Anti-PD-1×4-1BB binding protein

Abstract

Bispecific anti-4-1BB / anti-PD-1 binding proteins, including anti-4-1BB and anti-PD-1 binding proteins, and conditionally active derivatives thereof, are provided. Therapeutic and diagnostic methods using the binding proteins are provided.

Description

[Technical Field]

[0001] The present disclosure relates to immunoglobulin single variable domains (ISVs) directed against the costimulatory molecule 4-1BB. Through binding to 4-1BB, these proteins can activate 4-1BB-expressing cells, such as T cells. It also relates to anti-PD-1 binding proteins that can prevent inhibition of T-cell activation. It further relates to bispecific antigen-binding proteins that bind to both 4-1BB and PD-1, as well as conditionally active derivatives thereof. Such proteins are useful in the field of immuno-oncology. [Background technology]

[0002] The crucial factor in cancer treatment is the patient's own immune system. The immune system has the ability to attack and destroy tumor cells. However, some factors can prevent the immune system from responding effectively, for example, the tumor microenvironment inhibiting immune cells. In particular, tumors can prevent or hinder the activation of T cells.

[0003] T cell activation is a complex process that depends on several signals. This includes stimulation of T cells via T cell receptor (TCR) signaling and costimulatory signals via other receptors. Costimulation is important for effective T cell activation. One important costimulatory molecule is tumor necrosis factor receptor superfamily member 9 (TNFRSF9, also known as 4-1BB or CD137, encoded by the TNFRSF9 gene), a member of the tumor necrosis factor (TNF) receptor superfamily. Its expression is transiently induced by TCR signaling. 4-1BB was initially identified in mice by a modified differential screening procedure. The human homolog of 4-1BB was cloned from a library of activated human T-cell leukemia virus type 1-transformed human T lymphocytes.

[0004] 4-1BB is a costimulatory molecule that plays a role in the expansion of T cell memory, acquisition of effector function, survival, and development. It is involved in the activation of activated T cells, NK cells, NKT cells, and T reg In addition to being expressed on inflammatory cells, 4-1BB is also expressed on several cell types of the hematopoietic lineage, as well as endothelial and epithelial cells. The primary ligand of 4-1BB, 4-1BBL (TNFSF9), is expressed primarily on professional antigen-presenting cell (APC) populations, B cells, macrophages, and other cell types. Upon binding to 4-1BB, it induces signaling via TRAF1 and TRAF2, activating the NF-κB, AKT, p38 MAPK, and ERK pathways. These signaling pathways induce the expression of survival genes encoding survivin, Bcl-2, Bcl-XL, and Bfl-1, and reduce the expression of proapoptotic Bim. Thus, the 4-1BB / 4-1BBL signaling pathway may promote the survival of different cell types. Interestingly, the phenotypes responding to viral challenge differ between 4-1BB and its ligand. For example, 4-1BBL-deficient mice exhibit poor expression of memory CD8 + While showing reduced T cell accumulation, 4-1BB-deficient mice also exhibit reduced memory but acute CD8 + T cell accumulation is enhanced. This type of result indicates bidirectional signaling or distinct receptor signaling mechanisms. Other models of infection indicate that the effects of 4-1BB blockade depend on the context and disease characteristics. For example, infections that are quickly cleared or result in minimal inflammation are often unaffected by 4-1BB deficiency, whereas chronic or hyperinflammatory diseases require 4-1BB for memory development and / or viral clearance.

[0005] In tumor immunotherapy, it is desirable to activate the 4-1BB signaling pathway, which leads to T cell proliferation, increased effector activity (including cytokine production), memory formation, resistance to apoptosis, and methylation reprogramming. An obvious approach for such stimulation is agonistic antibodies. Several such antibodies have been developed by various companies, including urelumab and utomilumab. However, this approach faces several challenges due to the structure of 4-1BB: activated 4-1BB and its ligands exist primarily as trimers on the cell surface. This can complicate effective targeting of 4-1BB with conventional antibodies, which have only two binding sites; therefore, they may not provide agonistic stimulation that results in signal activation as effective as 4-1BB trimers.

[0006] In principle, this lack of stoichiometry can be compensated for by cross-linking multiple antibody molecules, for example, by binding to Fc receptors (FcRs). However, such an approach may cause severe side effects by increasing the systemic toxic effects of 4-1BB.

[0007] Therefore, it would be advantageous to have a molecule that acts as an effective agonist of 4-1BB, but does not require cross-linking, for example, FCR-mediated cross-linking or any other target-mediated cross-linking, such as tumor-associated antigen-mediated cross-linking, immune cell surface marker-mediated cross-linking, stromal antigen protein-mediated cross-linking, or cross-linking mediated by any other target expressed in cis or trans by tumor cells, immune cells, and / or normal cells.

[0008] The present invention provides such a molecule, whose activating effect is a pure agonist-like effect of anti-4-1BB V, whose activity is independent of any cross-linking. HH Based on.

[0009] In a further aspect, it has been observed that the efficacy of an anti-4-1BB binding moiety can be significantly enhanced when the anti-4-1BB binding moiety is conjugated to another moiety, such as one binding to the inhibitory receptor programmed death-1 (PD-1). Hypothetically, this may be explained by delivery of the anti-4-1BB binding moiety to PD-1 in cis, thereby allowing the anti-PD-1 binding moiety to bind to PD-1. + CD8 + It may serve to anchor on T cells or promote their clustering.

[0010] PD-1 (also called CD279) is a 288 amino acid protein receptor expressed on activated T and B cells, natural killer cells, and monocytes. PD-1 is a member of the CD28 / CTLA-4 (cytotoxic T lymphocyte antigen) / ICOS (inducible costimulator) family of T cell co-inhibitory receptors. Its primary function is to dampen immune responses. PD-1 has two ligands, PD-ligand 1 (PD-L1) and PD-L2. PD-L1 (also called CD274 or B7H1) binds to CD4 + and CD8 + PD-L2 is widely expressed on both lymphoid and non-lymphoid tissues, including T cells, macrophage-lineage cells, peripheral tissues, and tumor and virus-infected cells. PD-L2 (also known as CD273 or B7-DC) is more restricted in expression than PD-L1 and is expressed on activated dendritic cells and macrophages. PD-L1 is expressed in most human cancers, including melanoma, glioma, non-small cell lung cancer, head and neck squamous cell carcinoma, leukemia, pancreatic cancer, renal cell carcinoma, and hepatocellular carcinoma, and can be inducible in almost all cancer types. PD-1 binding to its ligand results in reduced T-cell proliferation and cytokine secretion, impairing humoral and cellular immune responses in diseases such as cancer or viral infection. Blockade of PD-1 binding for reverse immunosuppression is being investigated in viral immunotherapy and tumor immunotherapy.

[0011] T cell costimulatory and co-inhibitory molecules (collectively referred to as co-signaling molecules) play important roles in regulating T cell activation, subset differentiation, effector function, and survival. After TCR recognition of a cognate peptide-MHC complex on an APC, co-signaling receptors colocalize with the TCR at the immune synapse and synergize with TCR signaling to promote or inhibit T cell activation and function. The final immune response is regulated by the balance between costimulatory and co-inhibitory signals ("immune checkpoints"). PD-1 functions as one such "immune checkpoint" in mediating peripheral T cell tolerance and avoiding autoimmunity: PD-1 binding to PD-L1 or PD-L2 inhibits T cell activation. This ability of PD-1 to inhibit T cell activation is exploited by chronic viral infections and tumors to evade immune responses. In chronic viral infections, PD-1 is highly expressed on virus-specific T cells, and these T cells become "exhausted" with loss of effector function and proliferative capacity. PD-L1 is expressed on a wide variety of tumors, and studies in animal models have shown that PD-L1 on tumors can inhibit T cell activation and lysis of tumor cells, leading to increased killing of tumor-specific T cells. The PD-1 / PD-L1 complex also mediates the induction of T regulatory T cells (T reg ) cell development and T reg It plays an important role in maintaining function.

[0012] PD-1 plays an important role in autoimmunity, tumor immunity, and infection immunity, making it an ideal target for immunotherapy. Blockade of PD-1 with antagonists, including monoclonal antibodies, is being investigated in the treatment of cancer and chronic viral infections. Summary of the Invention [Problem to be solved by the invention]

[0013] The present invention provides such PD-1 antagonists. [Means for solving the problem]

[0014] In a first aspect, there is provided a multispecific antigen binding protein comprising at least one immunoglobulin single variable domain (ISV) that specifically binds to 4-1BB, wherein at least one ISV has pure agonist activity.

[0015] In some embodiments, pure agonist activity means that at least one ISV can activate T cells via 4-1BB signaling (i) under soluble conditions, and / or (ii) in the absence of a cross-linking reagent, and / or (iii) in an FcγR-independent manner (i.e., independent of Fcγ receptor engagement), and / or (iv) in the absence of target-mediated cross-linking of 4-1BB. In some embodiments, pure agonist activity can be determined in the absence of a cross-linking reagent, e.g., by an NF-κB pathway activation assay in the absence of an anti-human Fab antibody.

[0016] In some embodiments, at least one ISV competes with 4-1BBL for 4-1BB binding.

[0017] In some embodiments, at least one ISV interacts with the cysteine-rich domain 2 (CRD2) and / or cysteine-rich domain 3 (CRD3) domains of 4-1BB; preferably, the ISV interacts with the CRD2 and CRD3 domains of 4-1BB.

[0018] In some embodiments, the 4-1BB is human 4-1BB, the exemplary amino acid sequence of which is SEQ ID NO:13.

[0019] In some embodiments, at least one ISV interacts with at least one amino acid residue of 4-1BB selected from the group consisting of residues K69, G70, V71, F72, R73, F92, L95, S100, M101, C102, E103, Q104, K114, K115, and G116 of SEQ ID NO: 13.

[0020] In some embodiments, at least one ISV is HH is.

[0021] In some embodiments, at least one ISV comprises three complementarity determining regions CDR1, CDR2 and CDR3; CDR3 comprises or consists of the amino acid sequence ARGTRYKLST (SEQ ID NO: 14), ARGTRYKMST (SEQ ID NO: 15), or ARGTRYKIFA (SEQ ID NO: 62).

[0022] In some embodiments, CDR1 comprises or consists of the amino acid sequence GFTFSDHT (SEQ ID NO: 16), GFAFRDFT (SEQ ID NO: 66), GDTFSSYA (SEQ ID NO: 67), or GFTFANYR (SEQ ID NO: 68).

[0023] In some embodiments, CDR2 comprises or consists of the amino acid sequence ISSGGSRI (SEQ ID NO: 17), INPSGSQ (SEQ ID NO: 77), or IKKSGNRT (SEQ ID NO: 78).

[0024] In some embodiments, at least one ISV: (i) an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 58, 59, 60, and 61; or (ii) an amino acid sequence that shares at least 70% sequence identity across the non-CDR regions of SEQ ID NO: 2, 3, 58, 59, 60, or 61; It comprises or consists of:

[0025] In some embodiments, the ISV comprises or consists of the amino acid sequence of SEQ ID NO: 2 or 3. In some embodiments, the ISV comprises or consists of the amino acid sequence of SEQ ID NO: 3.

[0026] In some embodiments, the multispecific antigen binding protein comprises at least two ISVs that specifically bind to 4-1BB.

[0027] In some embodiments, at least two ISVs that specifically bind to 4-1BB are identical. Alternatively, at least two ISVs that specifically bind to 4-1BB can be different and then bind to (i) the same epitope, (ii) overlapping epitopes, or (iii) distinct epitopes of 4-1BB. In some embodiments, at least two ISVs that specifically bind to 4-1BB are different and bind to distinct epitopes of 4-1BB.

[0028] In some embodiments, the at least second ISV that specifically binds to 4-1BB comprises: an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 4, or - an amino acid sequence that shares at least 70% sequence identity over the non-CDR regions of SEQ ID NO: 1 or 4 It comprises or consists of:

[0029] In some embodiments, at least the second ISV comprises or consists of an amino acid sequence having SEQ ID NO:4.

[0030] In some embodiments, the multispecific antigen binding protein comprises at least two ISVs, one of which specifically binds to 4-1BB and another of which specifically binds to another target antigen, which in some embodiments may be a T cell antigen, a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA), or a non-self antigen.

[0031] In some embodiments, the multispecific antigen binding protein comprises at least four ISVs that specifically bind to 4-1BB. In some embodiments, these at least four ISVs are -(i) a first set of at least two identical ISVs that specifically bind to 4-1BB, and (ii) a second set of at least two other identical ISVs that specifically bind to 4-1BB; or - (i') a first set of at least two ISVs that specifically bind to a first epitope of 4-1BB, and (ii') a second set of at least two other ISVs that specifically bind to a second epitope of 4-1BB. Includes.

[0032] In some embodiments, the first set of at least two ISVs (i) or (i') above is an ISV that specifically binds to 4-1BB with pure agonist activity as defined above (for example, but not limited to, an ISV that includes or consists of the amino acid sequence of SEQ ID NO: 2 or 3).

[0033] In some embodiments, the second set of at least two ISVs of (ii) or (ii') above is an ISV that specifically binds to 4-1BB as defined above (for example, but not limited to, an ISV that includes or consists of the amino acid sequence of SEQ ID NO: 1 or 4).

[0034] In some embodiments, the multispecific antigen binding protein further comprises an antibody Fc region or fragment thereof. In some embodiments, the Fc region or fragment thereof is ADCC and / or ADCP silenced.

[0035] In some embodiments, the multispecific antigen binding protein further comprises at least one Fab fragment.

[0036] In some embodiments, the multispecific antigen binding protein comprises: (a) preferably from the N-terminus to the C-terminus: a first ISV that specifically binds to -4-1BB; a second ISV that specifically binds to -4-1BB, preferably different from the first ISV; - at least one C in the Fc region H domain; and - variable and constant domains of the Fab fragment a first polypeptide comprising: (b) a second polypeptide comprising the variable and constant domains of the Fab fragment; Includes; The variable and constant domains of the first and second polypeptides form a Fab fragment.

[0037] In some embodiments, the multispecific antigen binding protein further comprises a third polypeptide and a fourth polypeptide that are identical to the first polypeptide and the second polypeptide, respectively, and wherein at least one C of the first polypeptide and the third polypeptide is H The domains form the Fc region.

[0038] In some embodiments, The variable domain and the constant domain of the first polypeptide are V H and C H the variable and constant domains of the second polypeptide are V L and C L domain, or The variable domain and the constant domain of the first polypeptide are V L and C L domain, and the variable and constant domains of the second polypeptide are V H and C H It is one domain.

[0039] In some embodiments, at least one C of the first polypeptide H The domain is - IgG C H 2 and C H 3 domains; -IgD C H 2 and C H 3 domains; -IgA C H 2 and C H 3 domains; -IgM C H 2. C H 3 and C H 4 domains; or -IgE C H 2. CH 3, and C H 4 Domains Includes.

[0040] In some embodiments, at least one C of the first polypeptide H The domain is C of IgG H 2 domain and C H In some embodiments, at least one C of the first polypeptide comprises three domains. H The domain is C of IgG1 or IgG4. H 2 and C H In some embodiments, at least one C of the first polypeptide comprises three domains. H The domain is C of IgG1 H 2 domain and C H Includes 3 domains.

[0041] In some embodiments, the first polypeptide of the multispecific antigen binding protein preferably comprises, from N-terminus to C-terminus: a first ISV that specifically binds to -4-1BB; - a first linker; a second ISV that specifically binds to -4-1BB, preferably the second ISV is different from the first ISV; - a second linker; -IgG hinge region; -IgGC H 2 domains; - and IgGC H 3 domains; - a third linker; - V of Fab fragment H domain; and -Fab fragment C H 1 domain Includes.

[0042] In some embodiments, the second polypeptide of the multispecific antigen binding protein preferably comprises from N-terminus to C-terminus: - V of Fab fragment L domain; and -Fab fragment C L domain Includes.

[0043] In some embodiments, at least one Fab fragment specifically binds to a B cell surface protein and / or a T cell surface protein other than 4-1BB. In some embodiments, at least one Fab fragment specifically binds to an immune checkpoint molecule. In some embodiments, at least one Fab fragment is a PD-1 antagonist. In some embodiments, at least one Fab fragment specifically binds to PD-1. - the three light chain complementarity determining region (CDR) sequences found in SEQ ID NO: 7 or 5, and - the three heavy chain CDR sequences found in SEQ ID NO: 8 or 6 is an antigen-binding protein comprising:

[0044] In some embodiments, at least one Fab fragment specifically binds to PD-1; (i) the following three CDR sequences: - VL-CDR1: QSVPINF (SEQ ID NO: 18) or QSVSINF (SEQ ID NO: 19); VL-CDR2:EAS; and - VL-CDR3: GQYGSSPYT (SEQ ID NO: 20) or QQYGSSPYT (SEQ ID NO: 21) a light chain variable region comprising: (ii) the following three CDR sequences: - VH-CDR1: GGSISSSSYF (SEQ ID NO: 22) or GGSISTSSYF (SEQ ID NO: 23); VH-CDR2: IYRSGST (SEQ ID NO: 24); and - VH-CDR3: ARGITGDPGDY (SEQ ID NO: 25) a heavy chain variable region comprising is an antigen-binding protein comprising:

[0045] In some embodiments, at least one Fab fragment specifically binds to PD-1; (i) the following three CDR sequences: - VL-CDR1: QSVPINF (SEQ ID NO: 18); VL-CDR2:EAS; and -VL-CDR3: GQYGSSPYT (SEQ ID NO: 20) a light chain variable region comprising: (ii) the following three CDR sequences: - VH-CDR1:GGSISSSSYF (SEQ ID NO: 22); VH-CDR2: IYRSGST (SEQ ID NO: 24); and - VH-CDR3: ARGITGDPGDY (SEQ ID NO: 25) a heavy chain variable region comprising is an antigen-binding protein comprising:

[0046] In some embodiments, at least one Fab fragment specifically binds to PD-1; - a light chain variable region having SEQ ID NO: 7 or 5, or a light chain variable region sharing at least 70% sequence identity over the non-CDR regions of SEQ ID NO: 7 or 5; and - a heavy chain variable region having SEQ ID NO: 8 or 6, or a heavy chain variable region sharing at least 70% sequence identity across the non-CDR regions of SEQ ID NO: 8 or 6 is an antigen-binding protein comprising:

[0047] In some embodiments, at least one Fab fragment is an antigen binding protein that specifically binds to PD-1 and comprises a light chain variable region having SEQ ID NO:7 and a heavy chain variable region having SEQ ID NO:8.

[0048] In some embodiments, the multispecific antigen binding proteins provided herein comprise: - at least a first polypeptide having SEQ ID NO: 11 or 9 and at least a second polypeptide having SEQ ID NO: 12 or 10; or - at least a first polypeptide that shares at least 70% of sequence identity over a non-CDR region of SEQ ID NO: 11 or 9, and at least a second polypeptide that shares at least 70% of sequence identity over a non-CDR region of SEQ ID NO: 12 or 10. Includes.

[0049] In some embodiments, the multispecific antigen binding proteins provided herein comprise at least a first polypeptide having SEQ ID NO:11 and at least a second polypeptide having SEQ ID NO:12.

[0050] In some embodiments, the multispecific antigen binding proteins provided herein comprise at least a first polypeptide having SEQ ID NO:9 and at least a second polypeptide having SEQ ID NO:10.

[0051] In a second aspect, a conditionally active multispecific antigen binding protein is provided.

[0052] In some embodiments, the conditionally active multispecific antigen binding protein comprises: a multispecific antigen-binding protein as defined above, and at least one masking moiety that reduces or inhibits binding of the multispecific antigen-binding protein to at least one of its target antigens; Includes.

[0053] In some embodiments, at least one masking moiety comprises or consists of the amino acid sequence of SEQ ID NO: 97, or an amino acid sequence that shares at least 70% sequence identity with SEQ ID NO: 97. In some embodiments, at least one masking moiety comprises or consists of the amino acid sequence of SEQ ID NO: 44 or 45, or an amino acid sequence that shares at least 70% sequence identity with SEQ ID NO: 44 or 45.

[0054] In some embodiments, the conditionally active multispecific antigen-binding protein further comprises at least one linker between the multispecific antigen-binding protein and the masking moiety. In some embodiments, at least one linker is cleavable. In some embodiments, at least one linker is cleavable by at least one tumor-specific protease. In some embodiments, the at least one tumor-specific protease is selected from the group consisting of matrix metalloproteinase-9 (MMP-9), urokinase-type plasminogen activator (uPa), matrix metalloproteinase-2 (MMP-2), matriptase, regumain, kallikrein-related peptidase-3, human neutrophil elastase, proteinase 3 (Pr3), cathepsin B, and cathepsin K. In some embodiments, the at least one tumor-specific protease is MMP-9 or uPa, or a combination thereof. In some embodiments, at least one linker comprises the amino acid sequence of SEQ ID NO: 56 and / or 57. In some embodiments, at least one linker comprises or consists of the amino acid sequence of SEQ ID NO:46 or 47.

[0055] In some embodiments, the conditionally active multispecific antigen binding protein comprises: a. at least a first polypeptide having SEQ ID NO: 11 or 9; and b. at least a second polypeptide having SEQ ID NO: 52, 53, 54, or 55 Includes.

[0056] In some embodiments, the conditionally active multispecific antigen binding protein further comprises a third polypeptide and a fourth polypeptide that are identical to the first polypeptide and the second polypeptide, respectively.

[0057] In a third aspect, there is provided an immunoglobulin single variable domain (ISV) that specifically binds to 4-1BB, wherein the ISV has pure agonist activity.

[0058] In some embodiments, pure agonist activity means that the ISV can activate T cells via 4-1BB signaling (i) under soluble conditions, and / or (ii) in the absence of a cross-linking reagent, and / or (iii) in an FcγR-independent manner (i.e., independent of Fcγ receptor engagement), and / or (iv) in the absence of target-mediated cross-linking of 4-1BB. In some embodiments, pure agonist activity can be determined by an NF-κB pathway activation assay in the absence of a cross-linking reagent.

[0059] In some embodiments, the ISV competes with 4-1BBL for 4-1BB binding.

[0060] In some embodiments, the ISV interacts with the cysteine-rich domain 2 (CRD2) and / or cysteine-rich domain 3 (CRD3) domains of 4-1BB. In some embodiments, the ISV interacts with the CRD2 and CRD3 domains of 4-1BB.

[0061] In some embodiments, the 4-1BB is human 4-1BB, the exemplary amino acid sequence of which is SEQ ID NO:13.

[0062] In some embodiments, the ISV interacts with at least one amino acid residue of 4-1BB selected from the group consisting of residues K69, G70, V71, F72, R73, F92, L95, S100, M101, C102, E103, Q104, K114, K115 and G116 of SEQ ID NO: 13.

[0063] In some embodiments, the ISV is HH is.

[0064] In some embodiments, the ISV comprises three complementarity determining regions CDR1, CDR2 and CDR3, and CDR3 comprises or consists of the amino acid sequence ARGTRYKLST (SEQ ID NO: 14), ARGTRYKMST (SEQ ID NO: 15), or ARGTRYKIFA (SEQ ID NO: 62).

[0065] In some embodiments, CDR1 comprises or consists of the amino acid sequence GFTFSDHT (SEQ ID NO: 16), GFAFRDFT (SEQ ID NO: 66), GDTFSSYA (SEQ ID NO: 67), or GFTFANYR (SEQ ID NO: 68).

[0066] In some embodiments, CDR2 comprises or consists of the amino acid sequence ISSGGSRI (SEQ ID NO: 17), INPSGSQ (SEQ ID NO: 77), or IKKSGNRT (SEQ ID NO: 78).

[0067] In some embodiments, the ISV: (i) an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 58, 59, 60, and 61; or (ii) an amino acid sequence that shares at least 70% sequence identity across the non-CDR regions of SEQ ID NO: 2, 3, 58, 59, 60, or 61; It comprises or consists of:

[0068] In some embodiments, the ISV comprises or consists of the amino acid sequence of SEQ ID NO: 2 or 3. In some embodiments, the ISV comprises or consists of the amino acid sequence of SEQ ID NO: 3.

[0069] In some embodiments, the ISV is HH is.

[0070] In a fourth aspect, there is provided an immunoglobulin single variable domain (ISV) that specifically binds to 4-1BB, the ISV comprising: an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 4, or - an amino acid sequence that shares at least 70% sequence identity over the non-CDR regions of SEQ ID NO: 1 or 4 It comprises or consists of:

[0071] In some embodiments, the ISV comprises or consists of an amino acid sequence having SEQ ID NO:4.

[0072] In some embodiments, the ISV is HH is.

[0073] In a fifth aspect, there is provided a bivalent or bispecific antigen-binding protein comprising at least one immunoglobulin single variable domain (ISV), wherein the bivalent or bispecific antigen-binding protein comprises at least a first ISV that specifically binds to 4-1BB with pure agonist activity as defined above (for example, but not limited to, an ISV comprising or consisting of the amino acid sequence of SEQ ID NO: 2 or 3), and at least a second ISV that specifically binds to the same or another target antigen.

[0074] In some embodiments, the other target antigen may be a T cell antigen, a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA), or a non-self antigen.

[0075] In some embodiments, at least the second ISV is an ISV that specifically binds to 4-1BB as defined above (for example, but not limited to, an ISV that includes or consists of the amino acid sequence of SEQ ID NO: 1 or 4).

[0076] In a sixth aspect, there is provided a conditionally active immunoglobulin single variable domain (ISV) that specifically binds to 4-1BB, the conditionally active ISV comprising: - an ISV that specifically binds to 4-1BB as defined above (for example, but not limited to, an ISV that comprises or consists of the amino acid sequence of SEQ ID NO: 1 or 4), and - at least one masking moiety that reduces or inhibits binding of the ISV to its target antigen Includes.

[0077] In some embodiments, at least one masking moiety comprises or consists of the amino acid sequence of SEQ ID NO: 97, or an amino acid sequence that shares at least 70% sequence identity with SEQ ID NO: 97. In some embodiments, at least one masking moiety comprises or consists of the amino acid sequence of SEQ ID NO: 44 or 45, or an amino acid sequence that shares at least 70% sequence identity with SEQ ID NO: 44 or 45.

[0078] In some embodiments, the conditionally active ISV further comprises at least one linker between the ISV and the masking moiety. In some embodiments, the at least one linker is cleavable. In some embodiments, the at least one linker is cleavable by at least one tumor-specific protease. In some embodiments, the at least one tumor-specific protease is selected from the group consisting of matrix metalloproteinase-9 (MMP-9), urokinase-type plasminogen activator (uPa), matrix metalloproteinase-2 (MMP-2), matriptase, regumain, kallikrein-related peptidase-3, human neutrophil elastase, proteinase 3 (Pr3), cathepsin B, and cathepsin K. In some embodiments, the at least one tumor-specific protease is MMP-9 or uPa, or a combination thereof. In some embodiments, the at least one linker comprises the amino acid sequence of SEQ ID NO: 56 and / or 57. In some embodiments, at least one linker comprises or consists of the amino acid sequence of SEQ ID NO:46 or 47.

[0079] In some embodiments, the ISV is HH is.

[0080] In some embodiments, the conditionally active ISV comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 48, 49, 50 and 51.

[0081] In a seventh aspect, there is provided a conditionally active immunoglobulin single variable domain (ISV) that specifically binds to 4-1BB, the conditionally active ISV comprising: -4-1BB-specific binding ISVs; and at least one masking moiety that reduces or inhibits binding of the ISV to 4-1BB Includes.

[0082] In some embodiments, at least one masking moiety comprises or consists of the amino acid sequence of SEQ ID NO: 97, or an amino acid sequence that shares at least 70% sequence identity with SEQ ID NO: 97. In some embodiments, at least one masking moiety comprises or consists of the amino acid sequence of SEQ ID NO: 44 or 45, or an amino acid sequence that shares at least 70% sequence identity with SEQ ID NO: 44 or 45.

[0083] In some embodiments, the conditionally active ISV further comprises at least one linker between the ISV and the masking moiety. In some embodiments, the at least one linker is cleavable. In some embodiments, the at least one linker is cleavable by at least one tumor-specific protease. In some embodiments, the at least one tumor-specific protease is selected from the group consisting of matrix metalloproteinase-9 (MMP-9), urokinase-type plasminogen activator (uPa), matrix metalloproteinase-2 (MMP-2), matriptase, regumain, kallikrein-related peptidase-3, human neutrophil elastase, proteinase 3 (Pr3), cathepsin B, and cathepsin K. In some embodiments, the at least one tumor-specific protease is MMP-9 or uPa, or a combination thereof. In some embodiments, the at least one linker comprises the amino acid sequence of SEQ ID NO: 56 and / or 57. In some embodiments, at least one linker comprises or consists of the amino acid sequence of SEQ ID NO:46 or 47.

[0084] In some embodiments, the ISV isHH is.

[0085] In an eighth aspect, there is provided a conditionally active immunoglobulin single variable domain (ISV) that specifically binds to a target antigen, the conditionally active ISV comprising: - ISVs that specifically bind to a target antigen; and - at least one masking moiety that reduces or inhibits binding of the ISV to its target antigen Includes.

[0086] In some embodiments, at least one masking moiety comprises or consists of the amino acid sequence of SEQ ID NO: 97, or an amino acid sequence that shares at least 70% sequence identity with SEQ ID NO: 97. In some embodiments, at least one masking moiety comprises or consists of the amino acid sequence of SEQ ID NO: 44 or 45, or an amino acid sequence that shares at least 70% sequence identity with SEQ ID NO: 44 or 45.

[0087] In some embodiments, the conditionally active ISV further comprises at least one linker between the ISV and the masking moiety. In some embodiments, the at least one linker is cleavable. In some embodiments, the at least one linker is cleavable by at least one tumor-specific protease. In some embodiments, the at least one tumor-specific protease is selected from the group consisting of matrix metalloproteinase-9 (MMP-9), urokinase-type plasminogen activator (uPa), matrix metalloproteinase-2 (MMP-2), matriptase, regumain, kallikrein-related peptidase-3, human neutrophil elastase, proteinase 3 (Pr3), cathepsin B, and cathepsin K. In some embodiments, the at least one tumor-specific protease is MMP-9 or uPa, or a combination thereof. In some embodiments, the at least one linker comprises the amino acid sequence of SEQ ID NO: 56 and / or 57. In some embodiments, at least one linker comprises or consists of the amino acid sequence of SEQ ID NO:46 or 47.

[0088] In some embodiments, the ISV is HH is.

[0089] In a ninth aspect, there is provided an antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising: (i) three light chain complementarity determining region (CDR) sequences set forth in SEQ ID NO: 5 or 7, and (ii) the three heavy chain CDR sequences set forth in SEQ ID NO: 6 or 8 Includes.

[0090] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to PD-1. In some embodiments, the PD-1 is human PD-1, an exemplary amino acid sequence of which is SEQ ID NO:42.

[0091] In some embodiments, the antibody or antigen-binding fragment thereof is: (i) the following three CDR sequences: aV L CDR1: QSVPINF (SEQ ID NO: 18) or QSVSINF (SEQ ID NO: 19); bV L CDR2:EAS; and cV L - CDR3: GQYGSSPYT (SEQ ID NO: 20) or QQYGSSPYT (SEQ ID NO: 21) a light chain variable region comprising: (ii) the following three CDR sequences: aV H CDR1: GGSISSSSYF (SEQ ID NO: 22) or GGSISTSSYF (SEQ ID NO: 23); bV H CDR2: IYRSGST (SEQ ID NO: 24); and cV H -CDR3: ARGITGDPGDY (SEQ ID NO: 25) a heavy chain variable region comprising Includes.

[0092] In some embodiments, the antibody or antigen-binding fragment thereof is: (i) the following three CDR sequences: aV L - CDR1: QSVPINF (SEQ ID NO: 18); bV L CDR2:EAS; and cV L -CDR3: GQYGSSPYT (SEQ ID NO: 20) a light chain variable region comprising: (ii) the following three CDR sequences: aV H - CDR1:GGSISSSSYF (SEQ ID NO: 22); bV H CDR2: IYRSGST (SEQ ID NO: 24); and cV H -CDR3: ARGITGDPGDY (SEQ ID NO: 25) a heavy chain variable region comprising Includes.

[0093] In some embodiments, the antibody or antigen-binding fragment thereof is: (i) a light chain variable region having SEQ ID NO: 7 or 5, or a light chain variable region sharing at least 70% sequence identity over the non-CDR regions of SEQ ID NO: 7 or 5; and (ii) a heavy chain variable region having SEQ ID NO: 8 or 6, or a heavy chain variable region sharing at least 70% sequence identity across the non-CDR regions of SEQ ID NO: 8 or 6 Includes.

[0094] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region of SEQ ID NO:7 and a heavy chain variable region of SEQ ID NO:8.

[0095] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region of SEQ ID NO:5 and a heavy chain variable region of SEQ ID NO:6.

[0096] In some other aspects, there is provided a composition comprising a multispecific antigen binding protein as disclosed herein; or a conditionally active multispecific antigen binding protein as disclosed herein; or an immunoglobulin single variable domain (ISV) as disclosed herein; or a bivalent or bispecific antigen binding protein as disclosed herein; or a conditionally active immunoglobulin single variable domain (ISV) as disclosed herein; or an antibody or antigen-binding fragment thereof as disclosed herein; and a pharmaceutically acceptable carrier or excipient.

[0097] In some other aspects, provided are methods for treating a subject in need thereof, comprising administering an effective amount of one of the compositions disclosed herein. In some embodiments, the subject has cancer. In some embodiments, the subject is a human.

[0098] In some other aspects, one of the compositions disclosed herein is provided for use in treating cancer in a subject in need thereof. In some embodiments, the subject is a human.

[0099] Also provided herein is an isolated polynucleotide encoding any of the multispecific antigen-binding proteins disclosed herein; or the conditionally active multispecific antigen-binding proteins disclosed herein; or the immunoglobulin single variable domains (ISVs) disclosed herein; or the bivalent or bispecific antigen-binding proteins disclosed herein; or the conditionally active immunoglobulin single variable domains (ISVs) disclosed herein; or the antibodies or antigen-binding fragments thereof disclosed herein. Also provided herein are vectors comprising such polynucleotides, and host cells comprising such polynucleotides.

[0100] Also provided herein is a method of making any of the multispecific antigen binding proteins disclosed herein; or the conditionally active multispecific antigen binding proteins disclosed herein; or the immunoglobulin single variable domains (ISVs) disclosed herein; or the bivalent or bispecific antigen binding proteins disclosed herein; or the conditionally active immunoglobulin single variable domains (ISVs) disclosed herein; or the antibodies or antigen-binding fragments disclosed herein; the method comprising expressing one of the above polynucleotides in a cell.

