comparison of affinity chromatography product titers after transient gene expression in CHO cells. The expressed proteins contain the CD137L-RBD htCD137L-Var8 to htCD137L-Var12 fused to a trimerization domain (SEQ-ID: 51 ). The addition of surface glycosylation E128N increases expression titers also in Cysteine bridge containing variants (3.4-fold). Superior expression titers are observed for cysteine bridge variants V183C/G198C, A93C/L124C and V143C/L237C as compared to A93C/L237C.

Figure 5 Monomer content of cysteine bridge variants in homotrimeric CD137L- Variants. Determination of monomeric species in affinity chromatography product eluate by HP-SEC measurement. The expressed proteins contain the CD137L-RBD htCD137L-Var8 to htCD137L-Var12 fused to a trimerization domain (SEQ-ID: 51). The cysteine bridge variants V183C/G198C, A93C/L124C and V143C/L237C +/-E128N contain >95% monomeric species. Only the cysteine bridge variant A93C/L237C strongly aggregates and forms high molecular weight species (in addition to low AFC protein titers).

Figure 6 Surface glycosylation enhances expression titers in single-chain CD137L- variants. (A) Comparison of affinity chromatography product titers after transient gene expression in CHO cells. Introduction of surface glycosylation E128N in all modules of single-chain CD137L variants (black bars) increases expression titers as compared to variants without E128N mutation (gray bars). The titer increase due to E128N occurs in scCD137L-variants with and without incorporation of cysteine bridges. (B) Titer increase of scCD137L- variant pairs differing only in the absence and presence of E128N surface glycosylation. The expression titers are increased up to >8-fold. (A,B) The expressed proteins contain the trimeric single-chain CD137L-RBD variants scCD137L-Var1 to scCD137L-Var23 which are homodimerized via a linker/hinge-Fc-fusion (SEQ-ID: 53 and SEQ-ID: 77) to generate a hexavalent CD137 agonist (SEQ-ID:120 to SEQ-ID:142).

Figure 7 Single-module surface glycosylation is sufficient for increase in expression titer. Comparison of affinity chromatography product titers after transient gene expression in CHO cells. Introduction of surface glycosylation E128N in only one module (black bars) is sufficient to enhance titers to intermediate levels as compared to absence of E128N (gray bar). The expression titers are similar between variants with E128N in module 1 (Var25) or module 3 (Var26). The expressed proteins contain the trimeric single-chain CD137L- RBD variants scCD137L-Var24 to scCD137L-Var26 which are homodimerized via a linker/hinge-Fc-fusion (SEQ-ID: 53 and SEQ-ID: 77) to generate a hexavalent CD137 agonist (resulting in SEQ-IDs:143, 144, 145).

Figure 8 Schematic layout of bispecific, trivalent targeting constructs; construction based on direct or linker mediated fusion of one (A) or two (B) single-domain antibody moieties (VHH) to the trivalent scTNFSF-RBD.

Figure 9 Aggregate content of scCD137L-Fc variants which contain no cysteine bridges.

The degree of high-molecular weight species (HMWS) and monomer content of the ProteinA affinity-purified eluate was determined by analytical size exclusion chromatography HP-SEC (stable cell expression). The expressed proteins contain the trimeric single-chain CD137L-RBD variants scCD137L- Var1 to scCD137L-Var4 (SEQ-ID: 15 to SEQ-ID: 18) which are homodimerized via a linker/hinge-Fc-fusion (SEQ-ID: 53 and SEQ-ID: 77) to generate a hexavalent CD137 agonist (scCD137L-Fc) (resulting SEQ- IDs:120, 121 , 122, 123). The constructs differ in the presence or absence of the surface glycosylation site E128N and in the interprotomer linker variants (SEQ-ID: 44 and SEQ-ID: 46). In the absence of a disulfide bridge, the monomer content lies in the range of 71 % to 86%. In the scCD137L-Fc format for both interprotomer-linker pair variations, introduction of E128N based glycosylation slightly increases the presence of soluble multimeric species in the ProteinA affinity eluate (although at higher expression titers, data not shown). When shortening the interprotomer linker from SEQ-ID: 44 to SEQ- ID: 46, the scCD137L-Fc constructs become less flexible and form higher levels of monomeric species.

Figure 10 Aggregate content of scCD137L-Fc variants which contain cysteine bridge V143C/L237C.

The degree of high-molecular weight species (HMWS) and monomer content of the ProteinA affinity-purified eluate was determined by analytical size exclusion chromatography HP-SEC (stable cell expression). The expressed proteins contain the trimeric single-chain CD137L-RBD variants scCD137L- Var5 to scCD137L-Var8 (SEQ-ID: 19 to SEQ-ID: 22) which are homodimerized via a linker/hinge-Fc-fusion (SEQ-ID: 53 and SEQ-ID: 77) to generate a hexavalent CD137 agonist (scCD137L-Fc) (resulting SEQ- I Ds: 124, 125, 126, 127). The constructs differ in the presence or absence of the surface glycosylation site E128N and in the interprotomer linker variants (SEQ-ID: 44 and SEQ-ID: 46). In the presence of the disulfide bridge V143C/L237C, the monomer content is increased as compared to variants without disulfide bridges, and lies in the range of 88% to 92%. The cysteine bridge V143C/L237C stabilizes the scCD137L-Fc variant independent of the interprotomer linkers (SEQ-ID: 44 and SEQ-ID: 46) and the glycosylation site E128N, in contrast to the absence of a disulfide bridge (see Figure 9). The introduction of E128N glycosylation site even further increases the monomer content due to increased stability.

Figure 11 Aggregate content of scCD137L-Fc variants which contain cysteine bridge V183C/G198C.

The degree of high-molecular weight species (HMWS) and monomer content of the ProteinA affinity-purified eluate was determined by analytical size exclusion chromatography HP-SEC (stable cell expression). The expressed proteins contain the trimeric single-chain CD137L-RBD variants scCD137L- Var9 to scCD137L-Var12 (SEQ-ID: 23 to SEQ-ID: 26) which are homodimerized via a linker/hinge-Fc-fusion (SEQ-ID: 53 and SEQ-ID: 77) to generate a hexavalent CD137 agonist (scCD137L-Fc) (resulting SEQ- I Ds: 128, 129, 130, 131). The constructs differ in the presence or absence of the surface glycosylation site E128N and in the interprotomer linker variants (SEQ-ID: 44 and SEQ-ID: 46). In the presence of the disulfide bridge V183C/G198C, the monomer content is increased as compared to variants without disulfide bridges, and lies in the range of 87% to 92%, similar to disulfide bridge V143C/L237C. The cysteine bridge V183C/G198C stabilizes the scCD137L-Fc variant independent of the interprotomer linkers (SEQ-ID: 44 and SEQ-ID: 46) and the glycosylation site E128N, in contrast to the absence of a disulfide bridge (see Figure 9).

Figure 12 Aggregate content of scCD137L-Fc variants which contain cysteine bridge A93C/L124C.

The degree of high-molecular weight species (HMWS) and monomer content of the ProteinA affinity-purified eluate was determined by analytical size exclusion chromatography HP-SEC (stable cell expression). The expressed proteins contain trimeric single-chain CD137L-RBD variants containing disulfide bridge A93C/L124C with/without E128N glycosylation site and interprotomer linkers (SEQ-ID: 44 and SEQ-ID: 46). These scCD137L muteins are homodimerized via a linker/hinge-Fc-fusion (SEQ-ID: 53 and SEQ-ID: 77) to generate a hexavalent CD137 agonist (scCD137L-Fc). In contrast to the disulfide bridges V183C/G198C and V143C/L237C, the disulfide bridge A93C/L124C strongly destabilizes the scCD137L-Fc variants and leads to high level of HMWS and low levels of monomeric species (45% - 58%).

Figure 13 Introduction of E128N surface glycosylation site and disulfide bridge(s) increase monomer content in aPDL1-a-scCD137L-SAB variants.

The degree of monomeric species of the 2-Step affinity-purified eluate (ProteinA/CH1 -XL) was determined by analytical size exclusion chromatography HP-SEC (stable cell expression). The expressed bispecific SAB proteins are based on single-chain CD137L-RBD variants containing either no cysteine bridge or cysteine bridge V183C/G198C with/without E128N glycosylation site and interprotomer linkers (SEQ-ID:44 or SEQ- ID:46), fused to a hinge-Fc-knob region (SEQ-ID:64 and SEQ-ID:83). The resulting scCD137L-variants (SEQ-IDs:151 , 152, 153, 154, 160, 161) were co-expressed with the aPD-L1 antibody heavy and light chains SEQ-IDs:163 and 164, resulting in SAB constructs aPDL1 -a-scCD137L-Var1 -SAB, aPDL1 - a-scCD137L-Var3-SAB, aPDL1 -a-scCD137L-Var2-SAB, aPDL1-a- scCD137L-Var4-SAB, aPDL1-a-scCD137L-Var10-SAB and aPDL1-a- scCD137L-Var12-SAB.

