Multivalent and multispecific ox40-binding fusion proteins

Abstract

To provide a more potent agonist molecule of TNFRSF.SOLUTION: This invention relates generally to molecules that specifically engage OX40, a member of the TNF receptor superfamily (TNFRSF). More specifically, this invention relates to multivalent and multispecific molecules that bind at least OX40.SELECTED DRAWING: Figure 5

Description

Technical Field

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 277,027, filed on Jan. 11, 2016. The entire contents of each of the provisional applications are hereby incorporated by reference in their entirety.

[0002] The present invention generally relates to molecules that specifically engage OX40, i.e., a member of the Tumor Necrosis Factor Receptor Superfamily (TNFRSF). More specifically, the present invention relates to multivalent multispecific molecules that bind at least to OX40.

Background Art

[0003] The Tumor Necrosis Factor Receptor Superfamily consists of several structurally related cell surface receptors. Activation by multimeric ligands is a common feature of many of these receptors. Many members of the TNFRSF, when appropriately activated, have therapeutic utility in a very wide variety of pathologies. Agonism of this receptor family often requires higher order clustering and conventional bivalent antibodies are not suitable for this. Thus, there is a therapeutic need for more potent TNFRSF agonist molecules.

Summary of the Invention

[0004] The present disclosure provides a multivalent multispecific TNF receptor superfamily (TNFRSF) binding fusion polypeptide that binds to at least OX40 (also known as tumor necrosis factor receptor superfamily, member 4 (TNFRSF4) and / or CD134). The use of the term "OX40" is intended to encompass any variant thereof, for example, by way of non-limiting example, OX-40, and all variants are used interchangeably herein. These molecules that bind to at least OX40 are referred to herein as "OX40-targeting molecules" or "OX40-targeting fusions" or "OX40-targeting proteins" or "OX40-targeting fusion polypeptides" or "OX40-targeting fusion proteins". In some embodiments, the OX40-targeting molecule is a multivalent molecule, for example, a multivalent OX40-targeting fusion protein. In some embodiments, the OX40-targeting molecule is a multispecific molecule, for example, a multispecific OX40-targeting fusion protein. In some embodiments, the OX40-targeting molecule is a multivalent multispecific molecule, for example, a multivalent multispecific OX40-targeting fusion protein. As used herein, the terms "fusion protein" or "fusion polypeptide" or "OX40-targeting fusion protein" or "OX40-targeting fusion polypeptide" refer to any fusion protein embodiment of the present disclosure, including but not limited to multivalent fusion proteins, multispecific fusion proteins, or multivalent multispecific fusion proteins, unless otherwise specified.

[0005] The present disclosure also provides a multivalent multispecific fusion polypeptide that binds to at least programmed death ligand 1 (PDL1), also known as PD-L1, CD274, B7 homolog 1, and / or B7-H1. The use of the term "PDL1" is intended to encompass any variations thereof, for example, by way of non-limiting example, PD-L1 and / or PDL-1, and all variations are used interchangeably herein. These molecules that bind to at least PDL1 are referred to herein as "PDL1-targeting molecules" or "PDL1-targeting fusions" or "PDL1-targeting proteins" or "PDL1-targeting fusion polypeptides" or "PDL1-targeting fusion proteins". In some embodiments, the PDL1-targeting molecule is a multivalent molecule, for example, a multivalent PDL1-targeting fusion protein. In some embodiments, the PDL1-targeting molecule is a multispecific molecule, for example, a multispecific PD L1-targeting fusion protein. In some embodiments, the PDL1-targeting molecule is a multivalent multispecific molecule, for example, a multivalent multispecific PDL1-targeting fusion protein. As used herein, the terms "fusion protein" or "fusion polypeptide" or "PDL1-targeting fusion protein" or "PDL1-targeting fusion polypeptide" refer to any fusion protein embodiment of the present disclosure, including but not limited to multivalent fusion proteins, multispecific fusion proteins, or multivalent multispecific fusion proteins, unless otherwise specified.

[0006] The present disclosure also provides a multivalent multispecific fusion polypeptide that binds to at least PDL1 and OX40. These molecules that bind to at least PDL1 are referred to herein as "PDL1xOX40 targeting molecules" or "PDL1xOX40 targeting fusions" or "PDL1xOX40 targeting proteins" or "PDL1xOX40 targeting fusion polypeptides" or "PDL1xOX40 targeting fusion proteins". In some embodiments, the PDL1xOX40 targeting molecule is a multivalent molecule, such as a multivalent PDL1xOX40 targeting fusion protein. In some embodiments, the PDL1xOX40 targeting molecule is a multispecific molecule, such as a multispecific PDL1xOX40 targeting fusion protein. In some embodiments, the PDL1xOX40 targeting molecule is a multivalent multispecific molecule, such as a multivalent multispecific PDL1 targeting fusion protein. As used herein, the terms "fusion protein" or "fusion polypeptide" or "PDL1xOX40 targeting fusion protein" or "PDL1xOX40 targeting fusion polypeptide" refer to any fusion protein embodiment of the present disclosure including, but not limited to, multivalent fusion proteins, multispecific fusion proteins, or multivalent multispecific fusion proteins, unless otherwise specified.

[0007] Conventional antibodies targeting members of the TNF receptor superfamily (TNFRSF) have been shown to require exogenous cross-linking to achieve sufficient agonist activity, as is evident from the need for Fc gamma receptors (FcγR) for the activity of antibodies against DR4, DR5, GITR, and OX40 (Ichikawa et al 2001 al Nat.Med.7,954-960,Li et al 2008 Drug Dev. Res.69,69-82, Pukac et al 2005 Br.J.Cancer 92, 1430-1441, Yanda et al 2008 Ann.Oncol.19, 1060-1067, Yang et al 2007 Cancer Lett. 251:146 - 157, Bulliard et al 2013 JEM 210(9):1685, Bulliard et al 2014 (Immunol and Cell Biol 92:475 - 480). In addition to cross - linking via FcγR, other extrinsic factors including the addition of oligomeric ligands or antibody - binding entities (e.g., protein A and secondary antibodies) have been demonstrated to enhance the clustering and downstream signaling of anti - TNFRSF antibodies. For example, the addition of the DR5 ligand TRAIL enhanced the apoptosis - inducing ability of anti - DR5 antibodies (Graves et al 2014 Cancer Cell 26:177 - 189). These findings suggest the need for clustering beyond dimer formation of TNFRSF.

[0008] The present disclosure provides a multivalent TNFRSF - binding fusion protein comprising two or more TNFRSF - binding domains (TBDs), wherein at least one TBD binds to OX40. In some embodiments, the fusion protein of the present disclosure is useful for the treatment of neoplasms.

[0009] In some embodiments, the fusion protein comprises two or more different TBDs, each of which binds to OX40. In some embodiments, the fusion protein comprises multiple copies of a TBD that binds to OX40. For example, in some embodiments, the fusion protein comprises at least two copies of a TBD that binds to OX40. In some embodiments the fusion protein comprises at least three copies of a TBD that binds to OX40. In some embodiments, the fusion protein comprises at least four copies of a TBD that binds to OX40. In some embodiments, the fusion protein comprises at least five copies of a TBD that binds to OX40. In some embodiments, the fusion protein comprises at least six copies of a TBD that binds to OX40. In some embodiments, the fusion protein comprises six or more copies of a TBD that binds to OX40.

[0010] In other embodiments, the fusion proteins of the present disclosure bind to OX40 as well as a second TNFRSF member, such as GITR, CD137, CD27, TNFR2, and / or CD40. In these embodiments, the fusion proteins of the present disclosure regulate immune cells that lead to enhanced tumor destruction. In other embodiments, the fusion proteins of the present disclosure are useful for treating inflammatory conditions. In these embodiments, the fusion proteins of the present disclosure regulate immune cells that lead to suppression of inflammatory injury. For example, by specifically stimulating TNFR2, the proliferation of Tregs that leads to immunosuppression can be enhanced.

[0011] The fusion proteins of the present disclosure can enhance the clustering of TNFRSF members as compared to non-crosslinked bivalent antibodies. The enhanced clustering of TNFRSF members mediated by the fusion proteins of the present disclosure induces enhanced TNFRSF-dependent signaling as compared to non-crosslinked bivalent antibodies. In most embodiments, the fusion protein incorporates more than two TBDs, such as three, four, five, or six.

[0012] In some embodiments, the fusion protein is multispecific and comprises a binding domain for a TBD and a second antigen. In these embodiments, by binding to the second antigen, an additional crosslinking function can be provided, and TNFRSF activation can be achieved with only one or two TBDs. In these embodiments, TNFRSF signaling is enhanced and concentrated by the presence of the second antigen. These multispecific TBD-containing fusion proteins are useful means for achieving conditional signaling of a given TNFRSF member.

[0013] The present disclosure provides an isolated polypeptide that specifically binds to OX40. In some embodiments, the isolated polypeptide is a scFv, Fab, single domain antibody (sdAb), V NARor derived from an antibody or antibody fragment including VHH. In some embodiments, the isolated polypeptide is a human or humanized sdAb. The sdAb fragment may be derived from VHH, V NAR , or an artificially created VH or VK domain. VHH can be generated from the heavy-chain-only antibodies of camelids. V NAR can be generated from the heavy-chain-only antibodies of cartilaginous fish. Various methods have been implemented to generate monomeric sdAbs from the conventional heterodimeric VH and VK domains, including interface engineering and the selection of specific germline families. In other embodiments, the isolated polypeptide is derived from a non-antibody scaffold protein, such as, but not limited to, designed ankyrin repeat proteins (darpins), avimers, anticalins / lipocalins, centyrins, and fibronomers.

[0014] In some embodiments, the isolated polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-29 and 377-386.

[0015] In some embodiments, the isolated polypeptide comprises an amino acid sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-29 and 377-386.

[0016] In some embodiments, the isolated polypeptide comprises a complementarity-determining region 1 (CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 36, and 44, a complementarity-determining region 2 (CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 35, and 37, and a complementarity-determining region 3 (CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-34, 48-43, and 45.

[0017] The present disclosure provides a multivalent fusion protein, which comprises two or more binding domains (BDs), and at least one BD binds to PDL1. In some embodiments, the fusion protein of the present disclosure is useful for the treatment of neoplasms.

[0018] In some embodiments, the fusion protein comprises two or more different BDs, and each BD binds to PDL1. In some embodiments, the fusion protein comprises multiple copies of the BD that binds to PDL1. For example, in some embodiments, the fusion protein comprises at least two copies of the BD that binds to PDL1. In some embodiments, the fusion protein comprises at least three copies of the BD that binds to PDL1. In some embodiments, the fusion protein comprises at least four copies of the BD that binds to PDL1. In some embodiments, the fusion protein comprises at least five copies of the BD that binds to PDL1. In some embodiments, the fusion protein comprises at least six copies of the BD that binds to PDL1. In some embodiments, the fusion protein comprises six or more copies of the BD that binds to PDL1.

