A bispecific binding protein that binds CD137 and a tumor-associated antigen

Abstract

The present disclosure provides bispecific binding proteins and fragments thereof that bind to human CD137 and tumor-associated antigens (e.g., Claudin-6, Claudin18.2, or Nectin-4), polynucleotide sequences encoding these antibodies, and cells that produce them. The present disclosure further relates to therapeutic compositions comprising these antibodies, and methods of using them for cancer detection, prognosis, and antibody-based immunotherapy.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This international patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 240,402, filed September 3, 2021, and U.S. Provisional Patent Application No. 63 / 327,700, filed April 5, 2022, the entire contents of which are incorporated herein by reference.

[0002] Description of sequence listing The sequence listing associated with this application is provided in XML format in lieu of a paper copy and is incorporated herein by reference. The XML file containing the sequence listing is named 122863-5007_Sequence_Listing_ST.26.xml. This text file is approximately 111,345 bytes, was created on or about August 28, 2022, and has been submitted electronically via EFS-Web.

[0003] The present disclosure is in the field of immunotherapy and relates to bispecific binding proteins and fragments thereof that bind to human CD137 and tumor-associated antigens (e.g., Claudin-6, Claudin18.2, or Nectin-4), polynucleotide sequences encoding these antibodies, and cells that produce them. The disclosure further relates to therapeutic compositions comprising these bispecific binding proteins, and methods of their use for cancer detection, prognosis, and antibody-based immunotherapy. [Background technology]

[0004] T cell activation plays a central role in antitumor immunity. Two major signals are required to activate naive T cells. Signal 1 is provided via the T cell receptor (TCR), whereas signal 2 is a signal for costimulation. The CD28:B7 molecule is part of the most studied costimulatory pathway and is thought to be the main mechanism by which primary T cell stimulation occurs. However, numerous other molecules have been identified that function to amplify and diversify T cell responses after initial T cell activation. These include the CD137 / CD137 ligand (CD137L) molecule, also known as 4-1BB:4-1BB ligand (4-1BBL).

[0005] CD137 (4-1BB, tumor necrosis factor receptor superfamily 9) is a member of the TNF receptor superfamily (TNFRSF) and a costimulatory molecule expressed following activation of immune cells, both innate and adaptive. CD137 plays an important role in regulating the activity of various immune cells. Therapies targeting the CD137 / CD137L signaling pathway have been shown to have antitumor effects in multiple model systems, and agonistic anti-CD137 antibodies are also in clinical development (Yonezawa et al., Clin. Cancer Res. 2015 Jul.15;21(14):3113-20; Tolcher et al., Clin Cancer Res. 2017 Sep.15;23(18):5349-5357). CD137 agonists enhance immune cell proliferation, survival, cytokine secretion, and cytolytic activity of CD8 T cells. Numerous other studies have shown that activation of CD137 enhances immune responses to eliminate tumors in mice. In the clinic, CD137 monoclonal antibody therapy has shown promising antitumor effects, but systemic immune stimulation induces dose-limiting liver toxicity (Chester, C. et al., Cancer Immunol Immunother 65, 1243-1248 (2016); Segal, NH et al., Clin. Cancer Res. 2017, 23, 1929-1936).

[0006] To avoid the side effects of liver inflammation and to widen the therapeutic window, new CD137 agonist moieties are being developed with the aim of potent costimulation targeting the tumor microenvironment (TME). Different approaches are applied. The most advanced in clinical development are CD137-based bispecific constructs that bring CD137 costimulation specifically to the TME, such as bispecific antibodies targeting tumor antigens (e.g., TAAs or TSAs) and CD137. Antitumor activity may be achieved by directing the host immune system to tumor-associated antigens. Linking tumor cells with CD137-expressing T cells to increase cellular cytotoxicity represents a promising strategy in cancer therapy.

[0007] CD137-Her2 bispecific antibodies have demonstrated good tolerability of the constructs and evidence of clinical activity (Hinner MJ et al., Clin Cancer Res. 2019 Oct 1;25(19):5878-5889; Piha-Paul S et al., A Phase I Dose Escalation Study of the HER2 / 4-1BB Bispecific Molecule PRS-343 in Patients with HER2+ Malignancies, The Society for Cancer Immunotherapy's 34th Annual and Pre-Meeting Program, National Harbor, MD, 2019), and bispecific antibodies targeting the tumor antigens 5T4 and CD137 have also demonstrated good preclinical activity (Nelson M et al., Potent Tumor-Specific T Cell Activation and Tumor Inhibition Induced by the 4-1BB x 5T4 ADAPTIRTM Bispecific Antibody ALG.APV-527, The Society for Cancer Immunotherapy's 34th Annual and Pre-Meeting Program, National Harbor, MD, 2019).

[0008] Members of the Claudin superfamily are key components of tight junctions, which maintain cell polarity and seal the space between adjacent cells. Claudin6 (CLDN6) is an oncofetal protein that is expressed during early development but silenced in healthy adult human tissues. CLDN6 expression has been reported in a wide range of non-hematological cancers, including pediatric brain tumors, gastric adenocarcinoma and germ cell tumors, as well as ovarian and testicular cancer. Its expression often correlates with poor prognosis. Claudin18.2 is widely expressed in a wide range of human malignancies, including gastric, esophageal, pancreatic, lung, and ovarian cancers. Claudin18.2 has an excellent target safety profile. In normal tissues, Claudin18.2 expression is restricted to the stomach and only on short-lived differentiated cells. Nectin family proteins mediate cell-cell adhesion through homophilic and heterophilic trans interactions, with heterophilic trans interactions being much stronger than homophilic trans interactions. As a member of the nectin protein family, nectin-4 is a key driver of tumorigenesis and metastasis. Overexpression of nectin-4 in cancer tissues is associated with cancer progression and poor prognosis.

[0009] Cancers of epithelial origin represent a significant and global medical challenge affecting patients, their families, and healthcare systems. To address the unmet medical needs of patients with tumors overexpressing epithelial tumor antigens such as CLDN6, CLDN18.2, or Nectin-4, CD137 and specific binding proteins that bind to these tumor antigens can be used in antibody-based immunotherapy, alone or in combination with other agents, thus offering a potentially effective and safe therapeutic solution. Summary of the Invention

[0010] The present disclosure addresses the above needs by providing bispecific binding proteins that bind to CD137 and tumor-associated antigens (TAA). In certain embodiments, the present disclosure provides bispecific antibodies that bind to tumor-specific antigens CLDN18.2, CLDN6, or Nectin-4 and the costimulatory CD137 receptor. Such bispecific binding proteins may be useful in the treatment of diseases or disorders such as cancer.

[0011] Also provided herein is a bispecific binding protein that binds a tumor-associated antigen and CD137, the bispecific binding protein comprising (a) an antibody scaffold module comprising a first antigen-binding site that binds the tumor-associated antigen and a second antigen-binding site that binds the tumor-associated antigen, and (b) at least one first binding module comprising a third antigen-binding site that binds CD137.

[0012] In some embodiments, the tumor-associated antigen is selected from the group consisting of: Claudin6, Claudin18.2, and Nectin-4.

[0013] In some embodiments, the tumor-associated antigen is Claudin6.

[0014] In some embodiments, the tumor-associated antigen is Claudin 18.2.

[0015] In some embodiments, the tumor-associated antigen is Nectin-4.

[0016] In some embodiments, the antibody scaffold module is an IgG.

[0017] In some embodiments, the first antigen-binding site and the second antigen-binding site both bind to Claudin6 and comprise (i) a heavy chain variable region sequence comprising CDR1: SEQ ID NO: 45, CDR2: SEQ ID NO: 46, and CDR3: SEQ ID NO: 47, and a light chain variable region sequence comprising CDR1: SEQ ID NO: 48, CDR2: SEQ ID NO: 49, and CDR3: SEQ ID NO: 50, or (ii) a heavy chain variable region sequence comprising CDR1: SEQ ID NO: 51, CDR2: SEQ ID NO: 52, and CDR3: SEQ ID NO: 53, and a light chain variable region sequence comprising CDR1: SEQ ID NO: 54, CDR2: SEQ ID NO: 55, and CDR3: SEQ ID NO: 56.

[0018] In some embodiments, the first antigen-binding site and the second antigen-binding site both bind to Claudin6 and comprise a heavy chain variable region sequence set forth in SEQ ID NO:25 or SEQ ID NO:27 and a light chain variable region sequence set forth in SEQ ID NO:26 or SEQ ID NO:28.

[0019] In some embodiments, the first antigen-binding site and the second antigen-binding site both bind to Claudin6 and comprise (i) a heavy chain variable region sequence set forth in SEQ ID NO: 25 and a light chain variable region sequence set forth in SEQ ID NO: 26, or (ii) a heavy chain variable region sequence set forth in SEQ ID NO: 27 and a light chain variable region sequence set forth in SEQ ID NO: 28.

[0020] In some embodiments, the first antigen-binding site and the second antigen-binding site both bind to Claudin18.2 and comprise a heavy chain variable region sequence comprising CDR1: SEQ ID NO: 33, CDR2: SEQ ID NO: 34, and CDR3: SEQ ID NO: 35, and a light chain variable region sequence comprising CDR1: SEQ ID NO: 36, CDR2: SEQ ID NO: 37, and CDR3: SEQ ID NO: 38.

[0021] In some embodiments, the first antigen-binding site and the second antigen-binding site both bind to Claudin18.2 and comprise a heavy chain variable region sequence set forth in SEQ ID NO:21 and a light chain variable region sequence set forth in SEQ ID NO:22.

[0022] In some embodiments, the first antigen-binding site and the second antigen-binding site both bind to Nectin-4 and comprise a heavy chain variable region sequence comprising CDR1: SEQ ID NO: 57, CDR2: SEQ ID NO: 58, and CDR3: SEQ ID NO: 59, and a light chain variable region sequence comprising CDR1: SEQ ID NO: 60, CDR2: SEQ ID NO: 61, and CDR3: SEQ ID NO: 62.

[0023] In some embodiments, the first antigen-binding site and the second antigen-binding site both bind to Nectin-4 and comprise a heavy chain variable region sequence set forth in SEQ ID NO:29 or SEQ ID NO:31 and a light chain variable region sequence set forth in SEQ ID NO:30 or SEQ ID NO:32.

[0024] In some embodiments, the first antigen-binding site and the second antigen-binding site both bind to Nectin-4 and comprise (i) a heavy chain variable region sequence set forth in SEQ ID NO: 29 and a light chain variable region sequence set forth in SEQ ID NO: 30, or (ii) a heavy chain variable region sequence set forth in SEQ ID NO: 31 and a light chain variable region sequence set forth in SEQ ID NO: 32.

[0025] In some embodiments, the bispecific binding protein comprises one first binding module.

[0026] In some embodiments, the binding protein comprises two first link modules.

[0027] In some embodiments, the first binding module is an antibody fragment.

[0028] In some embodiments, the antibody fragment is an scFv.

[0029] In some embodiments, the first link module binds to CD137.

[0030] In some embodiments, the first link module is an scFV that binds CD137.

[0031] In some embodiments, an antibody scaffold module comprises two heavy chain sequences, each having a C-terminus and an N-terminus, and an antibody scaffold module comprises two light chain sequences, each having a C-terminus and an N-terminus, a first link module is covalently attached to the C-terminus of one or both of the antibody scaffold module heavy chain sequences, the C-terminus of one or both of the antibody scaffold module light chain sequences, the N-terminus of one or both of the antibody scaffold module heavy chain sequences, the N-terminus of one or both of the antibody scaffold module light chain sequences, or a combination thereof, and the first link module and the antibody scaffold module are covalently attached to each other directly or via an interlinker.

[0032] In some embodiments, the first link module and the antibody scaffold module are covalently linked to each other via an interlinker having the sequence set forth in SEQ ID NO:64 or SEQ ID NO:65.

[0033] In some embodiments, the first link module is covalently attached to both C-terminuses of the antibody scaffold module heavy chain sequence.

[0034] In some embodiments, the first link module is covalently attached to both C-terminuses of the antibody scaffold module light chain sequence.

[0035] In some embodiments, the first link module is covalently attached to both N-termini of the antibody scaffold module heavy chain sequence.

[0036] In some embodiments, the first link module binds to CD137 and comprises a heavy chain variable region sequence comprising CDR1: SEQ ID NO: 39, CDR2: SEQ ID NO: 40, and CDR3: SEQ ID NO: 41, and a light chain variable region sequence comprising CDR1: SEQ ID NO: 42, CDR2: SEQ ID NO: 43, and CDR3: SEQ ID NO: 44.

[0037] In some embodiments, the first link module binds to CD137 and comprises a heavy chain variable region sequence set forth in SEQ ID NO:23 and a light chain variable region sequence set forth in SEQ ID NO:24.

[0038] In some embodiments, the bispecific binding protein comprises two first link modules that bind to CD137, and the first antigen binding site and the second antigen binding site both bind to Claudin18.2 and comprise a heavy chain variable region sequence set forth in SEQ ID NO: 21 and a light chain variable region sequence set forth in SEQ ID NO: 22, and each of the first link modules comprises a heavy chain variable region sequence set forth in SEQ ID NO: 23 and a light chain variable region sequence set forth in SEQ ID NO: 24, and the first link modules are separately attached to the C-terminus of each heavy chain in the antibody scaffold module by a glycine-serine linker.

[0039] In some embodiments, the glycine-serine linker is a 3×G4S linker (SEQ ID NO:64).

[0040] In some embodiments, the heavy chain variable region sequence and the light chain variable region sequence in the first link module are linked by a glycine-serine linker.

[0041] In some embodiments, the glycine-serine linker is a 4×G4S linker (SEQ ID NO: 65).

[0042] In some embodiments, the heavy chain, the glycine-serine linker, and the first link module of the antibody scaffold module comprise the sequence set forth in SEQ ID NO:3.

[0043] In some embodiments, the bispecific binding protein comprises two first link modules that bind to CD137, and the first antigen binding site and the second antigen binding site both bind to Claudin18.2 and comprise a heavy chain variable region sequence set forth in SEQ ID NO: 21 and a light chain variable region sequence set forth in SEQ ID NO: 22, and each of the first link modules comprises a heavy chain variable region sequence set forth in SEQ ID NO: 23 and a light chain variable region sequence set forth in SEQ ID NO: 24, and the first link modules are separately attached to the C-terminus of each light chain in the antibody scaffold module by a glycine-serine linker.

[0044] In some embodiments, the glycine-serine linker is a 3×G4S linker (SEQ ID NO:64).

[0045] In some embodiments, the heavy chain variable region sequence and the light chain variable region sequence in the first link module are linked by a glycine-serine linker.

[0046] In some embodiments, the glycine-serine linker is a 4×G4S linker (SEQ ID NO: 65).

[0047] In some embodiments, the light chain, the glycine-serine linker, and the first link module of the antibody scaffold module comprise the sequence set forth in SEQ ID NO:5.

[0048] In some embodiments, the bispecific binding protein comprises two first link modules that bind to CD137, and the first antigen binding site and the second antigen binding site both bind to Claudin6 and comprise a heavy chain variable region sequence set forth in SEQ ID NO: 25 or 27 and a light chain variable region sequence set forth in SEQ ID NO: 26 or 28, and each of the first link modules comprises a heavy chain variable region sequence set forth in SEQ ID NO: 23 and a light chain variable region sequence set forth in SEQ ID NO: 24, and the first link modules are separately attached to the C-terminus of each heavy chain in the antibody scaffold module by a glycine-serine linker.

[0049] In some embodiments, the glycine-serine linker is a 3×G4S linker (SEQ ID NO:64).

[0050] In some embodiments, the heavy chain variable region sequence and the heavy chain variable region sequence in the first link module are linked by a glycine-serine linker.

[0051] In some embodiments, the glycine-serine linker is a 3×G4S linker (SEQ ID NO:64).

[0052] In some embodiments, the heavy chain, the glycine-serine linker, and the first link module of the antibody scaffold module comprise the sequence set forth in SEQ ID NO: 12, 13, or 72.

[0053] In some embodiments, the bispecific binding protein comprises two first link modules that bind to CD137, and the first antigen binding site and the second antigen binding site both bind to Nectin-4 and comprise a heavy chain variable region sequence set forth in SEQ ID NO: 29 or 31 and a light chain variable region sequence set forth in SEQ ID NO: 30 or 32, and each of the first link modules comprises a heavy chain variable region sequence set forth in SEQ ID NO: 23 and a light chain variable region sequence set forth in SEQ ID NO: 24, and the first link modules are separately attached to the C-terminus of each light chain in the antibody scaffold module by a glycine-serine linker.

[0054] In some embodiments, the glycine-serine linker is a 3×G4S linker (SEQ ID NO:64).

[0055] In some embodiments, the heavy chain variable region sequence and the light chain variable region sequence in the first link module are linked by a glycine-serine linker.

[0056] In some embodiments, the glycine-serine linker is a 4×G4S linker (SEQ ID NO: 65).

[0057] In some embodiments, the light chain, the glycine-serine linker, and the first link module of the antibody scaffold module comprise the sequence set forth in SEQ ID NO:17.

[0058] In some embodiments, the bispecific binding protein comprises two first link modules that bind to CD137, wherein the first antigen binding site and the second antigen binding site both bind to Nectin-4 and comprise a heavy chain variable region sequence set forth in SEQ ID NO: 29 or 31 and a light chain variable region sequence set forth in SEQ ID NO: 30 or 32, wherein the first link modules comprise a heavy chain variable region sequence set forth in SEQ ID NO: 23 and a light chain variable region sequence set forth in SEQ ID NO: 24, respectively, and the first link modules are separately attached to the N-terminus of each heavy chain in the antibody scaffold module by a glycine-serine linker.

[0059] In some embodiments, the glycine-serine linker is a 4×G4S linker (SEQ ID NO: 65).

[0060] In some embodiments, the heavy chain variable region sequence and the light chain variable region sequence in the first link module are linked by a glycine-serine linker.

[0061] In some embodiments, the glycine-serine linker is a 4×G4S linker (SEQ ID NO: 65).

[0062] In some embodiments, the first link module, the glycine-serine linker, and the heavy chain of the antibody scaffold module comprise the sequence set forth in SEQ ID NO:18.

[0063] In some embodiments, the bispecific binding protein comprises two first link modules that bind to CD137, and the first antigen binding site and the second antigen binding site both bind to Nectin-4 and comprise a heavy chain variable region sequence set forth in SEQ ID NO: 29 or 31 and a light chain variable region sequence set forth in SEQ ID NO: 30 or 32, and each of the first link modules comprises a heavy chain variable region sequence set forth in SEQ ID NO: 23 and a light chain variable region sequence set forth in SEQ ID NO: 24, and the first link modules are separately attached to the C-terminus of each heavy chain in the antibody scaffold module by a glycine-serine linker.

[0064] In some embodiments, the glycine-serine linker is a 3×G4S linker (SEQ ID NO:64).

[0065] In some embodiments, the heavy chain variable region sequence and the light chain variable region sequence in the first link module are linked by a glycine-serine linker.

[0066] In some embodiments, the glycine-serine linker is a 4×G4S linker (SEQ ID NO: 65).

[0067] In some embodiments, the heavy chain, the glycine-serine linker, and the first link module of the antibody scaffold module comprise the sequence set forth in SEQ ID NO:14.

[0068] In some embodiments, the antibody scaffold module further comprises a constant region.

[0069] In some embodiments, the constant region comprises one or more Fc-silencing mutations.

[0070] In some embodiments, the Fc silencing mutation can be L234A / L235A (LALA) alone or in combination with a P329A mutation (LALAP) or N297A.

[0071] In some embodiments, the constant region comprises SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, or SEQ ID NO:73.

[0072] Also disclosed herein is a bispecific binding protein that binds to a tumor associated antigen and CD137, the bispecific binding protein comprising (a) an antibody scaffold module comprising a means for binding to the tumor associated antigen via a first antigen binding site and a second antigen binding site, and (b) at least one first binding module comprising a means for binding to CD137 via a third antigen binding site.

[0073] The present disclosure also provides a pharmaceutical composition comprising a bispecific binding protein disclosed herein and a pharma- ceutically acceptable carrier.

[0074] The present disclosure also provides a method of treating or preventing cancer, the method comprising administering a bispecific binding protein disclosed herein to a patient in need thereof.

[0075] Also provided herein is an isolated polynucleotide comprising a sequence encoding the bispecific binding protein disclosed herein. The disclosure also provides a vector or cell comprising the polynucleotide disclosed herein. Also provided herein is a method for producing the bispecific binding protein disclosed herein, comprising culturing the cell disclosed herein. [Brief description of the drawings]

[0076] The foregoing summary, as well as the following detailed description of the present disclosure, will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the present disclosure, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the present disclosure is not limited to the precise arrangements, examples, and instrumentalities shown.