Introduction of the E128N in the SAB format increases the monomeric species content for aPDL1 -a-scCD137L-Var2-SAB and aPDL1 -a-scCD137L- Var4-SAB as compared to SAB variants lacking E128N (aPDL1-a- scCD137L-Var1 -SAB and aPDL1 -a-scCD137L-Var3-SAB). Furthermore, the introduction of the disulfide bridge V183C/G198C together with E128N glycosylation site even further increases the monomeric species up to 99% of monomer (aPDL1-a-scCD137L-Var10-SAB and aPDL1 -a-scCD137L- Var12-SAB compared to aPDL1-a-scCD137L-Var2-SAB and aPDL1-a- scCD137L-Var4-SAB). Surprisingly, shortening of the interprotomer linker from SEQ-ID:44 to SEQ-ID:46 increases the monomeric content due increased scCD137L stability (aPDL1-a-scCD137L-Var1-SAB, aPDL1-a- scCD137L-Var2-SAB, aPDL1 -a-scCD137L-Var10-SAB compared to aPDL1 - a-scCD137L-Var3-SAB, aPDL1 -a-scCD137L-Var4-SAB, aPDL1-a- scCD137L-Var12-SAB). As a result, the introduction of E128N + V183C/G198C with both interprotomer linkers (SEQ-ID:44, SEQ-ID:46) is highly preferred.

Figure 14 Introduction of disulfide bridge V143C/L237C increases monomer content in aPDL1-a-scCD137L-SAB variants.

The degree of monomeric species of the 2-Step affinity-purified eluate (ProteinA/CH1 -XL) was determined by analytical size exclusion chromatography HP-SEC (stable cell expression). The expressed bispecific SAB proteins are based on single-chain CD137L-RBD variants containing cysteine bridge V143C/L237C without E128N glycosylation site and interprotomer linker (SEQ-ID: 44), fused to a hinge-Fc-knob region (SEQ- ID:64 and SEQ-ID:83). The resulting scCD137L-variants (SEQ-IDs:151 , 155) were co-expressed with the aPD-L1 antibody heavy and light chains SEQ- IDs:163 and 164, resulting in SAB constructs aPDL1-a-scCD137L-Var1-SAB and aPDL1-a-scCD137L-Var5-SAB. Similar to Figure 13 using disulfide bridge V183C/G198C, the cysteine bridge V143C/L237C also increases the monomeric content in aPDL1 -a-scCD137L-Var5-SAB, thus making the use of V143C/L237C highly preferred for the construction of Ab-scCD137L-SAB formats.

Figure 15 Introduction of E128N glycosylation site together with V183C/G198C cysteine bridge in two interprotomer linker variations in the SAB format results in high degree of monomeric species for three different antibodies.

The applicability of introducing the glycosylation site E128N plus cysteine bridge V183C/G198C was demonstrated for three different antibodies (aPD- L1 -a, aPD-L1-b and aHER2) showing low levels of aggregate content and high levels of monomeric species by stabilizing the scCD137L-SAB domain in these variants. The degree of monomeric species of the 2-Step affinity- purified eluate (ProteinA/CH1-XL) was determined by analytical size exclusion chromatography HP-SEC (stable cell expression). The expressed bispecific SAB proteins are based on single-chain CD137L-RBD variants containing cysteine bridge V183C/G198C with E128N glycosylation site and interprotomer linkers (SEQ-ID: 44 and 46), fused to a hinge-Fc-knob region (SEQ-ID:64 and SEQ-ID:83). The resulting scCD137L-variants (SEQ- IDs:160, 161) were co-expressed with the aPD-L1 -a antibody heavy and light chains SEQ-IDs:163 and 164, resulting in SAB constructs aPDL1-a- scCD137L-Var10-SAB and aPDL1 -a-scCD137L-Var12-SAB, or with the aPD-L1 -b antibody heavy and light chains SEQ-IDs:165 and 166, resulting in SAB constructs aPDL1-b-scCD137L-Var10-SAB and aPDL1-b-scCD137L- Var12-SAB, or with the aHER2 antibody heavy and light chains SEQ-IDs:167 and 168, resulting in SAB constructs aHER2-scCD137L-Var10-SAB and aHER2-scCD137L-Var12-SAB. Taken together, the combination of E128N and V183C/G198C is highly preferred for the construction of Ab-scCD137L- SAB formats. Detailed Description of the Invention

Peptides comprising wildtype CD137L domains or functional fragments thereof, which have a high content of hydrophobic amino acids exposed at their surface, show limited expression rates in specific trivalent CD137L configurations, e.g. with very short linkers, when produced in eukaryotic cells. The inventors surprisingly found that introducing additional N-glycosylations on the outer surface of the CD137L domain and/or introducing functional intra-chain cystine(s) covalently connecting individual beta-barrels of the CD137L domain elevated not only the expression level of homotrimeric CD137L fusion peptides but also of single-chain based CD137L fusion proteins.

Therefore, one aspect of the present invention, relates to CD137L muteins comprising additional N-glycosylations on the outer surface and/or functional intra-chain cystine(s) covalently connecting individual beta-strands.

In addition, for individual combinations hydrophobic exchange mutations can be introduced.

CD137L muteins with N-glycosylation sites

In one aspect of the invention, the CD137L mutein comprises new N-glycosylation sites on its surface. The wildtype CD137L protein of SEQ-ID:01 lacks glycosylations on the outer surface leading to a more hydrophobic protein when compared to other TNFSF ligands. The inventors surprisingly found that introducing N-glycosylations enhanced expression titer of fusion proteins without effecting the binding ability of the mutein (Figures 3 and 6). For the generation of a glycosylation site, several surface exposed amino acid sequences were identified by visually inspecting the crystal structures of the human CD137L ectodomain published by Chin et al (2018) (Nat Commun. 2018 Nov 8;9(1):4679, doi: 10.1038/s41467-018-07136-7). Finally, amino acid sequences were chosen that potentially could be converted into the necessary consensus sequence NXS/T of a glycosylation site. With respect to their relative position on the CD137L surface, suitable mutations of SEQ-ID:01 are E128N; D129NG; Q227N; E156N; I103N in combination with G105S, or combinations thereof.

In a preferred embodiment, the CD137L mutein of SEQ-ID:01 comprises the E128N amino acid exchange to form a functional glycosylation site on the protein surface.

CD137L muteins with disulfide bridging

It is known to the skilled person that covalent disulfide bridges can stabilize the spatial structure of proteins. There are different kinds of disulfides present in natural proteins: surface or solvent exposed disulfides, e.g. in the hinge region of antibodies or those more or less buried inside the structures e.g. the disulfides stabilizing the CH3-domain of human IgGs. Unfortunately, natural CD137L lacks any stabilizing disulfide bridges. Therefore, to introduce potential intra-protomer cystines into the CD137L, amino acid positions with a spatial distance suitable for a disulfide bridge were identified by visually inspection of the published crystal structures of CD137L. Even with this restriction, there are multiple possibilities to introduce pairs of cysteines into the CD137L-ectodomain sequence. However, it is preferred to mutate pairs of amino acids having their side chains orientated into the hydrophobic core of the CD137L-protomer. Because the potential bridges are positioned inwards (in the internal structure of the protomer), they are not accessible to the neighboring protomers of the trimeric functional CD137L-RBD avoiding unspecific disulfide formation between them. In addition to its steric stabilizing effect (reciprocal stabilization), the disulfide bridge, when located in the core of the protein, also contributes, due to its own hydrophobicity, to the overall stability of the hydrophobic core. Also, while buried inside the structure, these mutations are not accessible to potential anti-drug antibodies.

Therefore, in one aspect of the invention the CD137L mutein comprises at least one stabilizing cysteine bridge. The cysteine bridge can either be located in anti-parallel, neighboring beta strands or in parallel beta-strands having a suitable steric distance.

In another preferred embodiment, the amino acids pairs selected for cysteine mutations are A93/L124, A93C/L237C, V143/L237 and V183/G198 of SEQ-ID:01 .

Surprisingly, the inventors found that mutating the positions V143 and L237 as well as positions V183 and G198 to cysteine resulted in the formation of C143-C237 as well as C183-C198 disulfide bridges in both protein formats investigated. This was not the case for the A93/L124 positions. Even though A93C/L124C is functional in the homotrimeric CD137L fusion protein format (Figure 5), it does not work in the single-chain based assembly of the CD137L protomers (scCD137L-Fc format) where these specific mutations lead to strong aggregation propensity and low monomer content; see Figure 12. Similar results for this specific cysteine bridge were obtained for the Ab-scCD137L- SAB format (data not shown). The A93C/L237C based CD137L mutein had a strong aggregation propensity already in the homotrimeric fusion protein format during transient expression experiments (see Figure 5).

In a preferred embodiment the cysteine bridge is formed between amino acid positions 143 und 237 of SEQ-ID:01 ; both positions mutated to cysteine (V143C and L237C).

In a highly preferred embodiment, the cysteine bridge is formed between amino acid positions 183 und 198 of SEQ-ID:01 ; both positions mutated to cysteine (V183C and G198C).