[0101] The above summary of the present disclosure is non-limiting, and other features and advantages of the disclosed antigen binding proteins and methods will be apparent from the following brief description of the drawings, detailed description of the disclosure, and claims. [Brief explanation of the drawings]

[0102] [Figure 1] Figure 1 shows a schematic of the "Fab-like" constructs used in Example 1. In each Fab-like construct, one VHH is fused to a human CL domain and the other to a human IgG1 CH1 domain: the two VHH associate via CH1-CL interactions. Human influenza hemagglutinin [HA] and 6-His [His] tags are used for purification purposes. These Fab-like constructs can be monospecific: either bivalent monoparatopic (i.e., containing the same VHH twice against the same target) or bivalent biparatopic (i.e., two different VHHs against the same target); or bispecific (i.e., one VHH against a first target and one VHH against another target). They can also be monovalent (i.e., one VHH against a first target and one VHH directed against an unrelated target). [Figure 2]Figure 2 is a graph showing the cross-reactivity of seven anti-4-1BB VHH clones (clone numbers 1 to 7) with human 4-1BB [h4-1BB], cynomolgus monkey 4-1BB [c4-1BB], and two other members of the tumor necrosis factor receptor superfamily / TNFRSF (human OX40 [hOX40] and human CD40 [CD40]). [Figure 3] FIG. 3 shows the results of a competition ELISA assay between three anti-4-1BB VHHs (clone numbers 2, 3 and 5) and the 4-1BB natural ligand, 4-1BBL. [Figure 4A] 4A-4F show 4-1BB-mediated activation of the NF-κB pathway in a reporter function assay, shown as signal-to-background ratio (S / B). The graphs show activation of the NF-κB pathway in genetically engineered Jurkat T cells constitutively expressing 4-1BB using increasing doses of anti-4-1BB VHH clones: - in a bivalent monoparatopic Fab-like format (i.e., with two identical anti-4-1BB VHHs) in the presence of a cross-linking reagent (anti-Fab antibody): Figure 4A; - in a bivalent monoparatopic Fab-like format (i.e., with two identical anti-4-1BB VHHs) in the absence of a cross-linking reagent: Figure 4B; - in a monovalent Fab-like format (i.e., with one anti-4-1BB VHH and one VHH) against an irrelevant target, e.g., FMDV, in the presence of a cross-linking reagent (anti-Fab antibody); Figure 4C; - in a monovalent Fab-like format (i.e., with one anti-4-1BB VHH and one VHH) in the absence of a cross-linking reagent (anti-Fab antibody) - in a bivalent monoparatopic Fab-like format (i.e., with two identical anti-4-1BB VHHs) versus a bivalent biparatopic Fab-like format (i.e., with two different anti-4-1BB VHHs) in the absence of a cross-linking reagent: Figure 4E; and - in a bispecific Fab-like format (i.e., with one anti-4-1BB VHH and one VHH against another target, e.g., CD28): Figure 4F. [Figure 4B-4C] Same as above. [Figure 4D] Same as above. [Figure 4E] Same as above. [Figure 4F] Same as above. [Figure 5A] Figures 5A-5B show 4-1BB-mediated NF-κB pathway activation in a reporter function assay, shown as signal-to-background ratio (S / B), using engineered Jurkat T cells constitutively expressing 4-1BB induced by increasing doses of anti-4-1BB VHH clones in a bispecific Fab-like format (i.e., one anti-4-1BB VHH and one VHH directed against another target, e.g., OX40), in the absence (Figure 5A) or presence (Figure 5B) of OX40+ cells. [Figure 5B] Figures 5A-5B show 4-1BB-mediated NF-κB pathway activation in a reporter function assay, shown as signal-to-background ratio (S / B), using engineered Jurkat T cells constitutively expressing 4-1BB induced by increasing doses of anti-4-1BB VHH clones in a bispecific Fab-like format (i.e., one anti-4-1BB VHH and one VHH directed against another target, e.g., OX40), in the absence (Figure 5A) or presence (Figure 5B) of OX40+ cells. [Figure 6] Figure 6 shows the X-ray crystallographic 3-D structure of 4-1BBL and anti-4-1BB clone no. 2 and clone no. 5 VHHs in complex with the extracellular domain of human 4-1BB (amino acid residues 24-186 of SEQ ID NO: 13). 4-1BB is shown in a "surface" representation, and 4-1BBL and the two anti-4-1BB VHHs are shown in a "cartoon" representation. Cysteine-rich domains 1 to 4 of 4-1BB (CRD1 to CRD4) are indicated by arrows. [Figures 7A-7B] Figures 7A-7B show the properties of four anti-4-1BB clone number 5 variants (clone numbers 5a-5d): binding assessed by ELISA (Figure 7A) and 4-1BB-mediated activation of the NF-κB pathway in a reporter function assay shown as signal-to-background ratio (S / B) (Figure 7B). [Figure 8] FIG. 8 illustrates a schematic representation of an anti-4-1BB / anti-PD-1 bispecific binding molecule according to certain exemplary embodiments. [Figure 9A]Figures 9A-9C show the activity of anti-4-1BB / anti-PD-1 bispecific binding molecules [constructs 1, 3, 5, and 6] relative to control antibodies and isotype controls in a T cell activation assay (Figure 9A), a mixed lymphocyte reaction (MLR; Figure 9B), and a CD3-PBMC activation assay (Figure 9C). [Figure 9B-9C] Same as above. [Figures 10A-10B] Figures 10A-10G show the results of binding assays using one of "Construct No. 3," the anti-PD-1 arm alone (clone T5), or control anti-4-1BB or anti-PD-1 antibodies in a dose-dependent manner on: human 300.19 pre-B cells expressing either human PD-1 (Figure 10A), cynomolgus PD-1 (Figure 10B), human 4-1BB (Figure 10C), or cynomolgus 4-1BB (Figure 10D); Jurkat T cells expressing either human PD-1 (Figure 10E) or human 4-1BB (Figure 10F); and stimulated primary T cells (Figure 10G). [Figures 10C-10D] Same as above [Figures 10E-10F] Same as above. [Figure 10G] Same as above. [Figures 11A-11B] Figures 11A-11B show the effect of the anti-4-1BB / anti-PD-1 bispecific binding molecule "Construct No. 3" relative to a control antibody and an isotype control in a reporter assay (4-1BB reporter assay: Figure 11A; PD-1 reporter assay: Figure 11B), shown as signal-to-noise ratio (S / N). [Figures 12A-12B] Figures 12A-12B show the effect of the anti-4-1BB / anti-PD-1 bispecific binding molecule "Construct No. 3" relative to control antibodies (one control anti-4-1BB antibody [Ctrl anti-4-1BB Ab], Figure 12A; two control anti-PD-1 antibodies [Ctrl anti-PD-1 Ab Nos. 1 and 2], Figure 12B) and an isotype control in an antibody-dependent cellular cytotoxicity (ADCC) assay. [Figures 13A-13B]Figures 13A-13B show the effect of the anti-4-1BB / anti-PD-1 bispecific binding molecule "Construct No. 3" relative to control antibodies (one control anti-4-1BB antibody [Ctrl anti-4-1BB Ab], Figure 13A; two control anti-PD-1 antibodies [Ctrl anti-PD-1 Ab Nos. 1 and 2], Figure 13B) and an isotype control in an antibody-dependent cellular phagocytosis (ADCP) assay. [Figures 14A-14B] Figures 14A-14B show the effect of the anti-4-1BB / anti-PD-1 bispecific binding molecule "Construct No. 3" relative to control antibodies (one control anti-4-1BB antibody [Ctrl anti-4-1BB Ab], Figure 14A; two control anti-PD-1 antibodies [Ctrl anti-PD-1 Ab Nos. 1 and 2], Figure 14B) and an isotype control in a complement-dependent cytotoxicity (CDC) assay. [Figures 15A-15B] Figures 15A-15C show the effect of the anti-4-1BB / anti-PD-1 bispecific binding molecule "Construct No. 3" relative to a control anti-4-1BB antibody [Ctrl anti-4-1BB Ab] and an isotype control in a T cell activation (TCA) assay. Figure 15A shows the release of interferon gamma [IFN-γ] from activated human T cells; Figure 15B shows the dose-dependent release of tumor necrosis factor-alpha [TNF-α] from activated human T cells; and Figure 15C shows the release of interleukin-2 [IL-2] from activated cynomolgus monkey T cells. [Figure 15C] Same as above. [Figures 16A-16B] Figures 16A-16C show the activity of the anti-4-1BB / anti-PD-1 bispecific binding molecule "Construct No. 3" versus a control anti-4-1BB antibody [Ctrl anti-4-1BB Ab] and a combination of one or two control anti-PD-1 antibodies [Ctrl anti-PD-1 Ab [No. 1 and No. 2] and an isotype control] in a mixed lymphocyte reaction (MLR) assay. Figure 16A shows the release of interleukin-2 [IL-2] from the coculture; Figure 16B shows the dose-dependent release of interleukin-2 [IL-2]; and Figure 16C shows the dose-dependent release of tumor necrosis factor-alpha [TNF-α]. [Figure 16C] Same as above. [Figure 17A]Figures 17A-17B show the activity of the anti-4-1BB / anti-PD-1 bispecific binding molecule "Construct No. 3" versus a control anti-4-1BB antibody [Ctrl anti-4-1BB Ab], a control anti-PD-1 antibody [Ctrl anti-PD-1 Ab], and their combination, and an isotype control in a CD3-PBMC activation assay. Interferon-gamma [IFN-γ] (Figure 17A) and tumor necrosis factor-alpha [TNF-α] (Figure 17B) release from PBMCs is shown. [Figure 17B] Figures 17A-17B show the activity of the anti-4-1BB / anti-PD-1 bispecific binding molecule "Construct No. 3" versus a control anti-4-1BB antibody [Ctrl anti-4-1BB Ab], a control anti-PD-1 antibody [Ctrl anti-PD-1 Ab], and their combination, and an isotype control in a CD3-PBMC activation assay. Interferon-gamma [IFN-γ] (Figure 17A) and tumor necrosis factor-alpha [TNF-α] (Figure 17B) release from PBMCs is shown. [Figure 18A] Figures 18A-18C show the activity of the anti-4-1BB / anti-PD-1 bispecific binding molecule "Construct No. 3" versus a control anti-4-1BB antibody [Ctrl anti-4-1BB Ab], a control anti-PD-1 antibody [Ctrl anti-PD-1 Ab], and their combination, and an isotype control in a CD3-PBMC activation assay. The dose-dependent release of interleukin-2 [IL-2] (Figure 18A), tumor necrosis factor-alpha [TNF-α] (Figure 18B), and interleukin-5 [IL-5] (Figure 18C) from PBMCs is shown. [Figures 18B-18C] Same as above. [Figure 19A]19A-19C show the reactivation of exhausted T cells in a modular immune in vitro construct (MIMIC) CD8+ T cell exhaustion assay using the anti-4-1BB / anti-PD-1 bispecific binding molecule "Construct No. 3" against one control anti-4-1BB antibody [Ctrl anti-4-1BB Ab], two control anti-PD-1 antibodies [Ctrl anti-PD-1 Ab Nos. 1 and 2], and their combinations, as well as its isotype control. Two "Construct No. 3" mutants were also tested: one lacking a functional PD-1 arm [Construct No. 3 ΔPD-1] and one lacking a functional 4-1BB arm [Construct No. 3 Δ4-1BB]. Figure 19A shows the total number of antigen-specific divided CD8+ T cells 19 days after treatment; Figure 19B shows the secretion of interferon gamma [IFN-γ] from reactivated CD8+ T cells; and Figure 19C shows the secretion of tumor necrosis factor alpha [TNF-α] from reactivated CD8+ T cells. [Figure 19B] Same as above. [Figure 19C] Same as above. [Figure 20] Figure 20 shows the results of a regulatory T cell (Treg) suppression assay using the anti-4-1BB / anti-PD-1 bispecific binding molecule "Construct No. 3" against a control anti-4-1BB antibody [Ctrl anti-4-1BB Ab] and an isotype control at several ratios of T effector cells [Tresp] to expanded T regulatory cells [eTreg]. [Figure 21] Figure 21 shows the results of an in vivo efficacy study in tumor-bearing mice (n=9 mice) using the anti-4-1BB / anti-PD-1 bispecific binding molecule "Construct No. 3" (low or high dose) versus a control anti-4-1BB antibody [Ctrl anti-4-1BB Ab] (0.3 mpk), a control anti-PD-1 antibody [Ctrl anti-PD-1 Ab] (3 mpk), and their combination (0.3 + 3 mpk), and an isotype control (3 mpk). DPI: days after implantation. [Figures 22A-22B]Figures 22A-22B show the dose-dependent binding densities of the anti-4-1BB / anti-PD-1 bispecific binding molecules "Construct No. 3" (Figure 22A) and "Optimized Construct No. 3" (Figure 22B), expressed as the number of molecules bound per μm of surface of Jurkat cells expressing 4-1BB and PD-1. Controls include mutants of "Construct No. 3" and "Optimized Construct No. 3" that lack a functional PD-1 arm [ΔPD-1], a functional 4-1BB arm [Δ4-1BB], or both [Δ4-1BB ΔPD-1]. [Figures 23A-23B] Figures 23A-23B show the effect of anti-4-1BB / anti-PD-1 bispecific binding molecules "Construct No. 3" and "Optimized Construct No. 3" relative to a control anti-4-1BB antibody [Ctrl anti-4-1BB Ab] and an isotype control in a T cell activation (TCA) assay. Figure 23A shows the release of interferon-gamma [IFN-γ] from activated human T cells; Figure 23B shows the release of interleukin-2 [IL-2] from activated human T cells. [Figures 24A-24B] Figures 24A-24B show the activity of anti-4-1BB / anti-PD-1 bispecific binding molecules "Construct No. 3" and "Optimized Construct No. 3" against a control anti-4-1BB antibody and a control anti-PD-1 antibody combination [Ctrl anti-4-1BB Ab + Ctrl anti-PD-1 Ab] and an isotype control in a mixed lymphocyte reaction (MLR) assay. Figure 24A shows the release of interferon-gamma [IFN-γ] from co-cultures, and Figure 24B shows the release of interleukin-2 [IL-2] from co-cultures. [Figure 25]Figure 25 shows the results of surface plasmon resonance (SPR) binding assays for human 4-1BB and human PD-1 using "Optimized Construct #3," or one of its four masked versions (MC1-MC4), or a protease-activated (i.e., unmasked) version of the masked compound ([MMP9-activated]), versus a control anti-4-1BB antibody [Ctrl anti-4-1BB Ab], a control anti-PD-1 antibody [Ctrl anti-PD-1 Ab], and an isotype control. Controls also included "Optimized Construct #3" mutants lacking a functional PD-1 arm [ΔT5_optimized] or partially or completely lacking a functional 4-1BB arm ([Δclone #2.1] or [Δclone #2.1 and 5.1], respectively). [Figures 26A-26B] Figures 26A-26D show the results of binding assays on human 300.19 pre-B cells expressing either human 4-1BB (Figure 26A), cynomolgus 4-1BB (Figure 26B), human PD-1 (Figure 26C), or cynomolgus PD-1 (Figure 26D) using one of "Optimized Construct #3," or one of its four masked versions (MC1-MC4), or a protease-activated (i.e., unmasked) version of Mass Compound ([MMP9-activated]) in a dose-dependent manner. [Figures 26C-26D] Same as above. [Figure 27] Figure 27 shows the results of a binding assay on Jurkat / NF-κB-4-1BB cells using "Optimized Construct No. 3," or one of its four masked versions (MC1-MC4), or a protease-activated (i.e., unmasked) version of the masked compound ([MMP9-activating] or [uPa-activating]). [Figure 28A] Figures 28A-28B show the results of binding assays on human Jurkat T cells expressing human 4-1BB (Figure 28A) or human PD-1 (Figure 28B) using one of "Optimized Construct No. 3," or one of its four masked versions (MC1-MC4), or a protease-activated (i.e., unmasked) version of the masked compound ([MMP9-activating]) in a dose-dependent manner. [Figure 28B]Figures 28A-28B show the results of binding assays on human Jurkat T cells expressing human 4-1BB (Figure 28A) or human PD-1 (Figure 28B) using one of "Optimized Construct No. 3," or one of its four masked versions (MC1-MC4), or a protease-activated (i.e., unmasked) version of the masked compound ([MMP9-activating]) in a dose-dependent manner. [Figure 29] Figure 29 shows the results of a luciferase-based reporter assay using "Optimized Construct No. 3," or one of its four masked versions (MC1-MC4), or a protease-activated (i.e., unmasked) version of the masked compound ([MMP9-activating] or [uPa-activating]). [Figure 30A-30B] Figures 30A-30B show the results of luciferase-based reporter assays, shown as signal-to-noise ratio (S / N), on 4-1BB+ Jurkat T cells (Figure 30A) or PD-1+ Jurkat T cells (Figure 30B) using "Optimized Construct No. 3," or one of its four masked versions (MC1-MC4), or a protease-activated (i.e., unmasked) version of the masked compound ([MMP9-activating]). [Figure 31] Figure 31 shows the results of a T cell activation (TCA) assay using "Optimized Construct No. 3," or one of its four masked versions (MC1-MC4), or a protease-activated (i.e., unmasked) version of the masked compound ([MMP9-activating] or [uPa-activating]). [Figure 32A] Figures 32A-32D show the results of a murine T cell activation (TCA) assay using "Optimized Construct No. 3," or one of its four masked versions (MC1-MC4), or a protease-activated (i.e., unmasked) version of the masked compound ([MMP9-activated]) versus an isotype control. The figures show the release of interleukin-4 ([IL-4]; Figure 32A); interleukin-2 ([IL-2]; Figure 32B); interferon-gamma ([IFN-γ]; Figure 32C); and tumor necrosis factor-alpha ([TNF-α]; Figure 32D). [Fig. 32B-32C] Same as above. [Figure 32D] Same as above. [Figure 33A] Figures 33A-33C show the activity of "Optimized Construct No. 3," or one of its four masked versions (MC1-MC4), or a protease-activated (i.e., unmasked) version of the masked compound ([MMP9-activated]) in a mixed lymphocyte reaction (MLR) assay. Figure 33A shows the release of interleukin-2 [IL-2] from cocultures; Figure 33B shows the donor-dependent activity, expressed as EC50, for interleukin-2 [IL-2] release; and Figure 33C shows the donor-dependent activity, expressed as EC50, for interferon-gamma [IFN-γ] release. [Figure 33B-33C] Same as above. [Figure 34A-34B] Figures 34A-34E show the activity of "Optimized Construct No. 3," or one of its four masked versions (MC1-MC4), or a protease-activated (i.e., unmasked) version of the masked compound ([MMP9-activated]) in a CD3-PBMC activation assay. Figure 34A shows the release of interleukin-2 [IL-2] from PBMCs; Figure 34B shows the donor-dependent activity expressed as EC50 for interleukin-2 [IL-2] release; Figure 34C shows the donor-dependent activity expressed as EC50 for interferon-gamma [IFN-γ] release; Figure 34D shows the donor-dependent activity expressed as EC50 for tumor necrosis factor-alpha [TNF-α] release; and Figure 34E shows the donor-dependent activity expressed as EC50 for interleukin-5 [IL-5] release. [Fig. 34C-34D] Same as above. [Figure 34E] Same as above. [Figure 35]Figure 35 shows the results of an in vivo efficacy study in tumor-bearing mice (n=11 mice / group) using the anti-4-1BB / anti-PD-1 bispecific binding molecule "Optimized Construct No. 3" (low, medium, or high dose) and its masked version "MC1" (same low, medium, and high doses) compared to a high dose isotype control [iso ctrl]. Data are presented as the median log2 tumor volume (mm3) of all mice per group. DPI: days after implantation. DETAILED DESCRIPTION OF THE INVENTION

[0103] Before the present disclosure is described, it is to be understood that this disclosure is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. The scope of the present disclosure will be limited only by the appended claims, and it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0104] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0105] As used herein, the term "about," when used in reference to a particular recited numerical value, means that the value may vary from the recited value by no more than 5%, preferably no more than 2%, and more preferably no more than 1%. For example, as used herein, the phrase "about 100" includes 99 and 101 and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0106] Although any methods and materials similar or equivalent to those described herein can be used in the practice of this disclosure, exemplary methods and materials are now described. All publications mentioned herein are incorporated by reference as if set forth in their entirety.

[0107] The term "PD-1" refers to the programmed death 1 protein, a T-cell co-inhibitor also known as CD279. Unless otherwise specified, the term "PD-1" refers to human PD-1. PD-1 is a member of the CD28 / CTLA-4 / ICOS family of T-cell co-inhibitors. PD-1 is a 288-amino acid protein with an IgV-like extracellular N-terminal domain, a transmembrane domain, and an intracellular domain containing immunoreceptor tyrosine-based inhibition (ITIM) motifs and immunoreceptor tyrosine-based switch (ITSM) motifs (Chattopadhyay et al., Immunol Rev. 2009 May;229(1):356-86). The PD-1 receptor has two ligands, PD-ligand-1 (PD-L1) and PD-L2. An exemplary amino acid sequence of PD-1 is set forth in SEQ ID NO: 42, which corresponds to human PD-1 (hPD-1), which has GenBank accession number NP_005009.2. [ka]

[0108] The term "PD-L1" refers to the ligand of the PD-1 receptor, also known as CD274 and B7H1. PD-L1 is a 290-amino acid protein with an extracellular IgV-like domain, a transmembrane domain, and a highly conserved intracellular domain of approximately 30 amino acids. PD-L1 is constitutively expressed on many cells, such as antigen-presenting cells (e.g., dendritic cells, macrophages, and B cells), as well as hematopoietic and non-hematopoietic cells (e.g., vascular endothelial cells, pancreatic islets, and immune-privileged sites). PD-L1 is also expressed on a wide variety of tumors and virus-infected cells and is a component of the immunosuppressive environment (Ribas, N Engl J Med. 2012 Jun 28;366(26):2517-9). An exemplary amino acid sequence of PD-L1 is set forth in SEQ ID NO: 43, which corresponds to human PD-L1 (hPD-L1), having GenBank accession number NP_054862.1. [ka]

[0109] As used herein, the term "4-1BB" or "CD137" refers to a surface glycoprotein belonging to the tumor necrosis factor receptor family (TNFRSF9). Its expression is induced upon activation on several leukocyte types. 4-1BB is expressed on primed T cells and natural killer (NK) cells and provides a potent costimulatory signal upon ligation. Activated CD8 + Perturbation of 4-1BB on T cells by 4-1BBL or agonistic monoclonal antibodies protects such antigen-specific cytotoxic T lymphocytes from apoptosis, enhances effector function, and promotes persistence and memory differentiation. 4-1BB is transiently expressed on activated T cells that encounter cognate antigen, activated NK cells, or mature dendritic cells (DCs). A unique functional ligand has been identified for 4-1BB, 4-1BBL, which is expressed on the surface of professional antigen-presenting cells such as DCs, macrophages, and B cells. 4-1BB trimerization leads to 4-1BBL receptor clustering and TRAF-mediated activation of NF-κB and MAPK intracellular signaling cascades, ultimately resulting in cell activation, proliferation, and survival. In T cells, T cell receptor (TCR) stimulation and subsequent CD3 signaling activate T cells upon ligation with agonistic antibodies or natural ligands. hThe transient expression of 4-1BB promotes T cell T cell response. In addition to inducing effector cytokine production, 4-1BB costimulation promotes T cell memory and effector differentiation, protects T cells from apoptosis, alters mitochondrial metabolism to increase T cell respiratory capacity, and induces global DNA demethylation and chromatin reprogramming. An exemplary amino acid sequence of 4-1BB is set forth in SEQ ID NO: 13, which corresponds to human 4-1BB (h4-1BB) with UniProt accession number Q07011. 4-1BB contains four cysteine-rich domains (CRDs) in its N-terminal extracellular region: CRD1 (amino acid residues 24-45 of SEQ ID NO: 13); CRD2 (amino acid residues 47-86 of SEQ ID NO: 13); CRD3 (amino acid residues 87-118 of SEQ ID NO: 13); and CRD4 (amino acid residues 119-159 of SEQ ID NO: 13). [ka]

[0110] As used herein, the term "T cell co-inhibitor" refers to a receptor expressed on a T cell and / or a ligand of such a receptor, which modulates immune responses through suppression of T cell receptor (TCR) signaling. The term "T cell co-inhibitor," also known as "T cell co-signaling molecule," includes, but is not limited to, PD-1; lymphocyte-activation gene 3 protein (LAG-3, also known as CD223); cytotoxic T lymphocyte antigen-4 (CTLA-4); attenuator of B and T lymphocytes (BTLA); 2B4; T cell immunoglobulin and mucin-3 (TIM-3); T cell immunoreceptor with immunoglobulin and ITIM (TIGIT; also known as VSIG9); leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1; also known as CD305); V-domain Ig suppressor of T cells (VISTA); PD-L1; PD-L2; CEACAM; B7-H3; B7-H4; KIR; A2aR; GAL9; and TGFR. For a review of T cell costimulation and costimulation, see Chen & Flies (Nat Rev Immunol 2013 Apr;13(4):227-42).

[0111] As used herein, the term "T cell costimulator" refers to a receptor expressed on a T cell and / or a ligand of such a receptor that modulates immune responses through T cell receptor (TCR) signaling activation. The term "T cell costimulator" includes, but is not limited to, CD28; inducible T cell costimulator (ICOS); OX40; CD27; 4-1BB (also known as CD137); death receptor 3 (DR3); B7; CD226; CRTAM; glucocorticoid-inducible TNFR-related protein (GITR); CD30; CD2; herpesvirus entry mediator (HVEM); BAFFR; BAFF; and Light. For a review of T cell costimulation and costimulation, see Chen & Flies (Nat Rev Immunol 2013 Apr;13(4):227-42).

[0112] The term "antigen-binding protein," as used herein, refers to a protein capable of specifically binding to at least one target via at least one immunoglobulin (Ig) variable domain. Examples of antigen-binding proteins include, but are not limited to, antibodies or fragments thereof, single-domain antibodies, Fab fragments, immunoglobulin single variable domains (ISVs), and combinations thereof. Antigen-binding proteins may be of non-human (e.g., murine) or human origin. If such antigen-binding proteins are of non-human (e.g., murine) origin, they may be "humanized" to reduce immunogenicity or increase stability.

[0113] The term "antibody," as used herein, is intended to refer to immunoglobulin (Ig) molecules (i.e., "intact antibody molecules") composed of four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds, and multimers thereof (e.g., IgM) or antigen-binding fragments thereof. Each heavy chain contains a heavy chain variable region ("HCVR" or "V"). H ”) and heavy chain constant region (“C H "; Domain CH 1. C H 2 and C H Each light chain is composed of a light chain variable region ("LCVR" or "VVR"). L ") and a light chain constant region ("LCCR" or "C L "). V H and V L The regions can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). H and V L is composed of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the present disclosure, the FRs of an antibody (or antigen-binding fragment thereof) can be identical to human germline sequences or can be naturally or artificially modified. An amino acid consensus sequence can be defined based on a parallel analysis of two or more CDRs.

[0114] The term "immunoglobulin single variable domain" (ISV) is used interchangeably with "single variable domain" and defines an immunoglobulin molecule in which the antigen-binding site resides on, and is formed by, a single immunoglobulin domain. This distinguishes immunoglobulin single variable domains from "conventional" immunoglobulins (e.g., monoclonal antibodies) or fragments thereof (Fab, Fab', F(ab')2, scFv, di-scFv, etc.), in which two immunoglobulin domains, particularly two variable domains, interact to form the antigen-binding site. Typically, in conventional immunoglobulins, a heavy chain variable domain (V H ) and the light chain variable domain (V L ) interact to form the antigen-binding site. In this case, V H and V LIn view of the above definition, the antigen-binding domain of a conventional four-chain antibody (e.g., an IgG, IgM, IgA, IgD, or IgE molecule known in the art) or an Fab fragment, an F(ab')2 fragment, an Fv fragment, such as a disulfide-linked Fv or scFv fragment, or a diabody derived from such a conventional four-chain antibody (all known in the art) is not typically considered to be an immunoglobulin single variable domain, because in these cases, binding to each epitope of an antigen is typically not by one (single) immunoglobulin domain, but by a pair of (associated) immunoglobulin domains such as a light and heavy chain variable domain, i.e., by the V of an immunoglobulin domain that together binds to each epitope of the antigen. H -V L This is because they arise in pairs.

[0115] In contrast, an immunoglobulin single variable domain can specifically bind to an epitope of an antigen without pairing with an additional immunoglobulin variable domain. The binding site of an immunoglobulin single variable domain consists of a single V H , a single V HH , or a single V L Thus, the antigen-binding site of an immunoglobulin single variable domain is formed by three or fewer CDRs. Therefore, a single variable domain may contain any light chain variable domain sequence (e.g., V), as long as it is capable of forming a single antigen-binding unit (i.e., a functional antigen-binding unit consisting essentially of a single variable domain, such that a single antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit). L -sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., V H Array or V HH sequence) or a suitable fragment thereof.

[0116] Immunoglobulin single variable domains (ISVs) can be, for example, heavy chain ISVs, e.g., camelized V Hor humanized V HH Contains V H , V HH In one embodiment, this may be a camelized V H or humanized V HH Contains V HH The heavy chain ISVs can be derived from a conventional four-chain antibody or from a heavy chain antibody. For example, immunoglobulin single variable domains can be (single) domain antibodies (or amino acid sequences suitable for use as single domain antibodies), "dAbs" or dAbs (or amino acid sequences suitable for use as dAbs), Nanobody® ISVs (as defined herein, V HH ; other single variable domains, or any suitable fragment of any one of these. In particular, the immunoglobulin single variable domain may be a Nanobody® ISV (e.g., a humanized VSV). HH Or Camelization V H Contains V HH ) or a suitable fragment thereof.

[0117] "V HH Domain" is "V HH "," "V HH antigen fragments,” and “V HH "V" is also known as a "heavy chain antibody" and was originally described as an antigen-binding immunoglobulin variable domain of a "heavy chain antibody" (i.e., an "antibody lacking light chains"; Hamers-Casterman et al., Nature. 1993 Jun 3;363(6428):446-8). HH The term "variable domain" refers to these variable domains, as compared to the heavy chain variable domains (herein referred to as "V" domains) present in conventional four-chain antibodies. H domain) present in conventional four-chain antibodies and the light chain variable domain (referred to herein as "V L The domain name was chosen to distinguish it from the domains referred to as "domains." HHFor further description, see the review article by Muyldermans (J Biotechnol. 2001 Jun;74(4):277-302). The terms "dAb" and "domain antibody" are also described, for example, in Ward et al. (Nature. 1989 Oct 12;341(6242):544-6), Holt et al. (Trends Biotechnol. 2003 Nov;21(11):484-90), and in, for example, WO 2004 / 068820, WO 2006 / 030220, WO 2006 / 003388, and other published patent applications of Domantis Ltd. It should also be noted that the single variable domains may be derived from certain species of sharks (e.g. the so-called "IgNAR domains", see e.g. WO 2005 / 18629), although these are less preferred as they are not of mammalian origin.

[0118] Typically, producing immunoglobulins involves immunizing laboratory animals, fusing immunoglobulin-producing cells to create hybridomas, and screening for the desired specificity. Alternatively, immunoglobulins can be produced by screening naive, immune, or synthetic libraries, e.g., by phage display. HH The generation of immunoglobulin sequences such as V has been widely described in various published literature, inter alia WO 1994 / 04678, Hamers-Casterman et al. (Nature. 1993 Jun 3;363(6428):446-8) and Muyldermans (J. Biotechnol. 2001 Jun;74(4):277-302). In these methods, camelids are immunized with a target antigen to induce an immune response against said target antigen. The V resulting from said immunization is HH The repertoire of V HHThe antibodies are then further screened for binding to the target antigen. In these instances, the generation of antibodies requires purified antigen for immunization and / or screening. The antigen can be purified from a natural source or during recombinant production. Immunization and / or screening for immunoglobulin sequences can be performed using peptide fragments of such antigens.

[0119] Immunoglobulin sequences of different origins can be used herein, including mouse, rat, rabbit, donkey, human, and camelid immunoglobulin sequences. Fully human, humanized, or chimeric sequences can also be used in the methods described herein. For example, camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences, or camelized domain antibodies, such as the camelized dAbs described by Ward et al. (Nature. 1989 Oct 12;341(6242):544-6), WO 1994 / 04678, and Davis et al. (FEBS Lett. 1994 Feb 21;339(3):285-90; and Protein Eng. 1996 Jun;9(6):531-7), can be used herein. Furthermore, ISVs can be fused to form multivalent and / or multispecific constructs (one or more V HH For multivalent and multispecific polypeptides containing domains and their preparation, see Conrath et al. (J Biol Chem. 2001 Mar 9;276(10):7346-50) and also, for example, WO 1996 / 34103 and WO 1999 / 23221).

[0120] "Humanized V HH " is a naturally occurring V HH domain, but is "humanized," i.e., HH One or more amino acid residues in the amino acid sequence (and particularly in the framework sequences) of the V HThe term "humanized" includes amino acid sequences that have been "humanized" by replacing one or more of the amino acid residues (e.g., as shown above) that occur at the corresponding positions in the domain. This can be done in a manner known per se, which will be clear to the skilled artisan, for example, based on the prior art (e.g., WO 2008 / 020079). Again, such humanized V HH can be obtained in any suitable manner known per se, and thus the naturally occurring V HH It should be noted that the invention is not strictly limited to polypeptides obtained using a polypeptide containing the domain as a starting material.

[0121] "Camelization V H " is a naturally occurring V H corresponding to the amino acid sequence of the naturally occurring V domain from a conventional four-chain antibody. H One or more amino acid residues in the amino acid sequence of the domain are replaced by the V HH The term "camelized" includes amino acid sequences that have been "camelized" by replacing one or more amino acid residues present at the corresponding positions in the V domain. This can be performed in a manner known per se, as will be clear to those skilled in the art, for example as described in the prior art (e.g., Davies et al., FEBS Lett. 1994 Feb 21;339(3):285-90; Davies et al., Biotechnology (NY). 1995 May;13(5):475-9; Davies et al., Protein Eng. 1996 Jun;9(6):531-7; and Riechmann et al., J Immunol Methods 1999 Dec 10;231(1-2):25-38). Such "camelized" substitutions can be performed in the V domain, as defined herein. H -V LIn some embodiments, camelized V residues are inserted at positions that form and / or are present at interfaces and / or so-called camelid hallmark residues (see, e.g., WO 1994 / 04678 and Davies et al. (1994 and 1996, supra)). H V, which is used as a starting material or starting point for generating or designing H The sequence is V from mammals H sequences, e.g., human V H Array, e.g. V H 3 sequence. However, such camelized V H can be obtained in any suitable manner known per se, and therefore can be obtained without using the naturally occurring V as starting material. H It should be noted that the polypeptide obtained using the polypeptide containing the domain is not strictly limited.

[0122] The structure of an immunoglobulin single variable domain (ISV) sequence can be considered to consist of four framework regions ("FR"), which are referred to in the art and herein as "framework region 1" ("FR1"); "framework region 2" ("FR2"); "framework region 3" ("FR3"); and "framework region 4" ("FR4"), respectively, interrupted by three complementarity-determining regions ("CDR"), which are referred to in the art and herein as "complementarity-determining region 1" ("CDR1"), "complementarity-determining region 2" ("CDR2"), and "complementarity-determining region 3" ("CDR3"), respectively. In such an immunoglobulin sequence, the framework sequences may be any suitable framework sequence; examples of suitable framework sequences will be clear to the skilled artisan, for example, on the basis of standard handbooks and the further disclosures and prior art referred to herein. The framework sequences are immunoglobulin framework sequences or (suitable combinations of) framework sequences derived from immunoglobulin framework sequences (e.g., by humanization or camelization). For example, the framework sequences may be those of a light chain variable domain (e.g., VL sequence) and / or heavy chain variable domain (e.g., V H Sequence or V HH In a particular embodiment, the framework sequences may be derived from V HH framework sequences derived from conventional V sequences (in which the framework sequences may optionally be partially or fully humanized), or camelized (as defined herein). H In particular, the framework sequences present in the ISV sequences described herein are any of the following: HH or Camelization V H Contains V HH Nanobody® ISVs such as, for example, AA ...

[0123] V H Domain and V HH The total number of amino acid residues in a domain will typically be in the range of 110-120, often 112-115. However, it should be noted that shorter and longer sequences may also be suitable for the purposes described herein. However, it should be noted that the ISVs described herein are not limited with respect to the origin of the ISV sequence (or the nucleotide sequence used to express it), nor are they limited with respect to the manner in which the ISV sequence or nucleotide sequence is generated or obtained. Thus, an ISV sequence may be a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence. In one specific, but non-limiting embodiment, the ISV sequence is a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence, including, but not limited to, a "humanized" (as defined herein) immunoglobulin sequence (e.g., partially or fully humanized mouse or rabbit immunoglobulin sequence, and particularly partially or fully humanized VHV sequences). HHsequences), "camelized" (as defined herein) immunoglobulin sequences (and in particular camelized V H ISVs include ISVs obtained by techniques such as ISVs derived from immunoglobulin sequences (e.g., starting from synthetic, random, or naturally occurring immunoglobulin sequences), affinity maturation (e.g., starting from synthetic, random, or naturally occurring immunoglobulin sequences), CDR grafting, veneering, joining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar immunoglobulin sequence engineering techniques well known to those skilled in the art, or any suitable combination of any of the foregoing. Similarly, the nucleotide sequence may be a naturally occurring nucleotide sequence or a synthetic or semi-synthetic sequence, such as a sequence isolated by PCR from a suitable naturally occurring template (e.g., DNA or RNA isolated from a cell), a nucleotide sequence isolated from a library (and in particular an expression library), a nucleotide sequence prepared by introducing mutations into a naturally occurring nucleotide sequence (using any suitable technique known per se, such as mismatch PCR), a nucleotide sequence prepared by PCR using overlapping primers, or a nucleotide sequence prepared using DNA synthesis techniques known per se.

[0124] Generally, Nanobody® ISVs (especially (partially) humanized V HH Sequence and camelized V H V containing arrays HH A Nanobody® ISV (a sequence) may be characterized by the presence of one or more "hallmark residues" (as described herein) in one or more of the framework sequences (also described further herein). Thus, in general, a Nanobody® ISV can be defined as an immunoglobulin sequence having the (general) structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and where one or more of the hallmark residues are as further defined herein.

[0125] In particular, a Nanobody® ISV may be an immunoglobulin sequence having the (general) structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and the framework sequences are as further defined herein.

[0126] More particularly, a Nanobody® ISV may be an immunoglobulin sequence having the (general) structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and where one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are selected from the hallmark residues mentioned in Table 1 below.

[0127] [Table 1]

[0128] [Table 2]

[0129] In some embodiments, the immunoglobulin single variable domains have certain amino acid substitutions in the framework regions effective to prevent or reduce binding of so-called "pre-existing antibodies" to the polypeptide, as described in WO 2015 / 173325, in which (i) the amino acid residue at position 112 is one of K or Q; and / or (ii) the amino acid residue at position 89 is T; and / or (iii) the amino acid residue at position 89 is L and the amino acid residue at position 110 is either K or Q; (iv) in each of (i) to (iii), the amino acid at position 11 is preferably V.

[0130] An immunoglobulin single variable domain (ISV) may comprise or consist essentially of one or more (at least one) immunoglobulin single variable domains and may form part of a protein or polypeptide, which may optionally further comprise one or more additional amino acid sequences (all optionally linked via one or more suitable linkers). The term "immunoglobulin single variable domain" may also encompass such polypeptides. One or more immunoglobulin single variable domains may be used as binding units in such proteins or polypeptides, which may optionally contain one or more additional amino acids capable of acting as binding units (one or more V HH For multivalent and multispecific polypeptides containing domains and their preparation, see Conrath et al. (J Biol Chem. 2001 Mar 9;276(10):7346-50), and for example WO 1996 / 34103, WO 1999 / 23221 and WO 2010 / 115998).