[0077] [Figure 1A-1]Figure 1. Figures 1-1C show the amino acid sequences of the heavy and light chain sequences of the CLDN18.2 / CD137, CLDN6 / CD137, and Nectin-4 / CD137 bispecifics. [Figure 1A-2] Same as above. [Figure 1B-1] Same as above. [Figure 1B-2] Same as above. [Figure 1C-1] Same as above. [Figure 1C-2] Same as above. [Figure 1C-3] Same as above. [Figure 1D-1] Figure ID shows the amino acid sequences of the VH and VL domains of binding proteins that bind to CD137, CLDN6, CLDN18.2, or Nectin-4. The sequences of other components of the TAA / CD137 bispecific protein are also shown. The CDR sequences (Kabat numbering) of the VH and VL domains are underlined in their respective variable domain sequences. Sequence identifiers are shown. [Figure 1D-2] Same as above. [Figure 1D-3] Same as above. [Figure 1D-4] Same as above. [Figure 1D-5] Same as above. [Figure 1D-6] Same as above.

[0078] [Diagram 2]FIG. 2 shows three exemplary bispecific binding protein formats, including: i) a first format (BsAb_A) having an antibody scaffold module having two antigen-binding sites that bind to a tumor-associated antigen and two first binding modules (e.g., scFvs) that bind to CD137, each separately bound to the C-terminus of the heavy chain constant region of the antibody scaffold module; ii) a second format (BsAb_B) having an antibody scaffold module having two antigen-binding sites that bind to a tumor-associated antigen and two first binding modules (e.g., scFvs) that bind to CD137, each separately bound to the C-terminus of the light chain constant region of the antibody scaffold module; and iii) a third format (BsAb_C) having an antibody scaffold module having two antigen-binding sites that bind to a tumor-associated antigen and two first binding modules (e.g., scFvs) that bind to CD137, each separately bound to the N-terminus of the heavy chain variable region of the antibody scaffold module.

[0079] [Diagram 3] FIG. 3 shows the composition of heavy and light chains of several binding proteins, including 1901Ab1, 1901Ab2, 1901Ab3, 1923Ab4, 1912Ab1, 1912Ab2, 1912Ab3, 1912Ab4, 1912Ab5, 1925Ab1, 1925Ab2, 1925Ab3, and 1925Ab4.

[0080] [Figure 4] FIG. 4 shows the composition of antibody scaffold modules and binding modules (if present) of multiple binding proteins, including 1901Ab1, 1901Ab2, 1901Ab3, 1923Ab4, 1912Ab1, 1912Ab2, 1912Ab3, 1912Ab4, 1912Ab5, 1925Ab1, 1925Ab2, 1925Ab3, and 1925Ab4.

[0081] [Diagram 5]5 shows sequence identifiers for sequences in multiple binding proteins, including 1901Ab1, 1901Ab2, 1901Ab3, 1923Ab4, 1912Ab1, 1912Ab2, 1912Ab3, 1912Ab4, 1912Ab5, 1925Ab1, 1925Ab2, 1925Ab3, and 1925Ab4. For 1901Ab2, 1912Ab3, 1912Ab4, 1912Ab5, 1925Ab1, and 1925Ab3, the heavy chain sequence includes the amino acid sequence of the heavy chain, glycine-serine linker, and first binding module of the antibody scaffold module. For 1901Ab3 and 1925Ab2, the light chain sequence includes the amino acid sequence of the light chain, a glycine-serine linker, and the first link module of the antibody scaffold module.

[0082] [Figure 6-1] Figures 6A-B show binding activity to the tumor antigen Claudin6. Figure 6A shows monospecific (1912Ab1 and 1912Ab2) and bispecific CLDN6 / CD137 (1912Ab3 and 1912Ab4) BsAbs against human Claudin6 on the cell surface of NEC8 wild-type cells compared to NEC8 CLDN6 knockout cells. Figure 6B shows bsAb 1912Ab5 binding to NEC8 wild-type cells. [Figure 6-2] FIG. 6C shows that 1912Ab5 selectively binds to Claudin 6 but not to Claudin 9.

[0083] [Figure 7-1] Figures 7A-B show CD137 binding activity. Figure 7A shows surface plasmon resonance (SPR) binding analysis of 1912Ab5 binding to CD137. Figure 7B shows human CD137 binding of 1912Ab3, 1912Ab4, and 1923Ab4 in a HEK-CD137 cell-based binding assay. [Figure 7-2]Figure 7C shows dose-response binding curves of 1912Ab5 and Urelumab-NR binding to human CD137. Urelumab-NR is an internal control anti-CD137 antibody based on publicly available information published in U.S. Patent No. 7,288,638.

[0084] [Figure 8-1] Figures 8A-D show Claudin-6-dependent activation of CD137 signaling by CLDN6 / CD137 BsAbs using CD137 NFKB reporter cells in Jurkat T cells. Figure 8A shows activity from 1912Ab3, 1912Ab4, or benchmark control Urelumab-NR in a co-culture assay in the presence of NEC8 wild-type cells or Claudin6 knockout NEC8 cells. Figure 8B shows dose-dependent activity of CLDN6 / CD137 BsAbs 1912Ab3, 1912Ab4, or benchmark control Urelumab-NR in a signaling assay in the presence of NEC8 wild-type cells. [Figure 8-2] Figure 8C shows NFKB activation by 1912Ab5 or urelumab-NR in a co-culture signaling assay using NEC8 target cells. Figure 8D shows NFKB activation by 1912Ab5 or urelumab-NR in a co-culture signaling assay using OV90 target cells.

[0085] [Figure 9-1] Figure 9 shows the Claudin6-dependent activation of CD8 T cells by CLDN6 / CD137 BsAb to induce IFNγ secretion. Figure 9A shows the activity from 1912Ab3, 1912Ab4, or benchmark control Urelumab-NR in a co-culture assay in the presence of NEC8 wild-type cells or Claudin6 knockout NEC8 cells. Figure 9B shows the dose-dependent activity of CLDN6 / CD137 BsAb 1912Ab3, 1912Ab4, and benchmark control Urelumab-NR in the presence of NEC8 wild-type cells. [Figure 9-2]Figure 9C shows IFNγ secretion by 1912Ab5 or Urelumab-NR in a co-culture signaling assay using NEC8 wild-type cells. Figure 9D shows NFKB activation by 1912Ab5 or Urelumab-NR in a co-culture signaling assay using Claudin6 knockout NEC8 cells.

[0086] [Figure 10] Figures 10A-B show T cell-derived killing, which is the killing of target cells. Figure 10A shows the killing of NEC8 cells by CLDN6 / CD137 bispecific antibodies 1912Ab3 and 1912Ab4. Figure 10B shows the killing of T cell-derived OV90 cells by CLDN6 / CD137 bispecific antibody 1912Ab5.

[0087] [Figure 11] Figures 11A-C show in vivo efficacy and safety data using a mouse MC38 tumor model. Figure 11A shows the inhibition of MC38-Claudin6 tumor growth in vivo by CLDN6 / CD137 BsAbs 1912Ab3 and 1912Ab4 with -Claudin6. Active enzyme activity in mice was measured using serum on day 21. ALT activity is shown in Figure 11B and AST activity in Figure 11C. Figure 11D shows the results of a rechallenge study using mice that were pre-treated with 1912Ab3 or 1912Ab4 and had complete tumor remission.

[0088] [Figure 12] FIG. 12 shows the anti-tumor growth effect of 1912Ab5 at 0.3 mpk, 1 mpk, and 3 mpk.

[0089] [Figure 13] FIG. 13 shows the anti-tumor growth effect of 1912Ab5 at 0.1 mpk and the benchmark antibody urelumab-NR at 0.1 mpk.

[0090] [Figure 14]FIG. 14 shows the antitumor efficacy of 1912Ab5 treating large established tumors.

[0091] [Figure 15] FIG. 15 shows fluorescent immunohistochemistry (IHC) data from tumors treated with control (FIG. 15A) or 1912Ab5 (FIG. 15B).

[0092] [Figure 16] Figures 16A-F show the results of tumor infiltrating lymphocytes from control or 1912Ab5 treated tumors. The data describes immune cell profiling comparing data from control and 1912Ab5 treated cells in CD4 (Figure 16A), CD8 (Figure 16B), Tcem (Figure 16C), Trm (Figure 16D), exhausted T cells (Figure 16E), and M2-like macrophage cells (Figure 16F).

[0093] [Figure 17] FIG. 17 shows the anti-tumor growth effect of 1912Ab5 in treating B16-F10 tumors.

[0094] [Figure 18] FIG. 18 shows the binding activity of monospecific 1901Ab1 and CLDN18.2 / CD137 BsAbs 1901Ab2 and 1901Ab3 to human Claudin18.2 on NUGC4 cells.

[0095] [Figure 19] FIG. 19 shows the binding activity of CLDN18.2 / CD137 BsAbs 1901Ab2 and 1901Ab3, and the monospecific anti-CD137 antibody 1923Ab4, to cell surface human CD137.

[0096] [Figure 20]Figures 20A-B show Claudin18.2-dependent activation of CD137 signaling by CLDN18.2 / CD137 BsAb using CD137 reporter cells in Jurkat T cells. Figure 20A shows a bar graph, and Figure 20B shows the dose-dependent activity of Claudin18.2-CD137 bispecific antibody.

[0097] [Figure 21] FIG. 21 shows the dose response curve of CLDN18.2 / CD137 BsAb to induce CD8 T cell activation in the presence of NUGC4 cells.

[0098] [Figure 22] FIG. 22 shows T cell-derived target cell killing by CLDN18.2 / CD137 BsAbs 1901Ab2 and 1901Ab3.

[0099] [Figure 23] FIG. 23 shows inhibition of MC38-Claudin18.2 tumor growth in vivo by CLDN18.2 / CD137 BsAb, 1901Ab2.

[0100] [Figure 24] FIG. 24 shows the binding activity of Nectin4 / CD137 BsAbs 1925Ab1, 1925Ab2, and 1925Ab3 to human Nectin4 on CHO cells compared to the binding activity of the parent mouse monoclonal antibody 1925Ab4.

[0101] [Diagram 25] FIG. 25 shows the binding activity of Nectin4 / CD137 BsAbs 1925Ab1, 1925Ab2, and 1925Ab3 to cell surface human CD137 compared to the binding activity of the parent murine monoclonal antibody 1925Ab4.

[0102] [Figure 26A]Figures 26A-B show that Nectin-4 / CD137 BsAb induces target cell-dependent CD137 agonism using CD137 reporter cells in Jurkat T cells. Figure 26A shows a bar graph, and Figure 26B shows the dose-dependent activity of Nectin4 / CD137 bispecific antibodies. [Figure 26B] Same as above.

[0103] [Figure 27-1] Figures 27A-C show immune cell infiltration induced by treatment with control antibody, urelumab-NR, or 1912Ab5. Mouse liver IHC sections stained with CD4 (A), CD8 (B), and F4 / 80 (C) were used to demonstrate T cell and macrophage infiltration. [Figure 27-2] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0104] The present disclosure provides bispecific binding proteins that bind to CD137 and tumor-associated antigens (TAAs). Exemplary TAAs include, but are not limited to, Claudin6, Claudin18.2, and Nectin4. Advantageously, the bispecific binding proteins disclosed herein can overcome on-target toxicity. For example, in tissues such as the liver where tumor-associated antigens are not expressed or accessible, the molecules of the present disclosure will be safe because they cannot activate CD137-mediated cytotoxicity. In contrast, in tumor tissues where tumor-associated antigens are overexpressed or accessible, the antibody undergoes tumor-associated antigen binding-dependent activation of CD137 signaling, resulting in CD137-mediated immune cell activation, thereby treating the tumor. The bispecific binding proteins can be used to treat cancer. Furthermore, the bispecific binding proteins disclosed herein can result in lower dose formulations, resulting in less frequent and / or more effective administration, leading to reduced costs and increased efficiency.

[0105] In the tumor microenvironment, full T cell activation depends on two signals, one of which is mediated through TCR / CD3 activation and the other is mediated by the costimulatory pathway. Among the surface receptors that provide T cell costimulation, CD137 is a key regulator. Tumor-targeted CD137 agonistic antibodies can be used alone or in combination with tumor-targeted CD3-independent antibodies to promote T cell proliferation, survival, memory formation, and tumor-killing function.

[0106] It has been reported that CD137 costimulation (i.e., agonism) leads to prolonged T cell proliferation, reactivation of anergic T cells, and promotion of the formation and maintenance of memory T cells (Hashimoto K. Cancers (Basel). 2021 May 11; 13(10): 2288; Chester C et al.. Blood 2018 Jan 4; 131(1): 49-57)). Activating CD137 with agonistic antibodies offers an opportunity to improve the therapeutic efficacy of immune checkpoint inhibitors (ICIs) or overcome resistance to ICIs. Furthermore, bispecific antibodies that activate CD137 signaling only in the presence of TAAs may help reduce the dose-dependent hepatotoxicity observed in clinical trials with monoclonal anti-CD137 agonistic antibodies against activation of CD137 signaling in liver-resident Kupffer cells. Thus, the present disclosure provides novel tetravalent TAA / CD137 binding proteins (i.e., bispecific antibodies) uniquely designed to activate the CD137 costimulatory pathway in the tumor microenvironment via TAA-mediated clustering of CD137.

[0107] So that the present disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0108] The following abbreviations shall be used throughout this disclosure: BsAb - bispecific antibody mAb or Mab or MAb- Monoclonal Antibody. CDR--complementarity determining region within an immunoglobulin variable region. VH or VH-immunoglobulin heavy chain variable region. VL or VL- immunoglobulin light chain variable region. FR-antibody framework region, immunoglobulin variable region excluding the CDR regions.

[0109] The term "CD137" refers to 4-1BB or TNFRSF9 (TNF receptor superfamily member 9), a member of the TNF receptor superfamily (TNFRSF), which is a costimulatory molecule expressed following activation of immune cells (both innate and adaptive immune cells). As used herein, 4-1BB can be derived from a mammal, for example, Homo sapiens (human) (NCBI accession number NP_001552). As described herein, the term CD137 includes variants, isoforms, homologs, orthologs, and paralogs. For example, an antibody specific for human CD137 protein may cross-react with CD137 protein from species other than human in some cases. In other embodiments, an antibody specific for human CD137 protein may be completely specific for human CD-137 protein, may exhibit species or other types of cross-reactivity, and may cross-react with CD137 from some other species but not all other species (e.g., cross-react with monkey CD137 but not with mouse 4-1BB). The term "cyno CD137" refers to cynomolgus monkey CD137, such as the complete amino acid sequence having NCBI accession number XP_005544945.1. The term "mouse CD137" refers to the mouse sequence 4-1BB, such as the complete amino acid sequence of mouse 4-1BB, having NCBI accession number NP_035742.1. The human CD137 sequence of the present disclosure can differ from human CD137 of NCBI Accession No. NP_001552, for example, by having conserved mutations or mutations in non-conserved regions, and CD137 of the present disclosure has substantially the same biological function as human CD137 of NCBI Accession No. NP_001552.

[0110] The term "tumor-associated antigen" or "TAA" refers to an antigen that is expressed on the surface of tumor cells in an amount greater than that observed on normal cells (i.e., non-tumor cells) (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more). The term "tumor-specific antigen" or "TSA" refers to an antigen that is unique to a tumor. Non-limiting examples of TAAs and TSAs include AFP, BAGE, BCMA, Claudin6, Claudin18.2, CAMEL, CEA, CD19, CD20, CD22, CD30, CD38, CD71, CD123, CD133, DAM-6, GPRC5D, PCMA, EGFR, cMET, HER2, HER3, TROP2, ROR1, ROR2, MSLN, B7H3, B7H4, PD-L1, MAGE, MUC1, MUC16, NY-ESO-1, PSM, TRP-2, Wt-1, PSA, and SART-1.

[0111] The term "Claudin6" or "CLDN6" (used interchangeably herein) preferably relates to human CLDN6, in particular to a protein comprising an amino acid sequence according to SEQ ID NO: 75 of the sequence listing, or to variants of said amino acid sequence. The term "CLDN6" includes any CLDN6 variants, such as post-translationally modified variants and conformational variants. The amino acid sequences of human, cynomolgus monkey, and mouse CLDN6 are shown in the NCBI reference sequences NP_067018.2 (human) (SEQ ID NO: 75), XP_005591080.1 (cynomolgus monkey) (SEQ ID NO: 76), and NP_061247.1 (mouse) (SEQ ID NO: 77). The orthologues of CLDN6 share more than 99% and about 88% identity with the human protein in cynomolgus monkey and mouse, respectively.

[0112] As used herein, the term "Claudin18 isoform 2" (used interchangeably with CLDN18.2) refers to a peptide comprising or consisting of the amino acid sequence provided in Claudin-18 isoform 2 of NCBI entry NP_001002026.1, including post-translationally modified variants and species homologs present on the surface of normal cells or transformed cancer cells or expressed on cells transfected with the CLDN18.2 gene. Claudin18.2 preferably has an amino acid sequence according to SEQ ID NO: 72.

[0113] The term "Nectin-4" (N4), or "Nectin-4 protein" includes human Nectin-4, specifically the native sequence polypeptide, isoforms, chimeric polypeptides, all homologs, fragments, and precursors of Nectin-4. The amino acid sequences of human, cynomolgus monkey, rat, and mouse Nectin-4 are shown in NCBI reference sequences NP_112178.2 (human) (SEQ ID NO: 78), XP_005541277.1 (cynomolgus monkey) (SEQ ID NO: 79), NP_001102546.1 (rat) (SEQ ID NO: 80), and NP_082169.2 (mouse) (SEQ ID NO: 81). The orthologs of Nectin-4 share greater than 99%, about 94%, and about 92% homology with the human protein in cynomolgus monkey, rat, and mouse, respectively.

[0114] The term "percent identity" is intended to indicate the percentage of amino acid residues that are identical between the two sequences to be compared, which percentage is obtained after optimal alignment and is entirely statistical, with the differences between the two sequences being distributed randomly and over their entire length. Sequence comparison between two amino acid sequences is conventionally carried out by comparing these sequences after optimal alignment, which is carried out by segments or "comparison windows" in order to identify and compare local regions of sequence similarity. Optimal alignments of sequences for comparison can be generated manually or by the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, by the local homology algorithm of Neddiernan and Wunsch, 1970, J. Mol. Biol. 48, 443, by the similarity search method of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 85, 2444, or by computer programs employing these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0115] The term "antibody" herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (eg, bispecific antibodies).

[0116] The term "antibody scaffold module" as used herein refers to a Y-shaped antibody having two heavy chains and two light chains. The two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. The antibody scaffold may have one or more binding modules attached to one or more of its heavy and / or light chains. The antibody binding scaffold includes two Fab and an Fc portion having two constant region sequences.

[0117] As used herein, the term "cross-reactive" refers to the ability of the anti-human CD137 antibody or anti-human TAA antibody described herein to bind to CD137 or TAA from different species, respectively.For example, the antibody described herein can also bind to CD137 or TAA from another species (e.g., rat or mouse CD137 or TAA).

[0118] An exemplary antibody, such as IgG, comprises two heavy chains and two light chains. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0119] Generally, the hypervariable region comprises amino acid residues from about amino acid residues 24-34 (LCDR1; "L" represents light chain), 50-56 (LCDR2), and 89-97 (LCDR3) in the light chain variable region, and about amino acid residues 31-35B (HCDR1; "H" represents heavy chain), 50-65 (HCDR2), and 95-102 (HCDR3) in the heavy chain variable region, as described in Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), and / or those described in Chothia and These include residues that form the hypervariable loops (e.g., residues 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3) in the light chain variable region, and residues 26-32 (HCDR1), 53-55 (HCDR2), and 96-101 (HCDR3) in the heavy chain variable region), as referenced in Lesk (1987) J. Mol. Biol. 196:901-917.

[0120] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. For example, the individual antibodies constituting this population are identical and / or bind to the same epitope, except for possible variant antibodies, which, for example, contain naturally occurring mutations or arise during the production of the monoclonal antibody preparation and are generally present in small amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" should not be interpreted as requiring the production of the antibody by any method, since it indicates the property of the antibody being obtained from a substantially homogeneous population of antibodies. For example, monoclonal antibodies used in accordance with the present disclosure may be made by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies are described herein.

[0121] The term "chimeric" antibody refers to recombinant antibodies and fragments thereof in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belongs to a particular antibody class or subclass, while the remaining portion of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belongs to another antibody class or subclass, so long as it exhibits the desired biological activity. In addition, complementarity determining region (CDR) grafting may be performed to alter certain properties of the antibody molecule, including affinity or specificity. Typically, the variable domains are obtained from an antibody derived from an experimental animal such as a rodent (the "parent antibody"), and the constant domain sequences are obtained from a human antibody, so that the resulting chimeric antibody is capable of directing effector functions in a human subject and is less likely to induce an adverse immune response than the parent (e.g., murine) antibody from which it is derived.