CD137L muteins with N-glycosylation sites and functional disulfide bridging

In one aspect, preferred embodiments of CD137L muteins comprise additional N- glycosylation sites on the outer surface in combination with cysteine bridges between amino acid positions 143 and 237 and/or amino acid positions 183 and 198 of SEQ-ID:01. In yet another preferred embodiment, the CD137L mutein comprises the mutations V143C and L237C and E128N of SEQ-ID:01.

In yet another preferred embodiment, the CD137L mutein comprises the mutations V183C and G198C and E128N of SEQ-ID:01.

In yet another preferred embodiment, the CD137L mutein comprises the mutations V143C and L237C and V183C and G198C and E128N of SEQ-ID:01.

Preferred N- and C-terminal ends of the CD137L muteins

In yet another preferred embodiment, the CD137L muteins disclosed above start with Q89 or G90 or M91 and end with V240 or T241 or P242 or E243 of SEQ-ID:01 as described in Table 2. CD137L muteins can be linked to trimerization domains

In another aspect of the invention, the CD137L muteins disclosed above can be employed to construct homotrimeric fusion proteins using protein linkers to fuse trimerization domains to their N- or C-terminal end. Each of those fusion proteins comprises three individual polypeptide chains to form a functional trivalent CD137L-receptor binding domain. Protein-domains or peptide sequences to generate stabilized homotrimeric TNFSF-RBD fusion protein are well known in the art, e.g. the bacteriophage RB69 foldon (SEQ-ID: 51 ) or human surfactant protein D derived domains (SEQ-ID: 52). In addition, WO/2009/007120 describes homotrimeric TNFSF collectin fusion proteins In a preferred embodiment, the homotrimeric CD137L-mutein fusion protein comprises htCD137L-Var2 or htCD137L-Var7 or htCD137L-Var8 or htCD137L-Var11 or htCD137L-Var12 listed in Table 1.

Multimerization of CD137L muteins

In another aspect of the invention, the CD137L muteins disclosed above can be employed to construct single-chain multimeric CD137L moieties either using protein linkers or by direct fusion of monomeric CD137L muteins. Preferred embodiments of the single chain CD137L fusion proteins include multiple (e.g. 2, 3, 4, 5, 6) CD137L muteins. Trimeric units are particularly preferred, as they conform to the natural arrangement of almost all TNFSF ligands. Suitable linkers for the construction of single-chain CD137L molecules are disclosed in Table 3 and Table 4 (SEQ ID 41-50). The invention demonstrates that the use of short linkers increases stability but decrease productivity of single-chain CD137L constructs. Surprisingly, the inventors found that outer glycosylation overcompensates many times over for the negative effects of short linkers on expression levels. Data on such a preferred combination of features (short linker + outer glycosylation at E128N) are exemplified in Figure 6.

E128N based glycosylation enables short interprotomer linkers

In some embodiments short linkers are better suited for the production of trimeric singlechain CD137L receptor binding domains but can only be expressed with sufficient productivity if the E128N based mutation and subsequent glycosylation is present in at least one of CD137L modules of the trimeric single-chain unit (see Figure 7).

In a preferred embodiment the N-terminus of each CD137L module starts with amino acid Q89, or G90, or M90 and ends with V240, or T241 , or P242 or E243 as C-terminus (see Table 2) and individual CD137L modules are connected with short glycine/serine rich linkers of Table 4 SEQ-ID:41-50.

Other embodiments of such single-chain CD137L modules combine advantageous linker lengths (in particular 1-12 amino acids) with outer surface glycosylation and/or stabilizing cysteine bridging.

Homomeric and/or heteromeric protein assemblies

Fusion proteins comprising above mentioned CD137L muteins can be employed for the construction of homomeric and/or heteromeric protein assemblies.

To generate such assemblies, the CD137L muteins of the invention may be fused, preferably via protein linkers, either as monomers or single-chain CD137L multimers (preferably single-chain trimers) to dimerizing or multimerizing protein domains.

In a preferred embodiment the fusion protein comprising the above mentioned CD137L muteins are fused to homo- or hetero-dimerization domains which allows for the formation of higher order protein assemblies.

Various suitable homo- or heterodimerization domains are known in the art and many of them are derived from human IgG-Fc-domains. Homodimerization occurs, when the wildtype-Fc-domain is used as a fusion partner and its CH3 domain comprises an unmodified dimerization interface. Heterodimerization is preferred, when the dimerization interface is modified by mutations giving two variants which preferentially form stable heterodimers; e.g. by the knob-into hole technology. Sequence examples and suitable sequence modules are given in PCT/EP2021/063005 and are incorporated by reference in their entirety. Multi-specific CD137L fusion protein assemblies

The multi-specific CD137L fusion protein assemblies comprise at least (i) one protein moiety which comprises a single-chain CD137L receptor binding domain and (ii) a protein moiety capable of specific binding to a cell surface antigen or an activity modulating effector.

In one aspect of the invention, the bispecific CD137L protein assembly comprises at least

(a) a trimeric single-chain CD137L receptor binding domain fused to

(b) a first peptide linker fused to

(c) a first hetero-dimerization domain and

(d) an antigen binding or interacting protein moiety fused to

(e) a second peptide linker fused to

(f) a second hetero-dimerization domain

A general overview of a CD137L fusion protein assembly of the invention is given in Figure 2A.

As depicted in Figure 2A, a typical multispecific CD137L fusion protein of the invention is a protein-unit comprising a typical IgG antibody-derived heavy and light chain assembly on one side and a trivalent single-chain CD137L-RBD-Fc fusion polypeptide on the other side. The heterodimerization of both halves of the protein-unit is enforced by the CH3- domains and additionally stabilized by the hinge inter-chain cysteines. The co-expression and correct assembly of three polypeptide-chains is necessary to form this functional bispecific protein unit.

Fusion proteins of this format are called Ab-scTNFSF-SAB (SAB=single-arm-bispecific), and their design and production are already disclosed in great detail in PCT/EP2021/063005.

Ab-scCD137L-SAB based immune modulators of the invention

As non-limiting examples preferred embodiments of the Ab-scCD137L-SAB multispecific immune modulator of the invention combines CD137 agonism with anti-PD-L1 targeting. Taking the technical descriptions from PCT/EP2021/063005 into account, Ab-scCD137L- SAB multispecific immune modulators with different targeting specificities’ can be easily generated by exchanging the VH and VL domains responsible for targeting. Non limiting examples for specific targets like PD-L1 , mesothelin, CD25, PD-1 , CEA, CD95L, and Her- 2 and their corresponding VH/VL protein-sequences are shown in table Table 4 (SEQ ID: 97-118). Examples for preferred scCD137L-muteins to construct the scCD137L-Fc-knob chain for bispecific Ab-scCD137L-SAB protein assemblies are listed in Table 7. scTNFSF-RBD-Fc based assemblies

In one aspect of the invention, the multi-specific CD137L fusion protein assembly comprises at least

(a) a single-chain CD137L receptor binding domain fused to

(b) a first peptide linker fused to

(c) a first hetero-dimerization domain

(d) and a non CD137L single-chain TNF-SF receptor binding domain fused to

(e) a second peptide linker fused to

(f) a second hetero-dimerization domain

A general overview of a multi-specific TNFSF fusion protein assembly of this aspect of the invention is given in Figure 1A.

As depicted in Figure 1A, a typical multispecific immune modulator of the invention can be achieved by combining a scCD137L-Fc fusion protein of the invention with a second non-CD137L scTNFSF-Fc fusion protein.

Fusion proteins of this format including their design and production are already disclosed in great detail in PCT/EP2021/063005. The teachings of PCT/EP2021/063005 can easily be applied by one of ordinary skill in the art to analogous CD137L fusion proteins of the present invention.

In a preferred embodiment the single-chain TNF-SF receptor binding domain of above step (d) is a second single-chain CD137L fusion protein (Figure 1A) resulting in a hexavalent scCD137L protein assembly. Examples for preferred scCD137L-mutein-Fc proteins are listed in Table 6. Fab-based targeting of scCD137L muteins of the invention

In one aspect of the invention, the multi-specific CD137L fusion protein assembly comprises at least

(a) a functional Fab domain of an antibody fused to

(b) a single-chain CD137L receptor binding domain, wherein the C-terminal end of the constant heavy chain domain of the Fab fragment (a) is fused to the single-chain CD137L receptor binding via a peptide linker.

A general overview of a multi-specific CD137L fusion protein assembly of this aspect of the invention is given in Figure 2B.

As depicted in Figure 2B, a typical multispecific CD137L immune modulator of this aspect of the invention combines CD137L agonism with anti-PD-L1 targeting (FAB targeting combined with single-chain based TNFSF receptor agonism). Fusion proteins of this format are called Ab-scTNFSF and their design and production are already disclosed in great detail in PCT/EP2021/063005.

As non-limiting examples preferred embodiments, of the Ab-scCD137L multispecific immune-Modulator of the invention combines CD137 agonism with anti-PD-L1 targeting. Taking the technical descriptions from PCT/EP2021/063005 into account, Ab-scCD137L multispecific immune modulators with different targeting specificities’ can be easily generated by exchanging the VH and VL domains responsible for targeting. Non limiting examples for specific targets like PD-L1 , mesothelin, CD25, PD-1 , CEA, CD95L, and Her- 2 and their corresponding VH/VL protein-sequences are shown in table Table 4 (SEQ ID: 97-118).