[0019] The present disclosure provides an isolated polypeptide that specifically binds to OX40. In some embodiments, the isolated polypeptide is derived from an antibody or antibody fragment including scFv, Fab, single domain antibody (sdAb), V NAR , or VHH. In some embodiments, the isolated polypeptide is a human or humanized sdAb. The sdAb fragment may be derived from VHH, V NAR , an artificially created VH or VK domain. VHH can be generated from the heavy-chain-only antibodies of camelids. V NARcan be generated from antibodies with only heavy chains of cartilaginous fish. Conventionally, various methods have been implemented to generate monomeric sdAbs from heterodimeric VH and VK domains, including interface engineering and the selection of specific germline families. In other embodiments, the isolated polypeptide is derived from a non-antibody scaffold protein, such as, but not limited to, designed ankyrin repeat proteins (darpins), avimers, anticalins / lipocalins, centyrins, and fibronomers.

[0020] In some embodiments, the isolated polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 46-57. In some embodiments, the isolated polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 52-57.

[0021] In some embodiments, the isolated polypeptide comprises an amino acid sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 46-57. In some embodiments, the isolated polypeptide comprises an amino acid sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 52-57.

[0022] In some embodiments, the isolated polypeptide comprises a complementarity-determining region 1 (CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 58, 61, and 64, a complementarity-determining region 2 (CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 59, 62, 65, and 69, and a complementarity-determining region 3 (CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 60, 63, 66-68, and 70.

[0023] In some embodiments, the present disclosure provides an isolated polypeptide that specifically binds to at least OX40 and PDL1. In some embodiments, each binding domain (BD) in the isolated polypeptide is derived from an antibody or antibody fragment, including scFv, Fab, single domain antibody (sdAb), V NAR , or an antibody or antibody fragment including VHH. In some embodiments, each BD is a human or humanized sdAb. The sdAb fragment may be derived from VHH, V NAR , an artificially created VH or VK domain. VHH can be generated from the heavy-chain-only antibodies of camelids. V NAR can be generated from the heavy-chain-only antibodies of cartilaginous fish. Various methods have been implemented to generate monomeric sdAbs from the conventional heterodimeric VH and VK domains, including interface engineering and the selection of specific germline families. In other embodiments, the isolated polypeptide is derived from a non-antibody scaffold protein, such as, but not limited to, designed ankyrin repeat protein (darpin), avimer, anticalin / lipocalin, centyrin, and fibronomer.

[0024] In some embodiments, the isolated polypeptide includes a first amino acid sequence that binds to 4B11 selected from the group consisting of SEQ ID NOs: 16-29 and 377-386, and a second amino acid sequence that binds to PDL1 selected from the group consisting of SEQ ID NOs: 46-57.

[0025] In some embodiments, the isolated polypeptide includes a first amino acid sequence that binds to 4B11 selected from the group consisting of SEQ ID NOs: 16-29 and 377-386, and a second amino acid sequence that binds to PDL1 selected from the group consisting of SEQ ID NOs: 52-57.

[0026] In some embodiments, the isolated polypeptide comprises an amino acid sequence that binds to 4B11 selected from the group consisting of SEQ ID NOs: 16-29 and 377-386, a first amino acid sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, and an amino acid sequence that binds to PDL1 selected from the group consisting of SEQ ID NOs: 46-57, a second amino acid sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical.

[0027] In some embodiments, the isolated polypeptide comprises an amino acid sequence that binds to 4B11 selected from the group consisting of SEQ ID NOs: 16-29 and 377-386, a first amino acid sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, and an amino acid sequence that binds to PDL1 selected from the group consisting of SEQ ID NOs: 52-57, a second amino acid sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical.

[0028] In some embodiments, the isolated polypeptide comprises (i) complementarity determining region 1 (CDR1) that binds to 4B11 and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 36, and 44, complementarity determining region 2 (CDR2) that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 35, and 37, and complementarity determining region 3 (CDR3) that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-34, 48-43, and 45 a first amino acid sequence, and (ii) a second amino acid sequence comprising a CDR1 that binds to PDL1 and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 58, 61, and 64, a CDR2 that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 59, 62, 65, and 69, and a CDR3 that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 60, 63, 66 - 68, and 70.

[0029] In some embodiments, the binding domain (BD) of the present disclosure is derived from an antibody or antibody fragment including, but not limited to, scFv, Fab, single domain antibody (sdAb), V NAR , or VHH. In some embodiments, the BD is a human or humanized sdAb. The sdAb fragment may be derived from VHH, V NAR , an artificially created VH or VK domain. VHH can be generated from the heavy chain only antibodies of camelids. V NAR can be generated from the heavy chain only antibodies of cartilaginous fish. Various methods have been implemented to generate monomeric sdAbs from the conventional heterodimeric VH and VK domains, including interface engineering and the selection of specific germline families. In other embodiments, the BDB is derived from a non - antibody scaffold protein, such as, but not limited to, designed ankyrin repeat protein (darpin), avimer, anticalin / lipocalin, centyrin, and fibronomer.

[0030] Generally, the fusion proteins of the present disclosure consist of at least two or more BDs operably linked via a linker polypeptide. The use of sdAb fragments as specific BDs within the fusion proteins of the present disclosure has the advantage of avoiding the heavy chain: light chain mispairing problems common to many bispecific / multispecific antibody approaches. Furthermore, the fusion proteins of the present disclosure avoid the use of long linkers required by many bispecific antibodies.

[0031] In some embodiments, all of the TBDs of the fusion protein recognize the same epitope on a given TNFRSF member. For example, the fusion proteins of the present disclosure may incorporate two, three, four, five, or six TBDs with the same specificity for OX40. In other embodiments, the fusion protein incorporates TBDs that recognize different epitopes on a given TNFRSF member. For example, the fusion proteins of the present disclosure may incorporate two, three, four, five, or six TBDs with different recognition specificities for different epitopes on OX40. In these embodiments, the fusion proteins of the present disclosure include multiple TBDs that target different regions of a specific TNFRSF member. In some embodiments, the TBDs may recognize different epitopes on the same TNFRSF member or on different TNFRSF members. For example, the present disclosure provides multispecific fusion proteins that incorporate TBDs that bind to GITR and OX40 or CD137 and OX40.

[0032] In some embodiments, all of the BDs of the fusion protein recognize the same epitope on PDL1. For example, the fusion proteins of the present disclosure may incorporate two, three, four, five, or six BDs with the same specificity for PDL1. In other embodiments, the fusion protein incorporates BDs that recognize different epitopes on PDL1. For example, the fusion proteins of the present disclosure may incorporate two, three, four, five, or six BDs with different recognition specificities for different epitopes on PDL1. In these embodiments, the fusion proteins of the present disclosure include multiple BDs that target different regions of PDL1. In some embodiments, the BDs may recognize different epitopes on PDL1.

[0033] In some embodiments, the multispecific fusion protein is a bispecific molecule that targets OX40 and PDL1 and includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 387-394.

[0034] In some embodiments, the multispecific fusion protein targets OX40 and PDL1 and is a bispecific molecule comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 387-394 and an amino acid sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical.

[0035] In some embodiments, the fusion protein of the present disclosure consists of a single polypeptide. In other embodiments, the fusion protein of the present disclosure consists of multiple polypeptides. For example, in that case, a heterodimerization domain is incorporated into the fusion protein to construct an asymmetric fusion protein. For example, when an immunoglobulin Fc region is incorporated into the fusion protein, the CH3 domain may be used as a homodimerization domain, or the CH3 dimer interface region may be mutated to enable heterodimerization.

[0036] In some embodiments, the fusion protein contains BDs at both ends. For example, the BDs are located at both the amino-terminal (N-terminal) portion and the carboxy-terminal (C-terminal) portion of the fusion protein. In other embodiments, all TBDs are present at the same end of the fusion protein. For example, the BD is present at either the amino or carboxy-terminal portion of the fusion protein.

[0037] In some embodiments, the linker polypeptide contains an immunoglobulin Fc region. In some embodiments, the immunoglobulin Fc region is an IgG isotype selected from the group consisting of the IgG1 isotype, IgG2 isotype, IgG3 isotype, and IgG4 isotype.

[0038] JPEG2025081649000002.jpg54168

[0039] In some embodiments, the immunoglobulin Fc region or an immunologically active fragment thereof comprises a human IgG1 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1.

[0040] In some embodiments, the Fc region of the human IgG1 is modified at amino acid Asn297 (boxed in SEQ ID NOs: 1-4, Kabat numbering), for example, Asn297Ala (N297A) or Asn297Asp (N297D), to prevent glycosylation of the fusion protein. In some embodiments, the Fc region of the fusion protein is modified at amino acid Leu235 (bold in SEQ ID NO: 1, Kabat numbering), for example, Leu235Glu (L235E) or Leu235Ala (L235A), to alter Fc receptor interaction. In some embodiments, the Fc region of the fusion protein is modified at amino acid Leu23 Modified at 4 (bold in Sequence No. 1, Kabat numbering), for example, at amino acid Leu234 (boxed, Kabat numbering) to modify, for example, Fc receptor interaction, e.g., to Leu235Glu (L235E). In some embodiments, the Fc region of the fusion protein is Leu234Ala (L234A). In some embodiments, the Fc region of the fusion protein is modified at both amino acids 234 and 235, e.g., to Leu234Ala and Leu235Ala (L234A / L235A) or Leu234Val and Leu235Ala (L234V / L235A). In some embodiments, the Fc region of the fusion protein lacks amino acids at one or more of the following positions to reduce Fc receptor binding: Glu233 (E233, bold in Sequence No. 1), Leu234 (L234), or Leu235 (L235). In some embodiments, the Fc region of the fusion protein is modified at Gly235 to reduce Fc receptor binding. For example, in that case, Gly235 is deleted from the fusion protein. In some embodiments, the Fc region of the human IgG1 is modified at amino acid Gly236 (boxed in Sequence No. 1), e.g., to Gly236Ala (G236A), to enhance the interaction with CD32A. In some embodiments, the Fc region of the human IgG1 lacks Lys447 (EU index of Kabat et al 1991 Sequences of Proteins of Immunological Interest).

[0041] In some embodiments, the Fc region of the fusion protein is modified at one or more of the following positions to reduce Fc receptor binding: Leu234 (L234), Leu235 (L235), Asp265 (D265), Asp270 (D270), Ser298 (S298), Asn297 (N297), Asn325 (N325), or Ala327 (A327). For example, Leu234Ala (L234A), Leu235Ala (L235A), Asp265Asn (D265N), Asp270Asn (D270N), Ser298Asn (S298N), Asn297Ala (N297A), Asn325Glu (N325E), or Ala327Ser (A327S). In a preferred embodiment, the modification within the Fc region reduces binding to the Fc receptor gamma receptor while having little effect on binding to the neonatal Fc receptor (FcRn).

[0042] JPEG2025081649000003.jpg62168

[0043] In some embodiments, the immunoglobulin Fc region or an immunologically active fragment thereof comprises a human IgG1 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 2.

[0044] JPEG2025081649000004.jpg51169

[0045] In some embodiments, the fusion or an immunologically active fragment thereof comprises a human IgG2 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 3.

[0046] In some embodiments, the Fc region of the human IgG2 is modified at amino acid Asn297 (boxed in SEQ ID NOs: 1, 3, 4, and 5), for example, to Asn297Ala (N297A), to prevent glycosylation of the antibody. In some embodiments, the Fc region of the human IgG2 lacks Lys447 corresponding to residue 217 of SEQ ID NO: 3 (EU index of Kabat et al 1991 Sequences of Proteins of Immunological Interest).

[0047] JPEG2025081649000005.jpg50169

[0048] In some embodiments, the antibody or its immunologically active fragment comprises a human IgG3 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 4.