[0122] The term "humanized antibody" refers to an antibody that has been engineered to contain one or more human framework regions in the variable regions, along with non-human (e.g., mouse, rat, or hamster) complementarity determining regions (CDRs) of the heavy and / or light chains. In certain embodiments, a humanized antibody contains sequences that are completely human except for the CDR regions. Humanized antibodies are typically less immunogenic in humans compared to non-humanized antibodies, and therefore provide therapeutic benefits in certain situations. Those skilled in the art will be aware of humanized antibodies, as well as techniques suitable for their production. See, e.g., Hwang, WYK, et al., Methods 36:35, 2005; Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033, 1989; Jones et al., Nature, 321:522-25, 1986; Riechmann et al., Nature, 332:323-27, 1988; Verhoeyen et al., Science, 239:1534-36, 1988; Orlandi et al., Nature, 321:522-25, 1986; al., Proc. Natl. Acad. Sci. USA, 86:3833-37, 1989; U.S. Patent Nos. 5,225,539, 5,530,101, 5,585,089, 5,693,761, 5,693,762, 6,180,370, and WO 90 / 07861 to Selick et al.

[0123] A "human antibody" is an antibody that has an amino acid sequence that corresponds to that of an antibody produced by a human and / or that has been produced using any of the techniques for producing human antibodies known to those skilled in the art. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including those described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985); Boerner et al., J. Immunol, 147(I):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol, 5:368-74 (2001). Human antibodies can be produced by administering antigen to transgenic animals that have been engineered to produce the antibody in response to antigen challenge, but in which the endogenous locus has been disabled, e.g., immunized HuMab mice (see, e.g., Nils Lonberg et al., 1994, Nature 2000, 144:1311-1325, for HuMab mice). 368:856-859, WO98 / 24884, WO94 / 25585, WO93 / 1227, WO92 / 22645, WO92 / 03918, and WO01 / 09187), xeno mice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 for XENOMOUSE™ technology), or Trianni mice (see, e.g., WO2013 / 063391, WO2017 / 035252, and WO2017 / 136734).

[0124] The "class" of an antibody refers to the type of constant domain or constant region that its heavy chain possesses. There are five main antibody classes, namely IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0125] The term "antigen-binding domain" (or simply "binding domain") of an antibody or similar terms refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen complex. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH domains; (ii) a F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341:544-546); (vi) an isolated complementarity determining region (CDR); and (vii) a combination of two or more isolated CDRs, which may be optionally joined by a synthetic linker.

[0126] The "variable domain" (V domain) of an antibody mediates binding and confers antigen specificity of a particular antibody. However, that variability is not evenly distributed across the 110 amino acid span of the variable domain. Instead, the V region consists of relatively invariant stretches of 15-30 amino acids called framework regions (FRs) separated by shorter regions of extreme variability, referred to herein as "hypervariable regions" or CDRs, each 9-12 amino acids in length. As will be appreciated by those of skill in the art, the exact numbering and arrangement of the CDRs may vary between different numbering systems. However, it should be understood that the disclosure of a variable heavy and / or variable light chain sequence includes the disclosure of the associated CDRs. Thus, the disclosure of each variable heavy chain region is a disclosure of the vhCDRs (e.g., vhCDR1, vhCDR2, and vhCDR3), and the disclosure of each variable light chain region is a disclosure of the vlCDRs (e.g., vlCDR1, vlCDR2, and vlCDR3).

[0127] "Complementarity determining region" or "CDR", as these terms are used herein, refers to short polypeptide sequences in the variable regions of both heavy and light chain polypeptides that are primarily responsible for mediating specific antigen recognition. Within each VL and each VH, there are three CDRs (designated CDR1, CDR2, and CDR3). Unless otherwise stated herein, CDR and framework regions are annotated according to the Kabat numbering convention (Kabat EA et al., 1991, Sequences of Proteins of Immunological Interest, In: NIH Publication No. 91-3242, US Department of Health and Human Services, Bethesda, Md).

[0128] In other embodiments, the CDRs of an antibody can be determined according to MacCallum RM et al, (1996) J Mol Biol 262:732-745, which is incorporated herein by reference in its entirety. In other embodiments, the CDRs of an antibody can be determined according to the AbM numbering convention, which refers to the AbM hypervariable regions, which represent a compromise between the Kabat CDRs and the Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.), which is incorporated herein by reference in its entirety. CDRs can also be determined according to the AbM numbering convention, which refers to the AbM hypervariable regions, which represent a compromise between the Kabat CDRs and the Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.), which is incorporated herein by reference in its entirety. thThe CDRs may be defined by sequence comparison as described in Ed. Public Health Service, National Institutes of Health, Bethesda, Md., whereas HVLs are structurally defined according to the three-dimensional structure of the variable domain as described in Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917. Where these two methods result in slightly different identification of the CDRs, the structural definition is preferred. As defined by Kabat, CDR-L1 is located at about residues 24-34 in the light chain variable domain, CDR-L2 at about residues 50-56, CDR-L3 at about residues 89-97, CDR-H1 is located at about residues 31-35 in the heavy chain variable domain, CDR-H2 at about residues 50-65, and CDR-H3 at about residues 95-102. IMGT and NORTH provide alternative definitions of CDRs (see Lefranc MP. Unique database numbering system for immunogenetic analysis. Immunol Today (1997) 18:509; and North B, Lehmann A, Dunbrack RLJ. A new clustering of antibody CDR loop conformations. J Mol Biol. (2011) 406:228-56). Additionally, CDRs may be defined according to Chemical Computing Group (CCG) numbering (Almagro et al., Proteins 2011; 79:3050-3066 and Maier et al., Proteins 2014; 82:1599-1610). Thus, CDR1, CDR2, CDR3 of the heavy and light chains define the unique and functional properties specific to a given antibody.

[0129] "Framework" or "Framework region" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains, FR1, FR2, FR3, and FR4.

[0130] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is made from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), Vols. 1-3. In one embodiment, for VL, the subgroup is subgroup kappa I as in Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III as in Kabat et al., supra.

[0131] The "hinge region" is generally defined as stretching from 216 to 238 (EU numbering) or 226 to 251 (Kabat numbering) of human IgG1. The hinge can be further divided into three distinct regions: the upper hinge, the middle (e.g., core) hinge, and the lower hinge.

[0132] The term "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0133] The term "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab)2, diabodies, linear antibodies, and single-chain antibody molecules (e.g., scFv). Papain digestion of an antibody produces two identical antigen-binding fragments called "Fab" fragments and a remaining "Fc" fragment, a name that reflects its ability to readily crystallize. The Fab fragment consists of an entire light (L) chain, the variable region domain (VH) of a heavy (H) chain, and the first constant domain (CH1) of one heavy chain. Pepsin treatment of an antibody produces a single large F(ab)2 fragment that roughly corresponds to two disulfide-linked Fab fragments that have bivalent antigen-binding activity and are still capable of cross-linking antigen. Fab fragments differ from Fab' fragments in that they have additional few residues at the carboxy terminus of the CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residues of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0134] "Fv" consists of a dimer of one heavy-chain variable domain and one light-chain variable domain in tight, non-covalent association. The folding of these two domains gives rise to six hypervariable loops (three loops each from the H and L chain) that contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody.

[0135] A "single-chain variable fragment" or "scFv" is a fragment of an immunoglobulin heavy chain (V H ) and light chain (V L) variable region fusion protein. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). In some embodiments, this region is joined with a short linker peptide of 10 to about 25 amino acids. The linker can be rich in glycine for flexibility or rich in serine or threonine for solubility and can connect the N-terminus of the VH to the C-terminus of the VL or vice versa. The protein retains the specificity of the original immunoglobulin even though the constant regions have been removed and the linker introduced. Disulfide-stabilized scFvs can be engineered by introducing pairwise cysteine ​​mutations at specific VH or VL residues. These residues are at the interface of VH and VL. See reference Weatherill, EE et al. Towards a universal disulphide stabilized single chain Fv format: importance of interchain disulphide bond location and VL-VH orientation. Protein Eng Des Sel 25, 321-329, NovaRock used VH44-VL100.

[0136] The term "multispecific antibody" is used in the broadest sense and specifically covers antibodies that contain a heavy chain variable domain (VH) and a light chain variable domain (VL), but the VH-VL unit has polyepitopic specificity (e.g., capable of binding to two different epitopes on one biomolecule or each epitope on a different biomolecule). Such multispecific antibodies include, but are not limited to, full-length antibodies, antibodies with two or more VL and VH domains, bispecific diabodies, and triabodies. "Polyepitopic specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different targets.

[0137] "Dual specificity" or "bispecificity" refers to the ability to specifically bind to two different epitopes on the same or different targets. However, in contrast to bispecific antibodies, bispecific antibodies have two antigen-binding arms that are identical in amino acid sequence, with each Fab arm having the ability to recognize two antigens. Bispecificity allows the antibody to interact with two different antigens with high affinity as a single Fab or IgG molecule. According to one embodiment, a multispecific antibody in the IgG1 format binds to each epitope with an affinity of 5 μM to 0.001 pM, 3 μM to 0.001 pM, 1 μM to 0.001 pM, 0.5 μM to 0.001 pM, or 0.1 μM to 0.001 pM. "Monospecificity" refers to the ability to bind only one epitope. Multispecific antibodies can have a structure similar to a complete immunoglobulin molecule and can include an Fc region, for example an IgG Fc region. Such structures include, but are not limited to, IgG-Fv, IgG-(scFv)2, DVD-Ig, (scFv)2-(scFv)2-Fc, and (scFv)2-Fc-(scFv)2. In the case of IgG-(scFv)2, the scFv can be added to either the N- or C-terminus of either the heavy or light chain.

[0138] As used herein, the term "bispecific antibody" (BsAb) refers to an antibody, often human or humanized, that has binding specificities for at least two different antigens. In the present disclosure, one of the binding specificities can be directed to CD137 and the other to CLDN6, CLDN18.2, or Nectin-4.

[0139] As used herein, the term "diabody" refers to a bivalent antibody comprising two polypeptide chains, in which each polypeptide chain comprises a VH domain and a VL domain joined by a linker (e.g., a linker consisting of five amino acids) that is too short to allow intramolecular association between the VH domain and the VL domain on the same peptide chain. In this configuration, each domain pairs with a complementary domain on another polypeptide chain to form a homodimeric structure. Thus, the term "triabody" refers to a trivalent antibody comprising three peptide chains, each of which contains one VH domain and one VL domain joined by a linker (e.g., a linker consisting of 1-2 amino acids) that is too short to allow intramolecular association between the VH domain and the VL domain on the same peptide chain.

[0140] The term "isolated antibody" as used to describe various antibodies disclosed herein means an antibody that has been identified and separated and / or recovered from a cell or cell culture in which it is expressed. An isolated antibody or antibody fragment may include variants of an antibody or antibody fragment that have one or more post-translational modifications that occur during production, purification, and / or storage of the antibody or antibody fragment. Contaminant components of its natural environment are materials that typically interfere with diagnostic or therapeutic uses of the polypeptide. They may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, an isolated antibody is purified to greater than 95% or 99% purity, for example, as determined by electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse-phase HPLC) methods. For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007). In a preferred embodiment, the antibody is purified (1) sufficiently to obtain at least 15 residues of N-terminal or internal amino acid sequence by using a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions with Coomassie blue staining or, preferably, silver staining.

[0141] With respect to the binding of an antibody to a target molecule, the term "specific binding to" or "specifically binds to" or "is specific for" an epitope on a particular polypeptide or particular polypeptide target means binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule relative to the binding of a control molecule. For example, specific binding can be determined by competition with a control molecule similar to the target, such as an excess of unlabeled target. In this case, specific binding is indicated when the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target. As used herein, the term "specific binding to" or "specifically binds to" or "is specific for" an epitope on a particular polypeptide or particular polypeptide target means binding that is measurably different from non-specific interactions. -4 M or less, or 10 -5 M or less, or 10 -6 M or less, or 10 -7 M or less, or 10 -8 M or less, or 10 -9 M or less, or 10 -10 M or less, or 10 -11 M or less, or 10 -12 Kd for targets less than or equal to M, or 10 -4 M~10 -6 M or 10 -6 M~10 -10 M or 10 -7 M~10 -9 The affinity can be shown by a molecule having a Kd in the range of 0.1 to 1.0 M. As will be understood by those skilled in the art, affinity and KD value are inversely proportional. A high affinity for an antigen is measured by a low KD value. In one embodiment, the term "specific binding" refers to binding where a molecule binds to CD137, CLDN6, CLDN18.2, or Nectin-4 (or an epitope of CD137, CLDN6, CLDN18.2, or Nectin-4) without substantially binding to any other polypeptide or polypeptide epitope.

[0142] As used herein, the terms "binds CD137," "binds CLDN6," "binds CLDN18.2," and "binds Nectin-4" refer to the ability of an antibody or antigen-binding fragment to recognize and bind endogenous human CD137, CLDN6, CLDN18.2, or Nectin-4, when occurring on the surface of normal or malignant cells, or when occurring on the surface of recombinant host cells engineered to overexpress CD137, CLDN6, CLDN18.2, or Nectin-4, respectively.

[0143] As used herein, the term "affinity" refers to the strength of binding of an antibody to an epitope. The affinity of an antibody is given by the dissociation constant Kd, defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of unbound antibody, and [Ag] is the molar concentration of unbound antigen. The affinity constant Ka is defined by 1 / Kd. Methods for determining the affinity of mAbs can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), which are incorporated herein by reference in their entireties. One standard method known in the art for determining the affinity of mAbs is the use of surface plasmon resonance (SPR) screening (such as by analysis with a BIAcore™ SPR analyzer).

[0144] "Epitope" refers to the site or sites of interaction between an antibody and its antigen. As described in Janeway, C, Jr., P. Travers, et al. (2001). Immunobiology: the immune system in health and disease. Part II, Sections 3-8. New York, Garland Publishing, Inc., "Antibodies generally recognize only a small region on the surface of a macromolecule such as a protein... [a particular epitope] is likely to be composed of amino acids from different parts of the [antigen] polypeptide chain that have been joined together by protein folding. This type of antigenic determinant is known as a conformational or discontinuous epitope because the structure recognized is discontinuous in the antigen's amino acid sequence but is composed of segments of the protein that have been joined together in three-dimensional structure. In contrast, an epitope that is composed of a single segment of a polypeptide chain is called a continuous or linear epitope" (Janeway, C. Jr., P. Travers, et al. (2001). Immunobiology: the immune system in health and disease. Part II, Sections 3-8. New York, Garland Publishing, Inc.).

[0145] As used herein, the term "KD" refers to the equilibrium dissociation constant obtained from the ratio of kd to ka (e.g., kd / ka) and expressed as molar concentration (M). The KD value of an antibody can be determined using methods well established in the art. Suitable methods for determining the KD of an antibody include biolayer interference (BLI) analysis, preferably using a Fortebio Octet RED instrument, surface plasmon resonance, using a biosensor system such as the BIACORE® surface plasmon resonance system, or flow cytometry and Scatchard analysis.

[0146] As used herein, the term "KD" is intended to refer to the dissociation constant of a particular antibody-antigen interaction. It is calculated by the following formula: Koff / Kon=KD. Binding kinetics describes how fast an antibody binds to its target (Kon) and how fast an antibody dissociates from its target (Koff). The residence time of an antibody on its target, for example on CD137, is determined by these kinetic characteristics (Schuetz, DA, et al. (2017) Kinetics for Drug Discovery: an industry-driven effort to target drug residence time. Drug Discov Today 22: 896-911).

[0147] As used herein, the term "IC 50 " is intended to refer to the effective concentration of a bispecific binding protein disclosed herein required to neutralize 50% of the biological activity of the antigen to which it binds.

[0148] EC for the agent and specific activity (e.g., binding to cells, inhibition of enzyme activity, activation or inhibition of immune cells). 50 " refers to the effective concentration of an agent that produces 50% of its maximum response or effect for such activity. 100 " refers to the effective concentration of an agent that produces substantially its maximal response for such activity.

[0149] As used herein, the term "antibody-drug conjugate (ADC)" refers to an immunoconjugate consisting of a recombinant monoclonal antibody covalently linked to a cytotoxic agent (known as the payload) via a synthetic linker. Immunoconjugates (antibody-drug conjugates, ADCs) are a class of highly potent antibody-based cancer therapeutics. ADCs consist of a recombinant monoclonal antibody covalently linked to a cytotoxic agent (known as the payload) via a synthetic linker. ADCs combine the specificity of monoclonal antibodies with the potency of small molecule chemotherapeutic agents to facilitate targeted delivery of highly cytotoxic small molecule drug moieties to tumor cells.

[0150] As used herein, the term "endocytosis" refers to the process by which eukaryotic cells internalize plasma membrane fragments, cell surface receptors, and components from extracellular fluids. Endocytic mechanisms include receptor-mediated endocytosis. The term "receptor-mediated endocytosis" refers to a biological mechanism in which a ligand binds to its target, triggering membrane invagination and pinching, and is internalized and delivered into the cytosol or transferred to the appropriate intracellular compartment.

[0151] The term "bystander effect" refers to target cell-mediated killing of healthy cells adjacent to tumor cells targeted by antibody-drug conjugates. The bystander effect is generally caused by cellular efflux of hydrophobic cytotoxic agents, which allows diffusion from antigen-positive target cells to adjacent antigen-negative healthy cells. The presence or absence of bystander effect may be due to the nature of the linker and conjugation chemistry used to generate the immunoconjugate.

[0152] The term "effector function" derived from the interaction of an antibody Fc region with a particular Fc receptor includes, but is not limited to, Clq binding, complement dependent cytotoxicity (CDC), Fc receptor binding, FcyR-mediated effector functions such as ADCC, antibody-dependent cell-mediated phagocytosis (ADCP), T-cell dependent cellular cytotoxicity (TCDD), and down-regulation of cell surface receptors. Such effector functions generally require the Fc region to be combined with an antigen-binding domain (e.g., an antibody variable domain).

[0153] As used herein, the terms "antibody-based immunotherapy" and "immunotherapy" are used broadly to refer to any form of therapy that relies on the targeting specificity of binding proteins that bind to CD137 and CLDN6, CD137 and CLDN18.2, or CD137 and Nectin-4 to mediate a direct or indirect effect on CD137-, CLDN6-, CLDN18.2-, and / or Nectin-4-expressing cells.

[0154] The term "Fc receptor" or "FcR" describes an antibody receptor that binds to the Fc region of immunoglobulins and is involved in antigen recognition located on the membrane of certain immune cells, including B lymphocytes, natural killer cells, macrophages, neutrophils, and mast cells. Fc receptors that recognize the Fc portion of IgG are called Fc gamma receptors (FcγR). The FcγR family includes allelic variants and alternatively spliced ​​forms of these receptors. Based on differences in structure, function, and affinity for IgG binding, FcγRs are classified into three major groups: FcγRI, FcγRII (FcγRIIa and FcγRIIb), and FcγRIII (FcγRIIIa and FcγRIIIb). Among them, FcγRI (CD64), FcγRIIa (CD32a), and FcγRIIIa (CD16a) are activating receptors that contain a signaling motif, an immunoreceptor tyrosine-based activation motif (ITAM), within the gamma subunit of FcγRI and FcγRIIIa or within the cytoplasmic tail of FcγRIIa. After binding of the antigen-antibody complex, activating Fcγ receptors (human: FcγRI, FcγRIIA, FcγRIIC, FcγRIIIA, FcγRIIIB, and mouse: FcγRI, FcγRIII, FcγRIV) elicit immune effector functions. In contrast, FcγRIIb (CD32b) is an inhibitory receptor. Cross-linking of FcγRIIb leads to phosphorylation of immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and inhibitory signaling (Patel et al. Front Immunol. 2019;10:223).

[0155] The term "Fc-silenced" refers to an Fc region that has been engineered to minimize / eliminate binding activity with FcγR and complement, leading to silencing or elimination of Fc-mediated effector functions. Strategies for engineering Fc include modifying Fc glycosylation, using hybrids of IgG subclasses, or introducing one or more mutations in the hinge and / or CH2 regions. The residues that are important for effector function and the respective mutations that silence Fc are known in the art, for example, in Strohl, WR and Strohl LM, "Antibody Fc engineering for optimal antibody performance" In Therapeutic Antibody Engineering, Cambridge: Woodhead Publishing (2012), pp 242, International Patent Application Publications WO2017 / 008169A1 and WO2021 / 055669.

[0156] Non-limiting examples of sites that can be engineered to silence human IgG1 Fc include L234, L235, G237, D265, N297, P329, P331, all in EU numbering.

[0157] As used herein, the term "bispecific" refers to a binding protein comprising an antibody scaffold module and a first binding module, which are derived from an antibody and / or receptor protein that have binding specificities for two different antigens. In one embodiment, the antibody scaffold module has binding specificity for a tumor-associated antigen (TAA) and the first binding module has binding specificity for CD137 (e.g., human CD137).

