Anti-PD-1x4-1BB binding proteins

Abstract

‑1BB and anti‑PD‑1 binding proteins, as well as bispecific anti‑4‑1BB / anti‑PD‑1 binding proteins, including conditionally‑active derivatives thereof, are provided. Therapeutic and diagnostic methods of using binding proteins are provided.

Description

[Technical Field]

[0001] The subject matter of this disclosure relates to the immunoglobulin single variable domain (ISV) that antagonizes the co-stimulatory receptor molecule 4-1BB. These proteins, by binding to 4-1BB, can activate cells expressing 4-1BB, such as T cells. It also relates to anti-PD-1 binding proteins that can prevent inhibition of T-cell activation. Furthermore, it relates to bispecific antigen-binding proteins that bind to both 4-1BB and PD-1, and their conditionally active derivatives. These proteins are applicable to the field of immuno-oncology. [Previous Technology]

[0002] The decisive factor in cancer treatment is the patient's own immune system. The immune system has the ability to invade and destroy tumor cells. However, several factors can prevent the immune system from responding effectively, such as suppressing the tumor microenvironment of immune cells. Specifically, tumors can prevent or interfere with the activation of T cells.

[0003] T-cell activation is a complex process dependent on several signals. This includes stimulation of T-cells via T-cell receptor (TCR) signaling and co-stimulatory signals from other receptors. Co-stimulatory action is crucial for the effective activation of T-cells. One important co-stimulatory molecule is tumor necrosis factor receptor superfamily member 9 (TNFRSF9, also known as 4-1BB or CD137, encoded by the TNFRSF9 gene), which belongs to the tumor necrosis factor (TNF) receptor superfamily. Its expression can be transitionally induced by TCR signaling. 4-1BB was first identified in mice using a modified differential screening process. Human homologs of 4-1BB were selected from a pool of human T-lymphocytes transformed with activated human type 1 human T-cell leukemia virus.

[0004] 4-1BB is a co-stimulatory molecule whose role is to amplify, acquire effector functions, survive, and develop T-cell memory. Besides being manifested in activated T cells, NK cells, NK T cells, and T reg cells, 4-1BB is also present in several cell types in hematopoietic cell lines, as well as endothelial and epithelial cells. 4-1BBL (TNFSF9), a secondary ligand of 4-1BB, is mainly present in professional antigen-presenting cell (APC) populations, B cells, macrophages, and other cell types. It binds to 4-1BB and induces signal transduction via TRAF1 and TRAF2 to activate the NF-κB, AKT, p38 MAPK, and ERK pathways. These signal transduction pathways induce the expression of survival genes encoding survivin, Bcl-2, Bcl-XL, and Bfl-1, and reduce the expression of Bim, a pro-apoptotic cell gene. Therefore, the 4-1BB / 4-1BBL signaling pathway can promote the survival of different cell morphologies. Notably, 4-1BB and its ligand exhibit different phenotypes in response to viral challenge. For example, 4-1BBL-deficient mice show reduced accumulation of memory CD8+ T- cells, while 4-1BB-deficient mice have reduced memory but enhanced accumulation of acute CD8+ T- cells. This result suggests bidirectional signaling or a unique receptor signaling mechanism. Other infectious disease models have shown that the 4-1BB blocking effect varies depending on the cause and disease characteristics. For example, infections that clear rapidly or produce minimal inflammation are often unaffected by 4-1BB deficiency, while chronic or highly inflammatory diseases require 4-1BB to develop memory and / or clear the virus.

[0005] In tumor immunotherapy, it is necessary to activate the 4-1BB signaling pathway to induce T-cell proliferation, enhance effector activity (including cytokine production), form memory, resist apoptosis, and methylation reprogramming. One well-established method for this stimulation is the use of agonist antibodies. Several different companies have developed several such antibodies, including urelumab and utomilumab. However, this approach faces some challenges because 4-1BB: activated 4-1BB and its ligands primarily exhibit a trimer structure on the cell surface. Therefore, it is difficult to effectively target 4-1BB when using conventional antibodies with only two binding sites; thus, it cannot provide the same potent agonist stimulation in signal activation as the 4-1BB trimer.

[0006] In principle, this missing stoichiometry can be compensated by cross-linking multiple antibody molecules, for example, by binding to Fc receptors (FcRs). However, such methods can cause serious side effects due to the multiplicative systemic toxicity of 4-1BB.

[0007] Therefore, there should be a molecule that can act as an effective agonist of 4-1BB, but does not require any cross-linking (e.g., 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, matrix antigen protein-mediated cross-linking, or any other target-mediated cross-linking mediated by cis or trans expression of tumor cells, immune cells, and / or normal cells).

[0008] This invention provides such molecules. The activation effect of these molecules is based on anti-4-1BB V HH having pure activator activity, which is independent of any cross-linking.

[0009] In another sample, it was observed that when the anti-4-1BB binding motif was coupled to another motif, such as binding to the inhibitory receptor planned death-1 (PD-1), the effect of the anti-4-1BB binding motif was significantly enhanced. This effect was hypothesized to be explained by the transfer of the cis-anti-4-1BB binding motif to PD-1, suggesting that the anti-PD-1 binding motif may assist in anchoring or promoting the clustering of anti-4-1BB binding motifs on PD-1+CD8+ T cells.

[0010] PD-1 (also known as CD279) is a 288-amino acid protein receptor expressed on activated T cells, B cells, natural killer cells, and monocytes. PD-1 is a member of the CD28 / CTLA-4 (cytotoxic T lymphocyte antigen) / ICOS (inducible co-stimulator) family of T cell co-inhibitory receptors. Its main function is to attenuate the immune response. PD-1 has two ligands: PD-ligand 1 (PD-L1) and PD-L2. PD-L1 (also known as CD274 or B7H1) is widely expressed in both lymphoid and non-lymphoid tissues, such as CD4+ and CD8+ T cells, macrophages, peripheral tissues, tumor cells, and virus-infected cells. PD-L2 (also known as CD273 or B7-DC) is more restricted in its expression than PD-L1, manifesting in 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 induced in almost all cancer morphologies. PD-1 binding to its ligand results in reduced T-cell proliferation and cytokine secretion, disrupting humoral and cellular immune responses in diseases such as cancer or viral infections. Blocking PD-1 binding to reverse immunosuppression has been investigated in viral and tumor immunotherapy.

[0011] Co-stimulatory and co-inhibitory molecules (collectively referred to as co-signaling molecules) play a crucial role in regulating T-cell activation, subset differentiation, effector function, and survival. After the TCR recognizes the homopeptide-MHC complex on the APC, the co-signaling receptor co-localizes with the TCR at the immune synapse, where it synergistically promotes or inhibits T-cell activation and function. The balance between co-stimulatory and co-inhibitory signals regulates the final immune response ("immune checkpoint"). PD-1 functions as such an "immune checkpoint" to mediate peripheral T-cell tolerance and avoid autoimmunity: PD-1 binds to PD-L1 or PD-L2 to inhibit T-cell activation. However, 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, which become "exhausted" and lose effector function and proliferative capacity. PD-L1 is present in a variety of tumors, and animal studies have shown that PD-L1 on tumors inhibits T-cell activation and lysis of tumor cells, potentially increasing tumor-specific T-cell death. The PD-1 / PD-L1 complex also plays an important role in inducing T-reg cell development and maintaining T-reg function.

[0012] Because PD-1 plays an important role in autoimmunity, tumor immunity, and infection immunity, it has become an ideal target for immunotherapy. Antagonists, including monoclonal antibodies, have been studied in the treatment of cancer and chronic viral infections to block PD-1.

[0013] The present invention provides such PD-1 antagonists. [Summary of the Invention]

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

[0015] In some embodiments, pure agonist activity means that the at least one ISV can (i) activate T cells via 4-1BB signaling in a soluble state, and / or (ii) in the absence of a crosslinking agent, and / or (iii) in an FcγR-independent manner (i.e., without the involvement of the Fcγ receptor), and / or (iv) in the absence of target-mediated 4-1BB crosslinking. In some embodiments, pure agonist activity can be determined by NF-κB pathway activation assay in the absence of a crosslinking agent, for example, in the absence of anti-human Fab antibody.

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

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

[0018] In some embodiments, 4-1BB is human 4-1BB, and an example of its amino acid sequence is SEQ ID NO: 13.

[0019] In some embodiments, at least one ISV interacts with at least one amino acid residue of 4-1BB, the residue being 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 V HH.

[0021] In some embodiments, at least one ISV includes three complementary determination regions CDR1, CDR2 and CDR3; and CDR3 includes or is composed 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), INPSGGSQ (SEQ ID NO: 77), or IKKSGNRT (SEQ ID NO: 78).

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

[0025] In some embodiments, the ISV comprises or is composed of the amino acid sequence SEQ ID NO: 2 or 3. In some embodiments, the ISV comprises or is composed of the amino acid sequence 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, the specific binding to at least two ISVs of 4-1BB is the same. Alternatively, the specific binding to at least two ISVs of 4-1BB may be different, and therefore may bind to (i) the same antigenic epitope, (ii) overlapping antigenic epitope, or (iii) a unique antigenic epitope of 4-1BB. In some embodiments, the specific binding to at least two ISVs of 4-1BB is different, and binds to a unique antigenic epitope of 4-1BB.

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

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

[0030] In some embodiments, the multispecific antigen-binding protein comprises at least two ISVs, wherein one of the at least two ISVs specifically binds to 4-1BB, and the other of the at least two ISVs specifically binds to another target antigen. 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-autoantigen.

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

[0032] In some embodiments, at least two ISVs in the first group of (i) or (i') above are ISVs that specifically bind to 4-1BB and have pure agonist activity as defined above (e.g., but not limited to: ISVs that contain or constitute an amino acid sequence SEQ ID NO: 2 or 3).

[0033] In some embodiments, at least two ISVs in the second group as described in (ii) or (ii') above are ISVs that specifically bind to 4-1BB as defined above (e.g., but not limited to: ISVs that contain or constitute an amino acid sequence SEQ ID NO: 1 or 4).

[0034] In some embodiments, the multispecific antigen-binding protein further comprises an antibody Fc region or a fragment thereof. In some embodiments, the Fc region or a 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) a first polypeptide, preferably comprising from the N-terminus to the C-terminus: - a first ISV specifically binding to 4-1BB; - a second ISV specifically binding to 4-1BB, preferably wherein the second ISV is different from the first ISV; - at least one CH domain of the Fc region; and - variable and constant domains of the Fab fragment; (b) a second polypeptide comprising the variable and constant domains of the Fab fragment; wherein the variable and constant domains of the first and second polypeptides form the Fab fragment.

[0037] In some embodiments, the multispecific antigen-binding protein further comprises a third and a fourth polypeptide that are identical to the first and second polypeptides, respectively, wherein at least one CH domain of the first and third polypeptides forms an Fc region.

[0038] In some embodiments, - the variable and constant structural domains of the first polypeptide are VH and CH1 domains, and the variable and constant structural domains of the second polypeptide are VL and CL domains; or - the variable and constant structural domains of the first polypeptide are VL and CL domains, and the variable and constant structural domains of the second polypeptide are VH and CH1 domains.

[0039] In some embodiments, at least one CH domain of the first polypeptide includes: - CH2 and CH3 domains of IgG; - CH2 and CH3 domains of IgD; - CH2 and CH3 domains of IgA; - CH2, CH3 and CH4 domains of IgM; or - CH2, CH3 and CH4 domains of IgE.

[0040] In some embodiments, at least one CH domain of the first polypeptide comprises the CH2 and CH3 domains of IgG. In some embodiments, at least one CH domain of the first polypeptide comprises the CH2 and CH3 domains of IgG1 or IgG4. In some embodiments, at least one CH domain of the first polypeptide comprises the CH2 and CH3 domains of IgG1.

[0041] In some embodiments, the first polypeptide of the multispecific antigen-binding protein preferably includes, from the N-terminus to the C-terminus: - a first ISV that specifically binds to 4-1BB; - a first linker; - a second ISV that specifically binds to 4-1BB, preferably wherein the second ISV is different from the first ISV; - a second linker; - an IgG hinge region; - an IgG CH2 domain; - and an IgG CH3 domain; - a third linker; - a VH domain of the Fab fragment; and - a CH1 domain of the Fab fragment.

[0042] In some embodiments, the second polypeptide of the multispecific antigen-binding protein preferably includes, from the N-terminus to the C-terminus: - a VL domain of the Fab fragment; and - a CL domain of the Fab fragment.

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

[0044] In some embodiments, at least one Fab fragment is an antigen-binding protein that specifically binds to PD-1, and comprises: (i) a light chain variable region comprising three of the following 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); and (ii) a heavy chain variable region comprising three of the following 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).

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

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

[0047] In some embodiments, at least one Fab fragment is an antigen-binding protein that specifically binds to PD-1 and includes 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 protein provided herein comprises - at least one first polypeptide having SEQ ID NO: 11 or 9, and at least one second polypeptide having SEQ ID NO: 12 or 10; or - at least one first polypeptide having at least 70% sequence identity with the non-CDR region of SEQ ID NO: 11 or 9, and at least one second polypeptide having at least 70% sequence identity with the non-CDR region of SEQ ID NO: 12 or 10.

[0049] In some embodiments, the multispecific antigen-binding protein provided herein comprises at least one first polypeptide having SEQ ID NO: 11 and at least one second polypeptide having SEQ ID NO: 12.

[0050] In some embodiments, the multispecific antigen-binding protein provided herein comprises at least one first polypeptide having SEQ ID NO: 9 and at least one second polypeptide having SEQ ID NO: 10.

[0051] In the second state sample, a conditionally-active multispecific antigen-binding protein is provided.

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

[0053] In some embodiments, at least one masking portion comprises or is composed of an amino acid sequence of SEQ ID NO: 97, or an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 97. In some embodiments, at least one masking portion comprises or is composed of an amino acid sequence of SEQ ID NO: 44 or 45, or an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 44 or 45.

[0054] In some embodiments, the conditionally active multispecific antigen-binding protein further includes at least one linker between the multispecific antigen-binding protein and the masking part. 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, legumain, kallikrein-related peptidase-3, human neutrophil elastase, protease 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 includes the amino acid sequence of SEQ ID NO: 56 and / or 57. In some embodiments, at least one linker comprises or constitutes an 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 one first polypeptide having SEQ ID NO: 11 or 9; and b. at least one second polypeptide having SEQ ID NO: 52, 53, 54 or 55.

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

[0057] In the third state, a single variable domain (ISV) of immunoglobulin specifically binds to 4-1BB, wherein the ISV has pure agonist activity.

[0058] In some embodiments, pure agonist activity means that the ISV can (i) activate T cells via 4-1BB signaling in soluble conditions, and / or (ii) in the absence of crosslinking agents, and / or (iii) in an FcγR-independent manner (i.e., without the involvement of Fcγ receptors), and / or (iv) in the absence of target-mediated 4-1BB crosslinking. In some embodiments, pure agonist activity can be determined by NF-κB pathway activation assay in the absence of crosslinking agents.

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

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

[0061] In some embodiments, 4-1BB is human 4-1BB, and an example of its amino acid sequence is SEQ ID NO: 13.

[0062] In some embodiments, the ISV interacts with one or more amino acid residues of 4-1BB, which are 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, ISV is V HH.

[0064] In some embodiments, the ISV includes three complementary determination regions CDR1, CDR2 and CDR3; and CDR3 includes or is composed 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 is composed 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), INPSGGSQ (SEQ ID NO: 77), or IKKSGNRT (SEQ ID NO: 78).

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

[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, ISV is V HH.

[0070] In the fourth state sample, an immunoglobulin single variable domain (ISV) specifically binding to 4-1BB is provided, wherein the ISV comprises or consists of: - an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and 4, or - an amino acid sequence having at least 70% sequence identity with the non-CDR region of SEQ ID NO: 1 or 4.

[0071] In some embodiments, the ISV contains or constitutes the amino acid sequence of SEQ ID NO: 4.

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

[0073] The fifth state provides 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 one first ISV that specifically binds to 4-1BB and has pure agonist activity as defined above (e.g., but not limited to: an ISV comprising or constituting an amino acid sequence SEQ ID NO: 2 or 3), and at least one 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-autogenous antigen.

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

[0076] In the sixth state sample, a conditionally active immunoglobulin single variable domain (ISV) specifically binding to 4-1BB is provided, wherein the conditionally active ISV comprises: - an ISV specifically binding to 4-1BB as defined above (e.g., but not limited to: an ISV comprising or constituting an amino acid sequence SEQ ID NO: 1 or 4), and - at least one masking motif that reduces or inhibits the binding of the ISV to its target antigen.

[0077] In some embodiments, at least one masking portion comprises or is composed of an amino acid sequence of SEQ ID NO: 97, or an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 97. In some embodiments, at least one masking portion comprises or is composed of an amino acid sequence of SEQ ID NO: 44 or 45, or an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 44 or 45.

[0078] In some embodiments, the conditionally active ISV further includes at least one linker between the ISV and the masking portion. In some embodiments, the at least one linker is cleavable. In some embodiments, the at least one linker can be cleaved 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), interstitial protease, legume protease, kallikrein-related peptidase-3, human neutrophil elastase, protease 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, the at least one linker comprises or constitutes the amino acid sequence of SEQ ID NO: 46 or 47.

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

[0080] In some embodiments, the conditionally active ISV comprises or consists of an amino acid sequence selected from the group consisting of: SEQ ID NO: 48, 49, 50 and 51.

[0081] In the seventh state, a conditionally active immunoglobulin single variable domain (ISV) specifically binding to 4-1BB is provided, wherein the conditionally active ISV comprises: - an ISV specifically binding to 4-1BB; and - at least one masking part that reduces or inhibits ISV binding to 4-1BB.

[0082] In some embodiments, at least one masking portion comprises or is composed of an amino acid sequence of SEQ ID NO: 97, or an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 97. In some embodiments, at least one masking portion comprises or is composed of an amino acid sequence of SEQ ID NO: 44 or 45, or an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 44 or 45.

[0083] In some embodiments, the conditionally active ISV further includes at least one linker between the ISV and the masking portion. In some embodiments, the at least one linker is cleavable. In some embodiments, the at least one linker can be cleaved 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), interstitial protease, bean protease, kallikrein-related peptidase-3, human neutrophil elastase, protease 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, the at least one linker comprises or constitutes the amino acid sequence of SEQ ID NO: 46 or 47.

[0084] In some embodiments, ISV is V HH.

[0085] In the eighth state, a conditionally active immunoglobulin single variable domain (ISV) specifically binding to a target antigen is provided, wherein the conditionally active ISV comprises: - an ISV specifically binding to a target antigen; and - at least one masking part that reduces or inhibits the binding of the ISV to its target antigen.

[0086] In some embodiments, at least one masking portion comprises or is composed of an amino acid sequence of SEQ ID NO: 97, or an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 97. In some embodiments, at least one masking portion comprises or is composed of an amino acid sequence of SEQ ID NO: 44 or 45, or an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 44 or 45.

[0087] In some embodiments, the conditionally active ISV further includes at least one linker between the ISV and the masking portion. In some embodiments, the at least one linker is cleavable. In some embodiments, the at least one linker can be cleaved 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), interstitial protease, legume protease, kallikrein-related peptidase-3, human neutrophil elastase, protease 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, the at least one linker comprises or constitutes the amino acid sequence of SEQ ID NO: 46 or 47.

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

[0089] In the ninth state sample, an antibody or an antigen-binding fragment thereof is provided, wherein the antibody or the antigen-binding fragment thereof comprises: (i) three light chain complementarity-determining region (CDR) sequences, as shown in SEQ ID NO: 5 or 7, and (ii) three heavy chain CDR sequences, as shown in SEQ ID NO: 6 or 8.

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

[0091] In some embodiments, the antibody or its antigen-binding fragment comprises: (i) a light chain variable region comprising three of the following 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); and (ii) a heavy chain variable region comprising three of the following 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).

[0092] In some embodiments, the antibody or its antigen-binding fragment comprises: (i) a light chain variable region comprising three of the following 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); and (ii) a heavy chain variable region comprising three of the following 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).

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

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

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

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

[0097] In some other embodiments, a method of providing treatment to an individual in need includes administering an effective amount of one of the components disclosed herein. In some embodiments, the individual has cancer. In some embodiments, the individual is a human.

[0098] In some other embodiments, one of the components disclosed herein is provided for treating cancer in an individual in need. In some embodiments, the individual is a human.

[0099] This document also provides isolated polynucleotides encoding any of the multispecific antigen-binding proteins disclosed herein; or conditionally active multispecific antigen-binding proteins disclosed herein; or immunoglobulin single variable domain (ISV) disclosed herein; or bivalent or bispecific antigen-binding proteins disclosed herein; or conditionally active immunoglobulin single variable domain (ISV) disclosed herein; or antibodies or antigen-binding fragments thereof disclosed herein. This document also provides carriers containing such polynucleotides; and host cells containing such polynucleotides.

[0100] This document also provides methods for manufacturing any multispecific antigen-binding protein disclosed herein; or conditionally active multispecific antigen-binding protein disclosed herein; or immunoglobulin single variable domain (ISV) disclosed herein; or bivalent or bispecific antigen-binding protein disclosed herein; or conditionally active immunoglobulin single variable domain (ISV) disclosed herein; or antibody or antigen-binding fragment thereof disclosed herein; the method includes expressing a polynucleotide as described above in cells.

[0101] The invention described above is not limited, and other features and advantages of the antigen-binding protein and method disclosed will be understood from the following illustrations, embodiments, and claims.

Implementation Method

[0137] Before describing this disclosure, it is understood that this disclosure is not limited to the specific methods and experimental conditions described herein, as such methods and conditions may vary. It is also understood that the terminology used herein is for illustrative purposes only and is not intended to be limiting, as the scope of the invention is limited only by the claims in the appendix. Definitions

[0138] Unless otherwise stated, all technical and scientific terms used herein have the same definitions as those skilled in the art to which this invention pertains.

[0139] The term “about” as used herein, when referring to a particularly extracted value, means that the value may vary from the extracted value by no more than 5%, preferably no more than 2%, and even more preferably no more than 1%. For example, the expression “about 100” as used herein includes 99 and 101 and all values ​​in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0140] Although similar or equivalent methods and materials may be used to work with the disclosure described herein, the methods and materials described herein will be described hereafter. The full contents of the descriptions and published documents herein are incorporated herein by reference.

[0141] The term “PD-1” refers to the planned death-1 protein, a T-cell co-inhibitor, also known as CD279. Unless otherwise specified from a non-human species, 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 intracellular domains containing immunoreceptor tyrosine inhibitory (ITIM) motifs and immunoreceptor tyrosine switching (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 example of the amino acid sequence of PD-1 is shown in SEQ ID NO: 42, which corresponds to human PD-1 (hPD-1), GenBank accession number NP_005009.2. SEQ ID NO: 42MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL

[0142] The term “PD-L1” refers to the ligand of the PD-1 receptor, also known as CD274 and B7H1. PD-L1 is a protein of 290 amino acids, possessing an extracellular IgV-like domain, a transmembrane domain, and a highly conserved intracellular domain of about 30 amino acids. PD-L1 is persistently expressed on many cells, such as antigen-presenting cells (e.g., dendritic cells, macrophages, and B cells) and on hematopoietic and non-hematopoietic cells (e.g., vascular endothelial cells, pancreatic islets, and immune-exempt sites). PD-L1 is also expressed on many types of tumor cells and cells infected with viruses, and is a component of the immunosuppressive environment (Ribas, N Engl J Med. 2012, 28 June; 366(26):2517-9). An example of the amino acid sequence of PD-L1 is shown in SEQ ID NO: 43, which corresponds to human PD-L1 (hPD-L1), GenBank accession number NP_054862.1. SEQ ID NO: 43MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET

[0143] The terms "4-1BB" or "CD137" as used herein refer to a surface glycoprotein belonging to the tumor necrosis factor receptor family (TNFRSF9). Activation on many leukocyte morphologies can induce its expression. 4-1BB is expressed on pre-sensitized T- and natural killer (NK) cells, providing a strong co-stimulatory signal upon binding. Perturbing 4-1BB with 4-1BBL or agonist monoclonal antibodies on activated CD8+ T- cells can protect these antigen-specific cytotoxic T- lymphocytes from apoptosis, enhance effector function, and promote sustained and memory differentiation. 4-1BB is also expressed on activated T- cells that have been exposed to homologous antigens, activated NK cells, or mature dendritic cells (DCs). The 4-1BB-specific functional ligand 4-1BBL is expressed on the surface of specialized antigen-presenting cells, such as DCs, macrophages, and B cells. 4-1BB trimerization leads to 4-1BBL receptor clustering and TRAF-mediated NF-κB activation, as well as the MAPK intracellular signaling cascade, ultimately achieving cell activation, proliferation, and survival. On T cells, T-cell receptor (TCR) stimulation and subsequent CD3 signaling induce a transition to 4-1BB, which, when bound to agonist antibodies or natural ligands, favors the Th1 response. Besides inducing the production of effector cytokines, 4-1BB co-stimulation also promotes T-cell memory and effector differentiation, protects T-cells from apoptosis, alters mitochondrial metabolism to enhance T-cell respiration, and induces overall DNA demethylation and chromatin reprogramming. An example of the amino acid sequence of 4-1BB is shown in SEQ ID NO: 13, which corresponds to human 4-1BB (h4-1BB), UniProt accession number Q07011. 4-1BB contains four cysteine-enriched 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).SEQ ID NO: 13MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFND QKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0144] The term “T-cell co-inhibitor” as used herein refers to receptors expressed on T-cells and / or ligands targeting such receptors that regulate immune responses by inhibiting T-cell receptor (TCR) signaling. The term "T-cell co-inhibitor" is also known as "T-cell co-signaling molecules," including (but not limited to): PD-1; lymphocyte activation gene 3 protein (LAG-3, also known as CD223); cytotoxic T-lymphocyte antigen 4 (CTLA-4); B and T lymphocyte attenuators (BTLA); 2B4; T-cell immunoglobulin and mucin-3 (TIM-3); T-cell immune receptor with immunoglobulin and ITIM (TIGIT; also known as VSIG9); leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1; also known as CD305); V-domain Ig repressor for T-cell activation (VISTA); PD-L1; PD-L2; CEACAM; B7-H3; B7-H4; KIR; A2aR; GAL9; and TGFR. For an overview of the co-stimulatory and co-inhibitory effects of T-cells, see Chen & Flies (Nat Rev Immunol. 2013 Apr;13(4):227-42).

[0145] As used herein, the term “T-cell costimulator” refers to receptors expressed on T-cells and / or ligands targeting such receptors that regulate immune responses through activation via T-cell receptor (TCR) signaling. The term “T-cell costimulator” includes (but is not limited to): CD28; inducible T-cell costimulators (ICOS); OX40; CD27; 4-1BB (also known as CD137); death receptor 3 (DR3); B7; CD226; CRTAM; glucocorticoid-induced TNFR-associated protein (GITR); CD30; CD2; herpesvirus entry vector (HVEM); BAFFR; BAFF; and light. For an overview of T-cell costimulatory and co-inhibitory effects, see Chen & Flies (Nat Rev Immunol. 2013 Apr;13(4):227-42).

[0146] As used herein, the term "antigen-binding protein" refers to a protein that can specifically bind 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., rodent) or human origin. If such antigen-binding proteins are of non-human (e.g., rodent) origin, they may be "humanized" to reduce immunogenicity or increase stability.

[0147] The term "antibody" as used herein refers to an immunoglobulin (Ig) molecule (i.e., a "complete antibody molecule") consisting of four polypeptide chains: two heavy (H) chains and two light (L) chains, linked by disulfide bonds, as well as its polymers (e.g., IgM) or its antigen-binding fragments. Each heavy chain consists of a heavy chain variable region ("HCVR" or "VH") and a heavy chain constant region ("HCCR" or "CH"; composed of domains CH1, CH2, and CH3). Each light chain consists of a light chain variable region ("LCVR" or "VL") and a light chain constant region ("LCCR" or "CL"). The VH and VL regions can be further subdivided into hypervariable regions, called complementarity-determining regions (CDRs), within which are scattered more conserved regions called framework regions (FRs). Each VH and VL lineage consists of three CDRs and four FRs, arranged sequentially from the amino terminus to the carboxyl terminus as follows: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments of the present invention, the FRs of the antibody (or their antigen-binding fragments) may be identical to the human germline sequence or may be naturally or artificially modified. The common amino acid sequence can be defined based on side-by-side analysis of two or more CDRs.

[0148] The term "single immunoglobulin variable domain" (ISV) is used interchangeably with "single variable domain." It defines an immunoglobulin molecule in which the antigen-binding site is located on and formed by a single immunoglobulin domain. Except for "conventional" immunoglobulins (e.g., monoclonal antibodies) or their fragments (e.g., Fab, Fab', F(ab')2, scFv, di-scFv), the two immunoglobulin domains in this group of single immunoglobulin variable domains, specifically the two variable domains, interact to form the antigen-binding site. Typically, in conventional immunoglobulins, the heavy chain variable domain (VH) and the light chain variable domain (VL) interact to form the antigen-binding site. In this case, the complementarity-determining regions (CDRs) of both VH and VL will generate the antigen-binding site, meaning a total of six CDRs are involved in forming the antigen-binding site. Based on the above definition, the antigen-binding domains of conventional 4-chain antibodies (such as IgG, IgM, IgA, IgD, or IgE molecules known in the art) or derived from such conventional 4-chain antibodies, such as Fab fragments, F(ab')2 fragments, Fv fragments (e.g., disulfide-linked Fv or scFv fragments), or bivalent antibodies (all known in the art), are generally not considered as single variable domains of immunoglobulins. This is because in these cases, binding to each epitope of the antigen usually does not occur in a single immunoglobulin domain, but requires a pair of (linked) immunoglobulin domains, such as light chain and heavy chain variable domains, i.e., paired V, H, V, L immunoglobulin domains, which together bind to the epitopes of each antigen.

[0149] Conversely, a single variable domain of an immunoglobulin can specifically bind to an antigenic epitope without pairing with an additional variable domain. The binding site of a single variable domain of an immunoglobulin is formed by a single VH, a single VHH, or a single VL domain. Therefore, the antigen binding site of a single variable domain of an immunoglobulin is formed by no more than three CDRs. Accordingly, a single variable domain can be a light chain variable domain sequence (e.g., a VL-sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH-sequence or a VHH-sequence) or a suitable fragment thereof; as long as it can form a single antigen-binding unit (i.e., a functional antigen-binding unit composed essentially of a single variable domain, such that the single antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit).

[0150] An immunoglobulin single variable domain (ISV) may be, for example, a heavy chain ISV, such as VH, VHH, including camel-derived VH or humanized VHH. In one embodiment, it is VHH, including camel-derived VH or humanized VHH. The heavy chain ISV may be derived from a conventional four-chain antibody or derived from a heavy chain antibody. For example, an immunoglobulin single variable domain may be a (single) domain antibody (or an amino acid sequence suitable for a single domain antibody), a "dAb" or dAb (or an amino acid sequence suitable for a dAb) or Nanobody® ISV (as defined herein, including (but not limited to): VHH); other single variable domains, or any suitable fragment of any of them. Specifically, an immunoglobulin single variable domain may be a Nanobody® ISV (such as: VHH, including humanized VHH or camel-derived VH) or a suitable fragment of it.

[0151] The “VHH domain” is also known as “VHH”, “VHH antibody fragment”, and “VHH antibody”. It was originally called the antigen-binding immunoglobulin variable domain of “heavy chain antibody” (i.e., “antibody without light chain”; Hamers-Casterman et al., Nature. 1993 Jun 3;363(6428):446-8). The term “VHH domain” has been chosen to distinguish this variable domain from the heavy chain variable domain (referred to as “VH domain” in this paper) found in conventional 4-chain antibodies and the light chain variable domain (referred to as “VL domain” in this paper) found in conventional 4-chain antibodies. Further information on VHH can be found in Muyldermans’s paper (J Biotechnol. 2001 Jun;74(4):277-302). The terms "dAb" and "domain antibody" can be found, 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, 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, although less applicable due to their non-mammal origin, a single variable domain can still be derived from certain shark species (e.g., the so-called "IgNAR domain," see, for example, WO 2005 / 18629).

[0152] Typically, immunoglobulin production involves immunizing laboratory animals, fusing immunoglobulin-producing cells to generate heterozygotes, and screening for the desired specificity. Alternatively, immunoglobulins can be generated by screening primitive, immunoglobulin-producing, or synthetic collections, for example, using phage display technology. Immunoglobulin sequences, such as V HHs, have been detailed in various publications, particularly 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 that target antigen. From all V HHs obtained from this immunization, further screening is performed for V HHs that bind to the target antigen. In these examples, antibody production requires purified antigens for immunization and / or screening. The antigens can be purified from natural sources or during the manufacture of recombinant antibodies. Immunization and / or screening of immunoglobulin sequences can be performed using peptide fragments of these antigens.

[0153] Immunoglobulin sequences from various sources may be used in this study, including those from mice, rats, rabbits, donkeys, humans, and camels. In addition, whole human, humanized, or chimeric sequences may be used in the methods described herein. For example, camel immunoglobulin sequences and humanized camel immunoglobulin sequences, or camel-derived domain antibodies may be used, such as camel-derived dAbs, which are described in 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). In addition, ISVs can fuse to form multivalent and / or multispecific constructs (for information on multivalent and multispecific peptides containing one or more V HH domains and their preparation methods, see Conrath et al. (J Biol Chem. 2001 Mar 9;276(10):7346-50) and, for example, WO 1996 / 34103 and WO 1999 / 23221).