[0049] In some embodiments, the Fc region of the human IgG3 is modified at amino acid Asn297 (boxed in SEQ ID NOs: 1 - 4, Kabat numbering), for example, to Asn297Ala (N297A), to prevent glycosylation of the antibody. In some embodiments, the Fc region of the human IgG3 is modified at amino acid 435, for example, to Arg435His (R435H, boxed in SEQ ID NO: 3), to extend the half - life. In some embodiments, the Fc region of the human IgG3 lacks Lys447 corresponding to residue 218 of SEQ ID NO: 4 (EU index of Kabat et al 1991 Sequences of Proteins of Immunological Interest).

[0050] JPEG2025081649000006.jpg50168

[0051] In some embodiments, the antibody or its immunologically active fragment comprises a human IgG4 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 5.

[0052] In other embodiments, the Fc region of the human IgG4 is modified at amino acid 235, for example, Leu235Glu (L235E), to alter Fc receptor interaction. In some embodiments, the Fc region of the human IgG4 is modified at amino acid Asn297 (boxed in SEQ ID NOs: 1-4, Kabat numbering), for example, Asn297Ala (N297A), to prevent glycosylation of the antibody. In some embodiments, the Fc region of the human IgG4 lacks Lys447 corresponding to residue 218 of SEQ ID NO: 4 (EU index of Kabat et al 1991 Sequences of Proteins of Immunological Interest).

[0053] JPEG2025081649000007.jpg50170

[0054] In some embodiments, the antibody or its immunologically active fragment comprises a human IgG4 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 6.

[0055] In some embodiments, the Fc region of the human IgG is modified to enhance FcRn binding. Examples of Fc mutations that enhance binding to FcRn are Met252Tyr, Ser254Thr, Thr256Glu (M252Y, S254T, T256E respectively) (Kabat numbering, Dall’Acqua et al 2006, J.Biol Chem Vol.281(33)23514-23524), Met428Leu and Asn434Ser (M428L, N434S) (Zalevsky et al 2010 Nature Biotech, Vol.28(2)157-159), and are Met252Ile, Thr256Asp, Met428Leu (M252I, T256D, M428L respectively), (EU index of Kabat et al 1991 Sequences of Proteins of Immunological Interest). Met252 corresponds to residue 23 in SEQ ID NOs: 1, 4, and 5 and residue 22 in SEQ ID NO: 3. Ser254 corresponds to residue 25 in SEQ ID NOs: 1, 4, and 5 and residue 24 in SEQ ID NO: 3. Thr256 corresponds to residue 27 in SEQ ID NOs: 1, 4, and 5 and residue 26 in SEQ ID NO: 3. Met428 corresponds to residue 199 in SEQ ID NOs: 1, 4, and 5 and residue 198 in SEQ ID NO: 3. Asn434 corresponds to residue 205 in SEQ ID NOs: 1, 4, and 5 and residue 204 in SEQ ID NO: 3. In some embodiments where the fusion protein of the present disclosure comprises an Fc polypeptide, the Fc polypeptide is mutated or modified. In these embodiments, the mutated or modified Fc polypeptide comprises the following mutations: using the Kabat numbering system, Met252Tyr and Met428Leu (M252Y, M428L).

[0056] In some embodiments, the Fc region of the human IgG is modified with amino acid modifications as described in, for example, Natsume et al., 2008 Cancer Res, 68(10):3863-72, Idusogie et al., 2001 J Immunol, 166(4):2571-5, Moore et al., 2010 mAbs, 2(2):181-189, Lazar et al., 2006 PNAS, 103(11):4005-4010, Shields et al., 2001 JBC, 276(9):6591-6604, Stavenhagen et al., 2007 Cancer Res, 67(18):8882-8890, Stavenhagen et al., 2008 Advan. Enzyme Regul., 48:152-164, Alegre et al., 1992 J Immunol, 148:3461-3468, Reviewed in Kaneko and Niwa, 2011 Biodrugs, 25(1):1-11 to alter antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC). Examples of mutations that enhance ADCC include modifications at Ser239 and Ile332, such as Ser239Asp and Ile332Glu (S239D, I332E). Examples of mutations that enhance CDC include modifications at Lys326 corresponding to residue 97 of SEQ ID NOs: 1, 4, and 5 and residue 96 of SEQ ID NO: 3, and at Glu333 corresponding to residue 104 of SEQ ID NOs: 1, 4, and 5 and residue 103 of SEQ ID NO: 3. In some embodiments, the Fc region is modified at at least one or both of these positions, for example, Lys326Ala and / or Glu333Ala (K326A and E333A).

[0057] In some embodiments, the Fc region of the human IgG is modified to induce heterodimerization. For example, having an amino acid modification at Thr366 within the CH3 domain, when it is substituted with a bulkier amino acid, such as Trp (T366W), can selectively pair with a second CH3 domain having amino acid modifications to less bulky amino acids, such as Ser, Ala, and Val (T366S / L368A / Y407V), at positions corresponding to Thr366 at residue 137 of SEQ ID NOs: 1, 4, and 5 and residue 136 of SEQ ID NO: 3, Leu368 at positions corresponding to residue 139 of SEQ ID NOs: 1, 4, and 5 and residue 138 of SEQ ID NO: 3, and Tyr407 at positions corresponding to residue 178 of SEQ ID NOs: 1, 4, and 5 and residue 177 of SEQ ID NO: 3. Heterodimerization via modification of CH3 can be further stabilized by introduction of disulfide bonds, for example, on opposite CH3 domains, by changing Ser354 corresponding to residue 125 of SEQ ID NOs: 1, 4, and 5 and residue 124 of SEQ ID NO: 3 to Cys (S354C), and Tyr349 corresponding to residue 120 of SEQ ID NOs: 1, 4, and 5 and residue 119 of SEQ ID NO: 3 to Cys (Y349C) (Carter, 2001 (summarized in Journal of Immunological Methods, 248:7-15). In some of these embodiments, the Fc region is modified at the protein A binding site of one member of the heterodimer to prevent binding of protein A, thereby allowing for more efficient purification of the heterodimeric fusion protein. An exemplary modification within this binding site is Ile253, which corresponds to residue 24 of SEQ ID NOs: 1, 4, and 5 and residue 23 of SEQ ID NO: 3, and is, for example, Ile253Arg (I253R). For example, the I253R modification may be combined with either the T366S / L368A / Y407V modification or the T366W modification. The T366S / L368A / Y407V modified Fc can form homodimers because there is no steric hindrance at the dimerization interface as in the case of T336W modified Fc. Thus, in some embodiments, the I253R modification is combined with the T366S / L368A / Y407V modified Fc so as not to allow purification of any homodimeric Fc that might be formed.

[0058] In some embodiments, the Fc region of the human IgG is modified to prevent dimerization. In these embodiments, the fusion proteins of the present disclosure are monomers. For example, modification of the charged residue at residue Thr366, such as Thr366Lys, Thr366Arg, Thr366Asp, or Thr366Glu (T366K, T366R, T366D, or T366E, respectively), prevents CH3-CH3 dimerization.

[0059] In some embodiments, the Fc region of the fusion protein is modified at one or more of the following positions to reduce Fc receptor binding: Leu234 (L234), Leu235 (L235), Asp265 (D265), Asp270 (D270), Ser298 (S298), Asn297 (N297), Asn325 (N325), or Ala327 (A327). For example, Leu234Ala (L234A), Leu235Ala (L235A), Asp265Asn (D265N), Asp270Asn (D270N), Ser298Asn (S298N), Asn297Ala (N297A), Asn325Glu (N325E), or Ala327Ser (A327S). In preferred embodiments, the modification within the Fc region reduces binding to the Fc receptor gamma receptor while having little effect on binding to the neonatal Fc receptor (FcRn).

[0060] In some embodiments, the fusion protein comprises a polypeptide derived from an immunoglobulin hinge region. The hinge region can be selected from any of the human IgG isotypes. For example, the fusion protein may comprise a modified IgG1 hinge having the sequence EPKSSDKTHTCPPC (SEQ ID NO: 7), in which case Cys220, which forms a disulfide with the light chain C-terminus cysteine, is mutated to serine, for example, Cys220Ser (C220S). In other embodiments, the fusion protein comprises a truncated hinge having the sequence DKTHTCPPC (SEQ ID NO: 8).

[0061] In some embodiments, the fusion protein has a modified hinge derived from IgG4 having the sequence ESKYGPPCPPC (SEQ ID NO: 9), which is modified, for example, to Ser228Pro (S228P) to prevent or reduce chain exchange. In some embodiments, the fusion protein comprises one or more linker polypeptides. In other embodiments, the fusion protein comprises one or more linkers and hinge polypeptides.

[0062] In some embodiments, the fusion protein of the present disclosure lacks or has reduced fucose linked to the N-linked glycan chain at N297. To prevent fucosylation, there are many methods including, but not limited to, the production of FUT8-deficient cell lines, the addition of inhibitors such as castanospermine, 2-deoxyfucose, 2-fluorofucose to mammalian cell culture media, production cell lines with naturally reduced fucosylation pathways, and the use of metabolic engineering of such production cell lines.

[0063] In some embodiments, the single domain antibody, VHH, or humanized single domain antibody, or human single domain antibody is engineered to exclude recognition by existing antibodies found in humans. In some embodiments, the single domain antibody of the present disclosure is modified by a mutation at the position of Leu11, such as Leu11Glu (L11E) or Leu11Lys (L11K). In other embodiments, the single domain antibody of the present disclosure is modified by a change in the carboxy-terminal region. For example, the terminal sequence consists of GQGTLVTVKPGG (SEQ ID NO: 14) or GQGTLVTVEPGG (SEQ ID NO: 15) or a modification thereof. In some embodiments, the single domain antibody of the present disclosure is modified by a mutation at position 11 and by a change in the carboxy-terminal region.

[0064] In some embodiments, the TBD of the fusion protein of the present disclosure is operably linked via an amino acid linker. In some embodiments, these linkers are mainly composed of the amino acids glycine and serine and are referred to herein as GS linkers. The GS linkers of the fusion proteins of the present disclosure can be of various lengths, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids in length.

[0065] In some embodiments, the GS linker is GGSGGS, i.e., (GGS) 2 (SEQ ID NO: 10), GGSGGSGGS, i.e., (GGS) 3 (SEQ ID NO: 11), GGSGGSGGSGGS, i.e., (GGS)4 (SEQ ID NO:12), and GGSGGSGGSGGSGGS, i.e., (GGS) 5 (SEQ ID NO:13) and comprises an amino acid sequence selected from the group consisting of

[0066] In some embodiments, the multivalent fusion protein is tetravalent. In some embodiments, the tetravalent fusion protein has the following structure: BD-linker-BD-linker-hinge-Fc. In some embodiments, the tetravalent fusion protein has the following structure: BD-linker-hinge-Fc-linker-BD.

[0067] In some embodiments, the BD of the tetravalent fusion protein is a single domain antibody or VHH. In some embodiments, each BD of the tetravalent fusion protein is a single domain antibody or VHH. In some embodiments, the tetravalent fusion protein has the following structure: VHH-linker-VHH-linker-hinge-Fc, wherein the VHH is a humanized or fully human VHH sequence. In some embodiments, the tetravalent fusion protein has the following structure: VHH-linker-hinge-Fc-linker-VHH, wherein the VHH is a humanized or fully human VHH sequence.