[0158] With respect to the binding of a bispecific binding protein to a target molecule, the term "specific binding to" or "specifically binds to" or "is specific for" an epitope on a particular polypeptide or particular polypeptide target means binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule relative to the binding of a control molecule. For example, specific binding can be determined by competition with a control molecule similar to the target, such as an excess of unlabeled target. In this case, specific binding is indicated when the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target. As used herein, the term "specific binding to" or "specifically binds to" or "is specific for" an epitope on a particular polypeptide or particular polypeptide target means binding that is measurably different from non-specific interactions. -4 M or less, or 10 -5 M or less, or 10 -6 M or less, or 10 -7 M or less, or 10 -8 M or less, or 10 -9 M or less, or 10 -10 M or less, or 10 -11 M or less, or 10 -12 Kd for targets less than or equal to M, or 10 -4 M~10 -6 M or 10 -6 M~10 -10 M or 10 -7 M~10 -9 Affinity can be shown by a molecule having a Kd in the range of M. As understood by those skilled in the art, affinity and KD value are inversely proportional. High affinity for an antigen is measured by a low KD value. In one embodiment, the term "specific binding" refers to binding in which a molecule binds to a specific polypeptide or an epitope on a specific polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

[0159] As used herein, the term "affinity" refers to the strength of binding of a bispecific binding protein to an epitope. The affinity of a bispecific binding protein is given by the dissociation constant Kd, defined as [bispecific binding protein] x [Ag] / [bispecific binding protein-Ag], where [bispecific binding protein-Ag] is the molar concentration of the bispecific binding protein-antigen complex, [bispecific binding protein] is the molar concentration of unbound bispecific binding protein, and [Ag] is the molar concentration of unbound antigen. The affinity constant Ka is defined by 1 / Kd. Methods for determining the affinity of binding proteins can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), which are incorporated herein by reference in their entireties. One standard method known in the art for determining the affinity of bispecific binding proteins is the use of surface plasmon resonance (SPR) screening (such as by analysis using a BIAcore™ SPR analyzer).

[0160] The term "linker" refers to at least one atom that forms a covalent bond between two chemical entities. The term "linker" may refer to at least one atom that forms a covalent bond between a scaffold module and another covalent bond to a binding module. If the scaffold module and the binding module are linked only through peptide bonds, the linker is called a "peptide linker". Otherwise, the linker is called a "chemical linker". Furthermore, "flexible peptide linkers" mostly contain small, non-polar or polar amino acids, while "rigid peptide linkers" contain alpha-helix-forming sequences and / or are rich in proline residues (Chen et al., 2013. Adv Drug Deliv Rev. 65(10):1357-1369).

[0161] CD137(4-1BB) CD137 (4-1BB) is an inducible costimulatory receptor expressed on activated T cells and natural killer (NK) cells. The 4-1BB protein has four extracellular cysteine-rich pseudorepeat (CRD) domains CRD1, CRD2, CRD3, and CRD4 (see amino acid sequence and CRD regions in the table below). The formation of 4-1BB trimer clusters by 4-1BB ligand (41BBL) trimers on T cells initiates a signaling cascade that results in the upregulation of antiapoptotic molecules, secretion of cytokines, and enhanced effector functions. On NK cells, 4-1BB signaling can increase antibody-dependent cell-mediated cytotoxicity.

[0162] CD137, a member of the TNF receptor superfamily, was first identified as an inducible molecule activated by T cells (Kwon and Weissman, 1989, Proc Natl Acad Sci USA 86, 1963-1967). Subsequent studies showed that many other immune cells also express 4-1BB, including NK cells, B cells, NKT cells, monocytes, neutrophils, mast cells, dendritic cells (DCs), and cells of non-hematopoietic origin such as endothelial and smooth muscle cells (Vinay and Kwon, 2011, Cell Mol Immunol 8, 281-284). Expression of 4-1BB in different cell types is primarily inducible and driven by various stimulatory signals, such as triggering of the T cell receptor (TCR) or B cell receptor, as well as signaling induced through costimulatory molecules or receptors for proinflammatory cytokines (Diehl et al., 2002, J Immunol 168, 3755-3762; Zhang et al., 2010, Clin Cancer Res 13, 2758-2767).

[0163] The 4-1BB ligand (4-1BBL or CD137L) was identified in 1993 (Goodwin et al., 1993, Eur J Immunol 23, 2631-2641). Expression of 4-1BBL has been shown to be restricted on professional antigen-presenting cells (APCs) such as B cells, DCs, and macrophages. Inducible expression of 4-1BBL is a feature of T cells, including both αβ and γδ T cell subsets, as well as endothelial cells (Shao and Schwarz, 2011, J Leukoc Biol 89, 21-29).

[0164] Costimulation via the 4-1BB receptor (e.g., by ligation of 4-1BBL) stimulates T cells (CD4 + Subsets and CD8 +4-1BB activates multiple signaling cascades in T cells (both in the primary and secondary subsets of T cells) and potently enhances T cell activation (Bartkowiak and Curran, 2015). In conjunction with TCR triggering, agonistic 4-1BB-specific antibodies enhance T cell proliferation, stimulate lymphokine secretion, and reduce the susceptibility of T lymphocytes to activation-induced cell death (Snell et al., 2011, Immunol Rev 244, 197-217). This mechanism was further advanced as the first proof of concept in cancer immunotherapy. In preclinical models, administration of agonistic antibodies against 4-1BB in tumor-bearing mice resulted in potent antitumor effects (Melero et al., 1997, Nat Med 3, 682-685). Accumulating evidence subsequently demonstrated that 4-1BB exerts its efficacy as an antitumor agent, usually only when administered in combination with other immunomodulatory compounds, chemotherapeutic drugs, tumor-specific vaccination, or radiation therapy (Bartkowiak and Curran, 2015, Front Oncol 5, 117).

[0165] Agonistic monoclonal antibodies targeting 4-1BB have been developed to exploit 4-1BB signaling for cancer immunotherapy. Preclinical results suggest that targeting 4-1BB with agonistic antibodies can result in tumor clearance and sustained antitumor immunity in a variety of induced and spontaneous tumor models. In addition, fusion proteins composed of one of the extracellular domains of the 4-1BB ligand and a single-chain antibody fragment (Homig et al., 2012, J Immunother 35, 418-429; Muller et al., 2008, J Immunother 31, 714-722) or a single 4-1BB ligand fused to the C-terminus of the heavy chain (Zhang et al., 2007, Clin Cancer Res 13, 2758-2767) have been generated. WO2010 / 010051 discloses the production of a fusion protein consisting of three TNF ligand ectodomains linked together and fused to an antibody moiety.

[0166] First generation immune agonist CD137 antibodies such as urelumab and utomirumab have not achieved the desired efficacy in the clinic. For T cell costimulatory agonists to function as cancer therapies, many factors must be considered, such as target engagement affinity, binding kinetics, binding valency, cluster formation, Fc receptor-mediated activity, etc. Design of a tailored tumor antigen-CD137 configuration has been used to improve the efficacy and safety of the disclosed bispecific antibodies. Specifically, highly specific tumor antigen binding antibodies were selected for tumor cell engagement, and bivalency for tumor antigen binding was used to maximize target engagement. 2) Suboptimal activation of T cells was thought to result in less T cell exhaustion and long-lasting antitumor effects (Stone JD et al., Immunology. 2009; 126(2): 165-176). CD137 antibodies with fast-on and fast-off characteristics were expected to avoid constant stimulatory signals to T cells and therefore perform better than slow-off antibodies (Garble K, Nature Reviews Drug Discovery 19, 3-5 (2020)). Furthermore, CD137 agonism with clustering dependency can avoid systemic activation of circulating T cells and activate only tumor cell-mediated T cells at the tumor site. This was achieved by using tumor antigen cluster-dependent CD137 agonism. Furthermore, silencing of Fc to eliminate effector function and formation of Fc-mediated receptor clusters can further reduce hepatotoxicity derived from Kupffer cell activation.

[0167] Claudin protein family The Claudin (CLDN) family consists of 27 members and exhibits distinct expression patterns in a cell- and tissue-type selective manner. Claudins are integral membrane proteins located within epithelial and endothelial tight junctions (TJs). CLDNs interact with each other both in the same cell (cis interactions) and on adjacent cells (trans interactions), resulting in the formation of TJs with tissue-specific barrier functions. Individual cell types express two or more of the Claudin family members. In normal physiology, Claudins interact with multiple proteins and are intimately involved in signaling to and from tight junctions (Lal-Nag, M and Morin, PJ, Genome Biol 10:235, 2009).

[0168] CLDN proteins contain four transmembrane (TM) helices (TM1, TM2, TM3, and TM4) and two extracellular loops (ELI and EL2). The extracellular loops of Claudins from adjacent cells interact with each other to seal the cell sheet and regulate paracellular transport between the lumen and basolateral space. The structure of Claudin proteins is highly conserved among different family members. CLDN6 contains 220 amino acids, is 23 kDa in size, and exhibits a Claudin-type protein structure.

[0169] The first Claudin protein family was first cloned in 1998 and named as the key structural and functional components of tight junctions. As a family, Claudins are a multigene family of four-transmembrane proteins involved in the barrier function of epithelial and endothelial cells, as well as in maintaining the cytoskeleton (Furuse et al., J. Cell. Biol. 141(7):1539-50, 1998). Claudins are integral membrane proteins that contain the major structural proteins of tight junctions, the apical most intercellular adhesive junctions in polarized cell types, such as those found in epithelial or endothelial cell sheets.

[0170] The first extracellular domain (ECD) of Claudin proteins typically consists of about 50 amino acids, whereas the second ECD is smaller than about 22 amino acids (Hashimoto, et al. Drug Discovery Today 21(10):1711-1718, 2016). The N-terminus is usually very short (e.g., about 4-10 amino acids), whereas the C-terminus ranges from 21 to about 63 amino acids and is required for localization of the protein at tight junctions.

[0171] The observation that tight junctions are often more permeable in tumour tissue than in normal tissue suggests that Claudin proteins may be more accessible on tumour cells than in normal tissue with intact tight junctions, making them attractive targets for therapeutic intervention in cancer.

[0172] The Claudin protein family in humans consists of at least 27 members, ranging in size from 22 to 34 kDa. All Claudins have a tetraspanin topology with both protein termini located on the intracellular face of the membrane, resulting in the formation of two extracellular (EC) loops, EC1 and EC2. Typically, EC1 is approximately 50-60 amino acids in size, while EC2 is smaller than EC1, usually containing approximately 25 amino acids. The EC loops mediate head-to-head homophilic interactions and, for certain combinations of Claudins, heterophilic interactions leading to the formation of tight junctions.

[0173] Claudin-6 Unlike the majority of Claudin proteins, which are ubiquitously expressed, CLDN6 is characterized by selective expression (Hewitt, et al., BMC Cancer, 6:186, 2006). CLDN6 is an oncofetal tight junction molecule expressed in several types of embryonic epithelial cells.

[0174] Impaired tight junctions and dysregulation of tight junction molecules are frequent features of cancer cells and are often associated with malignant transformation. CLDN6 expression is aberrantly activated in various cancer types, including gastric, lung and ovarian adenocarcinomas, endometrial and embryonal carcinomas, brain pediatric tumors (e.g., atypical teratomas / rhabdoid tumors), and germ cell tumors (Hassimoto et al., J Pharmacol Exp Ther 368:179-186, 2019; Kojima et al., Cancers 2020, 12, 2748). Increased expression of CLDN6 in several human malignancies is associated with poor prognosis, such as ovarian and gastric cancers (Zavala-Zendejas VE, et al., Cancer Invest. 29:1-11. 2011; Wang L, et al. Diagn Pathol. 8:1902013.). Therefore, CLDN6 is a promising tumor-associated antigen (TAA) for tumor-targeting therapeutics such as CART and T cell-engaging bispecific antibodies.

[0175] As a tumor-associated antigen, CLDN6 can be classified as a differentiation antigen due to its expression during the early stages of epithelial morphogenesis, which is important for epithelial differentiation and barrier formation. The distinct expression pattern of CLDN6 in cancer tissues but not in normal adult tissues, combined with the accessibility of the cancer cell surface to antibodies, qualifies CLDN6 as a promising target for diagnostic and immunotherapeutic approaches in a wide variety of cancer types.

[0176] There is a high degree of sequence conservation between CLDN6 and other Claudin proteins. Due to the high homology between CLDN6 and other Claudin proteins (e.g., CLDN9, CLDN4, and CLDN3), it is difficult to provide a CLDN6 antibody having properties suitable for therapeutic use, such as specificity, affinity, and safety.

[0177] CLDN6 is generally expressed in humans as a precursor protein of 220 amino acids, the first 21 amino acids of which constitute a signal peptide. The amino acid sequence of the CLDN6 precursor protein is publicly available at the National Center for Biotechnology Information (NCBI) website as NCBI reference sequence NP067018.2, and is set forth herein as SEQ ID NO:75.

[0178] Expression CLDN6 is highly expressed in germ cell tumors, including seminoma, embryonal carcinoma, and ovarian tumors, as well as in some cases of gastric, lung, ovarian, and endometrial cancers (Ushiku T et al.,Histopathology 61(6):1043-1056,2012,Hewitt KJ,Agarwal R,Morin PJ.The claudin gene family:expression in normal and neoplastic tissues.BMC Cancer 2006;6;186;Micke,P.et al.(2014)Aberrantly activated Claudin-6 and 18.2 as potential therapeutic targets in non-small-cell lung cancer.Int.J.Cancer 135,2206-2214;Lal-Nag,M.et al.(2012)Claudin-6:a novel receptor for CPE-mediated cytotoxicity in ovarian cancer.Oncogenesis 1,e33;Ben-David,U.et al.(2013)Immunologic and chemical targeting of the tight junction protein Claudin-6 eliminates tumorigenic human pluripotent stem cells.Nat.Commun.4,1992).

[0179] The human CLDN6 protein is very closely related to the human CLDN9 protein sequence in the extracellular domain (ECD), with >98% identity in ECD1 and >91% identity in ECD2. Human CLDN4 is also closely related to human CLDN6 in the ECD sequence, with >84% identity in ECD1 and >78% identity in ECD2. The search for monoclonal antibodies (MAbs) against CLDN6 has been hampered by the high homology of endogenously expressed Claudin-9 (CLDN9), which differs from CLDN6 by only three amino acids in the extracellular domain (two amino acids in ECD1 and one amino acid in ECD2). The predicted protein ECD sequences of cynomolgus monkey CLDN4, CLDN6, and CLDN9 proteins are 100% identical to the respective human ECD sequences. Furthermore, the Claudin-6 gene is highly conserved among different species, for example the human and mouse genes show 88% homology at the DNA and protein levels.

[0180] Claudin 18.2 Another member of the Claudin family of proteins, the tight junction molecule Claudin-18, is normally found in cellular tight junctions of the gastric mucosa and intestinal epithelium. Two human Claudin18 transcript variants alternatively spliced ​​in a promoter-dependent manner (Niimi et al., Mol. Cell. Biol. 21:7380-90, 2001) encoding distinct isoforms with expression restricted to the lung (CLDN18.1) and stomach (CLDN18.2) have been previously described. The major protein sequences of these splice variants differ in the N-terminal intracellular domain and in the N-terminal part including the first transmembrane domain (TMD1) and extracellular loop 1 (ECL1). CLDN18.2 is one of the few members of the human Claudin family that is strictly restricted to one cell lineage (Tureci et al.). More specifically, it provides a highly selective gastric lineage (e.g., gastric cell-specific) marker with an expression pattern restricted to short-lived differentiated epithelial cells and absent from the stem cell zone of gastric glands (Sahin et al., Clin. Cancer Res. 14(23)7624-7634, 2008).

[0181] CLDN18.2 is retained in malignant transformation and expressed in a significant portion of primary tumors and their metastases. Sahin et al. also reported that CLDN18.2, but not CLDN18.1, is frequently overexpressed in several different types of cancer, including pancreatic, gastric, esophageal, lung, and ovarian cancers. Thus, in the context of cancer, CLDN18.2 is not restricted to gastric cell lineages (Sahin et al.). Taken together, the findings of published reports demonstrate that CLDN18.2 provides both a diagnostic tool and a druggable target for the development of cancer immunotherapy for diseases associated with tumors of epithelial cell origin.

[0182] It has been reported that tight junction permeability is often higher in tumor tissues than in normal tissues, and it is therefore speculated that Claudin proteins on tumor cells may be more accessible than in normal tissues with intact tight junctions. This possibility makes Claudin proteins an attractive target for therapeutic intervention in cancer. Furthermore, published expression profiling results suggest that cancer therapies targeting CLDN18.2 have a favorable systemic toxicity profile because normal turnover and homeostatic processes replenish gastrointestinal epithelial cells every two to seven days (Sahin et al.). Transient gastrointestinal toxicity of limited duration is a common and manageable adverse event of cancer immunotherapy.

[0183] Pancreatic and gastroesophageal cancers are among the malignancies with the highest unmet medical needs (Sahin, et al.). Despite the fact that gastric and pancreatic cancers contribute significant cancer-related morbidity and mortality, treatment options are limited. Therefore, there is a need for anti-CLDN18.2 specific antibodies and binding agents for use in the immunotherapy of cancers associated with primary and metastatic solid tumors of epithelial cell origin.

[0184] CLDN18.2 contains four transmembrane domains with two small extracellular loops (loop 1 surrounded by hydrophobic regions 1 and 2, and loop 2 surrounded by hydrophobic regions 3 and 4). CLDN18.2 is a transmembrane protein, and therefore epitopes present within or formed by its extracellular loops represent desirable targets for antibody-based cancer immunotherapy. However, considering that CLDN18.1 is expressed by alveolar epithelial cells in normal lung tissue, a tissue highly relevant for toxicity, specificity for exclusive splice variants was recognized as a prerequisite for the use of CLDN18.2-specific antibodies in antibody-based cancer immunotherapy. Sahin et al. were the first to report proof-of-concept results validating CLDN18.2 as a druggable target for cancer immunotherapy based on the isolation of antibodies (polyclonal and monoclonal) that exclusively bind to CLDN18.2 but not CLDN18.1 (Sahin et al, Clin. Cancer Res. 14(23)7624-7634, 2008).

[0185] CLDN18.2 is expressed in multiple primary tumors and their metastases, including gastric cancer, esophageal cancer, pancreatic cancer, lung cancer such as non-small cell lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, and gallbladder cancer. Dysregulation of Claudin expression has been detected in many cancers and may contribute to tumorigenesis and cancer invasiveness (Singh et al, J Oncology 2010;2010:541957). CLDN18.2 expression is particularly elevated in pancreatic ductal adenocarcinoma (PDAC) (Tanaka et al, J Histochem Cytochem. 2011;59:942-952), esophageal tumors, non-small cell lung cancer (NSCLC), ovarian cancer (Sahin et al., Hu Cancer Biol. 2008;14:7624-7634), and bile duct adenocarcinoma (Keira et al, Virchows Arch. 2015;466:265-277).

[0186] Despite the fact that gastric cancer contributes to significant cancer-related morbidity and mortality, therapeutic options for gastric cancer are limited. Claudin is present in normal tissues, benign neoplasms, hyperplastic conditions, and cancers (Ding et al., Cancer Manag. Res. 5:367-375 (2013)). The expression pattern of Claudin is highly tissue-specific, and most tissues express multiple Claudins. Claudin proteins can interact with Claudins from adjacent cells in a homotypic or heterotypic manner to form tight junctions (Ding et al.). Alterations in Claudin expression and signaling pathways are known to be associated with cancer development, and the association between impaired tight junctions and tumor progression has been widely reported.

[0187] Nectin protein family Nectins (from the Latin "necto" meaning "to connect") interact with nectins on other cell surface molecules through the Ig-like V domains of their ECD. Nectins function to promote cell adhesion by first binding to form cis-dimers on the same cell and then forming homophilic or heterophilic trans-dimers with nectins or other members of the immunoglobulin superfamily (IgSF) on adjacent cells (Miyoshi et al., Am J Nephrol, 27:590, 2007). Heterophilic trans-dimers have been reported to form stronger cell-cell interactions than homophilic trans-dimers. The specificity of binding differs for each nectin (e.g., Nectin-4 binds to itself and to Nectin-1).

[0188] The human nectin family consists of nine homologs (Nectin-1 to Nectin-4 and Nectin-like 1 to 5) (Duraivelan et al., Sci Rep, 10:9434, 2020). Nectin proteins (Nectin-1, Nectin-2, Nectin-3, and Nectin-4) are calcium-independent immunoglobulin superfamily (IgSF) cell adhesion molecules that mediate cell-cell adhesion at adherens junctions in epithelial cells through homophilic or heterophilic trans interactions. In normal epithelia, adherens junctions define cell polarity, a property that is often lost during tumorigenesis.

[0189] Nectin-1, -2, -3, and -4 are expressed as single-pass type I glycoproteins and are characterized by a common domain organization consisting of an extracellular domain (ECD) with three tandem immunoglobulin-like domains / loops arranged as an N-terminal Ig-like variable domain (D1) followed by two Ig-like constant domains (D2 and D3). Nectins interact with each other through V-domain binding interactions, thereby creating a trans-heterointeraction network that supports cell-cell adhesion. Heterophilic interactions between Nectin-3 / Nectin-1, Nectin-3 / Nectin-2, and Nectin-1 / Nectin-4 have been reported (Harrison et al., Nat Struct Mol Biol, 19(9):906-915, 2012). In addition to their role in cell-cell adhesion, nectins play important roles in regulating various physiological cellular activities, viral entry, and immune regulation.