[0154] The amino acid sequence contained in the "humanized VHH" corresponds to the amino acid sequence of the natural VHH domain, but has been "humanized," that is, one or more amino acid residues in the amino acid sequence of the natural VHH (and specifically in the structural sequence) are replaced by one or more amino acid residues at the corresponding positions in the VH domain of a conventional 4-chain antibody from humans (e.g., as indicated above). This can be done in a manner known per se, as is understood by those skilled in the art, for example, according to the description of prior art (e.g., WO 2008 / 020079). Again, it should be noted that such humanized VHH can be obtained in any manner known per se, and therefore there is no strict limitation on using peptides containing the natural VHH domain as starting materials to obtain peptides.

[0155] The amino acid sequence contained in the "camel-derived VH" corresponds to the amino acid sequence of the natural VH domain, but has been "camel-derived", that is, one or more amino acid residues in the amino acid sequence of the natural VH domain of the traditional 4-chain antibody are replaced by one or more amino acid residues at the corresponding positions in the VHH domain of the (camel family) heavy chain antibody. This can be performed in a manner known to all those skilled in the art, such as by means of 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). These "camel-derived" substitutions are inserted at the amino acid sites that form and / or appear at the VH-VL interface and / or at so-called cameloid marker residues (as defined herein) (see, for example, WO 1994 / 04678 and Davies et al. (1994 and 1996, as cited above)). In some embodiments, the VH sequence used as the starting material or origin for generating or designing camel-derived VHs is a VH sequence derived from mammals, such as human VH sequences, such as the VH3 sequence. However, it should be noted that such camel-derived VHs can be obtained in any suitable manner known per se, and therefore there is no strict limitation on using peptides containing natural VH domains as starting materials.

[0156] The structure of an immunoglobulin single variable domain (ISV) sequence can be considered as consisting of four architecture regions ("FR"), which are referred to in related art and herein as "architecture region 1" ("FR1"), "architecture region 2" ("FR2"), "architecture region 3" ("FR3"), and "architecture region 4" ("FR4"); these architecture regions may be interspersed with three complementarity-determining regions ("CDR"), which are referred to in related art and herein as "complementarity-determining region 1" ("CDR1"), "complementarity-determining region 2" ("CDR2"), and "complementarity-determining region 3" ("CDR3"). In these immunoglobulin sequences, the architecture sequence can be any suitable architecture sequence, and examples of suitable architecture sequences are well known to those skilled in the art, such as those described in the standards manual and other disclosures and prior art. The architecture sequence is an immunoglobulin architecture sequence or a suitable combination of architecture sequences derived from immunoglobulin architecture sequences (e.g., through humanization or camelification). For example, the architecture sequence may be an architecture sequence derived from a light chain variable domain (e.g., a VL- sequence) and / or derived from a heavy chain variable domain (e.g., a VH- sequence or a VHH sequence). In a particular case, the architecture sequence is an architecture sequence derived from a VHH- sequence (wherein the architecture sequence may be partially or fully humanized as needed) or a conventional VH sequence that has been camelified (as defined herein). Specifically, the architecture sequence appearing in the ISV sequences described herein may contain one or more marker residues (as defined herein), and therefore the ISV sequence is a Nanobody® ISV, such as, for example, a VHH including humanized VHH or camelified VH. Non-limiting examples of such architecture sequences (suitable combinations) will be further understood from the contents herein.

[0157] The total number of amino acid residues in the VH domain and VHH domain is typically in the range of 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein. It should also be noted that the ISVs described herein do not limit the source of the ISV sequence (or the nucleotide sequence used to represent it), nor the manner in which the ISV sequence or nucleotide sequence has been (produced) or obtained. Therefore, ISV sequences can be natural sequences (from any suitable species) or synthetic or semi-synthetic sequences. In specific but not limited contexts, the ISV sequence is a natural sequence (from any suitable species) or a synthetic or semi-synthetic sequence, including (but not limited to): "humanized" (as defined herein) immunoglobulin sequences (e.g., partially or fully humanized mouse or rabbit immunoglobulin sequences, and specifically, partially or fully humanized VHH sequences), "camel-derived" (as defined herein) immunoglobulin sequences (and specifically, camel-derived VH sequences), and ISVs obtained using techniques such as: affinity maturation (e.g., starting with synthetic, random, or natural immunoglobulin sequences), CDR transplantation, mosaicking, combining fragments from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques known to those skilled in the art for the engineering of immunoglobulin sequences; or any suitable combination of any of the foregoing methods. Similarly, the nucleotide sequence can be a natural nucleotide sequence or a synthetic or semi-synthetic sequence, and can be, for example: a sequence isolated by PCR from a suitable natural template (e.g., DNA or RNA isolated from a cell), a nucleotide sequence isolated from a collection library (and specifically, from an expression collection library), a nucleotide sequence prepared by introducing a mutation into a natural nucleotide sequence (using any suitable technique known in itself, such as mismatch PCR), a nucleotide sequence prepared by PCR using overlapping primers, or a nucleotide sequence prepared using a technique known in itself for synthesizing DNA.

[0158] Typically, Nanobody® ISVs (specifically, V HH sequences, including (partially) humanized V HH sequences and camel-derived V H sequences) are characterized by the presence of one or more "marker residues" (again, as further explained herein) in one or more structural sequences (as described herein). Thus, Nanobody® ISVs can generally be defined as immunoglobulin sequences having the (general formula) structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to structural regions 1 to 4, and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3, and the one or more marker residues are further defined herein.

[0159] Specifically, Nanobody® ISV can be an immunoglobulin sequence having the (general formula) structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR1 to FR4 refer to architecture regions 1 to 4, and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3, and the architecture sequence is further defined as herein.

[0160] More specifically, Nanobody® ISV can be an immunoglobulin sequence having the (general formula) structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR1 to FR4 refer to framework regions 1 to 4, and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3, and one or more amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104, and 108 according to Kabat numbering are selected from the labeled residues described in Table 1 below. Table 1: Labeled Residues in Nanobody® ISV Location Human V H 3 Labeled residues 11 L, V; mainly L L, S, V, M, W, F, T, Q, E, A, R, G, K, Y, N, P, I; preferably L 37 V, I, F; usually V F (1) , Y, V, L, A, H, S, I, W, C, N, G, D, T, P, preferably F (1) Or Y 44 (8) G E (3) Q (3) G (2) , D, A, K, R, L, P, S, V, H, T, N, W, M, I; preferably G (2) E (3) Or Q (3) ; Best system G (2) Or Q (3) 。 45 (8) L L (2) R (3) ,P,H,F,G,Q,S,E,T,Y,C,I,D,V; preferably L (2) Or R(3) 47 (8) W, Y F (1) L (1) or W (2) G, I, S, A, V, M, R, Y, E, P, T, C, H, K, Q, N, D; preferably W (2) L (1) or F (1) 83 R or K; usually R R, K (5) , T, E (5) Q, N, S, I, V, G, M, L, A, D, Y, H; Preferred system: K or R; Best system: K 84 A, T, D; Mainly A P (5) ,S,H,L,A,V,I,T,F,D,R,Y,N,Q,G,E; preferably P 103 W W (4) R (6) ,G,S,K,A,M,Y,L,F,T,N,V,Q,P (6) E, C; Best-case W 104 G G, A, S, T, D, P, N, E, C, L; The preferred series is G. 108 L, M, or T; primarily L. Q, L (7) R, P, E, K, S, T, M, A, H; preferred series Q or L (7) Notes: (1) Specifically, but not excluded, it is combined with KERE or KQRE in positions 43-46. (2) Usually GLEW in positions 44-47. (3) Usually KERE or KQRE in positions 43-46, for example: KEREL, KEREF, KQREL, KQREF, KEREG, KQREW or KQREG in positions 43-47. Alternatively, it may be a sequence, such as: TERE (e.g., TEREL), TQRE (e.g., TQREL), KECE (e.g., KECEL or KECER), KQCE (e.g., KQCEL), RERE (e.g., REREG), RQRE (e.g., RQREL, RQREF or RQREW), QERE (e.g., QEREG), QQRE (e.g., QQREW, QQREL or QQREF), KGRE (e.g., KGREG), KDRE (e.g., KDREV). Other possible, but less desirable, sequences include, for example, DECKL and NVCEL. (4) GLEW at positions 44–47 and either KERE or KQRE at positions 43–46. (5) Often natural V HH The structural domain is located at KP or EP at positions 83-84. (6) Specifically, but not excluded, it is combined with GLEW at positions 44-47. (7) The condition is that it also contains W (non-humanized) V at position 103. HH In the sequence, when positions 44-47 are GLEW, then position 108 is always Q. (8) The GLEW group also contains GLEW-like sequences in positions 44-47, such as: for example: GVEW, EPEW, GLER, DQEW, DLEW, GIEW, ELEW, GPEW, EWLP, GPER, GLER and ELEW.

[0161] In some embodiments, the immunoglobulin single variable domain has certain amino acid substitutions in the architecture region that can prevent or reduce the binding of so-called "pre-existing antibodies" to the polypeptide. In the ISV, (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 one of K or Q; and (iv) in each of (i) to (iii), the amino acid at position 11 is preferably V, as described in WO 2015 / 173325.

[0162] An immunoglobulin single variable domain (ISV) may form a portion of a protein or polypeptide that may contain or substantially consist of one or more (at least one) immunoglobulin single variable domains, and may further contain one or more other amino acid sequences (all of which may be linked via one or more suitable linkers as needed). The term "immunoglobulin single variable domain" may also include such polypeptides. The one or more immunoglobulin single variable domains can be used as binding units in such proteins or polypeptides, and may contain one or more other amino acids that can be used as binding units as needed, in order to provide monovalent, multivalent or multispecific polypeptides of the present invention (for the preparation of multivalent and multispecific polypeptides containing one or more V HH domains and the like, 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).

[0163] A polypeptide may contain or substantially consist of a single variable immunoglobulin domain as described above. In this document, such polypeptides are also referred to as "monovalent" polypeptides. The term "multivalent" refers to the presence of multiple ISVs in a polypeptide. In one embodiment, the polypeptide is "divalent," meaning it contains or consists of two ISVs. In one embodiment, the polypeptide is "trivalent," meaning it contains or consists of three ISVs. In another embodiment, the polypeptide is "tetravalent," meaning it contains or consists of four ISVs. Polypeptides can therefore be "divalent," "trivalent," "tetravalent," "pentavalent," "hexavalent," "heptavalent," "octavalent," "nonavalent," etc., meaning the polypeptide contains or consists of two, three, four, five, six, seven, eight, nine, etc., ISVs respectively. In one embodiment, the multivalent ISV polypeptide is divalent. In one embodiment, the multivalent ISV polypeptide is trivalent. In another embodiment, the multivalent ISV polypeptide is tetravalent. In another embodiment, the multivalent ISV peptide is pentavalent.

[0164] Multivalent ISV peptides can also be monospecific or multispecific. The term "multispecific" refers to binding to multiple different target molecules (also known as antigens). Therefore, multivalent ISV peptides can be "bispecific," "trispecific," "quadrispecific," etc., meaning they can bind to two, three, four, etc., different target molecules. For example, a peptide can be bispecific-trivalent, such as a peptide containing or comprising three ISVs, where two ISVs bind to a first target and one ISV binds to a second target different from the first target. In another example, a peptide can be trispecific-quadrivalent, such as a peptide containing or comprising four ISVs, where one ISV binds to a first target, two ISVs bind to a second target different from the first target, and one ISV binds to a third target different from both the first and second targets. In another example, the polypeptide can be trispecific-pentavalent, such as a polypeptide containing or comprising five ISVs, wherein two ISVs bind to a first target, two ISVs bind to a second target different from the first target, and one ISV binds to a third target different from the first and second targets.

[0165] In one embodiment, the multivalent ISV peptide may further be a single complementary site or multiple complementary sites. The term "multiple complementary sites" refers to multiple different antigenic epitopes that bind to the same target molecule (also known as an antigen). The multivalent ISV peptide may therefore be a "double complementary site", a "triple complementary site", etc., that is, it may bind to two, three, etc., different antigenic epitopes on the same target molecule.

[0166] In another embodiment, the polypeptide of the present invention comprising or substantially consisting of one or more immunoglobulin single variable domains (or suitable fragments thereof) may further comprise one or more other groups, residues, portions, or binding units. These other groups, residues, portions, binding units, or amino acid sequences may provide or not provide additional functionality to the immunoglobulin single variable domain (and / or polypeptides containing such domains), and may or may not modify the properties of the immunoglobulin single variable domain. For example, these other groups, residues, portions, or binding units may be one or more additional amino acids, thus the compound, construct, or polypeptide is a (fusion) protein or (fusion) polypeptide. In a preferred but not limiting embodiment, the one or more other groups, residues, portions, or binding units are immunoglobulins. Even more preferably, the one or more other groups, residues, portions, or binding units are selected from: domain antibodies, amino acids suitable as domain antibodies, single-domain antibodies, amino acids suitable as single-domain antibodies, "dAbs", amino acids suitable as dAbs, or the group consisting of Nanobody® ISVs. Alternatively, these groups, residues, portions, or binding units may be, for example, chemical groups, residues, or portions, which may or may not be biologically and / or pharmaceutically active. For example (but not limited to): these groups may be linked to one or more immunoglobulin single variable domains to provide "derivatives" of immunoglobulin single variable domains. In another embodiment, the other residues may effectively prevent or reduce the binding of so-called "pre-existing antibodies" to the peptide. The polypeptide and its construct for this purpose may include a C-terminal elongation (X) n (where n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1)); and each X is (preferably native) amino acid residue, independently selected, and preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I), as can be seen in WO 2012 / 175741. Therefore, the polypeptide may further include a C-terminal elongation (X) n, where n is 1 to 5, such as 1, 2, 3, 4 or 5, and where X is a native amino acid, preferably without cysteine.

[0167] In the above-described polypeptides, one or more immunoglobulin single variable domains and one or more groups, residues, parts, or binding units may be directly linked to each other and / or linked via one or more suitable linkers or spacers. For example, when one or more groups, residues, parts, or binding units are amino acids, the linker may also be an amino acid, thus the resulting polypeptide is a fusion protein or fusion polypeptide. The term "linker" as used herein refers to a peptide that fuses two or more ISVs together to form a single molecule. This term also extends to any two amino acid sequences that are fused together to form a so-called "fusion protein" or "fusion polypeptide," such as, for example, an ISV as defined above with one or more other groups, residues, parts, or binding units.

[0168] The use of linkers to link two or more (poly)peptides is well known to those skilled in the art. One commonly used peptide linker is called a "Gly-Ser" or "GS" linker. These are linkers that are essentially composed of glycine (G) and serine (S) residues and typically contain one or more repeating sequences of peptide motifs, such as the GGGGS (SEQ ID NO: 26) motif (e.g., having the following formula (Gly-Gly-Gly-Gly-Ser)n, where n is 1, 2, 3, 4, 5, 6, 7 or more). Some commonly used examples of these 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). Other examples of peptide linkers are shown in Table 2 below. Table 2: Linker Sequences name SEQ IDNO amino acid sequence 3A connector --- AAA 5GS connector 26 GGGGS 7GS connector 27 SGGSGGS 8GS connector 28 GGGGSGGS 9GS connector 29 GGGGSGGGS 10GS connector 30 GGGGSGGGGS 15GS connector 31 GGGGSGGGGSGGGGS 18GS connector 32 GGGGSGGGGSGGGGSGGS 20GS connector 33 GGGGSGGGGSGGGGSGGGGS 25GS connector 34 GGGGSGGGGSGGGGSGGGGSGGGGS 30GS connector 35 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 35GS connector 36 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 40GS connector 37 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS G1 hinge 38 EPKSCDKTHTCPPCP 9GS-G1 hinge 39 GGGGSGGGSEPKSCDKTHTCPPCP Camel with long hinge area 40 EPKTPKPQPAAA G3 hinge 41 ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP

[0169] In all the antigen-binding proteins disclosed in this paper, one or more CDR residues may be substituted or one or more CDRs may be omitted. Antibodies have been described in the scientific literature in which one or two CDRs may be omitted for binding. Padlan et al. (FASB J. 1995;9(1):133-139) analyzed the contact area between the antibody and its antigen based on the published crystal structure and concluded that only about one-fifth to one-third of the CDR residues actually contact the antigen. Padlan also found that one or two CDRs in many antibodies do not have amino acids that contact the antigen (see also Vajdos et al., J Mol Biol. 2002;320(2):415-428).

[0170] Based on past research, molecular modeling and / or experiments can be used to determine the CDR residues that do not contact the antigen (e.g., residues H60-H65 of V H-CDR2 are often not required). If a CDR or its residue(s) is omitted, it is usually replaced by an amino acid occupying the corresponding position in another human antibody sequence or a common sequence of such sequences. The position of substitution in the CDR and the amino acid used for substitution can also be selected experimentally. Experimental substitutions can be conserved or non-conserved.

[0171] The anti-PD-1 / anti-4-1BB bispecific binding protein (or any individual component thereof) disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the architecture region and / or CDR region of the variable domain of the heavy chain and / or light chain, compared to the corresponding germline sequence. Such mutations can be readily confirmed by comparing the amino acid sequence disclosed herein with germline sequences from, for example, publicly available antibody sequence databases. This disclosure includes antibodies and their antigen-binding fragments derived from any amino acid sequence disclosed herein, wherein one or more amino acids in one or more architecture regions and / or CDR regions are mutated to corresponding residues (groups) of the germline sequence from which the antibody is derived, or mutated to corresponding residues (groups) of another human germline sequence, or mutated to conserved amino acid substitutions of the corresponding germline residues (groups of which are collectively referred to herein as "germline mutations"). Those skilled in the art can readily manufacture numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof, starting with the heavy and light chain variable region sequences disclosed herein. In some embodiments, all structural and / or CDR residues within the VH and / or VL domains are mutated back to residues present in the original germline sequence from which the antibody is derived. In other embodiments, only certain residues are mutated back to the original germline sequence; for example, the mutated residues are present only in the first eight amino acids of FR15 or the last eight amino acids of FR4, or the mutated residues are present only in CDR1, CDR2, or CDR3. In other embodiments, one or more structural and / or CDR residue groups are mutated to corresponding residue groups of a different germline sequence (i.e., a germline sequence different from the original germline sequence from which the antibody is derived). Furthermore, the antibodies disclosed herein may contain any combination of two or more germline mutations within the structural region and / or CDR region. For example, certain individual residues may be mutated to corresponding residues of a specific germline sequence, while other residues different from the original germline sequence may be maintained or mutated to corresponding residues of a different germline sequence. Once an antibody and antigen-binding fragment containing one or more germline mutations are obtained, it is easy to test one or more desired properties, such as: improved binding specificity, increased binding affinity, improved or enhanced antagonistic or activating biological properties (where feasible), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are all included in this disclosure.

[0172] This disclosure also includes anti-PD-1 / anti-4-1BB bispecific binding proteins (or any individual components thereof) containing variants of any HCVR, LCVR, and / or CDR amino acid sequences disclosed herein with one or more conserved substitutions. For example, this disclosure includes anti-PD-1 antibodies having HCVR, LCVR, and / or CDR amino acid sequences with, for example: 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc., conserved amino acid substitutions relative to any HCVR, LCVR, and / or CDR amino acid sequences disclosed herein; or anti-4-1BB immunoglobulin single variable domain having HCVR, LCVR, and / or CDR amino acid sequences with, for example: 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc., conserved amino acid substitutions relative to any HCVR, LCVR, and / or CDR amino acid sequences disclosed herein.

[0173] The term "human antibody" as used herein includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human mAbs, as disclosed herein, may include, for example, amino acid residues not encoded by human germline immunoglobulin sequences in the CDR and, specifically, in CDR3 (e.g., mutations introduced by random or site-specific in vitro mutagenesis or in vivo in vivo mutations). However, the term "human antibody" as used herein does not include mAbs in which a CDR sequence derived from the germline of another mammal (e.g., mouse) has been grafted onto a human FR sequence. This term includes antibodies generated in or recombined in non-human mammals. This term does not include antibodies isolated from or generated in human individuals.

[0174] As used herein, the term "recombinant" refers to the antibody or antigen-binding fragment thereof disclosed herein, produced, expressed, isolated, or obtained by techniques or methods known in the art, such as recombinant DNA techniques including, for example, DNA splicing and transgenic expression. The term also refers to an antibody or antigen-binding fragment thereof expressed in a non-human mammalian (including transgenic non-human mammals, such as transgenic mice) or cell (e.g., CHO cells) expression system or isolated from a recombinant human antibody library.

[0175] The term "multispecific antigen-binding molecule" as used herein refers to bispecific, trispecific, or multispecific antigen-binding molecules and their antigen-binding fragments. Multispecific antigen-binding molecules typically contain antigen-binding domains that specifically target more than one antigen. For antigen-binding molecules binding to more than one epitope of the same antigen, the term "multiple complementary sites" (e.g., bicomplementary sites, tricomplementary sites, etc.) is preferred, but there is inconsistency in the literature on this point, and authors may use the term "multispecific" to refer to antigen-binding molecules binding to more than one epitope of the same antigen. The use of such terminology in the text will clarify this discrepancy.

[0176] A multispecific antigen-binding molecule may be a single multifunctional polypeptide, or a polymeric complex of two or more polypeptides covalently or non-covalently linked together. The term "multispecific antigen-binding molecule" includes the antibody or antigen-binding fragment disclosed herein, which may be linked to or co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or antigen-binding fragment thereof may be functionally linked (e.g., by chemical coupling, gene fusion, non-covalent linking, or others) to one or more other molecular entities, such as proteins or fragments thereof, to produce a bispecific or multispecific antigen-binding molecule having a second binding specificity. According to this disclosure, the term "multispecific antigen-binding molecule" also includes bispecific, trispecific, or multispecific antibodies or antigen-binding fragments thereof. In some exemplary embodiments, the antibody or antigen-binding fragment thereof disclosed herein is functionally linked to another antibody or antigen-binding fragment thereof to produce a bispecific antibody having a second binding specificity.

[0177] In the exemplary embodiments, the antibody disclosed herein is a bispecific antibody. A bispecific antibody may be a monoclonal (e.g., human or humanized) antibody having binding specificity against at least two different antigens. In the exemplary embodiments, the bispecific antibody, fragments thereof, etc., have binding specificity against PD-1 and 4-1BB.

[0178] Methods for manufacturing bispecific antibodies are well known. Traditionally, recombinant methods for manufacturing bispecific antibodies are based on the co-expression of two paired immunoglobulin heavy / light chains, where the two heavy chains have different specificities (Milstein et al., Nature. 1983 Oct 6-12;305(5934):537-40). Due to the random mixing of immunoglobulin heavy and light chains, quadromas produce a possible mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule is usually accomplished 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 manufacturing bispecific antibodies are provided, for example, in Kufer et al., Trends Biotechnol. 2004 May;22(5):238-44.

[0179] The antibody variable domain with the desired binding specificity can be fused to an immunoglobulin constant domain sequence. Typically, it is fused to an immunoglobulin heavy chain constant domain comprising at least a portion of the hinge, CH2, and CH3 regions. It may have a first heavy chain constant region (CH1) containing sites necessary for light chain binding present in at least one fusion. The DNA encoding the immunoglobulin heavy chain fusion and, if necessary, the immunoglobulin light chain is embedded in separate expression vectors and co-transformed into a suitable host organism. Further details regarding the generation of bispecific antibodies can be found, for example: Suresh et al., Methods Enzymol. 1986;121:210-28.

[0180] The terms "specific binding," "specific binding," and any variations thereof mean that an antibody or its antigen-binding fragment forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding can be characterized by an equilibrium dissociation constant (denoted as "KD") of at least about 1 × 10⁻⁸ M or less (e.g., a lower KD indicates a tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasma resonance, and similar methods. Furthermore, multispecific antibodies that bind to one domain of PD-1 and one or more additional antigens, or bispecific antibodies that bind to two different regions of PD-1, can still be considered "specifically binding" antibodies as used herein.

[0181] The term "high affinity" for antibodies or their antigen-binding fragments refers to the binding affinity (expressed in KD) of such mAbs to antigens such as PD-1 and / or 4-1BB being at least 10⁻⁷ M; at least 10⁻⁸ M; at least 10⁻⁹ M; at least 10⁻¹⁰ M; or at least 10⁻¹¹ M, as measured by surface plasma resonance, such as BIACORE™ or solution-affinity ELISA.

[0182] The term "dissociation rate" or "K off" refers to a constant used to analyze the rate characteristic of the dissociation of an antibody or its antigen-binding fragment from its antigen (e.g., PD-1 and / or 4-1BB). "Slow dissociation rate" means that the rate constant of the dissociation of an antibody or its antigen-binding fragment from its antigen (e.g., PD-1 and / or 4-1BB) is 1 × 10⁻³ s⁻¹ or less, or 1 × 10⁻⁴ s⁻¹ or less, which is measured by surface plasma resonance, such as BIACORE™.

[0183] The term “surface plasma resonance” as used herein refers to an optical phenomenon that can analyze real-time biomolecular interactions, which detects changes in protein concentration in a biosensor array, for example, using the BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ).

[0184] As used herein, the terms “antigen-binding portion”, “antigen-binding fragment”, and similar terms include any natural, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that can specifically bind to an antigen to form a complex. As used herein, the terms “antigen-binding fragment” or “antibody fragment” refer to one or more fragments of an antibody that retain the ability to bind to PD-1 and / or 4-1BB. In specific embodiments, the disclosed antibodies or antibody fragments may bind to a portion of a ligand or medical part (“immunoconjugate”), such as an antibiotic, a secondary anti-PD-1 and / or anti-4-1BB antibody, or an antibody against another antigen (such as a tumor-specific antigen, a cell antigen of a virus, an Fc receptor, a T-cell receptor, or a T-cell co-inhibitor), or an immunotoxin, or any other medical part suitable for treating a disease or condition including cancer or a chronic viral infection.

[0185] The “isolated antibody” program used in this article refers to antibodies that do not actually have other antibodies (Abs) with different antigen specificities (e.g., isolated antibodies or their antigen-binding fragments that specifically bind to PD-1 and / or 4-1BB do not actually have Abs that specifically bind to antigens other than PD-1 and / or 4-1BB).

[0186] The term "blocking antibody," "neutralizing antibody," or "antagonist antibody" as used herein refers to an antibody that, upon binding to its target (e.g., PD-1), inhibits at least one biological activity of that target (e.g., PD-1). For example, this disclosure reveals that the antibody or its antigen-binding fragment can prevent or block the binding of a ligand (e.g., PD-L1) to PD-1.

[0187] The “activating antibody,” “enhancing antibody,” or “promoting antibody” program used in this paper refers to an antibody binding to its target (e.g., 4-1BB) that enhances or stimulates at least one biological activity of that target (e.g., 4-1BB). For example, this paper reveals that the antibody or its antigen-binding fragment can mimic the action of the target’s natural ligand (e.g., 4-1BBL) to promote the TRAF-mediated activation of the intracellular signaling cascade of NF-κB and MAPK, ultimately resulting in cell activation, proliferation, and survival.

[0188] In some exemplary embodiments, the disclosed antibody or its antigen-binding fragment may be both a blocking antibody and an activating antibody. For example, the disclosed anti-PD-1 / anti-4-1BB bispecific binding protein may serve as an antagonist anti-PD-1 binding protein and an activator anti-4-1BB binding protein.

[0189] As used herein, the term “pure agonist” means that an activating antibody or its antigen-binding fragment, as defined above herein, binds to its antigen in a manner that (i) enhances or stimulates the biological activity of at least one antigen (through 4-1BB signaling to activate T cells) in a manner independent of FcγR (i.e., independent of the participation of Fcγ receptor) and / or (iv) in the absence of target-mediated antigen cross-linking.

[0190] As used herein, the term "Fc-mediated crosslinking" refers to the crosslinking of a protein (e.g., an antibody) containing an Fc domain to an Fc-binding site via that Fc domain, such as an anti-Fc antibody or an Fc receptor.

[0191] 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, which has binding specificity to 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)].

[0192] As used herein, the term “target-mediated cross-linking” refers to the cross-linking of an antibody or its antigen-binding fragment with, for example, a tumor-associated antigen (TAA), an immune cell surface marker, a matrix antigen, or any other target expressed in cis or trans form by tumor cells, immune cells, and / or normal cells.

[0193] The term "antigenic epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site called a "complementary site" in the variable region of an antibody. A single antigen may have more than one antigenic epitope. Therefore, different antibodies can bind to different regions of the same antigen and may have different biological effects. The term "antigenic epitope" also refers to a site on an antigen that can respond to B- and / or T- cells. It also refers to a region of the antigen that is bound by an antibody. Antigenic epitopes can be defined by structure or function. Functional antigenic epitopes are usually a subgroup of structural antigenic epitopes and have residues that directly generate interaction affinity. Antigenic epitopes can also be conformational antigenic epitopes, i.e., composed of nonlinear amino acids. In some embodiments, the antigenic epitope may include a determinant, a chemically active surface group of its isomeric molecule, such as: amino acid, sugar side chain, phosphoyl, or sulfonylurea, and in some embodiments, may have specific three-dimensional structural characteristics and / or specific charge characteristics.

[0194] The terms "substantially identical" or "substantially identical" when referring to a nucleic acid or fragment thereof mean that, when optimally aligned with another nucleic acid (or its complementary strand) using appropriate nucleotide insertions or deletions, and measured by any known sequence identity algorithm discussed below, such as FASTA, BLAST, or GAP, at least about 90%, or at least about 95%, 96%, 97%, 98%, or 99% of the nucleotide bases are nucleotide sequence identical. In some examples, the polypeptide encoded by a nucleic acid molecule substantially identical to a reference nucleic acid molecule has the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0195] When the term "substantial similarity" or "substantial similarity" is applied to peptides, it means that two peptide sequences, when optimally aligned, such as using the GAP or BESTFIT algorithm with preset vacancy weights, have at least 90% sequence similarity, or at least 95%, 96%, 97%, 98%, or 99% sequence similarity. In the illustrated embodiments, the positions of inconsistent residues may differ due to conserved amino acid substitutions. A "conserved amino acid substitution" is the substitution of one amino acid residue by another amino acid residue with a side chain (R group) having similar chemical properties (e.g., in terms of charge or hydrophobicity). Generally, conserved amino acid substitutions do not substantially change the functional properties of a protein. If two or more amino acid sequences differ from each other due to conserved substitutions, the percentage or degree of similarity can be adjusted upwards to correct for the conservatism of the substitution. The manner in which this adjustment is made is known to those skilled in the art. See, for example, Pearson (Methods Mol Biol. 1994;24:307-31), the contents of which are incorporated herein by reference. Examples of amino acid groups having side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxy side chains: serine and threonine; 3) acetylamine side chains: aspartic acid and glutamic acid; 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. Examples of conservative amino acid substituents are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and glutamine-aspartic acid. Alternatively, a conservative substitution is any positive change in the PAM250 log-likelihood matrix as revealed by Gonnet et al. (Science. 1992 Jun 5;256(5062):1443-5), the contents of which are incorporated herein by reference. A "moderately conservative" substitution is any non-negative change in the PAM250 log-likelihood matrix.

[0196] Peptide sequence similarity is typically measured using sequence analysis software. Protein analysis software uses similarity measures for various substitutions, deletions, and other modifications (including conserved amino acid substitutions) to match similar sequences. For example, GCG software includes programs such as GAP and BESTFIT, which can use preset parameters to determine sequence homology or sequence identity between closely related peptides (e.g., homologous peptides from different species) or between wild-type proteins and their mutant proteins (muteins). See, for example, GCG version 6.1. Peptide sequences can also be aligned using FASTA; GCG version 6.1 with preset or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides the ratio of best overlap regions and the percentage of sequence identity between the lookup sequence and the search sequence (Pearson, Methods Mol Biol. 2000;132:185-219). When the sequences disclosed herein are compared with databases containing a large number of sequences from different organisms, another example of an algorithm is the use of computer programs BLAST with preset parameters, particularly BLASTP or TBLASTN. See, for example, 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, the contents of which are incorporated herein by reference.

[0197] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein may be "conditionally active". "Conditionally active" means that the antibody or antigen-binding fragment thereof can only bind to its antigen (i.e., is active) under certain conditions. In some exemplary embodiments, the conditionally active antibody or antigen-binding fragment thereof includes a masking portion.

[0198] As used herein, the terms “masking,” “masking domain,” or “masking motif” refer to a motif added to an antibody or its antigen-binding fragment to reduce the antibody’s ability to bind to its antigen. The masking effect is to prevent or reduce the binding of the antigen to one or more CDR sequences of the antibody or its antigen-binding fragment. Masking motifs include (but are not limited to): self-hinge domains, coiled-coil domains, non-antibody protein fragments, antibody fragments, affinity peptides, cross-masking antibodies, divalent peptide-double-stranded DNA conjugates, and the like. For an overview of suitable antibody masking motifs, see Lin et al. (J Biomed Sci. 2020 Jun 25;27(1):76).

[0199] In some exemplary embodiments, the masking portion is a polypeptide detachable from the antibody or its antigen-binding fragment. The antibody or its antigen-binding fragment can bind to its target antigen by cleaving the masking portion to a cleavable linker. In specific exemplary embodiments, the masking domain of the antibody or its antigen-binding fragment is cleaved at a tumor site, such as in a tumor bed or lymph node. The cleavable linker may be a linker that can be cleaved by a protease. In some exemplary embodiments, the cleavable linker includes at least one receptor for a tumor-specific protease.