VHH-based targeting of scCD137L muteins of the invention

In a further aspect of the invention, the multi-specific CD137L superfamily fusion protein assembly comprises at least

(a) a functional single VH (variable heavy chain) domain of an antibody fused to

(b) a single-chain CD137L-receptor binding domain, wherein the C-terminal end of the VH domain is fused to the single-chain TNF-SF receptor binding via a peptide linker (Figure 8).

Examples for functional single VH domains are the so-called VH derived single domain antibodies (VHH).

Further aspects and embodiments of the invention

As used herein, the terms single chain TNF-SF receptor binding domain, single chain TNFSF receptor binding domain and TNF-SF RBD and TNFSF RBD are used synonymously for the above mentioned trivalent non-aggregating TNF-SF receptor binding domains. In addition, when referring to said receptor binding domains, the expression ‘single chain’ is often abbreviated as ‘sc’, e.g. scTNFSF-RBD; accordingly, a trivalent non-aggregating single-chain CD137L receptor binding domains might be abbreviated as scCD137L-RBD.

The antigen binding or interacting moiety of some aspects of the invention can be an antibody fragment, for example a monospecific antibody fragment or a functional fragment thereof. Further suitable binding and interacting moieties are known in the art. Non-limiting examples are: single chain antibodies or functional fragments thereof, single domain antibodies, functional scFv fragments.

In specific embodiments, the functional antibody fragment is directed against a cell surface marker or an activity-modulating target. As a non-limiting example the antibody or antibody fragment is directed against: tyrosine- kinase-receptors (EGFR, HER2, HER3, HER4), VEGFRs, heteromeric integrin a- or p-receptor family, including VLA-4 and LFA- 1 , E-selectin, L-selectin, P-selectin, tumor stroma markers like fibroblast activation protein (FAP) endoglyx-1 , MCSP or endosialin, galectin, N-CAM (Myelin protein zero), ICAM (1 , 5), VCAM-1 , PE-CAM, L1 -CAM, Nectin (PVRL1 , PVRL2, PVRL3), EpCAM, tumor antigens, including NY-ESO-1 , MAGE1 , MAGE2, CA-125, Carcinoembryonic Antigen (CEA), CAMPATH-1 (CD52), CD44 and tumor specific variants thereof and other tumor selective cell surface markers, CD2, CD5, CD7, CD19, CD20, CD21 , CD22, CD24, CD25, CD30, CD33, CD38, CD40, CD52, CD56, CD71 , CD72, CD73, CD105, CD117, CD123, CD133, c-Met, PDGFR, IGF1-R, HMW-MAA, TAG-72, GD2, GD3, GM2, folate receptor, Lgr5, Ley, Muc-1 , Muc-2, PSMA, PSCA and uPAR. More preferably, the target molecule is FAP, EGFR, HER2 or HER, melanoma-associated chondroitin sulfate proteoglycan (MCSP).

The antibody or antibody fragment might also be directed against a member of the B7 family, including B7-1 (CD80), B7-2 (CD86), B7-DC (PDCD1 LG2, PD-L2, CD273), B7-H1 (PD-L1 , CD274), B7-H2 (ICOSLG, B7RP1 , CD275), B7-H3 (CD276), B7-H4 (VTCN1), B7-H5 (VISTA, Platelet receptor Gi24, SISP1), B7-H6 (NCR3LG1) and B7-H7 (HHLA2).

In a further embodiment, the antibody or antibody fragment might also be directed against activity modulating targets, including but not limited to CTLA-4, PD-1 , CD3, CD4, CD8, CD28, HLA Class I and Class II, LAG3 (CD223), ICOS (CD278), CD39, CD73, TIGIT, CD96, PTA1 (CD226), TIM-3, TIM-1 , CD47, SIRP-alpha, DNAM-1 , and Interleukins (antiinflammatory), including but not limited to IL4, IL6, IL9, IL10, IL11 , IL13, IL18, IL21 and IL22.

It has to be noted that all ectodomains of the TNF-SF and TNFR-SF are especially suited targets for functional antibody fragments as used in some aspects the invention. A preferred but not-limiting list comprises ectodomains of TNF-SF ligand domains like CD95L, TNF-alpha, CD40L, CD27L, LIGHT, TL1A and TWEAK and TNF-receptor domains like CD40, CD27, 4-1 BB, 0X40, GITR, HVEM, BCMA, LTBR and TWEAKR.

From a scientific and commercial point of view, combinations of trimeric single-chain CD137L of the invention with antibodies that bind already evaluated surface markers of cancer cells, such as CEA or HER2, or that intervene in the signaling cascade of checkpoint modulators (PD-1 , CTLA4, CD95) are particularly attractive. The peptides with aPD-L1 and aCD95L or anti-CEA activity shown in the examples and figures represent therefore further particularly preferred embodiments of the invention.

A further aspect of the present invention relates to nucleic acid molecules encoding CD137L muteins of the invention as well as fusion polypeptides comprising CD137L muteins of the invention. The nucleic acid molecule may be a DNA molecule, e.g. a double-stranded or single-stranded DNA molecule, or an RNA molecule. The nucleic acid molecule may encode the fusion protein or a precursor thereof, e.g. a pro- or pre-proform of the fusion protein which may comprise a signal sequence or other heterologous amino acid portions for secretion or purification which are preferably located at the N- and/or C- terminus of the fusion protein. The heterologous amino acid portions may be linked to the first and/or second domain via a protease cleavage site, e.g. a Factor X_a, thrombin or IgA protease cleavage site.

The nucleic acid molecule may be operatively linked to an expression control sequence, e.g. an expression control sequence that allows expression of the nucleic acid molecule in a desired host cell. The nucleic acid molecule may be located on a vector, e.g. a plasmid, a bacteriophage, a viral vector, a chromosomal integration vector, etc. Examples of suitable expression control sequences and vectors are described for example by Green and Sambrook et al. (2012) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, and Hacker et.al (2019) Recombinant Protein Expression in Mammalian Cells: Methods and Protocols (Methods in Molecular Biology, Band 1850, Humana Press) or more recent editions thereof.

Various expression vector/host cell systems may be used to express the nucleic acid sequences encoding the fusion proteins of the present invention. Suitable host cells include, but are not limited to prokaryotic cells such as bacteria, e.g. E.coli, eukaryotic host cells such as yeast cells, insect cells, plant cells or animal cells, preferably mammalian cells and, more preferably, human cells.

Further, the invention relates to a non-human organism transformed or transfected with a nucleic acid molecule as described above. Such transgenic organisms may be generated by known methods of genetic transfer including homologous recombination.

A further aspect of the present invention relates to a pharmaceutical or diagnostic composition comprising as the active agent at least one fusion protein, a respective nucleic acid encoding therefore, or a transformed or transfected cell, all as described herein. Fusion proteins of the invention, respective nucleic acids encoding said fusion proteins, transformed or transfected cell useful for the production of said fusion proteins may be used in therapy, e.g., in the prophylaxis and/or treatment of disorders caused by, associated with and/or accompanied by dysfunction of TNF-SF cytokines, particularly proliferative disorders, such as tumors, e.g. solid or lymphatic tumors; infectious diseases; inflammatory diseases; metabolic diseases; autoimmune disorders, e.g. rheumatoid and/or arthritic diseases; degenerative diseases, e.g. neurodegenerative diseases such as multiple sclerosis; apoptosis-associated diseases or transplant rejections.

Definitions

TNF-SF

TNF-SF: The term “TNF ligand family member” or “TNF family ligand” or “TNF superfamily” (TNF-SF) refers to a pro-inflammatory cytokine. Cytokines in general, and in particular the members of the TNF ligand superfamily, play a crucial role in the stimulation and coordination of the immune system. At present, nineteen cytokines have been identified as members of the TNF (tumor necrosis factor) ligand superfamily on the basis of sequence, functional, and structural similarities. All these ligands are type II transmembrane proteins with a C-terminal extracellular domain (ectodomain), N-terminal intracellular domain and a single transmembrane domain. The TNF-SF ectodomain comprises the stalk region and the C-terminal located sequence known as TNF homology domain (THD), which has 20-30% amino acid identity between the superfamily members. The C-terminal part of the TNF ectodomain is also responsible for the TNF ligands to form trimeric complexes that are recognized by their specific receptors. These trimeric complexes are the binding competent structures as the receptor binding takes place at the protomer interfaces of the so-called TNF-SF Receptor-binding-domain (RBD). In other words: the C-terminal regions of three individual TNF-SF polypeptides form a functional unit and trimer formation is a structural prerequisite for proper receptor recruitment of the human TNF-SF members.

In terms of its spatial structure, the CD137 ligand is a typical representative of the TNF- SF: Three individual CD137L polypeptides form the functional unit for receptor recruitment. In fusion proteins, this functional unit can either be assembled by three individual polypeptide chains or by a single polypeptide comprising a serial assembly of three CD137L-RBD protomer-modules interconnected with short linkers as described in WO 2017/068183.