[0131] A polypeptide can comprise or consist essentially of one immunoglobulin single variable domain, as outlined above. Such polypeptides are also referred to herein as "monovalent" polypeptides. The term "multivalent" refers to the presence of more than one ISV in a polypeptide. In one embodiment, a polypeptide is "bivalent," i.e., comprises or consists of two ISVs. In one embodiment, a polypeptide is "trivalent," i.e., comprises or consists of three ISVs. In another embodiment, a polypeptide is "tetravalent," i.e., comprises or consists of four ISVs. Thus, a polypeptide can be "bivalent," "trivalent," "tetravalent," "pentavalent," "hexavalent," "heptavalent," "octavalent," "nanovalent," etc., i.e., the polypeptide comprises or consists of 2, 3, 4, 5, 6, 7, 8, 9, etc. ISVs, respectively. In one embodiment, a multivalent ISV polypeptide is bivalent. In one embodiment, a multivalent ISV polypeptide is trivalent. In another embodiment, a multivalent ISV polypeptide is tetravalent. In yet another embodiment, a multivalent ISV polypeptide is pentavalent.

[0132] Multivalent ISV polypeptides can also be monospecific or multispecific. The term "multispecific" refers to binding to multiple different target molecules (also called antigens). Thus, multivalent ISV polypeptides can be "bispecific," "trispecific," "tetraspecific," etc., i.e., they can bind to two, three, four, etc. different target molecules, respectively. For example, a polypeptide can be bispecific trivalent, e.g., a polypeptide comprising or consisting of three ISVs, two ISVs binding to a first target and one ISV binding to a second target different from the first target. In another example, a polypeptide can be trispecific tetravalent, e.g., a polypeptide comprising or consisting of four ISVs, one ISV binding to a first target, two ISVs binding to a second target different from the first target, and one ISV binding to a third target different from the first and second targets. In yet another example, the polypeptide may be trispecific pentavalent, such as a polypeptide comprising or consisting of five ISVs, two ISVs binding to a first target, two ISVs binding to a second target different from the first target, and one ISV binding to a third target different from the first and second targets.

[0133] In one embodiment, the multivalent ISV polypeptide can also be monoparatopic or multiparatopic. The term "multiparatopic" refers to binding to multiple different epitopes on the same target molecule (also called an antigen). Thus, the multivalent ISV polypeptide can be "biparatopic," "triparatopic," etc., i.e., capable of binding to two, three, etc. different epitopes, respectively, on the same target molecule.

[0134] In another embodiment, a polypeptide of the invention comprising or consisting essentially of one or more immunoglobulin single variable domains (or suitable fragments thereof) may further comprise one or more other groups, residues, moieties or binding units. Such further groups, residues, moieties, binding units or amino acid sequences may or may not provide further functionality to the immunoglobulin single variable domain (and / or the polypeptide in which it is found) and may or may not modify the properties of the immunoglobulin single variable domain. For example, such further groups, residues, moieties or binding units may be one or more additional amino acids, such that the compound, construct or polypeptide is a (fusion) protein or (fusion) polypeptide. In a preferred, but non-limiting embodiment, said one or more other groups, residues, moieties or binding units are immunoglobulins. Even more preferably, said one or more other groups, residues, moieties or binding units are selected from the group consisting of a domain antibody, an amino acid suitable for use as a domain antibody, a single domain antibody, an amino acid suitable for use as a single domain antibody, a "dAb", an amino acid suitable for use as a dAb, or a Nanobody® ISV. Alternatively, such groups, residues, moieties or binding units may, for example, be chemical groups, residues, moieties which may or may not be biologically and / or pharmacologically active by themselves. For example, but not limited to, such groups may be linked to one or more immunoglobulin single variable domains so as to provide "derivatives" of immunoglobulin single variable domains. In another embodiment, said additional residues may be effective to prevent or reduce binding of so-called "pre-existing antibodies" to the polypeptide. To this end, polypeptides and constructs may contain a C-terminal extension (X) n (wherein n can be 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (preferably 1 or 2, e.g. 1), and each X is independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I), preferably an independently selected (preferably naturally occurring) amino acid residue, see WO 2012 / 175741. Thus, the polypeptide may comprise a C-terminal extension (X nwhere n is 1 to 5, for example 1, 2, 3, 4 or 5, and X is a naturally occurring amino acid, preferably not cysteine.

[0135] In the above polypeptides, the one or more immunoglobulin single variable domains and one or more groups, residues, moieties or binding units may be linked to each other directly and / or via one or more suitable linkers or spacers. For example, if one or more groups, residues, moieties or binding units are amino acids, the linker may also be an amino acid, such that the resulting polypeptide is a fusion protein or fusion polypeptide. As used herein, the term "linker" refers to a peptide that fuses two or more ISVs together into a single molecule. The term also extends to a peptide that fuses any two amino acid sequences together in a so-called "fusion protein" or "fusion polypeptide", such as an ISV and one or more other groups, residues, moieties or binding units as defined above.

[0136] The use of linkers to join two or more (poly)peptides is well known in the art. One commonly used class of peptide linkers is known as "Gly-Ser" or "GS" linkers. These are linkers consisting essentially of glycine (G) and serine (S) residues, usually containing a GGGGS (SEQ ID NO: 26) motif (e.g., a linker of the formula (Gly-Gly-Gly-Gly-Ser) n (wherein n can be 1, 2, 3, 4, 5, 6, 7 or more). Some frequently used examples of such GS linkers are the 9GS linker (GGGGSGGGS, SEQ ID NO: 29), the 15GS linker (n=3) and the 35GS linker (n=7). See, for example, Chen et al. (Adv Drug Deliv Rev, 2013 Oct;65(10):1357-69) and Klein et al. (Protein Eng Des Sel.27(10):325-30). Further exemplary peptide linkers are shown in Table 2 below.

[0137] [Table 3]

[0138] All antigen-binding proteins disclosed herein may also allow substitution of one or more CDR residues or omission of one or more CDRs. Antibodies have been described in the scientific literature that allow one or two CDRs to be dispensed for binding. Padlan et al. (FASEB J. 1995;9(1):133-139) analyzed the contact regions between antibodies and their antigens based on published crystal structures and concluded that only about one-fifth to one-third of the CDR residues actually contact the antigen. Padlan also discovered many antibodies in which one or two CDRs have no amino acids in contact with the antigen (Vajdos et al., J Mol Biol. 2002;320(2):415-428).

[0139] CDR residues that do not contact the antigen were selected based on previous studies (e.g., V H Residues H60-H65 in CDR2 are often not required, and can be identified empirically through molecular modeling and / or empirical analysis from regions of the Kabat CDRs outside the Chothia CDRs. When a CDR or its residues are omitted, the CDR is typically replaced with an amino acid occupying the corresponding position in another human antibody sequence or a consensus sequence for such a sequence. The positions for substitution within the CDR and the substituting amino acids can also be selected empirically. Empirical substitutions can be conservative or non-conservative.

[0140] The anti-PD-1 / anti-4-1BB bispecific binding proteins disclosed herein (or any of their individual components) may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy chain variable domain and / or light chain variable domain compared to the corresponding germline sequences. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The present disclosure includes antibodies, and antigen-binding fragments thereof, derived from any of the amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions have been mutated to the corresponding residue in the germline sequence from which the antibody is derived, or to the corresponding residue in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as "germline mutations"). Starting from the heavy chain and light chain variable region sequences disclosed herein, one of skill in the art can readily generate numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, V H and / or V LAll of the framework and / or CDR residues within a domain are mutated back to the residue found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only mutated residues found within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residue in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody was originally derived). Furthermore, an antibody may contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., certain individual residues are mutated to the corresponding residue in a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residue in a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be readily tested for one or more desired properties, e.g., improved binding specificity, increased binding affinity, improved or enhanced antagonist or agonist biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present disclosure.

[0141] The present disclosure also includes anti-PD-1 / anti-4-1BB bispecific binding proteins (or any of their individual components) comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein with one or more conservative substitutions. For example, the present disclosure includes anti-PD-1 antibodies with, e.g., no more than 10, no more than 8, no more than 6, no more than 4, etc., HCVR, LCVR, and / or CDR amino acid sequences, conservative amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein; or anti-4-1BB immunoglobulin single variable domains with, e.g., no more than 10, no more than 8, no more than 6, no more than 4, etc., HCVR, LCVR, and / or CDR amino acid sequences, conservative amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein.

[0142] The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human mAbs of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), e.g., in the CDRs, particularly CDR3. However, the term "human antibody," as used herein, is not intended to include mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., a mouse) have been grafted onto human FR sequences. This term includes antibodies recombinantly produced in non-human mammals or in the cells of non-human mammals. This term is not intended to include antibodies isolated from or generated in a human subject.

[0143] The term "recombinant," as used herein, refers to an antibody or antigen-binding fragment thereof of the present disclosure that is created, expressed, isolated, or otherwise obtained by techniques or methods known in the art, such as recombinant DNA technology, including, for example, DNA splicing and transgenic expression. The term also refers to antibodies or antigen-binding fragments thereof that are expressed in a non-human mammal (including a transgenic non-human mammal, e.g., a transgenic mouse), or cell (e.g., a CHO cell) expression system, or that are isolated from a recombinant combinatorial human antibody library.

[0144] The term "multispecific antigen-binding molecule" as used herein refers to bispecific, trispecific, or multispecific antigen-binding molecules and antigen-binding fragments thereof. Multispecific antigen-binding molecules typically contain antigen-binding domains specific for two or more antigens. For antigen-binding molecules that bind to two or more epitopes of the same antigen, the term "multiparatopic" (e.g., biparatopic, triparatopic, etc.) is preferred, although the literature is conflicting on this point, and authors sometimes use the term multispecific to refer to antigen-binding molecules that bind to two or more epitopes of the same antigen. The context in which these terms are used will clarify any ambiguity.

[0145] A multispecific antigen-binding molecule can be a single multifunctional polypeptide or a multimeric complex of two or more polypeptides covalently or noncovalently associated with each other. The term "multispecific antigen-binding molecule" includes an antibody or antigen-binding fragment thereof of the present disclosure that can be linked to or coexpressed with another functional molecule, such as another peptide or protein. For example, an antibody or antigen-binding fragment thereof can be operatively linked (e.g., by chemical bonding, genetic fusion, noncovalent bonding, or another method) to one or more other molecular entities, such as proteins or fragments thereof, to generate a bispecific or multispecific antigen-binding molecule with a second binding specificity. According to the present disclosure, the term "multispecific antigen-binding molecule" also includes a bispecific, trispecific, or multispecific antibody or antigen-binding fragment thereof. In certain exemplary embodiments, an antibody or antigen-binding fragment thereof of the present disclosure is operatively linked to another antibody or antigen-binding fragment thereof to generate a bispecific antibody with a second binding specificity.

[0146] In exemplary embodiments, the antibodies of the present disclosure are bispecific antibodies. Bispecific antibodies can be monoclonal antibodies, e.g., human or humanized antibodies, that have binding specificities for at least two different antigens. In exemplary embodiments, bispecific antibodies, fragments thereof, etc., have binding specificities for PD-1 and 4-1BB.

[0147] Methods for producing bispecific antibodies are well known. Traditionally, recombinant production of bispecific antibodies has been based on the coexpression of two immunoglobulin heavy / light chain pairs, with the two heavy chains having different specificities (Milstein et al., Nature. 1983 Oct 6-12; 305(5934):537-40). Due to the random assortment of immunoglobulin heavy and light chains, hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule is usually achieved by an affinity chromatography step. Similar procedures are disclosed in WO 1993 / 08829 and Traunecker et al. (EMBO J. 1991 Dec; 10(12):3655-9). Other methods for making bispecific antibodies are provided, for example, in Kufer et al., Trends Biotechnol. 2004 May;22(5):238-44.

[0148] Antibody variable domains with the desired binding specificities can be fused to immunoglobulin constant domain sequences. The fusion typically comprises a hinge, C H 2 and C H The fusion is performed with an immunoglobulin heavy-chain constant domain containing at least part of the three regions, with the first heavy-chain constant region (C) containing the site necessary for light-chain binding, present in at least one of the fusions. H 1). DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transformed into a suitable host organism. For further details on the production of bispecific antibodies, see, e.g., Suresh et al., Methods Enzymol. 1986;121:210-28.

[0149] The term "specifically binds" means that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding is at least about 1 x 10 -8 M or less (e.g., KD The equilibrium dissociation constant (K D "). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. Furthermore, multispecific antibodies that bind to one domain in PD-1 and one or more additional antigens or bispecific antibodies that bind to two different regions of PD-1 are nevertheless considered "specifically binding" antibodies as used herein.

[0150] The term "high affinity" refers to an antibody or antigen-binding fragment thereof that binds to an antigen, e.g., K D at least 10 for PD-1 and / or 4-1BB expressed as - 7M; at least 10 -8 M; at least 10 -9 M, at least 10 -10 M, or at least 10 -11 refers to a mAb with a binding affinity of M.

[0151] "Dissociation rate" or "K off The term "slow dissociation rate" refers to a constant used to characterize how quickly an antibody or antigen-binding fragment thereof dissociates from its antigen, e.g., PD-1 and / or 4-1BB. A "slow dissociation rate" refers to a constant used to characterize how quickly an antibody or antigen-binding fragment thereof dissociates from an antigen, e.g., PD-1 and / or 4-1BB, at a rate of 1 x 10 to 1 x 10, as determined by surface plasmon resonance, e.g., BIACORE™. -3 seconds -1 or less, or 1 x 10 -4 seconds -1 This means that the following rate constants are present:

[0152] The term "surface plasmon resonance," as used herein, refers to an optical phenomenon that allows for the analysis of real-time biomolecular interactions by detecting changes in protein concentration within a biosensor matrix, for example, using the BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ).

[0153] The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically derived, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. The term "antigen-binding fragment" of an antibody or "antibody fragment," as used herein, refers to one or more fragments of an antibody that retain the ability to bind to PD-1 and / or 4-1BB. In certain embodiments, an antibody or antibody fragment of the present disclosure may be conjugated to a ligand or therapeutic moiety (an "immunoconjugate"), e.g., an antibiotic, a second anti-PD-1 and / or anti-4-1BB antibody, or an antibody against another antigen, such as a tumor-specific antigen, a viral-infected cell antigen, an Fc receptor, a T-cell receptor, or a T-cell co-inhibitor, or an immunotoxin, or any other therapeutic moiety useful for treating a disease or condition, including cancer or chronic viral infection.

[0154] An "isolated antibody," as used herein, is an antibody that is substantially free of other antibodies (Abs) having different antigen specificities (e.g., an isolated antibody that specifically binds to PD-1 and / or 4-1BB, or an antigen-binding fragment thereof, is substantially free of Abs that specifically bind to antigens other than PD-1 and / or 4-1BB).

[0155] A "blocking antibody," "neutralizing antibody," or "antagonist antibody," as used herein, is intended to refer to an antibody whose binding to its target, e.g., PD-1, results in the inhibition of at least one biological activity of that target, e.g., PD-1. For example, an antibody or antigen-binding fragment thereof of the present disclosure may prevent or block a ligand, such as PD-L1, binding to PD-1.

[0156] As used herein, an "activating antibody," "enhancing antibody," or "agonist antibody" is intended to refer to an antibody that, upon binding to its target, e.g., 4-1BB, results in an increase or stimulation of at least one biological activity of the target, e.g., 4-1BB. For example, an antibody or antigen-binding fragment thereof of the present disclosure can mimic the action of the target's natural ligand, e.g., 4-1BBL, to promote TRAF-mediated activation of NF-κB and MAPK intracellular signaling cascades, ultimately resulting in cell activation, proliferation, and survival.

[0157] In certain exemplary embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure are both blocking and activating antibodies, for example, an anti-PD-1 / anti-4-1BB bispecific binding protein of the present disclosure can act as both an antagonist anti-PD-1 binding protein and an agonist anti-4-1BB binding protein.

[0158] The term "pure agonist," as used herein, is intended to refer to an activating antibody, or antigen-binding fragment thereof, as defined above, whose binding to an antigen increases or stimulates at least one biological activity of the antigen (by activating T cells via 4-1BB signaling) (i) under soluble conditions, and / or (ii) in the absence of a cross-linking reagent, and / or (iii) in an FcγR-independent manner (i.e., independent of Fcγ receptor engagement), and / or (iv) in the absence of target-mediated cross-linking of the antigen.

[0159] The term "Fc-mediated cross-linking," as used herein, refers to cross-linking of proteins comprising an Fc domain (e.g., an antibody) via binding to an Fc-binding portion of said Fc domain, e.g., an anti-Fc antibody or an Fc receptor.

[0160] As used herein, the term "Fc receptor" refers to a surface receptor protein found on immune cells, including B lymphocytes, natural killer cells, macrophages, basophils, neutrophils, and mast cells, that has binding specificity for the Fc region of an antibody. The term "Fc receptor" includes, but is not limited to, Fcγ receptors [e.g., FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and FcγRIIIB (CD16b)], Fcα receptors (e.g., FcαRI or CD89), and Fcε receptors [e.g., FcεRI and FcεRII (CD23)].

[0161] The term "target-mediated crosslinking," as used herein, refers to the crosslinking of an antibody or antigen-binding fragment thereof through the interaction of the antibody or antigen-binding fragment thereof with, for example, a tumor-associated antigen (TAA), an immune cell surface marker, a stromal antigen, or any other target expressed in cis or trans by tumor cells, immune cells, and / or normal cells.

[0162] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site within the variable region of an antibody, known as the "paratope." A single antigen can have more than one epitope. Thus, different antibodies may bind to different regions on the same antigen and have different biological effects. The term "epitope" also refers to the site on an antigen to which B cells and / or T cells respond. It also refers to the region of an antigen bound by an antibody. Epitopes can be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and contain residues that directly contribute to the affinity of the interaction. Epitopes can also be conformational, i.e., composed of nonlinear amino acids. In certain embodiments, epitopes contain epitopic determinants that are chemically active molecular surface groups such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, may have specific three-dimensional structural characteristics and / or specific charge characteristics.

[0163] The terms "substantial identity" or "substantially identical" when referring to a nucleic acid or fragment thereof indicates that when optimally aligned with another nucleic acid (or its complementary strand), with appropriate nucleotide insertions or deletions, there is nucleotide sequence identity over at least about 90%, or at least about 95%, 96%, 97%, 98%, or 99% of the nucleotide bases, as measured by any well-known sequence identity algorithm, such as FASTA, BLAST, or GAP, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain cases, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0164] When applied to polypeptides, the terms "substantial similarity" or "substantially similar" mean that two peptide sequences share at least 90% sequence identity, or at least 95%, 96%, 97%, 98%, or 99% sequence identity when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights. In exemplary embodiments, residue positions that are not identical may differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., in terms of charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent or degree of similarity may be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art. See, e.g., Pearson (Methods Mol Biol. 1994;24:307-31). Examples of groups of amino acids with side chains of similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains: cysteine ​​and methionine. Representative conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative replacement is any change that has a positive value in the PAM250 log likelihood matrix as disclosed in Gonnet et al. (Science. 1992 Jun 5;256(5062)1443-5, which is incorporated herein by reference. A "moderately conservative" replacement is any change that has a non-negative value in the PAM250 log likelihood matrix.

[0165] Sequence similarity of polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software contains programs such as GAP and BESTFIT, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species or between a wild-type protein and its mutein (see, e.g., GCG version 6.1). Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignment and percent sequence identity of the regions of best overlap between the query and search sequences (Pearson, Methods Mol Biol. 2000;132:185-219). Another exemplary algorithm for comparing the sequences of the present disclosure to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters (see, e.g., Altschul et al., J Mol Biol. 1990 Oct 5;215(3):403-10 and Altschul et al., Nucleic Acids Res. 1997 Sep 1;25(17):3389-402, each of which is incorporated herein by reference).

[0166] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein can be "conditionally active." "Conditionally active," as used herein, means that the antibody or antigen-binding fragment thereof is only capable of binding to its antigen (i.e., is active) under certain conditions. In some exemplary embodiments, the conditionally active antibody or antigen-binding fragment thereof comprises a masking moiety.

[0167] As used herein, the terms "mask," "masking domain," or "masking moiety" refer to a moiety added to an antibody or antigen-binding fragment thereof to reduce the antibody's ability to bind to its antigen. The mask serves to prevent or reduce antigen binding by one or more CDR sequences of the antibody or antigen-binding fragment thereof. Masking moieties include, but are not limited to, self-hinge domains, coiled-coil domains, non-antibody protein fragments, antibody fragments, affinity peptides, cross-masking antibodies, bivalent peptide-double-stranded DNA conjugates, and the like. For a review of suitable antibody masking moieties, see Lin et al. (J Biomed Sci. 2020 Jun 25;27(1):76).

[0168] In certain exemplary embodiments, the masking moiety is a polypeptide that can be removed from the antibody or antigen-binding fragment thereof by cleavage of a cleavable linker connecting the masking moiety to the antibody or antigen-binding fragment thereof, thereby allowing the antibody or antigen-binding fragment thereof to bind to its target antigen. In particularly exemplary embodiments, the masking domain of the antibody or antigen-binding fragment thereof is cleaved at the tumor site, e.g., the tumor bed or lymph node. The cleavable linker can be a protease-cleavable linker. In certain exemplary embodiments, the cleavable linker comprises at least one substrate for a tumor-specific protease.

[0169] The phrase "therapeutically effective amount" means the amount that produces the desired effect for which it is administered. The exact amount will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0170] The term "subject," as used herein, refers to an animal, e.g., a mammal, in need of amelioration, prevention, and / or treatment of a disease or disorder, such as cancer, or a chronic viral infection. In some embodiments, the subject is a human subject in need of amelioration, prevention, and / or treatment of a disease or disorder, such as cancer, or a chronic viral infection.

[0171] As used herein, "anti-cancer drug" means any agent useful in treating cancer, including, but not limited to, cytotoxins and antimetabolites, alkylating agents, anthracyclines, antibiotics, mitotic inhibitors, procarbazine, hydroxyurea, asparaginase, corticosteroids, mitotane (O,P'-(DDD)), biologics (e.g., antibodies and interferons), and radioactive agents. As used herein, "cytotoxin or cytotoxic agent" also refers to chemotherapeutic agents and means any agent that is detrimental to cells. Examples include TAXOL® (paclitaxel), temozolamide, cytochalasin B, gramicidin D, ethidium bromide, emetine, cisplatin, mitomycin, etoposide, tenoposide, vincristine, vinbiastine, coichicin, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologs thereof.

[0172] As used herein, the term "antiviral agent" refers to any drug or therapy used to treat, prevent, or ameliorate a viral infection in a host subject. The term "antiviral agent" includes, but is not limited to, zidovudine, lamivudine, abacavir, ribavirin, lopinavir, efavirenz, cobicistat, tenofovir, rilpivirine, analgesics, and corticosteroids. In the context of this disclosure, viral infections include long-term or chronic infections caused by viruses, including, but not limited to, human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), human papillomavirus (HPV), lymphocytic choriomeningitis virus (LCMV), and simian immunodeficiency virus (SIV).

[0173] The antibodies and antigen-binding fragments of the present disclosure specifically bind to PD-1 and modulate the interaction of PD-1 with a ligand, such as PD-L1, and specifically bind to 4-1BB and modulate the interaction of 4-1BB with a ligand, such as 4-1BBL. Anti-PD-1 / anti-4-1BB antibodies may bind to PD-1 and 4-1BB with high or low affinity. In certain embodiments, antibodies of the present disclosure may bind to PD-1 and block the interaction of PD-1 with PD-L1 (i.e., they are PD-1 antagonists), and may bind to 4-1BB and stimulate or enhance T cell activation (i.e., they are 4-1BB agonists). In some embodiments, the antibodies may be useful for stimulating or enhancing immune responses and / or treating subjects suffering from cancer or chronic viral infections. When administered to a subject in need thereof, the antibodies may reduce chronic infection by a virus, such as HIV, LCMV, or HBV, in the subject. They can be used to inhibit the growth of tumor cells in a subject. They can be used alone or as adjunctive therapy with other therapeutic moieties or modalities known in the art for treating cancer or viral infections.

[0174] The object of the present invention relates to immunoglobulin single variable domains (ISVs) that specifically bind to 4-1BB and have pure agonistic activity.

[0175] In some embodiments, pure agonist activity means that an ISV can activate T cells via 4-1BB signaling (i) under soluble conditions, and / or (ii) in the absence of a cross-linking reagent, and / or (iii) in an FcγR-independent manner (i.e., independent of Fcγ receptor engagement), and / or (iv) in the absence of target-mediated cross-linking of 4-1BB. In some embodiments, pure agonist activity is determined by an NF-κB pathway activation assay in the absence of a cross-linking reagent. An example of an NF-κB pathway activation assay for determining the pure agonist activity of an ISV is described in Example 1 herein, and those skilled in the art will be aware of other suitable NF-κB pathway activation assays that have already been widely described in the literature.

[0176] In some embodiments, the ISV competes with 4-1BBL for 4-1BB binding.

[0177] In some embodiments, 4-1BB is human 4-1BB, an exemplary amino acid sequence of which is shown in SEQ ID NO:13.

[0178] In some embodiments, the ISV cross-reacts with cynomolgus 4-1BB, ie, the ISV specifically binds to human 4-1BB and also to cynomolgus 4-1BB.

[0179] In some embodiments, the ISV interacts with the cysteine-rich domain 2 (CRD2) and / or cysteine-rich domain 3 (CRD3) domains of 4-1BB; preferably, the ISV interacts with the CRD2 and CRD3 domains of 4-1BB. In some embodiments, the ISV interacts with at least a first patch of amino acid residues from the CRD2 domain of 4-1BB, comprising amino acid residues K69, G70, V71, F72, and R73, and with at least a second and third patch of amino acid residues from the CRD3 domain of 4-1BB, comprising amino acid residues S100, M101, C102, E103, and Q104, and amino acid residues K114, K115, and G116, respectively (numbering based on SEQ ID NO: 13). In some embodiments, the ISV interacts with at least one amino acid residue of 4-1BB selected from the group consisting of residues K69, G70, V71, F72, R73, F92, L95, S100, M101, C102, E103, Q104, K114, K115 and G116 of SEQ ID NO: 13.

[0180] In some embodiments, the ISV comprises three complementarity determining regions (CDRs). As shown in the Examples section, ISVs with pure agonist activity as described herein interact with the target antigen 4-1BB only through their CDR3 among all three CDRs.

[0181] In some embodiments, the ISV comprises the CDR3 amino acid sequence found in SEQ ID NO: 2, 3, 58, 59, 60, or 61. In some embodiments, the ISV comprises the CDR3 amino acid sequence found in SEQ ID NO: 2 or 3.

[0182] In some embodiments, the CDR3 has the amino acid sequence: - ARGTRYKLST (SEQ ID NO: 14), ARGTRYKMST (SEQ ID NO: 15), or ARGTRYKIFA (SEQ ID NO: 62) according to IMGT numbering; or GTRYKMST (SEQ ID NO: 63), GTRYKLST (SEQ ID NO: 64), or GTRYKIFA (SEQ ID NO: 65) according to Kabat or Chothia numbering It comprises or consists of:

[0183] In some embodiments, the ISV comprises the CDR1 amino acid sequence found in SEQ ID NO: 2, 3, 58, 59, 60, or 61. In some embodiments, the ISV comprises the CDR1 amino acid sequence found in SEQ ID NO: 2 or 3.

[0184] In some embodiments, CDR1 has the amino acid sequence: - GFTFSDHT (SEQ ID NO: 16), GFAFRDFT (SEQ ID NO: 66), GDTFSSYA (SEQ ID NO: 67), or GFTFANYR (SEQ ID NO: 68) according to IMGT numbering; or - DHTMT (SEQ ID NO: 69), DFTMS (SEQ ID NO: 70), SYAMG (SEQ ID NO: 71), or NYRMS (SEQ ID NO: 72) according to Kabat numbering; or GFTFSDH (SEQ ID NO: 73), GFAFRDF (SEQ ID NO: 74), GDTFSSY (SEQ ID NO: 75), or GFTFANY (SEQ ID NO: 76) according to Chothia numbering It comprises or consists of:

[0185] In some embodiments, the ISV comprises the CDR2 amino acid sequence found in SEQ ID NO: 2, 3, 58, 59, 60, or 61. In some embodiments, the ISV comprises the CDR2 amino acid sequence found in SEQ ID NO: 2 or 3.

[0186] In some embodiments, CDR2 has the amino acid sequence: ISSGGSRI (SEQ ID NO: 17), INPSGGSQ (SEQ ID NO: 77) or IKKSGNRT (SEQ ID NO: 78) according to IMGT numbering; or - SISSGGSRIIYADSVKG (SEQ ID NO: 79), SINPSGGSQSYLPSVKG (SEQ ID NO: 80), SINPSGGSQSYHPSVKD (SEQ ID NO: 81), or SIKKSGNRTTYSDSVKG (SEQ ID NO: 82) according to Kabat numbering; or - SSGGSR (SEQ ID NO: 83), NPSGGS (SEQ ID NO: 84), or KKSGNR (SEQ ID NO: 85) according to Chothia numbering It comprises or consists of:

[0187] In some embodiments, the ISV comprises the three CDR amino acid sequences found in SEQ ID NO: 2, 3, 58, 59, 60, or 61. In some embodiments, the ISV comprises the three CDR amino acid sequences found in SEQ ID NO: 2 or 3.

[0188] In some embodiments, the ISV comprises three CDRs: - CDR1 comprises or consists of the amino acid sequence GFTFSDHT (SEQ ID NO: 16), GFAFRDFT (SEQ ID NO: 66), GDTFSSYA (SEQ ID NO: 67) or GFTFANYR (SEQ ID NO: 68); - CDR2 comprises or consists of the amino acid sequence ISSGGSRI (SEQ ID NO: 17), INPSGGSQ (SEQ ID NO: 77), or IKKSGNRT (SEQ ID NO: 78); and - CDR3 comprises or consists of the amino acid sequence ARGTRYKLST (SEQ ID NO: 14), ARGTRYKMST (SEQ ID NO: 15), or ARGTRYKIFA (SEQ ID NO: 62).

[0189] In some embodiments, the ISV comprises three CDRs: - CDR1 comprises or consists of the amino acid sequence DHTMT (SEQ ID NO: 69), DFTMS (SEQ ID NO: 70), SYAMG (SEQ ID NO: 71), or NYRMS (SEQ ID NO: 72); - CDR2 comprises or consists of the amino acid sequence SISSGGSRIIYADSVKG (SEQ ID NO: 79), SINPSGGSQSYLPSVKG (SEQ ID NO: 80), SINPSGGSQSYHPSVKD (SEQ ID NO: 81), or SIKKSGNRTTYSDSVKG (SEQ ID NO: 82); - CDR3 comprises or consists of GTRYKMST (SEQ ID NO: 63), GTRYKLST (SEQ ID NO: 64), or GTRYKIFA (SEQ ID NO: 65).

[0190] In some embodiments, the ISV comprises three CDRs: - CDR1 comprises or consists of the amino acid sequence GFTFSDH (SEQ ID NO: 73), GFAFRDF (SEQ ID NO: 74), GDTFSSY (SEQ ID NO: 75), or GFTFANY (SEQ ID NO: 76); - CDR2 comprises or consists of the amino acid sequence SSGGSR (SEQ ID NO: 83), NPSGGS (SEQ ID NO: 84), or KKSGNR (SEQ ID NO: 85); and - CDR3 comprises or consists of the amino acid sequence GTRYKMST (SEQ ID NO: 63), GTRYKLST (SEQ ID NO: 64), or GTRYKIFA (SEQ ID NO: 65).

[0191] In some embodiments, the ISV comprises three CDRs: - CDR1 comprises or consists of the amino acid sequence GFTFSDHT (SEQ ID NO: 16), - CDR2 comprises or consists of the amino acid sequence ISSGGSRI (SEQ ID NO: 17); - CDR3 comprises or consists of the amino acid sequence ARGTRYKMST (SEQ ID NO: 15), Or, - CDR1 comprises or consists of the amino acid sequence DHTMT (SEQ ID NO: 69), - CDR2 comprises or consists of the amino acid sequence SISSGGSRIIYADSVKG (SEQ ID NO: 79); - CDR3 comprises or consists of the amino acid sequence GTRYKMST (SEQ ID NO: 63), Or, - CDR1 comprises or consists of the amino acid sequence GFTFSDH (SEQ ID NO: 73), - CDR2 comprises or consists of the amino acid sequence SSGGSR (SEQ ID NO: 83); - CDR3 comprises or consists of the amino acid sequence GTRYKMST (SEQ ID NO: 63).

[0192] In some embodiments, the ISV comprises three CDRs: - CDR1 comprises or consists of the amino acid sequence GFTFSDHT (SEQ ID NO: 16), - CDR2 comprises or consists of the amino acid sequence ISSGGSRI (SEQ ID NO: 17); - CDR3 comprises or consists of the amino acid sequence ARGTRYKLST (SEQ ID NO: 14); Or, - CDR1 comprises or consists of the amino acid sequence DHTMT (SEQ ID NO: 69), - CDR2 comprises or consists of the amino acid sequence SISSGGSRIIYADSVKG (SEQ ID NO: 79); - CDR3 comprises or consists of the amino acid sequence GTRYKLST (SEQ ID NO: 64); Or, - CDR1 comprises or consists of the amino acid sequence GFTFSDH (SEQ ID NO: 73), - CDR2 comprises or consists of the amino acid sequence SSGGSR (SEQ ID NO: 83); - CDR3 comprises or consists of the amino acid sequence GTRYKLST (SEQ ID NO: 64).

[0193] In some embodiments, the ISV comprises three CDRs: - CDR1 comprises or consists of the amino acid sequence GFAFRDFT (SEQ ID NO: 66), - CDR2 comprises or consists of the amino acid sequence INPSGGSQ (SEQ ID NO: 77), - CDR3 comprises or consists of the amino acid sequence ARGTRYKMST (SEQ ID NO: 15), Or, - CDR1 comprises or consists of the amino acid sequence DFTMS (SEQ ID NO: 70), - CDR2 comprises or consists of the amino acid sequence SINPSGGSQSYLPSVKG (SEQ ID NO: 80); - CDR3 comprises or consists of the amino acid sequence GTRYKMST (SEQ ID NO: 63); Or, - CDR1 comprises or consists of the amino acid sequence GFAFRDF (SEQ ID NO: 74), - CDR2 comprises or consists of the amino acid sequence NPSGGS (SEQ ID NO: 84); - CDR3 comprises or consists of the amino acid sequence GTRYKMST (SEQ ID NO: 63).

[0194] In some embodiments, the ISV comprises three CDRs: - CDR1 comprises or consists of the amino acid sequence GDTFSSYA (SEQ ID NO: 67); - CDR2 comprises or consists of the amino acid sequence INPSGGSQ (SEQ ID NO: 77); - CDR3 comprises or consists of the amino acid sequence ARGTRYKIFA (SEQ ID NO: 62), Or, - CDR1 comprises or consists of the amino acid sequence SYAMG (SEQ ID NO: 71), - CDR2 comprises or consists of the amino acid sequence SINPSGGSQSYHPSVKD (SEQ ID NO: 81); - CDR3 comprises or consists of the amino acid sequence GTRYKIFA (SEQ ID NO: 65), Or, - CDR1 comprises or consists of the amino acid sequence GDTFSSY (SEQ ID NO: 75); - CDR2 comprises or consists of the amino acid sequence NPSGGS (SEQ ID NO: 84); - CDR3 comprises or consists of the amino acid sequence GTRYKIFA (SEQ ID NO: 65).

[0195] In some embodiments, the ISV comprises three CDRs: - CDR1 comprises or consists of the amino acid sequence GFTFANYR (SEQ ID NO: 68), - CDR2 comprises or consists of the amino acid sequence IKKSGNRT (SEQ ID NO: 78); - CDR3 comprises or consists of the amino acid sequence ARGTRYKMST (SEQ ID NO: 15), Or, - CDR1 comprises or consists of the amino acid sequence NYRMS (SEQ ID NO: 72), - CDR2 comprises or consists of the amino acid sequence SIKKSGNRTTYSDSVKG (SEQ ID NO: 82); - CDR3 comprises or consists of the amino acid sequence GTRYKMST (SEQ ID NO: 63); Or, - CDR1 comprises or consists of the amino acid sequence GFTFANY (SEQ ID NO: 76), - CDR2 comprises or consists of the amino acid sequence KKSGNR (SEQ ID NO: 85); - CDR3 comprises or consists of the amino acid sequence GTRYKMST (SEQ ID NO: 63).

[0196] In some embodiments, the ISV: an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 58, 59, 60, and 61, or - an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity over the non-CDR regions of SEQ ID NOs: 2, 3, 58, 59, 60, and 61 It comprises or consists of:

[0197] In some embodiments, the ISV: an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 3; or an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity over the non-CDR regions of SEQ ID NO: 2 or 3 It comprises or consists of:

[0198] In some embodiments, the ISV comprises or consists of the amino acid sequence of SEQ ID NO: 3. In some embodiments, the ISV comprises or consists of the amino acid sequence of SEQ ID NO:2.