[0068] In some embodiments, the multivalent TNFRSF-binding fusion protein is tetravalent. In some embodiments, the tetravalent TNFRSF-binding fusion protein has the following structure: TBD-linker-TBD-linker-hinge-Fc. In some embodiments, the tetravalent TNFRSF-binding fusion protein has the following structure: TBD-linker-hinge-Fc-linker-TBD.

[0069] In some embodiments, the TBD of the tetravalent TNFRSF-binding fusion protein is a single-domain antibody or VHH. In some embodiments, each TBD of the multivalent TNFRSF-binding fusion protein is a single-domain antibody or VHH. In some embodiments, the multivalent TNFRSF-binding fusion protein is tetravalent. In some embodiments, the tetravalent TNFRSF-binding fusion protein has the following structure: VHH-linker-VHH-linker-hinge-Fc, where the VHH is a humanized or fully human VHH sequence. In some embodiments, the tetravalent TNFRSF-binding fusion protein has the following structure : VHH-linker-hinge-Fc-linker-VHH, where the VHH is a humanized or fully human VHH sequence.

[0070] In some embodiments, the GS linker is GGSGGS, i.e., (GGS) 2 (SEQ ID NO: 10), GGSGGSGGS, i.e., (GGS) 3 (SEQ ID NO: 11), GGSGGSGGSGGS, i.e., (GGS) 4 (SEQ ID NO: 12), and GGSGGSGGSGGSGGS, i.e., (GGS) 5 (SEQ ID NO: 13), and comprises an amino acid sequence selected from the group consisting of.

[0071] In some embodiments, the TBD of the tetravalent TNFRSF-binding fusion protein is a single-domain antibody or VHH. In some embodiments, each TBD of the multivalent TNFRSF-binding fusion protein is a single-domain antibody or VHH. In some embodiments, the multivalent fusion protein is hexavalent. In some embodiments, the hexavalent fusion protein has the following structure: BD-linker-TBD-linker-BD-linker-hinge-Fc. In some embodiments, the hexavalent fusion protein has the following structure: BD-linker-BD-linker-hinge-Fc-linker-BD, or BD-linker-hinge-Fc-linker-BD-linker-BD.

[0072] In some embodiments, the BD of the hexavalent fusion protein is a single-domain antibody or VHH. In some embodiments, each BD of the hexavalent fusion protein is a single-domain antibody or VHH. In some embodiments, the hexavalent fusion protein has the following structure: VHH-linker-VHH-linker-VHH-linker-hinge-Fc, where VHH is a humanized or fully human VHH sequence. In some embodiments, the hexavalent fusion protein has the following structure: VHH-linker-VHH-linker-hinge-Fc-linker-VHH, or VHH-linker-hinge-Fc-linker-VHH-linker-VHH, where VHH is a humanized or fully human VHH sequence.

[0073] In some embodiments, the multivalent TNFRSF-binding fusion protein is hexavalent. In some embodiments, the hexavalent TNFRSF-binding fusion protein has the following structure: TBD-linker-TBD-linker-TBD-linker-hinge-Fc. In some embodiments, the hexavalent TNFRSF-binding fusion protein has the following structure: TBD-linker-TBD-linker-hinge-Fc-linker-TBD, or TBD-linker-hinge-Fc-linker-TBD-linker-TBD.

[0074] In some embodiments, the multivalent TNFRSF-binding fusion protein is hexavalent. In some embodiments, the hexavalent TNFRSF-binding fusion protein has the following structure: VHH-linker-VHH-linker-VHH-linker-hinge-Fc, where VHH is a humanized or fully human VHH sequence. In some embodiments, the hexavalent TNFRSF-binding fusion protein has the following structure: VHH-linker-VHH-linker-hinge-Fc-linker-VHH, or VHH-linker-hinge-Fc-linker-VHH-linker-VHH, where VHH is a humanized or fully human VHH sequence.

[0075] In some embodiments, the multivalent fusion protein lacks the Fc region. In some of these embodiments, the fusion protein is tetravalent and has the following structure: BD-linker-BD-linker-BD-linker-BD-linker. In some of these embodiments, the fusion protein is pentavalent and has the following structure: BD-linker-BD-linkerBD-linker-BD-linker-BD. In some of these embodiments, the fusion protein is hexavalent and has the following structure: BD-linker-BD-linker-BD-linker-BD-linker-BD-linkerBD.

[0076] In some embodiments, the multivalent TNFRSF-binding fusion protein lacks the Fc region. In some of these embodiments, the TNFRSF-binding fusion protein is tetravalent and has the following structure: TBD-linker-TBD-linker-TBD-linker-TBD-linker. In some of these embodiments, the TNFRSF-binding fusion protein is pentavalent and has the following structure: TBD-linker-TBD-linkerTBD-linker-TBD-linker-TBD. In some of these embodiments, the TNFRSF-binding fusion protein is hexavalent and has the following structure: TBD-linker-TBD-linker-TBD-linker-TBD-linker-TBD-linkerTBD.

[0077] In some embodiments, the BD of the multivalent fusion protein is a single domain antibody or VHH. In some embodiments, the multivalent fusion protein lacks an Fc region. In some of these embodiments, the fusion protein is tetravalent and has the following structure: VHH-linker-VHH-linker-VHH-linker-VHH-linker. In some of these embodiments, the fusion protein is pentavalent and has the following structure: VHH-linker-VHH-linker-VHH-linker-VHH-linker-VHH. In some of these embodiments, the fusion protein is hexavalent and has the following structure: VHH-linker-VHH-linker-VHH-linker-VHH-linker-VHH-linker-VHH. In any of these embodiments, the VHH is a humanized or fully human VHH sequence.

[0078] In some embodiments, the TBD of the multivalent TNFRSF-binding fusion protein is a single domain antibody or VHH. In some embodiments, the multivalent TNFRSF-binding fusion protein lacks an Fc region. In these embodiments, the TNFRSF-binding fusion protein is tetravalent and has the following structure: VHH-linker-VHH-linker-VHH-linker-VHH-linker. In these embodiments, the TNFRSF-binding fusion protein is pentavalent and has the following structure: VHH-linker-VHH-linker-VHH-linker-VHH-linker-VHH. In these embodiments, the TNFRSF-binding fusion protein is hexavalent and has the following structure: VHH-linker-VHH-linker-VHH-linker-VHH-linker-VHH-linker-VHH. In these embodiments, the VHH is a humanized or fully human VHH sequence.

[0079] In some embodiments, the GS linker is GGSGGS, i.e., (GGS) 2 (SEQ ID NO: 10), GGSGGSGGS, i.e., (GGS) 3 (SEQ ID NO: 11), GGSGGSGGSGGS, i.e., (GGS) 4(SEQ ID NO: 12), and GGSGGSGGSGGSGGS, i.e., (GGS) 5 comprises an amino acid sequence selected from the group consisting of (SEQ ID NO: 13).

[0080] In some embodiments, the fusion protein is multispecific and comprises a binding domain for TBD and a second antigen. In these embodiments, the binding domain for the second antigen can be located at multiple positions within the molecule relative to the TBD. In some embodiments, the binding domain for the second antigen is located at the N-terminal TBD. In other embodiments, the binding domain for the second antigen is located C-terminal to the TBD. In other embodiments, the binding domain for the second antigen is located on a separate polypeptide that associates with the first polypeptide comprising the TBD.

[0081] In some embodiments, the fusion protein is multispecific and comprises a binding domain for anti-OX40 and a binding domain for a second antigen. In these embodiments, the binding domain for the second antigen can be located at multiple positions within the molecule relative to the anti-OX40 binding domain. In some embodiments, the binding domain for the second antigen is located at the N-terminal anti-O X40 binding domain. In other embodiments, the binding domain for the second antigen is located C-terminal to the anti-OX40 binding domain. In other embodiments, the binding domain for the second antigen is located on a separate polypeptide that associates with the first polypeptide comprising the anti-OX40 binding domain.

[0082] In some embodiments, the fusion protein is multispecific and comprises an anti-PDL1 binding domain and a binding domain for a second antigen. In these embodiments, the binding domain for the second antigen can be positioned at multiple locations within the molecule relative to the anti-PDL1 binding domain. In some embodiments, the binding domain for the second antigen is positioned at the N-terminal anti-PDL1 binding domain. In other embodiments, the binding domain for the second antigen is positioned C-terminal to the anti-PDL1 binding domain. In other embodiments, the binding domain for the second antigen is positioned on a separate polypeptide that associates with a first polypeptide comprising the anti-PDL1 binding domain.

[0083] In some embodiments, the TBD within the multispecific TNFRSF-binding fusion protein is a single-domain antibody or VHH. In some embodiments, the TBD within the multispecific TNFRSF-binding fusion protein consists of antibody variable heavy (VH) and variable light (VL) chain regions. In some embodiments, the VH and VL of the TBD are formatted as a single-chain variable fragment (scFv) linked via a linker region. In some embodiments, the VH and VL of the TBD are formatted as a FAB fragment that associates via a constant heavy chain 1 (CH1) domain and a constant light chain (CL) domain. In some embodiments, a non-antibody heterodimerization domain is used to enable proper association of the VH and VL of the TBD. In some embodiments, the TBD within the multivalent TNFRSF-binding fusion protein is derived from a non-antibody scaffold protein such as, but not limited to, designed ankyrin repeat proteins (darpins), avimers, anticalins / lipocalins, centyrins, and fibronomers.

[0084] In some embodiments, the TBD within the multispecific TNFRSF-binding fusion protein is a single domain antibody or VHH that binds to OX40. In some embodiments, the anti-OX40 binding domain within the multispecific TNFRSF-binding fusion protein consists of antibody variable heavy (VH) and variable light (VL) chain regions. In some embodiments, the VH and VL of the anti-OX40 binding domain are formatted as a single-chain variable fragment (scFv) linked via a linker region. In some embodiments, the VH and VL of the anti-OX40 binding domain are formatted as a Fab fragment that associates via a constant heavy chain 1 (CH1) domain and a constant light chain (CL) domain. In some embodiments, a non-antibody heterodimerization domain is used to enable proper association of the VH and VL of the anti-OX40 binding domain. In some embodiments, the anti-OX40 binding domain within the multispecific TNFRSF-binding fusion protein is derived from a non-antibody scaffold protein such as, but not limited to, designed ankyrin repeat protein (darpin), avimer, anticalin / lipocalin, centyrin, and fibronomer.

[0085] In some embodiments, the binding domain within the multispecific fusion protein is a single domain antibody or VHH that binds to PDL1. In some embodiments, the anti-PDL1 binding domain within the multispecific TNFRSF-binding fusion protein consists of antibody variable heavy (VH) and variable light (VL) chain regions. In some embodiments, the VH and VL of the anti-PDL1 binding domain are formatted as a single-chain variable fragment (scFv) linked via a linker region. In some embodiments, the VH and VL of the anti-PDL1 binding domain are formatted as a Fab fragment that associates via a constant heavy chain 1 (CH1) domain and a constant light chain (CL) domain. In some embodiments, a non-antibody heterodimerization domain is used to enable proper association of the VH and VL of the anti-PDL1 binding domain. In some embodiments, the anti-PDL1 binding domain within the multispecific fusion protein is derived from a non-antibody scaffold protein, such as, but not limited to, designed ankyrin repeat proteins (darpins), avimers, anticalins / lipocalins, centyrins, and fibronomers.