As used herein, the term “homotrimeric CD137L” is used for the trivalent receptor binding domain unit formed by three individual polypeptides and htCD137L or (ht)CD137L are its common abbreviations.

As used herein, the terms single-chain CD137L receptor binding domain, single-chain CD137L are used synonymously for the trivalent but single polypeptide based CD137L receptor binding domains. In addition, when referring to said receptor binding domains, the expression ‘single-chain’ is often abbreviated as ‘sc’, e.g. scCD137L-RBD.

In terms of its spatial structure, the human CD137 ligand is a typical representative of the aforementioned TNF-SF: Three individual CD137L polypeptides form the functional unit for receptor recruitment. In fusion proteins, this functional unit can either be assembled by three individual polypeptide chains or by a single polypeptide comprising a serial assembly of three CD137L-RBD protomer-modules interconnected with short linkers as described in WO 2017/068183.

As used herein, the terms single-chain CD137L receptor binding domain, single-chain CD137L and are used synonymously for the trivalent but single polypeptide based CD137L receptor binding domains. In addition, when referring to said receptor binding domains, the expression ‘single-chain’ is often abbreviated as ‘sc’, e.g. scCD137L-RBD.

Dimer formation

Dimer formation: As used herein, dimerization means, that a polypeptide chain upon folding is capable to form a stable structure with a second polypeptide chain upon folding and that a certain dimerization domain implemented into the polypeptide chains is enforcing this process. Dimer formation takes places between these specific domains present in each of the both polypeptides. Examples for dimerization domains are well known in the art. In natural human IgA-, IgD- and IgG antibodies, the CH3-domain is the driving force for the dimerization of the heavy-chains. In natural IgE or IgM antibodies the CH4-domain is the structural and functional equivalent to the lgG-CH3 domain enforcing their heavy-chain dimerization. The CH3-domain or their equivalents are selective only for themselves. This means, that any polypeptide comprising a functional CH3-domain either by nature or by engineering approaches is capable to form a dimer with a second polypeptide comprising a functional CH3-domain due to the CH3/CH3 dimer formation.

Hetero-dimerization

Hetero-dimerization of two CH3-domain comprising polypeptides to a functional bispecific fusion protein is achieved by co-expression of both polypeptides in a suitable host cell ensuring the presence of both chains simultaneously during protein folding. During the protein synthesis in the host-cell, any CH3-domain combination of the present polypeptide chains will be formed: heterodimers as well as homodimers. The wanted heterodimeric protein product needs to be purified afterwards by suitable chromatographic procedures. Methods for co-expression of CH3-comprising polypeptides and subsequent purification concepts for the heterodimeric product are well known in the art. The CH3-domains used can be either wild-type or they can comprise point mutations stabilizing a certain assembly e.g. as described by Carter et al. (Merchant, A., Zhu, Z., Yuan, J. et al. An efficient route to human bispecific IgG. Nat Biotechnol 16, 677-681 (1998). https://doi.org/10.1038/nbt0798-677). For the generation of multi-specific immune modulators of the current invention, the usage of CH3-domain derived dimerization technologies is highly preferred. In a preferred embodiment, the CH3 domains implemented into both fusion protein polypeptides is a natural occurring sequence. In a preferred embodiment, the CH3 domains comprise point mutations, which are intended to stabilize the current dimerization product. It is highly preferred, that the stabilizing mutations result in covalent linkage of the both polypeptides, e.g. by cystines between the CH3-domains of a current assembly, thereby inhibiting the CH3-domain dissociation. As a consequence, interchain exchange reaction of the purified heterodimeric product and subsequent multimer and/or homodimer formation during the production are reduced. In a preferred embodiment, the CH3 domains comprise point mutations which preferentially lead to heterodimer formation during protein expression, e.g. knobs into hole (KiH) technology. In addition to the KiH technology, other more recent technologies to generate CH3 based heterodimerization domains have been developed employing either electrostatic steering or immunoglobulin domain interface exchange or a combination of both. The basic technologies present in the field are described in Skegro et al. J Biol Chem. 2017 Jun 9;292(23):9745-9759), Gunasekaran et al. J Biol Chem. 2010 Jun 18;285(25): 19637-46, Sampei et al. PLoS One. 2013;8(2):e57479, Von Kreudenstein et al. MAbs. 2013 Sep-Oct;5(5):646-54, Davis et al. Protein Eng Des Sei. 2010 Apr;23(4): 195-202.

Antibody

Antibody: The terms “full length antibody”, “intact antibody”, “whole antibody” and “natural antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure. “Native antibodies” refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG- class antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two light chains and two heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1 , CH2, and CH3), also called a heavy chain constant region. As used herein, typical IgG derived constant heavy chain domains used in the context of the invention are SEQ-ID:89, SEQ-ID:90, SEQ-ID:91 , SEQ-ID:92, SEQ-ID:93, and SEQ-ID:94 all defined to start with Ala118 according to the EU numbering. As used herein a typical IgG derived CL1 domain used in the context of the invention is SEQ-ID: 95 (CLkappa). The CH1 and CH2 domains are connected via a hinge region which stabilizes the antibody by cysteine bridges. Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a light chain constant domain (CL), also called a light chain constant region. The heavy chain of an antibody may be assigned to one of five types, called a (IgA), 5 (IgD), £ (IgE), y (IgG), or p (IgM), some of which may be further divided into subtypes, e.g. y1 ( I gG 1 ), y2 (I gG2) , y3 ( I gG3) , y4 ( I gG4) , a1 (lgA1) and a2 (lgA2). The light chain of an antibody may be assigned to one of two types, called kappa (K) and lambda (A). In addition, hybrid light chain formats can be engineered comprising lambda VL and kappa CL, and vice versa. In a preferred embodiment, a light chain is based on a kappa LC or a hybrid LC composed of VLIambda/CLkappa for improved solubility and faster folding kinetics. As used herein a typical CL kappa domain used in the context of the invention is SEQ-ID:95.

As used herein, anti PD-L1 antibodies or antibody fragments with anti-PD-L1 specificity are often referred to as “aPDL1” or“aPD-L1” antibodies or respective antibody fragments. The same is done for other antibody specificities; for example, for anti-HER2, aHER2 is also used and for anti-CEA, aCEA is also used.

Antibody fragment

Antibody fragment: An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies, triabodies, tetrabodies, cross-Fab fragments; linear antibodies; single-chain antibody molecules (e.g. scFv); and single domain antibodies (e.g. VHH). For a review of certain antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see e.g. Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer- Verlag, New York, pp. 269-315 (1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571 ,894 and 5,587,458. Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific, see, for example, EP 404,097; WO 1993/01161 ; Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). For a review on bispecific antibody fragment based constructs see, Brinkmann U, Kontermann RE. MAbs. 2017 Feb/Mar; 9(2): 182-212. Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. The first single domain antibodies were derived from the variable domain of the antibody heavy chain from camelids (nanobodies or VHH fragments). Furthermore, the term single domain antibody includes an autonomous human heavy chain variable domain (aVH) or VNAR fragments derived from sharks. In certain embodiments, a single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, Mass.; see e.g. U.S. Pat. No. 6,248,516 B1). Methods for the preparation of antibody fragments are familiar to those skilled in the art. Widely used methods include proteolytic digestion or recombinant production in host cells. A non-limiting overview of methods of preparation of antibodies and antibody fragments is shown in US20160200833A1.

Fab-Fragment

Fab-Fragment and scFv fragment: The term “Fab fragment” refers to an antibody fragment comprising a light chain fragment composed of a VL domain and a constant domain of a light chain (CL), and a VH domain and a first constant domain (CH1) of a heavy chain. The CH1 and CL domains can either contain wild-type sequences or point mutations for improved association (CH1 : L128F, EU numbering).

A “single-chain variable fragment (scFv)” is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an antibody, connected with a short linker peptide of ten to about 25 amino acids. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. This protein retains the specificity of the original antibody, despite removal of the constant regions and the introduction of the linker. scFv antibodies are, e.g. described in Houston, J. S., Methods in Enzymol. 203 (1991) 46-96).

Fc-domain

Fc-Domain: The term “Fc domain” or “Fc region” herein is used to define a C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. An IgG Fc region comprises an IgG CH2 and an IgG CH3 domain. However, as often used herein, the Fc extends from amino acid residue P230 to amino acid K447 (CH2: 230-340, CH3: 341 - 447). The “CH2 domain” of a human IgG Fc region usually extends from an amino acid residue at about position 231 to an amino acid residue at about position 340. In one embodiment, a carbohydrate chain is attached to the CH2 domain. The CH2 domain herein may be a native sequence CH2 domain or variant CH2 domain. The position N297 of the CH2 domain is glycosylated in a native sequence and required for Fc receptor binding. In one embodiment, a mutation at N297 abrogates Fc receptor binding. The “CH3 domain” comprises the stretch of residues C-terminal to a CH2 domain in an Fc region (i.e. from an amino acid residue at about position 341 to an amino acid residue at about position 447 of an IgG). The CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain (e.g. a CH3 domain with an introduced “protuberance” (“knob”) in one chain thereof and a corresponding introduced “cavity” (“hole”) in the other chain thereof; see U.S. Pat. No. 5,821 ,333, expressly incorporated herein by reference). Such variant CH3 domains may be used to promote hetero-dimerization of two non-identical antibody heavy chains as herein described. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Edelman, G.M. et al., Proc. Natl. Acad. USA, 63, 78-85 (1969).