[0199] In some embodiments, the ISV is HH is.

[0200] In some embodiments, the ISV is anti-4-1BB clone no. 5V, as described herein in the Examples section. HH , or a derivative thereof, for example, clone number 5.1, clone number 5a, clone number 5b, clone number 5c, or clone number 5d, and are also described in the Examples section.

[0201] Another object of the present invention relates to a bivalent or bispecific antigen-binding protein comprising at least one immunoglobulin single variable domain (ISV) that specifically binds to 4-1BB and has pure agonist activity.

[0202] In some embodiments, the bivalent or bispecific antigen binding protein comprises at least a second ISV that specifically binds to the same target antigen (i.e., 4-1BB) or to a different target antigen.

[0203] Where at least the second ISV specifically binds to another target antigen, said target antigen may be a T cell antigen, a tumor-associated or tumor-specific antigen, or a non-self antigen, or any other antigen deemed suitable by a person skilled in the art.

[0204] When the at least second ISV specifically binds to the same target antigen (i.e., 4-1BB), the at least second ISV may be the same ISV as the at least first ISV with pure agonist activity, or may be another ISV that specifically binds to 4-1BB and has pure agonist activity (including, but not limited to, the ISVs described above), or another ISV that specifically binds to 4-1BB but does not have pure agonist activity. For example, ISVs that do not have pure agonist activity but specifically bind to 4-1BB are described below.

[0205] Another object of the present invention relates to immunoglobulin single variable domains (ISVs) that specifically bind to 4-1BB. In some embodiments, the ISVs do not have pure agonist activity.

[0206] In some embodiments, the ISV does not compete with 4-1BBL for 4-1BB binding.

[0207] In some embodiments, 4-1BB is human 4-1BB, an exemplary amino acid sequence of which is shown in SEQ ID NO:13.

[0208] In some embodiments, the ISV does not cross-react or does not substantially cross-react with cynomolgus 4-1BB, i.e., the ISV specifically binds to human 4-1BB but does not bind or does not substantially bind to cynomolgus 4-1BB.

[0209] In some embodiments, the ISV comprises three complementarity determining regions (CDRs).

[0210] In some embodiments, the ISV comprises the CDR1 amino acid sequence found in SEQ ID NO:1 or 4.

[0211] In some embodiments, CDR1 comprises or consists of the amino acid sequence GGLFSINT (SEQ ID NO: 86; according to IMGT numbering); or INTGG (SEQ ID NO: 87; according to Kabat numbering); or GGLFSIN (SEQ ID NO: 88; according to Chothia numbering).

[0212] In some embodiments, the ISV comprises the CDR2 amino acid sequence found in SEQ ID NO:1 or 4.

[0213] In some embodiments, CDR2 comprises or consists of the amino acid sequence ITHDDRT (SEQ ID NO: 89; according to IMGT numbering); or TITHDDRTNYAESVKG (SEQ ID NO: 90; according to Kabat numbering); or THDDR (SEQ ID NO: 91; according to Chothia numbering).

[0214] In some embodiments, the ISV comprises the CDR3 amino acid sequence found in SEQ ID NO:1 or 4.

[0215] In some embodiments, the CDR3 comprises or consists of the amino acid sequence RLGSAAIRGY (SEQ ID NO: 92; according to IMGT numbering); or GSAAIRGY (SEQ ID NO: 93; according to Kabat or Chothia numbering).

[0216] In some embodiments, the ISV comprises the three CDR amino acid sequences found in SEQ ID NO:1 or 4.

[0217] In some embodiments, the ISV comprises three CDRs: - CDR1 comprises or consists of the amino acid sequence GGLFSINT (SEQ ID NO: 86), - CDR2 comprises or consists of the amino acid sequence ITHDDRT (SEQ ID NO: 89); - CDR3 comprises or consists of the amino acid sequence RLGSAAIRGY (SEQ ID NO: 92).

[0218] In some embodiments, the ISV comprises three CDRs: - CDR1 comprises or consists of the amino acid sequence INTGG (SEQ ID NO: 87), - CDR2 comprises or consists of the amino acid sequence TITHDDRTNYAESVKG (SEQ ID NO: 90); - CDR3 comprises or consists of the amino acid sequence GSAAIRGY (SEQ ID NO: 93).

[0219] In some embodiments, the ISV comprises three CDRs: - CDR1 comprises or consists of the amino acid sequence GGLFSIN (SEQ ID NO: 88), - CDR2 comprises or consists of the amino acid sequence THDDR (SEQ ID NO: 91); - CDR3 comprises or consists of the amino acid sequence GSAAIRGY (SEQ ID NO: 93).

[0220] In some embodiments, the ISV: an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 4, or an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity over the non-CDR regions of SEQ ID NO: 1 or 4 It comprises or consists of:

[0221] In some embodiments, the ISV comprises or consists of the amino acid sequence having SEQ ID NO: 4. In some embodiments, the ISV comprises or consists of the amino acid sequence having SEQ ID NO: 1.

[0222] In some embodiments, the ISV is HH is.

[0223] In some embodiments, the ISV is anti-4-1BB clone no. 2V, as described herein in the Examples section. HHor a derivative thereof such as clone no. 2.1, which is also described in the Examples section.

[0224] In some embodiments, the ISV further comprises at least one masking moiety, hi some embodiments, the masking moiety reduces or inhibits binding of the ISV to its target antigen, 4-1BB.

[0225] In some embodiments, the masking portion comprises or consists of the amino acid sequence CPELQGIFC (SEQ ID NO:94), or an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:94.

[0226] In some embodiments, the masking portion comprises or consists of the amino acid sequence CPELQGIFCYR (SEQ ID NO: 95), or an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 95.

[0227] In some embodiments, the masking portion comprises or consists of the amino acid sequence VEVCPELQGIFC (SEQ ID NO:96), or an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:96.

[0228] In some embodiments, the masking portion comprises or consists of the amino acid sequence VEVCPELQGIFCYR (SEQ ID NO:97), or an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:97.

[0229] In some embodiments, the masking moiety is the amino acid sequence X1X2X3X4X5X6X7X8X9X 10 X 11 CPELQGIFCX 12 X13 (SEQ ID NO: 98), wherein X1 to X 13 each of which represents any amino acid residue. In some embodiments, the masking moiety is 10 X 11 CPELQGIFCYR (SEQ ID NO: 99), 11 each represents any amino acid residue. In some embodiments, the masking moiety is 12 X 13 (SEQ ID NO: 100), wherein X1 to X8 and X 12 ~X 13 each of X1-X8 represents any amino acid residue. In some embodiments, the masking moiety comprises or consists of the amino acid sequence X1X2X3X4X5X6X7X8VEVCPELQGIFCYR (SEQ ID NO: 101), where each of X1-X8 represents any amino acid residue.

[0230] In some embodiments, the masking moiety comprises the amino acid sequence EVGSX5X6X7X8X9X 10 X 11 CPELQGIFCX 12 X 13 (SEQ ID NO: 102), 13 each represents any amino acid residue. In some embodiments, the masking moiety is 10 X 11 CPELQGIFCYR (SEQ ID NO: 103), 11 each represents any amino acid residue. In some embodiments, the masking moiety is 12 X 13 (SEQ ID NO: 104), 12 ~X 13each of X5-X8 represents any amino acid residue. In some embodiments, the masking moiety comprises or consists of the amino acid sequence EVGSX5X6X7X8VEVCPELQGIFCYR (SEQ ID NO: 105), where each of X5-X8 represents any amino acid residue.

[0231] In some embodiments, the masking portion comprises or consists of the amino acid sequence of SEQ ID NO: 44 or 45, or an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 44 or 45.

[0232] In some embodiments, the ISV further comprises at least one linker between the masking moiety and the ISV. In some embodiments, the at least one linker is cleavable. In some embodiments, the at least one linker is cleavable by at least one tumor-specific protease. In some embodiments, the at least one tumor-specific protease is selected from the group consisting of matrix metalloproteinase-9 (MMP-9), urokinase-type plasminogen activator (uPa), matrix metalloproteinase-2 (MMP-2), matriptase, regumain, kallikrein-related peptidase-3, human neutrophil elastase, proteinase 3 (Pr3), cathepsin B, and cathepsin K. In some embodiments, the at least one tumor-specific protease is MMP-9 or uPa, or a combination thereof.

[0233] In some embodiments, the linker comprises or consists of the amino acid sequence of SEQ ID NO: 56 and / or 57. However, one of skill in the art can readily appreciate that other amino acid sequences are suitable to function as linkers cleavable by tumor-specific proteases. Such amino acid sequences are well known in the art.

[0234] In some embodiments, the linker comprises or consists of the amino acid sequence of SEQ ID NO:46 or 47.

[0235] In some embodiments, cleavage of at least one linker releases the masking moiety and restores binding of the ISV to its target antigen 4-1BB.

[0236] In some embodiments, the masking moiety and cleavable linker may be fused to the N-terminus or C-terminus of the ISV; preferably, the masking moiety and cleavable linker are fused to the N-terminus of the ISV.

[0237] In some embodiments, the masked ISV (i.e., comprising the masking moiety and the cleavable linker) comprises or consists of the amino acid sequence of SEQ ID NO: 48, 49, 50, or 51, or an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 48, 49, 50, or 51; preferably the non-CDR sequences of SEQ ID NO: 48, 49, 50, or 51. an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity over the region; more preferably, comprising or consisting of an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity over the non-CDR, masking portion and cleavable linker region of SEQ ID NO: 48, 49, 50 or 51.

[0238] Another object of the present invention relates to an immunoglobulin single variable domain (ISV) that specifically binds to 4-1BB, the ISV comprising at least one masking moiety that reduces or inhibits binding of the ISV to its target antigen 4-1BB.

[0239] In some embodiments, the masking portion comprises or consists of the amino acid sequence CPELQGIFC (SEQ ID NO:94), or an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:94.

[0240] In some embodiments, the masking portion comprises or consists of the amino acid sequence CPELQGIFCYR (SEQ ID NO: 95), or an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 95.

[0241] In some embodiments, the masking portion comprises or consists of the amino acid sequence VEVCPELQGIFC (SEQ ID NO:96), or an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:96.

[0242] In some embodiments, the masking portion comprises or consists of the amino acid sequence VEVCPELQGIFCYR (SEQ ID NO:97), or an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:97.

[0243] In some embodiments, the masking moiety is the amino acid sequence X1X2X3X4X5X6X7X8X9X 10 X 11 CPELQGIFCX 12X13 (SEQ ID NO: 98), wherein X1 to X 13 each of which represents any amino acid residue. In some embodiments, the masking moiety is 10 X 11 CPELQGIFCYR (SEQ ID NO: 99), 11each represents any amino acid residue. In some embodiments, the masking moiety is 12 X 13 (SEQ ID NO: 100), wherein X1 to X8 and X 12 ~X 13 each of X1-X8 represents any amino acid residue. In some embodiments, the masking moiety comprises or consists of the amino acid sequence X1X2X3X4X5X6X7X8VEVCPELQGIFCYR (SEQ ID NO: 101), where each of X1-X8 represents any amino acid residue.

[0244] In some embodiments, the masking moiety comprises the amino acid sequence EVGSX5X6X7X8X9X 10 X 11 CPELQGIFCX 12 X 13 (SEQ ID NO: 102), 13 each represents any amino acid residue. In some embodiments, the masking moiety is 10 X 11 CPELQGIFCYR (SEQ ID NO: 103), 11 each represents any amino acid residue. In some embodiments, the masking moiety is 12 X 13 (SEQ ID NO: 104), 12 ~X 13 each of X5-X8 represents any amino acid residue. In some embodiments, the masking moiety comprises or consists of the amino acid sequence EVGSX5X6X7X8VEVCPELQGIFCYR (SEQ ID NO: 105), where each of X5-X8 represents any amino acid residue.

[0245] In some embodiments, the masking portion comprises or consists of the amino acid sequence of SEQ ID NO: 44 or 45, or an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 44 or 45.

[0246] In some embodiments, the ISV further comprises at least one linker between the masking moiety and the ISV. In some embodiments, the at least one linker is cleavable. In some embodiments, the at least one linker is cleavable by at least one tumor-specific protease. In some embodiments, the at least one tumor-specific protease is selected from the group consisting of matrix metalloproteinase-9 (MMP-9), urokinase-type plasminogen activator (uPa), matrix metalloproteinase-2 (MMP-2), matriptase, regumain, kallikrein-related peptidase-3, human neutrophil elastase, proteinase 3 (Pr3), cathepsin B, and cathepsin K. In some embodiments, the at least one tumor-specific protease is MMP-9 or uPa, or a combination thereof.

[0247] In some embodiments, the linker comprises or consists of the amino acid sequence of SEQ ID NO: 56 and / or 57. However, one of skill in the art can readily appreciate that other amino acid sequences are suitable to function as linkers cleavable by tumor-specific proteases. Such amino acid sequences are well known in the art.

[0248] In some embodiments, the linker comprises or consists of the amino acid sequence of SEQ ID NO:46 or 47.

[0249] In some embodiments, cleavage of at least one linker releases the masking moiety and restores binding of the ISV to its target antigen 4-1BB.

[0250] In some embodiments, the masking moiety and cleavable linker may be fused to the N-terminus or C-terminus of the ISV; preferably, the masking moiety and cleavable linker are fused to the N-terminus of the ISV.

[0251] In some embodiments, the ISV is HH is.

[0252] Another object of the invention relates to an immunoglobulin single variable domain (ISV) that specifically binds to a target antigen, wherein the ISV comprises at least one masking moiety that reduces or inhibits binding to the target antigen.

[0253] In some embodiments, the masking portion comprises or consists of the amino acid sequence CPELQGIFC (SEQ ID NO:94), or an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:94.

[0254] In some embodiments, the masking portion comprises or consists of the amino acid sequence CPELQGIFCYR (SEQ ID NO: 95), or an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 95.

[0255] In some embodiments, the masking portion comprises or consists of the amino acid sequence VEVCPELQGIFC (SEQ ID NO:96), or an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:96.

[0256] In some embodiments, the masking portion comprises or consists of the amino acid sequence VEVCPELQGIFCYR (SEQ ID NO:97), or an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:97.

[0257] In some embodiments, the masking moiety is the amino acid sequence X1X2X3X4X5X6X7X8X9X 10 X 11 CPELQGIFCX 12 X 13 (SEQ ID NO: 98), wherein X1 to X 13 each of which represents any amino acid residue. In some embodiments, the masking moiety is 10 X 11 CPELQGIFCYR (SEQ ID NO: 99), 11 each represents any amino acid residue. In some embodiments, the masking moiety is 12 X 13 (SEQ ID NO: 100), wherein X1 to X8 and X 12 ~X 13 each of X1-X8 represents any amino acid residue. In some embodiments, the masking moiety comprises or consists of the amino acid sequence X1X2X3X4X5X6X7X8VEVCPELQGIFCYR (SEQ ID NO: 101), where each of X1-X8 represents any amino acid residue.

[0258] In some embodiments, the masking moiety comprises the amino acid sequence EVGSX5X6X7X8X9X 10 X 11 CPELQGIFCX 12 X 13 (SEQ ID NO: 102), 13 each represents any amino acid residue. In some embodiments, the masking moiety is 10 X 11 CPELQGIFCYR (SEQ ID NO: 103), 11 each represents any amino acid residue. In some embodiments, the masking moiety is12 X 13 (SEQ ID NO: 104), 12 ~X 13 each of X5-X8 represents any amino acid residue. In some embodiments, the masking moiety comprises or consists of the amino acid sequence EVGSX5X6X7X8VEVCPELQGIFCYR (SEQ ID NO: 105), where each of X5-X8 represents any amino acid residue.

[0259] In some embodiments, the masking portion comprises or consists of the amino acid sequence of SEQ ID NO: 44 or 45, or an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 44 or 45.

[0260] In some embodiments, the ISV further comprises at least one linker between the masking moiety and the ISV. In some embodiments, the at least one linker is cleavable. In some embodiments, the at least one linker is cleavable by at least one tumor-specific protease. In some embodiments, the at least one tumor-specific protease is selected from the group consisting of matrix metalloproteinase-9 (MMP-9), urokinase-type plasminogen activator (uPa), matrix metalloproteinase-2 (MMP-2), matriptase, regumain, kallikrein-related peptidase-3, human neutrophil elastase, proteinase 3 (Pr3), cathepsin B, and cathepsin K. In some embodiments, the at least one tumor-specific protease is MMP-9 or uPa, or a combination thereof.

[0261] In some embodiments, the linker comprises or consists of the amino acid sequence of SEQ ID NO: 56 and / or 57. However, one of skill in the art can readily appreciate that other amino acid sequences are suitable to function as linkers cleavable by tumor-specific proteases. Such amino acid sequences are well known in the art.

[0262] In some embodiments, the linker comprises or consists of the amino acid sequence of SEQ ID NO:46 or 47.

[0263] In some embodiments, cleavage of at least one linker releases the masking moiety and restores binding of the ISV to its target antigen.

[0264] In some embodiments, the masking moiety and cleavable linker may be fused to the N-terminus or C-terminus of the ISV; preferably, the masking moiety and cleavable linker are fused to the N-terminus of the ISV.

[0265] In some embodiments, the ISV is HH is.

[0266] Another object of the present invention relates to an antibody or antigen-binding fragment thereof that specifically binds to PD-1.

[0267] In some embodiments, the PD-1 is human PD-1, an exemplary amino acid sequence of which is shown in SEQ ID NO:42.

[0268] In some embodiments, the antibody or antigen-binding fragment thereof has a human germinality index of 95% or greater for both the heavy and light variable regions.

[0269] "Human germinality index," a term coined by Pelat et al. (J Mol Biol. 2008 Dec 31;384(5):1400-7), refers to the percentage of framework residues that are identical between the variable region sequence of an antibody or antigen-binding fragment thereof and the closest human germline sequence.

[0270] In some embodiments, the antibody or antigen-binding fragment thereof is: (i) the three light chain CDR amino acid sequences found in SEQ ID NO: 7 or 5, and (ii) the three heavy chain CDR amino acid sequences found in SEQ ID NO: 8 or 6 Includes.

[0271] In some embodiments, the antibody or antigen-binding fragment thereof is: (i) the three light chain CDR amino acid sequences found in SEQ ID NO: 7, and (ii) the three heavy chain CDR amino acid sequences found in SEQ ID NO:8 Includes.

[0272] In some embodiments, the antibody or antigen-binding fragment thereof is: (i) the three light chain CDR amino acid sequences found in SEQ ID NO: 5, and (ii) the three heavy chain CDR amino acid sequences found in SEQ ID NO:6 Includes.

[0273] In some embodiments, the antibody or antigen-binding fragment thereof is: (i) the following three CDR amino acid sequences: aV L CDR1: QSVPINF (SEQ ID NO: 18) or QSVSINF (SEQ ID NO: 19), bV L CDR2:EAS, and cV L - CDR3: GQYGSSPYT (SEQ ID NO: 20) or QQYGSSPYT (SEQ ID NO: 21) a light chain variable region comprising: (ii) the following three CDR amino acid sequences: aV H - CDR1: GGSISSSSYF (SEQ ID NO: 22) or GGSISTSSYF (SEQ ID NO: 23), bV H CDR2: IYRSGST (SEQ ID NO: 24), and cV H -CDR3: ARGITGDPGDY (SEQ ID NO: 25) a heavy chain variable region comprising Includes.

[0274] In some embodiments, the antibody or antigen-binding fragment thereof is: (i) the following three CDR amino acid sequences: aV L CDR1: GASQSVPINFLA (SEQ ID NO: 106) or GASQSVSINFLA (SEQ ID NO: 107), bV L CDR2: EASSRHT (SEQ ID NO: 108) or EASSRAT (SEQ ID NO: 109), and cV L - CDR3: GQYGSSPYT (SEQ ID NO: 20) or QQYGSSPYT (SEQ ID NO: 21) a light chain variable region comprising: (ii) the following three CDR amino acid sequences: aV H CDR1: SSSYFWG (SEQ ID NO: 110) or TSSYFWG (SEQ ID NO: 111), bV H CDR2: SIYRSGSTYYNPSLKS (SEQ ID NO: 112), and cV H -CDR3: GITGDPGDY (SEQ ID NO: 113) a heavy chain variable region comprising Includes.

[0275] In some embodiments, the antibody or antigen-binding fragment thereof is: (i) the following three CDR amino acid sequences: aV L CDR1: GASQSVPINFLA (SEQ ID NO: 106) or GASQSVSINFLA (SEQ ID NO: 107); bV L CDR2: EASSRHT (SEQ ID NO: 108) or EASSRAT (SEQ ID NO: 109), and cV L - CDR3: GQYGSSPYT (SEQ ID NO: 20) or QQYGSSPYT (SEQ ID NO: 21) a light chain variable region comprising: (ii) the following three CDR amino acid sequences: aV H- CDR1: GGSISSSSY (SEQ ID NO: 114) or GGSISTSSY (SEQ ID NO: 115), bV H CDR2: YRSGS (SEQ ID NO: 116), and cV H -CDR3: GITGDPGDY (SEQ ID NO: 113) a heavy chain variable region comprising Includes.

[0276] In some embodiments, the antibody or antigen-binding fragment thereof is: (i) the following three CDR amino acid sequences: aV L - CDR1: QSVPINF (SEQ ID NO: 18), bV L CDR2:EAS, and cV L -CDR3: GQYGSSPYT (SEQ ID NO: 20) a light chain variable region comprising: (ii) the following three CDR amino acid sequences: aV H - CDR1:GGSISSSSYF (SEQ ID NO: 22), bV H CDR2: IYRSGST (SEQ ID NO: 24), and cV H -CDR3: ARGITGDPGDY (SEQ ID NO: 25) a heavy chain variable region comprising Or, (i) the following three CDR amino acid sequences: aV L - CDR1: GASQSVPINFLA (SEQ ID NO: 106), bV L CDR2: EASSRHT (SEQ ID NO: 108), and cV L -CDR3: GQYGSSPYT (SEQ ID NO: 20) a light chain variable region comprising: (ii) the following three CDR amino acid sequences: aV H -CDR1: SSSYFWG (SEQ ID NO: 110): bV H CDR2: SIYRSGSTYYNPSLKS (SEQ ID NO: 112), and cV H -CDR3: GITGDPGDY (SEQ ID NO: 113) a heavy chain variable region comprising Or, (i) the following three CDR amino acid sequences: aV L - CDR1: GASQSVPINFLA (SEQ ID NO: 106), bV L CDR2: EASSRHT (SEQ ID NO: 108), and cV L -CDR3: GQYGSSPYT (SEQ ID NO: 20) a light chain variable region comprising: (ii) the following three CDR amino acid sequences: aV H - CDR1:GGSISSSSY (SEQ ID NO: 114), bV H CDR2: YRSGS (SEQ ID NO: 116), and cV H -CDR3: GITGDPGDY (SEQ ID NO: 113) a heavy chain variable region comprising Includes.

[0277] In some embodiments, the antibody or antigen-binding fragment thereof is: (i) the following three CDR amino acid sequences: aV L - CDR1: QSVSINF (SEQ ID NO: 19), bV L CDR2:EAS, and cV L -CDR3: QQYGSSPYT (SEQ ID NO: 21) a light chain variable region comprising: (ii) the following three CDR amino acid sequences: aV H - CDR1: GGSISTSSYF (SEQ ID NO: 23), bV HCDR2: IYRSGST (SEQ ID NO: 24), and cV H -CDR3: ARGITGDPGDY (SEQ ID NO: 25) a heavy chain variable region comprising Or, (i) the following three CDR amino acid sequences: aV L - CDR1: GASQSVSINFLA (SEQ ID NO: 107), bV L CDR2: EASSRAT (SEQ ID NO: 109), and cV L -CDR3: QQYGSSPYT (SEQ ID NO: 21) a light chain variable region comprising: (ii) the following three CDR amino acid sequences: aV H - CDR1: TSSYFWG (SEQ ID NO: 111), bV H CDR2: SIYRSGSTYYNPSLKS (SEQ ID NO: 112), and cV H -CDR3: GITGDPGDY (SEQ ID NO: 113) a heavy chain variable region comprising Or, (i) the following three CDR amino acid sequences: aV L - CDR1: GASQSVSINFLA (SEQ ID NO: 107), bV L CDR2: EASSRAT (SEQ ID NO: 109), and cV L -CDR3: QQYGSSPYT (SEQ ID NO: 21) a light chain variable region comprising: (ii) the following three CDR amino acid sequences: aV H - CDR1: GGSISTSSY (SEQ ID NO: 115), bV H CDR2: YRSGS (SEQ ID NO: 116), and cV H -CDR3: GITGDPGDY (SEQ ID NO: 113) a heavy chain variable region comprising Includes.

[0278] In some embodiments, the antibody or antigen-binding fragment thereof is: (i) a light chain variable region having SEQ ID NO: 7 or 5, or a light chain variable region sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity over the non-CDR regions of SEQ ID NO: 7 or 5; and (ii) a heavy chain variable region having SEQ ID NO: 8 or 6, or a heavy chain variable region sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity over the non-CDR regions of SEQ ID NO: 8 or 6 Includes.

[0279] In some embodiments, the antibody or antigen-binding fragment thereof is: (i) a light chain variable region having SEQ ID NO: 7, or a light chain variable region sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity over the non-CDR regions of SEQ ID NO: 7; and (ii) a heavy chain variable region having SEQ ID NO: 8, or a heavy chain variable region sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity over the non-CDR regions of SEQ ID NO: 8 Includes.

[0280] In some embodiments, the antibody or antigen-binding fragment thereof is: (i) a light chain variable region having SEQ ID NO: 5, or a light chain variable region sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity over the non-CDR regions of SEQ ID NO: 5; and (ii) a heavy chain variable region having SEQ ID NO: 6, or a heavy chain variable region sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity over the non-CDR regions of SEQ ID NO: 6 Includes.

[0281] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region of SEQ ID NO:7 and a heavy chain variable region of SEQ ID NO:8.

[0282] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region of SEQ ID NO:5 and a heavy chain variable region of SEQ ID NO:6.

[0283] Another object of the present invention relates to a molecule, such as a multispecific antigen-binding protein, comprising at least one immunoglobulin single variable domain (ISV) that specifically binds to 4-1BB and has pure agonist activity as defined above.

[0284] In some embodiments, a molecule, e.g., a multispecific antigen-binding protein, comprises at least two ISVs that specifically bind to 4-1BB. In some embodiments, the at least two ISVs that specifically bind to 4-1BB are identical. Alternatively, the at least two ISVs that specifically bind to 4-1BB are different. If different, the at least two ISVs that specifically bind to 4-1BB may bind to the same epitope, an overlapping epitope, or distinct epitopes of 4-1BB. In some embodiments, the at least two ISVs that specifically bind to 4-1BB are different and bind to distinct epitopes of 4-1BB.

[0285] In some embodiments, at least one ISV that specifically binds to 4-1BB is an ISV with pure agonist activity as described herein (for example, but not limited to, an ISV comprising or consisting of the amino acid sequence of SEQ ID NO: 2, 3, 58, 59, 60, or 61). In some embodiments, at least a second ISV that specifically binds to 4-1BB is an ISV with pure agonist activity as described herein (for example, but not limited to, an ISV comprising or consisting of the amino acid sequence of SEQ ID NO: 2, 3, 58, 59, 60, or 61). In some embodiments, at least a second ISV that specifically binds to 4-1BB is an ISV without pure agonist activity as described herein (for example, but not limited to, an ISV comprising or consisting of the amino acid sequence of SEQ ID NO: 1 or 4).

[0286] In some embodiments, the molecule, e.g., a multispecific antigen-binding protein, comprises at least two ISVs, one of which specifically binds to 4-1BB and another of which specifically binds to another target antigen, which may be a T cell antigen, a tumor-associated or tumor-specific antigen, or a non-self antigen, or any other antigen deemed suitable by one skilled in the art.

[0287] In some embodiments, the molecule, e.g., the multispecific antigen binding protein, comprises at least four ISVs that specifically bind to 4-1BB. In some embodiments, the at least four ISVs are (i) a first set of two identical ISVs that specifically bind to 4-1BB, and (ii) a second set of two other identical ISVs that specifically bind 4-1BB; Or, (i') a first set of two ISVs that specifically bind to a first 4-1BB epitope; and (ii') a second set of two other ISVs that specifically bind to a second 4-1BB epitope Includes.

[0288] The first 4-1BB epitope and the second 4-1BB epitope may be identical, overlapping, or distinct. In some embodiments, the first 4-1BB epitope and the second 4-1BB epitope are different.

[0289] In some embodiments, the first set of two ISVs in (i) or (i') is an ISV with pure agonist activity as described herein (e.g., without limitation, an ISV comprising or consisting of the amino acid sequence of SEQ ID NO: 2, 3, 58, 59, 60 or 61).

[0290] In some embodiments, the second set of two ISVs (ii) or (ii') is an ISV that does not have pure agonist activity as described herein (e.g., without limitation, an ISV that includes or consists of the amino acid sequence of SEQ ID NO: 1 or 4).

[0291] In some embodiments, the molecule, e.g., the multispecific antigen-binding protein, further comprises an antibody Fc region or a fragment thereof. For example, the Fc region or a fragment thereof can be one of IgG, IgD, IgA, IgM, or IgE Fc regions; in particular, one of IgG Fc regions, such as an IgG1 or IgG4 Fc region. The Fc region can also be one of antibody-dependent cellular cytotoxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADCP) silenced. Examples of such silenced Fc regions are known in the art and include, but are not limited to, the IgG1 LALA Fc region, the IgG1 NNAS Fc region, and the IgG4 P-FALA Fc region. In some embodiments, the Fc region or a fragment thereof is one of IgG1 LALA Fc regions.

[0292] In some embodiments, the molecule, e.g., the multispecific antigen binding protein, further comprises at least one Fab fragment.

[0293] In some embodiments, the molecule, e.g., the multispecific antigen binding protein, -preferably from N-terminus to C-terminus: • The first ISV that specifically binds to 4-1BB; - a second ISV that specifically binds to 4-1BB, preferably different from the first ISV; At least one C in the Fc region H domain; and - variable and constant domains of the Fab fragment a first polypeptide (i.e., heavy chain) comprising: - a second polypeptide comprising the variable and constant domains of the Fab fragment (i.e., the light chain) Includes; The variable and constant domains of the first and second polypeptides form a Fab fragment.

[0294] In some embodiments, the molecule, e.g., the multispecific antigen binding protein, further comprises a third polypeptide and a fourth polypeptide that are identical to the first polypeptide and the second polypeptide, respectively, and at least one C of the first polypeptide and the third polypeptide. H The domains form the Fc region.

[0295] In some embodiments, the variable domain and the constant domain of the first polypeptide are V H Domain and C H the variable and constant domains of the second polypeptide are V L Domain and C L Alternatively, the variable domain and the constant domain of the first polypeptide are V L and C L domain, and the variable and constant domains of the second polypeptide are V H Domain and C H It is one domain.

[0296] In some embodiments, at least one C of the first polypeptide H The domain is - IgG CH 2 and C H 3 domains; -IgD C H 2 and C H 3 domains; -IgA C H 2 and C H 3 domains; -IgM C H 2. C H 3 and C H 4 domains; or -IgE C H 2. C H 3, and C H 4 Domains Includes.

[0297] In some embodiments, at least one C of the first polypeptide H The domain is C of IgG H 2 domain and C H Includes 3 domains.

[0298] In some embodiments, the first polypeptide (i.e., the heavy chain) preferably comprises, from N-terminus to C-terminus: a first ISV that specifically binds to -4-1BB; - a first linker; a second ISV that specifically binds to -4-1BB, preferably different from the first ISV; - a second linker; -IgG hinge region; -IgGC H 2 domains; - and IgGC H 3 domains; - a third linker; - V of Fab fragment H domain; and -Fab fragment C H 1 domain Includes.

[0299] In some embodiments, the second polypeptide (i.e., the light chain) preferably comprises, from N-terminus to C-terminus: - V of Fab fragmentL domain; and -Fab fragment C L domain Includes.

[0300] In some embodiments, at least one Fab fragment specifically binds to a B cell surface protein and / or a T cell surface protein other than 4-1BB. In some embodiments, at least one Fab fragment specifically binds to an immune checkpoint molecule. In some embodiments, at least one Fab fragment is a PD-1 antagonist.

[0301] In some embodiments, at least one Fab fragment is an antigen-binding fragment that specifically binds to PD-1 as described herein (for example, but not limited to, an antigen-binding fragment comprising or consisting of a light chain variable region having SEQ ID NO: 7 or 5 and a heavy chain variable region having SEQ ID NO: 8 or 6).

[0302] In some embodiments, the molecule, e.g., a multispecific antigen binding protein, comprises at least a first polypeptide having SEQ ID NO: 11 or 9 and at least a second polypeptide having SEQ ID NO: 12 or 10, or comprises at least a first polypeptide comprising an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 11 or 9, and at least a second polypeptide comprising an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 12 or 10.

[0303] In some embodiments, the molecule, e.g., the multispecific antigen binding protein, comprises at least a first polypeptide having SEQ ID NO:11 and at least a second polypeptide having SEQ ID NO:12.

[0304] In some embodiments, the molecule, e.g., the multispecific antigen binding protein, comprises at least a first polypeptide having SEQ ID NO:9 and at least a second polypeptide having SEQ ID NO:10.

[0305] In some embodiments, the molecule, e.g., the multispecific antigen binding protein, is capable of inducing T cell activation in a TCA and / or MLR assay.

[0306] In some embodiments, the molecule, e.g., the multispecific antigen binding protein, is capable of inducing IFN-γ and / or TNF-α secretion in a CD3-PBMC activation assay.

[0307] In some embodiments, the molecule, e.g., the multispecific antigen binding protein, binds to exhausted CD8 + T cells can be reactivated in vitro.

[0308] In some embodiments, the molecule, e.g., the multispecific antigen binding protein, is an in vitro T reg It is possible to induce suppressive activity.

[0309] In some embodiments, the molecule, e.g., a multispecific antigen-binding protein, comprises at least one masking moiety. In some embodiments, the masking moiety reduces or inhibits binding of the molecule to at least one of its targets. In some embodiments, the masking moiety reduces or inhibits binding of the molecule to 4-1BB.

[0310] In some embodiments, the masking portion comprises or consists of the amino acid sequence CPELQGIFC (SEQ ID NO:94), or an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:94.

[0311] In some embodiments, the masking portion comprises or consists of the amino acid sequence CPELQGIFCYR (SEQ ID NO: 95), or an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 95.

[0312] In some embodiments, the masking portion comprises or consists of the amino acid sequence VEVCPELQGIFC (SEQ ID NO:96), or an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:96.

[0313] In some embodiments, the masking portion comprises or consists of the amino acid sequence VEVCPELQGIFCYR (SEQ ID NO:97), or an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:97.

[0314] In some embodiments, the masking moiety is the amino acid sequence X1X2X3X4X5X6X7X8X9X 10 X 11 CPELQGIFCX 12 X 13 (SEQ ID NO: 98), wherein X1 to X 13 each of which represents any amino acid residue. In some embodiments, the masking moiety is 10 X 11 CPELQGIFCYR (SEQ ID NO: 99), 11 each represents any amino acid residue. In some embodiments, the masking moiety is 12 X 13 (SEQ ID NO: 100), wherein X1 to X8 and X 12 ~X 13each of X1-X8 represents any amino acid residue. In some embodiments, the masking moiety comprises or consists of the amino acid sequence X1X2X3X4X5X6X7X8VEVCPELQGIFCYR (SEQ ID NO: 101), where each of X1-X8 represents any amino acid residue.

[0315] In some embodiments, the masking moiety comprises the amino acid sequence EVGSX5X6X7X8X9X 10 X 11 CPELQGIFCX 12 X 13 (SEQ ID NO: 102), 13 each represents any amino acid residue. In some embodiments, the masking moiety is 10 X 11 CPELQGIFCYR (SEQ ID NO: 103), 11 each represents any amino acid residue. In some embodiments, the masking moiety is 12 X 13 (SEQ ID NO: 104), 12 ~X 13 each of X5-X8 represents any amino acid residue. In some embodiments, the masking moiety comprises or consists of the amino acid sequence EVGSX5X6X7X8VEVCPELQGIFCYR (SEQ ID NO: 105), where each of X5-X8 represents any amino acid residue.

[0316] In some embodiments, the masking portion comprises or consists of the amino acid sequence of SEQ ID NO: 44 or 45, or an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 44 or 45.

[0317] In some embodiments, the molecule, e.g., the multispecific antigen-binding protein, further comprises at least one linker between the masking moiety and the molecule. In some embodiments, the at least one linker is cleavable. In some embodiments, the at least one linker is cleavable by at least one tumor-specific protease. In some embodiments, the at least one tumor-specific protease is selected from the group consisting of matrix metalloproteinase-9 (MMP-9), urokinase-type plasminogen activator (uPa), matrix metalloproteinase-2 (MMP-2), matriptase, regumain, kallikrein-related peptidase-3, human neutrophil elastase, proteinase 3 (Pr3), cathepsin B, and cathepsin K. In some embodiments, the at least one tumor-specific protease is MMP-9 or uPa, or a combination thereof.