[0200] The phrase “medically effective dose” refers to the amount of drug administered to produce the desired effect. The exact dosage will depend on the therapeutic purpose and will be determined by a person skilled in the relevant art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0201] As used herein, the term "individual" refers to an animal, such as a mammal, that requires relief, prevention, and / or treatment of a disease or condition, such as cancer or a chronic viral infection. In some embodiments, the individual is a human individual that requires relief, prevention, and / or treatment of a disease or condition, such as cancer or a chronic viral infection.

[0202] As used herein, “anticancer drug” means any preparation suitable for the treatment of cancer, including (but not limited to): cytotoxins and preparations such as: antimetabolites, alkylating agents, anthracyclines, antibiotics, antimitotic agents, procarbazine, hydroxyurea, aspartate aminotransferase, corticosteroids, mytotane (O,P'-(DDD)), biological agents (e.g., antibodies and interferons), and radioactive agents. As used herein, “cytotoxin or cytotoxic agent” also refers to chemotherapeutic agents and means any preparation that is harmful to cells. Examples include TAXOL® (paclitaxel), temozolomide, cytochalasin B, gramicidin D, ethidium bromide, emetine, cisplatin, mitomycin, etoposide, tenoposide, vincristine, vinbiastine, coichicin, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, and actinomycin D. D) 1-Dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and their analogues or homologues.

[0203] As used herein, the term "antiviral drug" refers to any drug or therapy used to treat, prevent, or alleviate viral infection in a test host. The term "antiviral drug" includes (but is not limited to): zidovudine, lamivudine, abacavir, ribavirin, lopinavir, efavirenz, cobicistat, tenofovir, rilpivirine, analgesics, and corticosteroids. In this disclosure, viral infection includes long-term or chronic infection 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).

[0204] The antibodies and antigen-binding fragments disclosed herein specifically bind to PD-1 and regulate the interaction between PD-1 and ligands such as PD-L1; and specifically bind to 4-1BB and regulate the interaction between 4-1BB and ligands such as 4-1BBL. Anti-PD-1 / anti-4-1BB antibodies can bind to PD-1 and 4-1BB with high or low affinity. In some embodiments, the antibodies disclosed herein can bind to PD-1 and block the interaction between PD-1 and PD-L1 (i.e., they are PD-1 antagonists), and can 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 suitable for stimulating or enhancing immune responses and / or for treating individuals with cancer or chronic viral infections. When antibodies are administered to individuals in need, they can reduce chronic infection by viruses such as HIV, LCMV, or HBV. They can also be used to inhibit tumor cell growth in individuals. They can be used alone or as adjunctive therapy, and are used in conjunction with other medical procedures or therapies for treating cancer or viral infections, as well as in treatments known in related fields. Detailed Description of the Invention

[0205] The object of the present invention relates to a single variable domain (ISV) of an immunoglobulin that specifically binds to 4-1BB, wherein the ISV has pure agonist activity.

[0206] In some embodiments, pure agonist activity means that ISV can (i) activate T cells via 4-1BB signaling in soluble conditions, and / or (ii) in the absence of crosslinking agents, and / or (iii) in an FcγR-independent manner (i.e., without the involvement of Fcγ receptors), and / or (iv) in the absence of target-mediated 4-1BB crosslinking. In some embodiments, pure agonist activity is determined by NF-κB pathway activation assay in the absence of crosslinking agents. The NF-κB pathway activation assay for determining the pure agonist activity of ISV is described in Example 1 herein; those skilled in the art will also appreciate other suitable NF-κB pathway activation assays that have been described in detail in the literature.

[0207] In some embodiments, the ISV competes with 4-1BBL to bind to 4-1BB.

[0208] In some embodiments, 4-1BB is human 4-1BB, and an example of its amino acid sequence is shown in SEQ ID NO: 13.

[0209] In some embodiments, the ISV interacts with macaque 4-1BB, that is, the ISV specifically binds to human 4-1BB and also to macaque 4-1BB.

[0210] In some embodiments, the ISV interacts with the cysteine-enriched domain 2 (CRD2) and / or the cysteine-enriched domain 3 (CRD3) 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 one first amino acid residue from the CRD2 domain of 4-1BB containing amino acid residues K69, G70, V71, F72 and R73, and with at least one second and third amino acid residue from the CRD3 domain of 4-1BB containing amino acid residues S100, M101, C102, E103 and Q104, and amino acid residues K114, K115, and G116 (according to the numbering of SEQ ID NO: 13). In some embodiments, the ISV interacts with at least one amino acid residue of 4-1BB, which is 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.

[0211] In some embodiments, the ISV contains three complementary determinant regions (CDRs). As shown in the Examples section, all three CDRs of an ISV having the pure agonist activity described herein will interact with its target antigen 4-1BB only through its iso-CDR3.

[0212] In some embodiments, the ISV contains a CDR3 amino acid sequence, shown in: SEQ ID NO: 2, 3, 58, 59, 60, or 61. In some embodiments, the ISV contains a CDR3 amino acid sequence, shown in: SEQ ID NO: 2 or 3.

[0213] In some embodiments, CDR3 comprises or consists of an amino acid sequence: - ARGTRYKLST (SEQ ID NO: 14), ARGTRYKMST (SEQ ID NO: 15), or ARGTRYKIFA (SEQ ID NO: 62), according to the IMGT number; or - GTRYKMST (SEQ ID NO: 63), GTRYKLST (SEQ ID NO: 64), or GTRYKIFA (SEQ ID NO: 65), according to the Kabat or Chothia number.

[0214] In some embodiments, the ISV comprises a CDR1 amino acid sequence, shown in: SEQ ID NO: 2, 3, 58, 59, 60, or 61. In some embodiments, the ISV comprises a CDR1 amino acid sequence, shown in: SEQ ID NO: 2 or 3.

[0215] In some embodiments, CDR1 comprises or consists of an 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 the IMGT number; or - DHTMT (SEQ ID NO: 69), DFTMS (SEQ ID NO: 70), SYAMG (SEQ ID NO: 71), or NYRMS (SEQ ID NO: 72), according to the Kabat number; or - GFTFSDH (SEQ ID NO: 73), GFAFRDF (SEQ ID NO: 74), GDTFSSY (SEQ ID NO: 75), or GFTFANY (SEQ ID NO: 76), according to the Chothia number.

[0216] In some embodiments, the ISV comprises a CDR2 amino acid sequence, shown in: SEQ ID NO: 2, 3, 58, 59, 60, or 61. In some embodiments, the ISV comprises a CDR2 amino acid sequence, shown in: SEQ ID NO: 2 or 3.

[0217] In some embodiments, CDR2 comprises or consists of an amino acid sequence: - ISSGGSRI (SEQ ID NO: 17), INPSGGSQ (SEQ ID NO: 77), or IKKSGNRT (SEQ ID NO: 78), according to the IMGT number; or - SISSGGSRIIYADSVKG (SEQ ID NO: 79), SINPSGGSQSYLPSVKG (SEQ ID NO: 80), SINPSGGSQSYHPSVKD (SEQ ID NO: 81), or SIKKSGNRTTYSDSVKG (SEQ ID NO: 82), according to the Kabat number; or - SSGGSR (SEQ ID NO: 83), NPSGGS (SEQ ID NO: 84), or KKSGNR (SEQ ID NO: 85), according to the Chothia number.

[0218] In some embodiments, the ISV comprises three CDR amino acid sequences, shown in: SEQ ID NO: 2, 3, 58, 59, 60, or 61. In some embodiments, the ISV comprises three CDR amino acid sequences, shown in: SEQ ID NO: 2 or 3.

[0219] In some embodiments, the ISV comprises three CDRs, wherein: - CDR1 comprises or is composed 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 is composed 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 is composed of the amino acid sequence ARGTRYKLST (SEQ ID NO: 14), ARGTRYKMST (SEQ ID NO: 15), or ARGTRYKIFA (SEQ ID NO: 62).

[0220] In some embodiments, the ISV comprises three CDRs, wherein: - CDR1 comprises or is composed 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 is composed 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); and - CDR3 comprises or is composed of the amino acid sequence GTRYKMST (SEQ ID NO: 63), GTRYKLST (SEQ ID NO: 64), or GTRYKIFA (SEQ ID NO: 65).

[0221] In some embodiments, the ISV comprises three CDRs, wherein: - CDR1 comprises or is composed 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 is composed 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 is composed of the amino acid sequence GTRYKMST (SEQ ID NO: 63), GTRYKLST (SEQ ID NO: 64), or GTRYKIFA (SEQ ID NO: 65).

[0222] In some embodiments, the ISV comprises three CDRs, wherein: - CDR1 comprises or is composed of the amino acid sequence GFTFSDHT (SEQ ID NO: 16), - CDR2 comprises or is composed of the amino acid sequence ISSGGSRI (SEQ ID NO: 17), and - CDR3 comprises or is composed of the amino acid sequence ARGTRYKMST (SEQ ID NO: 15); or - CDR1 comprises or is composed of the amino acid sequence DHTMT (SEQ ID NO: 69), - CDR2 comprises or is composed of the amino acid sequence SISSGGSRIIYADSVKG (SEQ ID NO: 79), and - CDR3 comprises or is composed of the amino acid sequence GTRYKMST (SEQ ID NO: 63); or - CDR1 comprises or is composed of the amino acid sequence GFTFSDH (SEQ ID NO: 73), - CDR2 comprises or is composed of the amino acid sequence SSGGSR (SEQ ID NO: 83), and - CDR3 contains or constitutes the amino acid sequence GTRYKMST (SEQ ID NO: 63).

[0223] In some embodiments, the ISV comprises three CDRs, wherein: - CDR1 comprises or is composed of the amino acid sequence GFTFSDHT (SEQ ID NO: 16), - CDR2 comprises or is composed of the amino acid sequence ISSGGSRI (SEQ ID NO: 17), and - CDR3 comprises or is composed of the amino acid sequence ARGTRYKLST (SEQ ID NO: 14); or - CDR1 comprises or is composed of the amino acid sequence DHTMT (SEQ ID NO: 69), - CDR2 comprises or is composed of the amino acid sequence SISSGGSRIIYADSVKG (SEQ ID NO: 79), and - CDR3 comprises or is composed of the amino acid sequence GTRYKLST (SEQ ID NO: 64); or - CDR1 comprises or is composed of the amino acid sequence GFTFSDH (SEQ ID NO: 73), - CDR2 comprises or is composed of the amino acid sequence SSGGSR (SEQ ID NO: 83), and - CDR3 contains or constitutes the amino acid sequence GTRYKLST (SEQ ID NO: 64).

[0224] In some embodiments, the ISV comprises three CDRs, wherein: - CDR1 comprises or is composed of the amino acid sequence GFAFRDFT (SEQ ID NO: 66), - CDR2 comprises or is composed of the amino acid sequence INPSGGSQ (SEQ ID NO: 77), and - CDR3 comprises or is composed of the amino acid sequence ARGTRYKMST (SEQ ID NO: 15); or - CDR1 comprises or is composed of the amino acid sequence DFTMS (SEQ ID NO: 70), - CDR2 comprises or is composed of the amino acid sequence SINPSGGSQSYLPSVKG (SEQ ID NO: 80), and - CDR3 comprises or is composed of the amino acid sequence GTRYKMST (SEQ ID NO: 63); or - CDR1 comprises or is composed of the amino acid sequence GFAFRDF (SEQ ID NO: 74), - CDR2 comprises or is composed of the amino acid sequence NPSGGS (SEQ ID NO: 84), and - CDR3 contains or constitutes the amino acid sequence GTRYKMST (SEQ ID NO: 63).

[0225] In some embodiments, the ISV comprises three CDRs, wherein: - CDR1 comprises or is composed of the amino acid sequence GDTFSSYA (SEQ ID NO: 67); - CDR2 comprises or is composed of the amino acid sequence INPSGGSQ (SEQ ID NO: 77); and - CDR3 comprises or is composed of the amino acid sequence ARGTRYKIFA (SEQ ID NO: 62); or - CDR1 comprises or is composed of the amino acid sequence SYAMG (SEQ ID NO: 71), - CDR2 comprises or is composed of the amino acid sequence SINPSGGSQSYHPSVKD (SEQ ID NO: 81), and - CDR3 comprises or is composed of the amino acid sequence GTRYKIFA (SEQ ID NO: 65); or - CDR1 comprises or is composed of the amino acid sequence GDTFSSY (SEQ ID NO: 75), - CDR2 comprises or is composed of the amino acid sequence NPSGGS (SEQ ID NO: 84), and - CDR3 contains or constitutes the amino acid sequence GTRYKIFA (SEQ ID NO: 65).

[0226] In some embodiments, the ISV comprises three CDRs, wherein: - CDR1 comprises or is composed of the amino acid sequence GFTFANYR (SEQ ID NO: 68); - CDR2 comprises or is composed of the amino acid sequence IKKSGNRT (SEQ ID NO: 78); and - CDR3 comprises or is composed of the amino acid sequence ARGTRYKMST (SEQ ID NO: 15); or - CDR1 comprises or is composed of the amino acid sequence NYRMS (SEQ ID NO: 72), - CDR2 comprises or is composed of the amino acid sequence SIKKSGNRTTYSDSVKG (SEQ ID NO: 82), and - CDR3 comprises or is composed of the amino acid sequence GTRYKMST (SEQ ID NO: 63); or - CDR1 comprises or is composed of the amino acid sequence GFTFANY (SEQ ID NO: 76), - CDR2 comprises or is composed of the amino acid sequence KKSGNR (SEQ ID NO: 85), and - CDR3 contains or constitutes the amino acid sequence GTRYKMST (SEQ ID NO: 63).

[0227] In some embodiments, the ISV comprises or consists of: - an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 3, 58, 59, 60, and 61; or - an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the non-CDR region of SEQ ID NO: 2, 3, 58, 59, 60, or 61.

[0228] In some embodiments, the ISV comprises or consists of: - an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and 3; or - an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the non-CDR region of SEQ ID NO: 2 or 3.

[0229] In some embodiments, the ISV comprises or is composed of the amino acid sequence SEQ ID NO: 3. In some embodiments, the ISV comprises or is composed of the amino acid sequence SEQ ID NO: 2.

[0230] In some embodiments, ISV is V HH.

[0231] In some embodiments, the ISV is the anti-4-1BB pure line #5 V HH described in the Examples section of this document, or its derivatives, such as pure line #5.1, pure line #5a, pure line #5b, pure line #5c or pure line #5d also described in the Examples section.

[0232] Another aspect 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 as described above.

[0233] In some embodiments, the bivalent or bispecific antigen-binding protein includes at least one second ISV that specifically binds to the same target antigen (i.e., 4-1BB) or to another target antigen.

[0234] When the at least second ISV specifically binds to another target antigen, the target antigen may be a T-cell antigen, a tumor-associated or tumor-specific antigen, or a non-autogenous antigen, or any other antigen that a person skilled in the art deems appropriate.

[0235] 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 having pure agonist activity; or 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: an ISV that specifically binds to 4-1BB but does not have pure agonist activity, as described below.

[0236] Another aspect of the present invention relates to a single variable domain (ISV) of an immunoglobulin that specifically binds to 4-1BB. In some embodiments, the ISV does not have pure agonist activity.

[0237] In some embodiments, the ISV will not compete with 4-1BBL to bind to 4-1BB.

[0238] In some embodiments, 4-1BB is human 4-1BB, and an example of its amino acid sequence is shown in SEQ ID NO: 13.

[0239] In some embodiments, the ISV does not or substantially does not interact with macaque 4-1BB, that is, the ISV specifically binds to the human 4-1BB ISV, but does not or substantially does not bind to macaque 4-1BB.

[0240] In some embodiments, the ISV includes three complementary determination regions (CDRs).

[0241] In some embodiments, the ISV contains the CDR1 amino acid sequence, shown in: SEQ ID NO: 1 or 4.

[0242] In some embodiments, CDR1 comprises or is composed of the amino acid sequence GGLFSINT (SEQ ID NO: 86; according to IMGT number); or INTGG (SEQ ID NO: 87; according to Kabat number); or GGLFSIN (SEQ ID NO: 88; according to Chothia number).

[0243] In some embodiments, the ISV contains the CDR2 amino acid sequence, shown in: SEQ ID NO: 1 or 4.

[0244] In some embodiments, CDR2 comprises or consists of the amino acid sequence ITHDDRT (SEQ ID NO: 89; according to IMGT number); or TITHDDRTNYAESVKG (SEQ ID NO: 90; according to Kabat number); or THDDR (SEQ ID NO: 91; according to Chothia number).

[0245] In some embodiments, the ISV contains the CDR3 amino acid sequence, shown in: SEQ ID NO: 1 or 4.

[0246] In some embodiments, CDR3 comprises or is composed of the amino acid sequence RLGSAAIRGY (SEQ ID NO: 92; according to IMGT number); or GSAAIRGY (SEQ ID NO: 93; according to Kabat or Chothia number).

[0247] In some embodiments, the ISV contains three CDR amino acid sequences, shown in: SEQ ID NO: 1 or 4.

[0248] In some embodiments, the ISV comprises three CDRs, wherein: - CDR1 comprises or is composed of the amino acid sequence GGLFSINT (SEQ ID NO: 86); - CDR2 comprises or is composed of the amino acid sequence ITHDDRT (SEQ ID NO: 89); and - CDR3 comprises or is composed of the amino acid sequence RLGSAAIRGY (SEQ ID NO: 92).

[0249] In some embodiments, the ISV comprises three CDRs, wherein: - CDR1 comprises or is composed of the amino acid sequence INTGG (SEQ ID NO: 87); - CDR2 comprises or is composed of the amino acid sequence TITHDDRTNYAESVKG (SEQ ID NO: 90); and - CDR3 comprises or is composed of the amino acid sequence GSAAIRGY (SEQ ID NO: 93).

[0250] In some embodiments, the ISV comprises three CDRs, wherein: - CDR1 comprises or is composed of the amino acid sequence GGLFSIN (SEQ ID NO: 88); - CDR2 comprises or is composed of the amino acid sequence THDDR (SEQ ID NO: 91); and - CDR3 comprises or is composed of the amino acid sequence GSAAIRGY (SEQ ID NO: 93).

[0251] In some embodiments, the ISV comprises or consists of: - an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and 4, or - an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the non-CDR region of SEQ ID NO: 1 or 4.

[0252] In some embodiments, the ISV comprises or is composed of the amino acid sequence SEQ ID NO: 4. In some embodiments, the ISV comprises or is composed of the amino acid sequence SEQ ID NO: 1.

[0253] In some embodiments, ISV is V HH.

[0254] In some embodiments, the ISV is the anti-4-1BB pure line #2 V HH as described in the Examples section, or a derivative thereof, such as the pure line #2.1 also described in the Examples section.

[0255] In some embodiments, the ISV further comprises at least one masking portion. In some embodiments, the masking portion reduces or inhibits the binding of the ISV to its target antigen 4-1BB.

[0256] In some embodiments, the masking portion comprises or consists of the amino acid sequence CPELQGIFC (SEQ ID NO: 94), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 94.

[0257] In some embodiments, the masking portion comprises or consists of the amino acid sequence CPELQGIFCYR (SEQ ID NO: 95), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 95.

[0258] In some embodiments, the masking portion comprises or constitutes the amino acid sequence VEVCPELQGIFC (SEQ ID NO: 96), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 96.

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

[0260] In some embodiments, the masking portion comprises or is composed of the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11CPELQGIFCYR12X13 (SEQ ID NO: 98), wherein X1-X13 each represent any amino acid residue. In some embodiments, the masking portion comprises or is composed of the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11CPELQGIFCYR (SEQ ID NO: 99), wherein X1-X11 each represent any amino acid residue. In some embodiments, the masking portion comprises or is composed of the amino acid sequence X1X2X3X4X5X6X7X8VEVCPELQGIFCX12X13 (SEQ ID NO: 100), wherein X1-X8 and X12-X13 each represent any amino acid residue. In some embodiments, the masking portion comprises or is composed of the amino acid sequence X1X2X3X4X5X6X7X8VEVCPELQGIFCYR (SEQ ID NO: 101), wherein X1-X8 each represent any amino acid residue.

[0261] In some embodiments, the masking portion comprises or is composed of the amino acid sequence EVGSX 5X 6X 7X 8X 9X 10X 11CPELQGIFCX 12X 13 (SEQ ID NO: 102), wherein X5-X13 each represent any amino acid residue. In some embodiments, the masking portion comprises or is composed of the amino acid sequence EVGSX 5X 6X 7X 8X 9X 10X 11CPELQGIFCYR (SEQ ID NO: 103), wherein X5-X11 each represent any amino acid residue. In some embodiments, the masking portion comprises or is composed of the amino acid sequence EVGSX 5X 6X 7X 8VEVCPELQGIFCX 12X 13 (SEQ ID NO: 104), wherein X5-X8 and X12-X13 each represent any amino acid residue. In some embodiments, the masking portion comprises or is composed of the amino acid sequence EVGSX 5X 6X 7X 8VEVCPELQGIFCYR (SEQ ID NO: 105), wherein X 5-X 8 each represent any amino acid residue.

[0262] In some embodiments, the masking portion comprises or consists of an amino acid sequence of SEQ ID NO: 44 or 45, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 44 or 45.

[0263] In some embodiments, the ISV further includes at least one linker between the masking portion and the ISV. In some embodiments, the at least one linker is cleavable. In some embodiments, the at least one linker can be cleaved 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), interstitial protease, legume protease, kallikrein-related peptidase-3, human neutrophil elastase, protease 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.

[0264] In some embodiments, the linker comprises or constitutes the amino acid sequence of SEQ ID NO: 56 and / or 57. However, those skilled in the art will understand that other amino acid sequences are suitable as linkers that can be cleaved by tumor-specific proteases. Such amino acid sequences are known to those skilled in the art.

[0265] In some embodiments, the linker comprises or consists of an amino acid sequence of SEQ ID NO: 46 or 47.

[0266] In some embodiments, cleaving at least one linker releases the masking portion and restores ISV binding to its target antigen 4-1BB.

[0267] In some embodiments, the shielding portion and the detachable connector may be fused to the N-terminus or C-terminus of the ISV; preferably, the shielding portion and the detachable connector are fused to the N-terminus of the ISV.

[0268] In some embodiments, the masked ISV (i.e., including the masking portion and the cleavable linker) comprises or constitutes an amino acid sequence SEQ ID NO: 48, 49, 50 or 51, or an amino acid sequence having 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, an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the non-CDR region of SEQ ID NO: 48, 49, 50 or 51; more preferably, an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the non-CDR, masking portion and cleavable linker regions of SEQ ID NO: 48, 49, 50 or 51. 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identical amino acid sequences.

[0269] Another aspect of the present invention relates to a single variable domain (ISV) of an immunoglobulin that specifically binds to 4-1BB, wherein the ISV includes at least one masking part that reduces or inhibits the binding of the ISV to its target antigen 4-1BB.

[0270] In some embodiments, the masking portion comprises or consists of the amino acid sequence CPELQGIFC (SEQ ID NO: 94), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 94.

[0271] In some embodiments, the masking portion comprises or consists of the amino acid sequence CPELQGIFCYR (SEQ ID NO: 95), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 95.

[0272] In some embodiments, the masking portion comprises or consists of the amino acid sequence VEVCPELQGIFC (SEQ ID NO: 96), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 96.

[0273] In some embodiments, the masking portion comprises or consists of the amino acid sequence VEVCPELQGIFCYR (SEQ ID NO: 97), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 97.

[0274] In some embodiments, the masking portion comprises or is composed of the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11CPELQGIFCYR12X13 (SEQ ID NO: 98), wherein X1-X13 each represent any amino acid residue. In some embodiments, the masking portion comprises or is composed of the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11CPELQGIFCYR (SEQ ID NO: 99), wherein X1-X11 each represent any amino acid residue. In some embodiments, the masking portion comprises or is composed of the amino acid sequence X1X2X3X4X5X6X7X8VEVCPELQGIFCX12X13 (SEQ ID NO: 100), wherein X1-X8 and X12-X13 each represent any amino acid residue. In some embodiments, the masking portion comprises or is composed of the amino acid sequence X1X2X3X4X5X6X7X8VEVCPELQGIFCYR (SEQ ID NO: 101), wherein X1-X8 each represent any amino acid residue.

[0275] In some embodiments, the masking portion comprises or is composed of the amino acid sequence EVGSX 5X 6X 7X 8X 9X 10X 11CPELQGIFCX 12X 13 (SEQ ID NO: 102), wherein X5-X13 each represent any amino acid residue. In some embodiments, the masking portion comprises or is composed of the amino acid sequence EVGSX 5X 6X 7X 8X 9X 10X 11CPELQGIFCYR (SEQ ID NO: 103), wherein X5-X11 each represent any amino acid residue. In some embodiments, the masking portion comprises or is composed of the amino acid sequence EVGSX 5X 6X 7X 8VEVCPELQGIFCX 12X 13 (SEQ ID NO: 104), wherein X5-X8 and X12-X13 each represent any amino acid residue. In some embodiments, the masking portion comprises or is composed of the amino acid sequence EVGSX 5X 6X 7X 8VEVCPELQGIFCYR (SEQ ID NO: 105), wherein X 5-X 8 each represent any amino acid residue.

[0276] In some embodiments, the masking portion comprises or consists of an amino acid sequence of SEQ ID NO: 44 or 45, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 44 or 45.

[0277] In some embodiments, the ISV further includes at least one linker between the masking portion and the ISV. In some embodiments, the at least one linker is cleavable. In some embodiments, the at least one linker can be cleaved 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), interstitial protease, legume protease, kallikrein-related peptidase-3, human neutrophil elastase, protease 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.

[0278] In some embodiments, the linker comprises or constitutes the amino acid sequence of SEQ ID NO: 56 and / or 57. However, those skilled in the art will understand that other amino acid sequences are suitable as linkers that can be cleaved by tumor-specific proteases. These amino acid sequences are known to those skilled in the art.

[0279] In some embodiments, the linker comprises or consists of an amino acid sequence of SEQ ID NO: 46 or 47.

[0280] In some embodiments, cleaving at least one linker releases the masking portion and restores ISV binding to its target antigen 4-1BB.

[0281] In some embodiments, the shielding portion and the detachable connector may be fused to the N-terminus or C-terminus of the ISV; preferably, the shielding portion and the detachable connector are fused to the N-terminus of the ISV.

[0282] In some embodiments, ISV is V HH.

[0283] Another aspect of the present invention relates to a single variable domain (ISV) of an immunoglobulin that specifically binds to a target antigen, wherein the ISV includes at least one masking part that reduces or inhibits binding to the target antigen.

[0284] In some embodiments, the masking portion comprises or consists of the amino acid sequence CPELQGIFC (SEQ ID NO: 94), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 94.

[0285] In some embodiments, the masking portion comprises or consists of the amino acid sequence CPELQGIFCYR (SEQ ID NO: 95), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 95.

[0286] In some embodiments, the masking portion comprises or constitutes the amino acid sequence VEVCPELQGIFC (SEQ ID NO: 96), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 96.

[0287] In some embodiments, the masking portion comprises or consists of the amino acid sequence VEVCPELQGIFCYR (SEQ ID NO: 97), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 97.

[0288] In some embodiments, the masking portion comprises or is composed of the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11CPELQGIFCYR12X13 (SEQ ID NO: 98), wherein X1-X13 each represent any amino acid residue. In some embodiments, the masking portion comprises or is composed of the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11CPELQGIFCYR (SEQ ID NO: 99), wherein X1-X11 each represent any amino acid residue. In some embodiments, the masking portion comprises or is composed of the amino acid sequence X1X2X3X4X5X6X7X8VEVCPELQGIFCX12X13 (SEQ ID NO: 100), wherein X1-X8 and X12-X13 each represent any amino acid residue. In some embodiments, the masking portion comprises or is composed of the amino acid sequence X1X2X3X4X5X6X7X8VEVCPELQGIFCYR (SEQ ID NO: 101), wherein X1-X8 each represent any amino acid residue.

[0289] In some embodiments, the masking portion comprises or is composed of the amino acid sequence EVGSX 5X 6X 7X 8X 9X 10X 11CPELQGIFCX 12X 13 (SEQ ID NO: 102), wherein X5-X13 each represent any amino acid residue. In some embodiments, the masking portion comprises or is composed of the amino acid sequence EVGSX 5X 6X 7X 8X 9X 10X 11CPELQGIFCYR (SEQ ID NO: 103), wherein X5-X11 each represent any amino acid residue. In some embodiments, the masking portion comprises or is composed of the amino acid sequence EVGSX 5X 6X 7X 8VEVCPELQGIFCX 12X 13 (SEQ ID NO: 104), wherein X5-X8 and X12-X13 each represent any amino acid residue. In some embodiments, the masking portion comprises or is composed of the amino acid sequence EVGSX 5X 6X 7X 8VEVCPELQGIFCYR (SEQ ID NO: 105), wherein X 5-X 8 each represent any amino acid residue.

[0290] In some embodiments, the masking portion comprises or consists of an amino acid sequence of SEQ ID NO: 44 or 45, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 44 or 45.

[0291] In some embodiments, the ISV further includes at least one linker between the masking portion and the ISV. In some embodiments, the at least one linker is cleavable. In some embodiments, the at least one linker can be cleaved 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), interstitial protease, legume protease, kallikrein-related peptidase-3, human neutrophil elastase, protease 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.

[0292] In some embodiments, the linker comprises or constitutes the amino acid sequence of SEQ ID NO: 56 and / or 57. However, those skilled in the art will understand that other amino acid sequences are suitable as linkers that can be cleaved by tumor-specific proteases. These amino acid sequences are known to those skilled in the art.

[0293] In some embodiments, the linker comprises or consists of an amino acid sequence of SEQ ID NO: 46 or 47.

[0294] In some embodiments, at least one linker is cleaved to release the masking portion and restore the ability of ISV to bind to its target antigen.

[0295] In some embodiments, the shielding portion and the detachable connector may be fused to the N-terminus or C-terminus of the ISV; preferably, the shielding portion and the detachable connector are fused to the N-terminus of the ISV.

[0296] In some embodiments, ISV is V HH.

[0297] Another aspect of the present invention relates to an antibody or antigen-binding fragment thereof that specifically binds to PD-1.

[0298] In some embodiments, PD-1 is human PD-1, and an example of its amino acid sequence is shown in SEQ ID NO: 42.

[0299] In some embodiments, the antibody or its antigen-binding fragment has a human reproductive index of 95% or higher in the heavy and light variable regions.

[0300] "Human germinality index" refers to the proportion of identical structural residues between the variable region sequence of an antibody or its antigen-binding fragment and the closest related human germline sequence. It is established by Pelat et al. (J Mol Biol. 2008 Dec 31;384(5):1400-7).

[0301] In some embodiments, the antibody or its antigen-binding fragment comprises: (i) three light chain CDR amino acid sequences shown in: SEQ ID NO: 7 or 5, and (ii) three heavy chain CDR amino acid sequences shown in: SEQ ID NO: 8 or 6.

[0302] In some embodiments, the antibody or its antigen-binding fragment comprises: (i) three light chain CDR amino acid sequences, shown in: SEQ ID NO: 7, and (ii) three heavy chain CDR amino acid sequences, shown in: SEQ ID NO: 8.

[0303] In some embodiments, the antibody or its antigen-binding fragment comprises: (i) three light chain CDR amino acid sequences, shown in: SEQ ID NO: 5, and (ii) three heavy chain CDR amino acid sequences, shown in: SEQ ID NO: 6.

[0304] In some embodiments, the antibody or its antigen-binding fragment comprises: (i) a light chain variable region comprising three of the following CDR amino acid 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); and (ii) a heavy chain variable region comprising three of the following CDR amino acid 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).

[0305] In some embodiments, the antibody or its antigen-binding fragment comprises: (i) a light chain variable region comprising three of the following CDR amino acid sequences: a. V L-CDR1: GASQSVPINFLA (SEQ ID NO: 106) or GASQSVSINFLA (SEQ ID NO: 107), b. V L-CDR2: EASSRHT (SEQ ID NO: 108) or EASSRAT (SEQ ID NO: 109), and c. V L-CDR3: GQYGSSPYT (SEQ ID NO: 20) or QQYGSSPYT (SEQ ID NO: 21); and (ii) a heavy chain variable region comprising three of the following CDR amino acid sequences: a. V H-CDR1: SSSYFWG (SEQ ID NO: 110) or TSSYFWG (SEQ ID NO: 111), b. V H-CDR2: SIYRSGSTYYNPSLKS (SEQ ID NO: 108) or QQYGSSPYT (SEQ ID NO: 109), and c. V L-CDR3: GQYGSSPYT (SEQ ID NO: 20) or QQYGSSPYT (SEQ ID NO: 21); and (ii) a heavy chain variable region comprising three of the following CDR amino acid sequences: a. V H-CDR1: SSSYFWG (SEQ ID NO: 110) or TSSYFWG (SEQ ID NO: 111), b. V H-CDR2: SIYRSGSTYYNPSLKS (SEQ ID NO: 110) or QQYGSSPYT (SEQ ID NO: 109), and c. V H-CDR3: GQYGSSPYT (SEQ ID NO: 109). NO: 112), and c. V H-CDR3: GITGDPGDY (SEQ ID NO: 113).