[0190] Members of the nectin family are expressed as single-pass type I glycoproteins and are characterized by a common domain organization consisting of three Ig-like domains in the extracellular domain (a membrane-distal IgV domain followed by two IgC domains), a transmembrane region, and a cytoplasmic domain that binds to the actin cytoskeleton via the adaptor protein afadin (Samanta et al., Cell Mol Life Sci, 72(4):645-658, 2015).

[0191] Many viruses utilize IgSF member proteins to facilitate virus tropism, attachment, and subsequent entry into host cells. Several members of the nectin family were identified as viral receptors before their physiological functions as cell adhesion molecules were found. Initially, members of the nectin family were independently identified as virus entry receptors by multiple groups and assigned names based on their observed functions. Nectin-1, -2, and -3 were originally described as molecules homologous to the poliovirus receptor (PVR, necl-5, CD155) and were therefore named poliovirus receptor-related (PRR) proteins (nectin1 / PRR1 / CD111, nectin2 / PRR2 / CD112, and nectin3 / PRR3) (Reymond et al., J Biol Chem, 276(46):43205-15, 2001), and subsequently named CD111, CD112, and CD113, respectively. Nectin-4 was subsequently shown to recognize measles virus hemagglutinin (MV-H) and function as an epithelial cell receptor for measles virus entry (Samanta et al., Cell Mol Life Sci, 72(4):645-658, 2015).

[0192] Nectins function as cell adhesion molecules by first forming homocis-dimers on the cell surface and then trans-dimers on adjacent cells in both homophilic and heterophilic manners. The binding specificity differs for each nectin. Nectin-4 binds to itself and to Nectin-1 (Reymond et al., J Biol Chem, 276(46):43205-15, 2001, Fabre et al., J Biol Chem, 277(30):27006-27013, 2002). Cell-cell contact is thought to be initiated by interactions between nectins on adjacent cells. Cadherin-catenin complexes are then recruited to sites of nectin-based cell-cell adhesion, leading to trans-interactions of cadherins on adjacent cells, thereby forming adherens junctions (Boylan et al., Oncotarget, 8(6):9717-9738, 2017).

[0193] The ectodomains of nectin proteins share 30-55% amino acid sequence identity. Nectins are connected to the actin cytoskeleton through binding motifs in their cytoplasmic domains, afadin (an F-actin-binding protein), and are involved in the organization of epithelial and endothelial junctions. In complex interactions with other cell adhesion molecules (CAMs) and signaling molecules, they regulate several diverse physiological cellular activities, such as migration, proliferation, survival, differentiation, polarization, and viral entry.

[0194] The ability of nectin family members to interact with additional cell surface molecules significantly expands their interaction network in mammals. Nectins are known to interact in cis with other cell surface membrane receptors, such as platelet-derived growth factor receptor, fibroblast growth factor receptor, vascular endothelial growth factor receptor, prolactin receptor, ErbB2, ErbB3, and ErbB4, as well as integrins, such as integrin αvβ3 and integrin α6β4, regulating not only cell-cell adhesion but also cell migration, proliferation, differentiation, and survival (Kedashiro et al., Sci Rep, 9:18997, 2019).

[0195] Some members of the nectin family may exert immunomodulatory functions as a result of heterophilic trans-interactions with other members of the immunoglobulin superfamily. These interactions are known to affect the function of diverse immune cell types, including natural killer (NK) cells, monocytes, dendritic cells (DCs), and T lymphocytes. Several of the nectins, as well as some of the IgSF members that are known nectin family interactors, are known to recognize common binding partners. For example, Nectin-2 and PVR both recognize CD226, TIGIT, and Nectin-3 (Duraivelan et al., Sci Rep, 10:9434, 2020).

[0196] Bioinformatics analysis using algorithms to classify proteins into functionally related families predicted that five additional IgSF members, CD96 (TACTILE), CD226 (DNAM-1), TIGIT (WUCAM, VSTM3), CRTAM, and CD200, are functionally and evolutionarily related to nectin and nectin-like proteins and may represent binding partners for members of the nectin family (Rubinstein et al., Structure, 21(5):766-776, 2013). To date, all of these proteins, except for CD200, have been reported to bind members of the nectin / nectin-like family (Rubenstein, et al. ).

[0197] The ability of nectin family members to interact with additional cell surface molecules significantly expands their interaction network. Some members of the nectin family can exert immunomodulatory functions as a result of heterophilic trans-interactions with other members of the IgSF. These interactions are known to affect the function of various immune cell types, including natural killer (NK) cells, monocytes, dendritic cells (DCs), and T lymphocytes. Some of the nectins, as well as some of the IgSF members that are known nectin family partners, are known to recognize common binding partners. For example, Nectin-2 and PVR both recognize CD226, TIGIT, and Nectin-3 (Duraivelan et al., Sci Rep, 10:9434, 2020).

[0198] Nectin-4 Nectin-4 (also known as poliovirus receptor-like 4, PVRL4) was first identified through a bioinformatics search using sequences derived from known nectin protein extracellular domains to identify related sequences (Reymond et al., J Biol Chem, 276(46):43205-15, 2001). Human Nectin-4 was cloned from human trachea and described as an antigen with a restricted expression pattern in normal human tissues. More specifically, it has been described as an afadin-related member of the nectin family that interacts in trans with Nectin-1 through V-domain interactions, but not with Nectin-2, Nectin-3, or RVR (Reymond et al., J Biol Chem, 276(46):43205-15, 2001).

[0199] Reymond et al. identified Nectin-4 as a novel ligand for Nectin-1 based on their findings (Reymond et al., J Biol Chem, 276(46):43205-15, 2001): i) soluble chimeric recombinant Nectin-4 extracellular domain (Nectin-4-Fc) interacts with cells expressing Nectin-1 but not with cells expressing PVR / CD155, Nectin-2, or Nectin-3; conversely, Nectin-1Fc binds to cells expressing Nectin-4; ii) Nectin-1-Fc precipitates Nectin-4 expressed in COS cells; iii) complementary physical interactions were observed in vitro between Nectin-4-Fc and Nectin-1-Fc soluble recombinant proteins (Reymond, N et al.). Nectin-4-Fc / Nectin-4-Fc interactions were also detected, indicating that Nectin-4 has both homophilic and heterophilic properties.

[0200] The human Nectin-4 gene contains nine exons that code for the Nectin-4 adhesion receptor, a 55.5 kDa protein containing 510 amino acids. According to the protein knowledge database UniProtKb, Nectin-4 (Q96NY8) contains an N-terminal signal peptide (1-31 amino acids), an extracellular domain (32-349 amino acids) with three immunoglobulin-like subdomains (V-1 type 32-144 amino acids, C2-1 type 148-237 amino acids, C2-2 type 248-331 amino acids), a transmembrane domain (350-370 amino acids), and a cytoplasmic domain (371-510 amino acids).

[0201] It has been reported that the V-like domain of Nectin-4 is sufficient to mediate its trans-interaction with Nectin-1, and that the membrane-proximal Nectin-4 C-like domain contributes to increasing the affinity of the trans-interaction (Fabre et al., J Biol Chem, 277(30):27006-27013, 2002). Nectin-4 and Nectin-3 share a common binding region in the Nectin-1 V-like domain (Harrison et al., Nat Struct Mol Biol, 19(9):906-915, 2012).

[0202] It has also been reported that an anti-Nectin-1 monoclonal antibody (R1.302) with an epitope localized in the V-like domain of Nectin-1 blocks Nectin-4 / Nectin-1 trans-interaction (Reymond et al., J Biol Chem, 276(46):43205-15, 2001). A subsequent publication confirmed that a monoclonal antibody specific for the Ig-like V domain of Nectin-4 blocks the adhesion of Nectin-1 to an ovarian cancer cell line engineered to overexpress human Nectin-4 (NIH:OVCAR5) (Boylan et al., Oncotarget, 8(6):9717-9738, 2017).

[0203] Nectin-4 has been reported to be upregulated in various epithelial cell cancers, such as breast cancer (Fabre-Lafay et al., BMC Cancer, 7:73, 2007), lung cancer (Takano et al., Cancer Res, 69(16):6694-03, 2009), ovarian cancer (Derycke et al., Am J Clin Pathol, 5:835-845, 2010), pancreatic cancer (Nishiwada et al., J Exp Clin Cancer Res, 34(1):30, 2015), gallbladder cancer (Zhang et al., Cancer Lett, 375:179-189, 2016), and gastric cancer (Zhang et al., Hum Pathol, 72:107-116, 2018). These cancers frequently have copy number gains or focal amplifications of the Nectin-4 locus (Pavlova et al., Elife, 2:e00358, 2013).

[0204] Recently, accumulating evidence indicates that nectins contribute to tumorigenesis and metastasis-promoting functions. In particular, Nectin-4 is involved in cancer cell adhesion, migration, proliferation, and epithelial-mesenchymal transition. In breast, pancreatic, and lung cancer, overexpression of Nectin-4 or detection or soluble Nectin-4 in patient serum has been reported to be associated with tumor progression and / or poor survival (Fabre-Lafay et al., BMC Cancer, 7:73, 2007, Takano et al., Cancer Res, 69(16):6694-03, 2009, Derycke et al., Am J Clin Pathol, 5:835-845, 2010, Nishiwada et al., J Exp Clin Cancer Res, 34(1):30, 2015, and Lattanzio et al., Oncogenesis, 3:e118, 2014).

[0205] Targeting tumor-associated antigens for cancer immunotherapy In the past few years, more evidence has been established that tight junctions play a role in cancer cell proliferation, transformation, and metastasis. Dysregulation of Claudin leads to the breakdown of tight junctions in epithelial cells, which then results in loss of cell polarity and impairment of epithelial integrity. Overexpression of Claudin6 and / or Claudin18.2 by tumor cells may be associated with dysregulation of Claudin localization as a result of tumor cell dedifferentiation, or the need for cancerous tissue to grow rapidly to efficiently absorb nutrients within the tumor mass with abnormal angiogenesis (Morin PJ., Cancer Res.1;65(21):9603-6,2005). The reduction in cell-cell adhesion and increased mobility of cancer cells are suggested to be key events in the epithelial-to-mesenchymal transition (EMT), a key step in cancer progression and metastasis.

[0206] Nectin-4 was identified as a potential target using suppression subtractive hybridization due to its high levels of mRNA expression in bladder cancer (Challita-Eid et al., Cancer Res, 76(10):3003-13, 2016). Early publications have shown that Nectin-4 expression is restricted to endothelial cells in the human placenta (Reymond et al., J Biol Chem,276(46):43205-15,2001), is absent in normal adult tissues, and is re-expressed in a variety of cancer tissues, including breast, ovarian, pancreatic, and lung cancers (Fabre-Lafay et al., BMC Cancer,7:73,2007; Takano et al., Cancer Res,69(16):6694-03,2009; Derycke et al., Am J Clin Pathol,5:835-845,2010; Pavlova et al., Elife,2:e00358,2013; Nishiwada et al., J Exp Clin Cancer Res,34(1):30,2015; Challita-Eid et al., Cancer Nectin-4 was originally described as a tumor-specific antigen (TSA) because its expression in tumor-associated tumours was reported in mice (Res, 76(10):3003-13, 2016).

[0207] Results of immunohistochemistry (IHC) studies using a mouse antibody (M22-244b3) directed against the extracellular domain of human Nectin-4 and a panel of normal human tissue specimens (representing 36 human organs) showed more widespread expression in normal tissues at lower to moderate levels than previously reported (Challita-Eid et al.), identifying normal tissues that may be at increased risk of inducing on-target anti-Nectin-4 toxicity. Low levels of weak to moderate homogenous staining have been reported in human skin keratinocytes, skin appendages (sweat glands and hair follicles, and epithelium of the bladder, stomach, breast, esophagus, and salivary glands (ducts) (Challita-Eid et al., Reymond et al., J Biol Chem, 276(46):43205-15, 2001, Brancati et al., Am J Hum Gen, 87:265-273, 2010), suggesting that Nectin-4 is a tumor-associated antigen (TAA) rather than a TSA.

[0208] Nectin-4 is overexpressed in multiple cancers, particularly urothelial, lung, pancreatic, breast, and ovarian cancers (Challita-Eid et al., Cancer Res, 76(10):3003-13, 2016; Fabre-Lafay et al., BMC Cancer, 7:73, 2007; Takano et al., Cancer Res, 69(16):6694-03, 2009; Derycke et al., Am J Clin Pathol, 5:835-845, 2010). Extensive immunohistochemistry of Nectin-4 expression in human cancer tumor microarrays (TMAs) representing 34 tumors representing seven different indications (e.g., bladder, breast, pancreatic, lung, ovarian, head and neck, and esophageal cancers) confirmed that 69% of TMA specimens were positive for Nectin-4 across the cancer indications evaluated. The highest overall expression of Nectin-4 was observed for bladder, breast, and pancreatic tumors. The prevalence of Nectin-4 positive samples with moderate to strong staining was generally relatively low in ovarian, lung, head and neck, and esophageal cancer samples (Chalitta-Eid et al.). The relatively high Nectin-4 expression levels observed in cancer theoretically provide a therapeutic window for anti-Nectin-4 targeted ADCs and antibody-based immunotherapies characterized by an acceptable safety profile (Challita-Eid et al., Cancer Res, 76(10):3003-13, 2016, and Shim et al., Biomolecules, 10(3):360, 2020).

[0209] Early stages of epithelial cancer progression are characterized by genetic alterations that confer the ability to survive and grow in the absence of an extracellular matrix scaffold. The ability of cancer cells to tolerate loss of scaffold is critical for cancer cell survival and pathological progression of tumorigenesis, e.g., invasion of the underlying stroma, extravasation into blood vessels, and metastatic growth as distal sites (Pavlova et al., Elife, 2:e00358, 2013). Nectin-4 was identified in a gain-of-function screen for genes that enable matrix-anchor-independent cell growth in TL-HMECs (hTERT-immortalized human mammary epithelial cells transduced with SV40 large T antigen) (Pavlova et al., Elife, 2:e00358, 2013).

[0210] further reported that Nectin-4 drives the rapid assembly of TL-HMECs into multicellular clusters in suspension and that observable cluster formation could be blocked using an antibody directed against the extracellular domain of Nectin-4. Cell cluster formation was completely suppressed in the presence of anti-Nectin-4 antibodies. Similarly, an antibody targeting the extracellular region of Nectin-1 also inhibited Nectin-4-induced cell cluster formation.

[0211] further showed that Nectin-4 promotes the clustering of tumor cells with each other by engaging Nectin-1 receptors on neighboring cells, an interaction that triggers integrin β4 / SHP-2 / c-Src activation in a matrix attachment-independent manner. In the model presented by Pavlova et al., tumor-specific cell-cell contact and signaling through Nectin-4 / Nectin-1 interactions provides an alternative to cell-matrix signaling and confers a survival advantage by allowing cells to avoid anoikis (i.e., the induction of apoptosis in cells upon loss of attachment to the extracellular matrix (ECM) and neighboring cells).

[0212] Results of a study conducted to determine the biological significance of Nectin-4 in cellular functions underlying ovarian cancer progression (i.e., cell adhesion, spheroid formation, migration and proliferation) reported in vitro data showing that mAbs against the IgV-like domain of Nectin-4 almost completely blocked ovarian cancer cell adhesion to Nectin-1 (Boylan et al., Oncotarget, 8(6):9717-9738, 2017). Boylan et al. noted that Pavlova et al. used the same anti-Nectin-4 antibody in a mouse xenograft model of breast cancer and observed disruption of tumor cell adhesion and reduced tumor growth in vivo compared to tumors treated with control IgG, and based on their combined results, they speculate that blocking Nectin-4 cell adhesion may be a key component of the therapeutic efficacy of anti-Nectin-4 antibodies used in cancer immunotherapy (Boylan et al.).

[0213] Presentations reporting results from preclinical studies evaluating the use of anti-Nectin-4 ADCs as monotherapy for the treatment of Nectin-4-expressing tumors demonstrated the clinical development of anti-Nectin-4 antibody-based immunotherapy. For example, AGS-22M6E ADC monotherapy was reported to inhibit tumor growth in four mouse xenograft models of human bladder, pancreatic, breast, and lung cancer. A subsequent publication by M-Rabet et al. established Nectin-4 as a therapeutic target for primary and metastatic triple-negative breast cancer (TNBC), based on the observation that an ADC prepared with a different anti-Nectin-4 antibody (N41 mAb-vcMMAE) (WO 2017 / 042210) induced complete and durable responses in vitro and in vivo in three models of TNBC developed in immunodeficient NSG mice, namely against primary tumors, metastatic lesions, and local recurrences (M-Rabet et al., Annals of Oncology, 28(4):769-776, 2017).

[0214] A bispecific binding protein that binds CD137 and a tumor-associated antigen The present disclosure provides a bispecific binding protein that binds to CD137 and tumor-associated antigen (TAA) or tumor-specific antigen and its fragments.For example, tumor-specific antigen can be any antigen that is expressed on the surface of tumor cells in greater amounts than on non-tumor cells.In some embodiments, tumor-associated antigen can be Claudin6, Claudin18.2, or Nectin-4.

[0215] A bispecific binding protein that binds to a TAA and CD137 can comprise (a) an antibody scaffold module comprising a first antigen binding site that binds to the TAA and a second antigen binding site that binds to the TAA, and (b) at least one first binding module comprising a third antigen binding site that binds to CD137.

[0216] In some embodiments, a bispecific binding protein that binds a tumor associated antigen and CD137 comprises (a) an antibody scaffold module that comprises a means for binding to the tumor associated antigen via a first antigen binding site and a second antigen binding site, and (b) at least one first binding module that comprises a means for binding to CD137 via a third antigen binding site.

[0217] In some embodiments, the antibody scaffold module is a Y-shaped antibody having two heavy chains and two light chains. In yet another embodiment, the antibody scaffold module is an IgG, such as IgG1, IgG2, IgG3, or IgG4. In some embodiments, the first binding module is an antibody fragment, such as an scFv. In yet another embodiment, the scFv is stabilized by the introduction of a disulfide bond.

[0218] In some embodiments, the first link module binds to CD137 and has agonist activity.

[0219] In some embodiments, the antibody scaffold module is a bivalent monoclonal antibody. In some embodiments, the antibody scaffold module is a full-length antibody. In some embodiments, the antibody scaffold module is a murine antibody or a human antibody. In other embodiments, the antibody scaffold module is a chimeric antibody, a bispecific antibody, or a humanized antibody. In some embodiments, the antibody scaffold module is symmetric (e.g., homodimer) or asymmetric (e.g., heterodimer).

[0220] In other embodiments, the antibody scaffold module is an antibody fragment, such as an antibody fragment selected from the group consisting of Fab, Fab', F(ab)2, Fv, domain antibody (dAb), diabody, triabody, tetrabody, minibody, and single chain antibody (scFv). The antibody scaffold module may be a chimeric antibody or a bispecific antibody. In an alternative embodiment, the antibody scaffold module may be a polypeptide that contains at least a portion of an antibody sufficient to confer TAA selective binding to the polypeptide. is a human antibody.

[0221] In some embodiments, the antibody scaffold module comprises two heavy chain sequences, each having a C-terminus and an N-terminus, and two light chain sequences, each having a C-terminus and an N-terminus. In some embodiments, the first link module is covalently linked to one or both C-terminus of the antibody scaffold module heavy chain sequence, one or both C-terminus of the antibody scaffold module light chain sequence, one or both N-terminus of the antibody scaffold module heavy chain sequence, one or both N-terminus of the antibody scaffold module light chain sequence, or a combination thereof. In yet another embodiment, the first link module that binds CD137 and the antibody scaffold module that binds the TAA are covalently linked to each other directly or via an interlinker. In one embodiment, the first link module can be human or humanized.

[0222] The antibody scaffold module and the first link module may be directly conjugated (e.g., fused) or indirectly conjugated by a linker. Exemplary linkers include, for example, glycine-serine linkers, including 3xG4S linkers (e.g., GGGGSGGGGSGGGGGS (SEQ ID NO: 64)) and 4xG4S linkers (e.g., GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 65)).

[0223] In various embodiments, the bispecific binding proteins provided herein may comprise antibody scaffold modules with constant region (i.e., Fc region) substitutions or modifications, including but not limited to, amino acid residue substitutions, mutations, and / or modifications, that result in compounds with favorable properties, including but not limited to, altered pharmacokinetics, increased serum half-life, increased binding affinity, decreased immunogenicity, increased productivity, altered binding of Fc ligands to Fc receptors (FcRs), enhanced or reduced ADCC, CDC, ADCP, TDCC, modified glycosylation and / or disulfide bonds, and altered binding specificity.

[0224] Several publications have reported the successful use of protein engineering strategies to design variant human IgG1 Fc domains (CH regions) with optimized FcgR binding profiles and suitable activation / inhibition (A:I) ratios to optimize cell-mediated effector functions. Specifically, efforts have focused on increasing the affinity of the Fc domain for the low affinity receptor FcγIIIa. Several mutations within the Fc domain have been identified that directly or indirectly enhance Fc receptor binding, resulting in significantly enhanced cellular cytotoxicity (Lazar, GAPNAS 103:4005-4010 (2006); Shields, RL et al, J. Biol. Chem. 276:6591-6604 (2001); Stewart, R. et al., Protein Engineering Design and Selection 24:671-678 (2011); Richards, JO et al, Mol. Cancer Ther. 7:2517-2575 (2008)).