The “knob-into-hole” technology is described e.g. in U.S. Pat. No. 5,731 ,168; U.S. Pat. No. 7,695,936. Generally, the method involves introducing a protuberance (“knob”) at the interface of a first polypeptide and a corresponding cavity (“hole”) in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g. tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). The protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g. by site-specific mutagenesis, or by peptide synthesis. In a specific embodiment a knob modification comprises the amino acid substitution T366W in one of the two subunits of the Fc domain, and the hole modification comprises the amino acid substitutions T366S, L368A and Y407V in the other one of the two subunits of the Fc domain. In a further specific embodiment, the subunit of the Fc domain comprising the knob modification additionally comprises the amino acid substitution S354C, and the subunit of the Fc domain comprising the hole modification additionally comprises the amino acid substitution Y349C. Introduction of these two cysteine residues results in the formation of a disulfide bridge between the two subunits of the Fc region, thus further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)). The numbering is according to EU numbering. As used herein, typical IgG derived Fc-domains used in the context of the invention are SEQ-ID:83, SEQ-ID:84, SEQ-ID:85, SEQ-ID:86, SEQ-ID:87 and SEQ-ID:88, all defined to start with Pro230 according to the EU numbering.

A “region equivalent to the Fc region of an immunoglobulin” is intended to include naturally occurring allelic variants of the Fc region of an immunoglobulin (e.g. D356E/L358M) as well as variants having alterations which produce substitutions, additions, or deletions but which do not decrease substantially the ability of the immunoglobulin to mediate effector functions (such as antibody-dependent cellular cytotoxicity). For example, one or more amino acids can be deleted from the N-terminus or C-terminus of the Fc region of an immunoglobulin without substantial loss of biological function. Such variants can be selected according to general rules known in the art so as to have minimal effect on activity (see, e.g., Bowie, J. U. et al., Science 247:1306-10 (1990)).

As used herein, anti PD-L1 antibodies or antibody fragments with anti-PD-L1 specificity are often referred to as “aPDL1” or“aPD-L1” antibodies or respective antibody fragments. The same is done for other antibody specificities; for example, for anti-CD95L, aCD95L is also used and for anti-CEA, aCEA is also used.

Disulfide Bridge

Disulfide bonds usually can be formed between spatially proximal cysteine residues in proteins and peptides and contribute to their overall molecular architecture. The formation of a disulfide bond by two side chain S^Y atoms of cysteines constitutes a two-electron oxidation process leading from reduced sulfhydryl groups of cysteines (S-H) to the oxidized cystine (S-S) residue (fora mini review see Wiedemann et al. 2020, Front. Chem. DOI=10.3389/fchem.2020.00280). The terms “disulfide bridge” or “cysteine bridge” as used herein describe synonymously a cystine formation between spatially proximal cysteine residues. The cysteine residues can be positioned on the same or on two different polypeptide chains. Examples:

Example 1 : Transient Gene Expression

The folding kinetics and solubility of an individual protein in the ER during secretory pathway-based maturation translates into the level of expression titers observed during transient gene expression experiments. Therefore, the protein titer can be taken as a first surrogate parameter for an overall improvement of the producibility of the recombinant protein later on. Proteins, which aggregate during ER-passage, remain in the intracellular space and are directed to degradative pathways, thus resulting in low overall expression yields and potentially harm of the host cells.

To compare the relative titer of individual fusion proteins, recombinant expression and subsequent purifications were performed under identical conditions. This means, that the same CHO-S host cell line as well the same genetic elements and vectors were used for all proteins examined. Proteins were ranked by comparing the relative protein titers obtained. The recombinant protein titers were quantified by AFC and/or ELISA based methods.

For the transient expression of the aforementioned fusion proteins of the invention, synthetic DNA cassettes encoding the necessary polypeptides (e.g. htCD137L-RB69, scCD137L-Fc, antibody-HC, antibody-LC, VH-CH1-scCD137L) are inserted into eukaryotic expression vectors comprising appropriate genetic elements suitable to transiently express the protein of interest. To ensure secretory pathway based expression, an N-terminal signal peptide (SEQ-ID:02) is added to the protein sequence of interest . The plasmid DNAs carrying the gene cassettes of interest are prepared by method well known in the art.

One day prior to transfection, CHO-S cells were seeded at 1 x 10E6 cells/ml in fresh PowerCHO-2 CD + 4 mM Glutamax medium to achieve a cell density of 2 x 10E6 cells/ml (± 0.2 x 10E6 cells/ml) on the day of transfection by expected exponential growth. On the day of transfection, the required number of cells was centrifuged at 1000 rpm for 5 min at RT, washed in PBS and resuspended at a cell density of 4 x 10E6 cells/ml in the appropriate volume of fresh ProCHO5, supplemented with 4 mM Glutamax and 0.5x Anti/Anti. Transfection was performed in Coming Erlenmeyer Flasks. For each transfection, the appropriate plasmid DNAs (1.5 pg / 1 x 10E6 cells/ml) and linear PEI 25000 (2.5 pg / 1 x 10E6 cells/ml) were carefully mixed and incubated for 10 min at RT prior to addition to the cells. The transfected cultures were maintained for 6 days at 31 °C in 7 % CO₂ and 85 % humidity with agitation of 180 rpm.

To obtain cell free supernatants after 6-day incubation period, cultures were centrifuged at RT and 1000 rpm for 15 minutes. Subsequently, sodium azide was added with a ratio of 1 :1000, the supernatants were sterile filtrated, frozen at -85 °C for 1 day and stored at -20 °C until purification.

Example 2: Method for large scale expression and purification of recombinant monospecific and multispecific/bispecific CD137L fusion protein assemblies

For large scale expression of the aforementioned fusion proteins of the invention, synthetic DNA cassettes encoding the necessary polypeptides (e.g. htCD137L-RB69, scCD137L-Fc, antibody-HC, antibody-LC, VH-CH1-scCD137L) are inserted into eukaryotic expression vectors comprising appropriate selection markers (e.g. a functional expression cassette comprising a blasticidin, puromycin, hygromycin orzeocin resistance gene) and genetic elements suitable to enhance the number of transcriptionally active insertion sites within the host cell genome, e.g. the human p-globin matrix attachment region (MAR). The sequence verified expression vectors are introduced by electroporation into suspension adapted Chinese Hamster Ovary cells (CHO-S, Invitrogen). Appropriate selection pressure was applied three days post transfection to the transfected cells. Surviving cells carrying the vector derived resistance genes are recovered by subsequent cultivation under selection pressure. Upon stable growth of the selected cell pools in chemically defined medium (PowerCHO-2 CD, Lonza, supplemented with 4 mM glutamine/glutamax) at 37 °C and 7% CO₂ atmosphere in an orbital shaker incubator (100 rpm, 50 mm shaking throw), the individual supernatants are analyzed by ELISA assays detecting the aforementioned proteins. Cell pools with the highest specific productivity are expanded in shake flasks for protein production (orbital shaker, 100 rpm, shaking throw 50 mm). For lab-scale production, individual stable cell pools are cultured for 7-12 days in chemically defined medium (PowerCHO-2 CD, Lonza, supplemented with 4 mM glutamax) at 37 °C and 7% CO₂ atmosphere, either in shake flasks with orbital shaking (100 rpm, 55 mm shaking throw) or in a Wave bioreactor 20/50 EHT (GE Healthcare/Cytiva). The wave culture is started with a viable cell concentration of 0.3 x10e6 cells/ml and the following settings (for five or ten liter): shaking frequency 18 rpm, shaking angle 7°, gas current 0.2-0.3 L/min, 7% CO₂, 36.5 °C. During the wave run, the cell culture is fed twice with PowerFeed A (Lonza) with Lipids usually on day 3 (20 % feed) and on day 6 (30 % feed). After the second feed, shaking frequency is increased to 22 rpm and the shaking angle to 8°. The wave bioreactor is harvested between day 7 to day 11 when the cell viability drops below 80%. The culture supernatant containing CD137L-based fusion proteins is clarified using a depth filtration system (Millipore Millistak Pod MC0HC 0.054 m²), followed by sterile filtration of the clarified harvest using 0.22 pm bottle top filter (PES, Coming) and stored at 2-8 °C until further processing.