[0318] In some embodiments, the linker comprises or consists of the amino acid sequence of SEQ ID NO: 56 and / or 57. However, one of skill in the art can readily appreciate that other amino acid sequences are suitable to function as linkers cleavable by tumor-specific proteases. Such amino acid sequences are well known in the art.

[0319] In some embodiments, at least one linker comprises or consists of the amino acid sequence of SEQ ID NO:46 or 47.

[0320] In some embodiments, cleavage of at least one linker releases the masking moiety, restoring binding of the molecule, e.g., of the multispecific antigen binding protein, to at least one of its target antigens. In some embodiments, cleavage of at least one linker releases the masking moiety, restoring binding of the molecule, e.g., of the multispecific antigen binding protein, to 4-1BB.

[0321] In some embodiments, the masking moiety and cleavable linker may be fused to the N- or C-terminus of the heavy chain of a molecule, such as a multispecific antigen-binding protein. Alternatively, the masking moiety and cleavable linker may be fused to the N- or C-terminus of the light chain of a molecule, such as a multispecific antigen-binding protein.

[0322] In some embodiments, the masking moiety and cleavable linker are fused to the N-terminus of the heavy chain of a molecule, e.g., a multispecific antigen binding protein, e.g., the N-terminus of a first ISV that specifically binds 4-1BB.

[0323] In some embodiments, the molecule, e.g., the multispecific antigen binding protein, (i) at least a first polypeptide having SEQ ID NO: 11 or 9, or an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity over the non-CDR regions of SEQ ID NO: 11 or 9; and (ii) at least a second polypeptide having SEQ ID NO: 52, 53, 54 or 55, or an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity over the non-CDR region of SEQ ID NO: 52, 53, 54 or 55; preferably an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity over the non-CDR, masking and cleavable linker region of SEQ ID NO: 52, 53, 54 or 55. Includes.

[0324] In some embodiments, the molecule, e.g., the multispecific antigen binding protein, comprises at least a first polypeptide having SEQ ID NO:11 and at least a second polypeptide having SEQ ID NO:52.

[0325] In some embodiments, the molecule, e.g., the multispecific antigen binding protein, comprises at least a first polypeptide having SEQ ID NO:11 and at least a second polypeptide having SEQ ID NO:53.

[0326] In some embodiments, the molecule, e.g., the multispecific antigen binding protein, comprises at least a first polypeptide having SEQ ID NO:11 and at least a second polypeptide having SEQ ID NO:54.

[0327] In some embodiments, the molecule, e.g., the multispecific antigen binding protein, comprises at least a first polypeptide having SEQ ID NO:11 and at least a second polypeptide having SEQ ID NO:55.

[0328] In some embodiments, the molecule, e.g., the multispecific antigen binding protein, comprises at least a first polypeptide having SEQ ID NO:9 and at least a second polypeptide having SEQ ID NO:52.

[0329] In some embodiments, the molecule, e.g., the multispecific antigen binding protein, comprises at least a first polypeptide having SEQ ID NO:9 and at least a second polypeptide having SEQ ID NO:53.

[0330] In some embodiments, the molecule, e.g., the multispecific antigen binding protein, comprises at least a first polypeptide having SEQ ID NO:9 and at least a second polypeptide having SEQ ID NO:54.

[0331] In some embodiments, the molecule, e.g., the multispecific antigen binding protein, comprises at least a first polypeptide having SEQ ID NO:9 and at least a second polypeptide having SEQ ID NO:55.

[0332] In some embodiments, the molecule, e.g., the multispecific antigen binding protein, further comprises a third polypeptide and a fourth polypeptide that are identical to the first polypeptide and the second polypeptide, respectively.

[0333] Antigen-binding fragment of an antibody Unless otherwise specified, the term "antibody," as used herein, is understood to encompass antibody molecules comprising two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., "intact antibody molecules") and antigen-binding fragments thereof. Antibody fragments may include Fab fragments, F(ab')2 fragments, Fv fragments, dAb fragments, CDR-containing fragments, or isolated CDRs. In certain embodiments, the term "antigen-binding fragment" refers to a polypeptide fragment of a multispecific antigen-binding molecule. In such embodiments, the term "antigen-binding fragment" includes, for example, the extracellular domain of PD-L1 that specifically binds to PD-1 or the extracellular domain of 4-1BBL that specifically binds to 4-1BB. Antigen-binding fragments of antibodies can be derived from intact antibody molecules using any suitable standard techniques, such as, for example, proteolytic digestion or recombinant genetic engineering techniques, which involve the manipulation and expression of DNA encoding antibody variable and (optionally) constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or using molecular biology techniques, for example, to arrange one or more variable and / or constant domains in the appropriate configuration, or to introduce codons, create cysteine ​​residues, modify, add or delete amino acids, etc.

[0334] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic a hypervariable region of an antibody (e.g., an isolated complementarity-determining region (CDR), such as a CDR3 peptide, or a constraining FR3-CDR3-FR4 peptide). Domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other engineered molecules such as shark variable IgNAR domains are also encompassed by the term "antigen-binding fragment" as used herein.

[0335] Antigen-binding fragments of antibodies typically contain at least one variable domain. The variable domain may be of any size or amino acid composition and generally contains at least one CDR adjacent to or in-frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be positioned relative to each other in any suitable configuration. For example, the variable region may be dimeric and contain VH-VH, VH-VL, or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.

[0336] In certain embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody of the present disclosure include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) VH-CH1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2; (X) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids that provide a flexible or semi-flexible linkage between adjacent variable and / or constant domains within a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the present disclosure may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above in non-covalent association with each other and / or with one or more monomeric VH or VL domains (e.g., via disulfide bonds).

[0337] As with intact antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically comprise at least two different variable domains, each capable of specifically binding to a separate antigen or a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of antigen-binding fragments of antibodies of the present disclosure using routine techniques available in the art.

[0338] Preparation of human antibodies Methods for producing human antibodies in transgenic mice are known in the art. Such known methods can be used in connection with the present disclosure to produce human antibodies that specifically bind to PD-1 and / or 4-1BB.

[0339] Antibodies to PD-1 and / or 4-1BB can be generated using an immunogen comprising any one of the following: In certain embodiments, antibodies of the present disclosure are obtained from mice immunized with full-length, native PD-1 and / or 4-1BB, or recombinant PD-1 and / or 4-1BB peptides. Alternatively, PD-1 and / or 4-1BB or fragments thereof can be produced using standard biochemical techniques, modified, and used as immunogens. In certain embodiments, the immunogen can be a peptide from the N-terminus or C-terminus of PD-1 and / or 4-1BB.

[0340] In some embodiments, the immunogen may be recombinant PD-1 and / or 4-1BB peptides expressed in Escherichia coli (E. coli) or any other eukaryotic or mammalian cells, such as Chinese hamster ovary (CHO) cells.

[0341] In certain embodiments, antibodies that specifically bind to PD-1 and / or 4-1BB may be prepared using fragments of the above regions, or peptides extending beyond the designated regions by about 5 to about 20 amino acid residues from either the N-terminus or C-terminus of the regions described herein, or both. In certain embodiments, any combination of the above regions or fragments thereof may be used to prepare PD-1 and / or 4-1BB-specific antibodies.

[0342] Using VelocImmune® technology (see, e.g., U.S. Pat. No. 6,596,541, Regeneron Pharmaceuticals) or any other known method for generating monoclonal antibodies, high-affinity chimeric antibodies against PD-1 and / or 4-1BB with human variable regions and mouse constant regions are first isolated. VelocImmune® technology involves the generation of transgenic mice whose genomes contain human heavy and light chain variable regions operably linked to endogenous mouse constant region loci, such that the mice produce antibodies containing human variable regions and mouse constant regions in response to antigenic challenge. DNA encoding the heavy and light chain variable regions of the antibody is isolated and operably linked to DNA encoding human heavy and light chain constant regions. The DNA is then expressed in cells capable of expressing fully human antibodies. As will be appreciated by those skilled in the art, several other transgenic mouse systems can be used, such as Trianni® mice from Trianni Inc., Kymouse® mice from Kymab Limited, OmniMouse® from OmniAb, or HuMAb Mouse® from Medarex.

[0343] biological equivalent The anti-PD-1 / anti-4-1BB bispecific antibodies of the present disclosure (or any of their individual components) include proteins having amino acid sequences that differ from those of the described antibodies but retain the ability to bind to PD-1 and 4-1BB. Such variant antibodies and antigen-binding fragments thereof contain one or more additions, deletions, or substitutions of amino acids when compared to the parent sequence, but exhibit essentially the same biological activity as the described antibody. Similarly, antibody-encoding DNA sequences of the present disclosure include sequences that contain one or more additions, deletions, or substitutions of nucleotides when compared to the disclosed sequences, but encode antibodies or antibody fragments that are essentially biologically equivalent to the antibodies or antibody fragments of the present disclosure.

[0344] Two antigen-binding proteins, or antibodies, are considered bioequivalents, for example, if they are pharmaceutical equivalents or pharmaceutical substitutes and their absorption rates and extents do not differ significantly when administered at the same molar dose, either in single or multiple doses, under similar experimental conditions. Some antibodies may be considered bioequivalents or pharmaceutical substitutes if they are equivalent in their extent of absorption but not in their absorption rate, and yet such differences in absorption rate may be considered bioequivalents or pharmaceutical substitutes because they are intentional, reflected in labeling, and are not considered essential, for example, to achieving effective body drug concentrations in chronic use and are not considered medically significant for the particular drug product being studied.

[0345] In one embodiment, two antigen binding proteins are bioequivalent if there are no clinically meaningful differences in their safety, purity, or potency.

[0346] In one embodiment, two antigen binding proteins are bioequivalent if a patient can switch between the reference product and the biological product one or more times without a predicted increased risk of adverse effects, including clinically significant changes in immunogenicity or reduced efficacy, compared to continued treatment without such a switch.

[0347] In one embodiment, two antigen binding proteins are bioequivalent if they both act by a common mechanism or mode of action for the state or condition of use, so long as such mechanism is known.

[0348] Bioequivalence may be demonstrated by in vivo and / or in vitro methods. Bioequivalence measurements include, for example, (a) in vivo studies in humans or other mammals in which the concentration of the antibody or its metabolites is measured in blood, plasma, serum, or other biological fluids as a function of time; (b) in vitro studies that correlate with and are reasonably predictive of human in vivo bioavailability data; (c) in vivo studies in humans or other mammals in which the relevant acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) well-controlled clinical trials that establish the safety, efficacy, or bioavailability or bioequivalence of the antibody.

[0349] Bioequivalent variants of the antibodies of the present disclosure can be constructed, for example, by making various substitutions of residues or sequences or by deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity can be deleted or replaced with other amino acids to prevent the formation of unnecessary or erroneous intramolecular disulfide bridges upon renaturation. In other contexts, bioequivalent antibodies can include antibody variants containing amino acid changes that alter the glycosylation characteristics of the antibody, for example, mutations that eliminate or remove glycosylation.

[0350] Anti-PD-1 / anti-4-1BB antibodies containing Fc variants According to certain embodiments of the present disclosure, there are provided anti-PD-1 / anti-4-1BB antibodies comprising an Fc domain comprising one or more mutations that enhance or decrease antibody binding to the FcRn receptor at, for example, acidic pH compared to neutral pH. For example, the present disclosure provides H 2 or C HThe present invention also includes anti-PD-1 / anti-4-1BB antibodies containing mutations in the Fc domain or constant region, which increase the affinity of the Fc domain for FcRn in acidic environments (e.g., endosomes with a pH in the range of about 5.5 to about 6.0). Such mutations may result in an increase in the serum half-life of the antibody when administered to an animal. Hereinafter, unless otherwise specified, the numbering of amino acid residues in the Fc domain or constant region will be in accordance with Kabat et al., 1991 (Sequences of proteins of immunological interest). thThis is done by the so-called EU index, which is described in the Journal of the American Medical Association (Ed., Bethesda, MD: US Dept. of Health and Human Services, Public Health Service, National Institutes of Health). Non-limiting examples of such Fc modifications include, for example, modifications at positions 234 (e.g., A), 235 (e.g., A), 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T); or modifications at positions 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., A, W, H, F, or Y [N434A, N434W, N434H, N434F, or N434Y]); or modifications at positions 250 and / or 428; or modifications at positions 307 or 308 (e.g., 308F, V308F) and 434. In embodiments, the modifications include 234A (e.g., L234A) and 235A (e.g., L235A) modifications, 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications; 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; 25 2, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications; 250Q and 428L modifications (e.g., T250Q and M428L); and 307 and / or 308 modifications (e.g., 308F or 308P). In yet another embodiment, the modifications include a 265A (e.g., D265A) and / or a 297A (e.g., N297A) modification.

[0351] For example, the disclosure includes anti-PD-1 / anti-4-1BB antibodies comprising an Fc domain comprising one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T, and 256E (e.g., M252Y, S254T, and T256E); 428L and 434S (e.g., M428L and N434S); 257I and 311I (e.g., For example, P257I and Q311I; 257I and 434H (e.g., P257I and N434H); 376V and 434H (e.g., D376V and N434H); 307A, 380A and 434A (e.g., T307A, E380A and N434A); 433K and 434F (e.g., H433K and N434F); and 234A and 235A (e.g., L234A and L235A). In one embodiment, the present disclosure includes an anti-PD-1 antibody comprising an Fc domain containing the S108P mutation in the hinge region of IgG4 to promote dimer stabilization. All possible combinations of the above Fc domain mutations, as well as other mutations in the antibody variable domains disclosed herein, are contemplated within the scope of the present disclosure.

[0352] The present disclosure also provides chimeric heavy chain constant (C H ) region, and chimeric C H The region is composed of C H For example, the antibodies of the present disclosure may comprise segments derived from C regions derived from human IgG1, human IgG2, or human IgG4 molecules. H Chimeric C containing part or all of the 2 domains H C region derived from a human IgG1, human IgG2 or human IgG4 molecule H In certain embodiments, the antibodies of the present disclosure may be combined with any or all of the three domains. HFor example, the chimeric hinge may comprise an "upper hinge" amino acid sequence (amino acid residues 216-227 according to EU numbering) derived from a human IgG1, human IgG2, or human IgG4 hinge region in combination with a "lower hinge" sequence (amino acid residues 228-236 according to EU numbering) derived from a human IgG1, human IgG2, or human IgG4 hinge region. According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from a human IgG1 or human IgG4 upper hinge and amino acid residues derived from a human IgG2 lower hinge. The chimeric C described herein H Antibodies comprising the region may, in certain embodiments, exhibit modified Fc effector functions without adversely affecting the therapeutic or pharmacokinetic properties of the antibody (see, e.g., U.S. Pat. No. 9,359,437, the disclosure of which is incorporated herein by reference in its entirety).

[0353] Biological properties of antibodies Generally, the antibodies of the present disclosure function by binding to PD-1 and 4-1BB. The present disclosure includes anti-PD-1 / anti-4-1BB bispecific antibodies and antigen-binding fragments thereof (or any of their individual components) that bind with high affinity to soluble monomeric or dimeric PD-1 and 4-1BB molecules. For example, the present disclosure includes antibodies with a K of less than about 50 nM as measured by surface plasmon resonance. D In certain embodiments, the antibodies or antigen-binding fragments thereof have a K of less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 2 nM, or less than about 1 nM, as measured by surface plasmon resonance. D It binds to PD-1 and 4-1BB.

[0354] The present disclosure also includes antibodies and antigen-binding fragments thereof that bind to PD-1 and 4-1BB with a dissociation half-life (t) of greater than about 1.1 minutes as measured by surface plasmon resonance at 25° C. or 37° C. In certain embodiments, the antibodies or antigen-binding fragments of the disclosure bind to PD-1 with a t of greater than about 5 minutes, greater than about 10 minutes, greater than about 30 minutes, greater than about 50 minutes, greater than about 60 minutes, greater than about 70 minutes, greater than about 80 minutes, greater than about 90 minutes, greater than about 100 minutes, greater than about 200 minutes, greater than about 300 minutes, greater than about 400 minutes, greater than about 500 minutes, greater than about 600 minutes, greater than about 700 minutes, greater than about 800 minutes, greater than about 900 minutes, greater than about 1000 minutes, or greater than about 1200 minutes as measured by surface plasmon resonance at 25° C. or 37° C.

[0355] Species selectivity and species cross-reactivity According to certain embodiments of the present disclosure, anti-PD-1 / anti-4-1BB antibodies (or any of their individual components) bind to human PD-1 and human 4-1BB, but not to PD-1 and 4-1BB from other species. Alternatively, anti-PD-1 / anti-4-1BB antibodies of the present disclosure, in certain embodiments, bind to human PD-1 and human 4-1BB and PD-1 and 4-1BB from one or more non-human species. For example, anti-PD-1 / anti-4-1BB antibodies of the present disclosure may bind to human PD-1 and / or human 4-1BB, and optionally may or may not bind to PD-1 and / or 4-1BB from one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus monkey, marmoset, rhesus monkey, or chimpanzee. In certain embodiments, the anti-PD-1 / anti-4-1BB antibodies of the present disclosure may bind to human and cynomolgus monkey PD-1 and / or 4-1BB with the same or different affinities, but do not bind to rat and mouse PD-1 and / or 4-1BB.

[0356] Therapeutic Administration and Formulations The present disclosure provides therapeutic compositions comprising the anti-PD-1 / anti-4-1BB antibodies of the present disclosure (or any of their individual components). Therapeutic compositions according to the present disclosure are administered with suitable carriers, excipients, and other agents incorporated into the formulation to provide improved transport, delivery, tolerability, etc. Many suitable formulations can be found in formularies known to all medicinal chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsions of carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al., PDA J Pharm Sci Technol. 1998 Sep-Oct;52(5):238-311.

[0357] The antibody dosage may vary depending on the age and size of the recipient, the target disease, condition, route of administration, etc. When an antibody of the present disclosure is used to treat a disease or disorder in an adult patient or to prevent such a disease, it is typically advantageous to administer the antibody of the present disclosure at a single dose of about 0.1 to about 60 mg / kg body weight, about 5 to about 60 mg / kg body weight, about 10 to about 50 mg / kg body weight, or about 20 to about 50 mg / kg body weight. The frequency and duration of treatment can be adjusted depending on the severity of the condition. In certain embodiments, an antibody or antigen-binding fragment thereof of the present disclosure can be administered as an initial dose of at least about 0.1 mg to about 800 mg, about 1 mg to about 500 mg, about 5 mg to about 300 mg, or about 10 mg to about 200 mg, about 100 mg, or about 50 mg. In certain embodiments, the initial dose may be followed by administration of a second or multiple subsequent doses of the antibody or antigen-binding fragment thereof in an amount that may be about the same as or less than the initial dose, with the subsequent doses spaced apart by at least 1 to 3 days; at least 1 week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.

[0358] Various delivery systems are known, and the pharmaceutical compositions of the present disclosure can be administered by encapsulation in, for example, liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, or receptor-mediated endocytosis (see, e.g., Wu et al., J. Biol. Chem. 1987 Apr 5;262(10):4429-32). Methods of introduction include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intratumoral, intranasal, epidural, and oral routes. The compositions can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal, and intestinal mucosa, etc.), and can be administered together with other biologically active agents. Administration can be systemic or local. The pharmaceutical composition can be delivered in a vesicle, in particular a liposome (see, eg, Langer. Science. 1990 Sep 28;249(4976):1527-33).

[0359] The use of nanoparticles for delivering the antibodies of the present invention is also contemplated herein. Antibody-conjugated nanoparticles can be used for both therapeutic and diagnostic applications. Antibody-conjugated nanoparticles and methods for preparation and use are described in detail in Arruebo et al. (J Nanomat. 2009; pp. 1-24), which is incorporated herein by reference. Nanoparticles can be developed and conjugated to antibodies contained in pharmaceutical compositions to target tumor cells or virus-infected cells. Nanoparticles for drug delivery are also described, for example, in U.S. Pat. No. 8,257,740 or U.S. Pat. No. 8,246,995 (each of which is incorporated herein in its entirety).

[0360] In certain circumstances, the pharmaceutical composition may be delivered in a controlled release system. In one embodiment, a pump may be used. In another embodiment, a polymeric material may be used. In yet another embodiment, the controlled release system may be placed near the target of the composition, thus requiring only a fraction of the systemic dose.

[0361] Injectable preparations may include dosage forms for intravenous injection, subcutaneous injection, intradermal injection, intracranial injection, intraperitoneal injection, intramuscular injection, and infusion. These injectable preparations may be prepared by known methods. For example, injectable preparations may be prepared by dissolving, suspending, or emulsifying the antibody or a salt thereof in a sterile aqueous or oily medium conventionally used for injections. Aqueous injectable media include, for example, isotonic solutions containing saline, glucose, and other adjuvants, and may be used in combination with appropriate solubilizers such as alcohols (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)). Oily media include, for example, sesame oil and soybean oil, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The injectable preparations prepared in this manner are optionally filled into appropriate ampoules.

[0362] The pharmaceutical compositions of the present disclosure can be delivered subcutaneously or intravenously using a standard needle and syringe. Furthermore, for subcutaneous delivery, pen delivery devices are readily useful for delivering the pharmaceutical compositions of the present disclosure. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is emptied, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In disposable pen delivery devices, there is no replaceable cartridge. Rather, the disposable pen delivery device is pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0363] Numerous reusable pen and autoinjector delivery devices have utility for subcutaneous delivery of the pharmaceutical compositions of the present disclosure. Examples include, but are not limited to, the AUTOPEN™ (Owen Mumford, Inc. Woodstock, UK), the DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), the HUMALOG MIX 75 / 25™ pen, the HUMALOG™ pen, the HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, Ind.), the NOVOPEN™ I, II, and III (Novo Nordisk, Copenhagen, Denmark), the NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), the BD™ pen (Becton Dickinson, Franklin Lakes, NJ), the OPTIPEN™, the OPTIPEN PRO™, the OPTIPEN™, and the OPTIPEN™. Examples of disposable pen delivery devices that have application in the subcutaneous delivery of the pharmaceutical compositions of the present disclosure include, but are not limited to, the SOLOSTAR™ pen (Sanofi-Aventis), FLEXPEN™ (Novo Nordisk), and KWIKPEN™ (Eli Lilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, Calif.), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA™ Pen (Abbott Labs, Abbott Park, Ill.).

[0364] Advantageously, the above-mentioned pharmaceutical compositions for oral or parenteral use are prepared in a dosage form with a unit dose suitable for the dosage of the active ingredient. Examples of such dosage forms in unit doses include tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of antibody contained is generally about 5 to about 500 mg per dosage form in a unit dose, and particularly about 5 to about 100 mg of antibody in injection forms, and about 10 to about 250 mg in other dosage forms.

[0365] Therapeutic Uses of Antibodies The antibodies of the present disclosure (or any of their individual components) are useful, inter alia, for the treatment, prevention, and / or amelioration of any disease or disorder associated with or mediated by PD-1 and / or 4-1BB expression, signaling, or activity, or treatable by blocking the interaction between PD-1 and a PD-1 ligand (e.g., PD-L1 or PD-L2), and 4-1BB and a 4-1BB ligand (e.g., 4-1BBL), or otherwise inhibiting PD-1 and activating 4-1BB activity and / or signaling. For example, the present disclosure provides methods for treating cancer (tumor growth inhibition) and / or chronic viral infection by administering an anti-PD-1 / anti-4-1BB bispecific binding molecule (or a pharmaceutical composition comprising an anti-PD-1 / anti-4-1BB bispecific binding molecule) to a patient in need of such treatment as described herein. The antibodies of the present disclosure are useful for treating, preventing, and / or ameliorating a disease or disorder or condition, such as cancer or viral infection, and / or ameliorating at least one symptom associated with such disease, disorder, or condition. In connection with the methods of treatment described herein, the anti-PD-1 / anti-4-1BB bispecific binding molecules may be administered as monotherapy (i.e., as the only therapeutic agent) or in combination with one or more additional therapeutic agents (examples of which are described elsewhere herein).

[0366] In some embodiments of the present disclosure, the antibodies described herein are useful for treating subjects suffering from primary or recurrent cancer, including, but not limited to, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colon cancer, esophageal cancer, gastric cancer, head and neck cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, multiple myeloma, myelodysplastic syndrome, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney / renal cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer, and uterine cancer.

[0367] The antibodies may be used to treat early or late symptoms of cancer. In one embodiment, the antibodies or fragments thereof of the present disclosure may be used to treat metastatic cancer. The antibodies are useful for reducing, inhibiting, or shrinking tumor growth in both solid tumors and hematological cancers. In certain embodiments, treatment with the antibodies or antigen-binding fragments thereof of the present disclosure results in greater than 50% regression, greater than 60% regression, greater than 70% regression, greater than 80% regression, or greater than 90% regression of tumors in a subject. In certain embodiments, the antibodies may be used to prevent tumor recurrence. In certain embodiments, the antibodies are useful for extending overall survival in subjects with cancer. In some embodiments, the antibodies are useful for reducing toxicity resulting from chemotherapy or radiation therapy while maintaining long-term survival in patients afflicted with cancer.

[0368] In certain embodiments, antibodies of the present disclosure are useful for treating subjects suffering from chronic viral infections. In some embodiments, antibodies of the present disclosure are useful for reducing viral titers in a host and / or rescuing exhausted T cells. In certain embodiments, antibodies of the present disclosure or fragments thereof may be used to treat chronic viral infections caused by lymphocytic choriomeningitis virus (LCMV). In some embodiments, antibodies or antigen-binding fragments thereof may be administered in therapeutic doses to patients with infections caused by human immunodeficiency virus (HIV) or human papillomavirus (HPV) or hepatitis B / C virus (HBV / HCV). In a related embodiment, antibodies of the present disclosure or antigen-binding fragments thereof may be used to treat infections caused by simian immunodeficiency virus (SIV) in simian subjects, such as cynomolgus monkeys.

[0369] In certain embodiments, the antibodies of the present disclosure may be administered in a therapeutically effective amount to a subject suffering from cancer or a viral infection.

[0370] One or more antibodies of the disclosure may be administered to alleviate or prevent or reduce the severity of one or more symptoms or conditions of a disease or disorder.

[0371] Also contemplated herein is the prophylactic use of one or more antibodies of the present disclosure in patients at risk of developing a disease or disorder, such as cancer and chronic viral infections.

[0372] In a further embodiment of the disclosure, the antibody is used in the preparation of a pharmaceutical composition for treating a patient suffering from cancer or a viral infection. In another embodiment of the disclosure, the antibody is used as an adjunct therapy with any other drug or therapy known to those skilled in the art to be useful in the treatment of cancer or a viral infection.

[0373] Combination Therapies and Formulations The combination therapy may include any additional therapeutic agent that may be advantageously combined with the anti-PD-1 / anti-4-1BB bispecific binding molecules of the present disclosure (or any of their individual components) and the antibodies of the present disclosure.

[0374] Antibodies of the present disclosure may be synergistically combined with one or more anti-cancer drugs or therapies used to treat cancer, including, for example, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colon cancer, esophageal cancer, gastric cancer, head and neck cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, multiple myeloma, myelodysplastic syndrome, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, renal / kidney cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer, and uterine cancer. Contemplated herein is the use of anti-PD-1 / anti-4-1BB antibodies of the present disclosure in combination with immunostimulatory and / or immunosupportive therapy to inhibit tumor growth and / or enhance survival of cancer patients. Immunostimulatory therapy includes direct immunostimulatory therapy to enhance immune cell activity by either "releasing the brakes" on suppressed immune cells or "stepping on the gas" to activate the immune response. Examples include targeting other checkpoint receptors, adoptive cell therapy, vaccination, and adjuvants. Immune support modalities may increase tumor antigenicity by promoting immunogenic cell death, inflammation, or have other indirect effects that promote anti-tumor immune responses. Examples include radiation, chemotherapy, anti-angiogenic agents, and surgery.

[0375] In various embodiments, the one or more antibodies of the disclosure are an antibody against PD-L1; a second antibody against PD-1 (e.g., nivolumab); an antibody against 4-1BBL; a second antibody against 4-1BB; a LAG-3 inhibitor; a CTLA-4 inhibitor (e.g., ipilimumab); a TIM-3 inhibitor; a BTLA inhibitor; a TIGIT inhibitor; a CD47 inhibitor; an antagonist of another T cell co-inhibitor or ligand (e.g., PD-L2, CEACAM, VISTA, LAIR-1 , 2B4, B7-H3, B7-H4, KIR, A2aR, GAL9, or TGFR); agonists of T cell costimulators (e.g., antibodies or ligands against CD28, ICOS, OX40, CD27, B7, CD226, CRTAM, GITR, HVEM, BAFFR, BAFF, Light); adenosine; indoleamine-2,3-dioxygenase (IDO) inhibitors; vascular endothelial growth factor (VEGF) antagonists (e.g., U.S. Pat. No. 7,527,629); "VEGF-Trap" such as aflibercept or other VEGF-inhibiting fusion proteins, or anti-VEGF antibodies or antigen-binding fragments thereof (e.g., bevacizumab or ranibizumab), or small molecule kinase inhibitors of VEGF receptors (e.g., sunitinib, sorafenib, or pazopanib), as described in US Pat. No. 087,411; Ang2 inhibitors (e.g., nesbacumab); transforming growth factor beta (TGFβ) inhibitors, epidermal growth factor (EGFR) inhibitors agents (e.g., erlotinib, cetuximab); agonists for costimulatory receptors (e.g., agonists for glucocorticoid-inducible TNFR-related proteins); antibodies against tumor-specific antigens (e.g., CA9, CA125, melanoma-associated antigen 3 [MAGE3], carcinoembryonic antigen [CEA], vimentin, tumor M2-PK, prostate-specific antigen [PSA], mucin-1, MART-1, and CA19-9); vaccines (e.g., the cancer vaccine Bacillus Calmette-Guerin); adjuvants to increase antigen presentation (e.g., granulocyte-macrophage colony-stimulating factor); bispecific antibodies (e.g., CD3×CD20 bispecific antibody, PSMA×CD3 bispecific antibody); cytotoxins;chemotherapeutic agents (e.g., dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, and vincristine); cyclophosphamide; radiation therapy; IL-6R inhibitors (e.g., sarilumab); IL-4R inhibitors (e.g., dupilumab); IL-10 inhibitors; cytokines such as IL-2, IL-7, IL-12, IL-21, and IL-15; antibody-drug conjugates (ADCs) (e.g., anti-CD19-DM4 ADC, and anti-DS6-DM4 ADC) ADCs; immunocytokines (e.g., anti-FAP x IL-2v [e.g., RO6874281], anti-tenascin C x IL-2 [e.g., F16-IL2, also known as teleukin], anti-GD2 x IL-2 [e.g., hu14.18-IL2], anti-EDB x IL-2 [e.g., L19-IL2, also known as darleukin], anti-EDB x TNF [e.g., L19-TNF, also known as fibromun], anti-histone complex x IL-12 [e.g., NHS-IL12], anti-EDB×IL-12 [e.g., L19-IL12, also known as dodekin], anti-CSPG4×IL-2, anti-EpCAM×IL-2, anti-CD20×IL-2, anti-PD-1×IL-2, and anti-TNFα×IL-2; anti-inflammatory drugs (e.g., corticosteroids and nonsteroidal anti-inflammatory drugs); nutritional supplements such as antioxidants; or any palliative care for treating cancer. In certain embodiments, the anti-PD-1 antibodies of the present disclosure may be used in combination with cancer vaccines (including dendritic cell vaccines, oncolytic viruses, tumor cell vaccines, etc.) or adoptive cell therapy to enhance anti-tumor responses. Examples of cancer vaccines that can be used in combination with the anti-PD-1 antibodies of the present disclosure include MAGE3 vaccines for melanoma and bladder cancer, MUC1 vaccines for breast cancer, EGFRv3 (e.g., Rindopepimut) for brain cancer (including glioblastoma multiforme) or ALVAC-CEA (CEA; + (for cancer).

[0376] In certain embodiments, anti-PD-1 / anti-4-1BB antibodies of the present disclosure may be administered in combination with radiation therapy in a manner that generates long-term, durable anti-tumor responses and / or enhances survival of cancer patients. In some embodiments, anti-PD-1 / anti-4-1BB antibodies of the present disclosure may be administered before, concurrently with, or after radiation therapy to a cancer patient. For example, radiation therapy may be administered in one or more doses to a tumor lesion, followed by administration of one or more doses of an anti-PD-1 / anti-4-1BB antibody of the present disclosure. In some embodiments, radiation therapy may be administered locally to a tumor lesion to enhance the local immunogenicity of the patient's tumor (conditioning radiation) and / or to kill tumor cells (ablative radiation), followed by systemic administration of an anti-PD-1 / anti-4-1BB bispecific binding molecule of the present disclosure. For example, intracranial radiation may be administered to a patient with brain cancer (e.g., glioblastoma multiforme) in combination with systemic administration of an anti-PD-1 / anti-4-1BB bispecific binding molecule of the present disclosure. In certain embodiments, the anti-PD-1 / anti-4-1BB antibodies of the disclosure may be administered in combination with radiation therapy and a chemotherapeutic agent (e.g., temozolomide) or a VEGF antagonist (e.g., aflibercept).

[0377] In certain embodiments, anti-PD-1 / anti-4-1BB antibodies of the present disclosure may be administered in combination with one or more antiviral agents to treat chronic viral infections caused by LCMV, HIV, HPV, HBV, or HCV. Examples of antiviral agents include, but are not limited to, zidovudine, lamivudine, abacavir, ribavirin, lopinavir, efavirenz, cobicistat, tenofovir, rilpivirine, and corticosteroids. In some embodiments, anti-PD-1 / anti-4-1BB antibodies of the present disclosure may be administered in combination with any antagonist of a LAG3 inhibitor, a CTLA-4 inhibitor, or another T-cell co-inhibitor to treat chronic viral infections.

[0378] The additional therapeutically active agent / component may be administered prior to, concurrently with, or following administration of the anti-PD-1 / anti-4-1BB bispecific binding molecule of the present disclosure. For purposes of this disclosure, such administration regimens will be considered administration of the anti-PD-1 / anti-4-1BB bispecific binding molecule "in combination" with the second therapeutically active ingredient.

[0379] An additional therapeutically active ingredient may be administered to a subject prior to administration of an anti-PD-1 / anti-4-1BB bispecific binding molecule of the present disclosure. For example, a first ingredient may be considered to have been administered "before" the second ingredient if it is administered 1 week, 72 hours, 60 hours, 48 ​​hours, 36 hours, 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or less than 1 minute before administration of the second ingredient. In other embodiments, an additional therapeutically active ingredient may be administered to a subject after administration of an anti-PD-1 / anti-4-1BB bispecific binding molecule of the present disclosure. For example, a first component can be considered to be administered "after" the second component if it is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, or 72 hours after administration of the second component. In still other embodiments, the additional therapeutically active component can be administered to a subject simultaneously with administration of an anti-PD-1 / anti-4-1BB bispecific binding molecule of the present disclosure. "Concurrent" administration, for purposes of the present disclosure, includes, for example, administration of the anti-PD-1 / anti-4-1BB bispecific binding molecule and the additional therapeutically active component to a subject in a single dosage form (e.g., co-formulated), or administered in separate dosage forms that are administered to a subject within about 30 minutes of each other. When administered in separate dosage forms, each dosage form may be administered via the same route (e.g., both the anti-PD-1 / anti-4-1BB bispecific binding molecule and the additional therapeutically active ingredient may be administered intravenously, subcutaneously, intratumorally, etc.); alternatively, each dosage form may be administered via a different route (e.g., the anti-PD-1 / anti-4-1BB bispecific binding molecule may be administered intravenously and the additional therapeutically active ingredient may be administered subcutaneously or intratumorally; or the anti-PD-1 / anti-4-1BB bispecific binding molecule may be administered intratumorally and the additional therapeutically active ingredient may be administered intravenously or subcutaneously; etc.). In any event, administration of the ingredients in a single dosage, in separate dosage forms by the same route, or in separate dosage forms by different routes, are all considered "co-administration" for purposes of this disclosure.For purposes of this disclosure, administration of an anti-PD-1 / anti-4-1BB bispecific binding molecule "before," "concurrently with," or "after" the administration of an additional therapeutically active ingredient (as these terms are defined above) will be considered administration of the anti-PD-1 / anti-4-1BB bispecific binding molecule "in combination with" the additional therapeutically active ingredient.

[0380] The present disclosure includes pharmaceutical compositions in which the anti-PD-1 / anti-4-1BB bispecific binding molecules of the disclosure are co-formulated with one or more of the additional therapeutically active ingredients described elsewhere herein using various dosage combinations.