[0225] An antibody scaffold module may include an Fc region (e.g., two antibody heavy chain constant regions). In some embodiments, the Fc region includes at least one Fc silencing mutation, such as, for example, L234A L235A, or N297A. In some embodiments, the Fc region may include one heavy chain constant region with a knob-in-hole (KiH) mutation to promote dimerization of the heavy chains. Exemplary Fc constant regions for use in the antibody scaffold modules disclosed herein are set forth in SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, and SEQ ID NO:73. Exemplary constant regions of the light chains of the antibody scaffold modules disclosed herein are set forth in SEQ ID NO:70 and SEQ ID NO:71.

[0226] In one embodiment, the first link module that binds to CD137 comprises CDRs derived from an anti-CD137 antibody or fragment thereof. For example, the first link module may comprise a VH having the set of CDRs (HCDR1, HCDR2, and HCDR3) disclosed in Table 1. In one embodiment, the first link module comprises a heavy chain HCDR of an antibody that binds to CD137, such as an antibody comprising the variable heavy domain set forth in SEQ ID NO:23. [Table 1]

[0227] In one embodiment, the first link module that binds to CD137 comprises CDRs derived from an anti-CD137 antibody or a fragment thereof. For example, the first link module may comprise a VL having a set of CDRs (LCDR1, LCDR2, and LCDR3) disclosed in Table 1. In one embodiment, the first link module comprises an LCDR of an antibody that binds to CD137, such as an antibody comprising the variable light domain set forth in SEQ ID NO:24. [Table 2]

[0228] In one embodiment, the first link module comprises a VH and VL combination having a set of complementarity determining regions (CDR1, CDR2, and CDR3) selected from the group consisting of: (i) VH: CDR1: SEQ ID NO: 39, CDR2: SEQ ID NO: 40, CDR3: SEQ ID NO: 41, VL: CDR1: SEQ ID NO: 42, CDR2: SEQ ID NO: 43, CDR3: SEQ ID NO: 44.

[0229] In one embodiment, the first link module that binds to CD137 comprises CDRs derived from an anti-CD137 antibody or a fragment thereof. For example, the first link module may comprise a VH having a set of CDRs (HCDR1, HCDR2, and HCDR3) disclosed in Table 1 and a VL having a set of CDRs (LCDR1, LCDR2, and LCDR3) disclosed in Table 2.

[0230] In another embodiment, the first link module comprises a VH having the amino acid sequence set forth in SEQ ID NO: 23. In another embodiment, the first link module comprises a VL having the amino acid sequence set forth in SEQ ID NO: 24. In yet another embodiment, the first link module comprises a VH having the amino acid sequence set forth in SEQ ID NO: 23 and a VL having the amino acid sequence set forth in SEQ ID NO: 24.

[0231] In some embodiments, the bispecific binding protein comprises a first binding module that binds to CD137 with a KD of 1-10 nM or less. The binding association constant, ka, is between 1-10×10 6 (1 / Ms). The binding association constant kd is 1 to 10×10 -2 (1 / S).

[0232] An antibody scaffold module may comprise a set of CDRs from an antibody specific for a TAA.

[0233] In one embodiment, the antibody scaffold module comprises a VH that binds Claudin6 and has a set of CDRs (HCDR1, HCDR2, and HCDR3) disclosed in Table 3. In another embodiment, the antibody scaffold module comprises a VL that binds Claudin6 and has a set of CDRs (HCDR1, HCDR2, and HCDR3) disclosed in Table 4. In yet another embodiment, the antibody scaffold module comprises a VH that has a set of CDRs (HCDR1, HCDR2, and HCDR3) disclosed in Table 3 and a VL that has a set of CDRs (LCDR1, LCDR2, and LCDR3) disclosed in Table 4. [Table 3] [Table 4]

[0234] In one embodiment, an antibody scaffold module that binds to Claudin6 comprises a VH and VL combination having a set of complementarity determining regions (CDR1, CDR2 and CDR3) selected from the group consisting of: (i) VH: CDR1: SEQ ID NO: 45, CDR2: SEQ ID NO: 46, CDR3: SEQ ID NO: 47, VL: CDR1: SEQ ID NO: 48, CDR2: SEQ ID NO: 49, CDR3: SEQ ID NO: 50, and (ii) VH: CDR1: SEQ ID NO: 51, CDR2: SEQ ID NO: 52, CDR3: SEQ ID NO: 53, VL: CDR1: SEQ ID NO: 54, CDR2: SEQ ID NO: 55, CDR3: SEQ ID NO: 56.

[0235] In another embodiment, the antibody scaffold module comprises a VH having the amino acid sequence set forth in SEQ ID NO: 25 or SEQ ID NO: 27. In another embodiment, the antibody scaffold module comprises a VL having the amino acid sequence set forth in SEQ ID NO: 26 or SEQ ID NO: 28. In yet another embodiment, the antibody scaffold module comprises a VH having the amino acid sequence set forth in SEQ ID NO: 25 and a VL having the amino acid sequence set forth in SEQ ID NO: 26. In yet another embodiment, the antibody scaffold module comprises a VH having the amino acid sequence set forth in SEQ ID NO: 27 and a VL having the amino acid sequence set forth in SEQ ID NO: 28.

[0236] In one embodiment, the antibody scaffold module comprises a VH that binds to Claudin18.2 and has a set of CDRs (HCDR1, HCDR2, and HCDR3) disclosed in Table 5. In another embodiment, the antibody scaffold module comprises a VL that binds to Claudin18.2 and has a set of CDRs (HCDR1, HCDR2, and HCDR3) disclosed in Table 6. In yet another embodiment, the antibody scaffold module comprises a VH that has a set of CDRs (HCDR1, HCDR2, and HCDR3) disclosed in Table 5 and a VL that has a set of CDRs (LCDR1, LCDR2, and LCDR3) disclosed in Table 6. [Table 5] [Table 6]

[0237] In one embodiment, an antibody scaffold module that binds to Claudin18.2 comprises a VH and VL combination having a set of complementarity determining regions (CDR1, CDR2, and CDR3) selected from the group consisting of: (i) VH: CDR1: SEQ ID NO: 33, CDR2: SEQ ID NO: 34, CDR3: SEQ ID NO: 35, VL: CDR1: SEQ ID NO: 36, CDR2: SEQ ID NO: 37, CDR3: SEQ ID NO: 38.

[0238] In another embodiment, the antibody scaffold module comprises a VH having the amino acid sequence set forth in SEQ ID NO: 21. In another embodiment, the antibody scaffold module comprises a VL having the amino acid sequence set forth in SEQ ID NO: 22. In yet another embodiment, the antibody scaffold module comprises a VH having the amino acid sequence set forth in SEQ ID NO: 21 and a VL having the amino acid sequence set forth in SEQ ID NO: 22.

[0239] In one embodiment, the antibody scaffold module comprises a VH that binds to Nectin-4 and has a set of CDRs (HCDR1, HCDR2, and HCDR3) disclosed in Table 7. In another embodiment, the antibody scaffold module comprises a VL that binds to Nectin-4 and has a set of CDRs (HCDR1, HCDR2, and HCDR3) disclosed in Table 8. In yet another embodiment, the antibody scaffold module comprises a VH that has a set of CDRs (HCDR1, HCDR2, and HCDR3) disclosed in Table 7 and a VL that has a set of CDRs (LCDR1, LCDR2, and LCDR3) disclosed in Table 8. [Table 7] [Table 8]

[0240] In one embodiment, an antibody scaffold module that binds to Nectin-4 comprises a VH and VL combination having a set of complementarity determining regions (CDR1, CDR2, and CDR3) selected from the group consisting of: (i) VH: CDR1: SEQ ID NO: 57, CDR2: SEQ ID NO: 58, CDR3: SEQ ID NO: 59, VL: CDR1: SEQ ID NO: 60, CDR2: SEQ ID NO: 61, CDR3: SEQ ID NO: 62.

[0241] In another embodiment, the antibody scaffold module comprises a VH having the amino acid sequence set forth in SEQ ID NO: 29 or SEQ ID NO: 31. In another embodiment, the antibody scaffold module comprises a VL having the amino acid sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 32. In yet another embodiment, the antibody scaffold module comprises a VH having the amino acid sequence set forth in SEQ ID NO: 29 and a VL having the amino acid sequence set forth in SEQ ID NO: 30. In yet another embodiment, the antibody scaffold module comprises a VH having the amino acid sequence set forth in SEQ ID NO: 31 and a VL having the amino acid sequence set forth in SEQ ID NO: 32.

[0242] In another embodiment, the antibody scaffold module comprises a pair of variable heavy and variable light chain sequences selected from the following combinations: i) a variable heavy chain sequence that is 90%, 95% or 99% identical to SEQ ID NO:21, and a variable light chain sequence that is 90%, 95% or 99% identical to SEQ ID NO:22, and ii) a variable heavy chain sequence that is 90%, 95% or 99% identical to SEQ ID NO:23, and a variable light chain sequence that is 90%, 95% or 99% identical to SEQ ID NO:24; iii) a variable heavy chain sequence that is 90%, 95% or 99% identical to SEQ ID NO:25, and a variable light chain sequence that is 90%, 95% or 99% identical to SEQ ID NO:26; iv) a variable heavy chain sequence that is 90%, 95% or 99% identical to SEQ ID NO:27, and a variable light chain sequence that is 90%, 95% or 99% identical to SEQ ID NO:28; v) a variable heavy chain sequence that is 90%, 95% or 99% identical to SEQ ID NO:29, and a variable light chain sequence that is 90%, 95% or 99% identical to SEQ ID NO:30; vi) a variable heavy chain sequence that is 90%, 95% or 99% identical to SEQ ID NO:31, and a variable light chain sequence that is 90%, 95% or 99% identical to SEQ ID NO:32. One of ordinary skill in the art will further understand that the variable light and variable heavy chains may be independently selected or mixed and matched to prepare anti-CLDN6 antibodies containing variable heavy and variable light chain combinations that differ from the pairings specified above.

[0243] In some embodiments, the bispecific binding proteins contain one or more conservative amino acid substitutions. One of skill in the art will recognize that a conservative amino acid substitution is the replacement of one amino acid with another amino acid that has similar structural or chemical properties, such as a similar side chain. Exemplary conservative substitutions are described in the art, for example, in Watson et al., Molecular Biology of the Gene, The Benjamin / Cummings Publication Company, 4th Ed. (1987).

[0244] "Conservative modifications" refer to amino acid modifications that do not significantly affect or change the binding properties of the bispecific binding protein containing the amino acid sequence. Conservative modifications include amino acid substitutions, additions, and deletions. A conservative substitution is one in which an amino acid is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been well defined and include amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), uncharged polar side chains (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine, tryptophan), aromatic side chains (e.g., phenylalanine, tryptophan, histidine, tyrosine), aliphatic side chains (e.g., glycine, alanine, valine, leucine, isoleucine, serine, threonine), amides (e.g., asparagine, glutamine), beta-branched side chains (e.g., threonine, valine, isoleucine), and sulfur-containing side chains (cysteine, methionine). Additionally, any naturally occurring residue in the polypeptide may be substituted with alanine as previously described for alanine scanning mutagenesis (MacLennan et al. (1998) Acta Physiol Scand Suppl 643:55-67; ​​Sasaki et al. (1998) Adv Biophys 35:1-24). Amino acid substitutions into the bispecific binding proteins of the present disclosure may be made by known methods, such as PCR mutagenesis (U.S. Patent No. 4,683,195).

[0245] In some embodiments, the first link module that binds CD137 comprises a variable heavy chain sequence comprising an amino acid sequence having at least about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 23. In other embodiments, the first link module that binds CD137 retains the binding and / or functional activity of a link module that binds CD137 comprising the variable heavy chain sequence of SEQ ID NO: 23. In yet another embodiment, the first link module that binds CD137 comprises the variable heavy chain sequence of SEQ ID NO: 23 and has one or more conservative amino acid substitutions in the heavy chain variable sequence, e.g., 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, or 1-5 conservative amino acid substitutions. In yet another embodiment, the one or more conservative amino acid substitutions are contained within one or more framework regions of SEQ ID NO: 23 (based on the Kabat numbering system).

[0246] In certain embodiments, the first link module that binds to CD137 comprises a variable heavy chain sequence having at least about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variable region sequence set forth in SEQ ID NO:23 and contains one or more conservative amino acid substitutions in the framework regions (based on the Kabat numbering system), and retains the binding and / or functional activity of a first link module that binds to CD137 and comprises the variable heavy chain sequence set forth in SEQ ID NO:23 and the variable light chain sequence set forth in SEQ ID NO:24.

[0247] In some embodiments, the first link module that binds CD137 comprises a variable light chain sequence comprising an amino acid sequence having at least about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 24. In other embodiments, the first link module that binds CD137 retains the binding and / or functional activity of a link module that binds CD137 comprising the variable light chain sequence of SEQ ID NO: 24. In yet another embodiment, the first link module that binds CD137 comprises the variable light chain sequence of SEQ ID NO: 24 and has one or more conservative amino acid substitutions in the light chain variable sequence, e.g., 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, or 1-5 conservative amino acid substitutions. In yet another embodiment, the one or more conservative amino acid substitutions are contained within one or more framework regions of SEQ ID NO: 24 (based on the Kabat numbering system).

[0248] In certain embodiments, the first link module that binds to CD137 comprises a variable light chain sequence having at least about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variable region sequence set forth in SEQ ID NO:24, contains one or more conservative amino acid substitutions in the framework regions (based on the Kabat numbering system), and retains the binding and / or functional activity of a first link module comprising the variable heavy chain sequence set forth in SEQ ID NO:23 and the variable light chain sequence set forth in SEQ ID NO:24.

[0249] In some embodiments, the antibody scaffold module that binds to Claudin6 comprises a variable heavy chain sequence comprising an amino acid sequence having at least about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 25 or 27. In other embodiments, the antibody scaffold module that binds to Claudin6 retains the binding and / or functional activity of a binding module that binds to Claudin6 comprising a variable heavy chain sequence of SEQ ID NO: 25 or 27. In yet another embodiment, the antibody scaffold module that binds to Claudin6 comprises a variable heavy chain sequence of SEQ ID NO: 25 or 27 and has one or more conservative amino acid substitutions, for example, 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, or 1-5 conservative amino acid substitutions in the heavy chain variable sequence. In yet another embodiment, the one or more conservative amino acid substitutions are included within one or more framework regions of SEQ ID NO: 25 or 27 (based on the Kabat numbering system).

[0250] In certain embodiments, an antibody scaffold module that binds to Claudin6 comprises a variable heavy chain sequence having at least about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variable region sequence set forth in SEQ ID NO: 25 or 27, and comprises one or more conservative amino acid substitutions in the framework regions (based on the Kabat numbering system), and retains the binding and / or functional activity of an antibody scaffold module that binds to Claudin6 and comprises the variable heavy chain sequence set forth in SEQ ID NO: 25 or 27 and the variable light chain sequence set forth in SEQ ID NO: 26 or 28.

[0251] In some embodiments, the antibody scaffold module that binds to Claudin6 comprises a variable light chain sequence comprising an amino acid sequence having at least about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 26 or 28. In other embodiments, the antibody scaffold module that binds to Claudin6 retains the binding and / or functional activity of an antibody scaffold module that binds to Claudin6 comprising a variable light chain sequence of SEQ ID NO: 26 or 28. In yet another embodiment, the antibody scaffold module that binds to Claudin6 comprises a variable light chain sequence of SEQ ID NO: 26 or 28 and has one or more conservative amino acid substitutions, for example, 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, or 1-5 conservative amino acid substitutions in the light chain variable sequence. In yet another embodiment, the one or more conservative amino acid substitutions are included within one or more framework regions of SEQ ID NO: 26 or 28 (based on the Kabat numbering system).

[0252] In certain embodiments, an antibody scaffold module that binds to Claudin6 comprises a variable light chain sequence having at least about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variable region sequence set forth in SEQ ID NO: 26 or 28, contains one or more conservative amino acid substitutions in the framework regions (based on the Kabat numbering system), and retains the binding and / or functional activity of an antibody scaffold module comprising a variable heavy chain sequence set forth in SEQ ID NO: 25 or 27 and a variable light chain sequence set forth in SEQ ID NO: 26 or 28.

[0253] In some embodiments, an antibody scaffold module that binds to Claudin18.2 comprises a variable heavy chain sequence comprising an amino acid sequence having at least about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 21. In other embodiments, an antibody scaffold module that binds to Claudin18.2 retains the binding and / or functional activity of a binding module that binds to Claudin18.2 comprising the variable heavy chain sequence of SEQ ID NO: 21. In yet another embodiment, an antibody scaffold module that binds to Claudin18.2 comprises a variable heavy chain sequence of SEQ ID NO: 21 and has one or more conservative amino acid substitutions, for example, 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, or 1-5 conservative amino acid substitutions in the heavy chain variable sequence. In yet another embodiment, the one or more conservative amino acid substitutions are contained within one or more framework regions of SEQ ID NO: 21 (based on the Kabat numbering system).

[0254] In certain embodiments, an antibody scaffold module that binds to Claudin18.2 comprises a variable heavy chain sequence having at least about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variable region sequence set forth in SEQ ID NO:21, contains one or more conservative amino acid substitutions in the framework regions (based on the Kabat numbering system), and retains the binding and / or functional activity of an antibody scaffold module that binds to Claudin18.2 and comprises the variable heavy chain sequence set forth in SEQ ID NO:21 and the variable light chain sequence set forth in SEQ ID NO:22.

[0255] In some embodiments, an antibody scaffold module that binds to Claudin18.2 comprises a variable light chain sequence comprising an amino acid sequence having at least about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 22. In other embodiments, an antibody scaffold module that binds to Claudin18.2 retains the binding and / or functional activity of an antibody scaffold module that binds to Claudin18.2 comprising a variable light chain sequence of SEQ ID NO: 22. In yet another embodiment, an antibody scaffold module that binds to Claudin18.2 comprises a variable light chain sequence of SEQ ID NO: 22 and has one or more conservative amino acid substitutions in the light chain variable sequence, for example, 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, or 1-5 conservative amino acid substitutions. In yet another embodiment, the one or more conservative amino acid substitutions are included within one or more framework regions of SEQ ID NO: 22 (based on the Kabat numbering system).

[0256] In certain embodiments, an antibody scaffold module that binds to Claudin18.2 comprises a variable light chain sequence having at least about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variable region sequence set forth in SEQ ID NO:22, contains one or more conservative amino acid substitutions in the framework regions (based on the Kabat numbering system), and retains the binding and / or functional activity of an antibody scaffold module comprising the variable heavy chain sequence set forth in SEQ ID NO:21 and the variable light chain sequence set forth in SEQ ID NO:22.

[0257] In some embodiments, the antibody scaffold module that binds to Nectin-4 comprises a variable heavy chain sequence comprising an amino acid sequence having at least about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 29 or 31. In other embodiments, the antibody scaffold module that binds to Nectin-4 retains the binding and / or functional activity of a link module that binds to Nectin-4 comprising the variable heavy chain sequence of SEQ ID NO: 29 or 31. In yet another embodiment, the antibody scaffold module that binds to Nectin-4 comprises the variable heavy chain sequence of SEQ ID NO: 29 or 31 and has one or more conservative amino acid substitutions, for example, 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, or 1-5 conservative amino acid substitutions in the heavy chain variable sequence. In yet another embodiment, the one or more conservative amino acid substitutions are included within one or more framework regions of SEQ ID NO: 29 or 31 (based on the Kabat numbering system).

[0258] In certain embodiments, an antibody scaffold module that binds to Nectin-4 comprises a variable heavy chain sequence having at least about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variable region sequence set forth in SEQ ID NO: 29 or 31, and comprises one or more conservative amino acid substitutions in the framework regions (based on the Kabat numbering system), and retains the binding and / or functional activity of an antibody scaffold module that binds to Nectin-4 and comprises the variable heavy chain sequence set forth in SEQ ID NO: 29 or 31 and the variable light chain sequence set forth in SEQ ID NO: 30 or 32.

[0259] In some embodiments, the antibody scaffold module that binds to Nectin-4 comprises a variable light chain sequence comprising an amino acid sequence having at least about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 30 or 32. In other embodiments, the antibody scaffold module that binds to Nectin-4 retains the binding and / or functional activity of an antibody scaffold module that binds to Nectin-4 comprising a variable light chain sequence of SEQ ID NO: 30 or 32. In yet another embodiment, the antibody scaffold module that binds to Nectin-4 comprises a variable light chain sequence of SEQ ID NO: 30 or 32 and has one or more conservative amino acid substitutions, for example, 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, or 1-5 conservative amino acid substitutions in the light chain variable sequence. In yet another embodiment, the one or more conservative amino acid substitutions are included within one or more framework regions of SEQ ID NO: 30 or 32 (based on the Kabat numbering system).