[0306] In some embodiments, the antibody or its antigen-binding fragment comprises: (i) a light chain variable region comprising three of the following CDR amino acid sequences; a. V L-CDR1: GASQSVPINFLA (SEQ ID NO: 106) or GASQSVSINFLA (SEQ ID NO: 107); b. V L-CDR2: EASSRHT (SEQ ID NO: 108) or EASSRAT (SEQ ID NO: 109), and c. V L-CDR3: GQYGSSPYT (SEQ ID NO: 20) or QQYGSSPYT (SEQ ID NO: 21); and (ii) a heavy chain variable region comprising three of the following CDR amino acid sequences; a. V H-CDR1: GGSISSSSY (SEQ ID NO: 114) or GGSISTSSY (SEQ ID NO: 115), b. V H-CDR2: YRSGS (SEQ ID NO: 116), and c. V H-CDR3: GITGDPGDY (SEQ ID NO: 113).

[0307] In some embodiments, the antibody or its antigen-binding fragment comprises: (i) a light chain variable region comprising three of the following CDR amino acid 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); and (ii) a heavy chain variable region comprising three of the following CDR amino acid 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); or (i) a light chain variable region comprising three of the following CDR amino acid sequences; a. V L-CDR1: GASQSVPINFLA (SEQ ID NO: 106), b. V L-CDR2: GASQSVPINFLA (SEQ ID NO: 106), c. V L-CDR3: GASQSVPINFLA (SEQ ID NO: 106), c. V L-CDR2 ... L-CDR2: EASSRHT (SEQ ID NO: 108), and c. V L-CDR3: GQYGSSPYT (SEQ ID NO: 20); and (ii) heavy chain variable region comprising three of the following CDR amino acid sequences: a. V H-CDR1: SSSYFWG (SEQ ID NO: 110), b. V H-CDR2: SIYRSGSTYYNPSLKS (SEQ ID NO: 112), and c. V H-CDR3: GITGDPGDY (SEQ ID NO: 113); or (i) light chain variable region comprising three of the following CDR amino acid sequences: a. V L-CDR1: GASQSVPINFLA (SEQ ID NO: 106), b. V L-CDR2: EASSRHT (SEQ ID NO: 108), and c. V L-CDR3: GQYGSSPYT (SEQ ID NO: 20); and (ii) heavy chain variable region comprising three of the following CDR amino acid sequences: a. V L-CDR1: GASQSVPINFLA (SEQ ID NO: 106), b. V L-CDR2: EASSRHT (SEQ ID NO: 108), and c. V L-CDR3: GQYGSSPYT (SEQ ID NO: 20); NO: 20); and (ii) the heavy chain variable region comprising three of the following CDR amino acid sequences: a. V H-CDR1: GGSISSSSY (SEQ ID NO: 114); b. V H-CDR2: YRSGS (SEQ ID NO: 116); and c. V H-CDR3: GITGDPGDY (SEQ ID NO: 113).

[0308] In some embodiments, the antibody or its antigen-binding fragment comprises: (i) a light chain variable region comprising three of the following CDR amino acid sequences; a. V L-CDR1: QSVSINF (SEQ ID NO: 19), b. V L-CDR2: EAS, and c. V L-CDR3: QQYGSSPYT (SEQ ID NO: 21); and (ii) a heavy chain variable region comprising three of the following CDR amino acid sequences; a. V H-CDR1: GGSISTSSYF (SEQ ID NO: 23), b. V H-CDR2: IYRSGST (SEQ ID NO: 24), and c. V H-CDR3: ARGITGDPGDY (SEQ ID NO: 25); or (i) a light chain variable region comprising three of the following CDR amino acid sequences; a. V L-CDR1: GASQSVSINFLA (SEQ ID NO: 107), b. V L-CDR2: GASQSVSINFLA (SEQ ID NO: 19), c. V L-CDR3: QQYGSSPYT (SEQ ID NO: 21); and (ii) a heavy ...2: GASQSVSINFLA (SEQ ID NO: 19), c. V L-CDR3: GASQSVSINFLA (SEQ ID NO: 19), c. V L- (i) L-CDR2: EASSRAT (SEQ ID NO: 109), and (ii) L-CDR3: QQYGSSPYT (SEQ ID NO: 21); and (iii) a heavy chain variable region comprising three of the following CDR amino acid sequences: a. V H-CDR1: TSSYFWG (SEQ ID NO: 111), b. V H-CDR2: SIYRSGSTYYNPSLKS (SEQ ID NO: 112), and c. V H-CDR3: GITGDPGDY (SEQ ID NO: 113); or (i) a light chain variable region comprising three of the following CDR amino acid sequences: a. V L-CDR1: GASQSVSINFLA (SEQ ID NO: 107), b. V L-CDR2: EASSRAT (SEQ ID NO: 109), and c. V L-CDR3: QQYGSSPYT (SEQ ID NO: 21); and (ii) The heavy chain variable region contains three of the following CDR amino acid sequences: a. V H-CDR1: GGSISTSSY (SEQ ID NO: 115); b. V H-CDR2: YRSGS (SEQ ID NO: 116); and c. V H-CDR3: GITGDPGDY (SEQ ID NO: 113).

[0309] In some embodiments, the antibody or its antigen-binding fragment comprises: (i) a light chain variable region having a light chain variable region of SEQ ID NO: 7 or 5, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the non-CDR region of SEQ ID NO: 7 or 5; and (ii) a heavy chain variable region having a heavy chain variable region of SEQ ID NO: 8 or 6, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the non-CDR region of SEQ ID NO: 8 or 6.

[0310] In some embodiments, the antibody or its antigen-binding fragment comprises: (i) a light chain variable region having the light chain variable region of SEQ ID NO: 7, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the non-CDR region of SEQ ID NO: 7; and (ii) a heavy chain variable region having the heavy chain variable region of SEQ ID NO: 8, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the non-CDR region of SEQ ID NO: 8.

[0311] In some embodiments, the antibody or its antigen-binding fragment comprises: (i) a light chain variable region having the light chain variable region of SEQ ID NO: 5, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the non-CDR region of SEQ ID NO: 5; and (ii) a heavy chain variable region having the heavy chain variable region of SEQ ID NO: 6, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the non-CDR region of SEQ ID NO: 6.

[0312] In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region having SEQ ID NO: 7 and a heavy chain variable region having SEQ ID NO: 8.

[0313] In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region having SEQ ID NO: 5 and a heavy chain variable region having SEQ ID NO: 6.

[0314] Another aspect of the present invention relates to a molecule of a multispecific antigen-binding protein, comprising at least one immunoglobulin single variable domain (ISV) specifically binding to 4-1BB, wherein the molecule has pure agonist activity as defined above.

[0315] In some embodiments, for example, the molecule of the multispecific antigen-binding protein includes 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 the same. Alternatively, the at least two ISVs that specifically bind to 4-1BB are different. When they are different, the at least two ISVs that specifically bind to 4-1BB may bind to the same antigenic epitope, or an overlapping antigenic epitope, or a unique antigenic epitope of 4-1BB. In some embodiments, the at least two ISVs that specifically bind to 4-1BB are different and bind to a unique antigenic epitope of 4-1BB.

[0316] In some embodiments, at least one ISV specifically binding to 4-1BB is an ISV having pure agonist activity as described herein (e.g., but not limited to: an ISV comprising or constituting an amino acid sequence SEQ ID NO: 2, 3, 58, 59, 60, or 61). In some embodiments, at least one second ISV specifically binding to 4-1BB is an ISV having pure agonist activity as described herein (e.g., but not limited to: an ISV comprising or constituting an amino acid sequence SEQ ID NO: 2, 3, 58, 59, 60, or 61). In some embodiments, at least one second ISV specifically binding to 4-1BB is an ISV without pure agonist activity as described herein (e.g., but not limited to: an ISV comprising or constituting an amino acid sequence SEQ ID NO: 1 or 4).

[0317] In some embodiments, for example, the molecule of a multispecific antigen-binding protein comprises at least two ISVs, wherein one of the at least two ISVs specifically binds to 4-1BB, and the other of the at least two ISVs specifically binds to another target antigen. The other target antigen may be a T-cell antigen, a tumor-associated or tumor-specific antigen, or a non-autogenous antigen, or any other antigen that a person skilled in the art deems suitable.

[0318] In some embodiments, for example, the molecule of the multispecific antigen-binding protein includes at least four ISVs that specifically bind to 4-1BB. In some embodiments, the at least four ISVs include: (i) two identical ISVs of a first group that specifically bind to 4-1BB, and (ii) two other identical ISVs of a second group that specifically bind to 4-1BB; or (i') two ISVs of a first group that specifically bind to a first 4-1BB antigenic epitope, and (ii') two other ISVs of a second group that specifically bind to a second 4-1BB antigenic epitope.

[0319] The first 4-1BB antigenic epitope and the second 4-1BB antigenic epitope may be the same, overlap, or be unique. In some embodiments, the first 4-1BB antigenic epitope and the second 4-1BB antigenic epitope are unique.

[0320] In some embodiments, the first two ISVs of (i) or (i') are ISVs having pure agonist activity as described herein (e.g., but not limited to: ISVs comprising or constituting an amino acid sequence SEQ ID NO: 2, 3, 58, 59, 60 or 61).

[0321] In some embodiments, the two ISVs of the second group of (ii) or (ii') are ISVs that do not have the pure agonist activity as described herein (e.g., but not limited to: ISVs that comprise or constitute an amino acid sequence SEQ ID NO: 1 or 4).

[0322] In some embodiments, for example, the molecule of the multispecific antigen-binding protein further includes an antibody Fc region or a fragment thereof. For example, the Fc region or a fragment thereof may be one of IgG, IgD, IgA, IgM, or IgE Fc regions; specifically, one of the IgG Fc regions, such as IgG1 or IgG4 Fc regions. The Fc region may also be an antibody-dependent cytotoxicity (ADCC)-silencing and / or antibody-dependent phagocytosis (ADCP)-silencing region. Such silencing Fc regions are known in the art and include (but are not limited to): IgG1 LALA Fc region, IgG1 NNAS Fc region, and IgG4 P-FALA Fc region. In some embodiments, the Fc region or a fragment thereof is one of the IgG1 LALA Fc regions.

[0323] In some embodiments, for example, the molecule of a multispecific antigen-binding protein further comprises at least one Fab fragment.

[0324] In some embodiments, for example: the molecule of a multispecific antigen-binding protein comprises: - a first polypeptide (i.e., a heavy chain), preferably comprising 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 wherein the second ISV is different from the first ISV; § at least one CH domain of the Fc region; and § variable and constant domains of the Fab fragment; - a second polypeptide (i.e., a light chain), comprising the variable and constant domains of the Fab fragment; wherein the variable and constant domains of the first and second polypeptides form the Fab fragment.

[0325] In some embodiments, for example, the molecule of the multispecific antigen-binding protein further includes a third and a fourth polypeptide that are the same as the first and second polypeptides, respectively, wherein at least one CH domain of the first and third polypeptides forms an Fc region.

[0326] In some embodiments, the variable and constant domains of the first polypeptide are VH and CH1 domains, and the variable and constant domains of the second polypeptide are VL and CL domains. Alternatively, the variable and constant domains of the first polypeptide are VL and CL domains, and the variable and constant domains of the second polypeptide are VH and CH1 domains.

[0327] In some embodiments, at least one CH domain of the first polypeptide includes: - CH2 and CH3 domains of IgG; - CH2 and CH3 domains of IgD; - CH2 and CH3 domains of IgA; - CH2, CH3 and CH4 domains of IgM; or - CH2, CH3 and CH4 domains of IgE.

[0328] In some embodiments, at least one CH domain of the first polypeptide includes the CH2 and CH3 domains of IgG.

[0329] In some embodiments, the first polypeptide (i.e., the heavy chain) preferably comprises, from the N-terminus to the C-terminus: - a first ISV specifically binding to 4-1BB; - a first linker; - a second ISV specifically binding to 4-1BB, preferably wherein the second ISV is different from the first ISV; - a second linker; - an IgG hinge region; - an IgG CH2 domain; - and an IgG CH3 domain; - a third linker; - a VH domain of the Fab fragment; and - a CH1 domain of the Fab fragment.

[0330] In some embodiments, the second polypeptide (i.e., the light chain) preferably includes, from the N-terminus to the C-terminus: - a VL domain of the Fab fragment; and - a CL domain of the Fab fragment.

[0331] In some embodiments, at least one Fab fragment specifically binds to B- and / or T- cell surface proteins other than 4-1BB. In some embodiments, at least one Fab fragment specifically binds to immune checkpoint molecules. In some embodiments, at least one Fab fragment is a PD-1 antagonist.

[0332] In some embodiments, at least one Fab fragment is an antigen-binding fragment that specifically binds to PD-1 as described above (e.g., but not limited to: 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).

[0333] In some embodiments, for example: the molecule of the multispecific antigen-binding protein comprises: at least one first polypeptide having SEQ ID NO: 11 or 9, and at least one second polypeptide having SEQ ID NO: 12 or 10; or at least one first polypeptide comprising an amino acid sequence having 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 one second polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 12 or 10.

[0334] In some embodiments, for example, the molecule of the multispecific antigen-binding protein comprises at least one first polypeptide having SEQ ID NO: 11 and at least one second polypeptide having SEQ ID NO: 12.

[0335] In some embodiments, for example, the molecule of the multispecific antigen-binding protein includes at least one first polypeptide having SEQ ID NO: 9 and at least one second polypeptide having SEQ ID NO: 10.

[0336] In some embodiments, for example, molecules of multispecific antigen-binding proteins can initiate T-cell activation in TCA and / or MLR assays.

[0337] In some embodiments, for example, molecules of multispecific antigen-binding proteins can initiate the secretion of IFN-γ and / or TNF-α in a CD3-PBMC activation assay.

[0338] In some embodiments, for example, molecules of multispecific antigen-binding proteins can be used to resuscitate exhausted CD8+ T- cells in vitro.

[0339] In some embodiments, for example, molecules of multispecific antigen-binding proteins can induce in vitro T reg inhibitory activity.

[0340] In some embodiments, for example, the molecule of a multispecific antigen-binding protein includes at least one masking portion. In some embodiments, the masking portion reduces or inhibits the binding of the molecule to at least one of its targets. In some embodiments, the masking portion reduces or inhibits the binding of the molecule to 4-1BB.

[0341] In some embodiments, the masking portion comprises or consists of the amino acid sequence CPELQGIFC (SEQ ID NO: 94), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 94.

[0342] In some embodiments, the masking portion comprises or consists of the amino acid sequence CPELQGIFCYR (SEQ ID NO: 95), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 95.

[0343] In some embodiments, the masking portion comprises or constitutes the amino acid sequence VEVCPELQGIFC (SEQ ID NO: 96), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 96.

[0344] In some embodiments, the masking portion comprises or constitutes the amino acid sequence VEVCPELQGIFCYR (SEQ ID NO: 97), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 97.

[0345] In some embodiments, the masking portion comprises or is composed of the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11CPELQGIFCYR12X13 (SEQ ID NO: 98), wherein X1-X13 each represent any amino acid residue. In some embodiments, the masking portion comprises or is composed of the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11CPELQGIFCYR (SEQ ID NO: 99), wherein X1-X11 each represent any amino acid residue. In some embodiments, the masking portion comprises or is composed of the amino acid sequence X1X2X3X4X5X6X7X8VEVCPELQGIFCX12X13 (SEQ ID NO: 100), wherein X1-X8 and X12-X13 each represent any amino acid residue. In some embodiments, the masking portion comprises or is composed of the amino acid sequence X1X2X3X4X5X6X7X8VEVCPELQGIFCYR (SEQ ID NO: 101), wherein X1-X8 each represent any amino acid residue.

[0346] In some embodiments, the masking portion comprises or is composed of the amino acid sequence EVGSX 5X 6X 7X 8X 9X 10X 11CPELQGIFCX 12X 13 (SEQ ID NO: 102), wherein X5-X13 each represent any amino acid residue. In some embodiments, the masking portion comprises or is composed of the amino acid sequence EVGSX 5X 6X 7X 8X 9X 10X 11CPELQGIFCYR (SEQ ID NO: 103), wherein X5-X11 each represent any amino acid residue. In some embodiments, the masking portion comprises or is composed of the amino acid sequence EVGSX 5X 6X 7X 8VEVCPELQGIFCX 12X 13 (SEQ ID NO: 104), wherein X5-X8 and X12-X13 each represent any amino acid residue. In some embodiments, the masking portion comprises or is composed of the amino acid sequence EVGSX 5X 6X 7X 8VEVCPELQGIFCYR (SEQ ID NO: 105), wherein X 5-X 8 each represent any amino acid residue.

[0347] In some embodiments, the masking portion comprises or consists of an amino acid sequence of SEQ ID NO: 44 or 45, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 44 or 45.

[0348] In some embodiments, for example, the molecule of the multispecific antigen-binding protein further includes at least one linker between the masking portion and the molecule. In some embodiments, the at least one linker is cleavable. In some embodiments, the at least one linker can be cleaved 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), interstitial protease, legume protease, kallikrein-related peptidase-3, human neutrophil elastase, protease 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.

[0349] In some embodiments, the linker comprises or constitutes the amino acid sequence of SEQ ID NO: 56 and / or 57. However, those skilled in the art will understand that other amino acid sequences are suitable as linkers that can be cleaved by tumor-specific proteases. These amino acid sequences are known to those skilled in the art.

[0350] In some embodiments, at least one linker comprises or constitutes an amino acid sequence of SEQ ID NO: 46 or 47.

[0351] In some embodiments, cleaving at least one linker releases the masking portion and restores, for example, the binding of a multispecific antigen-binding protein molecule to at least one of its target antigens. In some embodiments, cleaving at least one linker releases the masking portion and restores, for example, the binding of a multispecific antigen-binding protein molecule to 4-1BB.

[0352] In some embodiments, the masking part and the cleavable linker may be fused to, for example, the N-terminus or C-terminus of the heavy chain of a multispecific antigen-binding protein molecule. Alternatively, the masking part and the cleavable linker may be fused to, for example, the N-terminus or C-terminus of the light chain of a multispecific antigen-binding protein molecule.

[0353] In some embodiments, the masking portion and the cleavable linker are fused to, for example, the N-terminus of the heavy chain of a molecule of a multispecific antigen-binding protein; such as, for example, the N-terminus of a first ISV that specifically binds to 4-1BB.

[0354] In some embodiments, for example: the molecule of the multispecific antigen-binding protein comprises: (i) at least one first polypeptide having SEQ ID NO: 11 or 9, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the non-CDR region of SEQ ID NO: 11 or 9; and (ii) at least one second polypeptide having SEQ ID NO: 52, 53, 54 or 55, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the non-CDR region of SEQ ID NO: 52, 53, 54 or 55; preferably having at least 70% sequence identity with the non-CDR, masking portion and cleavable linker region of SEQ ID NO: 52, 53, 54 or 55. 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identical amino acid sequences.

[0355] In some embodiments, for example, the molecule of the multispecific antigen-binding protein comprises at least one first polypeptide having SEQ ID NO: 11 and at least one second polypeptide having SEQ ID NO: 52.

[0356] In some embodiments, for example, the molecule of the multispecific antigen-binding protein comprises at least one first polypeptide having SEQ ID NO: 11 and at least one second polypeptide having SEQ ID NO: 53.

[0357] In some embodiments, for example, the molecule of the multispecific antigen-binding protein comprises at least one first polypeptide having SEQ ID NO: 11 and at least one second polypeptide having SEQ ID NO: 54.

[0358] In some embodiments, for example, the molecule of the multispecific antigen-binding protein comprises at least one first polypeptide having SEQ ID NO: 11 and at least one second polypeptide having SEQ ID NO: 55.

[0359] In some embodiments, for example, the molecule of the multispecific antigen-binding protein includes at least one first polypeptide having SEQ ID NO: 9 and at least one second polypeptide having SEQ ID NO: 52.

[0360] In some embodiments, for example, the molecule of the multispecific antigen-binding protein comprises at least one first polypeptide having SEQ ID NO: 9 and at least one second polypeptide having SEQ ID NO: 53.

[0361] In some embodiments, for example, the molecule of the multispecific antigen-binding protein comprises at least one first polypeptide having SEQ ID NO: 9 and at least one second polypeptide having SEQ ID NO: 54.

[0362] In some embodiments, for example, the molecule of the multispecific antigen-binding protein comprises at least one first polypeptide having SEQ ID NO: 9 and at least one second polypeptide having SEQ ID NO: 55.

[0363] In some embodiments, for example, the molecule of the multispecific antigen-binding protein further comprises third and fourth polypeptides, which are identical to the first and second polypeptides, respectively. Antigen-binding fragment of the antibody.

[0364] Unless otherwise stated, it is understood that the term "antibody" as used herein includes an antibody molecule comprising two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., a "complete antibody molecule") and its antigen-binding fragment. Antibody fragments may include Fab fragments, F(ab′)2 fragments, Fv fragments, dAb fragments, fragments containing a CDR, or isolated CDRs. In some embodiments, the term "antigen-binding fragment" refers to a polypeptide fragment of a multispecific antigen-binding molecule. In these embodiments, the term "antigen-binding fragment" includes, for example, the extracellular domain of PD-L1 specifically bound to PD-1 or the extracellular domain of 4-1BBL specifically bound to 4-1BB. Antigen-binding fragments of antibodies can be derived from, for example, complete antibody molecules using any suitable standard technique, such as protein hydrolysis or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding variable and (if desired) constant domains of the antibody. These DNAs are known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. They can be sequenced and chemically manipulated or manipulated using molecular biotechnology, such as arranging one or more variable and / or constant domains into a suitable configuration, or introducing codons, establishing cysteine ​​residues, modifying, adding, or deleting amino acids, etc.

[0365] Non-restricted 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) the smallest distinguishing unit consisting of amino acid residues of a hypervariable region of a mimicking antibody (e.g., a separated complementarity-determining region (CDR), such as a CDR3 peptide), or a restricted FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deficient antibodies, chimeric antibodies, CDR-transplanted antibodies, bivalent antibodies, trivalent antibodies, tetravalent antibodies, mini antibodies, nanoantibodies (e.g., monovalent nanoantibodies, bivalent nanoantibodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also included in the term "antigen-binding fragment" as used herein.

[0366] Antigen-binding fragments of antibodies typically contain at least one variable domain. The variable domain can be of any size or amino acid composition and typically contains at least one CDR, adjacent to or within one or more frame sequences. In antigen-binding fragments with a VH domain linked to a VL domain, the VH and VL domains can be arranged in any suitable relative position. For example, the variable region can be a dimer, and may 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.

[0367] In some embodiments, the antigen-binding fragment of the antibody may include at least one variable domain covalently linked to at least one constant domain. Non-limiting configuration examples of variable and constant domains that may appear in the antigen-binding fragment of the antibody disclosed herein 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 the variable and constant domains, including any of the configurations listed above, the variable and constant domains may be directly connected to each other or may be connected using complete or partial hinge or linker regions. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, creating flexible or semi-flexible bonds between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, the antigen-binding fragment of the antibody disclosed herein may comprise a homodimer or heterodimer (or other multimer) of any of the variable and constant domains listed above non-covalently linked to each other and / or linked to one or more monomeric VH or VL domains (e.g., using disulfide bonds(groups)).

[0368] For intact antibody molecules, the antigen-binding fragment can be monospecific or multispecific (e.g., bispecific). A multispecific antigen-binding fragment of an antibody typically contains at least two distinct variable domains, each of which can specifically bind to separate antigens or to different antigenic epitopes on the same antigen. Any multispecific antibody format, including the examples of bispecific antibody formats disclosed herein, can be applied to the antigen-binding fragment content of the antibodies disclosed herein using routine techniques available in the relevant art. Preparation of Human Antibodies

[0369] Methods for generating human antibodies in transgenic mice are well known in the art. Any of these known methods can be used in this disclosure to produce human antibodies that specifically bind to PD-1 and / or 4-1BB.

[0370] Antibodies against PD-1 and / or 4-1BB can be generated using an immunogen comprising any of the following: In some embodiments, the disclosed antibody system is derived from mice immunized with full-length original PD-1 and / or 4-1BB or with recombinant PD-1 and / or 4-1BB peptides. Alternatively, PD-1 and / or 4-1BB or fragments thereof can be manufactured using standard biochemical techniques, modified, and used as an immunogen. In some embodiments, the immunogen may be an N-terminal or C-terminal peptide of PD-1 and / or 4-1BB.

[0371] In some embodiments, the immunogen may be recombinant PD-1 and / or 4-1BB peptide expressed in Escherichia coli or any other eukaryotic or mammalian cell, such as Chinese hamster ovary (CHO) cells.

[0372] In some embodiments, antibodies that specifically bind to PD-1 and / or 4-1BB can be prepared using fragments of the aforementioned region or peptides extending from the N or C terminus or both ends of the region described herein beyond the designated region by about 5 to about 20 amino acid residues. In some embodiments, any combination of the aforementioned region or fragments thereof can be used to prepare PD-1 and / or 4-1BB specific antibodies.

[0373] Using Velocimmuno® technology (see, for example, U.S. Patent No. 6,596,541, Regeneron Pharmaceuticals) or any other known method for generating monoclonal antibodies, a high-affinity chimeric antibody against PD-1 and / or 4-1BB containing both human variable and mouse constant regions can be isolated. Velocimmuno® technology involves generating transgenic mice having a genome containing human heavy and light chain variable regions operably linked to an endogenous mouse constant region locus, such that the mice produce antibodies containing both human variable and mouse constant regions that respond to antigen stimulation. The DNA encoding the variable regions of the heavy and light chains of this antibody is isolated and operably linked to DNA encoding the human heavy and light chain constant regions. This DNA is then expressed in cells capable of expressing fully human antibodies. Those skilled in this art are aware that several other transgenic mouse systems can be used, such as the Trianni® mouse from Trianni Inc., the Kymouse® mouse from Kymab Limited, the OmniMouse® from OmniAb, or the HuMAb Mouse® from Medarex. Bioequivalents

[0374] This disclosure describes anti-PD-1 / anti-4-1BB bispecific antibodies (or any individual components thereof) comprising proteins having amino acid sequences different from those of the aforementioned antibodies, but still retaining the ability to bind to PD-1 and 4-1BB. These variant antibodies and their antigen-binding fragments, when aligned with the parental sequence, contain one or more added, deleted, or substituted amino acids, but essentially retain the same biological activity as the aforementioned antibodies. Similarly, the DNA sequence encoding antibodies disclosed herein includes sequences containing one or more added, deleted, or substituted nucleotides when aligned with the disclosed sequence, but the encoded antibody or antibody fragment is essentially biologically equivalent to the antibody or antibody fragment disclosed herein.

[0375] Two antigen-binding proteins or antibodies that are, for example, pharmacologically equivalent or pharmacological substitutes, are considered bioequivalent if, under similar experimental conditions and at the same molar dose, their absorption rates and extent do not show significant differences, whether administered as a single dose or multiple doses. Some antibodies that have the same extent of absorption but different absorption rates will be considered equivalent or pharmacological substitutes. Because these differences in absorption rates are intentional and will be reflected on the label, they may not necessarily reach an effective drug concentration in the body. For example, they can still be considered bioequivalent if used long-term and without significantly affecting the specific drug product under investigation.

[0376] In one embodiment, two antigen-binding proteins are bioequivalent if there are no clinically significant differences in their safety, purity, or potency.

[0377] In one embodiment, the two antigen-binding proteins are bioequivalent if a patient can switch between the reference product and the biological product one or more times without an expected increased risk of side effects, including clinically significant changes in immunogenicity or reduced efficacy, compared to continuous therapy without such switching.

[0378] In one embodiment, a bioequivalence is defined as follows: if the two antigen-binding proteins act on one or more conditions of use through one or more combined mechanisms of action, and such mechanisms are known to the extent that they are bioequivalent.

[0379] Bioequivalence can be demonstrated by in vivo and / or in vitro methods. Bioequivalence assays include, for example: (a) in vivo studies in humans or other mammals, wherein the concentration of the antibody or its metabolites in blood, plasma, serum, or other biological fluids is measured as a function of time; (b) in vitro studies that have correlated with and are reasonably estimated to be equivalent to in vivo bioavailability data in humans; (c) in vivo studies in humans or other mammals, wherein the appropriate acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) well-controlled clinical trials that have established the safety, potency, or bioavailability or bioequivalence of the antibody.

[0380] This disclosure discloses that the construction of bioequivalent variants of antibodies can be achieved, for example, by creating various substitutions or deletions of biologically unwanted terminal or internal residues or sequences. For example, biologically unwanted cysteine ​​residues can be deleted or replaced by other amino acids to prevent unwanted or incorrect intramolecular disulfide bridging bonds while restoring the original properties. In other contexts, bioequivalent antibodies may include antibody variants containing altered amino acids that modify the glycosylation characteristics of the antibody, such as by eliminating or removing glycosylation mutations. Examples include anti-PD-1 / anti-4-1BB antibodies containing Fc variants.

[0381] According to certain embodiments of this disclosure, the provided anti-PD-1 / anti-4-1BB antibody includes an Fc domain containing one or more mutations that, for example, enhance or eliminate antibody binding to the FcRn receptor at acidic pH compared to neutral pH. For example, the anti-PD-1 / anti-4-1BB antibody included in this disclosure contains mutations in the CH2 or CH3 region of the Fc domain, wherein the mutation(s) increase the affinity of the Fc domain for FcRn in an acidic environment (e.g., in endosomes, where the pH range is from about 5.5 to about 6.0). When the antibody is administered to animals, these mutations can prolong the serum half-life of the antibody. In the following text, unless otherwise stated, the amino acid residue numbering of the Fc domain or constant region is based on the so-called EU index, which is described in Kabat et al., 1991 (Sequences of proteins of immunological interest. 5th 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: modification 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 modification 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 modification positions 250 and / or 428; or modification positions 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modification includes 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; 252, 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 another embodiment, the modification includes 265A (e.g., D265A) and / or 297A (e.g., N297A) modifications.

[0382] For example: The anti-PD-1 / anti-4-1BB antibody disclosed herein contains an Fc domain containing one or more pairs or groups of mutations selected from the following groups: 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., P257). I 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 anti-PD-1 antibody disclosed herein includes an Fc domain containing an S108P mutation in the hinge region of IgG4 to promote dimer stabilization. All possible combinations of the above-described Fc domain mutations, and other mutations within the variable domains of the antibodies disclosed herein, are included within the scope of this disclosure.

[0383] This disclosure also includes anti-PD-1 / anti-4-1BB antibodies comprising a chimeric heavy chain constant (CH) region, wherein the chimeric CH region comprises segments derived from CH regions of more than one immunoglobulin isotype. For example, the antibody disclosed herein may comprise a chimeric CH region comprising a portion or all of the CH2 domains derived from human IgG1, human IgG2, or human IgG4 molecules, combined with a portion or all of the CH3 domains derived from human IgG1, human IgG2, or human IgG4 molecules. According to some embodiments, the antibody disclosed herein comprises a chimeric CH region having a chimeric hinge region. For example, the chimeric hinge may comprise an "upper hinge" amino acid sequence (amino acid residues at positions 216 to 227 according to EU number) derived from the hinge region of human IgG1, human IgG2, or human IgG4, combined with a "lower hinge" sequence (amino acid residues at positions 228 to 236 according to EU number) derived from the hinge region of human IgG1, human IgG2, or human IgG4. According to some embodiments, the chimeric hinge region comprises amino acid residues derived from the upper hinge of human IgG1 or human IgG4 and amino acid residues derived from the lower hinge of human IgG2. Antibodies containing the chimeric CH region described herein may, in some embodiments, have modified Fc effector function without side effects that negatively affect the medical or pharmacokinetic properties of the antibody (see, for example, U.S. Patent No. 9,359,437, the entire contents of which are incorporated herein by reference). Biocharacterization of Antibodies

[0384] Typically, the antibodies disclosed herein function to bind to PD-1 and 4-1BB. This disclosure includes anti-PD-1 / anti-4-1BB bispecific antibodies and their antigen-binding fragments (or any individual components thereof) that bind with high affinity to soluble monomeric or dimerized PD-1 and 4-1BB molecules. For example, this disclosure includes antibodies that bind to PD-1 and 4-1BB and their antigen-binding fragments, with a KD (e.g., at 25°C or 37°C) less than about 50 nM as measured by surface plasma resonance. In some embodiments, the KD of the antibody or its antigen-binding fragment bound to PD-1 and 4-1BB, as measured by surface plasma resonance, is 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.