[0086] In some embodiments, the anti-OX40 binding domain of the multispecific TNFRSF binding fusion protein is a bispecific antibody or an antigen-binding fragment thereof.

[0087] In some embodiments, the anti-PDL1 binding domain of the multispecific fusion protein is a bispecific antibody or an antigen-binding fragment thereof.

[0088] In any of these embodiments, the bispecific antibody or antigen fragment thereof is, by way of non-limiting example, an antibody fragment such as an X-Link Fab, a cross-linked Fab fragment, a tascFv / BiTE, a tandem scFv / bispecific T cell engager, a Db, a bispecific antibody, a taDb, a format based on a tandem bispecific antibody, an Fc fusion such as a Db-Fc, a bispecific antibody-Fc fusion, a taDb-Fc fusion, a tandem bispecific antibody-Fc fusion, a taDb-CH3, a tandem bispecific antibody-CH3 fusion, a (scFv)4-Fc, a tetra scFv-Fc fusion, a DVD-Ig, a format based on a dual variable domain immunoglobulin, an IgG format such as knob-hole and SEED, a chain exchange operation domain, a CrossMab, a knob-hole in combination with heavy and light chain domain exchange, a bsAb, a bispecific antibody derived from a quadroma, an sdAb, a single domain-based antibody, and a kappa-lambda body, such as any suitable bispecific format known in the art including those described in PCT Publication No. WO2012 / 023053.

[0089] In any of the above embodiments, at least one TBD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-29 and 377-386.

[0090] In any of the above embodiments, at least one TBD includes a complementarity-determining region 1 (CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 36, and 44, a complementarity-determining region 2 (CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 35, and 37, and a complementarity-determining region 3 (CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-34, 48-43, and 45.

[0091] In any of the above embodiments, at least one BD includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 46-57.

[0092] In any of the above embodiments, at least one BD includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 52-57.

[0093] In any of the above embodiments, at least one BD includes a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 58, 61, and 64, a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 59, 62, 65, and 69, and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 60, 63, 66-68, and 70.

[0094] In any of the above embodiments, at least one TBD includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-29 and 377-386, and at least one BD includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 46-57.

[0095] In any of the above embodiments, at least one TBD includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-29 and 377-386, and at least one BD includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 52-57.

[0096] In any of the above embodiments, at least one TBD comprises a complementarity-determining region 1 (CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 36, and 44, a complementarity-determining region 2 (CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 35, and 37, and a complementarity-determining region 3 (CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-34, 48-43, and 45, and at least one BD comprises a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 58, 61, and 64, a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 59, 62, 65, and 69, and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 60, 63, 66-68, and 70.

Brief Description of the Drawings

[0097]

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Mode for Carrying Out the Invention

[0098] Detailed Description of the Invention All patents and publications referred to herein are incorporated herein by reference to the same extent as if each individual patent and publication were specifically and individually indicated to be incorporated by reference.

[0099] Definitions Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless the context specifically requires otherwise, singular terms shall include the plural, and plural terms shall include the singular. Generally, the nomenclature and techniques used in connection with cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those commonly used and well known in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzyme reactions and purification techniques are performed according to the manufacturer's specifications, or as commonly achieved in the art, or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclature and experimental procedures and techniques used in connection with analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are those commonly used and well known in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0100] The following terms used in accordance with the present disclosure shall be understood to have the following meanings unless otherwise indicated.

[0101] As used herein, the terms "bispecific fusion protein" and "antibody" may be synonymous. As used herein, the term "antibody" refers to an immunoglobulin molecule and an immunologically active portion of an immunoglobulin (Ig) molecule, i.e., a molecule that specifically binds (immunoreacts) with an antigen, including an antigen-binding site. "Specifically binds" or "immunoreacts with" or "against" means that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides, or binds with much lower affinity (K d >10 -6 ). Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, dAb (domain antibody), single-chain, Fab, Fab', and F(ab') 2 fragments, F v , scFv, Fab expression libraries, as well as single-domain antibody (sdAb) fragments, e.g., V H H, V NAR , artificially created V H or V K .

[0102] The basic antibody structural unit is known to comprise a tetramer. Each tetramer consists of two identical pairs of polypeptide chains, and each pair has one "light" (about 25 kDa) and one "heavy" chain (about 50 - 70 kDa). The amino-terminal portion of each chain contains a variable region of about 100 - 110 or more amino acids that is mainly involved in antigen recognition. The carboxy-terminal portion of each chain defines a constant region that is mainly involved in effector functions. Generally, antibody molecules obtained from humans are related to one of the classes IgG, IgM, IgA, IgE, and IgD, which differ from each other in the nature of the heavy chains contained in the molecule. A particular class still has subclasses (also known as isotypes), e.g., IgG 1 , IgG 2 , etc. Furthermore, in humans, the light chain can be either a kappa chain or a lambda chain.

[0103] As used herein, the term "monoclonal antibody" (MAb) or "monoclonal antibody composition" refers to a population of antibody molecules that includes only one molecular species of an antibody molecule consisting of a specific light chain gene product and a specific heavy chain gene product. In particular, the complementarity determining regions (CDRs) of a monoclonal antibody are identical in all molecules of the population. An MAb can immunoreact with a specific epitope of the antigen and includes an antigen-binding site characterized by a specific binding affinity therefor.

[0104] The term "antigen-binding site" or "binding portion" refers to the portion of an immunoglobulin molecule involved in antigen binding. The antigen-binding site is formed by the amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") chain and the light ("L") chain. Three highly divergent regions within the V regions of the heavy and light chains, called "hypervariable regions", are interposed between more conserved adjacent regions known as "framework regions" or "FRs". Thus, the term "FR" refers to the amino acid sequences found naturally between and adjacent to the hypervariable regions of an immunoglobulin. In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged in relation to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and each of the three hypervariable regions of the heavy and light chains is called a "complementarity determining region" or "CDR". The amino acid assignments to each domain follow the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Maryland (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature 342:878-883 (1989). The single domain antibody (sdAb) fragment portion of the fusion protein of the present disclosure is interchangeably referred to herein as the targeted polypeptide of the present specification.

[0105]

[0106] As used herein, the term "epitope" includes any protein determinant capable of specifically binding to / by an immunoglobulin or a fragment thereof, or a T cell receptor. The term "epitope" includes any protein determinant capable of specifically binding to / by an immunoglobulin or a T cell receptor. Epitope determinants usually consist of a chemically active surface population of molecules such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics and specific charge characteristics. An antibody is said to specifically bind an antigen when the dissociation constant is ≤ 1 mM, for example, in some embodiments, ≤ 1 μM, for example, ≤ 100 nM, ≤ 10 nM, or ≤ 1 nM.

[0107] As used herein, the terms "immunological binding" and "immunological binding properties" refer to the type of non-covalent binding interactions that occur between an immunoglobulin molecule and an antigen to which the immunoglobulin is specific. The strength or affinity of an immunological binding interaction can be expressed in terms of the dissociation constant (K d ) of the interaction, with a smaller K d representing a greater affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method involves measuring the rates of antigen-binding site / antigen complex formation and dissociation, which rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally affect the rates in both directions. Thus, both the "on-rate constant" (k on ) and the "off-rate constant" (k off ) can be determined by calculation of the concentrations and the actual rates of association and dissociation (see Nature 361:186-87 (1993)). The ratio of k off / k on allows cancellation of all parameters not related to affinity and gives the dissociation constant K dEqual to (see generally Davies et al. (1990) Annual Rev Biochem 59:439-473). The antibodies of the present disclosure are measured in an assay such as a radio-ligand binding assay, surface plasmon resonance (SPR), flow cytometry binding assay, or a similar assay known in the art, and the equilibrium binding constant (K d ) is said to specifically bind to the antigen when it is ≦ 1 mM, and in some embodiments, ≦ 1 μM, ≦ 100 nM, ≦ 10 nM, or ≦ 100 pM to about 1 pM.

[0108] As used herein, the term "isolated polynucleotide" refers to a polynucleotide of genomic, cDNA, or synthetic origin, or a combination of some of them, and based on its origin, the "isolated polynucleotide" is (1) not associated with all or part of the polynucleotide, in which case the "isolated polynucleotide" is found in nature, (2) operably linked to a polynucleotide that is not naturally bound, or (3) does not naturally occur as part of a larger sequence. It is intended to mean something that does not occur.

[0109] As used herein, the term "isolated protein" refers to a protein of cDNA, recombinant RNA, or synthetic origin, or a combination of some of them, and based on its origin or source of derivation, the "isolated protein" is (1) not associated with a protein found in nature, (2) does not contain other proteins from the same origin, for example, does not contain marine proteins, (3) is expressed by cells from different species, or (4) does not occur in nature.

[0110] The term "polypeptide" as a genus name is used herein to refer to natural proteins, fragments, or analogs of polypeptide sequences. Thus, natural protein fragments, and analogs are species of the polypeptide genus.

[0111] As used herein, the term "naturally occurring" as applied to an object refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence contained in an organism (including viruses) that can be isolated from a natural origin and that has not been intentionally modified by humans in a laboratory or otherwise is a naturally occurring one.

[0112] As used herein, the term "operably linked" refers to the situation where the components so described are in a relationship that intentionally enables them to function. A control sequence "operably linked" to a coding sequence is joined in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequence.

[0113] As used herein, the term "control sequence" refers to a polynucleotide sequence essential for achieving expression and processing of a coding sequence to which it is ligated. The nature of such control sequences varies depending on the host organism in prokaryotes, and such control sequences generally include a promoter, a ribosome binding site, and a transcription termination sequence in eukaryotic cells, and generally, such control sequences include a promoter and a transcription termination sequence. The term "control sequence" is intended to include at a minimum all components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, such as a leader sequence and a sequence of a fusion partner. As used herein, the term "polynucleotide" refers to a polymer of nucleotides, either ribonucleotides or deoxynucleotides, or a modified form of either type of nucleotide, having a length of at least 10 bases. The term includes DNA in both single-stranded and double-stranded forms.

[0114] As used herein, the term "oligonucleotide" includes naturally occurring nucleotides and modified nucleotides linked by naturally occurring and non-naturally occurring oligonucleotide linkages. Oligonucleotides are generally a subset of polynucleotides having a length of 200 bases or less. In some embodiments, the oligonucleotide has a length of 10 to 60 bases, and in some embodiments, a length of 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases. Oligonucleotides are usually single-stranded, but for example, with respect to probes, such as for constructing gene variants, oligonucleotides may be double-stranded. The oligonucleotides of the present disclosure are either sense or antisense oligonucleotides.

[0115] As used herein, the term "naturally occurring nucleotide" includes deoxyribonucleotides and ribonucleotides. As used herein, the term "modified nucleotide" includes nucleotides having modified or substituted sugar groups, etc. As used herein, the term "oligonucleotide linkage" includes oligonucleotide linkages such as phosphorothioate, phosphorodithioate , phosphoroserellorate, phosphorodiselenoate, phosphorothioanilato, phosphororaniladate, phosphoramidate, etc. See, for example, LaPlanche et al. Nucl. Acids Res. 14:9081 (1986), Stec et al. J. Am. Chem. Soc. 106:6077 (1984), Stein et al. Nucl. Acids Res. 16:3209 (1988), Zon et al. Anti Cancer Drug Design 6:539 (1991), Zon et al. Oligonucleotides and Analogues: A Practical See Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University Press, Oxford, England (1991)), Stec et al., U.S. Patent No. 5,151,510, and Uhlmann and Peyman, Chemical Reviews 90:543 (1990). The oligonucleotide can optionally include a label for detection.

