WO2023088889A1 - Ligand cd137 - Google Patents

Ligand cd137 Download PDF

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WO2023088889A1
WO2023088889A1 PCT/EP2022/081974 EP2022081974W WO2023088889A1 WO 2023088889 A1 WO2023088889 A1 WO 2023088889A1 EP 2022081974 W EP2022081974 W EP 2022081974W WO 2023088889 A1 WO2023088889 A1 WO 2023088889A1
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cd137l
sccd137l
mutein
seq
domain
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PCT/EP2022/081974
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Katharina BILLIAN-FREY
Oliver Hill
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Apogenix Ag
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • 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.
  • TNFSF tumor necrosis factor superfamily
  • TNFRSF TNF receptor superfamily
  • 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.
  • TNFRSF TNF receptor superfamily
  • CD137L fusion proteins of PCT/EP2021/063005 and PCT/EP2016/075543 are well suited for therapeutic use.
  • 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.
  • 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.
  • infectious diseases e.g. bacterial infections
  • metabolic diseases e.g. bacterial infections
  • 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 e.g. pulmonary embosis.
  • FIG 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).
  • FIG. 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.
  • FIG. 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.
  • FIG. 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).
  • FIG. 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.
  • 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).
  • FIG 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).
  • 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).
  • 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).
  • SEQ-ID: 44 and SEQ-ID: 46 interprotomer linkers
  • 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).
  • 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%).
  • the degree of monomeric species of the 2-Step affinity-purified eluate 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.
  • 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).
  • 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).
  • the degree of monomeric species of the 2-Step affinity-purified eluate 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • hydrophobic exchange mutations can be introduced.
  • 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 (2016) (Nat Commun.
  • amino acid sequences were chosen that potentially could be converted into the necessary consensus sequence NXS/T of a glycosylation site.
  • suitable mutations of SEQ-ID:01 are E128N; D129NG; Q227N; E156N; I103N in combination with G105S, or combinations thereof.
  • the CD137L mutein of SEQ-ID:01 comprises the E128N amino acid exchange to form a functional glycosylation site on the protein surface.
  • covalent disulfide bridges can stabilize the spatial structure of proteins.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • amino acids pairs selected for cysteine mutations are A93/L124, A93C/L237C, V143/L237 and V183/G198 of SEQ-ID:01 .
  • cysteine bridge is formed between amino acid positions 143 und 237 of SEQ-ID:01 ; both positions mutated to cysteine (V143C and L237C).
  • 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 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.
  • the CD137L mutein comprises the mutations V143C and L237C and E128N of SEQ-ID:01.
  • the CD137L mutein comprises the mutations V183C and G198C and E128N of SEQ-ID:01.
  • the CD137L mutein comprises the mutations V143C and L237C and V183C and G198C and E128N of SEQ-ID:01.
  • 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
  • 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).
  • 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.
  • 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.
  • 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).
  • 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.
  • Such single-chain CD137L modules combine advantageous linker lengths (in particular 1-12 amino acids) with outer surface glycosylation and/or stabilizing cysteine bridging.
  • Fusion proteins comprising above mentioned CD137L muteins can be employed for the construction of homomeric and/or heteromeric protein 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.
  • 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.
  • 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.
  • the bispecific CD137L protein assembly comprises at least
  • FIG. 2A A general overview of a CD137L fusion protein assembly of the invention is given 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.
  • 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).
  • the multi-specific CD137L fusion protein assembly comprises at least
  • FIG. 1A A general overview of a multi-specific TNFSF fusion protein assembly of this aspect of the invention is given 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.
  • 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.
  • Figure 1A second single-chain CD137L fusion protein
  • the multi-specific CD137L fusion protein assembly comprises at least
  • 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.
  • Ab-scCD137L multispecific immune-Modulator of the invention combines CD137 agonism with anti-PD-L1 targeting.
  • 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 VHH-based targeting of scCD137L muteins of the invention
  • the multi-specific CD137L superfamily fusion protein assembly comprises at least
  • VHH VH derived single domain antibodies
  • single chain TNF-SF receptor binding domain 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.
  • 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.
  • the functional antibody fragment is directed against a cell surface marker or an activity-modulating target.
  • 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),
  • CEA Carcinoembry
  • 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).
  • 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 Gi
  • 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.
  • 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 Inter
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • TNF tumor necrosis factor
  • 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.
  • TNF homology domain TNF homology domain
  • 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).
  • TNF-SF Receptor-binding-domain RBD
  • 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.
  • CD137L is used for the trivalent receptor binding domain unit formed by three individual polypeptides and htCD137L or (ht)CD137L are its common abbreviations.
  • single-chain CD137L receptor binding domain single-chain CD137L are used synonymously for the trivalent but single polypeptide based CD137L receptor binding domains.
  • single-chain is often abbreviated as ‘sc’, e.g. scCD137L-RBD.
  • 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.
  • single-chain CD137L receptor binding domain 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.
  • single-chain is often abbreviated as ‘sc’, e.g. scCD137L-RBD.
  • 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.
  • 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 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.
  • 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.
  • CH3-domain derived dimerization technologies For the generation of multi-specific immune modulators of the current invention, the usage of CH3-domain derived dimerization technologies is highly preferred.
  • the CH3 domains implemented into both fusion protein polypeptides is a natural occurring sequence.
  • the CH3 domains comprise point mutations, which are intended to stabilize the current dimerization product.
  • 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.