[0385] This disclosure also includes antibodies and antigen-binding fragments that bind to PD-1 and 4-1BB, whose dissociation half-life (t½) measured at 25°C or 37°C by surface plasma resonance exceeds approximately 1.1 minutes. In some embodiments, the t½ for binding of the disclosed antibody or antigen-binding fragment to PD-1, measured at 25°C or 37°C by surface plasma resonance, is more than approximately 5 minutes, more than approximately 10 minutes, more than approximately 30 minutes, more than approximately 50 minutes, more than approximately 60 minutes, more than approximately 70 minutes, more than approximately 80 minutes, more than approximately 90 minutes, more than approximately 100 minutes, more than approximately 200 minutes, more than approximately 300 minutes, more than approximately 400 minutes, more than approximately 500 minutes, more than approximately 600 minutes, more than approximately 700 minutes, more than approximately 800 minutes, more than approximately 900 minutes, more than approximately 1000 minutes, or more than approximately 1200 minutes. Species selectivity and species cross-reactivity

[0386] According to certain embodiments of this disclosure, the anti-PD-1 / anti-4-1BB antibody (or any individual component thereof) binds to human PD-1 and human 4-1BB, but not to PD-1 and 4-1BB from other species. Alternatively, in some embodiments of this disclosure, the anti-PD-1 / anti-4-1BB antibody binds to human PD-1 and human 4-1BB and PD-1 and 4-1BB from one or more non-human species. For example, the anti-PD-1 / anti-4-1BB antibody of this disclosure may bind to human PD-1 and / or human 4-1BB and may or may not bind (as the case may) to one or more PD-1 and / or 4-1BB from mice, rats, guinea pigs, hamsters, gerbils, pigs, cats, dogs, rabbits, goats, sheep, cattle, horses, camels, macaques, marmosets, rhesus monkeys, or chimpanzees. In some embodiments, the disclosed anti-PD-1 / anti-4-1BB antibodies can bind to human and macaque PD-1 and / or 4-1BB with the same or different affinities, but not to rat and mouse PD-1 and / or 4-1BB. Medical dosing and formulations.

[0387] This disclosure provides medical compositions comprising the anti-PD-1 / anti-4-1BB antibody (or any other individual component thereof) disclosed herein. Medical compositions according to this disclosure utilize suitable carriers, excipients, and other formulations incorporated to provide improved transport, delivery, tolerability, etc. Many suitable formulations can be found in all pharmacologically known prescriptions: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example: powders, pastes, ointments, gels, waxes, oils, lipids, lipid-containing (cationic or anionic) capsules (e.g., LIPOFECTIN™), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsified carbowax (various molecular weights of polyethylene glycol), 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.

[0388] The dosage of the antibody may vary depending on the age and body size of the individual receiving the drug, the target disease, the condition, the route of administration, etc. When the antibody disclosed herein is used to treat or prevent diseases in adult patients, the antibody disclosed herein is generally administered in a single dose of about 0.1 to about 60 mg / kg body weight, or 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 may be adjusted depending on the severity of the condition. In some embodiments, the antibody disclosed herein or its antigen-binding fragment is administered at 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 some embodiments, a second or plurality of subsequent doses of the antibody or its antigen-binding fragment may be administered following the initial dose, in amounts approximately equal to or less than the initial dose, wherein the subsequent doses may be spaced at intervals of at least 1 to 3 days; at least one 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.

[0389] Various delivery systems are known and can be used to deliver the pharmaceutical composition disclosed herein, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells that can express mutant viral strains, and receptor-mediated endocytosis (see, for example, 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 composition can be administered via any suitable route, such as infusion or bolus injection, absorption through the epithelial or mucosal layer (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other bioactive agents. Administration can be systemic or local. Pharmaceutical components can also be in the form of cysts, specifically liposomes (see, for example, Langer. Science. 1990 Sep 28;249(4976):1527-33).

[0390] This document also includes the use of nanoparticles in the delivery of the antibodies of the present invention. The antibody-conjugated nanoparticles can be used in both medical and diagnostic applications. The antibody-conjugated nanoparticles, their manufacturing methods, and their uses have been described in detail in Arruebo et al. (J Nanomat. 2009; pp. 1-24), the contents of which are incorporated herein by reference. Nanoparticles can be developed and conjugated to antibodies contained in pharmaceutical compositions to target tumor cells or virus-infected cells. Drug delivery nanoparticles have also been described, for example, in U.S. Patent No. 8,257,740 or U.S. Patent No. 8,246,995, which are incorporated herein by reference in their entirety.

[0391] In some cases, the pharmaceutical composition can be delivered in a controlled-release system. In one embodiment, a pump can be used. In another embodiment, a polymeric material can be used. In yet another embodiment, the composition can be placed near a target by the controlled-release system, thus requiring only a portion of the systemic dose.

[0392] Injectable formulations may include dosage forms for intravenous, subcutaneous, intradermal, intracranial, intraperitoneal, intramuscular, and intratumoral injection, infusion, etc. These injectable formulations may be prepared using known methods. For example, the preparation of injectable formulations may involve, for instance, dissolving, suspending, or emulsifying the aforementioned antibodies or their salts in a sterile aqueous or oily medium conventionally used for injection. Aqueous media for injection may include, for example, physiological saline, isotonic solutions containing glucose and other excipients, etc., which may be combined with appropriate solvents such as alcohols (e.g., ethanol), polyols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (a 50 mol polyoxyethylene adduct of hydrogenated castor oil)], etc. Oily media that may be used include, for example, sesame oil, soybean oil, etc., which may be combined with solvents such as methyl benzoate, benzyl alcohol, etc. The prepared injection can be filled into appropriate ampoules as needed.

[0393] The pharmaceutical composition disclosed herein can be delivered subcutaneously or intravenously using standard needles and syringes. Furthermore, when delivered subcutaneously, the pharmaceutical composition disclosed herein can be easily delivered using a pen delivery device. These pen delivery devices can be reused or are disposable. Reusable pen delivery devices typically utilize a replaceable cartridge containing the pharmaceutical composition. Once all the pharmaceutical composition in the cartridge has been dispensed and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. Thus, the pen delivery device can be reused. In disposable pen delivery devices, there is no replaceable cartridge. Instead, the disposable pen delivery device is pre-filled with the pharmaceutical composition and stored in the device's reservoir. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0394] Many reusable pen-type and auto-injector delivery devices have been used for subcutaneous delivery of the pharmaceutical ingredients disclosed herein. Examples include (but are not limited to): AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ injection pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25™ injection pen, HUMALOG™ injection pen, HUMALIN 70 / 30™ injection pen (Eli Lilly and Co., Indianapolis, Ind.), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ injection pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany), and the like. Examples of disposable pen delivery systems for subcutaneous delivery of the disclosed pharmaceutical ingredients include (but are not limited to): SOLOSTAR™ injection pen (Sanofi-Aventis), FLEXPEN™ (Novo Nordisk), and KWIKPEN™ (Eli Lilly), SURECLICK™ autoinjector (Amgen, Thousand Oaks, Calif.), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA™ injection pen (Abbott Labs, Abbott Park, Ill.), and the like.

[0395] The above-mentioned oral or non-enteral pharmaceutical compositions should be formulated into unit-dose dosage forms in accordance with the dosage of the active ingredient. Such unit-dose dosage forms include, for example, tablets, pills, capsules, ampoules, suppositories, etc. The antibody content in a unit dose is typically about 5 to about 500 mg per dose; particularly, the antibody content in injectable forms is about 5 to about 100 mg, and in other dosage forms it is about 10 to about 250 mg. Medical Uses of Antibodies

[0396] This disclosure discloses antibodies (or any individual components thereof) that are particularly suitable for the treatment, prevention, and / or relief of diseases or conditions associated with or mediated by PD-1 and / or 4-1BB expression, signaling, or activity, or that can be treated by blocking the interaction between PD-1 and PD-1 ligands (e.g., PD-L1, or PD-L2), and between 4-1BB and 4-1BB ligands (e.g., 4-1BBL), or by inhibiting PD-1 and activating 4-1BB activity and / or signaling. For example, this disclosure provides a method for treating cancer (inhibiting tumor growth) and / or chronic viral infections by administering to patients requiring such treatment the anti-PD-1 / anti-4-1BB bispecific binding molecule described herein (or a pharmaceutical composition containing the anti-PD-1 / anti-4-1BB bispecific binding molecule). This disclosure discloses antibodies suitable for the treatment, prevention, and / or relief of diseases or conditions such as cancer or viral infections, and / or relief of at least one symptom associated with such diseases, conditions, or illnesses. In the context of the treatments described herein, the anti-PD-1 / anti-4-1BB bispecific binding molecule may be administered as a monotherapy (i.e., as the sole medical agent) or in combination with one or more additional medical agents (examples of which are illustrated elsewhere in this document).

[0397] In some embodiments of this disclosure, the antibodies described herein are applicable to the treatment of individuals with 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, myelohematopoietic syndrome, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer, and uterine cancer.

[0398] Antibodies can be used to treat symptoms of early or late-stage cancer. In one embodiment, the disclosed antibody or a fragment thereof can be used to treat metastatic cancer. Antibodies are suitable for reducing or inhibiting or shrinking tumor growth in both solid tumors and hematologic malignancies. In some embodiments, treatment with the disclosed antibody or its antigen-binding fragment results in tumor regression of more than 50%, more than 60%, more than 70%, more than 80%, or more than 90% in an individual. In some embodiments, antibodies can be used to prevent tumor recurrence. In some embodiments, antibodies are suitable for prolonging the overall survival of an individual with cancer. In some embodiments, antibodies are suitable for reducing toxicity attributed to chemotherapy or radiotherapy while maintaining long-term survival in cancer patients.

[0399] In some embodiments, the disclosed antibody is suitable for treating individuals suffering from chronic viral infections. In some embodiments, the antibody of the present invention is suitable for reducing viral titers in the host and / or rescuing exhausted T cells. In some embodiments, the disclosed antibody or a fragment thereof can be used to treat chronic viral infections caused by lymphocytic choriomeningitis virus (LCMV). In some embodiments, the disclosed antibody or an antigen-binding fragment thereof can be used to administer a medical dose to patients suffering from human immunodeficiency virus (HIV), human papillomavirus (HPV), or hepatitis B / C virus (HBV / HCV) infection. In related embodiments, the disclosed antibody or an antigen-binding fragment thereof can be used to treat simian immunodeficiency virus (SIV)-infected ape individuals, such as macaques.

[0400] In some embodiments, the antibodies disclosed herein can deliver a medically effective amount to individuals suffering from cancer or viral infections.

[0401] One or more of the antibodies disclosed herein may be administered to relieve or prevent or reduce the severity of one or more symptoms or conditions of a disease or ailment.

[0402] This article also includes patients who are developing diseases or conditions such as cancer and chronic viral infections and who are using one or more of the antibodies disclosed herein for preventive treatment.

[0403] In a further embodiment of this disclosure, the antibody is used to prepare a pharmaceutical composition for treating patients suffering from cancer or viral infections. In another embodiment of this disclosure, the antibody is used as an adjunct therapy to any other formulation or therapy known to those skilled in the art as suitable for treating cancer or viral infections. Combination therapies and formulations

[0404] Combination therapy may include the anti-PD-1 / anti-4-1BB bispecific binding molecule disclosed herein (or any individual component thereof) and any additional medical agents that may be beneficial in combination with the antibodies disclosed herein.

[0405] This disclosure discloses antibodies that can be synergistically combined with one or more anticancer drugs or therapies for the treatment of cancers, 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, myelohematopoietic syndrome, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, renal / kidney cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer, and uterine cancer. This article covers the use of the disclosed anti-PD-1 / anti-4-1BB antibodies in combination with immunostimulatory and / or immunosupportive therapies to inhibit tumor growth and / or enhance cancer patient survival. Immunostimulatory therapies include direct immunostimulatory therapies, which activate the immune response by "releasing the brakes" or "accelerating" suppressed immune cells to enhance immune cell activity. Examples include targeting other checkpoint receptors, adoptive cell therapy, vaccination, and adjuvants. Immune supportive approaches can enhance tumor antigenicity by promoting immunogenic cell death, inflammation, or other indirect effects that promote antitumor immune responses. Examples include radiation, chemotherapy, anti-angiogenic agents, and surgery.

[0406] In various embodiments, one or more of the antibodies disclosed herein may be used in combination: an antibody against PD-L1; a secondary antibody against PD-1 (e.g., nivolumab); an antibody against 4-1BBL; a secondary antibody against 4-1BB; a LAG-3 inhibitor; and CTLA. T-4 inhibitors (e.g., ipilimumab); TIM-3 inhibitors; BTLA inhibitors; TIGIT inhibitors; CD47 inhibitors; antagonists of another T-cell co-inhibitor or ligand (e.g., antibodies against PD-L2, CEACAM, VISTA, LAIR-1, 2B4, B7-H3, B7-H4, KIR, A2aR, GAL9, or TGFR); agonists of T-cell co-stimulators (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., VEGF-trap (VEGF-1) inhibitors). Anti-VEGF antibodies (such as aflibercept) or other VEGF-inhibitory fusion proteins as shown in U.S. Patent No. 7,087,411, or their antigen-binding fragments [e.g., bevacizumab or ranibizumab], or small molecule kinase inhibitors of VEGF receptors [e.g., sunitinib, sorafenib, or pazopanib]; Ang2 inhibitors (e.g., nesvacumab); transformed growth factor β (TGFβ) inhibitors; epidermal growth factor receptor (EGFR) inhibitors (e.g., erlotinib, cetuximab); agonists of co-stimulatory receptors (e.g., agonists of glucocorticoid-induced TNFR-related proteins); and tumor-specific antigens (e.g., CA9, CA125, melanoma-associated antigen 3). Antibodies against MAGE3, carcinoembryonic antigen (CEA), vimentin, tumor-M2-PK, prostate-specific antigen (PSA), mucin-1, MART-1, and CA19-9; vaccines (e.g., Bacillus Calmette-Guerin, cancer vaccines); adjuvants that enhance antigen presentation (e.g., granulocyte-macrophage community-stimulating factor); bispecific antibodies (e.g., CD3×CD20 bispecific antibody, PSMA×CD3 bispecific antibody); cytotoxins;Chemotherapy agents (e.g., dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, and vincristine); cyclophosphamide; radiotherapy; 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-12. -15; Antibody-drug conjugates (ADCs) (e.g., anti-CD19-DM4 ADC and anti-DS6-DM4 ADC); Immunocytocins (e.g., anti-FAP×IL-2v [e.g., RO6874281], anti-tendinin (anti-Atenascin)C×IL-2 [e.g., F16-IL2, aka teleukin], anti-GD2×IL-2 [e.g., hu14.18-IL2], anti-EDB×IL-2 [e.g., L19-IL2, aka darleukin], anti-EDB×TNF [e.g., L19-TNF, aka fibromun], anti-histone complex×IL-12 [e.g., NHS-IL12], anti-EDB×IL-12 [e.g., L19-IL12, aka descarboxin] [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 non-steroidal anti-inflammatory drugs); dietary supplements, such as antioxidants;Or any palliative care for cancer treatment. In some embodiments, the anti-PD-1 antibody disclosed herein can be used in combination with cancer vaccines (including dendritic cell vaccines, oncolytic viruses, tumor cell vaccines, etc.) or adoptive cell therapy to enhance the anti-tumor response. Examples of cancer vaccines that can be used in combination with the anti-PD-1 antibody disclosed herein include the MAGE3 vaccine for melanoma and bladder cancer, the MUC1 vaccine for breast cancer, EGFRv3 (e.g., Rindopepimut) for brain cancer (including glioblastoma multiforme), or ALVAC-CEA (for CEA+ cancer).

[0407] In some embodiments, the disclosed anti-PD-1 / anti-4-1BB antibody can be administered in combination with radiotherapy to produce a long-term sustained anti-tumor response and / or enhance cancer patient survival. In some embodiments, the disclosed anti-PD-1 / anti-4-1BB antibody can be administered to cancer patients before, simultaneously with, or after radiotherapy. For example, one or more doses of radiotherapy can be administered to the tumor lesion, followed by one or more doses of the disclosed anti-PD-1 / anti-4-1BB antibody. In some embodiments, radiotherapy can be administered locally to the tumor lesion to enhance the local immunogenicity of the patient's tumor (adjuvant irradiation) and / or kill tumor cells (ablation irradiation), followed by systemic administration of the disclosed anti-PD-1 / anti-4-1BB bispecific binding molecule. For example, intracranial irradiation can be administered to patients with brain cancer (e.g., glioblastoma multiforme) in combination with systemic administration of the disclosed anti-PD-1 / anti-4-1BB bispecific binding molecule. In some embodiments, the anti-PD-1 / anti-4-1BB antibody disclosed herein can be combined with radiotherapy and chemotherapeutic agents (e.g., temozolomide) or VEGF antagonists (e.g., aflibercept).

[0408] In some embodiments, the anti-PD-1 / anti-4-1BB antibody disclosed herein may be administered in combination with one or more antiviral drugs to treat chronic viral infections caused by LCMV, HIV, HPV, HBV, or HCV. Examples of antiviral drugs include (but are not limited to): zidovudine, lamivudine, abacavir, ribavirin, lopinavir, efavirenz, cobicistat, tenofovir, rilpivirine, and corticosteroids. In some embodiments, the anti-PD-1 / anti-4-1BB antibody disclosed herein may be administered in combination with any antagonist of a LAG3 inhibitor, a CTLA-4 inhibitor, or another T-cell co-inhibitor that can treat chronic viral infections.

[0409] Additional therapeutic agents(groups) / components(groups) may be administered before, simultaneously with, or after administration of the anti-PD-1 / anti-4-1BB bispecific binding molecule disclosed herein. For the purposes of this disclosure, such administration procedures may be considered as a "combined administration" of a second therapeutic active component by the anti-PD-1 / anti-4-1BB bispecific binding molecule.

[0410] The additional medically active ingredient(s) may be administered to an individual prior to administration of the disclosed anti-PD-1 / anti-4-1BB bispecific binding molecule. For example, if the first component 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 1 minute prior to administration of the second component, the first component is considered to be administered "before" the second component. In other embodiments, the additional medically active ingredient(s) may be administered to an individual after administration of the disclosed anti-PD-1 / anti-4-1BB bispecific binding molecule. For example, if the first component 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 the second component is administered, then the first component is considered to have been administered "after" the second component. In other embodiments, the additional medically active ingredient(s) may be administered to the individual simultaneously with the anti-PD-1 / anti-4-1BB bispecific binding molecule disclosed herein. For the purposes of this disclosure, "simultaneously" includes, for example, administering the anti-PD-1 / anti-4-1BB bispecific binding molecule and the additional medically active ingredient to the individual in a single dosage form (e.g., co-formulated), or in separate dosage forms, administered to the individual at intervals of approximately 30 minutes or less. When administered in separate dosage forms, each dosage form can be administered via the same route (e.g., both the anti-PD-1 / anti-4-1BB bispecific binding molecule and the additional active ingredient can be administered intravenously, subcutaneously, intratumorally, etc.); or, each dosage form can be administered via different routes (e.g., the anti-PD-1 / anti-4-1BB bispecific binding molecule can be administered intravenously, and the additional active ingredient can be administered subcutaneously or intratumorally; or the anti-PD-1 / anti-4-1BB bispecific binding molecule can be administered intratumorally, and the additional active ingredient can be administered intravenously or subcutaneously; etc.). In any case, whether the component is administered in a single dosage form, in separate dosage forms administered via the same route, or in separate dosage forms administered via different routes, it is considered "simultaneous administration" for the purposes of this disclosure. For the purposes of this disclosure, administration of the anti-PD-1 / anti-4-1BB bispecific binding molecule "before," "simultaneously," or "after" the additional medically active ingredient (as defined above herein) is considered as "combined administration" of the additional medically active ingredient by the anti-PD-1 / anti-4-1BB bispecific binding molecule.

[0411] This disclosure includes a pharmaceutical composition wherein the anti-PD-1 / anti-4-1BB bispecific binding molecule of this disclosure is co-formulated with one or more additional medically active ingredients (groups) described herein in various dosage combinations. Treatment course

[0412] According to certain embodiments of this disclosure, multiple doses of the disclosed anti-PD-1 / anti-4-1BB antibody (or any individual component thereof) – or a pharmaceutical composition comprising an anti-PD-1 antibody and any additional medically active agent described herein – may be administered to an individual over a specified period of time. The method of this manner according to this disclosure includes sequentially administering multiple doses of the disclosed anti-PD-1 / anti-4-1BB antibody to an individual. As used herein, "sequentially administered" means that each dose of the disclosed anti-PD-1 / anti-4-1BB antibody is administered to the individual at different time points, for example, at predetermined intervals (e.g., hours, days, weeks, or months). The method included in this disclosure includes sequentially administering an initial dose of anti-PD-1 / anti-4-1BB antibody to a patient, followed by one or more second doses of anti-PD-1 / anti-4-1BB antibody, and, if necessary, one or more third doses of anti-PD-1 antibody. Anti-PD-1 / anti-4-1BB antibodies can be administered at doses ranging from 0.1 mg / kg to 100 mg / kg.

[0413] As used herein, the terms “initial dose,” “second dose,” and “third dose” refer to the temporal sequence of administration of the disclosed anti-PD-1 / anti-4-1BB antibody. Therefore, “initial dose” is the dose administered at the start of treatment (also known as the “baseline dose”); “second dose” is the dose administered following the initial dose; and “third dose” is the dose administered following the second dose. The initial, second, and third doses may all contain the same amount of anti-PD-1 / anti-4-1BB antibody, but may typically differ in their administration frequencies. However, in some embodiments, the anti-PD-1 / anti-4-1BB antibody content in the initial, second, and / or third doses may vary during treatment (e.g., adjusted upwards or downwards as appropriate). In some embodiments, two or more doses (e.g., 2, 3, 4, or 5) are administered at the start of treatment as a “load dose,” followed by subsequent doses at a lower frequency (e.g., a “maintenance dose”).

[0414] In certain exemplary embodiments of this disclosure, each second and / or third dose is administered immediately after the previous dose for 1 to 26 weeks (e.g., 1, 1½, 2, 2½, 3, 3½, 4, 4½, 5, 5½, 6, 6½, 7, 7½, 8, 8½, 9, 9½, 10, 10½, 11, 11½, 12, 12½, 13, 13½, 14, 14½, 15, 15½, 16, 16½, 17, 17½, 18, 18½, 19, 19½, 20, 20½, 21, 21½, 22, 22½, 23, 23½, 24, 24½, 25, 25½, 26, 26½ weeks, or longer). The phrase "immediately following the previous dose" used in this article means that in a multiple dosing sequence, the dose of anti-PD-1 / anti-4-1BB antibody given to the patient is given immediately before the next dose, without any other doses in between.

[0415] The method according to this embodiment of the invention may include administering a second and / or third dose of anti-PD-1 / anti-4-1BB antibody to a patient at any number of times. For example, in some embodiments, only one second dose is administered to the patient. In other embodiments, two or more doses (e.g., 2, 3, 4, 5, 6, 7, 8, or more) of the second dose are administered to the patient. Similarly, in some embodiments, only one third dose is administered to the patient. In other embodiments, two or more doses (e.g., 2, 3, 4, 5, 6, 7, 8, or more) of the third dose are administered to the patient.

[0416] In embodiments involving multiple second doses, the dosing frequency of each second dose may be the same as that of the other second doses. For example, each second dose may be administered to the patient 1 to 2 weeks or 1 to 2 months immediately following the previous dose. Similarly, in embodiments involving multiple third doses, the dosing frequency of each third dose may be the same as that of the other third doses. For example, each third dose may be administered to the patient 2 to 12 weeks immediately following the previous dose. In some embodiments of the invention, the frequency of administering the second and / or third doses to the patient may vary during treatment. Alternatively, during treatment, the dosing frequency may be adjusted by the physician according to the individual patient's needs after a clinical examination.

[0417] In the treatment regimen disclosed herein, patients are administered 2 to 6 loading doses at a first frequency (e.g., once a week, once every two weeks, once every three weeks, once a month, once every two months, etc.), followed by two or more maintenance doses at a lower frequency. For example, according to this disclosure, if the loading dose is administered once a month (e.g., two, three, four, or more loading doses once a month), then maintenance doses can be administered to patients 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. Diagnostic uses of antibodies

[0418] This disclosure discloses an anti-PD-1 / anti-4-1BB bispecific binding molecule (or any individual component thereof, i.e., a single variable domain of anti-4-1BB immunoglobulin, or an anti-PD-1 antibody or its antigen-binding fragment) that can be used to detect and / or measure PD-1 and / or 4-1BB in a sample, for example, for diagnostic purposes. Some embodiments contemplate the use of one or more binding molecules of this invention in analytical methods to detect diseases or conditions, such as cancer or chronic viral infections. Examples of diagnostic analytical methods for PD-1 and / or 4-1BB may include, for example, contacting a sample obtained from a patient with the disclosed anti-PD-1 / anti-4-1BB bispecific binding molecule (or one of its individual components), wherein the anti-PD-1 / anti-4-1BB bispecific binding molecule (or one of its individual components) is labeled with a detectable marker or reporter molecule or used as a capture ligand to selectively separate 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 invention (or one of its individual components) can be combined with a second antibody that already possesses a detectable marker for diagnostic purposes. This detectable marker or reporter molecule can be a radioisotope, such as 3H, 14C, 32P, 35S, or 125I; a fluorescent or chemiluminescent part, such as luciferin isocyanate or rhodamine; or an enzyme, such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Specific analytical methods for detecting or measuring PD-1 and / or 4-1BB in a sample include enzyme-binding immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell screening (FACS).

[0419] Samples that can be used for the diagnostic analysis of PD-1 and / or 4-1BB according to this disclosure include any tissue or fluid sample from patients in normal or pathological states, containing detectable amounts of PD-1 protein and / or 4-1BB protein, or fragments thereof. Typically, PD-1 and / or 4-1BB are first measured in specific samples from healthy patients (e.g., patients without cancer) to establish baseline or standard values ​​for PD-1 and / or 4-1BB. This baseline value for PD-1 and / or 4-1BB can then be compared with PD-1 and / or 4-1BB measured in samples from individuals suspected of having cancer-related conditions or symptoms associated with such conditions.

[0420] This disclosure discloses that anti-PD-1 / anti-4-1BB bispecific binding molecules (or any individual component thereof, i.e., a single variable domain of anti-4-1BB immunoglobulin, or an anti-PD-1 antibody or its antigen-binding fragment) may not contain additional markers or demerits, or may contain N-terminal or C-terminal markers or demerits. In one embodiment, the marker or demerit is biotin. In binding assays, the position of the marker (if present) can determine the orientation of the peptide relative to the binding of the peptide. For example, if avidin is coated on a surface, the orientation of a peptide containing N-terminal biotin will cause the C-terminal portion of the peptide to be away from the surface.

[0421] This disclosure further utilizes the following examples, which should not constitute further limitations. All illustrations in this application and all excerpts from references, patent cases, and published patent applications are incorporated herein by reference for all purposes.

[0422] Furthermore, according to this disclosure, conventional biotechnology, microbiology, and recombinant DNA technology known to those skilled in the art can be employed. These techniques have been fully described in the literature. See, for example: Green & Sambrook, *Molecular Cloning: A Laboratory Manual*, 4th edition (2012), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; *DNA Cloning: A Practical Approach*, Volumes I & II (edited by DN Glover, 1985); *Oligonucleotide Synthesis* (edited by MJ Gait, 1984); *Nucleic Acid Hybridization* (edited by BD Hames & SJ Higgins, 1985); *Transcription And Translation* (edited by BD Hames & SJ Higgins, 1984); *Animal Cell Culture* (edited by RI Freshney, 1986); *Immobilized Cells And Enzymes* (IRL Press, 1986); B. Perbal, *A Practical Guide to Molecular Cloning* (1984); FM Ausubel et al. (editors), *Current Protocols in Molecular Biology*, John Wiley & Sons, Inc. (1994). Examples

[0423] The following examples are provided to those skilled in the art to fully disclose and explain how to manufacture and use the features of the present invention, and are not intended to limit the scope of the inventors' invention. Efforts have been made to ensure the accuracy of the numerical values ​​used (e.g., dosage, temperature, etc.), but some experimental errors and deviations should still be considered. Unless otherwise stated, parts are by weight, molecular weight is the average molecular weight, temperature is in Celsius, and pressure is at or near atmospheric pressure. Example 1: Anti-4-1BB V HH Materials and Methods: Alpaca Immunization and Establishment of Collection Bank

[0424] Immunization and library establishment were performed at the "VIB Nanobody Core" (Vrije Universiteit Brussel, Brussels, Belgium). Alpacas were injected intradermally four times, each time with approximately 2 mg of a vector containing the 4-1BB gene. After each injection, the animals underwent electroporation to introduce the vector into animal cells. Three weeks after the last vector injection, recombinant 4-1BB protein was injected subcutaneously. Four days after the protein injection, anticoagulated blood was collected to establish the VHH library.

[0425] Establishment of a VHH library. Total ribonucleic acid was extracted from peripheral blood lymphocytes and used as a template to synthesize the first complementary DNA using an oligo(dT) primer. The VHH coding sequence was amplified from the complementary DNA by polymerase chain reaction (PCR), digested with PstI and NotI, and the site between PstI and NotI upstream of the decapeptide human influenza hemagglutinin-tagged phage plasmid vector pHEN4 was selected. A VHH library of approximately 10⁸ independent transformants was obtained. Phage presentation was used for panning.

[0426] The bacterial collection was grown in 2YTAG medium (2×YT medium, 100 µg / mL ampicillin, 2% glucose) until the absorbance at 600 nm (OD 600) reached 0.5, and then infected with M13K07 helper phage (Invitrogen). After centrifugation, the bacteria were resuspended in 2YTAK medium (2×YT medium, 100 µg / mL ampicillin, 50 µg / mL kanamycin) and grown overnight. 20% w / v polyethylene glycol 8000 (PEG8000) and 2.5 M NaCl were added to precipitate the phage particles from the culture supernatant. The phages were centrifuged and resuspended in phosphate-buffered saline (PBS). The phages underwent another washing and precipitation process, and finally resuspended in cold PBS / 15% v / v glycerol. Cells were panned.

[0427] FreeStyle™ HEK293-FS cells transfected with 4-1BB were panned at 4°C. The phage-V HH library was saturated in PBS / 2% w / v BSA and cultured at 2 × 10⁷ cells / 2 hours at 4°C. After washing twice with PBS, the cell clumps were resuspended in PBS and loaded into a fetal bovine serum / Percoll gradient. After centrifugation, the cell layer was collected and washed twice with PBS. Cells conjugated with phage were recovered and added to a second portion of the fetal bovine serum / Percoll gradient. After washing, mechanical lysis was performed using Dynabeads (Invitrogen). The recovered phage-V HH was used to infect exponentially growing *Escherichia coli* TG1 bacteria and amplified overnight in 2YTAG medium for several panning cycles, or plated onto 2YTAG culture plates. Fab-like construction, production, and purification.

[0428] After amplification by polymerase chain reaction, complementary DNA of anti-4-1BB V HH was selected and inserted into the reading frame containing the human CL domain or human IgG1 CH1 domain in a specific mammalian expression vector, and fused with human influenza hemagglutinin and a 6-His tag (both tags were used for purification purposes). The same treatment was performed using anti-OX40 and anti-CD28 V HH, as well as anti-foot-and-mouth disease virus (FMDV) V HH, the latter serving as a control group, which will be described in detail below.

[0429] Plasmids were purified using the NucleoBond Macherey-Nagel kit and subjected to Sanger sequencing. FreeStyle™ HEK293-FS cells were co-transfected with a mixture of two plasmids encoding two different V HHs (one fused to the human CL domain and the other to the human IgG1 CH1 domain) to generate bivalent monocomplementary (i.e., two identical anti-4-1BB V HHs), bivalent bicomplementary (i.e., two different anti-4-1BB V HHs), and bispecific (i.e., one anti-4-1BB V HH and one V HH targeting another) Fab-like constructs. The supernatant was collected after 7 days, purified on a nickel affinity column, and analyzed on a CALIPER GXII (Perkin Elmer). Phage-V HHs were generated in 96-well plates for ELISA.

[0430] Individual TG1 colonies of the 4-1BB V HH of interest were collected and grown in 2YTA medium at 37°C until the OD 600 reached 0.5. Cells were then infected with M13K07 helper phage and grown overnight at 30°C. The supernatant containing phage-V HH was collected for the assay. ELISA combined with analytical method.

[0431] ELISA analysis was performed on a Nunc® MaxiSorp™ 96-well plate (Sigma) pre-coated with 1 µg / mL of human His-tagged 4-1BB recombinant protein in PBS, incubated overnight at 4°C, and then saturated for 1 hour at room temperature using PBS / 2% milk. Bacterial supernatant containing phage-V HH, purified Fab-like constructs, or 4-1BBL-Fc was incubated for 1 hour at room temperature. After several washes in PBS / 0.1% Tween, the following HRP-conjugating antibodies were added: anti-HA-tagged mAb for detecting bound Fab-like constructs (Sigma), anti-M13 mAb for detecting bound phage-V HH (Santa Cruz Biotechnology), and anti-human Fc mAb for detecting bound 4-1BBL-His. Peroxidase activity was detected using TMB (3,3',5,5'-tetramethylbenzidine, KPL) as a reagent, and OD at 450 nm was measured using a SpectraMax microanalyzer after adding sulfuric acid as a stop solution. ELISA competitive assay.

[0432] ELISA analysis was performed on a Nunc® MaxiSorp™ 96-well disc (Sigma) pre-coated with 1 µg / mL of human His-tagged 4-1BB recombinant protein in PBS, incubated overnight at 4°C, and then saturated for 1 hour at room temperature using PBS / 2% milk. For antigen epitope compartmentalization competition, serially diluted bivalent Fab-like constructs were incubated at room temperature for 1 hour, followed by the addition of EC90 phage-V HH, and incubated for an extended 45 minutes at room temperature. Alternatively, in the competitive assay, serially diluted control anti-4-1BB antibody or 4-1BBL-Fc was incubated at room temperature for 1 hour, followed by the addition of bivalent Fab-like constructs at EC90 concentrations, and incubated for 45 minutes at room temperature. After washing several times with PBS / 0.1% Tween, the following HRP-conjugating antibodies were added: anti-HA-tag mAb (Sigma) for detecting bound Fab-like constructs and anti-M13 mAb (Santa Cruz Biotechnology) for detecting bound phage-V HH. Peroxidase activity was detected using TMB assays, and OD 450 nm activity was measured on a SpectraMax microanalyzer after adding sulfuric acid to stop the reaction. Reporton functional analysis crosslinking assays were performed.