[0116] As used herein, the term "selectively hybridizes" means to detectably and specifically bind. The polynucleotides, oligonucleotides, and fragments thereof of the present disclosure selectively hybridize to nucleic acid strands under hybridization and wash conditions that minimize a detectable amount of binding to non-specific nucleic acids. As is known in the art and as discussed herein, selective hybridization conditions can be achieved using high stringency conditions. Generally, the nucleic acid sequence homology between the polynucleotides, oligonucleotides, and fragments of the present disclosure and the nucleic acid sequence of interest is at least 80%, more typically with high homology of at least 85%, 90%, 95%, 99%, and 100%. Two amino acid sequences are homologous if there is partial or complete identity between their sequences. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum match. Gaps (in either of the two sequences being aligned) are allowed in maximizing the match, and the gap length is preferably 5 or less, more preferably 2 or less. Alternatively, two protein sequences (or polypeptide sequences derived therefrom of at least 30 amino acids in length) are homologous as the term is used herein if they have an alignment score of greater than 5 (in standard deviation units) using the program ALIGN with a mutation data matrix and a gap penalty of 6 or more. See Dayhoff, M.O., in Atlas of Protein Sequence and Structure, pp. 101-110 (Volume 5, National Biomedical Research Foundation (1972)) and Supplement 2 to this volume, pp. 1-10. The two sequences or portions thereof are more preferably homologous if they are at least 50% identical when their amino acids are optimally aligned using the ALIGN program. The term "corresponding to" is used herein to mean that a polynucleotide sequence is homologous (i.e., identical, strictly not evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to a reference polypeptide sequence.In contrast, the term "complementary to" is used herein to mean that the complementary sequence is homologous to all or a portion of the reference polynucleotide sequence. By way of example, the nucleotide sequence "TATAC" corresponds to the reference sequence "TATAC" and is complementary to the reference sequence "GTATA".

[0117] The following terms are used to describe sequence relationships between two or more polynucleotides or amino acid sequences: "reference sequence", "comparison window", "sequence identity", "percentage of sequence identity", and "substantial identity". A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length cDNA or gene sequence listed in a sequence listing, or a complete cDNA It may contain A or a gene sequence. Generally, the reference sequence is at least 18 nucleotides or 6 amino acids in length, often at least 24 nucleotides or 8 amino acids in length, and in many cases at least 48 nucleotides or 16 amino acids in length. Since two polynucleotide or amino acid sequences each may (1) contain a similar sequence (i.e., a portion of the complete polynucleotide or amino acid sequence) between the two molecules, and (2) further contain sequences that differ between the two polynucleotide or amino acid sequences, sequence comparison between two (or more) molecules is usually performed by comparing the sequences of the two molecules over a "comparison window" to identify and compare local regions of sequence similarity. As used herein, a "comparison window" refers to at least 18 consecutive nucleotide positions or a conceptual segment of 6 amino acids in which a polynucleotide sequence or amino acid sequence can be compared to a reference sequence consisting of at least 18 consecutive nucleotides or 6 amino acid sequences, and the portion of the polynucleotide sequence within the comparison window may contain no more than 20 percent additions, deletions, substitutions, and the like (i.e., gaps) when compared to the reference sequence (which contains no additions or deletions) for an optimal alignment of the two sequences.Optimal alignment of arrays to align comparison windows may be performed by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch J. Mol. Biol. 48:443 (1970), by the search for similarity methods of Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85:2444 (1988), by computer implementation of these algorithms (GAP, BESTFIT, FASTA, and TFASTA of the Wisconsin Genetics Software Package Release 7.0 (Genetics Computer Group, 575 Science Dr., Madison, Wisconsin), Geneworks, or MacVector software packages), or by visual inspection, and the best alignment (i.e., the one that yields the highest percentage of homology over the comparison window) generated by these various methods is selected.

[0118] The term "sequence identity" means that two polynucleotide or amino acid sequences are identical (i.e., on a nucleotide-by-nucleotide or residue-by-residue basis) over a comparison window. The term "percent sequence identity" is calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions at which the identical nucleic acid bases (e.g., A, T, C, G, U, or I) or residues occur in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percent sequence identity. As used herein, the term "substantially identical" means a characteristic of a polynucleotide or amino acid sequence, where the polynucleotide or amino acid includes a sequence having at least 85 percent sequence identity, e.g., at least 90-95 percent sequence identity, more typically at least 99 percent sequence identity, over a comparison window of at least 18 nucleotides (6 amino acids) positions, often over a window of at least 24-48 nucleotides (8-16 amino acids) positions, when compared to a reference sequence, and where the percent sequence identity is calculated by comparing the reference sequence to a sequence that may include deletions or additions totaling up to 20 percent of the reference sequence over the comparison window. The reference sequence may be a subset of a larger sequence.

[0119] As used herein, the 20 conventional amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)). Stereoisomers (e.g., D-amino acids), unnatural amino acids, e.g., α,α-Disubstituted amino acids, N-alkyl amino acids, lactic acid, and other non-conventional amino acids can also be suitable components for the polypeptides of the present disclosure. Examples of non-conventional amino acids include 4-hydroxyproline, γ-carboxyglutamic acid, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, according to standard usage and convention, the left-handed direction is the amino-terminal direction, and the right-handed direction is the carboxy-terminal direction.

[0120] Similarly, unless otherwise specified, the left end of a single-stranded polynucleotide sequence is the 5' end, and the left-handed direction of a double-stranded polynucleotide sequence is called the 5' direction. The 5' to 3' addition direction of a nascent RNA transcript is called the transcription direction, and the sequence region of the DNA strand having the same sequence as the RNA, which is 5' relative to the 5' end of the RNA transcript, is called the "upstream sequence", and the sequence region of the DNA strand having the same sequence as the RNA, which is 3' relative to the 3' end of the RNA transcript, is called the "downstream sequence".

[0121] The term "substantial identity" as applied to polypeptides means that two peptide sequences share at least 80 percent sequence identity, e.g., at least 90 percent sequence identity, at least 95 percent sequence identity, or at least 99 percent sequence identity when optimally aligned by, for example, the GAP or BESTFIT programs using the default gap weights.

[0122] In some embodiments, non-identical residue positions differ by conservative amino acid substitutions.

[0123] Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains are glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains are serine and threonine; a group of amino acids having amide-containing side chains are asparagine and glutamine; a group of amino acids having aromatic side chains are phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains are lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains are cysteine and methionine. Groups of suitable conservative amino acid substitutions are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine.

[0124] Minor changes in the amino acid sequence of an antibody or immunoglobulin molecule as discussed herein are contemplated to be encompassed by the present disclosure provided that the change in the amino acid sequence maintains at least 75%, for example, at least 80%, 90%, 95%, or 99%. In particular, conservative amino acid substitutions are contemplated. Conservative substitutions are made within families of amino acids that have related side chains. Genetically encoded amino acids are generally classified into families. That is, (1) acidic amino acids are aspartic acid, glutamic acid; (2) basic amino acids are lysine, arginine, histidine; (3) nonpolar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Hydrophilic amino acids include arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, and threonine. Hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine. Other amino acid families include (i (i) The aliphatic-hydroxy family, serine and threonine, (ii) the amide-containing family, asparagine and glutamine, (iii) the aliphatic family, alanine, valine, leucine, and isoleucine, and (iv) the aromatic family, phenylalanine, tryptophan, and tyrosine. For example, a single substitution of leucine by isoleucine or valine, a single substitution of aspartic acid by glutamic acid, a single substitution of threonine by serine, or a similar substitution of an amino acid by a structurally related amino acid is not expected to have a major impact on the binding or properties of the resulting molecule, particularly if the substitution does not involve an amino acid within the framework site. Whether an amino acid change results in a functional peptide can be readily determined by assaying the specific activity of the polypeptide derivative. The assay is described in detail herein. Antibodies or fragments or analogs of immunoglobulin molecules can be readily prepared by one of ordinary skill in the art. The appropriate amino and carboxy termini of the fragment or analog occur near the boundaries of the functional domain. The structural and functional domains can be identified by comparison of the nucleotide and / or amino acid sequence data to publicly available or proprietary sequence databases. In some embodiments, computer-based comparison methods are used to identify sequence motifs or predicted protein structural domains that occur in other proteins of known structure and / or function. Methods for identifying protein sequences that fold into known three-dimensional structures are known. Bowie et al. Science 253:164 (1991). Thus, the above examples demonstrate that one of ordinary skill in the art can recognize sequence motifs and structural arrangements that can be used to determine structural and functional domains in accordance with the present invention.

[0125] Suitable amino acid substitutions are those that (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter the binding affinity for protein complex formation, (4) alter the binding affinity, and (4) confer or modify other physicochemical or functional properties of such analogs. Analogs can include various mutant proteins of sequences other than the naturally occurring peptide sequences. For example, single or multiple amino acid substitutions (e.g., conservative amino acid substitutions) may be made in a naturally occurring sequence (e.g., a portion of a polypeptide outside of the domain(s) that form intermolecular contacts). Conservative amino acid substitutions should not substantially change the structural properties of the parental sequence (e.g., the substituted amino acid should not tend to break the helices that occur in the parental sequence nor break other types of secondary structure that characterize the parental sequence). Examples of secondary and tertiary structures of polypeptides that are recognized in the art are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W.H. Freeman and Company, New York (1984)), Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et al. Nature 354:105 (1991).

[0126] As used herein, the term "polypeptide fragment" refers to a polypeptide having deletions at the amino and / or carboxy termini, but in which the remaining amino acid sequence is identical to the corresponding position in a naturally occurring sequence, such as that deduced from a full-length cDNA sequence. Fragments are typically at least 5, 6, 8, or 10 amino acids in length, for example, at least 14 amino acids in length, at least 20 amino acids in length, at least 50 amino acids in length, or at least 70 amino acids in length. As used herein, the term "analogue" refers to a polypeptide consisting of at least a 25-amino acid segment that has substantial identity to a portion of the putative amino acid sequence and has specific binding to CD47 under appropriate binding conditions. Typically, polypeptide analogues contain conservative amino acid substitutions (or additions or deletions) with respect to the naturally occurring sequence. Analogues are typically at least 20 amino acids in length, for example, at least 50 amino acids in length or more and can often be the same length as the full-length naturally occurring polypeptide.