  • the CH3 domains comprise point mutations which preferentially lead to heterodimer formation during protein expression, e.g. knobs into hole (KiH) technology.
  • KiH knobs into hole
  • 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.
  • VH variable region
  • CH2 constant domains
  • 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.
  • 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.
  • 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.
  • VL variable region
  • CL light chain constant domain
  • 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).
  • hybrid light chain formats can be engineered comprising lambda VL and kappa CL, and vice versa.
  • a light chain is based on a kappa LC or a hybrid LC composed of VLIambda/CLkappa for improved solubility and faster folding kinetics.
  • a typical CL kappa domain used in the context of the invention is SEQ-ID:95.
  • 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 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.
  • 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).
  • scFv single domain antibodies
  • 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).
  • 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).
  • the term single domain antibody includes an autonomous human heavy chain variable domain (aVH) or VNAR fragments derived from sharks.
  • 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 and scFv 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.
  • CL light chain
  • 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 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.
  • 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.
  • 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.
  • a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain.
  • the C-terminal lysine (Lys447) of the Fc region may or may not be present.
  • 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.
  • 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.
  • a knob modification comprises the amino acid substitution T366W in one of the two subunits of the Fc domain
  • the hole modification comprises the amino acid substitutions T366S, L368A and Y407V in the other one of the two subunits of the Fc domain.
  • the subunit of the Fc domain comprising the knob modification additionally comprises the amino acid substitution S354C
  • the subunit of the Fc domain comprising the hole modification additionally comprises the amino acid substitution Y349C.
  • 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).
  • allelic variants of the Fc region of an immunoglobulin e.g. D356E/L358M
  • 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.
  • 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 bonds usually can be formed between spatially proximal cysteine residues in proteins and peptides and contribute to their overall molecular architecture.
  • 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:
  • 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.
  • synthetic DNA cassettes encoding the necessary polypeptides are inserted into eukaryotic expression vectors comprising appropriate genetic elements suitable to transiently express the protein of interest.
  • 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.
  • 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.
  • 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.
  • the appropriate plasmid DNAs 1.5 pg / 1 x 10E6 cells/ml
  • linear PEI 25000 2.5 pg / 1 x 10E6 cells/ml
  • Example 2 Method for large scale expression and purification of recombinant monospecific and multispecific/bispecific CD137L fusion protein assemblies
  • synthetic DNA cassettes encoding the necessary polypeptides 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).
  • appropriate selection markers e.g. a functional expression cassette comprising a blasticidin, puromycin, hygromycin orzeocin resistance gene
  • 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).
  • MAR human p-globin matrix attachment region
  • 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% CO 2 atmosphere in an orbital shaker incubator (100 rpm, 50 mm shaking throw), the individual supernatants are analyzed by ELISA assays detecting the aforementioned proteins.
  • chemically defined medium PowerCHO-2 CD, Lonza, supplemented with 4 mM glutamine/glutamax
  • Cell pools with the highest specific productivity are expanded in shake flasks for protein production (orbital shaker, 100 rpm, shaking throw 50 mm).
  • 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% CO 2 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% CO 2 , 36.5 °C.
  • the cell culture is fed twice with PowerFeed A (Lonza) with Lipids usually on day 3 (20 % feed) and on day 6 (30 % 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 m 2 ), 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.
  • a depth filtration system Millipore Millistak Pod MC0HC 0.054 m 2
  • PES bottle top filter
  • scCD137L-Fc-based immune modulators 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 SuReTM 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.
  • 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.
  • the protein is then eluted from the column with two column volumes of a suitable elution buffer at pH3.5 (e.g.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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 KappaSelectTM 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.
  • the second affinity step employs Capture SelectTM CH1-XL Resin (Thermo Scientific) which binds the CH1 domain of the Fab domain with high affinity.
  • 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).
  • 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.
  • a C-terminal Streptag e.g. htCD137L-RB69
  • Size exclusion chromatography 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.
  • 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.
  • HP-SEC high-performance size exclusion chromatography
  • 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.
  • 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).
  • 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).
  • the monomer content lies between 71 % to 86% depending on the module configuration ( Figure 9).
  • 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.
  • 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).
  • 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.
  • 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).
  • disulfide bridge V143C/L237C shows a more stable protein with increased levels of monomeric species ( Figure 14).
  • 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).
  • Table 2 N-and C-terminal residues of CD137L modules for generation of htCD137L-variants and scCD137L-variants.
  • scCD137L-variants a combination of different modules can be used which are connected via interprotomer linkers.
  • Glycosite sc single-chain trimer
  • N-term N-terminus
  • C-term C-terminus
  • Var8 Var10, Var12, Var14, Var16, Vari 8, Vari 9, Var21 and Var23.
  • E128N surface glycosylation is only present in one module of the single-chain trimer.
  • 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.
  • HMWS High-molecular weight species
  • 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

Abstract

L'invention concerne des fractions de ligand CD137 présentant des propriétés protéiques améliorées. Les ligands de l'invention présentent une solubilité et une stabilité de pH accrues, ainsi qu'une cinétique de repliement améliorée, conduisant à des titres d'expression plus élevés pendant la production dans des cellules eucaryotes. Les ligands CD137 de l'invention sont bien appropriés pour la construction de protéines de fusion CD137L multivalentes thérapeutiques employant un assemblage homotrimérique ou à chaîne unique des protomères CD137L. L'invention concerne en outre des acides nucléiques et des cellules hôtes transfectées pour la production desdites protéines de fusion CD137L améliorées.
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