[0433] The test compound and saturated concentration of anti-human Fab (Sigma, I5260) were pre-cultured at room temperature for 30 minutes. During the isoexponential growth phase, Jurkat-4-1BB-NF-κB (Promega) cells were collected, and 25 µL of cell suspension was added to 96-well plates (50,000 cells / well) containing 25 µL of cross-linked or non-cross-linked test compound. The cells were then subjected to OX40-expression conditions.

[0434] Jurkat-4-1BB-NF-κB cells were collected during the exponential growth phase and mixed with OX40-expressing cells to obtain a final reporter-to-accessory cell ratio of 1:1. 25 µL of the cell suspension was added to a 96-well plate (50,000 cells / well) containing 25 µL of the test compound. All conditions were maintained.

[0435] The culture dishes were incubated in a humid incubator at 37°C and 5% CO2 for 6 hours. Then, 50 μL of Bio-Glo™ (Promega, G7941) reagent, prepared according to the manufacturer's instructions, was added to each well and mixed. Intact cells were allowed to lyse for at least 5 minutes, and then luminescence was measured using an Envision multi-function reader (Perkin Elmer). The results showed the production of anti-4-1BB V HH.

[0436] Anti-4-1BB V HH was obtained from alpacas. More specifically, V HH was selected from alpaca immunization and the original V HH collection library. After inoculating an alpaca with 4-1BB-DNA, a V HH phage collection library (i.e., an immune collection library) was established from PBMCs. During alpaca immunization, six injections of plastids representing the full length of human 4-1BB were administered; a final injection of recombinant human 4-1BB (SinoBiological) was given.

[0437] VHHs selected from recombinant 4-1BB protein or 4-1BB-expressing cells were screened for their binding to 4-1BB and further sequenced. The characteristics of seven pure lines (pure lines #1 to #7) were further analyzed in the binding agent. These seven anti-4-1BB V HHs were reorganized into bivalent monocomplementary sites (i.e., two identical anti-4-1BB V HHs), bivalent bicomplementary sites (i.e., two different anti-4-1BB V HHs), bispecific (i.e., one anti-4-1BB V HH and one V HH targeting another target), or monovalent (i.e., one anti-4-1BB V HH and one V HH targeting an unrelated target, such as FMDV) molecules, forming a "Fab-like" format. The first V HH is fused to the CH1 human IgG constant domain, and the second V HH is fused to the CLλ human IgG constant domain; both the first and second V HHs are linked via CH1-CL interactions (Figure 1). The molecule contains hemagglutinin (HA) and polyhistamine (His) tags for purification purposes. Specificity of anti-4-1BB V HHs.

[0438] Seven V HHs (each containing two identical anti-4-1BB V HHs in a bivalent monocomplementary "Fab-like" construct) were evaluated, and their binding affinity to human 4-1BB, macaque ("cyno") 4-1BB, and two TNFRSF members (human OX40 and human CD40) was tested.

[0439] Results are shown in Figure 2: The seven VHHs exhibit varying degrees of cross-reactivity with human 4-1BB and macaque 4-1BB. None of the seven VHHs showed binding to human OX40 or human CD40. Competition and epitope binning.

[0440] In a competitive assay of the natural ligand 4-1BB 4-1BBL against anti-4-1BB and the anti-control anti-4-1BB antibody, the diversity of 4-1BB binding sites for seven V HHs (bivalent monocomplementary "Fab-like" constructs, each containing two identical anti-4-1BB V HHs) was evaluated.

[0441] Based on the competitive patterns of seven V HH antibodies against 4-1BBL and anti-4-1BB, the three different epitope compartments (compartment A, compartment B, and compartment C) were identified using this analytical method. The results are shown in Table 3 and Figure 3. Table 3: Epitope Compartments V HH Antigen epitope compartments Competing with 4-1BBL? Competition with control anti-4-1BB antibody? V HH source Pure Series #1 Warehouse A no yes Alpaca Immune Collection Library Pure Series #2 Warehouse A no yes Pure Series #3 Warehouse B no no Pure Series #4 Warehouse B no no Original collection library Pure Series #5 C warehouse yes no Pure Series #6 Warehouse A no yes Pure Series #7 Warehouse A no It is the activating effect of anti-4-1BB V HH on T cells.

[0442] Activation of 4-1BB results in activation of the NF-κB pathway. NF-κB reporter assays were performed to detect activation of the 4-1BB signaling pathway mediated by anti-4-1BB V HH. NF-κB activation was measured using a bioluminescent cell-based reporter assay (Promega). This assay consisted of genetically engineered Jurkat T-cell lines that consistently expressed 4-1BB, in which the luciferase system was controlled by NF-κB responsive elements. Therefore, 4-1BB activation resulted in luciferase expression.

[0443] Anti-4-1BB VHH in the previously described "Fab-like" format was evaluated in the presence or absence of the cross-linking agent (anti-Fab antibody) (soluble conditions), either as a monocomplementary bivalent (each containing two identical anti-4-1BB V HH) or as a monocomplementary monovalent (containing one anti-4-1BB V HH and one V HH against an unrelated target, i.e., the FMDV protein). The results are shown in Figures 4A-D. All bivalent constructs (except the negative control [anti-FMDV]) exhibited dose-dependent activity upon cross-linking (Figure 4A), but in the absence of cross-linking, only pure line #5 maintained a high activation potential (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 that of their individual bivalent constructs (Figure 4C). Finally, in the NF-κB reporter assay, uncrosslinked monovalent molecules showed no activation (Figure 4D).

[0444] Next, we investigated the bivalent double-complementary-site constructs (each containing two different "Fab-like" constructs of anti-4-1BB V HH). These molecules were tested under soluble conditions (i.e., without crosslinking) and compared with the bivalent monocomplementary-site and monovalent constructs. The results are shown in Figure 4E. In the presence of any other tested anti-4-1BB V HH, the bivalent double-complementary-site construct containing pure line #5 demonstrated the highest activation activity under soluble conditions.

[0445] In summary, these results confirm that the bivalent monocomplementary "Fab-like" construct containing two anti-4-1BB pure line #5 V HHs is highly active under soluble conditions (i.e., without cross-linking agents, such as anti-Fab antibodies, making it a "pure agonist") and competes with 4-1BBL, but not with the control anti-4-1BB antibody. Notably, when used monovalently (in combination with unrelated V HHs, such as anti-FMDV V HHs), pure line #5 showed no activity; however, pure line #5 was active when combined with a V HH targeting another antigen expressed by the same cells in a bispecific configuration (e.g., CD28 – Figure 4F). When in a bivalent bicomplementary configuration (in combination with an anti-4-1BB V HH targeting another 4-1BB epitope), pure line #5 even showed a modified and potent agonist effect in the activation of the NF-κB pathway.

[0446] Finally, as shown in Figures 5A-B, the bispecific "Fab-like" construct, comprising a combination of anti-4-1BB pureline #5 V HH and a V HH targeting an antigen expressed by another cell (e.g., OX40 expressed on helper cells), converts the anti-4-1BB pureline #5 V HH into a cell adaptor and T-cell activator. 4-1BB binds to antigens (e.g., OX40) on both Jurkat T cells and helper cells, inducing immune cell activation. This feature presents noteworthy perspectives, such as enabling T-cells to target pathogenic cells. Structural Feature Analysis

[0447] The 3-D structure of anti-4-1BB pure line #2 and pure line #5 V HH in the complex with the extracellular domain of human 4-1BB (amino acid residues 24-186 of SEQ ID NO: 13) was determined by X-ray crystallography.

[0448] The 3-D structure (Figure 6) highlights the different parts of the two V HH pure lines that bind to 4-1BB: - The anti-4-1BB pure line #2 V HH mainly binds to the first enriched cysteine ​​domain (CRD) of 4-1BB; - The anti-4-1BB pure line #5 V HH binds to both the second and third CRDs of 4-1BB and overlaps with the 4-1BBL binding antigen epitope.

[0449] All three CDRs 1-3 of the anti-4-1BB pure series #2 V HH have been found to interact with 4-1BB; however, according to the 3-D structure of X-ray crystallography, of all CDRs, only CDR 3 of the anti-4-1BB pure series #5 V HH shows interaction with 4-1BB. Anti-4-1BB pure series #5 V HH variants

[0450] Among all the previously isolated 4-1BB binders, several V HHs with sequences similar to the anti-4-1BB pure series #5 V HH were identified. These were also prepared in a bivalent monocomplementary "Fab-like" format as described above. Their binding and functional properties were evaluated: the anti-4-1BB pure series #5a, #5b, #5c, and #5d V HHs showed that, compared to the anti-4-1BB pure series #5 V HH, they had a similar binding EC50 against 4-1BB (Figure 7A) without improvement, and compared to the anti-4-1BB pure series #5 V HH, they exhibited higher activity under soluble conditions (i.e., without cross-linking agents, such as anti-Fab antibodies) (Figure 7B). Sequence

[0451] The sequences of anti-4-1BB pure line #2 and pure line #5 V HH, and the sequences of variants of anti-4-1BB pure line #5 V HH (pure lines #5a, #5b, #5c and #5d) are shown below, with their isochronous CDR sequences specifically marked (IMGT numbers are in bold; Kabat numbers are underlined; Chothia numbers are in italics).

[0452] Pure line #2 (SEQ ID NO: 1): QVQLQESGGGLVQPGGSLRLSCAAS GGLFS IN T GGWYRQAPGKQRELVA TI THDDRTNYAESVKGRFTLSRDNAKNTVYLQMNSLKPEDTAVYYC RL GSAAIRGYWGQGTQVTVSS

[0453] Pure Line #5 (SEQ ID NO: 2): QVQLQESGGGLVQPGGSLRLSCAAS GFTFS DH T MTWVRQAPGKGLEWVS SI SSGGSRIIYADSVKGRFTISRDNAKNTLYLQMNNLRPEDTAVYFC AR GTRYKMSTSGPGTQVTVSS

[0454] Pure Line #5a (SEQ ID NO: 58): QVQLQESGGGWVQPGGSLRLSCAAS GFAFR DF T MSWARQAPGERFEWIS SI NPSGGSQSYLPSVKGRFTISRDNAKNTMFLQMDNLTPEDTAVYFC AR GTRYKMSTSGPGTQVTVSS

[0455] Pure line #5b (SEQ ID NO: 59): QVQLQESGGGWVQPGGSLRLSCAAS GFAFR DF T MSWARQAPGERFEWIS SI NPSGGSQSYLPSVKGRFTISRDNAKNTMFLQMDNLTPEDTAVYFC AR GTRYKMSTSGSGTQVTVSS

[0456] Pure line #5c (SEQ ID NO: 60): QVQLQESGGGLVQAGGSLRLSCAAS GDTFS SY A MGWFRQAPGERLEWVA SI NPSGGSQSYHPSVKDRFTISRDNGKNILFLQLDKLNPEDTAVYVC AR GTRYKIFASGQGTQVTVSS

[0457] Pure line #5d (SEQ ID NO: 61): QVQLQESGGGLVQPGGSLRLSCVAS GFTFA NY R MSWVRQAPGKGLEWVS SI KKSGNRTTYSDSVKGRFTISRDNAKNTMFLQMDNLTPEDTAVYFC AR GTRYKMSTSGPGTQVTVSS Example 2 Anti-PD-1 antibody

[0458] Our goal is to produce monoclonal anti-PD-1 antibodies that bind to PD-1 in humans and macaques and compete with the PD-1 ligand PD-L1.

[0459] In short, Trianni® mice were immunized to obtain 3,556 IgGs, which were then screened for binding to human and macaque PD-1. Redundant sequences were eliminated, and the remaining IgGs were reassembled into IgG1 LALA. Hit point characteristics were analyzed, and 59 pure lines were selected for further in vitro functional analysis (including PD-1 / PD-L1 blocking bioanalysis and allogeneic MLR analysis). After these in vitro analyses, 8 functional pure lines were selected; these were then reassembled into Fab format and produced, and anti-PD-1 antibody activity in monomeric form was selected.

[0460] Finally, we retain an anti-PD-1 antibody, hereinafter referred to as the pure line "T5," in which the two variable regions exhibit a human reproductive index (i.e., the percentage of amino acid sequence identity of the architectural regions relative to the closest human VH and VL germlines) exceeding 95% (Table 4). The pure anti-PD-1 antibody T5 was also confirmed to be active in the monomeric Fab format in PD-1 / PD-L1 interaction assays (Table 5). Table 4 Heavy chain Light chain The closest human V H Reproductive system Fertility Index (%) The closest human V H Reproductive system Fertility Index (%) Pure T5 IGHV4-39 96.97 IGKV3D-20 95.83 Control anti-PD-1 antibody #1 IGHV3-33 90.82 IGKV3-11 98.95 Control anti-PD-1 antibody #2 IGHV1-2 79.59 IGKV3-20 Table 5, 84.95 Relative to EC 50 (M) Fab:IgG1 ratio IgG1 LALA Fab Pure T5 2.78×10 -10 4.88×10 -8 176 Control anti-PD-1 antibody #1 1.81×10 -10 >1.00×10 -6 >1.00×10 -4 Control anti-PD-1 antibody #2 4.95×10 -10 1.48×10 -8 30 sequences

[0461] The anti-PD-1 pure line T5 has a light chain variable region (LCVR) of the amino acid sequence shown in SEQ ID NO: 5: EIVLTQSPATLSLSPGERATLSC GAS QSVSINFLAWYQQKPGLAPRLLIY EAS SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYGSSPYTFGQGTKLEIK (SEQ ID NO: 5). The CDR sequence is specially marked: IMGT is in bold; Kabat is underlined; Chothia is in italics.

[0462] The anti-PD-1 pure T5 has a heavy chain variable region (HCVR) of the amino acid sequence shown in SEQ ID NO: 6: QLQLQESGPGLVKPSETLSLTCTVS GGSIS TSSY F WGWIRQPPGKGLEWIG SI YRSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYC AR GITGDPGDYWGQGTLVTVSS (SEQ ID NO: 6). The CDR sequence is specifically marked: IMGT is in bold; Kabat is underlined; Chothia is in italics. Example 3 Anti-4-1BB / Anti-PD-1 Bispecific Binding Protein

[0463] The inventors aim to develop an anti-4-1BB / anti-PD-1 bispecific binding molecule comprising the pure agonist anti-4-1BB V HH (pure line #5) described in Example 1 and the specific anti-PD-1 binding protein (pure line "T5") described in Example 2.

[0464] Several structures were designed and tested, including 1 to 2 types of anti-PD-1 Fab (± one Fc region) and 3 to 6 types of anti-4-1BB V HH, depending on the orientation. From these structures, four bispecific structures were selected first (Figure 8).

[0465] Construct #1 contains, from N to C-terminus: - Anti-PD-1 pure line T5 Fab; - IgG1-LALA* Fc region; - Variant of anti-4-1BB pure line #2 V HH as described in Example 1 (i.e., pure line #2.1); and - Variant of anti-4-1BB pure line #5 V HH as described in Example 1 (i.e., pure line #5.1).

[0466] Constructs #3, #5, and #6 contain the following from the N to C-terminus: - A variant of the anti-4-1BB pure line #2 V HH described in Example 1 (i.e., pure line #2.1); - A variant of the anti-4-1BB pure line #5 V HH described in Example 1 (i.e., pure line #5.1); - An IgG1-LALA* Fc region (constructor #3), an IgG1-NNAS** Fc region (constructor #5), or an IgG4-P-FALA*** Fc region (constructor #6); and - A reverse anti-PD-1 pure line T5 Fab. * LALA refers to an ADCC-silent mutation (L234A / L235A) that disrupts the function of the IgG1 Fc effector. ** NNAS refers to an ADCC-silent mutation (S298N / T299A / Y300S) that abolishes the function of the IgG1 Fc effector. *** P-FALA refers to an ADCC-silent mutation (S228P / F234A / L235A) that disrupts the function of the IgG4 Fc effector. Materials and Methods: Performance and Purification of HEK293-FS Cells

[0467] The phenotypes of the heavy and light chains of the test compound were propagated in Escherichia coli (E. coli) DH5α. In E. coli, plastids for transfection were prepared using the EndoFree® Plasmid Mega kit (Qiagen, ref. 12381).

[0468] HEK293-FS cells grown in F17 serum-free suspension medium (Invitrogen) were transfected with polyethyleneimine (PEI) transfection reagent to accept heavy and light chain plastids. After culturing at 37°C for 7 days, the cells were removed by centrifugation, and the supernatant was passed through a 0.22 µm filter to remove particles.

[0469] During purification, the test compounds were captured on a HiTrap® MabSelect SuRe™ column (GE Healthcare, Ref. 11-0034-93), dissociated using 0.1 M citrate buffer at pH 3.0, and directly desalted using a HiPrep 26 / 10 desalting column (GE Healthcare, Ref. 17-05087-02). Proteins were purified by size exclusion chromatography using a HiLoad® 26 / 600 Superdex® 200 (GE Healthcare, Ref. 28-9893-36) followed by a final ultrafiltration concentration step, and then further characterized using the test compounds. CHO cells

[0470] Plastids encoding the heavy and light chains of the test compound were selected and implanted into Evitria's (Zurich, Switzerland) proprietary expression vector system using conventional non-PCR-based selection techniques. The expression vector system was synthesized from the gene. Plastid DNA was prepared by anion exchange chromatography under low endotoxin conditions. The absorbance at 260 nm was measured to determine the DNA concentration. Sequence accuracy was verified using Sanger sequencing (up to two sequencing reactions per plastid, depending on the size of the cDNA).

[0471] CHO K1 cells were suspension-acclimated for production. The bacterial strain was grown in eviGrow medium (Evitria), a chemically defined, animal-free, and serum-free medium. Cells were transfected with a custom-designed transfection reagent, eviFect (Evitria), and the transfected cells were grown in eviMake2 (Evitria), an animal-free and serum-free medium.

[0472] After centrifugation to collect the supernatant, it was then filtered through a 0.2-μm filter.

[0473] The 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), following the same procedure as the purification of HEK293-FS described above. Binding assays for stable cell lines: human PD-1 and 4-1BB.

[0474] Objective: To evaluate the affinity of the anti-4-1BB / anti-PD-1 bispecific binding molecule relative to the control antibody and isotype control for their respective targets (separate 4-1BB and PD-1 groups). Calculate the binding EC50 and Emax values ​​of the test compound.

[0475] The assay was performed on ice in 96-well plates using PD-1 NFAT-luc2 Jurkat cells (Promega #J1252), or 4-1BB NF-κB-luc2P Jurkat cells (Promega #J2332), or self-made transfected 300.19 (pre-B) cells expressing human PD-1 (hPD-1) or human 4-1BB (h4-1BB).

[0476] Spread the cell suspension at a density of 50 × 10³ cells per well in a 96-well U-shaped dish. Add a series of diluted test compounds to the cells for 1 hour. After washing, add a fluorescently labeled secondary antibody targeting the Fc region to each well for 30 minutes. Then, measure the MFI signal by flow cytometry.

[0477] Data from flow cytometry were analyzed using FlowJo (V10.8.1), and then graphs of binding curves, Emax, and EC50 values ​​were plotted using GraphPad Prism (V9.1.2). Rhesus monkey PD-1 and 4-1BB...

[0478] Objective: To evaluate the affinity of the anti-4-1BB / anti-PD-1 bispecific binding molecule relative to the control antibody and isotype control for their respective targets (separate 4-1BB and PD-1 groups). Calculate the binding EC50 and Emax values ​​of the test compound.

[0479] The assay was performed on ice in 96-well plates using self-made transfections of 300.19 (pre-B) cells expressing either macaque PD-1 (cyPD-1) or macaque 4-1BB (cy4-1BB).

[0480] Spread the cell suspension at a density of 50 × 10³ cells per well in a 96-well U-shaped dish. Add a series of diluted test compounds to the cells for 1 hour. After washing, add a fluorescently labeled secondary antibody targeting the Fc region to each well for 30 minutes. Then, measure the MFI signal using flow cytometry.

[0481] Data from flow cytometry were analyzed using FlowJo (V10.8.1), and then graphs of binding curves, Emax, and EC50 values ​​were plotted using GraphPad Prism (V9.1.2). Binding analysis of first-generation human T-cells.

[0482] Objective: To evaluate the affinity of the anti-4-1BB / anti-PD-1 bispecific binding molecule for its equivalent targets (simultaneously targeting 4-1BB and PD-1) relative to the control antibody and isotype control. Calculate the binding EC50 and Emax values ​​of the test compound.

[0483] Human PBMCs were isolated from fresh erythrocyte sedimentation rate (ESR) buffy coat using a Ficoll gradient. Total CD3+ T-cells were isolated using negative selection. T-cells were plated at a density of 50 × 10³ cells / well onto 96-well U-bottom culture dishes pre-coated with 5 µg / mL anti-human CD3 and cultured in X-Vivo 15 medium (Lonza #BE02-061Q) supplemented with 1% penicillin / streptomycin.

[0484] A series of diluted test compounds were added to the cells for 1 hour. After washing, fluorescently labeled secondary antibodies targeting the Fc region were added to each well for 30 minutes. MFI signals were then measured by flow cytometry.

[0485] Data from flow cytometry were analyzed using FlowJo (V10.8.1), and then graphs of binding curves, Emax, and EC50 values ​​were plotted using GraphPad Prism (V9.1.2). Reporter analysis method 4-1BB

[0486] Objective: To evaluate the activity of the anti-4-1BB / anti-PD-1 bispecific binding molecule relative to the control antibody and isotype control in the 4-1BB group. Calculate the EC50 and Emax of each test compound.

[0487] Data were read using 4-1BB reporter Jurkat NF-κB-luc2P Promega cell line (GloResponse NF-κB-luc2P Jurkat Cell, Promega #J2332) cultured in RPMI 1640, 10% SVF, 1% glutamic acid, 1% NEAA, 1 mM sodium pyruvate, 800 µg / mL G418, and 500 µg / mL hygromycin, according to the manufacturer’s instructions.

[0488] Spread cells at a density of 50 × 10³ cells per well in a 96-well white flat-bottomed dish. Add a series of diluted test compounds. After incubating at 37°C for 6 hours, add Bio-Glo reagent to each well. Measure luminescence using an Infinit Pro M1000 or Spark Tecan reader.

[0489] GraphPad Prism (V9.1.2) was used to plot the combined curve, E max, and EC 50 value. PD-1

[0490] Objective: To evaluate the activity of the anti-4-1BB / anti-PD-1 bispecific binding molecule relative to the control antibody and isotype control in the PD-1 group. Calculate the IC50 and Imax for each test compound.

[0491] Data were read from the manufacturer’s instructions in the presence of PD-1 reporter Jurkat NFAT-luc2 Promega cell line (GloResponse PD-1 NFAT-luc2 Jurkat cells, Promega #J1252) cultured in PMI 1640, 10% SVF, 1% glutamic acid, 1% NEAA, 1 mM PyNa, 500 µg / mL G418, and 200 µg / mL hygromycin.

[0492] PD-L1 aAPC / CHO-K1 cells were spread at a density of 40 × 10³ cells per well on 96-well white flat-bottomed culture dishes and cultured overnight at 37°C in Ham / F12 medium supplemented with 1% FCS. The next day, the medium was removed, and PD-1 NFAT-luc2 Jurkat cell suspension at a density of 50 × 10³ cells / well, along with a series of diluted test compounds, were added to the wells. After culturing at 37°C for 6 hours, Bio-Glo reagent was added to each well. The luminescence intensity was measured using an Infinit Pro M1000 or SPARK TECAN reader.

[0493] GraphPad Prism (V9.1.2) was used to plot the binding curve, Imax, and IC50 values. Antibody-dependent cytotoxicity (ADCC) analysis was performed using the ADCC / ADCP / CDC assay.

[0494] Objective: To evaluate the ADCC effect of the anti-4-1BB / anti-PD-1 bispecific binding molecule relative to the control antibody and isotype control using a dose-response assay. Calculate the EC50 and Emax values ​​of the test compound.

[0495] The ADCC assay uses NFAT-luc2 Jurkat reporter cells (Promega #G7102) overexpressing the highly sensitive variant of human FcγRIIIa-V158 as effector cells, co-cultured with self-made rodent 300.19 (pre-B) target cells expressing human PD-1 or human 4-1BB. In 96-well U-bottom suspension trays, 1.5 × 10⁵ Jurkat effector cells and 7.5 × 10⁴ 300.19 target cells per well (E:T ratio = 2:1) were co-co-coated with the test compound, diluted nine times in a 1:4 series to achieve concentrations from 200 nM to 0.003 nM, with triple replicates. After culturing at 37°C, 5% CO2 and 95% Rh for 24 hours, data were read using a Bio-Glo luciferase analysis system (Promega #G7940) and a Tecan Spark microdisk photometric reader.

[0496] The EC50 and Emax values ​​of the test compounds were calculated using the Biostat-Speed ​​statistical tool. The results were obtained using the 4-parameter logistic model based on Ratkovsky & Reedy (Biometrics. 1986 Sep;42(3):575-82). The Levenberg-Marquardt algorithm in SAS v9.1.3 software was used to adjust the results using nonlinear regression. Antibody-dependent phagocytosis (ADCP) analysis.

[0497] Objective: To evaluate the ADCP effect of the anti-4-1BB / anti-PD-1 bispecific binding molecule relative to the control antibody and isotype control using a dose-response assay. Calculate the EC50 and Emax values ​​of the test compound.

[0498] The ADCP assay uses NFAT-luc2 Jurkat reporter cells (Promega #G9871) overexpressing the highly sensitive variant of human FcγRIIa-H131 as effector cells, co-cultured with self-made rodent 300.19 (pre-B) target cells expressing human PD-1 or human 4-1BB. In 96-well U-bottom suspension trays, each well contains 3 × 10⁴ Jurkat effector cells and 1.5 × 10⁴ 300.19 4-1BB target cells (E:T ratio = 2:1) or 10⁵ Jurkat effector cells and 10⁵ 300.19 PD-1 target cells (E:T ratio = 1:1) coated with the test compound at concentrations ranging from 200 nM to 0.003 nM achieved through nine 1:4 dilution steps, with triple replicates. After culturing at 37°C, 5% CO2 and 95% rH for 24 hours, data were read using the Bio-Glo luciferase assay system (Promega #G7940) and the Tecan Spark microdisk photometric reader.

[0499] The EC50 and Emax values ​​of the test compounds were calculated using the Biostat-Speed ​​statistical tool. The results were obtained using the 4-parameter Rogers model based on Ratkovsky & Reedy (Biometrics. 1986 Sep;42(3):575-82). The Levenberg-Marquardt algorithm in SAS v9.1.3 software was used to obtain adjusted results via nonlinear regression. Complement-dependent cytotoxicity (CDC) analysis.

[0500] Objective: To evaluate the CDC effect of the anti-4-1BB / anti-PD-1 bispecific binding molecule relative to the control antibody and isotype control using a dose-response assay. Calculate the IC50 and Imax values ​​of the test compound.

[0501] The CDC assay was performed in a 96-well U-bottom suspension dish. 1.5 × 10⁴ self-made rodent 300.19 (pre-B) target cells expressing human PD-1 or human 4-1BB were coated with 10% human serum complement (Quidel #A113) and the test compound, diluted nine times in a 1:4 series to achieve concentrations ranging from 200 nM to 0.003 nM, with triple overlap. After incubation at 37°C, 5% CO₂, and 95% rH for 24 hours, data were read using a CellTiter-Glo Luminescent Cell Viability analysis system (Promega #G7571) and a Tecan Spark microdisk spectrophotometer.

[0502] The IC50 and Imax values ​​of the test compounds were calculated using the Biostat-Speed ​​statistical tool. The results were obtained using the 4-parameter Rogers model based on Ratkovsky & Reedy (Biometrics. 1986 Sep;42(3):575-82). The adjusted results were obtained using the Levenberg-Marquardt algorithm in SAS v9.1.3 software via nonlinear regression. T-cell activation (TCA) in humans

[0503] Objective: To evaluate the activity of the anti-4-1BB / anti-PD-1 bispecific binding molecule relative to the control antibody and isotype control. To observe the release of cytokines from activated human T cells.

[0504] PBMCs were isolated from the fresh erythrocyte sedimentation rate (ESR) layer using a Ficoll gradient. T cells were isolated using negative selection. T cells were spread at a density of 100 × 10³ cells / well onto 96-well flat-bottomed plates pre-coated with 5 µg / mL anti-human CD3 and cultured in X-Vivo 15 medium (Lonza #BE02-061Q) supplemented with 1% penicillin / streptomycin.

[0505] Add a series of diluted test compounds to the culture medium. After 4 or 6 days of culture, assess cytokine secretion in the collected supernatant using either the CBA human TH1 / TH2 cytokine kit on a flow cytometer according to the manufacturer’s instructions (BD Bioscience #550749) or using homogeneous time-resolved fluorescence (HTRF) human IFN-γ / TNF-α cytokine kits (Cisbio #62HIFNGPEH and #62HTNFAPEH) on a Pherastar FSX multi-disc reader (BMG Labtech) according to the manufacturer’s instructions.

[0506] Cytokine secretion in CBA samples was analyzed using an FCAP array (V3.0.19.2091), followed by plotting using GraphPad Prism (V9.1.2). HTRF cytokine secretion was plotted directly using GraphPad Prism (V9.1.2). (Macaque)

[0507] Objective: To evaluate the activity of the anti-4-1BB / anti-PD-1 bispecific binding molecule relative to the control antibody and isotype control. To observe the release of cytokines from activated macaque T cells.

[0508] Frozen rhesus monkey PBMCs were obtained from Primacyt. Total CD3+ T- cells were isolated by negative selection. T- cells were spread at a density of 50 × 10³ cells / well on 96-well V-bottom culture dishes and cultured in X-Vivo 15 medium (Lonza #BE02-061Q) supplemented with 1% penicillin / streptomycin.

[0509] A series of diluted test compounds were added to a culture medium containing an NHP T-cell activation / expansion kit (Miltenyi #130-092-919) at a cell:bead ratio of 1:1. After 2 days of culture, cytokine secretion in the collected supernatant was assessed using MSD according to the manufacturer's instructions (Meso QuickPlex SQ120 #1300).

[0510] Cytokine secretion was analyzed using MSD Discovery Workbench (V4.0.12), and then plotted using GraphPad Prism (V9.1.2). Mixed lymphocyte reaction (MLR) analysis.

[0511] Objective: To evaluate the activity of the anti-4-1BB / anti-PD-1 bispecific binding molecule relative to the control antibody and isotype control using MLR analysis. To observe the release of cytokines from the co-culture.

[0512] PBMCs were isolated from fresh erythrocyte sedimentation rate (ESR) amber layer using a Ficoll gradient. Monocytes were isolated using positive selection. Monocytes were cultured for 6 days in RPMI 1640, 10% FCS, 2 mM glutamic acid, and 1% penicillin / streptomycin in the presence of 50 ng / mL GM-CSF and 10 ng / mL IL-4 to induce isodifferentiation into Mo-DCs. Simultaneously, PBMCs were isolated from fresh ESR amber layer from another donor using a parallel Ficoll gradient. T-cells were isolated using negative selection.

[0513] A mixture of heterologous T-cells and Mo-DC at a ratio of 10:1 was spread onto a 96-well U-shaped tray and cultured in X-Vivo 15 medium (Lonza #BE02-061Q) supplemented with 1% penicillin / streptomycin.

[0514] Add a series of diluted test compounds to the co-culture. After 4 or 6 days of culture, assess cytokine secretion in the collected supernatant using either the CBA Human Th1 / Th2 cytokine kit on a flow cytometer according to the manufacturer’s instructions (BD Bioscience #550749) or using Homogeneous Time Resolved Fluorescence (HTRF) Human IFN-γ / TNF-α cytokine kits (Cisbio #62HIFNGPEH and #62HTNFAPEH) on a Pherastar FSX multi-disk reader (BMG Labtech) according to the manufacturer’s instructions.

[0515] Cytokine secretion in CBA samples was analyzed using an FCAP array (V3.0.19.2091), followed by plotting using GraphPad Prism (V9.1.2). HTRF cytokine secretion was plotted directly using GraphPad Prism (V9.1.2). CD3-PBMC activation analysis.

[0516] Objective: To evaluate the activity of the anti-4-1BB / anti-PD-1 bispecific binding molecule relative to the control antibody and isotype control in a CD3-PBMC activation assay. To observe the cytokines released from PBMCs.

[0517] PBMCs were isolated from the fresh erythrocyte sedimentation rate (ESR) layer and plated at a density of 150 × 10³ cells / well. They were then cultured in the presence of 0.04 µg / mL soluble anti-CD3 and a series of diluted test compounds. After 4 or 6 days of culture, cytokine secretion in the collected supernatant was assessed using either the CBA human Th1 / Th2 cytokine kit on a flow cytometer according to the manufacturer's instructions (BD Bioscience #550749) or homogeneous time-resolved fluorescence (HTRF) human IFN-γ / TNF-α cytokine kits (Cisbio #62HIFNGPEH and #62HTNFAPEH) on a Pherastar FSX multi-disk reader (BMG Labtech) according to the manufacturer's instructions.