[0127] Peptide analogues are commonly used in the pharmaceutical industry as non-peptide drugs with properties similar to those of the template peptide. These types of non-peptide compounds are referred to as "peptide mimetic" or "peptidomimetic". Fauchere, J. Adv. Drug Res. 15:29 (1986), Veber and Freidinger TINS p. 392 (1985), and Evans et al. J. Med. Chem. 30:1229 (1987). Such compounds are often developed using computer-aided molecular modeling. Equivalent therapeutic or prophylactic effects may be obtained using peptidomimetics that are structurally similar to a therapeutically useful peptide. Generally, peptidomimetics are structurally similar to a paradigm polypeptide (i.e., a polypeptide having biochemical properties or pharmacological activity), such as a human antibody, but by methods well known in the art, --CH 2 NH--, --CH 2 S-, --CH 2 -CH2 --, --CH=CH-- (cis and trans), --COCH 2 --, CH(OH)CH 2 --, and -CH 2 It has one or more peptide bonds optionally substituted with a bond selected from the group consisting of --SO--. Systematic substitution of one or more amino acids of the consensus sequence with the corresponding D-amino acids (e.g., D-lysine instead of L-lysine) may be used to generate a more stable polypeptide. Further, a constrained peptide comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods known in the art (Rizo and Gierasch Ann.Rev.Biochem. 61:387 (1992)), for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges that cyclize the peptide.

[0128] The term "agent" is used herein to denote a compound, a mixture of compounds, a biopolymer, and / or an extract consisting of a biological material.

[0129] The term "label" or "labeled" as used herein refers to the introduction of a detectable marker, for example, by the introduction of a radiolabeled amino acid or by the attachment of a biotinyl moiety to a polypeptide that can be detected by a labeled avidin (e.g., streptavidin containing a fluorescent marker or enzyme activity that can be detected by optical or calorimetric methods). In certain situations, the label or marker may also be therapeutic. Various labeling methods for polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to: radioisotopes or radionuclides (e.g., 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131I), Fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), biotinyl groups by chemiluminescence, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, the label is attached by spacer arms of various lengths to reduce potential steric hindrance. As used herein, the term "pharmaceutical or formulation" refers to a compound or composition that can induce a desired therapeutic effect when appropriately administered to a patient.

[0130] As used herein, the term "antineoplastic agent" is used to refer to an agent having functional properties that inhibit the development or progression of neoplasms in humans, particularly malignant (cancerous) lesions, such as epithelial malignancies, non-epithelial malignancies, lymphomas, or leukemias. Inhibition of metastasis is often a property of antineoplastic agents.

[0131] As used herein, terms such as "treat", "treating", "treatment", etc. refer to reducing and / or ameliorating a disorder and / or its associated symptoms. "Alleviate" and / or "alleviating" mean, for example, reducing, suppressing, alleviating, decreasing, halting, and / or stabilizing the development or progression of a disease such as cancer. It will be understood that treating a disorder or condition does not require, but does not exclude, complete elimination of the disorder, condition, or its associated symptoms.

[0132] Other chemical terms herein are used according to their conventional usage in the art, as exemplified in The McGraw-Hill Dictionary of Chemical Terms (Parker, S., Ed., McGraw-Hill, San Francisco (1985)).

[0133] As used herein, "substantially pure" means that the target species is the dominant species present (i.e., on a molar basis, it is more abundant than any other individual species in the composition), and in some embodiments, the substantially purified fraction is a composition in which the target species constitutes at least about 50 percent (on a molar basis) of all polymeric species present.

[0134] Generally, a substantially pure composition contains more than about 80 percent of all polymeric species contained in the composition, for example, more than about 85%, 90%, 95%, and 99%. In some embodiments, the target species is purified to the point of essential homogeneity (where no contaminating species can be detected in the composition by conventional detection methods) such that the composition consists essentially of a single polymeric species.

[0135] In the present disclosure, "comprises", "comprising", "containing", "having", etc. can have the meanings ascribed to them in U.S. patent law, can mean "include", "including", etc., and the terms "consisting essentially of" or "consists essentially" likewise have the meanings ascribed to them in U.S. patent law, and these terms are open-ended and permit the presence of elements other than those recited, provided that the basic or novel features of the recited elements are not changed by the presence of elements other than those recited, except for embodiments of the prior art.

[0136] "Effective amount" means an amount necessary to improve the symptoms of a disease as compared to an untreated patient. The effective amount of the active compound(s) used to practice the present invention for the therapeutic treatment of a disease will vary depending on the method of administration, the age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosing schedule. Such amount is referred to as an "effective" amount.

[0137] "Subject" means a human or non-human mammal, such as, but not limited to, cows, horses, dogs, rodents, sheep, primates, camels, or cats.

[0138] As used herein, the term "administer" refers to any method of moving, delivering, introducing, or transporting a therapeutic agent to a subject in need of treatment with such therapeutic agent. Such methods include, but are not limited to, oral, topical, intravenous, intraperitoneal, intramuscular, intradermal, intranasal, and subcutaneous administration.

[0139] "Fragment" means a portion of a polypeptide or nucleic acid molecule. This portion includes, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the full length of the reference nucleic acid molecule or polypeptide. Fragments can contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0140] The ranges provided herein are to be understood as being shorthand for all of the values within the range. For example, a range of 1 to 50 is to be understood as including any number, combination of numbers, or sub-ranges from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0141] Unless otherwise specified or clear from the context, the terms "a", "an", and "the" as used herein are to be understood to be in the singular or plural. Unless otherwise specified or clear from the context, the term "or" as used herein is to be understood to be inclusive.

[0142] Unless otherwise specified or not apparent from the context, the term "about" as used herein is understood to be within the normal tolerance range in the art, for example, within two standard deviations of the mean. About can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the recited value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term "about".

[0143] OX40 (TNFRSF4, CD134) targeting OX40 is a member of the TNF receptor superfamily that is mainly expressed on activated T cells and functions as a co-stimulatory molecule. Engagement of OX40 has been shown to induce downregulation of CTLA-4. Blocking agents of OX40 can suppress the immune response, while agonists of OX40 enhance the immune response. Agonists of OX40 have the potential to enhance anti-tumor immunity. Crystallographic studies have revealed that the ligand of OX40 (OX40L) exists as a trimer. Furthermore, mouse studies comparing the anti-tumor activities of OX40 agonist antibodies using FcγR-expressing and deficient mice have shown the necessity of FcγR engagement and suggested the necessity of antibody cross-linking. Signaling of OX40 has been suggested to suppress the inhibitory ability of regulatory T cells and co-stimulate effector T cells. Agonism of OX40 has been shown to be important for promoting the differentiation into memory T cells and protecting memory T cells.

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[0145] In some embodiments, the fusion protein is multispecific and comprises at least a first binding domain, such as, for example, TBD, and a second binding domain for programmed death ligand 1 (PD-L1). In these embodiments, the binding to PD-L1 can provide an additional cross-linking function, and the activation of TNFRSF is achieved by only one or two TBDs. In these embodiments, the signaling of the TNFRSF is enhanced and concentrated by the presence of PD-L1-expressing cells.

[0146] PDL1 is a 40 kDa type I transmembrane protein that forms a complex with its receptor programmed cell death protein 1 (PD1), also known as CD279. Engagement of PDL1 with its receptor PD1 on T cells sends signals that inhibit TCR-mediated activation of IL-2 production and T cell proliferation. Aberrant expression and / or activity of PDL1 and PDL1-related signaling are involved in the pathogenesis of many diseases and disorders, such as cancer, inflammation, and autoimmunity.

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[0149] In some embodiments, the PDL1 binding domain comprises or is derived from a known anti-PDL1 antibody sequence or an antigen-binding fragment thereof. In some embodiments, the PDL1 binding domain comprises or is derived from the antibody sequence disclosed in PCT Publication No. WO2016 / 149201. The contents of such publication are hereby incorporated by reference in their entirety.

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[0168] In some embodiments, the fusion protein is multispecific, comprising a TBD and a binding domain for folate receptor alpha (FRα). In these embodiments, binding to FRα can provide an additional cross-linking function, and activation of TNFRSF can be achieved with only one or two TBDs. In these embodiments, the signaling of the TNFRSF is enhanced and focused by the presence of FRα-expressing cells.

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[0170] In some embodiments, the fusion protein is a tumor, bacterial, or viral antigen (such as a virion, In some embodiments, the vaccine comprises a TBD fused to a TBD (a cutin sequence). In these embodiments, binding to the TBD promotes enhanced immunogenicity of the vaccine sequence, thereby promoting adaptive immunity against tumors, bacteria, or viruses expressing the vaccine sequence. In some embodiments, the TBD and vaccine are not fused and may be introduced separately. In these embodiments, the vaccine may be a nucleic acid sequence, a protein sequence, or a whole cell, e.g., a tumor cell, a bacterial cell, or a virus. Vaccines may be monovalent (also called univalent) or multivalent (also called polyvalent). Monovalent vaccines are designed to immunize against a single antigen or a single microorganism or cancer type. Multivalent vaccines are designed to immunize against two or more strains of the same microorganism or cancer type, or against two or more different microorganisms or cancer types.

[0171] The present disclosure is further described in the following examples, which do not limit the scope of the disclosure, which is described in the claims. EXAMPLES

[0172] Example 1. The OX40-targeted single-domain antibody binds to OX40 As used herein, the OX40-targeted single-domain antibodies (sdAbs) designated 1A06 (SEQ ID NO: 16), 2B07, 2C09 (SEQ ID NO: 19), 1D10 (SEQ ID NO: 22), 2E4 (SEQ ID NO: 18), 2H06 (SEQ ID NO: 17), 3E11 (SEQ ID NO: 23), 3G9 (SEQ ID NO: 24), and G3 (SEQ ID NO: 25) bind to cell surface OX40 expressed on CHO cells (Figs. 2A-2B). Binding was evaluated by flow cytometry using CHO cells expressing OX40, and the data are shown as the median fluorescence intensity.

[0173] Similarly, the OX40-targeted sdAbs designated 1D10, G3, and 3E11 herein were evaluated for their ability to bind to cynomolgus OX40 expressed on CHO cells (Fig. 4). Binding was evaluated by flow cytometry using CHO cells expressing cynoOX40, and the data are shown as the median fluorescence intensity.

[0174] Similarly, various humanized forms of the OX40-targeted sdAbs conjugated to the Fc region were evaluated for their ability to bind to human OX40 and cynomolgus OX40 (Figs. 12A and 12B). Binding was evaluated by flow cytometry using CHO cells expressing OX40, and the data are shown as the median fluorescence intensity.

[0175] Example 2. The OX40-targeted single-domain antibody blocks OX40

[0176] As used herein, the OX40-targeted single-domain antibodies (sdAbs) designated 1D10 (SEQ ID NO: 22), 2E4 (SEQ ID NO: 18), G3 (SEQ ID NO: 25), 3E11 (SEQ ID NO: 23), and H11 block the interaction between OX40 and OX40L. As shown in Fig. 3, 2E4 blocks the interaction between OX40 and OX40L, while the other single-domain antibodies do not. A single-domain antibody that binds to GITR was included as a negative control. Blocking was evaluated by flow cytometry using an OX40L citrine fusion protein, and the data are shown as the median fluorescence intensity.

[0177] Example 3. Multivalent OX40-targeting molecules For example, multiple copies of a binding domain such as a single domain antibody (sdAb) can be operably linked to produce a multivalent OX-40-targeting molecule. In some embodiments, multiple OX40-targeting VHHs are operably linked to a polypeptide of the Fc region to produce a multivalent OX40-targeting molecule.

[0178] Figure 5A is a schematic diagram showing the formats of various embodiments of a multivalent OX40-binding fusion protein and their respective estimated molecular weights. Figure 5B is a photograph of a Coomassie blue-stained SDS-PAGE gel of the multivalent OX40-binding fusion protein under reducing and non-reducing conditions.