[0381] Dosing regimen According to certain embodiments of the present disclosure, multiple doses of an anti-PD-1 / anti-4-1BB antibody of the present disclosure (or any of its individual components)—or a pharmaceutical composition comprising a combination of an anti-PD-1 antibody and any of the additional therapeutically active agents mentioned herein—can be administered to a subject over a defined time course. A method according to this aspect of the present disclosure comprises sequentially administering multiple doses of an anti-PD-1 / anti-4-1BB antibody of the present disclosure to a subject. As used herein, "sequentially administering" means that each dose of an anti-PD-1 / anti-4-1BB antibody is administered to a subject at different times, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The present disclosure includes methods comprising sequentially administering a single initial dose of an anti-PD-1 / anti-4-1BB antibody to a patient, followed by one or more secondary doses of the anti-PD-1 / anti-4-1BB antibody to the patient, optionally followed by one or more tertiary doses of the anti-PD-1 antibody. The anti-PD-1 / anti-4-1BB antibody may be administered at a dose of 0.1 mg / kg to 100 mg / kg.

[0382] The terms "initial dose," "secondary dose," and "tertiary dose" refer to the temporal order of administration of anti-PD-1 / anti-4-1BB antibody. Thus, an "initial dose" is a dose administered at the beginning of a treatment regimen (also referred to as a "baseline dose"); a "secondary dose" is a dose administered after the initial dose; and a "tertiary dose" is a dose administered after the secondary dose. The initial, secondary, and tertiary doses may all contain the same amount of anti-PD-1 / anti-4-1BB antibody or may differ from one another in terms of administration frequency. However, in certain embodiments, the amount of anti-PD-1 / anti-4-1BB antibody contained in the initial, secondary, and / or tertiary doses differs from one another (e.g., adjusted up or down as needed) over the course of treatment. In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered as "loading doses" at the beginning of a treatment regimen, followed by subsequent doses (e.g., "maintenance doses") administered at a less frequent frequency.

[0383] In certain exemplary embodiments of the present disclosure, each secondary and / or tertiary dose is administered 1 to 26 (e.g., 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5 or more) weeks after the immediately preceding administration. The phrase "immediately preceding dose" as used herein means the dose of anti-PD-1 / anti-4-1BB antibody administered to a patient prior to the administration of the very next dose in a multiple dose series, without any intervening doses.

[0384] Methods according to this aspect of the invention can include administering any number of secondary and / or tertiary doses of anti-PD-1 / anti-4-1BB antibody to the patient. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient. Similarly, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient.

[0385] In embodiments involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1-2 weeks or 1-2 months after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2-12 weeks after the immediately preceding dose. In certain embodiments of the invention, the frequency with which the secondary and / or tertiary doses are administered to the patient may vary over the course of the treatment regimen. The administration frequency may also be adjusted by a physician during the course of treatment based on clinical testing and the needs of the individual patient.

[0386] The present disclosure includes dosing regimens in which two to six loading doses are administered to a patient at a first frequency (e.g., once per week, once every two weeks, once per three weeks, once per month, once per two months, etc.), followed by two or more maintenance doses administered to the patient less frequently. For example, according to this aspect of the disclosure, if the loading dose is administered at a frequency of, for example, once per month (e.g., two, three, four, or more loading doses administered monthly), the maintenance doses may be administered to the patient once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every ten weeks, once every twelve weeks, etc.

[0387] Diagnostic Uses of Antibodies The anti-PD-1 / anti-4-1BB bispecific binding molecules of the present disclosure (or any of their individual components, i.e., anti-4-1BB immunoglobulin single variable domains, or anti-PD-1 antibodies or antigen-binding fragments thereof) may be used to detect and / or measure PD-1 and / or 4-1BB in a sample, e.g., for diagnostic purposes. Some embodiments contemplate the use of one or more binding molecules of the present invention in assays for detecting diseases or disorders, such as cancer or chronic viral infections. An exemplary diagnostic assay for PD-1 and / or 4-1BB may comprise, for example, contacting a sample obtained from a patient with an anti-PD-1 / anti-4-1BB bispecific binding molecule of the present disclosure (or one of its individual components), which is labeled with a detectable label or reporter molecule or used as a capture ligand to selectively isolate PD-1 and / or 4-1BB from the patient sample. Alternatively, the unlabeled anti-PD-1 / anti-4-1BB bispecific binding molecule of the present disclosure (or one of its individual components) can be used for diagnostic applications in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule can be: 3 H, 14 C. 32 P, 35 S or 125 The PD-1 and / or 4-1BB antibody may be a radioisotope such as I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine; or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, luciferase, etc. Certain exemplary assays that can be used to detect or measure PD-1 and / or 4-1BB in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

[0388] Samples that can be used in PD-1 and / or 4-1BB diagnostic assays according to the present disclosure include any tissue or bodily fluid sample obtainable from a patient that contains a detectable amount of PD-1 protein and / or 4-1BB protein, or fragments thereof, under normal or pathological conditions. Generally, the level of PD-1 and / or 4-1BB in a particular sample obtained from a healthy patient (e.g., a patient not afflicted with cancer) is measured to first establish a baseline, or standard, level of PD-1 and / or 4-1BB. This baseline level of PD-1 and / or 4-1BB can then be compared with the level of PD-1 and / or 4-1BB measured in a sample obtained from an individual suspected of having a cancer-related condition or symptoms associated with such a condition.

[0389] The anti-PD-1 / anti-4-1BB bispecific binding molecules of the present disclosure (or any of their individual components, i.e., anti-4-1BB immunoglobulin single variable domains, or anti-PD-1 antibodies or antigen-binding fragments thereof) may contain no additional labels or moieties, or they may contain N- or C-terminal labels or moieties. In one embodiment, the label or moiety is biotin. In binding assays, the location of the label (if present) may determine the orientation of the peptide relative to the surface to which it is bound. For example, if the surface is coated with avidin, a peptide containing an N-terminal biotin will be oriented such that the C-terminal portion of the peptide is distal from the surface.

[0390] The present disclosure is further illustrated by the following examples, which should not be construed as further limiting. The contents of the figures and all references, patents and published patent applications cited throughout this application are expressly incorporated herein by reference for all purposes.

[0391] Furthermore, in accordance with the present disclosure there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art, such techniques being fully explained in the literature. For example, Green & Sambrook, Molecular Cloning: A Laboratory Manual, Fourth Edition (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; DNA Cloning: A Practical Approach, Volumes I and II (DNGlover ed.1985); Oligonucleotide Synthesis (MJ Gait ed.1984); eds.(1985)];Transcription And Translation[BDHames&S.J.Higgins,eds.(1984)];Animal Cell Culture[RIFreshney,ed.(1986)];Immobilized Cells And Enzymes[IRL Press,(1986)];B.Perbal,A Practical Guide To Molecular Cloning(1984);FMAusubel et al.(eds.),Current Protocols in Molecular Biology,John See Wiley & Sons, Inc. (1994). [Example]

[0392] The following examples are put forth to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions featured in this invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise specified, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0393] Example 1 Anti-4-1BB V HH Materials and Methods Llama immunization and library construction Immunization and library construction were performed at the "VIB Nanobody Core" (Vrije Universiteit Brussels, Brussels, Belgium). Llamas were injected intradermally four times, each time with approximately 2 mg of vector carrying the 4-1BB gene. After each injection, the animals were electroporated to introduce the vector into the animal cells. Three weeks after the last vector injection, the animals were boosted subcutaneously with recombinant 4-1BB protein. Four days after the protein boost, anticoagulated blood was collected from the VIB Nanobody Core. HH were taken for library construction.

[0394] V HH The library was constructed. Total ribonucleic acid was extracted from peripheral blood lymphocytes and used as a template for first-strand complementary DNA synthesis with an oligo(dT) primer. HH The coding sequence was amplified by polymerase chain reaction from complementary DNA, digested with PstI and NotI, and cloned between the PstI and NotI sites of the phagemid vector pHEN4 upstream of the human influenza hemagglutinin tag. 8 V of independent transformants HH A library was obtained.

[0395] Phage display panning The bacterial library was analyzed by optical density (OD) analysis at 600 nm (OD 600 The cells were grown in 2YTAG medium (2xYT medium, 100 μg / mL ampicillin, 2% glucose) until the absorbance at 1000 u / s reached 0.5 and then infected with M13K07 helper phage (Invitrogen). After centrifugation, the bacteria were resuspended in 2YTAK medium (2xYT medium, 100 μg / mL ampicillin, 50 μg / mL kanamycin) and grown overnight. Phage particles were precipitated from the culture supernatant by adding 20% ​​w / v polyethylene glycol 8000 (PEG8000) and 2.5 M NaCl, centrifuged, and resuspended in phosphate-buffered saline (PBS). The phage were subjected to another washing and precipitation step and finally resuspended in 15% v / v cold PBS / glycerol.

[0396] Panning against cells Panning was performed at 4°C on FreeStyle™ HEK293-FS cells transfected with 4-1BB. HH The library was saturated with PBS / 2% w / v BSA and diluted to 2 × 10 7 The cells were incubated with 1000 cells for 2 hours at 4°C. After washing twice with PBS, the cell pellet was resuspended in PBS and loaded onto a fetal bovine serum / Percoll gradient. After centrifugation, the cell layer was harvested and washed twice with PBS. The recovered cells with bound phage were added to a second fetal bovine serum / Percoll gradient and washed before mechanical lysis using beads (Dynabeads, Invitrogen). The recovered phage-V HH was used to infect exponentially growing Escherichia coli TG1 bacteria and amplified overnight in 2YTAG medium or plated onto 2YTAG plates for a new round of panning.

[0397] Fab-like construction, production, and purification After amplification by polymerase chain reaction, anti-4-1BB V HHThe complementary DNA of human C fused to human influenza hemagglutinin and a 6-His tag (both tags for purification purposes) was used. L Domain or human IgG1 C H The same was done for anti-OX40 and anti-CD28 V domains, which were cloned into their own mammalian expression vectors in frame with either the V domain or the V domain. HH , and anti-foot-and-mouth disease virus (FMDV) V HH (which was later used as a control, as detailed below).

[0398] Plasmids were purified using the NucleoBond Macherey-Nagel kit and Sanger sequenced. HH 2x), bivalent biparatopic (i.e., two different anti-4-1BB V HH ) and bispecific (i.e., one anti-4-1BB V HH and one V HH Fab-like constructs (one directed against a different target) were transfected into FeeStyle™ HEK293-FS cells with two V HH A mixture of two plasmids encoding human C L fused to the human IgG1 C domain H The IgG1A1 gene was produced by co-transfection with the IgG1A1 gene (fused to the IgG1A1 domain). Supernatants were collected after 7 days, purified on a nickel affinity column, and analyzed on a CALIPER GXII (Perkin Elmer).

[0399] Phage-V in 96-well plates for ELISA HH production The desired 4-1BB V HH Individual TG1 colonies were cultured at OD 600 The cells were grown in 2YTA medium at 37°C until the β-actin (β) reached 0.5. The cells were then infected with M13K07 helper phage and grown overnight in 2YTAK at 30°C. Phage-V HH The supernatant containing was collected and used for testing.

[0400] ELISA binding assay ELISA was performed in PBS at 4°C on Nunc® MaxiSorp™ 96-well plates (Sigma) pre-coated overnight with 1 μg / mL human His-tagged 4-1BB recombinant protein and further saturated with PBS / 2% milk for 1 hour at room temperature. HH Bacterial supernatants containing purified Fab-like constructs or 4-1BBL-Fc were incubated for 1 h at room temperature. After several washes with PBS / 0.1% Tween, the following HRP-conjugated antibodies were added: anti-HA tag mAb (Sigma) to detect bound Fab-like constructs, anti-phage-V (Sigma) to detect bound phage-V (Sigma) to detect bound Fab-like constructs, and anti-HA tag mAb (Sigma) to detect bound phage-V ... HH Anti-M13 mAb (Santa Cruz Biotechnology) to detect 4-1BBL-His, and anti-human Fc mAb to detect bound 4-1BBL-His. Detection of peroxidase activity was performed using TMB (3,3',5,5'-tetramethylbenzidine, KPL) substrate, and OD was measured in a SpectraMax microplate reader after adding a sulfuric acid stop solution. 450nm was measured.

[0401] ELISA competition assay ELISA was performed in PBS at 4°C on Nunc® MaxiSorp™ 96-well plates (Sigma) pre-coated overnight with 1 μg / mL human His-tagged 4-1BB recombinant protein and further saturated with PBS / 2% milk for 1 h at room temperature. For epitope binning competition, serial dilutions of bivalent Fab-like constructs were incubated for 1 h at room temperature and then their EC 90 Phage-V HH Alternatively, for competition assays, serial dilutions of control anti-4-1BB antibody or 4-1BBL-Fc were incubated at room temperature for 1 hour, followed by addition of the bivalent Fab-like construct to EC 90After several washes with PBS / 0.1% Tween, the following HRP-conjugated antibodies were added: anti-HA tag mAb (Sigma) to detect bound Fab-like constructs and anti-phage-V (Sigma) to detect bound phage-V. HH Anti-M13 mAb (Santa Cruz Biotechnology) for detecting peroxidase activity. Detection of peroxidase activity was performed using TMB substrate, and after adding a sulfuric acid stop solution, OD was measured using a SpectraMax microplate reader. 450nm was measured.

[0402] Reporter function assay Cross-linking experiments Test compounds were pre-incubated with a saturating concentration of anti-human Fab (Sigma, I5260) for 30 min at room temperature. Jurkat-4-1BB-NF-κB (Promega) cells were harvested during their exponential growth phase, and 25 μL of the cell suspension was added to a 96-well plate (50,000 cells / well) containing 25 μL of cross-linked or non-cross-linked test compound.

[0403] For conditions using OX40-expressing cells Jurkat-4-1BB-NF-κB cells were harvested during their exponential growth phase and mixed with OX40-expressing cells to obtain a final ratio of 1:1 between reporter cells and accessory cells. 25 μL of the cell suspension was added to a 96-well plate (50,000 cells / well) containing 25 μL of test compound.

[0404] All conditions Plates were incubated for 6 hours in a humidified incubator at 37°C with 5% CO2. Next, 50 μL of Bio-Glo™ (Promega, G7941) reagent, prepared according to the manufacturer's instructions, was added to each well and mixed. Complete cell lysis was allowed to occur for at least 5 minutes, after which luminescence was measured using an Envision multimode plate reader (Perkin Elmer).

[0405] result Anti-4-1BB V HH Generation of Anti-4-1BB V HH was obtained from llamas. More specifically, V HH llama immune library and naive V HH After immunization of one llama with 4-1BB-DNA, V was isolated from PBMCs. HH A phage library (i.e., immune library) was constructed. Llama immunization was performed with six injections of a plasmid expressing full-length human 4-1BB, and a final boost was administered with one injection of recombinant human 4-1BB (SinoBiological).

[0406] V selected by library panning on recombinant 4-1BB protein or 4-1BB-expressing cells HH were screened for binding to 4-1BB and further sequenced. Among the binders, seven clones (clone numbers 1 to 7) were further characterized. These seven anti-4-1BB V HH , bivalent monoparatopic (i.e., identical anti-4-1BB V HH 2x), bivalent biparatopic (i.e., two different anti-4-1BB V HH ), bispecific (i.e., one anti-4-1BB V HH and one V aimed at another target. HH ), or monovalent (i.e., one anti-4-1BB V directed against an unrelated target, e.g., FMDV HH and one V HH ) molecule in a "Fab-like" format, HH C H 1 fused to a human IgG constant domain and a second V HH C L fused to the lambda human IgG constant domain; the first and second V HH Both are C H 1-C L They associate through interactions (Figure 1). For purification purposes, the molecules contain a hemagglutinin (HA) tag and a polyhistidine (His) tag.

[0407] Anti-4-1BB V HH Specificity of Seven Vs were identified by testing their binding to human 4-1BB, cynomolgus monkey ("cyno") 4-1BB, and two TNFRSF members (human OX40 and human CD40). HH (Bivalent monoparatopic "Fab-like" constructs each containing two copies of the same anti-4-1BB VHH) were evaluated for their specificity.

[0408] The results are shown in Figure 2: HH showed binding to human 4-1BB and varying levels of cross-reactivity with cyno 4-1BB. HH None of the antibodies showed binding to human OX40 or human CD40.

[0409] Competition and epitope binning Seven Vs HH The diversity of binding sites to 4-1BB (each of the same anti-4-1BB V HH (in a bivalent monoparatopic "Fab-like" construct containing two times) were evaluated in a competition assay against 4-1BB, the natural ligand of 4-1BBL, and a control anti-4-1BB antibody.

[0410] These assays identified seven V with 4-1BBL and anti-4-1BB antibodies. HH According to the competition profile of the three epitope bins, three distinct epitope bins (bin A, bin B, and bin C) could be identified. The results are shown in Table 3 and Figure 3.

[0411] [Table 4]

[0412] Anti-4-1BB V HH T cell activation by Activation of 4-1BB leads to activation of the NF-κB pathway. NF-κB reporter assays were performed to detect the activity of anti-4-1BB V. HHActivation of the 4-1BB signaling pathway mediated by NF-κB was detected. NF-κB activation was measured using a bioluminescent cell-based reporter assay (Promega). This assay consists of a genetically engineered Jurkat T cell line that constitutively expresses 4-1BB and whose luciferase is regulated by an NF-κB response element. Thus, activation of 4-1BB results in luciferase expression.

[0413] Anti-4-1BB V HH and the previously described "Fab-like" format, monoparatopic bivalent format (each of which is the same anti-4-1BB V HH a construct containing two anti-4-1BB V HH and one V directed against an unrelated target, i.e., FMDV protein. HH The constructs were evaluated either in the presence or absence (soluble state) of a cross-linking reagent (anti-Fab antibody). The results are shown in Figures 4A-D. All bivalent constructs (except the negative control [anti-FMDV]) were active in a dose-dependent manner when cross-linked (Figure 4A), but only clone #5 retained a high activation potential without cross-linking (Figure 4B). The cross-linked monovalent constructs showed a reduced activation potential compared to the bivalent molecules, ranging from no activation to approximately 25% of the activation potential of the respective bivalent constructs (Figure 4C). Finally, the monovalent molecules without cross-linking showed no activation in the NF-κB reporter assay (Figure 4D).

[0414] Next, bivalent biparatopic constructs (each containing two different anti-4-1BB V HH These molecules were tested under soluble conditions (i.e., in the absence of cross-linking) and compared to bivalent monoparatopic and monovalent constructs. The results are shown in Figure 4E. The bivalent biparatopic construct containing clone no. 5 was significantly superior to any other anti-4-1BB V construct tested under soluble conditions. HH The highest activation was observed in the presence of

[0415] Overall, these results demonstrate that two anti-4-1BB clones, number 5V HH This demonstrates that a bivalent, monoparatopic "Fab-like" construct containing is highly active under soluble conditions (i.e., without cross-linking reagents such as anti-Fab antibodies, rendering it a "pure agonist") and competes with 4-1BBL but not with a control anti-4-1BB antibody. Interestingly, clone #5 is monovalent ( 例えば , unrelated VHHs such as anti-FMDV VHHs HH However, clone number 5 was shown to be inactive in combination with V, which targets a different antigen expressed by the same cells. HH It was active in a bispecific format when combined with another anti-4-1BB V targeting a different 4-1BB epitope (e.g., CD28 - Figure 4F). HH ), clone No. 5 showed a further improved and potent agonistic effect on the activation of the NF-κB pathway.

[0416] Finally, as shown in Figure 5A-B, V targets an antigen expressed by another cell (e.g., OX40 expressed on an accessory cell). HH Anti-4-1BB clone no. 5 V in combination with HH The V of anti-4-1BB clone no. 5 was obtained by a bispecific "Fab-like" construct containing HH 4-1BB was transformed into a cell engager and T cell activator, and simultaneous binding of 4-1BB to Jurkat T cells and antigens (e.g., OX40) on accessory cells induced immune cell activation. This feature offers interesting prospects for, for example, targeting T cells to pathogenic cells.

[0417] Structural characterization V of anti-4-1BB clone No. 2 and clone No. 5 in complex with the extracellular domain of human 4-1BB (amino acid residues 24 to 186 of SEQ ID NO: 13) HH The 3D structure of was solved by X-ray crystallography.

[0418] The 3D structure (Figure 6) shows both V HHIt was highlighted that the clones bind to different parts of 4-1BB: -Anti-4-1BB clone number 2V HH binds primarily to the first cysteine-rich domain (CRD) of 4-1BB; -Anti-4-1BB clone number 5V HH binds to both the second and third CRDs of 4-1BB and overlaps with the 4-1BBL binding epitope.

[0419] Anti-4-1BB clone number 2V HH All three CDRs 1-3 of the antibody were found to interact with 4-1BB. However, among all the CDRs, the anti-4-1BB clone no. 5V HH Only the CDR3 of 4-1BB was shown to interact with 4-1BB according to the X-ray crystallography 3-D structure.

[0420] Anti-4-1BB clone number 5V HH variant Among all the 4-1BB binders initially isolated, anti-4-1BB clone no. 5V HH A few Vs with sequence similarity to HH These were also produced in a bivalent, monoparatopic "Fab-like" format as described above. Their binding and functional properties were evaluated: V of anti-4-1BB clones no. 5a, no. 5b, no. 5c, and no. 5d. HH Anti-4-1BB clone number 5V HH Similar, if not improved, EC2 binding to 4-1BB compared to 50 (Figure 7A) and anti-4-1BB clone number 5V HH The activity of the antibody was comparable to that of the antibody of the present invention under soluble conditions (i.e., without cross-linking reagents such as anti-Fab antibodies) (Fig. 7B).

[0421] array Anti-4-1BB clone number 2 and clone number 5V HH and anti-4-1BB clone number 5V HHThe sequences of the variants (clone no. 5a, no. 5b, no. 5c and no. 5d) are shown below with their CDR sequences highlighted (IMGT numbering is in bold; Kabat numbering is underlined; Chothia numbering is in italics):

[0422] Clone No. 2 (SEQ ID NO: 1): [ka] Clone No. 5 (SEQ ID NO: 2): [ka] Clone No. 5a (SEQ ID NO: 58): [ka] Clone No. 5b (SEQ ID NO: 59): [ka] Clone No. 5c (SEQ ID NO: 60): [ka] Clone No. 5d (SEQ ID NO: 61): [ka]

[0423] Example 2 Anti-PD-1 antibody The inventors aimed to generate antagonistic monoclonal anti-PD-1 antibodies that bind to human and cynoPD-1 and compete with the PD-1 ligand, PD-L1.

[0424] Briefly, after immunization of Trianni® mice, 3,556 IgGs were obtained and screened for binding to human and cynoPD-1. Redundant sequences were eliminated, and the remaining IgGs were reformatted to IgG1 LALA. Hits were characterized, and 59 clones were selected for further in vitro functional assays (including PD-1 / PD-L1 blocking bioassays and allogeneic MLR assays). Eight functional clones were selected after these in vitro assays, which were reformatted and generated in Fab format to select for active anti-PD-1 antibodies in a monomeric format.

[0425] Finally, we achieved a human germinality index (i.e., the closest human V) of greater than 95% for both variable regions. H and V L One anti-PD-1 antibody, designated clone "T5," was retained, which has a similar sequence identity (percentage of amino acid sequence identity in the framework regions compared to germline) (Table 4). Anti-PD-1 antibody clone T5 was also active in the monomeric Fab format, as evidenced by the PD-1 / PD-L1 interaction assay (Table 5).

[0426] [Table 5]

[0427] [Table 6]

[0428] array Anti-PD-1 clone T5 has a light chain variable region (LCVR) having the amino acid sequence set forth in SEQ ID NO:5. [ka] CDR sequences are highlighted: IMGT numbering is in bold; Kabat numbering is underlined; Chothia numbering is in italics.

[0429] Anti-PD-1 clone T5 has a heavy chain variable region (HCVR) having the amino acid sequence set forth in SEQ ID NO:6. [ka] CDR sequences are highlighted: IMGT numbering is in bold; Kabat numbering is underlined; Chothia numbering is in italics.

[0430] Example 3 Anti-4-1BB / anti-PD-1 bispecific binding protein We have used the pure agonist anti-4-1BB V described in Example 1. HH The aim of this study was to develop an anti-4-1BB / anti-PD-1 bispecific binding molecule comprising the antibody (clone number 5) and a unique anti-PD-1 binding protein (clone "T5") described in Example 2.

[0431] 1-2 anti-PD-1 Fab (±Fc region) and 3-6 anti-PD-1 BB V HH Several constructs containing the nucleotides 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,

[0432] Construct No. 1 contains, from N-terminus to C-terminus: -Anti-PD-1 clone T5 Fab; -IgG1-LALA*Fc region; - Anti-4-1BB clone number 2V described in Example 1 HH a variant of (i.e., clone number 2.1); and - Anti-4-1BB clone number 5 V described in Example 1 HH variant (i.e., clone number 5.1) Includes.

[0433] Constructs no. 3, no. 5 and no. 6 contain, from N-terminus to C-terminus: - Anti-4-1BB clone number 2 V described in Example 1 HH variant (i.e., clone no. 2.1); - Anti-4-1BB clone number 5 V described in Example 1 HH variants of (i.e., clone no. 5.1); IgG1-LALA*Fc region (construct no. 3), IgG1-NNAS**Fc region (construct no. 5), or IgG4-P-FALA***Fc region (construct no. 6); and -Reverse anti-PD-1 clone T5 Fab Includes. *LALA refers to ADCC silencing mutations (L234A / L235A) that impair IgG1 Fc effector function. **NNAS refers to ADCC-silencing mutations (S298N / T299A / Y300S) that abrogate IgG1 Fc effector function. ***P-FALA refers to ADCC-silencing mutations (S228P / F234A / L235A) that impair IgG4 Fc effector function.

[0434] Materials and Methods Expression and purification HEK293-FS cells Expression plasmids encoding the heavy and light chains of the test compounds were propagated in E. coli DH5α. Plasmids used for transfection were prepared from E. coli using the EndoFree® Plasmid Mega kit (Qiagen, ref. 12381).

[0435] HEK293-FS cells grown in F17 serum-free suspension culture (Invitrogen) were transfected with the heavy and light chain plasmids using polyethyleneimine (PEI) transfection reagent. After 7 days of culture at 37°C, the cells were removed by centrifugation, and the supernatant was passed through a 0.22 μm filter to remove particles.

[0436] For purification, test compounds were captured on a HiTrap® MabSelect SuRe™ column (GE Healthcare, ref. 11-0034-93), eluted with 0.1 M citrate buffer pH 3.0, and desalted using a HiPrep 26 / 10 desalting column (GE Healthcare, ref. 17-05087-02). After protein polishing by size exclusion chromatography using a HiLoad® 26 / 600 Superdex® 200 (GE Healthcare, ref. 28-9893-36) and a final ultrafiltration concentration step, test compounds were used for further characterization.

[0437] CHO cells Expression plasmids encoding the heavy and light chains of test compounds were cloned into a proprietary expression vector system from Evitria (Zurich, Switzerland) using conventional, non-PCR-based cloning techniques. Expression vectors were gene synthesized. Plasmid DNA was prepared under low-endotoxin conditions based on anion-exchange chromatography. DNA concentration was determined by measuring absorbance at 260 nm. Sequence accuracy was verified by Sanger sequencing (with up to two sequencing reactions per plasmid depending on the size of the cDNA).

[0438] Suspension-adapted CHO K1 cells were used for production. Seeds were grown in eviGrow medium (Evitria), a chemically defined, animal-component-free, serum-free medium. Cells were transfected with a custom-made transfection reagent, eviFect (Evitria), and cells were expanded after transfection in eviMake2 (Evitria), an animal-component-free, serum-free medium.

[0439] The supernatant was collected by centrifugation and subsequent filtration through a 0.2 μm filter.

[0440] Test compounds were purified using a HiTrap® MabSelect SuRe™ column (GE Healthcare, ref. 11-0034-93) and a HiLoad® 26 / 600 Superdex® 200 (GE Healthcare, ref. 28-9893-36) using the same protocol as the HEK293-FS purification described above.

[0441] Binding assays on stable cell lines Human PD-1 and 4-1BB Objective: To evaluate the affinity of the anti-4-1BB / anti-PD-1 bispecific binding molecule for its target (4-1BB and PD-1 arms separately) relative to a control antibody and an isotype control. Binding EC 50 value and E max Calculate the value.

[0442] Binding assays were performed in 96-well plates using PD-1 NFAT-luc2 Jurkat cells (Promega #J1252), or 4-1BB NF-κB-luc2P Jurkat cells (Promega #J2332), or in-house transfected 300.19(pre-B) expressing either human PD-1 (hPD-1) or human 4-1BB (h4-1BB).

[0443] The cell suspension was plated at 50 x 10 cells per well in a 96-well U-bottom plate. 3 The cells were plated at a density of 1000 cells / well. Serially diluted concentrations of test compounds were added to the cells for 1 hour. After washing, a fluorescently labeled secondary antibody targeting the Fc portion was added to each well for 30 minutes. The MFI signal was then measured using a flow cytometer.

[0444] Data from the flow cytometer was analyzed using FlowJo (V10.8.1), and then binding curves, E max , and E.C. 50 Values ​​were plotted using GraphPad Prism (V9.1.2).

[0445] Cynomolgus monkey PD-1 and 4-1BB Objective: To evaluate the affinity of the anti-4-1BB / anti-PD-1 bispecific binding molecule for its target (4-1BB and PD-1 arms separately) relative to a control antibody and an isotype control. Binding EC 50 value and E max Calculate the value.

[0446] Binding assays were performed in 96-well plates on ice using in-house transfected 300.19 (pre-B) cells expressing either cynoPD-1 (cyPD-1) or cyno4-1BB (cy4-1BB).

[0447] The cell suspension was plated at 50 x 10 cells per well in a 96-well U-bottom plate. 3 The cells were plated at a density of 1000 cells / well. Serially diluted concentrations of test compounds were added to the cells for 1 hour. After washing, a fluorescently labeled secondary antibody targeting the Fc portion was added to each well for 30 minutes. The MFI signal was then measured using a flow cytometer.

[0448] Data from the flow cytometer was analyzed using FlowJo (V10.8.1), and then binding curves, E max , and E.C. 50 Values ​​were plotted using GraphPad Prism (V9.1.2).

[0449] Binding assays on primary human T cells Objective: To evaluate the affinity of the anti-4-1BB / anti-PD-1 bispecific binding molecule for its target (4-1BB and PD-1 simultaneously) relative to a control antibody and an isotype control. Binding EC 50 value and E max Calculate the value.

[0450] Human PBMCs were isolated from fresh buffy coats using a Ficoll gradient. +T cells were isolated by negative selection. T cells were cultured at 50 × 10 in 96-well U-bottom culture plates pre-coated with 5 μg / mL anti-human CD3. 3 The cells were plated at a density of 10 cells / well and cultured in X-Vivo15 medium (Lonza No. BE02-061Q) supplemented with 1% penicillin / streptomycin.

[0451] Serially diluted concentrations of test compounds were added to the cells for 1 hour. After washing, a fluorescently labeled secondary antibody targeting the Fc portion was added to each well for 30 minutes. The MFI signal was then measured using a flow cytometer.

[0452] Data from the flow cytometer was analyzed using FlowJo (V10.8.1), and then binding curves, E max , and E.C. 50 Values ​​were plotted using GraphPad Prism (V9.1.2).

[0453] Reporter assay 4-1BB Objective: To evaluate the activity of the 4-1BB arm of the anti-4-1BB / anti-PD-1 bispecific binding molecule relative to a control antibody and an isotype control. 50 and E max Calculate.

[0454] Readout using the 4-1BB reporter Jurkat NF-κB-luc2P Promega cell line (GloResponse NF-κB-luc2P Jurkat cells, Promega #J2332) cultured in RPMI 1640, 10% SVF, 1% glutamine, 1% NEAA, 1 mM sodium pyruvate, 800 μg / mL G418, 500 μg / mL hygromycin according to the manufacturer's instructions.

[0455] Cells were plated at 50 x 10 per well in a 96-well white flat-bottom plate. 3Cells were plated at a density of 1 / 4 of a cell. Serially diluted concentrations of test compounds were added. After 6 hours of incubation at 37°C, Bio-Glo reagent was added to each well. Luminescence was measured using an Infinit Pro M1000 or SPARK TECAN reader.

[0456] Bonding curve, E max and EC 50 Values ​​were plotted using GraphPad Prism (V9.1.2).

[0457] PD-1 Objective: To evaluate the activity of the PD-1 arm of the anti-4-1BB / anti-PD-1 bispecific binding molecule relative to a control antibody and an isotype control. To evaluate the IC for each of the test compounds. 50 and I max Calculate.

[0458] Readout using the PD-1 reporter Jurkat NFAT-luc2 Promega cell line (GloResponse PD-1 NFAT-luc2 Jurkat cells, Promega #J1252) cultured in RPMI 1640, 10% SVF, 1% glutamine, 1% NEAA, 1 mM PyNa, 500 μg / mL G418, 200 μg / mL hygromycin in the presence of PD-L1 aAPC / CHO-K1 accessory cells (Promega #J1252) cultured in HAM / F12, 10% SVF, 200 μg / mL hygromycin, 250 μg / mL G418 according to the manufacturer's instructions.

[0459] PD-L1 aAPC / CHO-K1 were plated at 40 × 10 per well in a 96-well white flat-bottom culture plate. 3 The cells were plated at a density of 50 × 10 cells / well and incubated overnight at 37°C in Ham / F12 medium supplemented with 1% FCS. The next day, the medium was removed and 50 × 10 cells / well were plated at a density of 50 × 10 cells / well. 3A suspension of PD-1 NFAT-luc2 Jurkat cells was added to the wells at a density of cells / well, followed by serially diluted concentrations of test compounds. After 6 hours of incubation at 37°C, Bio-Glo reagent was added to each well. Luminescence was measured using an Infinit Pro M1000 or SPARK TECAN reader.

[0460] Bonding curve, I max value, and IC 50 Values ​​were plotted using GraphPad Prism (V9.1.2).

[0461] ADCC / ADCP / CDC assay Antibody-dependent cellular cytotoxicity (ADCC) assay Objectives: Evaluate the ADCC efficacy of anti-4-1BB / anti-PD-1 bispecific binding molecules relative to a control antibody and an isotype control in a dose-response experiment. EC 50 value and E max Calculate the value.

[0462] For ADCC assays, NFAT-luc2 Jurkat reporter cells overexpressing the human FcγRIIIa-V158 hypersensitive variant were used as effector cells (Promega #G7102) cocultured with in-house generated mouse 300.19 (pre-B) target cells expressing either human PD-1 or human 4-1BB. 1.5 × 10 5 Jurkat effector cells and 7.5 x 10 4 300.19 target cells (E:T ratio = 2:1) were plated in 96-well U-bottom suspension plates with test compounds in triplicate wells at concentrations ranging from 200 nM to 0.003 nM in nine serial 1:4 dilution steps. Incubation was carried out at 37°C, 5% CO2, and 95% rH2O for 24 hours, followed by readout using the Bio-Glo Luciferase Assay System (Promega #G7940) and a Tecan Spark luminescence microplate reader.

[0463] EC of test compound50 value and E max The values ​​were calculated by applying the Biostat-Speed ​​statistical calculation tool. The results were obtained using a four-parameter logistic model by Ratkovsky & Reedy (Biometrics. 1986 Sep;42(3):575-82). Adjustments were obtained by nonlinear regression using the Levenberg-Marquardt algorithm in SAS v9.1.3 software.

[0464] Antibody-dependent cellular phagocytosis (ADCP) assay Objectives: Evaluate the ADCP efficacy of anti-4-1BB / anti-PD-1 bispecific binding molecules relative to control antibodies and isotype controls in dose-response experiments. EC 50 value and E max Calculate the value.

[0465] In the ADCP assay, NFAT-luc2 Jurkat reporter cells overexpressing the human FcγRIIa-H131 hypersensitive variant were used as effector cells (Promega #G9871) co-cultured with in-house generated mouse 300.19 (pre-B) target cells expressing either human PD-1 or human 4-1BB. 3 × 10 cells per well were cultured. 4 Jurkat effector cells and 1.5 x 10 4 300.19 4-1BB target cells (E:T ratio = 2:1) or 10 5 Jurkat effector cells and 10 5 300.19 PD-1 target cells (E:T ratio = 1:1) were plated in 96-well U-bottom suspension plates with test compounds in triplicate wells at concentrations ranging from 200 nM to 0.003 nM in nine serial 1:4 dilutions. Incubation was carried out at 37°C, 5% CO2, and 95% RH for 24 hours, followed by readout using the Bio-Glo Luciferase Assay System (Promega #G7940) and a Tecan Spark luminescence microplate reader.

[0466] EC of test compound50 value and E max The values ​​were calculated by applying the Biostat-Speed ​​statistical calculation tool. The results were obtained using the four-parameter logistic model by Ratkovsky & Reedy (Biometrics. 1986 Sep; 42(3):575-82). Adjustments were obtained by nonlinear regression using the Levenberg-Marquardt algorithm in SAS v9.1.3 software.

[0467] Complement-dependent cytotoxicity (CDC) assay Objectives: Evaluate the CDC effect of anti-4-1BB / anti-PD-1 bispecific binding molecules against a control antibody and an isotype control in a dose-response experiment. IC of test compounds 50 Value and I max Calculate the value.