[0518] Cytokine secretion in CBA samples was analyzed using an FCAP array (V3.0.19.2091), followed by plotting using GraphPad Prism (V9.1.2). HTRF cytokine secretion was plotted directly using GraphPad Prism (V9.1.2). Modular Immuno-In Vivo Construct (MIMIC) CD8+T-cell Exhaustion Analysis

[0519] Objective: To evaluate the activity of the anti-4-1BB / anti-PD-1 bispecific binding molecule relative to the control antibody and isotype control in a CD8+ T-cell exhaustion assay. To observe the proliferation of exhausted CD8+ T- cells and the release of cytokines.

[0520] Monocytes were isolated from frozen PBMCs using positive selection and cultured in CellGro medium (CellGenix) for 6 days in the presence of 500 ng / mL GM-CSF and 125 ng / mL IL-4 to induce isodifferentiation into cytokine-derived dendritic cells (CDDCs). CDDCs were collected and resuspended in X-Vivo 15 medium (Lonza #BE02-061Q) in 24-well plates at a density of 3.34 × 10⁴ cells / well. CDDCs were pre-sensitized with 1 µg / mL HLA-restricted peptide (BioSynthesis) for 2–3 hours.

[0521] CD8+ T- cells were isolated from the same autologous PBMC donor using negative selection. CD8+ T- cells were plated at a density of 2 × 10⁶ cells / well on culture plates containing pulsed CDDCs (CD8+ T- cells: CDDC ratio of 60:1) and cultured in X-Vivo 15 medium (Lonza #BE02-061Q) for 12 days.

[0522] Functional CD8+ T-cells: CDDC cocultures were kept immobile for 12 days. Depleted CD8+ T-cells: CDDC cocultures were pulsed again on days 4 and 8 with 1 µg / mL HLA-restricted peptide.

[0523] On day 6, autologous CDDCs were prepared again as described above. On day 12, CDDCs were collected and plated in X-Vivo 15 medium (Lonza #BE02-061Q) in 96-well plates at a density of 5 × 10⁴ cells / well. CDDCs were pre-sensitized with 1 µg / mL HLA-restriction peptide for 2–3 hours.

[0524] Collect CD8+T- cells:CDDC co-culture. Take CFSE-stained CD8+T- cells and spread them at a density of 5 × 10 5 cells / well in a culture dish containing pulsed CDDC (CD8+T- cells:CDDC ratio of 10:1).

[0525] A series of diluted test compounds were added to the co-culture. After 5 days of culture, IFN-γ and TNF-α secretion in the collected supernatant was analyzed 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 on a BioPlex / Luminex system (BioRad). After washing, the MFI of CFSE-stained CD8+ T- cells was measured by flow cytometry. Pentamer-positive CD8+ T- cells were detected according to the manufacturer's instructions (BioSynthesis).

[0526] 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). Regulation of T-cell (Treg) inhibition analysis.

[0527] Objective: To evaluate the activity of the anti-4-1BB / anti-PD-1 bispecific binding molecule relative to the control antibody and isotype control in a T-reg inhibition assay. To observe the proliferation of CD4+ effector T-cells (T-eff) in the presence of inhibitory T-regs.

[0528] PBMCs were isolated from the fresh erythrocyte sedimentation rate (ESR) layer using a Ficoll gradient. Total CD4+ T- cells were isolated by negative selection, followed by CD4+CD25+ T-reg fractions by positive selection. These fractions were then expanded for 14 days in TexMACS medium (Miltenyi #130-097-196) supplemented with 5% FBS and 1% penicillin / streptomycin, in the presence of CD3 / CD28 MACSi beads (Miltenyi #130-095-353). Another fraction of T-eff CD4+CD25- cells was frozen for later use. T-eff proliferation data were read according to the manufacturer's instructions using the carboxylucosamine succinimide (CFSE) dilution method.

[0529] Expanded T regs (eT regs) and CFSE-stained T effs from the same donor at different ratios (2:1, 4:1, 8:1, 16:1, 32:1, 64:1) were cultured in the presence of CD2 / CD3 / CD28 MACSi beads (Miltenyi #130-092-909) at a bead:cell ratio of 1:1. A series of dilutions of the test compound were added to the culture medium. After 5 days of culture, the MFI of CFSE staining was measured by flow cytometry.

[0530] Flow cytometry results were analyzed using FlowJo (v.10.8.1) and then plotted using GraphPad Prism (v.9.1.2). In vivo potency study.

[0531] C57BL / 6-Pdcd1 tm1 (PDCD1)Tnfrsf9 tm1 (TNFRSF9) / Bcgen mice (humanized hPD-1 / h4-1BB dual KI) from Biocytogen (Ref. 120516) were subcutaneously implanted with MC38 tumor cells. Once the tumor was established (≈100 mm³), the mice were randomly assigned to different groups. Following randomization, the test compound was administered via intraperitoneal (ip) for Q3D cycles. Results

[0532] As shown in Figures 9A-9C, all four bispecific binding molecules (constructs #1, 3, 5 and 6) activated T cells to a greater extent than the individual monospecific control antibodies against 4-1BB or PD-1, and also to a greater extent than the combination of the two monospecific control antibodies.

[0533] Construct #3 was selected for further experiments. Binding analysis on stable cell lines.

[0534] Table 6 summarizes the EC50 and Emax values ​​of "Construct #3" in 300.19 (pre-B) cells transfected with self-made PD-1 (human, Fig. 10A; or macaque, Fig. 10B) or 4-1BB (human, Fig. 10C; or macaque, Fig. 10D) cells, relative to the control antibody. Table 6: EC50 and Emax values ​​in 300.19 (pre-B) cells Front-B h4-1BB front-B cy4-1BB Pre-B hPD-1 Pre-B cyPD-1 Structure #3 EC 50 (nM) 0.2994 0.3122 3.686 7.773 E max (nM) 483.5 829.6 3752 13815 anti-PD-1 antibody pure line T5 EC 50 (nM) nb nb 0.2842 0.4175 E max (nM) nb nb 6241 17731 Ctrl anti-4-1BB antibody EC 50 (nM) 0.5303 1.689 nb nb E max (nM) 1079 18.59 nb nb Ctrl anti-PD-1 antibody EC 50 (nM) nb nb 0.2831 0.4436 E max (nM) nb nb 6722 17587Nb: Not bound

[0535] Table 7 summarizes the EC50 and Emax values ​​of "Construct #3" in Jurkat cells expressing human PD-1 (Fig. 10E) or human 4-1BB (Fig. 10F), measured relative to the control antibody based on physiological antigen density. Table 7: EC50 and Emax values ​​in Jurkat cells Jurkat h4-1BB Jurkat hPD-1 Structure #3 EC 50 (nM) 0.1907 12.35 E max (nM) 1232 320.8 anti-PD-1 antibody pure line T5 EC 50 (nM) Nb 0.2776 E max (nM) Nb 280.3 Ctrl anti-4-1BB antibody EC 50 (nM) 0.1349 nb E max (nM) 1505 nb Ctrl anti-PD-1 antibody EC 50 (nM) Nb 0.3788 E max (nM) Nb 356.6 Nb: Binding assay of unbound primary human T cells

[0536] Table 8 summarizes the EC50 and Emax values ​​of "Construct #3" in stimulated primary T-cells relative to the control antibody (Figure 10G). Table 8: EC50 and Emax values ​​in stimulated primary T-cells Stimulated T cells Structure #3 EC 50 (nM) 1.534 E max (nM) 51.52 anti-PD-1 antibody pure line T5 EC 50 (nM) 0.205 E max (nM) 33.16 Ctrl anti-4-1BB antibody EC 50 (nM) 0.046 E max (nM) 17.09 Ctrl anti-PD-1 antibody #1 EC 50 (nM) 0.044 E max (nM) 34.81 Ctrl anti-PD-1 antibody #2 EC 50 (nM) 0.007 E max (nM) 31.33Nb: Not combined with report sub-analysis

[0537] Construct #3, used as a soluble agent, exhibited similar EC50 and IC50 values ​​to the control antibody in both 4-1BB and PD-1 reporter assays (Figures 11A and 11B, respectively; and Table 9). Table 9: EC50 and IC50 values ​​in 4-1BB and PD-1 reporter assays. test compounds EC 50 (nM)4-1BB Report Sub-analysis IC 50 (nM)PD-1 reporter sub-analysis Ctrl anti-4-1BB antibody 0.4 Nb Ctrl anti-PD-1 antibody nb 1.8 anti-PD-1 antibody pure line T5 nb 2.8 Structure #3 0.1 6.7nb: Not combined with ADCC / ADCP / CDC analysis method

[0538] Construct #3 showed no residual ADCC (Fig. 12A-12B) or ADCP (Fig. 13A-13B) Fc backbone activity, and only weak CDC Fc backbone activity (Fig. 14A-14B). T-cell activation (TCA)

[0539] Construct #3, as a soluble agent, initiated excellent T-cell activation in a T-cell activation assay (Figures 15A-15B, human T-cell activation; Figure 15C, macaque T-cell activation). Mixed lymphocyte reaction (MLR) assay.

[0540] In the MLR assay, construct #3, as a soluble agent, induced T-cell activation superior to the combination of two monospecific control antibodies, and also superior to the combination of each individual monospecific group constituting "construct #3" (Figures 16A-16C). CD3-PBMC activation assay

[0541] Construct #3 also initiates the secretion of multiple cytokines in the CD3-PBMC activation assay (Figures 17A-17B).

[0542] In the dose-response CD3-PBMC activation assay, "Construct #3" showed higher potency than the combination of two monospecific control antibodies (Figures 18A-18C). MIMIC CD8+T-cell exhaustion assay

[0543] The resuscitation of exhausted T-cells by construct #3 was also higher than that of the combination of two monospecific control antibodies (Fig. 19A). The resuscitation of exhausted CD8+ T-cells restored their function, as they were able to secrete IFN-γ and TNF-α after treatment (Figs. 19B-19C). T-cell (Treg) inhibition assay.

[0544] When the Teff:eTreg cell ratio was as low as 2:1, the in vitro Treg inhibitory activity induced by construct #3 was higher than that of the control anti-4-1BB antibody (Figure 20). In vivo efficacy study

[0545] In hu 4-1BB + PD-1 dKI mice with MC38 tumor pattern, excellent in vivo efficacy of a single formulation was observed at both high (8 / 9 CR) and low (6 / 9 CR) doses (Figure 21). Sequence

[0546] Anti-4-1BB pure line #5.1 V HH has the following amino acid sequence: EVQLVESGGGVVQPGGSLRLSCAAS GFTFS DH T MTWVRQAPGKGLEWVS SI SSGGSRIIYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYC AR GTRYKLSTSGQGTLVTVSS (SEQ ID NO: 3) The CDR sequence is specially marked: IMGT is in bold; Kabat is underlined; Chothia is in italics.

[0547] Anti-4-1BB pure line #2.1 V HH has the following amino acid sequence: EVQLVESGGGVVQPGGSLRLSCAAS GGLFS IN T GGWYRQAPGKQRELVA TI THDDRTNYAESVKGRFTISRDNAKNTVYLQMNSLRPEDTALYYC RL GSAAIRGYWGQGTLVTVSS (SEQ ID NO: 4) The CDR sequence is specially marked: IMGT is in bold; Kabat is underlined; Chothia is in italics.

[0548] The light chain (LC) sequence of “Construct #3” is shown below: EIVLTQSPATLSLSPGERATLS CGAS QSVSINFLAWYQQKPGLAPRLLIY EASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYY C QQYGSSPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVV CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGE C (SEQ ID NO: 9) Where C means intermediate disulfide bond; C means internal disulfide bond; and the bolded residue segments are the light chain CDRs 1, 2 and 3 of anti-PD-1 Fab “T5”, according to IMGT numbering.

[0549] The heavy chain (HC) sequence of "Construct #3" is as follows: EVQLVESGGGVVQPGGSLRLS CAAS GGLFSINTGGWYRQAPGKQRELVAT ITHDDRTNYAESVKGRFTISRDNAKNTVYLQMNSLRPEDTALYY C RLGSAAIRGYWGQGTLVTVSSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLS CAAS GFFTFSDHTMTWVRQAPGKGLEWVSSISSGGSRIIYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYY C ARGTRYKLSTSGQGTLVTVSSGGGGSDKTHT CPP CPAPEAAGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSGGGGSQLQLQESGPGLVKPSETLSLT CTVS GGSISTSSYFWGWIRQPPGKGLEWIGS IYRSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYY C ARGITGDPGDYWGQGTLVTVSSASTKGSSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKS C (SEQ ID NO: 10) where C represents the intermediate disulfide bond; C represents the inner disulfide bond; the first segment of three bolded residues represents CDRs 1, 2, and 3 of the anti-4-1BB pure series #2.1 V HH (according to IMGT number); the second segment of three bolded residues represents CDRs 1, 2, and 3 of the anti-4-1BB pure series #5.1 V HH (according to IMGT number); and the third segment of three bolded residues represents CDRs 1, 2, and 3 of the heavy chain of anti-PD-1 Fab "T5" (according to IMGT number). Example 4 Optimized Construct #3

[0550] The anti-PD-1 Fab "T5" from the original "Constructor #3" (described in Example 2) underwent sequence optimization ("T5_optimized" pure line). The resulting T5_optimized pure line was then embedded into a new "optimized construct #3," replacing the original T5 pure line. Material and method performance and purification.

[0551] For manifestations and purification, please refer to Example 3. Surface plasma resonance (SPR) combined analysis method.

[0552] Objective: To evaluate the affinity of the original “Construction #3” with respect to the equivalent target as opposed to “Optimized Construction #3”.

[0553] The SPR binding assay was performed on a Biacore 8K instrument using a CM5 anti-Fc wafer and human PD-1 or human 4-1BB as the analyte. The original "construct #3" and the "optimized construct #3" were used as ligands.

[0554] The anti-Fc capture antibody was diluted 1:20 in the transport buffer and coupled to the CM5 chip (Cytiva, catalog number 29149603) using the standard amine conjugation method with the amine conjugation kit (Cytiva, catalog number BR-100-50), producing approximately 8000 response units (RU).

[0555] Using a ligand at a concentration of 0.5 µg / mL, inject at a rate of 10 µg / mL for 60 seconds. For analytes at concentrations of 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, and 100 nM, inject at a rate of 30 µL / min for 240 seconds, followed by a dissociation period of 1200 seconds in HBS-EP+ buffer at a rate of 30 µL / min. Regeneration is then performed using 3 M MgCl2, injected at a rate of 30 µL / min for 60 seconds.

[0556] Binding kinetics data were evaluated using Biacore Insight Evaluation software (Cytiva) in 1:1 binding mode. Single-molecule localization microscopy (SMLM) was also employed.

[0557] The objective of this experiment is to evaluate and compare the compatibility of “Construction #3” and “Optimized Construction #3” with respect to constructions without PD-1, without 4-1BB, or both (i.e., the relevant elements in the construction are replaced by unrelated elements).

[0558] Jurkat cells expressing 4-1BB and PD-1 were activated at 37°C for 3 hours over a 5-day period using flasks pre-coated with OKT3 (CD3) antibody (Invitrogen 16-0037-85) at a final concentration of 5 µg / mL. The culture medium consisted of RPMI 1640 (Gibco 21875), 10% fetal bovine serum (Gibco 16629525), 2 mM L-glutamic acid (Stemcell 07100), 1% non-essential amino acids (Gibco 11140-035), 1 mM sodium pyruvate (Gibco 12539059), hygromycin B at a final concentration of 400 µg / mL (Invitrogen 10687010), and geneticin at a final concentration of 600 µg / mL (Gibco 11811-023).

[0559] On the 5th day, two analytical methods were performed in parallel: combined analysis and receptor density analysis.

[0560] Assay: 1.10 × 10⁶ cells were placed in an Eppendorf tube, centrifuged, resuspended with the test compound (200 nM, 300 nM, 600 nM, and 1200 nM, diluted in RPMI 1640), and incubated at 37°C for 30 minutes. After incubation, the cells were fixed with 4% formaldehyde solution at 4°C for 20 minutes, washed, and then labeled at room temperature with Fcγ fragment-specific Alexa Fluor® 647 AffiniPure Fab fragment goat anti-human IgG (Jackson 109-607-008) 1 / 1000 for 1 hour. After washing, labeled cells were spread onto MW6 slides (Marienfeld 0117650) pre-coated with poly-D-lysine (Corning 354210) diluted 1 / 400 in PBS and incubated at 37°C for 30 minutes. After 10 minutes, the slides were mounted and SMLM was performed using Smart kit super resolution buffer (Abbelight). TCA & MLR analysis were also performed.

[0561] For information on TCA and MLR analysis methods, please refer to Example 3. Results: Surface Plasma Resonance (SPR) combined with analysis method

[0562] As shown in Table 10, "Optimized Construct #3" demonstrates an improved KD (Kill / Durability) when combined with Human PD-1 compared to the original "Construct #3". The effectiveness when combined with 4-1BB remains unchanged. Table 10: KD Values ligands Analytes K D (M) K D (nM) Original structure #3 Human PD-1 1.60 × 10 -8 16.04 Original structure #3 Human 4-1BB 7.82 × 10 -9 7.82 Optimize Structure #3 Human PD-1 4.54 × 10 -9 4.54 Optimize Structure #3 Human 4-1BB 7.23 × 10 -9 7.23 Single-molecule localization microscope (SMLM)

[0563] Figures 22A-22B show the densities of “Construction #3” and “Optimized Construction #3”, respectively (expressed as the number of bound test compounds per µm² Jurkat cell surface).

[0564] The binding of "Constructor #3 ΔPD-1" and "Constructor #3 Δ4-1BB" (representing the groups lacking functional PD-1 and 4-1BB, respectively) increased in a dose-dependent manner, reaching a peak density at 600 nM. "Constructor #3" reached a peak density at 300 nM. Notably, the density of construct #3 at 300 nM was significantly different from that of the two ΔPD-1 and Δ4-1BB constructs, with the density of "Constructor #3" being higher than the combined density of the two ΔPD-1 and Δ4-1BB constructs (Fig. 22A). When comparing the density of binding proteins on Jurkat cells with the density of receptors (PD-1 and 4-1BB), we observed that the 4-1BB group had a good binding rate on the cell membrane, resulting in 4-1BB antigen saturation, while the PD-1 group showed the opposite, with only about 30% of the available PD-1 antigen binding. We hypothesize that in “Construction #3”, group 4-1BB drives this binding activity.

[0565] The binding affinity of "Optimized Construct #3", "Optimized Construct #3 ΔPD-1" and "Optimized Construct #3 Δ4-1BB" all increased to the highest density at 600 nM in a dose-dependent manner. This is contrary to the observation of "Construct #3" above, where we did not observe a significant density change among these three compounds (Fig. 22B).

[0566] When comparing the density of binding proteins and receptors (PD-1 and 4-1BB) on Jurkat cells, we observed that both the PD-1 and 4-1BB groups exhibited good binding rates to the cell membrane, resulting in PD-1 and 4-1BB antigen saturation. We hypothesize that in "Optimized Construct #3," the 4-1BB group is no longer the sole driver of binding activity, contrary to "Construct #3." TCA & MLR analysis

[0567] The "Optimized Construct #3" outperformed the original "Construct #3" in the T-cell activation assay (Figures 23A-23B), and showed similar performance in the MLR assay (Figures 24A-24B). In both assays, the original "Construct #3" and the "Optimized Construct #3" outperformed the single-specific control antibody. Sequence

[0568] The optimized anti-PD-1 Fab "T5_optimized" has the following light chain variable region (LCVR) amino acids: EIVLTQSPATLSLSPGERATLSC GAS QSVPINFLAWYQQKPGLAPRLLIY EAS SRHTGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC GQYGSSPYTFGQGTKLEIK (SEQ ID NO: 7) The CDR sequences are specially marked: IMGT number is in bold; Kabat number is underlined; Chothia number is in italics.

[0569] Optimized anti-PD-1 Fab "T5_Optimized" has the following heavy chain variable region (HCVR) amino acids: QLQLQESGPGLVKPSETLSLTCTVS GGSIS SSSY F WGWIRQPPGKGLEWIG SI YRSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYC AR GITGDPGDYWGQGTLVTVSS (SEQ ID NO: 8) The CDR sequences are specially marked: IMGT number is in bold; Kabat number is underlined; Chothia number is in italics.

[0570] The light chain (LC) sequence of "Optimized Build #3" is as follows: EIVLTQSPATLSLSPGERATLS CGAS QSVPINFLAWYQQKPGLAPRLLIY EASSRHTGIPDRFSGSGSGTDFTLTISRLEPEDFAVYY C GQYGSSPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVV CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGE C (SEQ ID NO: 11) where C means intermediate disulfide bond; C means internal disulfide bond; and the bold residues of anti-PD-1 Fab "T5_Optimized" are light chain CDR1, 2 and 3 (according to IMGT number).

[0571] The heavy chain (HC) sequence of "Optimized Construct #3" is as follows: EVQLVESGGGVVQPGGSLRLS CAAS GGLFSINTGGWYRQAPGKQRELVAT ITHDDRTNYAESVKGRFTISRDNAKNTVYLQMNSLRPEDTALYY C RLGSAAIRGYWGQGTLVTVSSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLS CAAS GFTFSDHTMTWVRQAPGKGLEWVSSISSGGSRIIYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYY C ARGTRYKLSTSGQGTLVTVSSGGGGSDKTHT CPP CPAPEAAGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGGGGSGGGSGGGGSGGGGSGGGGSQLQLQESGPGLVKPSETLSLT CTVS GGSISSSSYFWGWIRQPPGKGLEWIGS IYRSGSTYYNPSLKSRVTISSVDTSKNQFSLKLSSVTAADTAVYY C ARGITGDPGDYWGQGTLVTVSSASTKGSSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKS C(SEQ ID NO: 12) where C refers to the intermediate disulfide bond; C refers to the inner disulfide bond; the three bolded residues in the first segment are CDR1, 2, and 3 of the anti-4-1BB pure series #2.1 V HH (according to IMGT number); the three bolded residues in the second segment are CDR1, 2, and 3 of the anti-4-1BB pure series #5.1 V HH (according to IMGT number); and the three bolded residues in the third segment are CDR1, 2, and 3 of the heavy chain of the anti-PD-1 Fab "T5_optimized" (according to IMGT number).Example 5: Conditional active anti-4-1BB / anti-PD-1 bispecific binding protein.

[0572] We have generated conditionally active anti-4-1BB / anti-PD-1 bispecific binding proteins from the "optimized construct #3" described in Example 3.

[0573] In short, the conditionally active bispecific binding protein contains, from the N to the C-terminus: - masked anti-4-1BB V HH (pure line #2.1); - anti-4-1BB V HH (pure line #5.1); - IgG1-LALA Fc region; and - anti-PD-1 Fab (pure line "T5_optimized").

[0574] The masked anti-4-1BB V HH pure line #2.1 itself contains, from the N-terminus to the C-terminus: the masking part (MM), the cleavable linker and the anti-4-1BB V HH pure line #2.1.

[0575] Cleavable linkers typically contain a short amino acid sequence that serves as a protease target. For example, in cancer therapy, it is preferable that the protease is a tumor-specific protease, that is, a protease that, if not unique, is at least predominantly present in the in vivo tumor environment. Therefore, in the absence of the protease, the masking moiety remains fused to the bispecific binding protein, thereby reducing, inhibiting, or abolishing the binding of the bispecific binding protein to its target (in this case, the binding of the masked anti-4-1BB group to its target 4-1BB). However, when a conditionally active bispecific binding protein co-colonizes with a protease capable of cleaving the cleavable linker in, for example, the tumor microenvironment, the masking moiety is released from the bispecific binding protein, and the latter's binding to the target antigen (e.g., 4-1BB) is restored.

[0576] Using the synthetic collection library described in WO 2019 / 149282 A1, two alternative masking moieties (MM1 and MM2) were identified according to Adagene's procedures; MM1 or MM2 was linked to the N-terminus of anti-4-1BB V HH pure line #2.1 via one of two cleavable linkers (cleavable linker #1 or cleavable linker #2). Combining a masking moiety with a cleavable linker formed four different conditionally active (masked) bispecific binding proteins: - MC1, which contains MM1 and cleavable linker #1; - MC2, which contains MM2 and cleavable linker #1; - MC3, which contains MM1 and cleavable linker #2; and - MC4, which contains MM2 and cleavable linker #2. Materials and Methods: Performance and Purification

[0577] For manifestations and purification, please refer to Example 3. MMP9-mediated cleavage.

[0578] The experiment requires the cleavage of the masking part by MMP9 protease (hereinafter referred to as "MMP9-activated compound").

[0579] In the first step, recombinant human MMP9 protein (R&D Systems, Ref. 911-MP-010) was activated by adding 100 μg / mL of p-aminophenylmercuric acetate (APMA; Calbiochem, Ref. 164610-700MG) to the MMP9 solution, resulting in a final concentration of 1 mM, and incubating at 37°C for 24 hours. The activated MMP9 was then aliquoted and stored at -80°C for further use.

[0580] Then, the test (masked) compound was diluted to 1 mg / mL and incubated with activated MMP9 at 37°C and gentle shaking (300 rpm) for 24 hours at a final concentration of 5 nM.

[0581] If necessary, exclude excess masking peptides and purify the sample using a HiLoad® 26 / 600 Superdex® 200 (GE Healthcare, Ref. 28-9893-36). Surface plasma resonance (SPR) binding assay.

[0582] Objective: To evaluate the binding affinity of "Optimized Construct #3" to its conditionally active anti-4-1BB / anti-PD-1 bispecific binding proteins MC1-MC4 and its unmasked form to their respective targets.

[0583] The SPR binding assay was performed on a Biacore 8K instrument using a CM5 anti-Fc wafer and human PD-1 or human 4-1BB as the analyte. The test compound was used as the ligand.

[0584] The anti-Fc capture antibody was diluted 1:20 in transport buffer and coupled to a CM5 wafer (Cytiva, catalog number 29149603) using the standard amine coupling method with an amine coupling kit (Cytiva, catalog number BR-100-50), producing approximately 8,000 response units (RU).

[0585] Using a ligand at a concentration of 0.5 µg / mL, inject at a rate of 10 µg / mL for 60 seconds. For analytes at concentrations of 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, and 100 nM, inject at a rate of 30 µL / min for 240 seconds, followed by a dissociation period of 1200 seconds in HBS-EP+ buffer at a rate of 30 µL / min. Regeneration is then performed by injecting 3 M MgCl2 at a rate of 30 µL / min for 60 seconds.

[0586] Binding kinetic data were analyzed using Biacore Insight Evaluation software (Cytiva) in a 1:1 binding mode. Binding analysis was performed on stable cell lines for human PD-1 and 4-1BB.

[0587] Objective: To evaluate the affinity of "Optimized Construct #3" for its targets relative to the conditionally active anti-4-1BB / anti-PD-1 bispecific binding proteins MC1-MC4 and its unmasked form. Calculate the binding EC50 and Emax values ​​of the test compounds.

[0588] The assay was performed on ice in a 96-well plate using PD-1 NFAT-luc2 Jurkat cells (Promega #J1252) or 4-1BB NF-κB-luc2P Jurkat cells (Promega #J2332), or self-made transfected 300.19 (pre-B) cells expressing human PD-1 (hPD-1) or human 4-1BB (h4-1BB).

[0589] The cell suspension was spread into 96-well U-shaped trays at a density of 50 × 10³ cells per well. A series of diluted test compounds were added to the cells for 1 hour. After washing, fluorescently labeled secondary antibodies targeting the Fc region were added to each well for 30 minutes. The MFI signal was then measured by flow cytometry.

[0590] Data from flow cytometry were analyzed using FlowJo (V10.8.1), and then graphs of binding curves, Emax, and EC50 values ​​were plotted using GraphPad Prism (V9.1.2). Rhesus monkey PD-1 and 4-1BB...

[0591] Objective: To evaluate the affinity of "Optimized Construct #3" for its targets relative to the conditionally active anti-4-1BB / anti-PD-1 bispecific binding proteins MC1-MC4 and its unmasked form. Calculate the binding EC50 and Emax values ​​of the test compounds.

[0592] The assay was performed on ice in 96-well plates using self-made transfected 300.19 (pre-B) cells expressing macaque PD-1 (cyPD-1) or macaque 4-1BB (cy4-1BB).

[0593] The cell suspension was spread into 96-well U-shaped trays at a density of 50 × 10³ cells per well. A series of diluted test compounds were added to the cells for 1 hour. After washing, fluorescently labeled secondary antibodies targeting the Fc region were added to each well for 30 minutes. The MFI signal was then measured by flow cytometry.

[0594] Data from flow cytometry were analyzed using FlowJo (V10.8.1), and then graphs of binding curves, Emax, and EC50 values ​​were plotted using GraphPad Prism (V9.1.2). Reporter analysis method 4-1BB

[0595] Objective: To evaluate the activity of "Optimized Construct #3" relative to conditionally active anti-4-1BB / anti-PD-1 bispecific binding proteins MC1-MC4 and their demasked 4-1BB groups. Calculate the EC50 and Emax of each test compound.

[0596] Data were read 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% glutamic acid, 1% NEAA, 1 mM sodium pyruvate, 800 µg / mL G418, and 500 µg / mL hygromycin, according to the manufacturer’s instructions.

[0597] Spread cells at a density of 50 × 10³ cells per well in a 96-well white flat-bottomed dish. Add a series of diluted test compounds. After incubating at 37°C for 6 hours, add Bio-Glo reagent to each well. Measure luminescence using an Infinit Pro M1000 or Spark Tecan reader.

[0598] Plot the combined curve, E max, and EC 50 value using GraphPad Prism (V9.1.2). PD-1

[0599] Objective: To evaluate the activity of "Optimized Construct #3" relative to conditionally active anti-4-1BB / anti-PD-1 bispecific binding proteins MC1-MC4 and their demasked PD-1 groups. Calculate the IC50 and Emax of each test compound.

[0600] Data were read from the manufacturer’s instructions in the presence of 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% glutamic acid, 1% NEAA, 1 mM PyNa, 500 µg / mL G418, and 200 µg / mL hygromycin.

[0601] PD-L1 aAPC / CHO-K1 cells were spread at a density of 40 × 10³ cells per well in 96-well white flat-bottomed culture dishes and incubated overnight at 37°C in Ham / F12 medium supplemented with 1% FCS. The next day, the medium was removed, and PD-1 NFAT-luc2 Jurkat cell suspension was added to the wells at a density of 50 × 10³ cells / well, along with a series of diluted test compounds. After incubation at 37°C for 6 hours, Bio-Glo reagent was added to each well. The luminescence intensity was measured using an Infinit Pro M1000 or SPARK TECAN reader.

[0602] GraphPad Prism (V9.1.2) was used to plot the binding curve, Imax, and IC50 values. T-cell activation (TCA) analysis; human TCA analysis.

[0603] The in vitro bioactivity of "optimized construct #3" (unmasked parts), each MC1-MC4 (masked), and protease-activated compounds (unmasked) was measured using the CellTiter-Glo (CTG) method.

[0604] In short, peripheral blood mononuclear cells (PBMCs) were first isolated from fresh human blood, and then total T cells were purified using a StemCell kit. These cells (1 × 10⁵ cells per well) were cultured in 96-well tissue culture dishes pre-coated with anti-human CD3 at a concentration lower than the optimal level (5 µg / mL) under a series of dilutions with or without the test substance. T-cell activation was then measured by T-cell proliferation using the CTG method. Rodent TCA assay.

[0605] Objective: To evaluate the activity of "Optimized Construct #3" relative to conditionally active anti-4-1BB / anti-PD-1 bispecific binding proteins MC1-MC4 and its unmasked form relative to isotype controls. Observe the release of cytokines from activated rodent T cells.

[0606] A novel h4-1BB KI mouse was produced by crossing C57BL / 6-Tnfrsf9 tm1 (TNFRSF9) / Bcgen mice (humanized h4-1BB KI) from Biocytogen (Ref. 110004) with hPD-1 KI mice from CIPHE (Center for Immunophenomics, Marseille, France). This dKI mouse model was validated genotype and phenotype. The bridging of the new mouse model was also verified.

[0607] Rodent T-cell lines were isolated from spleen cells of dKI mice using negative selection. T-cells were plated at a density of 200 × 10³ cells / well in 96-well flat-bottomed dishes pre-coated with 1 µg / mL anti-mouse CD3 and cultured in complete medium supplemented with RPMI 1640 (Gibco 31870-025), 2 mM L-glutamic acid (Gibco 25030-081), 10% FCS (Eurobio CVFSF00-01), 1× non-essential amino acid (Gibco 11140-035), 1 mM sodium pyruvate (Gibco 11360-070), 0.05 mM 2-hydrothioethanolamine (Gibco 31350-010), and 1% penicillin / streptomycin.

[0608] A series of diluted test compounds were added to the culture medium. After 2 days of culture, the supernatant was collected and the cytokine secretion was assessed on a flow cytometer using a mouse Th1 / Th2 cytokine flow cytometry microarray (CBA) kit, according to the manufacturer's instructions (BD Bioscience 551287).

[0609] Cytokine secretion was analyzed using an FCAP array (v3.0.19.2091), followed by plotting using GraphPad Prism (v9.5.0). Mixed lymphocyte response (MLR) analysis was also performed.