[0260] In certain embodiments, an antibody scaffold module that binds to Nectin-4 comprises a variable light chain sequence having at least about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variable region sequence set forth in SEQ ID NO: 30 or 32, contains one or more conservative amino acid substitutions in the framework regions (based on the Kabat numbering system), and retains the binding and / or functional activity of an antibody scaffold module comprising a variable heavy chain sequence set forth in SEQ ID NO: 29 or 31 and a variable light chain sequence set forth in SEQ ID NO: 30 or 32.

[0261] In some embodiments, the bispecific binding protein comprises SEQ ID NO:3 and SEQ ID NO:2 (1901 Ab2), SEQ ID NO:4 and SEQ ID NO:5 (1901 Ab3), SEQ ID NO:12 and SEQ ID NO:9 (1912 Ab3), SEQ ID NO:13 and SEQ ID NO:11 (1912 Ab4), SEQ ID NO:72 and SEQ ID NO:9 (1912 Ab5), SEQ ID NO:14 and SEQ ID NO:15 (1925 Ab1), SEQ ID NO:16 and SEQ ID NO:17 (1925 Ab2), or SEQ ID NO:18 and SEQ ID NO:15 (1925 Ab3).

[0262] In another embodiment, the bispecific binding protein comprises SEQ ID NO:3 and SEQ ID NO:2 (1901 Ab2) and binds to CD137 and Claudin18.2. In another embodiment, the bispecific binding protein comprises SEQ ID NO:4 and SEQ ID NO:5 (1901 Ab3) and binds to CD137 and Claudin18.2. In another embodiment, the bispecific binding protein comprises SEQ ID NO:12 and SEQ ID NO:9 (1912 Ab3) and binds to CD137 and Claudin6. In another embodiment, the bispecific binding protein comprises SEQ ID NO:13 and SEQ ID NO:11 (1912 Ab4) and binds to CD137 and Claudin6. In another embodiment, the bispecific binding protein comprises SEQ ID NO:72 and SEQ ID NO:9 (1912 Ab5) and binds to CD137 and Claudin6. In another embodiment, the bispecific binding protein comprises SEQ ID NO:14 and SEQ ID NO:15 (1925 Ab1) and binds to CD137 and Nectin-4. In another embodiment, the bispecific binding protein comprises SEQ ID NO: 16 and SEQ ID NO: 17 (1925 Ab2) and binds to CD137 and Nectin-4. In another embodiment, the bispecific binding protein comprises SEQ ID NO: 18 and SEQ ID NO: 15 (1925 Ab3) and binds to CD137 and Nectin-4.

[0263] In one embodiment, the bispecific binding protein that binds CD137 and Claudin18.2 is i) an antibody scaffold module that binds to Claudin18.2, the antibody scaffold module being an IgG having two heavy chains and two light chains, the IgG comprising an Fc region comprising two constant chains having an N-terminus and a C-terminus; and ii) two first link modules that bind to CD137, the first link modules being scFvs, the scFvs comprising a VH and a VL linked by a 4×(G4S) linker, each of the first link modules comprising two first link modules separately linked to the C-terminus of an Fc constant chain by a 3×(G4S) linker.

[0264] In some embodiments, the 3×(G4S) linker has an N-terminus and a C-terminus, the N-terminus of the 3×(G4S) linker is attached to the C-terminus of the two Fc constant chains, and the C-terminus of the 3×(G4S) linker is attached to the N-terminus of the VH in the first link module. The scFv may be stabilized. In some embodiments, the two heavy chains consisting of the antibody scaffold module, the 3×(G4S) linker, and the first link module both comprise the amino acid sequence from N-terminus to C-terminus set forth in SEQ ID NO:3. In yet another embodiment, the antibody scaffold module comprises two light chains each having the amino acid sequence set forth in SEQ ID NO:2.

[0265] In one embodiment, the bispecific binding protein that binds CD137 and Claudin18.2 is i) an antibody scaffold module that binds to Claudin18.2, the antibody scaffold module being an IgG having two heavy chains and two light chains, the IgG comprising an Fc region comprising two constant chains having an N-terminus and a C-terminus; and ii) two first link modules that bind to CD137, wherein the first link modules are scFvs, the scFvs comprising a VH and a VL linked by a 4×(G4S) linker, and each of the first link modules comprises two first link modules separately linked to the C-terminus of the light chain by a 3×(G4S) linker.

[0266] In some embodiments, the 3×(G4S) linker has an N-terminus and a C-terminus, the N-terminus of the 3×(G4S) linker is attached to the C-terminus of the two light chains, and the C-terminus of the 3×(G4S) linker is attached to the N-terminus of the VH in the first link module. The scFv may be stabilized. In some embodiments, the two light chains consisting of the antibody scaffold module, the 3×(G4S) linker, and the first link module each separately comprise an amino acid sequence from N-terminus to C-terminus set forth in SEQ ID NO:5. In yet another embodiment, the antibody scaffold module comprises two heavy chains each having an amino acid sequence set forth in SEQ ID NO:4.

[0267] In one embodiment, a bispecific binding protein that binds CD137 and Claudin6 is i) an antibody scaffold module that binds to Claudin6, the antibody scaffold module being an IgG having two heavy chains and two light chains, the IgG comprising an Fc region comprising two constant chains having an N-terminus and a C-terminus; and ii) two first link modules that bind to CD137, the first link modules being scFvs, the scFvs comprising a VH and a VL linked by a 4×(G4S) linker, each of the first link modules comprising two first link modules separately linked to the C-terminus of an Fc constant chain by a 3×(G4S) linker.

[0268] In some embodiments, the 3×(G4S) linker has an N-terminus and a C-terminus, the N-terminus of the 3×(G4S) linker is attached to the C-terminus of the two Fc constant chains, and the C-terminus of the 3×(G4S) linker is attached to the N-terminus of the VH in the first link module. The scFv may be stabilized. In some embodiments, the two heavy chains consisting of the antibody scaffold module, the 3×(G4S) linker, and the first link module each separately comprise an amino acid sequence from N-terminus to C-terminus set forth in SEQ ID NO: 12 or SEQ ID NO: 72. In yet another embodiment, the antibody scaffold module comprises two light chains each having an amino acid sequence set forth in SEQ ID NO: 9.

[0269] In one embodiment, a bispecific binding protein that binds CD137 and Claudin6 is i) an antibody scaffold module that binds to Claudin6, the antibody scaffold module being an IgG having two heavy chains and two light chains, the IgG comprising an Fc region comprising two constant chains having an N-terminus and a C-terminus; and ii) two first link modules that bind to CD137, wherein the first link modules are scFvs, the scFvs comprising a VH and a VL linked by a 4×(G4S) linker, and each of the first link modules comprises two first link modules separately linked to the C-terminus of the light chain by a 3×(G4S) linker.

[0270] In some embodiments, the 3×(G4S) linker has an N-terminus and a C-terminus, the N-terminus of the 3×(G4S) linker is attached to the C-terminus of the two light chains, and the C-terminus of the 3×(G4S) linker is attached to the N-terminus of the VH in the first link module. The scFv may be stabilized. In some embodiments, the two light chains consisting of the antibody scaffold module, the 3×(G4S) linker, and the first link module each separately comprise an amino acid sequence from N-terminus to C-terminus set forth in SEQ ID NO: 11. In yet another embodiment, the antibody scaffold module comprises two heavy chains each having an amino acid sequence set forth in SEQ ID NO: 13.

[0271] In one embodiment, the bispecific binding protein that binds CD137 and Nectin-4 is i) an antibody scaffold module that binds to Nectin-4, the antibody scaffold module being an IgG having two heavy chains and two light chains, the IgG comprising an Fc region comprising two constant chains having an N-terminus and a C-terminus; and ii) two first link modules that bind to CD137, the first link modules being scFvs, the scFvs comprising a VH and a VL linked by a 4×(G4S) linker, each of the first link modules comprising two first link modules separately linked to the C-terminus of an Fc constant chain by a 3×(G4S) linker.

[0272] In some embodiments, the 3×(G4S) linker has an N-terminus and a C-terminus, the N-terminus of the 3×(G4S) linker is linked to the C-terminus of the two Fc constant chains, and the C-terminus of the 3×(G4S) linker is linked to the N-terminus of the VH in the first link module. The scFv may be stabilized. In some embodiments, the two heavy chains consisting of the antibody scaffold module, the 3×(G4S) linker, and the first link module each separately comprise an amino acid sequence from N-terminus to C-terminus set forth in SEQ ID NO: 14. In another embodiment, the antibody scaffold module comprises two light chains each having an amino acid sequence set forth in SEQ ID NO: 15.

[0273] In one embodiment, the bispecific binding protein that binds CD137 and Nectin-4 is i) an antibody scaffold module that binds to Nectin-4, the antibody scaffold module being an IgG having two heavy chains and two light chains, the IgG comprising an Fc region comprising two constant chains having an N-terminus and a C-terminus; and ii) two first link modules that bind to CD137, wherein the first link modules are scFvs, the scFvs comprising a VH and a VL linked by a 4×(G4S) linker, and each of the first link modules comprises two first link modules separately linked to the C-terminus of the light chain by a 3×(G4S) linker.

[0274] In some embodiments, the 3×(G4S) linker has an N-terminus and a C-terminus, the N-terminus of the 3×(G4S) linker is attached to the C-terminus of the two light chains, and the C-terminus of the 3×(G4S) linker is attached to the N-terminus of the VH in the first link module. The scFv may be stabilized. In some embodiments, the two light chains consisting of the antibody scaffold module, the 3×(G4S) linker, and the first link module each separately comprise an amino acid sequence from N-terminus to C-terminus set forth in SEQ ID NO: 17. In yet another embodiment, the antibody scaffold module comprises two heavy chains each having an amino acid sequence set forth in SEQ ID NO: 16.

[0275] In one embodiment, the bispecific binding protein that binds CD137 and Nectin-4 is i) an antibody scaffold module that binds to Nectin-4, the antibody scaffold module being an IgG having two heavy chains and two light chains, the IgG comprising an Fc region comprising two constant chains having an N-terminus and a C-terminus; and ii) two first link modules that bind to CD137, the first link modules being scFvs, the scFvs comprising a VH and a VL linked by a 4×(G4S) linker, each of the first link modules comprising two first link modules separately linked to the N-terminus of the two heavy chains by a 4×(G4S) linker.

[0276] In some embodiments, the 4x(G4S) linker has an N-terminus and a C-terminus, and the C-terminus of the 4x(G4S) linker is attached to the N-terminus of the two heavy chains, and the N-terminus of the 4x(G4S) linker is attached to the C-terminus of the VL in the first link module. The scFv may be stabilized. In some embodiments, the first link module, the 4x(G4S) linker, and the two heavy chains of the antibody scaffold module each separately comprise an N- to C-terminal amino acid sequence set forth in SEQ ID NO: 18. In yet another embodiment, the antibody scaffold module comprises two light chains each having an N- to C-terminal amino acid sequence set forth in SEQ ID NO: 15.

[0277] The therapeutic value of the bispecific binding proteins of the disclosure can be enhanced by conjugation to cytotoxic drugs or agents that improve their efficacy and potency, such as cytotoxic effector agents, such as radioisotopes, drugs, or cytotoxins.

[0278] In some embodiments, the bispecific binding proteins disclosed herein exhibit one or more of the following structural and functional characteristics, alone or in combination: (a) capable of binding to human CD137 and tumor-associated antigens (TAAs); (b) cross-reacts with cynomolgus CD137 and one of the tumor-associated antigens (TAA); (c) blocking (e.g., reducing or preventing) human CD137L binding to CD137; (d) exhibiting fast on and fast off properties for CD137; (e) having TAA-dependent agonistic activity against CD137 signaling; (f) activating T cells in a TAA-dependent manner; and (g) Killing TAA-expressing cells by activating CD8 T cells.

[0279] In some embodiments, the bispecific binding protein is a Claudin-6 / CD137 BsAb that exhibits one or more of the following structural and functional characteristics, alone or in combination: (a) Bivalency for Claudin6 binding, (b) Fast-on / fast-off CD137 binding kinetics; (c) enhancing lymphocytic infiltration in tumors; (d) promoting T cell proliferation / activation in tumors; (e) protecting T cells from exhaustion in tumors; (f) promoting the formation of T cell memory from tumor-transduced T cells; (g) decreasing the Treg / CD8 ratio in the tumor microenvironment (TME); and (h) Reducing M2-like macrophages in the TME.

[0280] Methods for Producing Monoclonal Antibodies for Use as Scaffold or Binding Modules Bispecific binding proteins that bind CD137 and TAA may be produced by any method known in the art. For example, a recipient may be immunized with a soluble recombinant CD137 protein or a fragment of a CD137 peptide conjugated to a carrier protein. Similarly, a recipient may be immunized with a soluble recombinant TAA protein or a fragment of a tumor-associated antigen peptide conjugated to a carrier protein. Any suitable immunization method may be used. Such methods may include the use of adjuvants, other immune stimulants, repeated booster immunizations, and one or more immunization routes. CDRs or VH / VL derived from an antibody may be used in the antibody scaffold module and / or the first binding module.

[0281] Any suitable source of human CD137 or TAA can be used as an immunogen for generating non-human or human anti-CD137 and / or anti-TAA antibodies of the compositions and methods disclosed herein.

[0282] Different forms of CD137 and / or TAAs may be used to induce immune responses to identify biologically active anti-CD137 or anti-TAA antibodies. Thus, the inducing CD137 antigen or TAA may be a single epitope, multiple epitopes, or the entire protein alone or in combination with one or more immunogenic stimulants. In some embodiments, the inducing antigen is an isolated soluble full-length protein or a soluble protein smaller than the full-length sequence (e.g., immunization with peptides comprising the extracellular domain / loop, ECD1 and / or ECD2 of CD137 or TAA, alone or in combination). As used herein, the term "portion" refers to the minimum number of amino acids or nucleic acids that constitute an immunogenic epitope of the antigen of interest, as appropriate. Any genetic vector suitable for transformation of the cells of interest may be employed, including, but not limited to, adenoviral vectors, plasmids, and non-viral vectors such as cationic lipids.

[0283] It is desirable to prepare monoclonal antibodies (mAbs) from a variety of mammalian hosts, including mice, rodents, primates, and humans. Descriptions of techniques for preparing such monoclonal antibodies can be found, for example, in Sties et al. (eds.) BASIC AND CLINICAL IMMUNOLOGY (4th ed.) Lance Medical Publication, Los Altos, CA, and references cited therein; Harlow and Lane (1988) ANTIBODIES: A LABORATORY MANUAL CSH Press; Goding (1986) MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2nd ed.) Academic Press, New York, NY. Typically, spleen cells from an animal immunized with a desired antigen are immortalized, usually by fusion with a myeloma cell. See Kohler and Milstein (196) Eur. J. Immunol. 6:511-519. Alternative methods of immortalization include transformation with Epstein-Barr virus, oncogenes, or retroviruses, or other methods known in the art. See, for example, Doyle et al. (eds. 1994 and regular supplements) CELL AND TISSUE CULTURE: LABORATORY PROCEDURES, John Wiley and Sons, New York, NY. Colonies arising from a single immortalized cell are screened for production of antibodies of the desired specificity and affinity to the antigen, and the yield of monoclonal antibodies produced by such cells can be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host. Alternatively, DNA sequences encoding monoclonal antibodies or antigen-binding fragments thereof may be isolated by screening DNA libraries from human B cells, for example, following the general protocol outlined by Huse et al. (1989) Science 246:1275-1281. Thus, antibodies can be obtained by various techniques well known to those skilled in the art.

[0284] Other suitable techniques include the selection of antibody libraries in phage, yeast, virus, or similar vectors.See, for example, Huse et al. and Ward et al. (1989) Nature 341:544-546, supra. The polypeptides and antibodies disclosed herein can be used with or without modification, including chimeric or humanized antibodies.In many cases, the polypeptides and antibodies will be labeled by covalently or non-covalently binding a substance that provides a detectable signal.A wide variety of labeling and conjugation techniques are known and widely reported in both scientific and patent literature.Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Patents teaching the use of such labels include U.S. Patent Nos. 3,817,837, 3,850,752, 3,9396,345, 4,277,437, 4,275,149, and 4,366,241. Recombinant immunoglobulins may also be produced. See U.S. Patent No. 4,816,567 to Cabilly, and Queen et al. (1989) Proc. Nat'l Acad. Sci. USA 86:10029-10023. Alternatively, for production in transgenic mice, see Nils Lonberg et al. (1994), Nature 368:856-859, and Mendez et al. (1997) Nature Genetics 15:146-156; TRANSGENIC ANIMALS AND METHODS OF USE (WO2012 / 62118), Medarex, Trianni, Abgenix, Ablexis, OminiAb, Harbour, and other technologies.

[0285] In some embodiments, the ability of the produced antibodies to bind to CD137 or a TAA can be assessed using standard binding assays such as surface plasmon resonance (SPR), FoteBio (BLI), Gator (BLI), ELISA, Western blot, immunofluorescence, flow cytometric analysis (FACS), or internalization assays.

[0286] Antibody compositions prepared from hybridomas or host cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being a typical purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human gamma 1, gamma 2, or gamma 4 heavy chains (see, for example, Lindmark et al., 1983 J. Immunol. Meth. 62:1-13). Protein G is recommended for all mouse isotypes and human gamma 3 (see, for example, Guss et al., 1986 EMBO J. 5:1567-1575). The matrix to which the affinity ligand is attached is most often agarose, although other matrices are available. Physically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. If the antibody contains a CH3 domain, Bakerbond ABX™ resin (JT Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification, such as fractionation on ion exchange columns, ethanol precipitation, reversed-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™ chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, are also available depending on the antibody to be recovered.

[0287] Following any preliminary purification steps, the mixture containing the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH of about 2.5-4.5, typically performed at a low salt concentration (e.g., about 0-0.25 M salt).

[0288] Polynucleotides, Vectors, and Host Cells Other embodiments include isolated polynucleotides comprising sequences encoding the bispecific binding proteins disclosed herein, vectors and host cells comprising said polynucleotides, and recombinant techniques for the production of said bispecific binding proteins. The isolated polynucleotides may encode any desired form of bispecific binding protein, including its components, such as a scaffold module and / or a first binding module.

[0289] In one embodiment, the isolated polynucleotide sequence encodes a first binding module comprising a VH and VL combination having a set of complementarity determining regions (CDR1, CDR2, and CDR3) selected from VH: CDR1: SEQ ID NO: 39, CDR2: SEQ ID NO: 40, CDR3: SEQ ID NO: 41, and VL: CDR1: SEQ ID NO: 42, CDR2: SEQ ID NO: 43, CDR3: SEQ ID NO: 44.

[0290] In one embodiment, the isolated polynucleotide sequence encodes an antibody scaffold module that binds to Claudin6 and includes a VH and VL combination having a set of complementarity determining regions (CDR1, CDR2, and CDR3) selected from VH: CDR1: SEQ ID NO: 45, CDR2: SEQ ID NO: 46, CDR3: SEQ ID NO: 47, and VL: CDR1: SEQ ID NO: 48, CDR2: SEQ ID NO: 49, CDR3: SEQ ID NO: 50.

[0291] In one embodiment, the isolated polynucleotide sequence encodes an antibody scaffold module that binds to Claudin6 and includes a VH and VL combination having a set of complementarity determining regions (CDR1, CDR2, and CDR3) selected from VH: CDR1: SEQ ID NO: 51, CDR2: SEQ ID NO: 52, CDR3: SEQ ID NO: 53, and VL: CDR1: SEQ ID NO: 54, CDR2: SEQ ID NO: 55, CDR3: SEQ ID NO: 56.

[0292] In one embodiment, the isolated polynucleotide sequence encodes an antibody scaffold module that binds to Claudin18.2 and includes a VH and VL combination having a set of complementarity determining regions (CDR1, CDR2, and CDR3) selected from VH: CDR1: SEQ ID NO: 33, CDR2: SEQ ID NO: 34, CDR3: SEQ ID NO: 35, and VL: CDR1: SEQ ID NO: 36, CDR2: SEQ ID NO: 37, CDR3: SEQ ID NO: 38.

[0293] In one embodiment, the isolated polynucleotide sequence encodes an antibody scaffold module that binds to Nectin-4 and comprises a VH and VL combination having a set of complementarity determining regions (CDR1, CDR2, and CDR3) selected from VH: CDR1: SEQ ID NO: 57, CDR2: SEQ ID NO: 58, CDR3: SEQ ID NO: 59, and VL: CDR1: SEQ ID NO: 60, CDR2: SEQ ID NO: 61, CDR3: SEQ ID NO: 62.

[0294] In another embodiment, the isolated polynucleotide sequence encodes an antibody scaffold module comprising a VH having the amino acid sequence set forth in SEQ ID NO:29 or SEQ ID NO:31. In another embodiment, the isolated polynucleotide sequence encodes an antibody scaffold module comprising a VL having the amino acid sequence set forth in SEQ ID NO:30 or SEQ ID NO:32. In yet another embodiment, the isolated polynucleotide sequence encodes an antibody scaffold module comprising a VH having the amino acid sequence set forth in SEQ ID NO:29 and a VL having the amino acid sequence set forth in SEQ ID NO:30. In yet another embodiment, the isolated polynucleotide sequence encodes an antibody scaffold module comprising a VH having the amino acid sequence set forth in SEQ ID NO:31 and a VL having the amino acid sequence set forth in SEQ ID NO:32.