[0468] For the CDC assay, 1.5 x 104 in-house generated murine 300.19 (pre-B) target cells expressing either human PD-1 or human 4-1BB were plated in nine serial 1:4 dilutions per well in a 96-well U-bottom suspension plate with 10% human serum complement (Quidel #A113) and test compound in triplicate wells at concentrations ranging from 200 nM to 0.003 nM. The cells were incubated at 37°C, 5% CO2, and 95% rH2O for 24 hours, followed by readout using the CellTiter-Glo Luminescent Cell Viability Assay System (Promega #G7571) and a Tecan Spark luminescence microplate reader.

[0469] I C 50 and I of the test compound maxThe values ​​were calculated by applying the Biostat-Speed ​​statistical calculation tool. The results were obtained using the four-parameter logistic model by Ratkovsky & Reedy (Biometrics. 1986 Sep; 42(3):575-82). Adjustments were obtained by nonlinear regression using the Levenberg-Marquardt algorithm in SAS v9.1.3 software.

[0470] T cell activation (TCA) Human Objectives: To evaluate the activity of anti-4-1BB / anti-PD-1 bispecific binding molecules against control antibodies and isotype controls. To monitor cytokine release from activated human T cells.

[0471] PBMCs were isolated from fresh buffy coats using a Ficoll gradient. T cells were isolated by negative selection. T cells were plated at 100 x 10 in 96-well flat-bottom well plates pre-coated with 5 μg / mL anti-human CD3. 3 The cells were plated at a density of 10 cells / well and cultured in X-Vivo15 medium (Lonza No. BE02-061Q) supplemented with 1% penicillin / streptomycin.

[0472] Serially diluted concentrations of test compounds were added to the cultures. After 4 or 6 days of incubation, cytokine secretion in the collected supernatants was analyzed by flow cytometry using CBA human T cells according to the manufacturer's instructions (BD Bioscience #550749). h 1 / T h 2 cytokine kit, or Homogeneous Time Resolved Fluorescence (HTRF) human IFN-γ / TNF-α cytokine kits (Cisbio no. 62HIFNGPEH and no. 62HTNFAPEH) on a PHERAstar FSX multimode reader (BMG Labtech) according to the manufacturer's instructions.

[0473] Cytokine secretion was analyzed by FCAP array (V3.0.19.2091) for CBA samples and then plotted using GraphPad Prism (V9.1.2). HTRF cytokine secretion was plotted directly using GraphPad Prism (V9.1.2).

[0474] Cynomolgus monkeys Objectives: To evaluate the activity of anti-4-1BB / anti-PD-1 bispecific binding molecules against control antibodies and isotype controls. To monitor cytokine release from activated cynoT cells.

[0475] Frozen cyno PBMCs were obtained from Primacyt. Total CD3 + T cells were isolated by negative selection. T cells were cultured at 50 × 10 in 96-well V-bottom culture plates. 3 The cells were plated at a density of 10 cells / well and cultured in X-Vivo15 medium (Lonza No. BE02-061Q) supplemented with 1% penicillin / streptomycin.

[0476] Serially diluted concentrations of test compounds were added to the cultures in the presence of a 1:1 ratio of cells to beads from the NHP T Cell Activation / Expansion Kit (Miltenyi No. 130-092-919). After 2 days of incubation, cytokine secretion was assessed in the supernatants collected by MSD according to the manufacturer's instructions (Meso QuickPlex SQ120 No. 1300).

[0477] Cytokine secretion was analyzed with MSD Discovery Workbench (V4.0.12) and then plotted using GraphPad Prism (V9.1.2).

[0478] Mixed lymphocyte reaction (MLR) assay Objectives: To evaluate the activity of anti-4-1BB / anti-PD-1 bispecific binding molecules against control antibodies and isotype controls in an MLR assay. To monitor cytokine release from co-cultures.

[0479] PBMCs were isolated from fresh buffy coats using a Ficoll gradient. Monocytes were isolated by positive selection. Monocytes were cultured in RPMI 1640, 10% FCS, 2 mM glutamine, 1% penicillin / streptomycin in the presence of 50 ng / mL GM-CSF and 10 ng / mL IL-4 for 6 days to differentiate into Mo-DCs. In parallel, PBMCs were isolated from fresh buffy coats from another donor using a Ficoll gradient. T cells were isolated by negative selection.

[0480] The heterologous T cell and Mo-DC mixture at a T cell:Mo-DC ratio of 10:1 was plated in 96-well U-bottom plates and cultured in X-Vivo15 medium (Lonza no. BE02-061Q) supplemented with 1% penicillin / streptomycin.

[0481] Serially diluted concentrations of test compounds were added to the co-cultures. After 4 or 6 days of incubation, cytokine secretion in the collected supernatants was analyzed by flow cytometry using CBA human T cells according to the manufacturer's instructions (BD Bioscience #550749). h 1 / T h 2 cytokine kit, or Homogeneous Time Resolved Fluorescence (HTRF) human IFN-γ / TNF-α cytokine kits (Cisbio no. 62HIFNGPEH and no. 62HTNFAPEH) on a PHERAstar FSX multimode reader (BMG Labtech) according to the manufacturer's instructions.

[0482] Cytokine secretion was analyzed by FCAP array (V3.0.19.2091) for CBA samples and then plotted using GraphPad Prism (V9.1.2). HTRF cytokine secretion was plotted directly using GraphPad Prism (V9.1.2).

[0483] CD3-PBMC activation assay Objectives: To evaluate the activity of anti-4-1BB / anti-PD-1 bispecific binding molecules versus control antibodies and isotype controls in a CD3-PBMC activation assay. To monitor cytokine release from PBMCs.

[0484] PBMCs were isolated from fresh buffy coats and 150 × 10 3 CBA human T cells were plated at a density of 100 cells / well and cultured in the presence of 0.04 μg / mL soluble anti-CD3 and serially diluted concentrations of test compound. After 4 or 6 days of incubation, cytokine secretion in the collected supernatants was analyzed by flow cytometry according to the manufacturer's instructions (BD Bioscience #550749). h 1 / T h 2 cytokine kit, or Homogeneous Time Resolved Fluorescence (HTRF) human IFN-γ / TNF-α cytokine kits (Cisbio no. 62HIFNGPEH and no. 62HTNFAPEH) on a PHERAstar FSX multimode reader (BMG Labtech) according to the manufacturer's instructions.

[0485] Cytokine secretion was analyzed by FCAP array (V3.0.19.2091) for CBA samples and then plotted using GraphPad Prism (V9.1.2). HTRF cytokine secretion was plotted directly using GraphPad Prism (V9.1.2).

[0486] Modular IMmune In vitro Construct(MIMIC)CD8 + T cell exhaustion assay Goal: CD8 + Evaluate the activity of anti-4-1BB / anti-PD-1 bispecific binding molecules relative to control antibodies and isotype controls in T cell exhaustion assays. + The proliferation of T cells and their cytokine release are monitored.

[0487] Monocytes were isolated from frozen PBMCs by positive selection and differentiated into cytokine-derived dendritic cells (CDDCs) by culturing them for 6 days in the presence of 500 ng / mL GM-CSF and 125 ng / mL IL-4 in CellGro medium (CellGenix). CDDCs were harvested and cultured at 3.34 × 10 in 24-well plates in X-Vivo15 medium (Lonza #BE02-061Q). 4 CDDCs were resuspended at a density of 10 cells / well and primed with 1 μg / mL of HLA-restricted peptide (BioSynthesis) for 2–3 hours.

[0488] CD8 + T cells were isolated from the same autologous PBMC donor via negative selection. + T cells in a 60:1 ratio with CD8 + 2 × 10 T cells in plates containing pulsed CDDC at a CDDC ratio of 6 The cells were plated at a density of 10 cells / well and cultured for 12 days in X-Vivo15 medium (Lonza No. BE02-061Q).

[0489] functional CD8 + T cell:CDDC co-cultures remained untouched for the 12-day period. + T cell:CDDC co-cultures were pulsed again on days 4 and 8 with 1 μg / mL of HLA-restricted peptide.

[0490] On day 6, autologous CDDCs were re-prepared as described above. On day 12, CDDCs were harvested and plated at 5 x 10 in 96-well plates in X-Vivo15 medium (Lonza #BE02-061Q). 4 CDDCs were plated at a density of 10 cells / well. CDDCs were primed with 1 μg / mL of HLA-restricted peptide for 2–3 hours.

[0491] CD8 + T cell:CDDC cocultures were collected and CFSE-stained CD8 + T cells in a 10:1 ratio with CD8 + 5 × 10 T cells in plates containing pulsed CDDC at a CDDC ratio of5 Cells were plated at a density of 10 cells / well.

[0492] Serially diluted concentrations of test compounds were added to the co-cultures. After 5 days of incubation, IFN-γ and TNF-α secretion was assessed in the collected supernatants using the Milliplex Human Cytokine Custom 10 Plex detection system (EMD Millipore) according to the manufacturer's instructions. IFN-γ and TNF-α secretion were measured using the BioPlex / Luminex system (BioRad). After washing, CFSE-stained CD8 + The MFI of T cells was measured by flow cytometry. Pentamer-positive CD8 + T cells were detected according to the manufacturer's instructions (BioSynthesis).

[0493] Cytokine secretion was analyzed using BioPlex Manager Software and then plotted using GraphPad Prism (V9.1.2). Flow cytometry results were analyzed using FlowJo (V10.8.1) and then plotted using GraphPad Prism (V9.1.2).

[0494] Regulatory T cell (Treg) suppression assay Goal:T reg The activity of the anti-4-1BB / anti-PD-1 bispecific binding molecules is assessed relative to a control antibody and an isotype control in an inhibitory assay. reg CD4 in the presence of + Effector T cells (T eff ) Observe growth.

[0495] PBMCs were isolated from fresh buffy coats using a Ficoll gradient. + T cells were isolated by negative selection and then CD4 + CD25 + T regFractions were isolated by positive selection and expanded for 14 days in TexMACS medium (Miltenyi No. 130-097-196) supplemented with 5% FBS and 1% penicillin / streptomycin in the presence of CD3 / CD28 MACSiBeads (Miltenyi No. 130-095-353). eff CD4 + The CD25 fraction was frozen for later use. Readout: T using carboxyfluorescein succinimidyl ester (CFSE) dilutions according to the manufacturer's instructions. eff Proliferation.

[0496] Enlarged T reg (eT reg ) in the presence of CD2 / CD3 / CD28 MACSiBeads (Miltenyi number 130-092-909) at a bead:cell ratio of 1:1, and T eff :eT reg CFSE stained T cells from the same donor eff Serially diluted concentrations of test compounds were added to the cultures. After 5 days of incubation, the MFI of CFSE staining was measured by flow cytometry.

[0497] Flow cytometry results were analyzed using FlowJo (v.10.8.1) and plotted using GraphPad Prism (v.9.1.2).

[0498] In vivo efficacy testing C57BL / 6-Pdcd1 from Biocytogen (reference number 120516) tm1(PDCD1) Tnfrsf9 tm1(TNFRSF9) MC38 tumor cells were implanted subcutaneously into Bcgen mice (humanized hPD-1 / h4-1BB double KI) and randomized once tumors were established (approximately 100 mm 3 ). Test compounds were administered directly after randomization by ip route in a Q3D regimen.

[0499] result As shown in Figures 9A-9C, all four bispecific binding molecules (constructs Nos. 1, 3, 5, and 6) were able to activate T cells to a greater extent than the monospecific control antibodies directed against 4-1BB or PD-1 separately, but also to a greater extent than the combination of the two monospecific control antibodies.

[0500] Construct number 3 was selected for further experiments.

[0501] Binding assays on stable cell lines Table 6 shows the EC values ​​of "Construct No. 3" relative to control antibodies measured in in-house transfected 300.19 (pre-B) cells overexpressing either PD-1 (human, Figure 10A; or cyno, Figure 10B) or 4-1BB (human, Figure 10C or cyno, Figure 10D). 50 value and E max Summarize the values.

[0502] [Table 7]

[0503] Table 7 shows the EC values ​​of "Construct No. 3" relative to control antibodies measured in Jurkat cells expressing either human PD-1 (FIG. 10E) or human 4-1BB (FIG. 10F) at physiological antigen densities. 50 value and E max Summarize the values.

[0504] [Table 8]

[0505] Binding assay on primary human T cells Table 8 shows the EC of "Construct No. 3" relative to a control antibody measured in stimulated primary T cells. 50 value and E max The values ​​are summarized (Figure 10G).

[0506] [Table 9]

[0507] Reporter assay Construct No. 3, used as a soluble agent, has similar EC50 and IC50 values ​​as the control antibody in both the 4-1BB and PD-1 reporter assays (Figures 11A and -11B, respectively; and Table 9).

[0508] [Table 10]

[0509] ADCC / ADCP / CDC assay Construct No. 3 has no residual ADCC (FIGS. 12A-12B) or ADCP (FIGS. 13A-13B) Fc scaffold activity, and only weak CDC Fc scaffold activity (FIGS. 14A-14B).

[0510] T cell activation (TCA) Construct #3 induced excellent T cell activation as a soluble agent in T cell activation assays (Figures 15A-15B for human T cell activation; Figure 15C for cyno T cell activation).

[0511] Mixed lymphocyte reaction (MLR) assay Construct No. 3, as a soluble agent, induced superior T cell activation in MLR assays to the combination of two monospecific control antibodies, but also to the combination of each of the monospecific arms that make up "Construct No. 3" (Figures 16A-16C).

[0512] CD3-PBMC activation assay Construct #3 also induced multicytokine secretion in a CD3-PBMC activation assay (FIGS. 17A-17B).

[0513] In a dose-response CD3-PBMC activation assay, "construct no. 3" appeared to be more potent than the combination of two monospecific control antibodies (FIGS. 18A-18C).

[0514] MIMIC CD8 + T cell exhaustion assay Construct #3 also reactivated more exhausted T cells than the combination of two monospecific control antibodies (Figure 19A). + The T cells were again functional, as they were able to secrete IFN-γ and TNF-α after treatment (FIGS. 19B-19C).

[0515] Regulatory T cell (Treg) suppression assay Construct number 3 has a low T of 2:1. eff Cell:eT reg T cells in vitro to a greater extent than the control anti-4-1BB antibody. reg suppressive activity (Fig. 20).

[0516] In vivo efficacy testing Excellent single-agent in vivo efficacy was observed in the hu4-1BB+PD-1 dKI mouse bearing MC38 tumor model at both high dose (8 / 9CR) and low dose (6 / 9CR) (Figure 21).

[0517] array Anti-4-1BB clone number 5.1V HH has the amino acid sequence set forth below: [ka] CDR sequences are highlighted: IMGT numbering is in bold; Kabat numbering is underlined; Chothia numbering is in italics.

[0518] Anti-4-1BB clone number 2.1V HH has the amino acid sequence set forth below: [ka] CDR sequences are highlighted: IMGT numbering is in bold; Kabat numbering is underlined; Chothia numbering is in italics.

[0519] The light chain (LC) sequence of "construct no. 3" is written as follows: [ka] (In the formula, [ka] denotes inter-disulfide bond; [ka] denotes intradisulfide bond; bolded residues section are light chain CDR1, 2 and 3 of anti-PD-1 Fab "T5" according to IMGT numbering).

[0520] The heavy chain (HC) sequence of "construct no. 3" is written as follows: [ka] (In the formula, [ka] denotes inter-disulfide bond; [ka] indicates an intradisulfide bond; the first three bolded residues are from anti-4-1BB clone no. 2.1V HH The second section of three bolded residues is CDR1, 2 and 3 (according to IMGT numbering) of anti-4-1BB clone no. 5.1V HH the third section of three bolded residues are the heavy chain CDRs 1, 2, and 3 (according to IMGT numbering) of anti-PD-1 Fab "T5."

[0521] Example 4 Optimized construct number 3 The anti-PD-1 Fab "T5" from the original "Construct #3" (described in Example 2) was sequence optimized (the "T5_optimized" clone). This T5_optimized clone was inserted into the new "Optimized Construct #3" in place of the original T5 clone.

[0522] Materials and Methods Expression and purification For expression and purification, see Example 3.

[0523] Surface plasmon resonance (SPR) binding assay Goal: To evaluate the binding affinity of the original "construct no. 3" versus "optimized construct no. 3" for their targets.

[0524] SPR binding assays were performed on a Biacore 8K instrument using a CM5 anti-Fc chip and either human PD-1 or human 4-1BB as the analyte. The original "Construct No. 3" and "Optimized Construct No. 3" served as ligands.

[0525] The anti-Fc capture antibody was diluted 1:20 in running buffer and coupled to a CM5 chip (Cytiva, catalog no. 29149603) using standard amine coupling, yielding approximately 8000 response units (RU) using an amine coupling kit (Cytiva, catalog no. BR-100-50).

[0526] Ligands were used at a concentration of 0.5 μg / mL and injected at 10 μg / mL for 60 seconds. Analyte concentrations of 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, and 100 nM were injected at 30 μL / min for 240 seconds, followed by a 1200-second dissociation phase at 30 μL / min in HBS-EP+ buffer. Regeneration was performed with a 60-second injection of 3 M MgCl2 at 30 μL / min.

[0527] Binding kinetic data were evaluated with Biacore Insight Evaluation Software (Cytiva) using a 1:1 binding model.

[0528] Single molecule localization microscopy (SMLM) The goal of this experiment was to evaluate and compare the binding of "Construct No. 3" and "Optimized Construct No. 3" to constructs lacking the PD-1 arm, the 4-1BB arm, or both (i.e., constructs in which the relevant arm was replaced with an irrelevant arm).

[0529] Jurkat cells expressing 4-1BB and PD-1 were activated for 5 days using flasks pre-coated with OKT3 (CD3) antibody (Invitrogen 16-0037-85) at a final concentration of 5 μg / mL for 3 hours at 37°C. The culture medium consisted of RPMI 1640 (Gibco 21875), 10% fetal bovine serum (Gibco 16629525), 2 mM L-glutamine (Stemcell 07100), 1% non-essential amino acids (Gibco 11140-035), 1 mM sodium pyruvate (Gibco 12539059), 400 μg / mL hygromycin B (Invitrogen 10687010), and 600 μg / mL geneticin (Gibco 11811-023).

[0530] On day 5, two assays were performed in parallel: a binding assay and a receptor density assay.

[0531] Binding assay: 1.10 x 10 6Cells were placed in Eppendorf tubes, centrifuged, and resuspended in test compounds (200 nM, 300 nM, 600 nM, and 1200 nM, diluted in RPMI 1640) and incubated for 30 minutes at 37°C. After incubation, cells were fixed with 4% formol for 20 minutes at 4°C, washed, and then labeled with Fcγ fragment-specific Alexa Fluor® 647 AffiniPure Fab Fragment Goat anti-human IgG (Jackson 109-607-008) at 1 / 1000 for 1 hour at room temperature. After washing, the labeled cells were spread onto MW6 slides (Marienfeld 0117650) precoated with poly-D-lysine (Corning 354210) diluted at 1 / 400 in PBS for 30 minutes at 37°C. After 10 minutes, slides were mounted for SMLM acquisition using Smart Kit Super Resolution Buffer (Abbelight).

[0532] TCA and MLR assays For TCA and MLR assays, see Example 3.

[0533] result Surface plasmon resonance (SPR) binding assay As shown in Table 10, "Optimized Construct No. 3" exhibited improved K as compared to the original "Construct No. 3" for binding to human PD-1. D The binding efficacy to 4-1BB remains unchanged.

[0534] [Table 11]

[0535] Single molecule localization microscopy (SMLM) Figures 22A-22B show the density of "Construct No. 3" and "Optimized Construct No. 3," respectively (μm on the surface of Jurkat cells). 2 (expressed as number of test compounds bound per antibody).

[0536] The binding of "Construct No. 3ΔPD-1" and "Construct No. 3Δ4-1BB" (which lack functional PD-1 and 4-1BB arms, respectively) increased dose-dependently, reaching a maximum density at 600 nM. For "Construct No. 3," the maximum density was reached at 300 nM. Interestingly, the density achieved for Construct No. 3 at 300 nM was significantly different for the two ΔPD-1 and Δ4-1BB constructs, with the density of "Construct No. 3" being higher than the sum of the densities obtained for the two ΔPD-1 and Δ4-1BB constructs combined (Figure 22A). Comparing the binding protein density and receptor (PD-1 and 4-1BB) density on Jurkat cells, we could observe that the 4-1BB arm had a good binding kinetics on the cell membrane, leading to saturation of the 4-1BB antigen, in contrast to the PD-1 arm, which bound only approximately 30% of the available PD-1 antigen. We hypothesize that in "construct no. 3," the 4-1BB arm drives binding activity.

[0537] The binding of "Optimized Construct No. 3," "Optimized Construct No. 3ΔPD-1," and "Optimized Construct No. 3Δ4-1BB" all increased dose-dependently, reaching a maximum density at 600 nM. In contrast to the observations made with "Construct No. 3" above, the inventors did not observe any significant changes in density among these three compounds (FIG. 22B).

[0538] Comparing the binding protein density and receptor (PD-1 and 4-1BB) density on Jurkat cells, we could observe that both the PD-1 arm and the 4-1BB arm had good binding kinetics on the cell membrane, leading to saturation of the PD-1 antigen and the 4-1BB antigen. We hypothesize that in "Optimized Construct No. 3," the 4-1BB arm was no longer the sole driver of binding activity, in contrast to "Construct No. 3."

[0539] TCA and MLR assays "Optimized Construct No. 3" outperformed the original "Construct No. 3" in T cell activation assays (Figures 23A-23B) and performed essentially similarly in MLR assays (Figures 24A-24B). In both assays, the original "Construct No. 3" and "Optimized Construct No. 3" outperformed the monospecific control antibody.

[0540] array The optimized anti-PD-1 Fab "T5_optimized" has the light chain variable region (LCVR) amino acids listed as follows: [ka] CDR sequences are highlighted: IMGT numbering is in bold; Kabat numbering is underlined; Chothia numbering is in italics.

[0541] The optimized anti-PD-1 Fab "T5_optimized" has the heavy chain variable region (HCVR) amino acids listed as follows: [ka] CDR sequences are highlighted: IMGT numbering is in bold; Kabat numbering is underlined; Chothia numbering is in italics.

[0542] The light chain (LC) sequence of "Optimized Construct No. 3" is written as follows: [ka] (In the formula, [ka] denotes inter-disulfide bond; [ka] denotes intradisulfide bond; the section of bolded residues are light chain CDRs 1, 2 and 3 (according to IMGT numbering) of anti-PD-1 Fab "T5_optimized".

[0543] The heavy chain (HC) sequence of "Optimized Construct No. 3" is written as follows: [ka] (In the formula, [ka] denotes inter-disulfide bond; [ka] indicates an intradisulfide bond; the first three bolded residues are from anti-4-1BB clone no. 2.1V HH The second section of three bolded residues is CDR1, 2 and 3 (according to IMGT numbering) of anti-4-1BB clone no. 5.1V HH the third section of three bolded residues are the heavy chain CDRs 1, 2, and 3 (according to IMGT numbering) of anti-PD-1 Fab "T5_optimized".

[0544] Example 5 Conditionally active anti-4-1BB / anti-PD-1 bispecific binding protein We generated a conditionally active anti-4-1BB / anti-PD-1 bispecific binding protein from "Optimized Construct 3" described in Example 3.

[0545] Briefly, the conditionally active bispecific binding protein comprises, from N- to C-terminus: -Masked anti-4-1BB V HH (clone no. 2.1); -Anti-4-1BB V HH (clone no. 5.1); -IgG1-LALA Fc region; and -Anti-PD-1 Fab (clone "T5_optimized") Includes.

[0546] Masked anti-4-1BB V HHClone number 2.1 itself contains, from the N-terminus to the C-terminus, a masking moiety (MM), a cleavable linker, and an anti-4-1BB V HH Contains clone number 2.1.

[0547] Cleavable linkers typically contain a short amino acid sequence that is a target for a protease. In the case of cancer treatment, the protease is preferably a tumor-specific protease, i.e., a protease that is found at least primarily, if not exclusively, in the tumor microenvironment in vivo. Thus, in the absence of the protease, the masking moiety remains fused to the bispecific binding protein, thereby reducing, inhibiting, or abrogating binding of the bispecific binding protein to its target (here, the masked anti-4-1BB arm to its target, 4-1BB). However, when the conditionally active bispecific binding protein colocalizes with a protease capable of cleaving the cleavable linker, for example, in the tumor microenvironment, the masking moiety is released from the bispecific binding protein, and binding of the latter to its target antigen (e.g., 4-1BB) is restored.

[0548] Two alternative masking moieties (MM1 and MM2) were identified according to Adagene's protocol using the synthetic library described in WO 2019 / 149282 A1; MM1 or MM2 were linked to the anti-4-1BBV antibody via one of two cleavable linkers (cleavable linker no. 1 or cleavable linker no. 2). HH The masking moiety was linked to the N-terminus of clone no. 2.1. Combining one masking moiety with one cleavable linker resulted in four different conditionally active (masked) bispecific binding proteins: - comprising MC1, MM1 and cleavable linker number 1; - comprising MC2, MM2 and cleavable linker number 1; - comprising MC3, MM1 and cleavable linker number 2; and - containing MC4, MM2 and cleavable linker number 2.

[0549] Materials and Methods Expression and purification For expression and purification, see Example 3.

[0550] MMP9-mediated cleavage As experiments requiring cleavage of the masking moiety by MMP9 protease (compounds hereafter identified as “MMP9 activation”).

[0551] In the first step, recombinant human MMP9 protein (R&D Systems, ref. 911-MP-010) was added to a 100 μg / mL MMP9 solution containing p-aminophenylmercuric acetate (APMA; Calbiochem, ref. 164610-700MG) to a final concentration of 1 mM and incubated for 24 h at 37 °C. The activated MMP9 was then aliquoted and stored at -80 °C until further use.

[0552] Test (masked) compounds were then diluted to 1 mg / mL and incubated with activated MMP9 at a final concentration of 5 nM for 24 hours at 37° C. under gentle shaking (300 rpm).

[0553] Where necessary, samples were purified using HiLoad® 26 / 600 Superdex® 200 (GE Healthcare, ref. 28-9893-36) to remove excess masking peptides.

[0554] Surface plasmon resonance (SPR) binding assay Objective: To evaluate the binding affinity of "Optimized Construct No. 3" to the conditionally active anti-4-1BB / anti-PD-1 bispecific binding proteins MC1-MC4 and their unmasked versions for their targets.

[0555] SPR binding assays were performed on a Biacore 8K instrument using a CM5 anti-Fc chip and either human PD-1 or human 4-1BB as the analyte. Test compounds served as ligands.

[0556] The anti-Fc capture antibody was diluted 1:20 in running buffer and coupled to a CM5 chip (Cytiva, catalog no. 29149603) using standard amine coupling, yielding approximately 8000 response units (RU) using an amine coupling kit (Cytiva, catalog no. BR-100-50).

[0557] Ligands were used at a concentration of 0.5 μg / mL and injected at 10 μg / mL for 60 seconds. Analyte concentrations of 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, and 100 nM were injected at 30 μL / min for 240 seconds, followed by a 1200-second dissociation phase at 30 μL / min in HBS-EP+ buffer. Regeneration was performed with a 60-second injection of 3 M MgCl2 at 30 μL / min.

[0558] Binding kinetic data were evaluated with Biacore Insight Evaluation Software (Cytiva) using a 1:1 binding model.

[0559] Binding assays on stable cell lines Human PD-1 and 4-1BB Objective: To evaluate the affinity of "Optimized Construct No. 3" for the conditionally active anti-4-1BB / anti-PD-1 bispecific binding proteins MC1-MC4 and their unmasked versions for their targets. Binding EC of test compounds 50 value and E max Calculate the value.

[0560] Binding assays were performed in 96-well plates using PD-1 NFAT-luc2 Jurkat cells (Promega #J1252), or 4-1BB NF-κB-luc2P Jurkat cells (Promega #J2332), or in-house transfected 300.19(pre-B) expressing either human PD-1 (hPD-1) or human 4-1BB (h4-1BB).

[0561] The cell suspension was plated at 50 x 10 cells per well in a 96-well U-bottom plate.3 The cells were plated at a density of 1000 cells / well. Serially diluted concentrations of test compounds were added to the cells for 1 hour. After washing, a fluorescently labeled secondary antibody targeting the Fc portion was added to each well for 30 minutes. The MFI signal was then measured using a flow cytometer.

[0562] Data from the flow cytometer was analyzed using FlowJo (V10.8.1), and then binding curves, E max , and E.C. 50 Values ​​were plotted using GraphPad Prism (V9.1.2).

[0563] Cynomolgus monkey PD-1 and 4-1BB Objective: To evaluate the affinity of "Optimized Construct No. 3" for the conditionally active anti-4-1BB / anti-PD-1 bispecific binding proteins MC1-MC4 and their unmasked versions for their targets. Binding EC of test compounds 50 value and E max Calculate the value.

[0564] Binding assays were performed in 96-well plates on ice using in-house transfected 300.19 (pre-B) cells expressing either cynoPD-1 (cyPD-1) or cyno4-1BB (cy4-1BB).

[0565] The cell suspension was plated at 50 x 10 cells per well in a 96-well U-bottom plate. 3 The cells were plated at a density of 1000 cells / well. Serially diluted concentrations of test compounds were added to the cells for 1 hour. After washing, a fluorescently labeled secondary antibody targeting the Fc portion was added to each well for 30 minutes. The MFI signal was then measured using a flow cytometer.

[0566] Data from the flow cytometer was analyzed using FlowJo (V10.8.1), and then binding curves, E max , and E.C. 50 Values ​​were plotted using GraphPad Prism (V9.1.2).

[0567] Reporter assay 4-1BB Objective: To evaluate the activity of the 4-1BB arm of "Optimized Construct No. 3" against the conditionally active anti-4-1BB / anti-PD-1 bispecific binding proteins MC1-MC4 and their unmasked versions. EC 50 and E max Calculate.

[0568] Readout using the 4-1BB reporter Jurkat NF-κB-luc2P Promega cell line (GloResponse NF-κB-luc2P Jurkat cells, Promega #J2332) cultured in RPMI1640, 10% SVF, 1% glutamine, 1% NEAA, 1 mM sodium pyruvate, 800 μg / mL G418, 500 μg / mL hygromycin according to the manufacturer's instructions.

[0569] Cells were plated at 50 x 10 per well in a 96-well white flat-bottom plate. 3 Cells were plated at a density of 1 / 4 of a cell. Serially diluted concentrations of test compounds were added. After 6 hours of incubation at 37°C, Bio-Glo reagent was added to each well. Luminescence was measured using an Infinit Pro M1000 or SPARK TECAN reader.

[0570] Bonding curve, E max and EC 50 Values ​​were plotted using GraphPad Prism (V9.1.2).

[0571] PD-1 Objective: To evaluate the PD-1 arm activity of "Optimized Construct No. 3" for the conditionally active anti-4-1BB / anti-PD-1 bispecific binding proteins MC1-MC4 and their unmasked versions. IC for each of the test compounds. 50 and E max Calculate.

[0572] Readout using the PD-1 reporter Jurkat NFAT-luc2 Promega cell line (GloResponse PD-1 NFAT-luc2 Jurkat cells, Promega #J1252) cultured in RPMI1640, 10% SVF, 1% glutamine, 1% NEAA, 1 mM PyNa, 500 μg / mL G418, 200 μg / mL hygromycin in the presence of PD-L1 aAPC / CHO-K1 accessory cells (Promega #J1252) cultured in HAM / F12, 10% SVF, 200 μg / mL hygromycin, 250 μg / mL G418 according to the manufacturer's instructions.

[0573] PD-L1 aAPC / CHO-K1 were plated at 40 × 10 per well in a 96-well white flat-bottom culture plate. 3 The cells were plated at a density of 50 × 10 cells / well and incubated overnight at 37°C in Ham / F12 medium supplemented with 1% FCS. The next day, the medium was removed and 50 × 10 cells / well were plated at a density of 50 × 10 cells / well. 3 A suspension of PD-1 NFAT-luc2 Jurkat cells was added to the wells at a density of cells / well, followed by serially diluted concentrations of test compounds. After 6 hours of incubation at 37°C, Bio-Glo reagent was added to each well. Luminescence was measured using an Infinit Pro M1000 or SPARK TECAN reader.

[0574] Bonding curve, I max value, and IC 50 Values ​​were plotted using GraphPad Prism (V9.1.2).

[0575] T cell activation (TCA) assay Human TCA assay The in vitro bioactivity of "optimized construct number 3" (without masking moiety), each of MC1 to MC4 (masked), and the protease-activating compound (unmasked) was measured using the CellTiter-Glo (CTG) method.

[0576] Briefly, peripheral blood mononuclear cells (PBMCs) were first isolated from fresh human blood, and then total T cells were purified with the StemCell kit. These cells (1 × 10 5 T cells (cells / well) were incubated in 96-well tissue culture plates pre-coated with a suboptimal concentration (5 μg / mL) of anti-human CD3 with or without serial dilutions of test articles. The level of T cell activation was then measured by T cell proliferation using the CTG method.

[0577] Mouse TCA assay Objectives: To evaluate the activity of "Optimized Construct No. 3" against the conditionally active anti-4-1BB / anti-PD-1 bispecific binding proteins MC1-MC4 and its unmasked version against isotype controls. To observe cytokine release from activated murine T cells.

[0578] C57BL / 6-Tnfrsf9 derived from Biocytogen (reference number 110004) tm1(TNFRSF9) We crossed Bcgen mice (humanized h4-1BB KI) with hPD-1 KI mice from CIPHE (Center for Immunophenomics, Marseille, France) to generate novel h4-1BB / hPD-1 double KI (dKI) mice. This dKI mouse model was validated genotypically and phenotypically. We also validated cross-linking of the new mouse model.

[0579] Mouse T cells were isolated from spleen cells of dKI mice via negative selection. T cells were cultured at 200 × 10 in 96-flat-bottom well plates previously coated with 1 μg / mL anti-mouse CD3. 3Cells were plated at a density of 10 cells / well and cultured in complete medium containing RPMI 1640 supplemented with 1% penicillin / streptomycin (Gibco 31870-025), 2 mM L-glutamine (Gibco 25030-081), 10% FCS (Eurobio CVFSF00-01), 1x non-essential amino acids (Gibco 11140-035), 1 mM sodium pyruvate (Gibco 11360-070), and 0.05 mM 2-mercaptoethanol (Gibco 31350-010).

[0580] Serially diluted concentrations of test compounds were added to the cultures. After 2 days of incubation, mouse T cells were analyzed using a flow cytometer according to the manufacturer's instructions (BD Bioscience 551287). h 1 / T h Cytokine secretion was assessed in the collected supernatants using a 2 cytokine cytometry bead array (CBA) kit.

[0581] Cytokine secretion was analyzed by FCAP array (v3.0.19.2091) and then plotted using GraphPad Prism (v9.5.0).

[0582] Mixed lymphocyte reaction (MLR) assay Objectives: To evaluate the activity of "optimized construct no. 3" against the conditionally active anti-4-1BB / anti-PD-1 bispecific binding proteins MC1-MC4 and their unmasked versions in an MLR assay. To observe cytokine release from co-cultures.

[0583] PBMCs were isolated from fresh buffy coats using a Ficoll gradient. Monocytes were isolated by positive selection. Monocytes were cultured in RPMI 1640, 10% FCS, 2 mM glutamine, 1% penicillin / streptomycin in the presence of 50 ng / mL GM-CSF and 10 ng / mL IL-4 for 6 days to differentiate into Mo-DCs. In parallel, PBMCs were isolated from fresh buffy coats from another donor using a Ficoll gradient. T cells were isolated by negative selection.

[0584] The heterologous T cell and Mo-DC mixture at a T cell:Mo-DC ratio of 10:1 was plated in 96-well U-bottom plates and cultured in X-Vivo15 medium (Lonza no. BE02-061Q) supplemented with 1% penicillin / streptomycin.

[0585] Serially diluted concentrations of test compounds were added to the co-cultures. After 4 or 6 days of incubation, cytokine secretion in the collected supernatants was analyzed by flow cytometry using CBA human T cells according to the manufacturer's instructions (BD Bioscience #550749). h 1 / T h 2 cytokine kit, or Homogeneous Time Resolved Fluorescence (HTRF) human IFN-γ / TNF-α cytokine kits (Cisbio no. 62HIFNGPEH and no. 62HTNFAPEH) on a PHERAstar FSX multimode reader (BMG Labtech) according to the manufacturer's instructions.

[0586] Cytokine secretion was analyzed by FCAP array (V3.0.19.2091) for CBA samples and then plotted using GraphPad Prism (V9.1.2). HTRF cytokine secretion was plotted directly using GraphPad Prism (V9.1.2).

[0587] CD3-PBMC activation assay Objectives: To evaluate the activity of "Optimized Construct No. 3" against the conditionally active anti-PD-1 / anti-PD-1 bispecific binding proteins MC1-MC4 and their unmasked versions in a CD3+ PBMC activation assay. To observe cytokine release from PBMCs.

[0588] PBMCs were isolated from fresh buffy coats and 150 × 10 3CBA human T cells were plated at a density of 100 cells / well and cultured in the presence of 0.04 μg / mL soluble anti-CD3 and serially diluted concentrations of test compound. After 4 or 6 days of incubation, cytokine secretion in the collected supernatants was analyzed by flow cytometry according to the manufacturer's instructions (BD Bioscience #550749). h 1 / T h 2 cytokine kit, or Homogeneous Time Resolved Fluorescence (HTRF) human IFN-γ / TNF-α cytokine kits (Cisbio no. 62HIFNGPEH and no. 62HTNFAPEH) on a PHERAstar FSX multimode reader (BMG Labtech) according to the manufacturer's instructions.