Example 3: Purification of monospecific scCD137L-Fc fusion proteins

For affinity purification of the monospecific scCD137L-Fc-based immune modulators a purification process on an AKTA chromatography system (GE Healthcare/Cytiva) is performed which makes use of the Fc domain of the scCD137L-Fc fusion protein. MabSelect SuRe™ ProteinA (GE Healthcare/Cytiva) as solid phase affinity ligand is used which binds with high binding capacity to the Fc domain of the scCD137L-Fc fusion protein. Briefly, the sterile filtered clarified cell culture supernatant/harvest is loaded on a HiTrap MabSelect SuRe column (CV=5 ml) which was equilibrated in wash buffer 1 (20 mM Pi, 95 mM NaCI, pH 7.2) not exceeding a load of 10 mg fusion protein per ml column volume. The column is washed with 10 column volumes (10 CV) of wash buffer 1 followed by at least four column volumes (4 CV) of wash buffer 2 (20 mM Pi, 95 mM NaCI, pH 8.0) to deplete host-cell proteins and host-cell DNA. After a series of washing steps, the protein is then eluted from the column with two column volumes of a suitable elution buffer at pH3.5 (e.g. 20 mM Pi, 95 mM NaCI, pH 3.5 or 50 mM NaAc, 150 mM NaCI, pH3,5). The eluate is collected in fractions and immediately neutralized with 1 M Tris-HCI pH 8.0 to neutral pH. The linear velocity is set to 150 cm/h and kept constant during the aforementioned affinity chromatography method.

Example 4: Purification of multispecific scCD137L-Fc fusion proteins

The affinity purification of multispecific scCD137L-Fc fusion proteins is based on a 2-step AFC purification procedure which makes use of the different properties of the hetero-Fc domain in the aforementioned bispecific scCD137L-Fc fusion protein. The multispecific immune modulators contain a heterodimerized Fc domain in which one of the hetero-Fc sequences comprises a reduced affinity towards protein A introduced by specific mutations (H435R,Y436F).

For affinity purification of the multispecific immune modulators, a purification process on an AKTA chromatography system (GE Healthcare/Cytiva) is performed which makes use of the different properties of the aforementioned bispecific scCD137L-Fc fusion proteins introduced by specific mutations in each of the both Fc-scaffolds used. First, MabSelect SuRe™ ProteinA (GE Healthcare/Cytiva) as solid phase affinity ligand is used which binds with high binding capacity to the Fc domain of the bispecific scCD137L-Fc fusion protein. Briefly, the sterile filtered clarified cell culture supernatant/harvest is loaded on a HiTrap MabSelect SuRe column (CV=5 ml) which was equilibrated in wash buffer 1 (20 mM Pi, 95 mM NaCI, pH 7.2) not exceeding a load of 10 mg fusion protein per ml column volume. The column is washed with 10 column volumes (10 CV) of wash buffer 1 followed by at least four column volumes (4 CV) of wash buffer 2 (20 mM Pi, 95 mM NaCI, pH 8.0) to deplete host-cell proteins and host-cell DNA. Also the homodimeric contaminant which is lacking a protein A binding site is removed as it remains in the column flowthrough and does not bind to the column. After a series of washing steps, the protein is then eluted from the column with two column volumes at pH3.5 (e.g. 20 mM Pi, 95 mM NaCI, pH 3.5 or 50 mM NaAc, 150 mM NaCI, pH3,5). The eluate is collected in fractions and immediately neutralized with 1 M Tris-HCI pH 8.0 to neutral pH. The linear velocity is set to 150 cm/h and kept constant during the aforementioned affinity chromatography method.

In the case of the purification of the multispecific immune modulators comprising two different TNFSF-ligands, the heterodimeric fusion protein present in the eluate is polished by a combination of SEC and ion-exchange chromatography. The second affinity step for the purification of the multispecific immune modulators in the SAB format employs KappaSelect™ Resin (GE Healthcare/Cytiva) which binds the CL- kappa domain of the Fab domain of the bispecific CD137 agonist and depletes the homodimeric agonist Fc-fusion protein. Alternatively, the second affinity step employs Capture Select™ CH1-XL Resin (Thermo Scientific) which binds the CH1 domain of the Fab domain with high affinity. This also leads to the depletion of the homodimeric agonist Fc-fusion protein. The eluate of the first MabSelect SuRe™ ProteinA-based affinity chromatography is loaded either on the Capture Select CH1-XL (Thermo Scientific) or on KappaSelect Resin (GE Healthcare/Cytiva) (CV = 5 ml) equilibrated with wash buffer (PBS pH 7.4 = 10 mM Pi, 2.7 mM KCI, 140 mM NaCI), not exceeding 10 mg Fab per ml column volume. After a washing step with wash buffer (6 CV), the aforementioned bispecific CD137 agonist was eluted with 2 CV elution buffer (0.1 M glycine, pH 3.5) and immediately neutralized with 1 M Tris-HCI pH 8.0 to neutral pH (0.4 CV). The protein amount of eluate fractions was quantified by OD 280 measurements and concentrated by ultrafiltration for subsequent size exclusion chromatography (SEC).

For the affinity purification of the multispecific immune modulators of the Ab-scCD137L format, only the aforementioned CH 1 -based affinity purification is employed and the protein is polished by subsequent size exclusion chromatography.

Example 5: Purification of Strep-tagged CD137L-proteins

Proteins comprising a C-terminal Streptag (e.g. htCD137L-RB69) were purified by Streptactin-Sepharose based affinity chromatography as essentially described below: prior to purification, the cell culture supernatant was sterile filtrated and Biolock (IBA, 1 ml/l supernatant) was added. Streptactin matrix was washed and equilibrated to pH 7.4 with 10 column volumes (CV) of PBS. Cell culture supernatant was loaded, the flow through collected and unspecifically bound components were removed by washing with 10 CV PBS. Subsequently, elution of bound proteins followed with approximately 5 CV elution buffer (100 mM Arginine, 20 mM Tris, 2.5 mM Desthiobiotin; pH 7.4). The protein concentration of each elution fraction was measured (OD 280) and samples were stored below -20°C for further analytics and/or subsequent purification steps. Example 6: Size exclusion chromatography and subsequent analysis

Size exclusion chromatography (SEC) is performed on HiLoad 26/600 Superdex 200 pg or Superdex 200 Increase 10/300 GL columns (GE Healthcare/Cytiva) using an AKTA chromatography system. The columns are either equilibrated with phosphate buffered saline or an equivalent Tris based buffer system at neutral pH (pH 7.4).

The concentrated, affinity-purified protein is loaded onto the SEC column with the sample volume not exceeding 2% (v/v) of the column volume. A flow rate of 2.5 ml per min (HiLoad 26/600 Superdex 200 pg) or 0.5 ml per min (Superdex 200 Increase 10/300 GL) is applied and the elution profile monitored by absorbance at 280 nm. For determination of the apparent molecular weight of the purified protein under native conditions, the SEC columns are loaded with standard proteins of known molecular weight. Based on the elution volume of the standard proteins a calibration curve is plotted and the molecular weight of the purified protein is determined. The CD137 agonist fusion proteins (either the SAB-format or Fc-format) elute from the Superdex SEC columns with an apparent molecular weight of around 150-180 kDa, whereas the bispecific Ab-scCD137L-format elutes at an apparent molecular weight of around 100 kDa and the htCD137L-Rb69 format at 60-80 kDa. Fractions containing the purified fusion protein are pooled and concentrated. Finally, the concentrated purified fusion protein solution is stored at -20°C until further usage. HP-SEC, ELISA-based sandwich assays with both targets and TNFRSF reporter-cell based activity assays are used to determine the agonistic and/or bispecific nature of the aforementioned monospecific and bispecific TNFRSF agonists.

Example 7: Analytical size exclusion chromatography HP-SEC

Analytical high-performance size exclusion chromatography (HP-SEC) of protein samples was performed employing the HPLC 1260 Infinity device from Agilent. 20 pl of protein at a concentration of 1 mg/ml (20 pg protein in total) was injected to the column (TSKgel G3000SWxl, 250 A, 5pm, 7.8 mm ID x 30 cm length, Tosoh Bioscience GmbH) for each analytical SEC run. A buffer containing 200 mM Arginine, 20 mM TRIS, 109 mM Succinate, pH 7.2, was applied as running buffer with a flow rate of 0.5 ml/min. Peak heights and peak areas for the main peak as well as for HMWS and LMWS peaks were determined, and relative quantities were calculated. Example 8: Introduction of E128N glycosylation site increases expression titers in homotrimeric htCD137L and trivalent single-chain CD137L-Fc variants

In transient gene expression experiments, which directly monitor the folding kinetics of proteins during secretory pathway and which is measure for titer, the introduction of the surface glycosylation site E128N strongly enhances the expression titers in the homotrimeric CD137L variants (Figure 1 B, Figure 3) as well as in single-chain hexavalent scCD137L-Fc formats (Figure 1 A, Figure 6) up to ca. 9-fold. In addition, only E128N surface glycosylation as compared to other surface glycosylation sites tested, shows a complete and uniform glycosylation pattern and shift towards higher molecular weight (SDS-PAGE, Figure 3C) assuming that all E128N positions are accessible to be glycosylated inside the cell. Improved solubility of the protein and routing through the glycoprotein secretion pathway in the ER leads to increased expression titers and higher production yields. Also, the expression titer improvement by E128N based glycosylation can be already achieved by mutating only one of the three CD137L modules in the scCD137 L-Fc format (Figure 7).