[0179] The OX40 multivalent molecule shows that the enhancement of OX40 signaling is mediated by higher valency binding. Figure 6 shows a comparison between tetravalent and bivalent binding of OX40 using the fusion proteins of the present disclosure. OX40 signaling was observed using an NF-kB reporter 293 cell line expressing OX40. A fusion protein incorporating the 1D10 binding domain (SEQ ID NO: 22) as the TBD was used in these assays.

[0180] Figure 7A shows a comparison between tetravalent and hexavalent binding of OX40 using the fusion proteins of the present disclosure. Figure 7B shows a comparison between the hexavalent binding of OX40 using the fusion proteins of the present disclosure and a known hexameric OX40L fusion protein described in U.S. Patent No. 7,959,925. The multivalent OX40-binding fusion protein of the present disclosure showed equivalent or enhanced OX40 agonist activity compared to the hexameric OX40L fusion protein. OX40 signaling was observed using an NF-kB reporter 293 cell line expressing OX40. A fusion protein incorporating 1D10 as the TBD was used in these assays.

[0181] Figures 8A and 8B are a series of graphs showing the elution profiles from a size exclusion chromatography (SEC) column (Superdex 200) for tetravalent (Figure 8A) and hexavalent (Figure 8B) 1D10. The molecular weights calculated based on the retention volume (standard curve) are also shown.

[0182] Figure 9 is a graph showing the signal transduction of OX40 mediated by various multivalent formats of the fusion proteins of the present disclosure, including hexavalent with three tandem OX40 VHHs linked to the Fc region, tetravalent with two tandem OX40 VHHs linked to the Fc region, and tetravalent with N-terminal and C-terminal OX40 VHHs separated in the Fc region. The signal transduction of OX40 was observed using an NF-κB reporter 293 cell line expressing OX40. Fusion proteins incorporating 1D10 as the TBD were used in these assays.

[0183] Figures 14A and 14B are a pair of graphs showing that the signal transduction of OX40 is mediated by various multivalent formats of the fusion proteins of the present disclosure, including hexavalent with three tandem OX40 VHHs linked to the Fc region, tetravalent with two tandem OX40 VHHs linked to the Fc region, and tetravalent with N-terminal and C-terminal OX40 VHHs separated in the Fc region. The signal transduction of OX40 was observed using an NF-κB reporter 293 cell line expressing OX40. For these assays, fusion proteins incorporating hzG3v9 as the TBD into the tetravalent and hexavalent molecules were used.

[0184] Figures 10A, 10B, and 10C show PDL1-dependent OX40 agonism mediated by the bispecific PDL1-OX40 targeting fusion protein of the present disclosure. Figures 10A and B are conceptual diagrams showing that the bispecific fusion protein has only slight OX40 agonist properties (Figure 10A) unless PDL1-expressing cells bind (Figure 10B). Figure 10C is a graph showing that PDL1-positive cells (here PDL1-transfected CHO cells) have the ability to mediate OX40 signaling and PDL1-negative cells (here non-transfected CHO cells) do not have the ability to mediate OX40 signaling. OX40 signaling was observed using an NF-kB reporter 293 cell line expressing OX40. This figure shows many different bispecific fusion proteins, each containing a different OX40-binding VHH (e.g., G3, 2E4, 3G9, 1D10) and the same PDL1 VHH, namely 28A10.

[0185] Figure 11 shows the signaling of an FRα-dependent OX40 agonist mediated by the bispecific FRa-OX40 targeting fusion protein of the present disclosure. An ovarian cancer cell line expressing FRα, namely SKOV3, was used in these assays. OX40 signaling was observed using an NF-kB reporter 293 cell line expressing OX40. This figure shows many different bispecific fusion proteins, each containing a different FRα-binding VHH (e.g., 1G10, 1A3, 57, 5) and the same OX40 VHH, namely 1D10.

[0186] Figures 13A and 13B are a pair of graphs showing whether various humanized OX40 single-domain antibodies bind to various TNFRSF members. For these assays, various humanized sdAbs of the present disclosure were used as TBDs in tetravalent and hexavalent molecules.

Claims

1. An isolated polypeptide that binds to OX40 and comprises multiple tumor necrosis factor receptor superfamily (TNFRSF) binding domains (TBDs), wherein at least a first TBD (TBD1) binds to OX40.

2. 1. An isolated polypeptide that binds to PDL1 and comprises multiple polypeptide binding domains (BDs), wherein at least a first BD (BD1) binds to PDL1.

3. An isolated polypeptide that binds to PLD1 and OX40 and comprises multiple polypeptide binding domains (BDs) and multiple tumor necrosis factor receptor superfamily (TNFRSF) binding domains (TBDs), wherein at least a first binding domain (BD1) binds to PLD1 and at least a first TNFRSF binding domain (TBD1) binds to OX40.

4. The isolated polypeptide of any one of claims 1 to 3, which is monospecific.

5. The isolated polypeptide of any one of claims 1 to 3, which is multispecific.

6. The isolated polypeptide of any one of claims 1 to 3, which is bispecific.

7. 4. The isolated polypeptide of claim 1, wherein the polypeptide comprises at least a second TBD (TBD2), which binds to a second TNFRSF member.

8. The isolated polypeptide of claim 1 , wherein each of the TBDs in the plurality of TBDs binds to OX40.

9. The isolated polypeptide of claim 8 , wherein the multiple TBDs bind to the same epitope on OX40.

10. The isolated polypeptide of claim 8 , wherein at least two of the TBDs bind to different epitopes on OX40.

11. The isolated polypeptide of any one of claims 8 to 10, wherein the multiple TBDs comprise at least four TBDs.

12. The isolated polypeptide of any one of claims 8 to 10, wherein the multiple TBDs comprise at least six TBDs.

13. The isolated polypeptide of claim 2 , wherein the polypeptide comprises at least a second BD (BD2), which binds to a second antigen.

14. The isolated polypeptide of claim 2, wherein each BD in the plurality of BDs binds to PDL1.

15. 15. The isolated polypeptide of claim 14, wherein the multiple BDs bind to the same epitope on PDL1.

16. 15. The isolated polypeptide of claim 14, wherein at least two BDs in the plurality of BDs bind to different epitopes on PDL1.

17. The isolated polypeptide according to any one of claims 13 to 16, wherein the multiple BDs comprise at least four BDs.

18. The isolated polypeptide according to any one of claims 13 to 16, wherein the plurality of BDs comprises at least six BDs.

19. The isolated polypeptide of any one of claims 1 to 3, wherein each of the TBDs in the plurality of TBDs, or each of the BDs in the plurality of BDs, is operably linked via a linker polypeptide.

20. 4. An isolated polypeptide according to any one of claims 1 to 3, comprising at least one binding domain that binds to a target, with the proviso that the target is not a TNFRSF member.

21. 2. The isolated polypeptide of claim 1, wherein at least one of the TBDs among the plurality of TBDs binds to a second TNFRSF member selected from the group consisting of DR5, GITR, and CD137.

22. 4. The isolated polypeptide of claim 1, comprising a heterodimerization domain.

23. The isolated polypeptide of any one of claims 1 to 3, comprising an immunoglobulin Fc region polypeptide.

24. 24. The isolated polypeptide of claim 23, wherein the immunoglobulin Fc region polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6.

25. 25. The isolated polypeptide of any one of claims 1 to 24, wherein at least one TBD of the plurality of TBDs or at least one BD of the plurality of BDs comprises an antibody or an antigen-binding fragment thereof.

26. 25. The isolated polypeptide of any one of claims 1 to 24, wherein each TBD in the plurality of TBDs or each BD in the plurality of BDs comprises an antibody or an antigen-binding fragment thereof.

27. The antibody or antigen-binding fragment thereof may be an scFv, Fab, single domain antibody (sdAb), V ＮＡＲ 27. The isolated polypeptide of claim 25 or claim 26, which is a VHH.

28. 27. The isolated polypeptide of claim 25 or claim 26, wherein the antibody or antigen-binding fragment thereof is an sdAb.

29. 29. The isolated polypeptide of claim 28, wherein the sdAb is a human or humanized sdAb.

30. The sdAb is ＮＡＲ 29. The isolated polypeptide of claim 28, which is an artificially created VH domain or an artificially created VK domain.

31. 31. The isolated polypeptide of claim 30, wherein the sdAb is generated from a cartilaginous heavy chain only antibody.

32. 2. The isolated polypeptide of claim 1, wherein at least one of the binding domains comprises a non-antibody scaffold protein.

33. 33. The isolated polypeptide of claim 32, wherein the non-antibody scaffold protein is an ankyrin repeat protein, a darpin, an avimer, an anticalin / lipocalin, centirin, or a finomer.

34. 4. The isolated polypeptide of claim 1 or claim 3, wherein the polypeptide comprises an amino acid sequence that binds to OX40, selected from the group consisting of SEQ ID NOs: 16-29 and 377-386.

35. 4. The isolated polypeptide of claim 1 or claim 3, wherein the polypeptide binds to OX40 and comprises an amino acid sequence comprising a complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 36, and 44, a complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 35, and 37, and a complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-34, 48-43, and 45.

36. 4. The isolated polypeptide of claim 2 or claim 3, wherein the polypeptide comprises an amino acid sequence that binds to PDL1, selected from the group consisting of SEQ ID NOs: 52-57.

37. 4. The isolated polypeptide of claim 2 or claim 3, wherein the polypeptide binds to PDL1 and comprises an amino acid sequence comprising a complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 58, 61, and 64, a complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 59, 62, 65, and 69, and a complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 60, 63, 66-68, and 70.

38. 4. The isolated polypeptide of claim 3, wherein the polypeptide comprises a first amino acid sequence that binds to OX40, selected from the group consisting of SEQ ID NOs:52-57, and a second amino acid sequence that binds to PDL1, selected from the group consisting of SEQ ID NOs:52-57.

39. 4. The isolated polypeptide of claim 3, wherein the polypeptide binds to OX40 and comprises an amino acid sequence comprising a complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 36, and 44, a complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 35, and 37, and a complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-34, 48-43, and 45, and wherein the polypeptide binds to PDL1 and comprises an amino acid sequence comprising a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 58, 61, and 64, a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 59, 62, 65, and 69, and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 60, 63, 66-68, and 70.

40. The isolated polypeptide of any one of claims 1 to 3, wherein the polypeptide is tetravalent.

41. 41. The isolated polypeptide of claim 40, wherein the polypeptide comprises the structure: VHH-linker-VHH-linker-hinge-Fc, wherein the VHH is a humanized or fully human VHH sequence.

42. The isolated polypeptide of any one of claims 1 to 3, wherein the polypeptide is hexavalent.

43. 43. The isolated polypeptide of claim 42, wherein the polypeptide comprises the structure: VHH-linker-VHH-linker-VHH-linker-hinge-Fc, wherein the VHH is a humanized or fully human VHH sequence.

44. 1. An isolated polypeptide that binds to OX40 and PDL1, said polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 387-394.

45. Use of a polypeptide according to any one of claims 1 to 44 for the treatment of a neoplasm.

46. Use of a polypeptide according to any one of claims 1 to 44 for modulating immune cells to enhance the destruction of tumors.

47. Use of a polypeptide according to any one of claims 1 to 44 for the treatment of an inflammatory disease.

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