[0589] Cytokine secretion was analyzed by FCAP array (V3.0.19.2091) for CBA samples and then plotted using GraphPad Prism (V9.1.2). HTRF cytokine secretion was plotted directly using GraphPad Prism (V9.1.2).

[0590] plasma stability The stability of each of MC1 to MC4 (masks) was evaluated in mouse, cynomolgus monkey, and human plasma over a 7-day period.

[0591] Briefly, each of MC1 to MC4 was diluted to a final concentration of 100 μg / mL in mouse, cynomolgus monkey, or human EDTA plasma. Samples were incubated at 37°C for 7 days (i.e., 168 hours) and then analyzed by ELISA. Total fractions (cleaved and uncleaved compounds) were detected using immobilized anti-human IgG Fc antibody and anti-human IgG Fab-HRP conjugate for detection; immobilized cleaved 4-1BB (anti-4-1BB clone no. 2.1V) for detection. HH The cleaved fraction was detected using an anti-human IgG Fab-HRP conjugate (which can only bind to IgG).

[0592] In vivo efficacy testing C57BL / 6-Tnfrsf9 derived from Biocytogen (reference number 110004) tm1(TNFRSF9) We generated a novel h4-1BB / hPD-1 double KI (dKI) mouse model by crossing Bcgen mice (humanized h4-1BB KI) with hPD-1 KI mice from CIPHE (Center for Immunophenomics, Marseille, France). This dKI mouse model was validated genotypically and phenotypically. These dKI mice were implanted subcutaneously with MC38 tumor cells and randomized once tumors were established (approximately 100 mm). 3 ). Test compounds were administered directly after randomization by the ip route in a Q3D regimen.

[0593] result Surface plasmon resonance (SPR) binding assay As can be seen in Figure 25, the four conditionally active anti-4-1BB / anti-PD-1 bispecific binding proteins MC1-MC4 exhibited 4-1BB binding only after MMP9-mediated cleavage of their masking moieties (Table 11).

[0594] In contrast, PD-1 binding was not affected by the masking moiety (Table 12).

[0595] All positive and negative controls exhibited the expected target binding behavior.

[0596] [Table 12]

[0597] [Table 13]

[0598] Binding assays on stable cell lines Pre-B cells In human 300.19 pre-B cells, conditionally active anti-4-1BB / anti-PD-1 bispecific binding proteins with cleavable linker number 1 (MC1 and MC2) had higher EC values ​​for 4-1BB than those with cleavable linker number 2 (MC3 and MC4). 50 was high.

[0599] The conditionally active anti-4-1BB / anti-PD-1 bispecific binding protein is a human 4-1BB + It had a 1.5-2 log masking efficacy in cells (Figure 26A), and cyno 4-1BB + After MMP9-mediated cleavage of their masking moieties, all four bispecific binding proteins had an EC20 equivalent to "optimized construct no. 3" (unmasked) in cells (Figure 26B). 50 (Table 13).

[0600] In contrast, PD-1 binding was not affected by the masking moiety (Figures 26C-26D; Table 14).

[0601] All positive and negative controls exhibited the expected target binding behavior.

[0602] [Table 14]

[0603] [Table 15]

[0604] Jurkat T cells As shown in Table 15 and Figure 27, the protease-activated (i.e., unmasked) forms (MMP9-activated and uPA-activated) were significantly reduced in EC 50The masked compounds MC1-MC4 show similar binding activity to Jurkat / NF-κB-4-1BB cells compared to the parental "Optimized Construct No. 3" based on the α- and β-blockers values. Binding is reduced by at least 166-fold for all masked compounds MC1-MC4 and up to 553-fold for MC1 compared to "Optimized Construct No. 3."

[0605] [Table 16]

[0606] Further experiments with Jurkat T cells showed that the conditionally active anti-4-1BB / anti-PD-1 bispecific binding proteins with cleavable linker number 1 (MC1 and MC2) had a higher EC2 response to 4-1BB than those with cleavable linker number 2 (MC3 and MC4). 50 was high.

[0607] The conditionally active anti-4-1BB / anti-PD-1 bispecific binding protein is a human 4-1BB + After MMP9-mediated cleavage of their masking moieties, all four bispecific binding proteins had an EC20 equivalent ...

Claims

1. 1. A multispecific antigen-binding protein comprising at least one immunoglobulin single variable domain (ISV) that specifically binds to 4-1BB, wherein said at least one ISV that specifically binds to 4-1BB has pure agonist activity.

2. Pure agonist activity means that the ISV is capable of activating T cells via 4-1BB signaling (i) under soluble conditions, and / or (ii) in the absence of a cross-linking reagent, and / or (iii) in an FcγR-independent manner, and / or (iv) in the absence of target-mediated cross-linking of 4-1BB; Optionally, the pure agonist activity is determined by an NF-κB pathway activation assay in the absence of a cross-linking reagent.

3. 3. The multispecific antigen-binding protein of claim 1 or 2, wherein said at least one ISV that specifically binds to 4-1BB competes with 4-1BBL for 4-1BB binding.

4. the at least one ISV that specifically binds to 4-1BB interacts with the cysteine-rich domain 2 (CRD2) and / or cysteine-rich domain 3 (CRD3) domain of 4-1BB; The multispecific antigen-binding protein according to any one of claims 1 to 3, wherein said at least one ISV that specifically binds to 4-1BB preferably interacts with the CRD2 and CRD3 domains of 4-1BB.

5. 5. The multispecific antigen-binding protein of any one of claims 1 to 4, wherein said at least one ISV that specifically binds to 4-1BB interacts with one or more amino acid residues of 4-1BB selected from the group consisting of residues K69, G70, V71, F72, R73, F92, L95, S100, M101, C102, E103, Q104, K114, K115 and G116 of SEQ ID NO:

13.

6. the at least one ISV that specifically binds to 4-1BB comprises three complementarity determining regions CDR1, CDR2 and CDR3; 6. The multispecific antigen-binding protein of any one of claims 1 to 5, wherein CDR3 comprises or consists of the amino acid sequence ARGTRYKLST (SEQ ID NO: 14), ARGTRYKMST (SEQ ID NO: 15), or ARGTRYKIFA (SEQ ID NO: 62).

7. 7. The multispecific antigen-binding protein of claim 6, wherein CDR1 comprises or consists of the amino acid sequence GFTFSDHT (SEQ ID NO: 16), GFAFRDFT (SEQ ID NO: 66), GDTFSSYA (SEQ ID NO: 67), or GFTFANYR (SEQ ID NO: 68).

8. 8. The multispecific antigen-binding protein of claim 6 or 7, wherein CDR2 comprises or consists of the amino acid sequence ISSGGSRI (SEQ ID NO: 17), INPSGGSQ (SEQ ID NO: 77) or IKKSGNRT (SEQ ID NO: 78).

9. The at least one ISV that specifically binds to 4-1BB is (i) an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 58, 59, 60, and 61; or (ii) an amino acid sequence that shares at least 70% sequence identity across the non-CDR regions of SEQ ID NO: 2, 3, 58, 59, 60, or 61 9. The multispecific antigen-binding protein of any one of claims 1 to 8, comprising or consisting of:

10. the at least one ISV that specifically binds to 4-1BB comprises or consists of the amino acid sequence of SEQ ID NO: 2 or 3; The multispecific antigen-binding protein according to any one of claims 1 to 9, wherein said at least one ISV that specifically binds to 4-1BB preferably comprises or consists of the amino acid sequence of SEQ ID NO:

3.

11. 11. The multispecific antigen-binding protein of any one of claims 1 to 10, comprising at least two ISVs that specifically bind to 4-1BB.

12. 12. The multispecific antigen-binding protein of claim 11, wherein the at least two ISVs that specifically bind to 4-1BB are identical.

13. the at least two ISVs that specifically bind to 4-1BB are different and bind to (i) the same epitope, (ii) overlapping epitopes, or (iii) distinct epitopes of 4-1BB; 12. The multispecific antigen-binding protein of claim 11, wherein the at least two ISVs that specifically bind to 4-1BB are different and bind to distinct epitopes of 4-1BB.

14. at least a second ISV that specifically binds to 4-1BB, (i) an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 4; or (ii) an amino acid sequence that shares at least 70% sequence identity over the non-CDR regions of SEQ ID NO: 1 or 4; 14. The multispecific antigen-binding protein of any one of claims 11 to 13, comprising or consisting of:

15. 15. The multispecific antigen-binding protein of claim 14, wherein the ISV comprises or consists of an amino acid sequence having SEQ ID NO:

4.

16. 11. The multispecific antigen-binding protein of any one of claims 1 to 10, comprising at least two ISVs, one of said at least two ISVs specifically binding to 4-1BB and one of said at least two ISVs specifically binding to another target antigen.

17. 17. The multispecific antigen-binding protein of claim 16, wherein the other target antigen is a T cell antigen, a tumor-associated or tumor-specific antigen, or a non-self antigen.

18. 18. The multispecific antigen-binding protein of any one of claims 1 to 17, comprising at least four ISVs that specifically bind to 4-1BB.

19. The at least four ISVs: (i) a first set of at least two identical ISVs that specifically bind to 4-1BB, and (ii) a second set of at least two other identical ISVs that specifically bind to 4-1BB; or (i') a first set of at least two ISVs that specifically bind to a first epitope of 4-1BB, and (ii') a second set of at least two other ISVs that specifically bind to a second epitope of 4-1BB.

19. The multispecific antigen-binding protein of claim 18, comprising:

20. 20. The multispecific antigen-binding protein of claim 19, wherein said first set of at least two ISVs of (i) or (i') are ISVs as defined in any one of claims 1 to 10.

21. 21. The multispecific antigen-binding protein of claim 19 or 20, wherein said second set of at least two ISVs of (ii) or (ii') are ISVs as defined in claim 14 or 15.

22. further comprising an antibody Fc region or a fragment thereof; The multispecific antigen-binding protein of any one of claims 1 to 21, wherein the Fc region or fragment thereof is ADCC and / or silenced ADCP.

23. 23. The multispecific antigen-binding protein of any one of claims 1 to 22, further comprising at least one Fab fragment.

24. the multispecific antigen-binding protein comprising: a) preferably from the N-terminus to the C-terminus: i. a first ISV that specifically binds to 4-1BB; ii. a second ISV that specifically binds to 4-1BB, preferably different from said first ISV; iii. At least one C of the Fc region Ｈ domain; and iv. Variable and constant domains of the Fab fragment a first polypeptide comprising: b) a second polypeptide comprising the variable and constant domains of the Fab fragment; Including; 24. The multispecific antigen-binding protein of any one of claims 1 to 23, wherein the variable and constant domains of the first and second polypeptides form a Fab fragment.

25. and a third polypeptide and a fourth polypeptide identical to the first polypeptide and the second polypeptide, respectively, wherein the at least one C of the first polypeptide and the third polypeptide is Ｈ 25. The multispecific antigen-binding protein of claim 24, wherein the domains form an Fc region.

26. - the variable and constant domains of the first polypeptide are V Ｈ Domain and C Ｈ one domain, and the variable and constant domains of the second polypeptide are V Ｌ Domain and C Ｌ is a domain; or - the variable and constant domains of the first polypeptide are V Ｌ Domain and C Ｌ domain, and the variable domain and constant domain of the second polypeptide are V Ｈ Domain and C Ｈ 26. The multispecific antigen-binding protein of claim 24 or 25, which is one domain.

27. the at least one C of the first polypeptide Ｈ The domain is - IgG C Ｈ 2 and C Ｈ 3 domains; -IgD C Ｈ 2 and C Ｈ 3 domains; -IgA C Ｈ 2 and C Ｈ 3 domains; -IgM C Ｈ 2. C Ｈ 3 and C Ｈ 4 domains; or -IgE C Ｈ 2. C Ｈ 3, and C Ｈ 4 Domains 27. The multispecific antigen-binding protein of any one of claims 24 to 26, comprising:

28. the at least one C of the first polypeptide Ｈ The domain is C of IgG Ｈ 2 domain and C Ｈ Contains 3 domains; Preferably, the IgG is IgG1 or IgG4; More preferably, the multispecific antigen-binding protein of any one of claims 24 to 27, wherein the IgG is IgG1.

29. The first polypeptide preferably comprises, from N-terminus to C-terminus: - a first ISV that specifically binds to 4-1BB; - a first linker; a second ISV that specifically binds to -4-1BB, preferably different from the first ISV; - a second linker; - IgG hinge region; - IgG C Ｈ 2 domains; - and IgG C Ｈ 3 domains; - a third linker; - V of Fab fragment Ｈ domain; and -C of Fab fragment Ｈ 1 domain 29. The multispecific antigen-binding protein of any one of claims 24 to 28, comprising:

30. The second polypeptide preferably comprises, from N-terminus to C-terminus: - V of Fab fragment Ｌ domain; and -C of Fab fragment Ｌ domain 30. The multispecific antigen-binding protein of any one of claims 24 to 29, comprising:

31. 31. The multispecific antigen-binding protein of any one of claims 23 to 30, wherein said at least one Fab fragment specifically binds to a B and / or T cell surface protein other than 4-1BB.

32. 32. The multispecific antigen-binding protein of any one of claims 23 to 31, wherein said at least one Fab fragment specifically binds to an immune checkpoint molecule.

33. 33. The multispecific antigen-binding protein of any one of claims 23 to 32, wherein said at least one Fab fragment is a PD-1 antagonist.

34. the at least one Fab fragment is an antigen-binding protein that specifically binds to PD-1; (i) the three light chain complementarity determining region (CDR) sequences found in SEQ ID NO: 7 or 5, and (ii) the three heavy chain CDR sequences found in SEQ ID NO: 8 or 6 34. The multispecific antigen-binding protein of any one of claims 23 to 33, comprising:

35. the at least one Fab fragment is an antigen-binding protein that specifically binds to PD-1; (i) the following three CDR sequences: a.V Ｌ CDR1: QSVPINF (SEQ ID NO: 18) or QSVSINF (SEQ ID NO: 19); b.V Ｌ CDR2: EAS; and c.V Ｌ CDR3: GQYGSSPYT (SEQ ID NO: 20) or QQYGSSPYT (SEQ ID NO: 21) a light chain variable region comprising: (ii) the following three CDR sequences: a.V Ｈ CDR1: GGSISSSSYF (SEQ ID NO: 22) or GGSISTSSYF (SEQ ID NO: 23); b.V Ｈ CDR2: IYRSGST (SEQ ID NO: 24); and c.V Ｈ - CDR3: ARGITGDPGDY (SEQ ID NO: 25) a heavy chain variable region comprising 35. The multispecific antigen-binding protein of any one of claims 23 to 34, comprising:

36. the at least one Fab fragment is an antigen-binding protein that specifically binds to PD-1; (i) the following three CDR sequences: a.V Ｌ - CDR1: QSVPINF (SEQ ID NO: 18); b.V Ｌ CDR2: EAS; and c.V Ｌ - CDR3: GQYGSSPYT (SEQ ID NO: 20) a light chain variable region comprising: (ii) the following three CDR sequences: a.V Ｈ - CDR1: GGSISSSSYF (SEQ ID NO: 22); b.V Ｈ CDR2: IYRSGST (SEQ ID NO: 24); and c.V Ｈ - CDR3: ARGITGDPGDY (SEQ ID NO: 25) a heavy chain variable region comprising 36. The multispecific antigen-binding protein of any one of claims 23 to 35, comprising:

37. the at least one Fab fragment is an antigen-binding protein that specifically binds to PD-1; (i) a light chain variable region having SEQ ID NO: 7 or 5, or a light chain variable region sharing at least 70% sequence identity over the non-CDR regions of SEQ ID NO: 7 or 5; and (ii) a heavy chain variable region having SEQ ID NO: 8 or 6, or a heavy chain variable region sharing at least 70% sequence identity across the non-CDR regions of SEQ ID NO: 8 or 6 37. The multispecific antigen-binding protein of any one of claims 23 to 36, comprising:

38. 38. The multispecific antigen-binding protein of any one of claims 23 to 37, wherein the at least one Fab fragment is an antigen-binding protein that specifically binds to PD-1 and comprises a light chain variable region having SEQ ID NO:7 and a heavy chain variable region having SEQ ID NO:

8.

39. 39. The multispecific antigen-binding protein of any one of claims 1 to 38, comprising at least a first polypeptide having SEQ ID NO: 11 or 9 and at least a second polypeptide having SEQ ID NO: 12 or 10; or comprising at least a first polypeptide that shares at least 70% sequence identity over the non-CDR regions of SEQ ID NO: 11 or 9 and at least a second polypeptide that shares at least 70% sequence identity over the non-CDR regions of SEQ ID NO: 12 or 10.

40. 40. The multispecific antigen-binding protein of any one of claims 1 to 39, comprising at least a first polypeptide having SEQ ID NO: 11 and at least a second polypeptide having SEQ ID NO:

12.

41. 40. The multispecific antigen-binding protein of any one of claims 1 to 39, comprising at least a first polypeptide having SEQ ID NO:9 and at least a second polypeptide having SEQ ID NO:

10.

42. (i) a multispecific antigen-binding protein according to any one of claims 1 to 41, and (ii) at least one masking moiety that reduces or inhibits binding of said multispecific antigen-binding protein to at least one of its target antigens.

1. A conditionally active multispecific antigen-binding protein comprising:

43. 43. The conditionally active multispecific antigen-binding protein of claim 42, wherein said at least one masking moiety comprises or consists of the amino acid sequence VEVCPELQGIFCYR (SEQ ID NO:97), or an amino acid sequence sharing at least 70% sequence identity with SEQ ID NO:

97.

44. 44. The conditionally active multispecific antigen-binding protein of claim 42 or 43, wherein said at least one masking moiety comprises or consists of the amino acid sequence of SEQ ID NO: 44 or 45, or an amino acid sequence sharing at least 70% sequence identity with SEQ ID NO: 44 or 45.

45. 45. The conditionally active multispecific antigen-binding protein of any one of claims 42 to 44, further comprising at least one linker between the multispecific antigen-binding protein and the masking moiety.

46. 46. ​​The conditionally active multispecific antigen-binding protein of claim 45, wherein said at least one linker is cleavable.

47. 47. The conditionally active multispecific antigen-binding protein of claim 45 or 46, wherein said at least one linker is cleavable by at least one tumor-specific protease.

48. 48. The conditionally active multispecific antigen-binding protein of claim 47, wherein said at least one tumor-specific protease is selected from the group consisting of matrix metalloproteinase-9 (MMP-9), urokinase-type plasminogen activator (uPa), matrix metalloproteinase-2 (MMP-2), matriptase, regumain, kallikrein-related peptidase-3, human neutrophil elastase, proteinase 3 (Pr3), cathepsin B, and cathepsin K.

49. 49. The conditionally active multispecific antigen-binding protein of claim 47 or 48, wherein said at least one tumor-specific protease is MMP-9 or uPa, or a combination thereof.

50. 50. The conditionally active multispecific antigen-binding protein of any one of claims 45 to 49, wherein said at least one linker comprises the amino acid sequence of SEQ ID NO: 56 and / or 57.

51. 51. The conditionally active multispecific antigen-binding protein of any one of claims 45 to 50, wherein said at least one linker comprises or consists of the amino acid sequence of SEQ ID NO: 46 or 47.

52. a. at least a first polypeptide having SEQ ID NO: 11 or 9; and b. at least a second polypeptide having SEQ ID NO: 52, 53, 54 or 55 52. The conditionally active multispecific antigen-binding protein of any one of claims 42 to 51, comprising:

53. 53. The conditionally active multispecific antigen-binding protein of claim 52, further comprising a third polypeptide and a fourth polypeptide identical to said first polypeptide and second polypeptide, respectively.

54. An immunoglobulin single variable domain (ISV) that specifically binds to 4-1BB and has pure agonist activity.

55. 55. The ISV of claim 54, wherein pure agonist activity means that the ISV is able to activate T cells via 4-1BB signaling (i) under soluble conditions, and / or (ii) in the absence of a cross-linking reagent, and / or (iii) in an FcγR-independent manner, and / or (iv) in the absence of target-mediated cross-linking of 4-1BB.

56. 56. An ISV according to claim 54 or 55, wherein the pure agonist activity is determined by an NF-κB pathway activation assay in the absence of a cross-linking reagent.

57. 57. An ISV according to any one of claims 54 to 56, wherein the ISV competes with 4-1BBL for 4-1BB binding.

58. 58. An ISV according to any one of claims 54 to 57, which interacts with the cysteine ​​rich domain 2 (CRD2) and / or cysteine ​​rich domain 3 (CRD3) domain of 4-1BB; preferably interacts with the CRD2 and CRD3 domains of 4-1BB.

59. 59. An ISV according to any one of claims 54 to 58, which interacts with one or more amino acid residues of 4-1BB selected from the group consisting of residues K69, G70, V71, F72, R73, F92, L95, S100, M101, C102, E103, Q104, K114, K115 and G116 of SEQ ID NO:

13.

60. comprising three complementarity determining regions CDR1, CDR2 and CDR3, 60. An ISV according to any one of claims 54 to 59, wherein CDR3 comprises or consists of the amino acid sequence ARGTRYKLST (SEQ ID NO: 14), ARGTRYKMST (SEQ ID NO: 15), or ARGTRYKIFA (SEQ ID NO: 62).

61. 61. The ISV of claim 60, wherein CDR1 comprises or consists of the amino acid sequence GFTFSDHT (SEQ ID NO: 16), GFAFRDFT (SEQ ID NO: 66), GDTFSSYA (SEQ ID NO: 67), or GFTFANYR (SEQ ID NO: 68).

62. 62. An ISV according to claim 60 or 61, wherein CDR2 comprises or consists of the amino acid sequence ISSGGSRI (SEQ ID NO: 17), INPSGGSQ (SEQ ID NO: 77), or IKKSGNRT (SEQ ID NO: 78).

63. (i) an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 58, 59, 60, and 61; or (ii) an amino acid sequence that shares at least 70% sequence identity across the non-CDR regions of SEQ ID NO: 2, 3, 58, 59, 60, or 61 63. An ISV according to any one of claims 54 to 62, comprising or consisting of:

64. 64. An ISV according to any one of claims 54 to 63 comprising or consisting of the amino acid sequence of SEQ ID NO: 2 or 3.

65. 65. An ISV according to any one of claims 54 to 64 comprising or consisting of the amino acid sequence of SEQ ID NO:

3.

66. V ＨＨ 66. The ISV of any one of claims 54 to 65,

67. An immunoglobulin single variable domain (ISV) that specifically binds to 4-1BB, (i) an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 4; or (ii) an amino acid sequence that shares at least 70% sequence identity over the non-CDR regions of SEQ ID NO: 1 or 4 An ISV comprising or consisting of:

68. 68. An ISV as described in claim 67 comprising or consisting of an amino acid sequence having SEQ ID NO:

4.

69. V ＨＨ 69. The ISV of claim 67 or 68,

70. 67. A bivalent or bispecific antigen-binding protein comprising at least one immunoglobulin single variable domain (ISV) according to any one of claims 54 to 66 and at least a second ISV which specifically binds to the same or another target antigen.

71. 71. The bivalent or bispecific antigen-binding protein of claim 70, wherein said other target antigen is a T cell antigen, a tumor-associated or tumor-specific antigen, or a non-self antigen.

72. 71. A bivalent or bispecific antigen-binding protein according to claim 70, wherein said at least a second ISV is an ISV according to any one of claims 67 to 69.

73. A conditionally active immunoglobulin single variable domain (ISV), comprising: (i) an ISV according to any one of claims 67 to 69, and (ii) at least one masking moiety that reduces or inhibits binding of said ISV to its target antigen; Conditionally active ISVs, including:

74. 74. The conditionally active ISV of claim 73, wherein the at least one masking moiety comprises or consists of the amino acid sequence VEVCPELQGIFCYR (SEQ ID NO:97), or an amino acid sequence sharing at least 70% sequence identity with SEQ ID NO:

97.

75. 75. The conditionally active ISV of claim 73 or 74, wherein the at least one masking moiety comprises or consists of an amino acid sequence of SEQ ID NO: 44 or 45, or an amino acid sequence sharing at least 70% sequence identity with SEQ ID NO: 44 or 45.

76. 76. A conditionally active ISV according to any one of claims 73 to 75, further comprising at least one linker between the ISV and the masking moiety.

77. 77. The conditionally active ISV of claim 76, wherein said at least one linker is cleavable.

78. 78. The conditionally active ISV of claim 76 or 77, wherein the at least one linker is cleavable by at least one tumor-specific protease.

79. 79. The conditionally active ISV of claim 78, wherein the at least one tumor-specific protease is selected from the group consisting of matrix metalloproteinase-9 (MMP-9), urokinase-type plasminogen activator (uPa), matrix metalloproteinase-2 (MMP-2), matriptase, regumain, kallikrein-related peptidase-3, human neutrophil elastase, proteinase 3 (Pr3), cathepsin B, and cathepsin K.

80. 80. The conditionally active ISV of claim 78 or 79, wherein the at least one tumor-specific protease is MMP-9 or uPa, or a combination thereof.

81. 81. The conditionally active ISV of any one of claims 76 to 80, wherein the at least one linker comprises the amino acid sequence of SEQ ID NO: 56 and / or 57.

82. 82. The conditionally active ISV of any one of claims 76 to 81, wherein the at least one linker comprises or consists of the amino acid sequence of SEQ ID NO: 46 or 47.

83. 83. The conditionally active ISV of any one of claims 73 to 82, comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 48, 49, 50 and 51.

84. A conditionally active immunoglobulin single variable domain (ISV) that specifically binds to 4-1BB, a. an ISV that specifically binds to 4-1BB; and b. at least one masking moiety that reduces or inhibits binding of said ISV to 4-1BB Conditionally active ISVs, including:

85. 85. The conditionally active ISV of claim 84, wherein the at least one masking moiety comprises or consists of the amino acid sequence VEVCPELQGIFCYR (SEQ ID NO:97), or an amino acid sequence sharing at least 70% sequence identity with SEQ ID NO:

97.

86. 86. The conditionally active ISV of claim 84 or 85, wherein the at least one masking moiety comprises or consists of an amino acid sequence of SEQ ID NO: 44 or 45, or an amino acid sequence sharing at least 70% sequence identity with SEQ ID NO: 44 or 45.

87. 87. A conditionally active ISV according to any one of claims 84 to 86, further comprising at least one linker between the ISV and the masking moiety.

88. 88. The conditionally active ISV of claim 87, wherein said at least one linker is cleavable.

89. 89. The conditionally active ISV of claim 87 or 88, wherein the at least one linker is cleavable by at least one tumor-specific protease.

90. 90. The conditionally active ISV of claim 89, wherein the at least one tumor-specific protease is selected from the group consisting of matrix metalloproteinase-9 (MMP-9), urokinase-type plasminogen activator (uPa), matrix metalloproteinase-2 (MMP-2), matriptase, regumain, kallikrein-related peptidase-3, human neutrophil elastase, proteinase 3 (Pr3), cathepsin B, and cathepsin K.

91. 91. The conditionally active ISV of claim 89 or 90, wherein the at least one tumor-specific protease is MMP-9 or uPa, or a combination thereof.

92. 92. The conditionally active ISV of any one of claims 87 to 91, wherein the at least one linker comprises the amino acid sequence of SEQ ID NO: 56 and / or 57.

93. 93. The conditionally active ISV of any one of claims 87 to 92, wherein the at least one linker comprises or consists of the amino acid sequence of SEQ ID NO: 46 or 47.

94. V ＨＨ 94. The conditionally active ISV of any one of claims 84 to 93, wherein

95. A conditionally active immunoglobulin single variable domain (ISV) that specifically binds to a target antigen, comprising: a. an ISV that specifically binds to a target antigen; and b. at least one masking moiety that reduces or inhibits binding of said ISV to its target antigen Conditionally active ISVs, including:

96. 96. The conditionally active ISV of claim 95, wherein the at least one masking moiety comprises or consists of the amino acid sequence VEVCPELQGIFCYR (SEQ ID NO:97), or an amino acid sequence sharing at least 70% sequence identity with SEQ ID NO:

97.

97. 97. The conditionally active ISV of claim 95 or 96, wherein the at least one masking moiety comprises or consists of an amino acid sequence of SEQ ID NO: 44 or 45, or an amino acid sequence sharing at least 70% sequence identity with SEQ ID NO: 44 or 45.

98. 98. A conditionally active ISV according to any one of claims 95 to 97, further comprising at least one linker between the ISV and the masking moiety.

99. 99. The conditionally active ISV of claim 98, wherein said at least one linker is cleavable.

100. 100. The conditionally active ISV of claim 98 or 99, wherein the at least one linker is cleavable by at least one tumor-specific protease.

101. 101. The conditionally active ISV of claim 100, wherein the at least one tumor-specific protease is selected from the group consisting of matrix metalloproteinase-9 (MMP-9), urokinase-type plasminogen activator (uPa), matrix metalloproteinase-2 (MMP-2), matriptase, regumain, kallikrein-related peptidase-3, human neutrophil elastase, proteinase 3 (Pr3), cathepsin B, and cathepsin K.

102. 102. The conditionally active ISV of claim 100 or 101, wherein the at least one tumor-specific protease is MMP-9 or uPa, or a combination thereof.

103. 103. The conditionally active ISV of any one of claims 98 to 102, wherein the at least one linker comprises the amino acid sequence of SEQ ID NO: 56 and / or 57.

104. 104. The conditionally active ISV of any one of claims 98 to 103, wherein the at least one linker comprises or consists of the amino acid sequence of SEQ ID NO: 46 or 47.

105. V ＨＨ 105. The conditionally active ISV of any one of claims 95 to 104, wherein

106. (i) the three light chain complementarity determining region (CDR) sequences found in SEQ ID NO: 7 or 5, and (ii) the three heavy chain CDR sequences found in SEQ ID NO: 8 or 6 An antibody or antigen-binding fragment thereof comprising:

107. The antibody or antigen-binding fragment thereof of claim 106, which specifically binds to PD-1.

108. (i) the following three CDR sequences: a.V Ｌ CDR1: QSVPINF (SEQ ID NO: 18) or QSVSINF (SEQ ID NO: 19); b.V Ｌ CDR2: EAS; and c.V Ｌ CDR3: GQYGSSPYT (SEQ ID NO: 20) or QQYGSSPYT (SEQ ID NO: 21) a light chain variable region comprising: (ii) the following three CDR sequences: a.V Ｈ CDR1: GGSISSSSYF (SEQ ID NO: 22) or GGSISTSSYF (SEQ ID NO: 23); b.V Ｈ CDR2: IYRSGST (SEQ ID NO: 24); and c.V Ｈ - CDR3: ARGITGDPGDY (SEQ ID NO: 25) a heavy chain variable region comprising 108. The antibody or antigen-binding fragment thereof of claim 106 or 107, comprising:

109. (i) the following three CDR sequences: a.V Ｌ - CDR1: QSVPINF (SEQ ID NO: 18); b.V Ｌ CDR2: EAS; and c.V Ｌ - CDR3: GQYGSSPYT (SEQ ID NO: 20) a light chain variable region comprising: (i) the following three CDR sequences: a.V Ｈ - CDR1: GGSISSSSYF (SEQ ID NO: 22); b.V Ｈ CDR2: IYRSGST (SEQ ID NO: 24); and c.V Ｈ - CDR3: ARGITGDPGDY (SEQ ID NO: 25) a heavy chain variable region comprising The antibody or antigen-binding fragment thereof according to any one of claims 106 to 108, comprising:

110. (i) a light chain variable region having SEQ ID NO: 7 or 5, or a light chain variable region sharing at least 70% sequence identity over the non-CDR regions of SEQ ID NO: 7 or 5; and (ii) a heavy chain variable region having SEQ ID NO: 8 or 6, or a heavy chain variable region sharing at least 70% sequence identity across the non-CDR regions of SEQ ID NO: 8 or 6 The antibody or antigen-binding fragment thereof according to any one of claims 106 to 109, comprising:

111. 111. The antibody or antigen-binding fragment thereof according to any one of claims 106 to 110, comprising a light chain variable region having SEQ ID NO: 7 and a heavy chain variable region having SEQ ID NO:

8.

112. 111. The antibody or antigen-binding fragment thereof according to any one of claims 106 to 110, comprising a light chain variable region having SEQ ID NO:5 and a heavy chain variable region having SEQ ID NO:

6.

113. 42. A composition comprising the multispecific antigen-binding protein of any one of claims 1 to 41 and a pharmaceutically acceptable carrier or excipient.

114. 54. A composition comprising the conditionally active multispecific antigen-binding protein of any one of claims 42 to 53 and a pharmaceutically acceptable carrier or excipient.

115. 67. A composition comprising an immunoglobulin single variable domain according to any one of claims 54 to 66 and a pharmaceutically acceptable carrier or excipient.

116. 70. A composition comprising an immunoglobulin single variable domain according to any one of claims 67 to 69 and a pharmaceutically acceptable carrier or excipient.

117. 73. A composition comprising the bivalent or bispecific antigen-binding protein of any one of claims 70 to 72 and a pharmaceutically acceptable carrier or excipient.

118. 84. A composition comprising the conditionally active immunoglobulin single variable domain of any one of claims 73 to 83 and a pharmaceutically acceptable carrier or excipient.

119. 95. A composition comprising the conditionally active immunoglobulin single variable domain of any one of claims 84 to 94 and a pharmaceutically acceptable carrier or excipient.

120. 106. A composition comprising the conditionally active immunoglobulin single variable domain of any one of claims 95 to 105 and a pharmaceutically acceptable carrier or excipient.

121. A composition comprising the antibody or antigen-binding fragment thereof of any one of claims 106 to 112 and a pharmaceutically acceptable carrier or excipient.

122. 122. A method of treating a subject in need thereof, comprising administering to said subject an effective amount of a composition according to any one of claims 113 to 121.

123. 114. A method of treating a subject in need thereof, comprising administering to the subject an effective amount of the composition of claim 113.

124. 115. A method of treating a subject in need thereof, comprising administering to the subject an effective amount of the composition of claim 114.

125. The method of any one of claims 122 to 124, wherein the subject has cancer.

126. 122. The composition of any one of claims 113 to 121 for use in treating cancer in a subject in need thereof.

127. 114. The composition of claim 113 for use in treating cancer in a subject in need thereof.

128. 115. The composition of claim 114 for use in treating cancer in a subject in need thereof.

129. 42. An isolated polynucleotide encoding the multispecific antigen-binding protein of any one of claims 1 to 41.

130. 54. An isolated polynucleotide encoding the conditionally active multispecific antigen-binding protein of any one of claims 42 to 53.

131. 67. An isolated polynucleotide encoding an immunoglobulin single variable domain according to any one of claims 54 to 66.

132. 70. An isolated polynucleotide encoding an immunoglobulin single variable domain according to any one of claims 67 to 69.

133. 73. An isolated polynucleotide encoding the bivalent or bispecific antigen-binding protein of any one of claims 70 to 72.

134. 84. An isolated polynucleotide encoding the conditionally active immunoglobulin single variable domain of any one of claims 73 to 83.

135. 95. An isolated polynucleotide encoding the conditionally active immunoglobulin single variable domain of any one of claims 84 to 94.

136. 106. An isolated polynucleotide encoding the conditionally active immunoglobulin single variable domain of any one of claims 95 to 105.

137. 113. An isolated polynucleotide comprising the antibody or antigen-binding fragment thereof of any one of claims 106 to 112.

138. A vector comprising the polynucleotide of any one of claims 129 to 137.

139. A host cell comprising the polynucleotide of any one of claims 129 to 137.

140. 130. A method of making a multispecific antigen-binding protein of any one of claims 1 to 41, said method comprising expressing the polynucleotide of claim 129 in a cell.

141. 130. A method of making the conditionally active multispecific antigen binding protein of any one of claims 42 to 53, said method comprising expressing the polynucleotide of claim 130 in a cell.

142. 131. A method for producing an immunoglobulin single variable domain according to any one of claims 54 to 66, comprising expressing the polynucleotide according to claim 131 in a cell.

143. 132. A method for producing an immunoglobulin single variable domain according to any one of claims 67 to 69, comprising expressing the polynucleotide according to claim 132 in a cell.

144. 134. A method of making a bivalent or bispecific antigen-binding protein according to any one of claims 70 to 72, said method comprising expressing the polynucleotide of claim 133 in a cell.

145. 134. A method of producing a conditionally active immunoglobulin single variable domain according to any one of claims 73 to 83, comprising expressing the polynucleotide of claim 134 in a cell.

146. 136. A method of making a conditionally active immunoglobulin single variable domain according to any one of claims 84 to 94, comprising expressing the polynucleotide of claim 135 in a cell.

147. 136. A method of producing a conditionally active immunoglobulin single variable domain according to any one of claims 95 to 105, comprising expressing the polynucleotide of claim 136 in a cell.

148. 137. A method for producing the antibody or antigen-binding fragment thereof of any one of claims 106 to 112, comprising expressing the polynucleotide of claim 137 in a cell.

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