Example 9: The introduction of intraprotomeric cysteine bridges increases stability and the degree of monomeric species

Wildtype CD137L has the tendency to form aggregates resulting in lower product titers during expression and purification processes. Therefore, we aimed to improve the CD137L module to be better suited for a production process with higher expression yields, higher degree of monomeric species, lower aggregation propensity and therefore increased production yields. This was achieved by the introduction of intraprotomeric cysteine bridges which are located in the hydrophobic core of the CD137L protein, and therefore not accessible for inter-protomer disulfide bridge formation or potential anti-drug antibodies thereby reducing the mutation related immunogenicity risks. We identified four potential positions to introduce functional cysteine bridges: A93C/L124C, A93C/L237C, V143C/L237C and V183C/G198C, and evaluated them in the homotrimeric CD137L format (Figure 4). Superior expression titers are observed for cysteine bridge variants A93C/L124C, V143C/L237C and V183C/G198C as compared to A93C/L237C. Furthermore, the addition of surface glycosylation E128N to cysteine bridge V143C/L237C increases expression titers by 3.4 fold (Figure 4). In addition to the expression titers, the cysteine bridges A93C/L124C, V143C/L237C and V183C/G198C are highly preferred in the homotrimeric CD137L format since these variants contain >95% monomeric species (Figure 5). Again, the disulfide bridge A93C/L237C seems to be disfunctional as it induces high levels of aggregation (Figure 5).

We further introduced the above mentioned cysteine bridges into the hexavalent scCD137L-Fc format in order to increase stability and monomer content (results depicted in Figure 9 to Figure 12 and Table 5). As expected, in the absence of a cysteine bridge, the monomer content lies between 71 % to 86% depending on the module configuration (Figure 9). However, the monomer content can be significantly increased by the incorporation of cysteine bridge V143C/L237C (Figure 10) or cysteine bridge V183C/G198C (Figure 11) up to >92% and both are highly preferred. Surprisingly, cysteine bridge A93C/L124C, which was functional and non-aggregating in the homotrimeric CD137L format, shows in the scCD137L-Fc format high tendency to aggregation and reduced levels of monomeric species (Figure 12).

When shortening the interprotomer linker from SEQ-ID: 44 to SEQ-ID: 46, the scCD137L- Fc constructs become less flexible and form higher levels of monomeric species, thus favoring a shorter interprotomer linker (Figure 9 to Figure 11). When combining the disulfide bridges V143C/L237C (Figure 10) or cysteine bridge V183C/G198C (Figure 11) with the E128N glycosylation site, the monomeric species content is increased as compared to variants without cysteine bridges, and equal or better as compared to the cysteine bridge alone and therefore these specific combinations are highly preferred.

Example 10: The introduction of intraprotomeric cysteine bridges and E128N glycosylation site increases stability and the degree of monomeric species in the SAB format

The improved properties of the scCD137L-Fc modules described in Example 9 were transferred to the SAB format which employs the same scCD137L module but is fused to a Hinge-Fc containing a heterodimerization domain to build up a bispecific format described in Figure 2A. Similar to the scCD137L-Fc format, in the SAB format the introduction of the E128N glycosylation site increases the monomeric species content in aPDL1-a-scCD137L-SAB variants which can even further be enhanced by the introduction of E128N together with the disulfide bridge V183C/G198C up to 99% monomeric species (Figure 13). Furthermore, also the disulfide bridge V143C/L237C shows a more stable protein with increased levels of monomeric species (Figure 14). As for the scCD137L-Fc format, also in the SAB format a shortening of the interprotomer linker from SEQ-ID:44 to SEQ-ID:46 increases the monomeric content due increased scCD137L stability in aPD-L1-a-scCD137L-SAB variants (Figure 13).

Finally, the applicability of the improved scCD137L modules (by introduction of E128N glycosylation site and the cysteine bridges V183C/G198C or V143C/L237C to increase monomeric species, stability and yield) was demonstrated in the SAB format. The observed improvements are is independent of the specific VH/VL domain composition of the antibody-arm. As shown in Figure 15, introduction of E128N+V183C/G198C using interprotomer linker variants (SEQ-ID:44, SEQ-ID:46) in the scCD137L module which is then coexpressed as bispecific SAB format (Figure 2A) shows a very high degree of monomeric species (92% - 99%) for two different anti-PD-L1 antibodie arms (aPDL1-a and aPDL1 -b) and an anti-HER2 antibody arm.

Table 1: Module composition of homotrimeric CD137L variants

Cysteine Surface SEQ-ID

Name N-Module C-Module bridge Glycosite

htCD137L-Var12 Q89 V240 V143C/L237C E128N SEQ-ID:14 ht = homotrimeric; CD137L annotation according to SEQ-ID:01

Table 2: N-and C-terminal residues of CD137L modules for generation of htCD137L-variants and scCD137L-variants.

For scCD137L-variants, a combination of different modules can be used which are connected via interprotomer linkers. CD137L annotation according to SEQ-ID:01.

Table 3: Module composition of trimeric single-chain CD137L variants

.. N-term C-term N-term C-term Interprotomer Cysteine Surface a^me Modulel Modulel Module2/3 Module2/3 linker bridge Glycosite

sc = single-chain trimer; N-term = N-terminus; C-term = C-terminus; CD137L annotation according to SEQ-ID:01

Three modules (Modi , Mod2, Mod3) are connected by two interprotomer linkers to build up one singlechain trimer. E128N surface glycosylation is present in each module of the single-chain trimer in Var2, Var4, Var6,

Var8, Var10, Var12, Var14, Var16, Vari 8, Vari 9, Var21 and Var23. For Var25 and Var26, E128N surface glycosylation is only present in one module of the single-chain trimer.

Table 4: Sequences of the invention

Table 5: Aggregate content of scCD137L-Fc Variants is influenced by the absence or presence of disulfide bridges.

The disulfide bridges V143C/L237C or V183C/G198C stabilize the scCD137L-Fc variants leading to high degree of monomeric species. scCD137L-Variant- Disulfide Interprotomer Surface % HMWS % Monomer

Fc bridge linker glycosylation

E128N

HMWS = High-molecular weight species; CD137L annotation according to SEQ-ID:01

Table 6: Preferred scCD137L-mutein-Fc fusion proteins of the invention

Preferred scCD137L-mutein-Fc fusion proteins of the invention and their composition are listed in Table6. From left to right, 5 their three different functional components (as listed in Table 3 and Table 4), their unique SEQ:ID (in Table 4) as well as the resulting individual names are shown. The features of the individual scCD137L-Variants can be found in Table 3.

Table 7: Preferred scCD137L-muteins to construct the scCD137L-Fc-knob chain for bispecific Ab-scCD137L-SAB protein assemblies

Claims

Claims

1. An N-terminally and C-terminally truncated CD137L mutein of SEQ-ID:01 , wherein the CD137L starts at amino acid position Q89, G90, or M91 and ends at amino acid position V240, T241 , P242, or E243.

2. A CD137L mutein of claim 1 , wherein the CD137L mutein comprises additional N- glycosylations on the outer surface.

3. A CD137L mutein of claim 2, wherein the N-glycosylation on the outer surface is selected from the group consisting of E128N, D129NG, Q227N and I103N.

4. A CD137L mutein of claim 2, wherein the mutein is N-glycosylated at position E128N.

5. A CD137L mutein of claim 1 , wherein the CD137L mutein comprises additional cysteine bridges.

6. A CD137L mutein of claim 5, wherein the cysteine bridges are formed between positions V183C and G198C and/or between positions V143C and L237C.

7. A CD137L mutein of claim 5, wherein a cysteine bridge is formed between positions V143C and L237C

8. A CD137L mutein of claim 5, wherein the cysteine bridges are formed between positions V183C and G198C and/or between positions V143C and L237C and wherein the mutein comprises N-glycosylations on the outer surface.

9. A CD137L mutein of claim 8, wherein the N-glycosylations are selected from the group consisting of E128N, D129NG, Q227N and I103N.

10. A CD137L mutein of claim 9, wherein the mutein is N-glycosylated at position E128N.

11 . A fusion protein comprising a CD137L mutein of any one of claims 1 -9.

12. A fusion protein of claim 11 , comprising three CD137L muteins in a homotrimeric configuration.

13. A fusion protein of claim 11 , comprising CD137L muteins in a single-chain

63 configuration.

14. A fusion protein of claim 13, comprising three CD137L muteins in a single-chain configuration.

15. A fusion protein of claim 14, comprising three linker-separated CD137L muteins, wherein the length of the linkers is selected from linkers having 1-12 amino acids.

16. A fusion protein of claim 15, comprising a dimerization domain

17. A bispecific CD137L protein assembly comprising at least

(a) a trimeric single-chain CD137L mutein domain fused to

(b) a first peptide linker fused to

(c) a first hetero-dimerization domain and (d) an antigen binding or interacting protein moiety fused to

(e) a second peptide linker fused to

(f) a second hetero-dimerization domain

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