[0610] Objective: To evaluate the activity of "Optimized Construct #3" relative to conditionally active anti-4-1BB / anti-PD-1 bispecific binding proteins MC1-MC4 and their unmasked forms using MLR analysis. Cytokines released from the co-culture were observed.

[0611] PBMCs were isolated from fresh erythrocyte sedimentation rate (ESR) amber layer using a Ficoll gradient. Monocytes were isolated using positive selection. Monocytes were cultured for 6 days in RPMI 1640, 10% FCS, 2 mM glutamic acid, and 1% penicillin / streptomycin in the presence of 50 ng / mL GM-CSF and 10 ng / mL IL-4 to induce isodifferentiation into Mo-DCs. Simultaneously, PBMCs were isolated from fresh ESR amber layer from another donor using a parallel Ficoll gradient. T-cells were isolated using negative selection.

[0612] A mixture of heterologous T-cells and Mo-DC at a ratio of 10:1 was spread on a 96-well U-shaped tray and cultured in X-Vivo 15 medium (Lonza #BE02-061Q) supplemented with 1% penicillin / streptomycin.

[0613] Add a series of diluted test compounds to the co-culture. After 4 or 6 days of culture, assess cytokine secretion in the collected supernatant using either the CBA human TH1 / TH2 cytokine kit on a flow cytometer according to the manufacturer’s instructions (BD Bioscience #550749) or homogeneous time-resolved fluorescence (HTRF) human IFN-γ / TNF-α cytokine kits (Cisbio #62HIFNGPEH and #62HTNFAPEH) on a Pherastar FSX multi-disk reader (BMG Labtech) according to the manufacturer’s instructions.

[0614] Cytokine secretion in CBA samples was analyzed using an FCAP array (V3.0.19.2091), followed by plotting using GraphPad Prism (V9.1.2). HTRF cytokine secretion was plotted directly using GraphPad Prism (V9.1.2). CD3-PBMC activation analysis.

[0615] Objective: To evaluate the activity of "Optimized Construct #3" relative to conditionally active anti-4-1BB / anti-PD-1 bispecific binding proteins MC1-MC4 and their unmasked forms in a CD3-PBMC activation assay. Cytokines released from PBMCs will be observed.

[0616] PBMCs were isolated from the fresh erythrocyte sedimentation rate (ESR) layer and plated at a density of 150 × 10³ cells / well. They were then cultured in the presence of 0.04 µg / mL soluble anti-CD3 and a series of diluted test compounds. After 4 or 6 days of culture, cytokine secretion in the collected supernatant was assessed using either the CBA human Th1 / Th2 cytokine kit on a flow cytometer according to the manufacturer's instructions (BD Bioscience #550749) or homogeneous time-resolved fluorescence (HTRF) human IFN-γ / TNF-α cytokine kits (Cisbio #62HIFNGPEH and #62HTNFAPEH) on a Pherastar FSX multi-disk reader (BMG Labtech) according to the manufacturer's instructions.

[0617] Cytokine secretion in CBA samples was analyzed using an FCAP array (V3.0.19.2091), followed by plotting using GraphPad Prism (V9.1.2). HTRF cytokine secretion was plotted directly using GraphPad Prism (V9.1.2). Plasma stability

[0618] The stability of each MC1-MC4 (masked type) was analyzed in mouse, macaque and human plasma over a period of 7 days.

[0619] In short, each MC1-MC4 was diluted in mouse, macaque, or human EDTA plasma to a final concentration of 100 µg / mL. Samples were incubated at 37°C for 7 days (168 hours) and then analyzed by ELISA. Total amounts (lysed and unlysed compounds) were detected using immobilized anti-human IgG Fc antibody and the anti-human IgG Fab-HRP conjugate for detection; lysed fractions were detected using immobilized truncated 4-1BB (binding only to the anti-4-1BB pure line #2.1 V HH) and the anti-human IgG Fab-HRP conjugate for detection. In vivo potency studies.

[0620] A novel h4-1BB KI mouse model was generated by mating C57BL / 6-Tnfrsf9 tm1 (TNFRSF9) / Bcgen mice (humanized h4-1BB KI) from Biocytogen (Ref. 110004) with hPD-1 KI mice from CIPHE (Center for Immunophenomics, Marseille, France). This dKI mouse model was validated genotype and phenotype. These dKI mice were subcutaneously implanted with MC38 tumor cells and randomly assigned to groups when tumors were established (≈100 mm³). Immediately after randomization, the test compound was administered via intraperitoneal (ip) according to a Q3D treatment regimen. Results were obtained using surface plasma resonance (SPR) binding assay.

[0621] As shown in Figure 25, the four conditionally active anti-4-1BB / anti-PD-1 bispecific binding proteins MC1-MC4 showed that 4-1BB can only bind after MMP9 mediates the cleavage of its isomasking parts (Table 11).

[0622] On the other hand, PD-1 binding is not affected by the masking component (Table 12).

[0623] All positive and negative controls showed the expected target binding status. Table 11: SPR binding to human and macaque 4-1BB. sample K D (nM)h4-1BB K D (nM)cy4-1BB Macaque / Human K D ratio Control anti-4-1BB antibody 6.06 296* 48.8* Structure #3 11.84 25.1 2.1 Optimize Structure #3 10.87 21.1 1.9 MC1 nb nb n / a MC1 MMP9 - Activation 6.65 29.9 4.5 MC2 nb nb n / a MC2 MMP9 - Activation 7.57 28.7 3.8 MC3 nb nb n / a MC3 MMP9 - Activation 7.79 30.1 3.9 MC4 nb nb n / a MC4 MMP9 - Activation 6.51 21.7 3.3* The control anti-4-1BB antibody showed no cross-reactivity with macaques. h4-1BB: human 4-1BB; cy4-1BB: macaque 4-1BB; nb: no binding; n / a: not applicable. Table 12: SPR binding to human and macaque PD-1 sample K D (nM)hPD-1 K D (nM)cyPD-1 Macaque / Human K D ratio Control anti-PD-1 antibody 7.36 3.12 0.4 Structure #3 7.05 18.2 2.6 Optimize Structure #3 7.29 14.9 2.0 MC1 6.42 14.9 2.3 MC1 MMP9 - Activation 6.89 10.7 1.6 MC2 7.73 17.3 2.2 MC2 MMP9 - Activation 7.40 15.0 2.0 MC3 6.73 13.4 2.0 MC3 MMP9 - Activation 7.07 13.0 1.8 MC4 6.82 18.3 2.7 MC4 MMP9 - Activation 26.5 57.4 2.2hPD-1: Human PD-1; cyPD-1: Macaque PD-1. Pre-B cell binding assay for stable cell lines.

[0624] For human 300.19 pre-B- cells, conditionally active anti-4-1BB / anti-PD-1 bispecific binding proteins with cleavable linkers #1 (MC1 and MC2) have a higher EC50 for 4-1BB than those with cleavable linkers #2 (MC3 and MC4).

[0625] Conditionally active anti-4-1BB / anti-PD-1 bispecific binding proteins exhibited 1.5 to 2 log masking efficacy in human 4-1BB+ cells (Fig. 26A) and 0.5 to 1 log masking efficacy in macaque 4-1BB+ cells (Fig. 26B). After MMP9-mediated cleavage of their isomasking portions, all four bispecific binding proteins exhibited EC50 equivalent to "Optimized Construct #3" (unmasked) (Table 13).

[0626] On the other hand, the binding of PD-1 is not affected by the shielding part (Figs. 26C-26D; Table 14).

[0627] All positive and negative controls showed the expected target binding status. Table 13: Binding to h4-1BB+ or cy4-1BB+ 300.19 pre-B- cells. h4-1BB peak value (MFI) h4-1BBEC 50 (nM) cy4-1BB highest value (MFI) cy4-1BBEC 50 (nM) EC 50 Ratio cy / h h / cy4-1BB masking efficacy** h / cy4-1BB binding recovery rate (%)*** Control anti-4-1BB antibody* 5807 0.25 125 Unstable n / a n / a n / a Optimize Structure #3 2841 0.27 3566 0.91 0.30 1 / 1 100 / 100 MC1 5139 83.79 4183 6.24 0.07 310 / 7 n / a MC1 MMP9 - Activation 3043 0.18 3741 0.70 3.89 n / a 100 / 100 MC2 2537 6.81 4366 16.64 2.44 25 / 18 n / a MC2 MMP9 - Activation 2824 0.14 3636 0.94 6.71 n / a 99 / 100 MC3 5744 25.57 4631 5.90 0.23 95 / 6 n / a MC3 MMP9 - Activation 3050 0.18 3581 0.61 3.39 n / a 100 / 100 MC4 3775 9.70 4254 9.81 1.01 36 / 11 n / a MC4 MMP9 - Activation 3245 0.15 3810 0.94 6.27 n / a 100 / 100* No cross-reactivity in mammoths with the control anti-4-1BB antibody. ** Masking potency corresponds to the ratio of EC50 of the masked type (i.e., MC1-MC4) to the EC50 value of the naked type (i.e., "optimized construct #3"). *** Binding recovery rate corresponds to the difference between the highest values ​​of the naked type (i.e., "optimized construct #3") and the unmasked type (i.e., MC1-MC4 MMP9-activated). h4-1BB: Human 4-1BB; cy4-1BB: Mammoth 4-1BB; n / a: Not applicable. Table 14: Binding on hPD-1+ or cyPD-1+ 300.19 pre-B-cells. hPD-1 peak value (MFI) hPD-1EC 50 (nM) Maximum cyPD-1 value (MFI) cyPD-1EC 50 (nM) EC50 Ratio cy / h Control anti-PD-1 antibody 22620 0.15 38204 0.71 4.73 Optimize Structure #3 19725 1.40 17494 1.45 1.04 MC1 19295 1.65 19869 1.67 0.99 MC1 MMP9 - Activation 19833 1.74 17368 1.18 0.68 MC2 20165 2.97 20915 1.50 0.51 MC2 MMP9 - Activation 18618 1.62 17342 1.98 1.22 MC3 19188 1.76 18962 1.40 0.80 MC3 MMP9 - Activation 19481 1.36 17882 1.55 1.14 MC4 19166 2.68 23131 4.80 1.79 MC4 MMP9 - Activation 18733 1.56 18174 1.87 1.20hPD-1: Human PD-1; cyPD-1: Macaque PD-1. Jurkat T cells

[0628] As shown in Table 15 and Figure 27, based on EC50 values, protease-activated (i.e., unmasked) types (MMP9-activated and uPA-activated) exhibited similar binding activity against Jurkat / NF-κB-4-1BB cells compared to the parental "Optimized Construct #3". All masked compounds MC1-MC4 showed a binding coefficient decrease of at least 166-fold relative to "Optimized Construct #3", while MC1 showed a decrease of up to 553-fold. Table 15: EC50 and Masking Potential Values test items EC 50 (nM) Relative shielding effectiveness Optimize structure #3 (no shading) 0.48 1 MC1 266.2 553 MC2 123.0 256 MC3 79.6 166 MC4 79.7 166 MMP9 - Activation (MC1 / MC2) 0.52 1.1 uPA - Activation (MC3 / MC4) 0.67 1.4 V HH Empty vector control group na na

[0629] In further experiments with Jurkat T cells, conditionally active anti-4-1BB / anti-PD-1 bispecific binding proteins with cleavable linkers #1 (MC1 and MC2) had higher EC50 against 4-1BB than those with cleavable linkers #2 (MC3 and MC4).

[0630] Conditionally active anti-4-1BB / anti-PD-1 bispecific binding proteins exhibited 1.5 to 2-log masking efficacy in human 4-1BB+ cells (Fig. 28A). After MMP9-mediated cleavage of their isomasking mesosomes, all four bispecific binding proteins had EC50 equivalent to "Optimized Construct #3" (unmasked) (Table 16).

[0631] On the other hand, the binding of PD-1 is not affected by the masking part (Fig. 28B; Table 17).

[0632] All positive and negative controls showed the expected target binding status. Table 16: Binding to 4-1BB+Jurkat T-cells test items Maximum value (MFI) EC 50 Value (nM) Concealing effect* Combined with recovery rate (%)** Control anti-4-1BB antibody 645 0.22 n / a n / a Optimize Structure #3 525 0.48 1 100% MC1 661 111.6 233 n / a MC1 MMP9 - Activation 587 0.54 n / a 100% MC2 575 114.0 238 n / a MC2 MMP9 - Activation 594 0.61 n / a 100% MC3 604 24.41 51 n / a MC3 MMP9 - Activation 590 0.55 n / a 100% MC4 649 36.09 75 n / a MC4 MMP9 - Activation 592 0.50 n / a 100%* Masking efficacy corresponding to the ratio of EC50 of the masked type (i.e., MC1-MC4) to the EC50 value of the naked type (i.e., "optimized construct #3").** Binding recovery rate corresponding to the difference % between the highest values ​​of the naked type ("optimized construct #3") and the unmasked type (i.e., MC1-MC4 MMP9-activated). Table 17: Binding to PD-1 + Jurkat T-cells test items Maximum value (MFI) EC 50 Value (nM) Control anti-PD-1 antibody 488 0.06 Optimize Structure #3 457 0.50 MC1 483 0.70 MC1 MMP9 - Activation 493 0.85 MC2 466 0.78 MC2 MMP9 - Activation 457 0.46 MC3 502 0.89 MC3 MMP9 - Activation 472 0.55 MC4 476 0.72 MC4 MMP9 - Activation 451 0.40 Report Sub-analysis

[0633] As shown in Table 18 and Figure 29, based on EC50 values, the protease-activated (i.e., cleavage-type) (MMP9-activated and uPA-activated) proton gene activities are similar to those of the parental "optimized construct #3". Compared to "optimized construct #3", the EC50 values ​​of the masked compounds MC1-MC4 shifted by at least 67-fold, and MC1 by as much as 121-fold. Table 18: EC50 and Masking Efficacy Values test items EC 50 (nM) Relative shielding effectiveness Optimize structure #3 (no shading) 0.95 1 MC1 114.3 121 MC2 83.3 88 MC3 89.7 95 MC4 63.0 67 MMP9 - Activation (MC1 / MC2) 1.1 1.1 uPA - Activation (MC3 / MC4) 1.1 1.2 V HH Empty vector control group 139 147

[0634] In further experiments, "Optimized Construct #3" showed significantly superior 4-1BB activity compared to the control anti-4-1BB antibody; and significantly superior PD-1 activity compared to the control anti-PD-1 antibody.

[0635] Conditionally active anti-4-1BB / anti-PD-1 bispecific binding proteins with cleavable linkers #1 (MC1 and MC2) have higher EC50 values ​​than those with cleavable linkers #2 (MC3 and MC4) in the 4-1BB reporter assay, but are still within the same nM range.

[0636] Conditionally active anti-4-1BB / anti-PD-1 bispecific binding proteins exhibit 1.5 to 2-log masking efficacy (Fig. 30A). After MMP9-mediated cleavage of their isomasking portions, all four bispecific binding proteins exhibit EC50 equivalent to that of "Optimized Construct #3" (unmasked) (Table 19).

[0637] On the other hand, the shielding components and the cleavable linkers do not affect the functionality of the PD-1 group (Fig. 30B; Table 20).

[0638] All positive and negative controls showed the expected target binding status. Table 19: 4-1BB reporter subanalysis test items EC 50 Value (nM) Concealing effect* Control anti-4-1BB antibody 0.33 n / a Optimize Structure #3 0.18 1 MC1 18.11 101 MC1 MMP9 - Activation 0.25 n / a MC2 15.13 84 MC2 MMP9 - Activation 0.22 n / a MC3 7.28 40 MC3 MMP9 - Activation 0.24 n / a MC4 5.79 32 MC4 MMP9 - Activation 0.23 n / a* corresponds to the shading effectiveness of EC 50 for the shaded type (i.e., MC1-MC4) and EC 50 for the bare type (i.e., "optimized construct #3"). Table 20: PD-1 reporter sub-analysis method test items Maximum value (S / N) EC 50 Value (nM) Control anti-PD-1 antibody 3.44 0.38 Optimize Structure #3 3.20 1.39 MC1 3.15 2.43 MC1 MMP9 - Activation 2.99 0.88 MC2 2.99 2.54 MC2 MMP9 - Activation 2.72 0.84 MC3 3.25 2.62 MC3 MMP9 - Activation 3.01 1.16 MC4 3.13 2.81 MC4 MMP9 - Activation 2.95 1.01 Human T-cell proliferation and activation analysis

[0639] As shown in Table 21 and Figure 31, in the T-cell proliferation analysis, the EC50 values ​​of each masked compound MC1-MC4 relative to the parental "optimized construct #3" were observed to be at least 105 times shif...

Claims

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

2. The multispecific antigen-binding protein as claimed in claim 1, wherein pure agonist activity means that the ISV can (i) activate T cells via 4-1BB signaling in soluble conditions, and / or (ii) in the absence of a cross-linking agent, and / or (iii) in an FcγR-independent manner, and / or (iv) in the absence of target-mediated 4-1BB cross-linking; wherein, if necessary, the pure agonist activity is determined by NF-κB pathway activation assay in the absence of a cross-linking agent.

3. For the multispecific antigen-binding proteins of claim 1 or 2, at least one ISV that specifically binds to 4-1BB will compete with 4-1BBL for binding to 4-1BB.

4. A multispecific antigen-binding protein as claimed in any of claims 1 to 3, wherein at least one ISV specifically binding to 4-1BB interacts with the cysteine ​​enrichment domain 2 (CRD2) and / or cysteine ​​enrichment domain 3 (CRD3) of 4-1BB; preferably, at least one ISV specifically binding to 4-1BB interacts with the CRD2 and CRD3 domains of 4-1BB.

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

7. The multispecific antigen-binding protein of claim 6, wherein CDR1 comprises or is composed 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. The multispecific antigen-binding protein of claim 6 or 7, wherein CDR2 comprises or is composed of the amino acid sequence ISSGGSRI (SEQ ID NO: 17), INPSGGSQ (SEQ ID NO: 77), or IKKSGNRT (SEQ ID NO: 78).

9. A multispecific antigen-binding protein as claimed in any of claims 1 to 8, wherein at least one ISV specifically binds to 4-1BB comprises or consists of: (i) an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, 3, 58, 59, 60 and 61; or (ii) an amino acid sequence having at least 70% sequence identity with the non-CDR region of SEQ ID NO: 2, 3, 58, 59, 60 or 61.

10. A multispecific antigen-binding protein of any one of claims 1 to 9, wherein at least one ISV specifically binding to 4-1BB comprises or constitutes an amino acid sequence SEQ ID NO: 2 or 3; preferably, at least one ISV specifically binding to 4-1BB comprises or constitutes an amino acid sequence SEQ ID NO:

3.

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

12. The multispecific antigen-binding protein of claim 11, wherein at least two of them specifically bind to the same ISV of 4-1BB.

13. The multispecific antigen-binding protein of claim 11, wherein at least two of them specifically bind to different ISVs of 4-1BB, and (i) bind to the same antigenic epitope of 4-1BB, (ii) bind to an overlapping antigenic epitope of 4-1BB, or (iii) bind to a unique antigenic epitope of 4-1BB; preferably, the at least two of them specifically bind to different ISVs of 4-1BB and bind to a unique antigenic epitope of 4-1BB.

14. A multispecific antigen-binding protein of any one of claims 11 to 13, wherein at least one second ISV specifically binds to 4-1BB comprises or consists of: (i) an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and 4, or (ii) an amino acid sequence having at least 70% sequence identity with the non-CDR region of SEQ ID NO: 1 or 4.

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

4.

16. A multispecific antigen-binding protein as claimed in any of claims 1 to 10, comprising at least two ISVs, wherein one of the at least two ISVs specifically binds to 4-1BB, and the other of the at least two ISVs specifically binds to another target antigen.

17. A multispecific antigen-binding protein as claimed in claim 16, wherein another target antigen is a T-cell antigen, a tumor-associated or tumor-specific antigen, or a non-autoantigen.

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

19. The multispecific antigen-binding protein of claim 18, wherein at least four ISVs comprise: (i) at least two identical ISVs of a first group that specifically bind to 4-1BB, and (ii) at least two other identical ISVs of a second group that specifically bind to 4-1BB; or (i') at least two ISVs of a first group that specifically bind to a first antigenic epitope of 4-1BB, and (ii') at least two other ISVs of a second group that specifically bind to a second antigenic epitope of 4-1BB.

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

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

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

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

24. A multispecific antigen-binding protein as claimed in any one of claims 1 to 23, wherein the multispecific antigen-binding protein comprises: a) a first polypeptide, preferably comprising, from the N-terminus to the C-terminus: i. a first ISV specifically binding to 4-1BB; ii. a second ISV specifically binding to 4-1BB, preferably wherein the second ISV is different from the first ISV; iii. at least one CH domain of the Fc region; and iv. variable and constant domains of the Fab fragment; b) a second polypeptide comprising the variable and constant domains of the Fab fragment; wherein the variable and constant domains of the first and second polypeptides form the Fab fragment.

25. The multispecific antigen-binding protein of claim 24, further comprising a third and a fourth polypeptide identical to the first and second polypeptides, respectively, wherein at least one CH domain of the first and third polypeptides forms an Fc region.

26. A multispecific antigen-binding protein as claimed in claim 24 or 25, wherein: - The variable and constant domains of the first polypeptide are VH and CH1 domains, and the variable and constant domains of the second polypeptide are VL and CL domains; or - The variable and constant domains of the first polypeptide are VL and CL domains, and the variable and constant domains of the second polypeptide are VH and CH1 domains.

27. A multispecific antigen-binding protein as claimed in any of claims 24 to 26, wherein at least one CH domain of the first polypeptide comprises: - CH2 and CH3 domains of IgG; - CH2 and CH3 domains of IgD; - CH2 and CH3 domains of IgA; - CH2, CH3 and CH4 domains of IgM; or - CH2, CH3 and CH4 domains of IgE.

28. A multispecific antigen-binding protein of any one of claims 24 to 27, wherein at least one CH domain of the first polypeptide comprises the CH2 and CH3 domains of IgG; preferably wherein the IgG is IgG1 or IgG4; more preferably wherein the IgG is IgG1.

29. A multispecific antigen-binding protein of any one of claims 24 to 28, wherein the first polypeptide preferably comprises, from the N-terminus to the C-terminus: - a first ISV specifically binding to 4-1BB; - a first linker; - a second ISV specifically binding to 4-1BB, preferably wherein the second ISV is different from the first ISV; - a second linker; - an IgG hinge region; - an IgG CH2 domain; - and an IgG CH3 domain; - a third linker; - a VH domain of the Fab fragment; and - a CH1 domain of the Fab fragment.

30. A multispecific antigen-binding protein of any one of claims 24 to 29, wherein the second polypeptide preferably comprises, from the N-terminus to the C-terminus: - a VL domain of the Fab fragment; and - a CL domain of the Fab fragment.

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

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

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

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

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

36. A multispecific antigen-binding protein of any one of claims 23 to 35, wherein at least one Fab fragment is an antigen-binding protein that specifically binds to PD-1, and comprising: (i) a light chain variable region comprising three of the following 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); and (ii) a heavy chain variable region comprising three of the following 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).

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

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

8.

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

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

12.

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

10.

42. A conditionally active multispecific antigen-binding protein comprising: (i) a multispecific antigen-binding protein as claimed in any one of claims 1 to 41, and (ii) at least one masking part that reduces or inhibits the binding of the multispecific antigen-binding protein to at least one of its target antigens.

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

97.

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

46. ​​A conditionally active multispecific antigen-binding protein as claimed in claim 45, wherein at least one linker is cleavable.

47. A conditionally active multispecific antigen-binding protein as claimed in claim 45 or 46, wherein at least one linker is cleavable by at least one tumor-specific protease.

48. The conditionally active multispecific antigen-binding protein of claim 47, wherein 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, legumain, kallikrein-related peptidase-3, human neutrophil elastase, protease 3 (Pr3), cathepsin B, and cathepsin K.

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

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

51. A conditionally active multispecific antigen-binding protein as claimed in any of claims 45 to 50, wherein at least one linker comprises or constitutes an amino acid sequence of SEQ ID NO: 46 or 47.

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

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

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

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

56. The ISV of request item 54 or 55, wherein the pure activator activity is determined by NF-κB pathway activation assay in the absence of crosslinking reagent.

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

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

59. The ISV of any one of claims 54 to 58, wherein the ISV 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. The ISV of any one of claims 54 to 59, wherein the ISV comprises three complementarity determination regions CDR1, CDR2 and CDR3; and wherein CDR3 comprises or constitutes an amino acid sequence ARGTRYKLST (SEQ ID NO: 14), ARGTRYKMST (SEQ ID NO: 15) or ARGTRYKIFA (SEQ ID NO: 62).

61. The ISV of claim 60, wherein CDR1 comprises or constitutes 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. The ISV of claim 60 or 61, wherein CDR2 comprises or constitutes the amino acid sequence ISSGGSRI (SEQ ID NO: 17), INPSGGSQ (SEQ ID NO: 77), or IKKSGNRT (SEQ ID NO: 78).

63. An ISV of any one of claims 54 to 62, wherein the ISV comprises or consists of: (i) an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, 3, 58, 59, 60 and 61; or (ii) an amino acid sequence having at least 70% sequence identity with the non-CDR region of SEQ ID NO: 2, 3, 58, 59, 60 or 61.

64. An ISV of any one of claims 54 to 63, wherein the ISV comprises or constitutes an amino acid sequence SEQ ID NO: 2 or 3.

65. An ISV of any one of claims 54 to 64, wherein the ISV comprises or constitutes an amino acid sequence SEQ ID NO:

3.

66. If the ISV is any of the requests in items 54 to 65, then it is V HH.

67. An immunoglobulin single variable domain (ISV) that specifically binds to 4-1BB, wherein the ISV comprises or consists of: (i) an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and 4, or (ii) an amino acid sequence having at least 70% sequence identity with the non-CDR region of SEQ ID NO: 1 or 4.

68. The ISV of claim 67, wherein the ISV comprises or constitutes an amino acid sequence SEQ ID NO:

4.

69. If the ISV of request item 67 or 68 is V HH.

70. A bivalent or bispecific antigen-binding protein comprising at least one immunoglobulin single variable domain (ISV) as claimed in any one of claims 54 to 66, and at least one second ISV that specifically binds to the same or another target antigen.

71. A bivalent or bispecific antigen-binding protein, as requested in claim 70, wherein the other target antigen is a T-cell antigen, a tumor-associated or tumor-specific antigen, or a non-autoantigen.

72. A bivalent or bispecific antigen-binding protein as claimed in claim 70, wherein at least the second ISV is an ISV of any one of claims 67 to 69.

73. A conditionally active immunoglobulin single variable domain (ISV) comprising: (i) an ISV as claimed in any one of claims 67 to 69, and (ii) at least one masking portion that reduces or inhibits the binding of the ISV to its target antigen.

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

97.

75. The conditional active ISV of claim 73 or 74, wherein at least one masking portion comprises or constitutes an amino acid sequence of SEQ ID NO: 44 or 45, or an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 44 or 45.

76. The conditionally active ISV of any of claims 73 to 75 further includes at least one connector between the ISV and the shielding part.

77. A conditionally active ISV as claimed in claim 76, wherein at least one linker is cleavable.

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

79. The conditionally active ISV of claim 78, wherein 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), interstitial protease, soybean protease, kallikrein-associated peptidase-3, human neutrophil elastase, protease 3 (Pr3), cathepsin B, and cathepsin K.

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

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

82. A conditionally active ISV as claimed in any of claims 76 to 81, wherein at least one linker comprises or constitutes an amino acid sequence of SEQ ID NO: 46 or 47.

83. The conditional active ISV of any one of claims 73 to 82, wherein the ISV comprises or constitutes an amino acid sequence selected from the group consisting of SEQ ID NO: 48, 49, 50 and 51.

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

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

97.

86. The conditional active ISV of claim 84 or 85, wherein at least one masking portion comprises or constitutes an amino acid sequence of SEQ ID NO: 44 or 45, or an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 44 or 45.

87. The conditionally active ISV of any of claims 84 to 86 further includes at least one connector between the ISV and the shielding part.

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

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

90. The conditionally active ISV of claim 89, wherein 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), interstitial protease, soybean protease, kallikrein-associated peptidase-3, human neutrophil elastase, protease 3 (Pr3), cathepsin B, and cathepsin K.

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

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

93. A conditionally active ISV as claimed in any of claims 87 to 92, wherein at least one linker comprises or constitutes an amino acid sequence of SEQ ID NO: 46 or 47.

94. The conditional active ISV as described in any of claims 84 to 93, wherein the ISV is V HH.

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

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

97.

97. The conditional active ISV of claim 95 or 96, wherein at least one masking portion comprises or constitutes an amino acid sequence of SEQ ID NO: 44 or 45, or an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 44 or 45.

98. The conditionally active ISV of any of claims 95 to 97 further includes at least one connector between the ISV and the shielding part.

99. A conditionally active ISV as claimed in claim 98, wherein at least one linker is cleavable.

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

101. The conditionally active ISV of claim 100, wherein 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), interstitial protease, soybean protease, kallikrein-associated peptidase-3, human neutrophil elastase, protease 3 (Pr3), cathepsin B, and cathepsin K.

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

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

104. A conditionally active ISV as claimed in any of claims 98 to 103, wherein at least one linker comprises or constitutes an amino acid sequence of SEQ ID NO: 46 or 47.

105. The conditional active ISV as described in any of claims 95 to 104, wherein the ISV is V HH.

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

107. The antibody or its antigen-binding fragment, as requested in claim 106, specifically binds to PD-1.

108. The antibody or antigen-binding fragment thereof as claimed in claim 106 or 107, comprising: (i) a light chain variable region comprising three of the following 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); and (ii) a heavy chain variable region comprising three of the following 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).

109. An antibody or antigen-binding fragment thereof of any one of claims 106 to 108, comprising: (i) a light chain variable region comprising three of the following 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); and (i) a heavy chain variable region comprising three of the following 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).

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

111. An antibody or antigen-binding fragment thereof of 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. An antibody or antigen-binding fragment thereof of 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. A composition comprising a multispecific antigen-binding protein as claimed in any one of claims 1 to 41, and a pharmaceutically acceptable carrier or excipient.

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

115. A composition comprising a single variable immunoglobulin domain as claimed in any one of claims 54 to 66, and a pharmaceutically acceptable carrier or excipient.

116. A composition comprising a single variable immunoglobulin domain as claimed in any one of claims 67 to 69, and a pharmaceutically acceptable carrier or excipient.

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

118. A composition comprising a conditionally active immunoglobulin single variable domain as claimed in any of claims 73 to 83, and a pharmaceutically acceptable carrier or excipient.

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

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

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

122. A method of treating an individual in need, comprising administering to the individual an effective amount of any one of the components of claims 113 to 121.

123. A method of treating an individual in need, comprising administering to the individual an effective amount of the component as requested in claim 113.

124. A method of treating an individual in need, comprising administering to the individual an effective amount of the component as requested in claim 114.

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

126. A component of any of claims 113 to 121, used to treat cancer in an individual in need.

127. A component of claim 113, used to treat cancer in an individual in need.

128. A component of claim 114, used to treat cancer in an individual in need.

129. An isolated polynucleotide encoding a multispecific antigen-binding protein as claimed in any one of claims 1 to 41.

130. An isolated polynucleotide encoding a conditionally active multispecific antigen-binding protein as claimed in any of claims 42 to 53.

131. An isolated polynucleotide encoding an immunoglobulin single variable domain as claimed in any of claims 54 to 66.

132. An isolated polynucleotide encoding an immunoglobulin single variable domain as claimed in any of claims 67 to 69.

133. An isolated polynucleotide encoding a bivalent or bispecific antigen-binding protein as claimed in any of claims 70 to 72.

134. An isolated polynucleotide encoding a conditionally active immunoglobulin single variable domain as claimed in any of claims 73 to 83.

135. An isolated polynucleotide encoding a conditionally active immunoglobulin single variable domain as claimed in any of claims 84 to 94.

136. An isolated polynucleotide encoding a conditionally active immunoglobulin single variable domain as claimed in any of claims 95 to 105.

137. An isolated polynucleotide encoding an antibody or antigen-binding fragment thereof as claimed in any one of claims 106 to 112.

138. A carrier comprising a polynucleotide as claimed in any one of claims 129 to 137.

139. A host cell comprising a polynucleotide as claimed in any one of claims 129 to 137.

140. A method for manufacturing a multispecific antigen-binding protein as claimed in any one of claims 1 to 41, comprising expressing a polynucleotide as claimed in claim 129 in a cell.

141. A method for manufacturing a conditionally active multispecific antigen-binding protein as claimed in any one of claims 42 to 53, comprising expressing a polynucleotide as claimed in claim 130 in a cell.

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

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

144. A method for manufacturing a bivalent or bispecific antigen-binding protein as claimed in any one of claims 70 to 72, comprising expressing a polynucleotide as claimed in claim 133 in a cell.

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

146. A method for producing a single variable domain of a conditionally active immunoglobulin as claimed in any one of claims 84 to 94, comprising expressing a polynucleotide as claimed in claim 135 in a cell.

147. A method for manufacturing a conditionally active immunoglobulin single variable domain as claimed in any one of claims 95 to 105, comprising expressing a polynucleotide as claimed in claim 136 in a cell.

148. A method of manufacturing an antibody or antigen-binding fragment thereof as claimed in any one of claims 106 to 112, comprising expressing a polynucleotide as claimed in claim 137 in cells.

Images