[0295] In another embodiment, the isolated polynucleotide sequence encodes an antibody scaffold module comprising a pair of variable heavy and variable light chain sequences selected from the following combinations: i) a variable heavy chain sequence that is 90%, 95% or 99% identical to SEQ ID NO:21, and a variable light chain sequence that is 90%, 95% or 99% identical to SEQ ID NO:22, and ii) a variable heavy chain sequence that is 90%, 95% or 99% identical to SEQ ID NO:23, and a variable light chain sequence that is 90%, 95% or 99% identical to SEQ ID NO:24; iii) a variable heavy chain sequence that is 90%, 95% or 99% identical to SEQ ID NO:25, and a variable light chain sequence that is 90%, 95% or 99% identical to SEQ ID NO:26; iv) a variable heavy chain sequence that is 90%, 95% or 99% identical to SEQ ID NO:27, and a variable light chain sequence that is 90%, 95% or 99% identical to SEQ ID NO:28; v) a variable heavy chain sequence that is 90%, 95% or 99% identical to SEQ ID NO:29, and a variable light chain sequence that is 90%, 95% or 99% identical to SEQ ID NO:30; vi) a variable heavy chain sequence that is 90%, 95% or 99% identical to SEQ ID NO:31, and a variable light chain sequence that is 90%, 95% or 99% identical to SEQ ID NO:32.

[0296] In some embodiments, the isolated polynucleotide sequence encodes a bispecific binding protein comprising SEQ ID NO:3 and SEQ ID NO:2 (1901 Ab2), SEQ ID NO:4 and SEQ ID NO:5 (1901 Ab3), SEQ ID NO:12 and SEQ ID NO:9 (1912 Ab3), SEQ ID NO:13 and SEQ ID NO:11 (1912 Ab4), SEQ ID NO:72 and SEQ ID NO:9 (1912 Ab5), SEQ ID NO:14 and SEQ ID NO:15 (1925 Ab1), SEQ ID NO:16 and SEQ ID NO:17 (1925 Ab2), or SEQ ID NO:18 and SEQ ID NO:15 (1925 Ab3).

[0297] In another embodiment, the isolated polynucleotide sequence encodes a bispecific binding protein comprising SEQ ID NO:3 and / or SEQ ID NO:2 (1901 Ab2) and binds to CD137 and Claudin18.2. In another embodiment, the isolated polynucleotide sequence encodes a bispecific binding protein comprising SEQ ID NO:4 and / or SEQ ID NO:5 (1901 Ab3) and binds to CD137 and Claudin18.2. In another embodiment, the isolated polynucleotide sequence encodes a bispecific binding protein comprising SEQ ID NO:12 and / or SEQ ID NO:9 (1912 Ab3) and binds to CD137 and Claudin6. In another embodiment, the isolated polynucleotide sequence encodes a bispecific binding protein comprising SEQ ID NO:13 and / or SEQ ID NO:11 (1912 Ab4) and binds to CD137 and Claudin6. In another embodiment, the isolated polynucleotide sequence encodes a bispecific binding protein comprising SEQ ID NO:72 and / or SEQ ID NO:9 (1912 Ab5) and binds to CD137 and Claudin6. In other embodiments, the isolated polynucleotide sequence comprises SEQ ID NO: 14 and / or SEQ ID NO: 15 (1925 Ab1) and encodes a bispecific binding protein that binds to CD137 and Nectin-4. In other embodiments, the isolated polynucleotide sequence comprises SEQ ID NO: 16 and / or SEQ ID NO: 17 (1925 Ab2) and encodes a bispecific binding protein that binds to CD137 and Nectin-4. In other embodiments, the isolated polynucleotide sequence comprises SEQ ID NO: 18 and / or SEQ ID NO: 15 (1925 Ab3) and encodes a bispecific binding protein that binds to CD137 and Nectin-4.

[0298] Also included are nucleic acids that hybridize under low, medium, and high stringency conditions, as defined herein, for all or a portion of the nucleotide sequence represented by the isolated polynucleotide sequence encoding the bispecific binding protein of the present disclosure (e.g., the portion encoding the variable region). The hybridizing portion of the hybridizing nucleic acid is typically at least 15 (e.g., 20, 25, 30, or 50) nucleotides in length. The hybridizing portion of the hybridizing nucleic acid is at least 80%, e.g., at least 90%, at least 95%, or at least 98% identical to the sequence of a portion or all of the nucleic acid encoding the polypeptide chain of the bispecific binding protein (e.g., the variable region of the heavy or light chain of the antibody scaffold module and / or the first binding module) or its complementary chain. Hybridizing nucleic acids of the type described herein can be used, for example, as cloning probes, primers, e.g., PCR primers, or diagnostic probes.

[0299] A polynucleotide comprising a sequence encoding a bispecific binding protein disclosed herein can be fused to one or more regulatory or control sequences known in the art and can be contained in a suitable expression vector or cell known in the art. Each polynucleotide molecule encoding a heavy or light chain variable domain of an antibody binding scaffold can be independently fused to a polynucleotide sequence encoding a constant domain, such as a human constant domain, to form an antibody scaffold module. Alternatively, the polynucleotides or portions thereof can be fused together to provide a template for the production of a first binding module.

[0300] For recombinant production, a polynucleotide encoding a bispecific binding protein disclosed herein (e.g., its antibody scaffold module, including two heavy chains and two light chains, and a first binding module) is inserted into a replicable vector for cloning (amplification of DNA) or expression. Many suitable vectors for expressing bispecific binding proteins are available. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0301] A bispecific binding protein (e.g., its antibody scaffold module, including two heavy chains and two light chains, and a first binding module) may also be produced as a fusion polypeptide, in which the bispecific binding protein is fused to a heterologous polypeptide, such as a signal sequence, or other polypeptide that has a specific cleavage site at the amino terminus of the mature protein or polypeptide. The heterologous signal sequence selected is typically one that is recognized and processed by the host cell (i.e., cleaved by a signal peptidase). For prokaryotic host cells that do not recognize and process the bispecific binding protein signal sequence, the signal sequence can be replaced by a prokaryotic signal sequence. The signal sequence may be, for example, an alkaline phosphatase, penicillinase, lipoprotein, heat-stable enterotoxin II leader, or the like. For secretion in yeast, the native signal sequence can be replaced by, for example, leader sequences derived from yeast invertase alpha factor (including the α-factor leaders of Saccharomyces and Kluyveromyces), acid phosphatase, C. albicans glucoamylase, or the signals described in WO90 / 13646. In mammalian cells, mammalian signal sequences as well as viral secretory leaders, such as the herpes simplex gD signal, can be used. The DNA of such a precursor region is ligated in reading frame to the DNA encoding the bispecific binding protein (e.g., its antibody scaffold module including two heavy chains and two light chains, and the first binding module).

[0302] Expression and cloning vectors contain a nucleic acid sequence that allows the vector to replicate in one or more selected host cells. In general, in cloning vectors, this sequence allows the vector to replicate independently of the host chromosomal DNA and includes an origin of replication or an autonomously replicating sequence. Such sequences are well known for various bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 is suitable for most gram-negative bacteria, the 2-υ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV, and BPV) are useful for cloning vectors in mammalian cells. Generally, the origin of replication element is not required for mammalian expression vectors (the SV40 origin may typically be used only because it contains the early promoter).

[0303] Expression and cloning vectors may contain a gene encoding a selection marker to allow easy identification of expression. Typical selection marker genes encode proteins that confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline, or that are complementary auxotrophic defects, or alternatively, that supply a particular nutrient not present in complex media, such as the gene encoding D-alanine racemase in Bacillus.

[0304] Also provided herein are host cells comprising one or more polynucleotides encoding the bispecific binding proteins. The cells used to produce the bispecific binding proteins disclosed herein may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma-Aldrich), Minimum Essential Medium ((MEM), Sigma-Aldrich), RPMI-1640 (Sigma-Aldrich), FreeStyle™ (Gibco), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma-Aldrich) are suitable for culturing the host cells. Any of these or other media may be supplemented as necessary with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as gentamicin), trace elements (such as inorganic compounds that are usually present at final concentrations in the submicromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to one of skill in the art. Culture conditions such as temperature, pH, etc. will include those previously used with the cells selected for expression and will be apparent to one of skill in the art.

[0305] Non-therapeutic use The bispecific binding protein described herein is useful as an affinity purification agent. In this process, the bispecific binding protein is immobilized on a solid phase, such as a protein A resin, using methods well known in the art. The immobilized bispecific binding protein is contacted with a sample containing the CD137 and TAA protein (or fragments thereof) to be purified, and then the support is washed with a suitable solvent that removes substantially all of the material in the sample except for the CD137 and TAA protein bound to the immobilized bispecific binding protein. Finally, the support is washed with another suitable solvent that releases the CD137 and TAA protein from the bispecific binding protein.

[0306] The bispecific binding proteins disclosed herein are also useful in diagnostic assays for detecting and / or quantifying CD137 and / or TAA proteins, e.g., detecting expression of CD137 and / or TAA in specific cells, tissues, or serum. Bispecific binding proteins can be used diagnostically, e.g., as part of a clinical testing procedure to monitor the development or progression of a disease, e.g., to determine the effectiveness of a given therapeutic and / or prophylactic regimen. Detection can be facilitated by coupling the bispecific binding protein to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, various positron-emitting metals using positron emission tomography, and non-radioactive paramagnetic metal ions. See, e.g., U.S. Pat. No. 4,741,900 for metal ions that can be conjugated to bispecific binding proteins for use as diagnostics according to the present disclosure.

[0307] The bispecific binding proteins can be used in methods for diagnosing disorders associated with CD137 and / or TAAs (e.g., disorders characterized by abnormal expression of CD137 and / or TAAs) or for determining whether a subject is at high risk for developing a disorder associated with CD137 and / or TAAs. Such methods include contacting a biological sample from a subject with a bispecific binding protein disclosed herein and detecting binding of the molecule to CD137 and / or TAAs. By "biological sample" is intended any biological sample obtained from an individual, cell line, tissue culture, or other source of cells potentially expressing CD137 and / or TAAs. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art.

[0308] In some embodiments, the method may further include comparing the levels of CD137 and / or TAA in the patient sample with a control sample (e.g., a subject not having a CD137 and / or TAA associated disorder) to determine whether the patient has a CD137 and / or TAA associated disorder or is at risk of developing a CD137 and / or TAA associated disorder.

[0309] In some embodiments, it is advantageous for diagnostic purposes to label the bispecific binding protein, for example, with a detectable moiety. Numerous detectable labels are available, including radioisotopes, fluorescent labels, enzyme substrate labels, and the like. The label may be indirectly conjugated to the bispecific binding protein using various known techniques. For example, the bispecific binding protein can be conjugated with biotin, and any of the three broad categories of labels mentioned above can be conjugated with avidin, or vice versa. Biotin selectively binds to avidin, and therefore the label can be conjugated to the bispecific binding protein in this indirect manner. Alternatively, to achieve indirect conjugation of the label to the bispecific binding protein, the bispecific binding protein can be conjugated with a small hapten (such as digoxin), and one of the different types of labels mentioned above is conjugated with an anti-hapten antibody (e.g., anti-digoxin antibody). Thus, indirect conjugation of the label to the bispecific binding protein can be achieved.

[0310] Exemplary radioisotope labels include: 35 S, 14 C. 125 I, 3 H, and 131Bispecific binding proteins can be labeled with radioisotopes, for example, using techniques described in Current Protocols in Immunology, Volumes 1 and 2, 1991, Coligen et al., Ed. Wiley-Interscience, New York, NY, Pubs. Radioactivity can be measured, for example, by scintillation counting.

[0311] Exemplary fluorescent labels include and are available from rare earth chelates (europium chelates) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, Lissamine, phycoerythrin, and Texas Red. Fluorescent labels can be conjugated to bispecific binding proteins via known techniques, such as those disclosed in Current Protocols in Immunology. Fluorescence can be quantified using a fluorometer.

[0312] There are a variety of well-characterized enzyme-substrate labels known in the art (see, e.g., U.S. Pat. No. 4,275,149). The enzyme generally catalyzes a chemical change in a chromogenic substrate that can be measured using a variety of techniques. For example, the change can be a color change in the substrate that can be measured spectrophotometrically. Alternatively, the enzyme can change the fluorescence or chemiluminescence of the substrate. Techniques for quantifying the change in fluorescence are described above. The chemiluminescent substrate becomes electronically excited by the chemical reaction and can then emit light that can be measured, for example, using a chemiluminometer, or donate energy to a fluorescent acceptor.

[0313] Examples of enzyme labels include luciferases, such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidases, such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. Techniques for conjugating enzymes to proteinaceous molecules are described, for example, in O'Sullivan et al., 1981, Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in Methods in Enzym. (J. Langone & H. Van Vunakis, eds.), Academic Press, NY, 73:147-166.

[0314] Examples of enzyme-substrate combinations include, for example, horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate, which oxidizes a dye precursor such as orthophenylenediamine (OPD) or 3,3,5,5-tetramethylbenzidine hydrochloride (TMB); alkaline phosphatase (AP) with paranitrophenyl phosphate as a chromogenic substrate; β-D-galactosidase (β-D-Gal) with a fluorogenic substrate such as p-nitrophenyl-β-D-galactosidase or β-D-Gal with a chromogenic substrate such as 4-methylumbelliferyl-β-D-galactosidase.

[0315] In another embodiment, the bispecific binding proteins disclosed herein are used unlabeled and are detected with a labeled antibody that binds to the bispecific binding protein.

[0316] The bispecific binding proteins described herein may be used in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays, see, e.g., Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

[0317] The bispecific binding proteins disclosed herein can be used to inhibit the binding of CD137 and / or TAAs to their respective receptors. Such methods include administering the bispecific binding proteins disclosed herein to a cell (e.g., a mammalian cell) or cellular environment, thereby inhibiting receptor-mediated signaling. These methods can be performed in vitro or in vivo. By "cellular environment" is intended the tissue, medium, or extracellular matrix surrounding the cell.

[0318] Therapeutic Compositions and Methods The present disclosure also provides compositions, including, for example, pharmaceutical compositions comprising the bispecific binding proteins disclosed herein. Such compositions have numerous therapeutic applications for the treatment, prevention, or amelioration of diseases or disorders, such as cancer.

[0319] Activation of CD137 in TME by TAA-CD137 antibody can enhance immune response to cancer cells in patients.Cancers that can be inhibited by the bispecific antibody of the present disclosure include cancers that typically respond to immunotherapy and cancers that typically do not respond to immunotherapy, including immune checkpoint resistant tumors.Cancer can be solid tumor or liquid tumor.

[0320] Non-limiting examples of cancers for treatment include bone cancer, skin cancer, uterine cancer, squamous cell carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), glioma, gastrointestinal cancer, renal cancer, ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, pancreatic cancer, cervical cancer, gastric cancer, bladder cancer, hepatoma, breast cancer, colon cancer, head and neck cancer, germ cell tumors, melanoma, testicular cancer, cancer of the fallopian tubes, cancer of the endometrium, cancer of the cervix, cancer of the vagina, cancer of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the ureter, cancer of the renal pelvis, primary C NS lymphoma, spinal axis tumor, brain cancer, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, all types of leukemia, lymphoma, and myeloma, such as acute leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myelogenous leukemia (CML), anaplastic AML (MO), myeloblastic leukemia (Ml), lymphomas, such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B-cell hematological malignancies, such as B-cell lymphoma, T-cell lymphoma, lymphoplasmacytoid lymphoma, monocytic B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g. Ki1+) Large cell lymphoma, adult T-cell lymphoma / leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, hemangiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T-lymphoblastic lymphoma / leukemia (T-Lbly / TALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, true histiocytic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, B-cell lymphoma, lymphoblastic lymphoma (LBL), hematopoietic tumors of lymphoid lineage , acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC), myeloma, such as IgG myeloma, light chain myeloma, non-secretory myeloma, smoldering myeloma (also called indolent myeloma), solitary plasmacytoma, and multiple myeloma, chronic lymphocytic leukemia (CLL), hairy cell lymphoma, and any combination of the above cancers.

[0321] The antibodies described herein may also be used to treat metastatic cancers, unresectable and / or refractory cancers (e.g., cancers that are refractory to previous immunotherapy), as well as recurrent cancers. In certain embodiments, TAA-CD137 Abs are administered to patients with cancers that have responded inadequately to previous treatments, such as previous treatments with immuno-oncology drugs, or to patients with refractory or resistant cancers that are either inherently refractory or resistant (e.g., refractory to PD-1 pathway antagonists), or to patients with cancers that have acquired a resistant or refractory state. For example, subjects that do not respond or do not respond adequately to a first therapy, or whose disease progresses after a treatment, such as an anti-PD-1 therapy, may be treated by administering TAA-CD137 antibodies alone or in combination with another therapy (e.g., anti-PD-1 therapy). In certain embodiments, TAA-CD137 antibodies are administered to patients who have not previously received (i.e., have been treated with) an immuno-oncology agent, such as a PD-1 pathway antagonist. TAA-CD137 antibody may be administered in conjunction with standard treatment.TAA-CD137 antibody may be administered as a maintenance therapy, for example, a therapy intended to prevent tumor development or recurrence.Anti-GITR antibody may be administered in conjunction with another therapy, for example, radiation, surgery, or chemotherapy.

[0322] In humans, some tumors, such as melanoma, have been shown to be immunogenic. Lowering the threshold for T cell activation through activation of CD137 can activate tumor responses in the host, allowing the treatment of non-immunogenic or tumors with limited immunogenicity.

[0323] In some embodiments, compositions are provided, including pharmaceutical compositions comprising bispecific binding proteins that bind CD137 and tumor-associated antigens, for use as therapeutic agents for treating patients with cancer. In a specific embodiment, the compositions described herein are administered to cancer patients to kill tumor cells. For example, the compositions described herein can be used to treat patients with solid tumors characterized by the presence of cancer cells that express or overexpress tumor-associated antigens. In some aspects, the disclosed compositions can be used to treat breast cancer, lung cancer, ovarian cancer, testicular cancer, pancreatic cancer, gastric cancer, gallbladder cancer, and urothelial cancer.

[0324] The present disclosure also provides methods for the treatment or prevention of cancer comprising administering to a subject in need thereof a composition or formulation comprising a bispecific binding protein disclosed herein and, optionally, another immune-based therapy.

[0325] The disclosed bispecific binding proteins are also useful in methods of treating cancer, either alone (eg, as monotherapy) or in combination with other immunotherapeutic agents and / or chemotherapy.

[0326] The bispecific binding proteins can be administered alone or in combination with other compositions useful for the treatment of immune-mediated inflammatory disorders or autoimmune diseases.

[0327] In some aspects, pharmaceutical compositions are provided, for example pharmaceutical compositions comprising one or more bispecific binding proteins disclosed herein. Pharmaceutical compositions can be formulated in combination with a pharma- ceutical acceptable carrier or diluent, as well as any other known adjuvants and excipients, according to conventional techniques, such as those disclosed in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 1995.

[0328] Typically, compositions for administration by injection are solutions in sterile isotonic aqueous buffer. Optionally, the pharmaceutical agent may also include a solubilizing agent and a local anesthetic, such as lignocaine, to reduce pain at the injection site. Generally, the ingredients are supplied separately or mixed together in unit dosage form, for example as a dry lyophilized powder or water-free concentrate in a sealed container, such as an ampoule or sachet, labeled with the amount of active agent. When the pharmaceutical agent is administered by injection, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. When the pharmaceutical agent is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0329] As used herein, "pharmaceutical acceptable carriers" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., the bispecific binding protein, may be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0330] The composition can be administered by various methods known in the art. As understood by those skilled in the art, the route and / or mode of administration will vary depending on the desired results. The bispecific binding protein can be prepared with a carrier that protects the compound from rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. The method of preparing such formulations is generally known to those skilled in the art. For example, see Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0331] Dosage levels of the bispecific binding protein in a pharmaceutical composition may be varied to obtain an amount of the bispecific binding protein effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration without being toxic to the subject. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular composition employed, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, overall health, and prior medical history of the patient being treated, and similar factors well known in the medical arts.

[0332] The pharmaceutical compositions described herein can be administered in an effective amount. "Effective amount" refers to an amount that achieves a desired reaction or a desired effect, either alone or together with additional doses. In the case of treating a specific disease or a specific condition, the desired reaction is preferably related to the inhibition of the course of the disease, which includes slowing the progression of the disease, and in particular, halting or reversing the progression of the disease.

[0333] In some aspects, the compositions described herein are administered to a patient, e.g., in vivo, to treat or prevent various disorders, such as those described herein. Suitable patients include human patients having a disorder that can be corrected or ameliorated by administering the bispecific binding proteins disclosed herein.

[0334] In some aspects, conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acid encoding bispecific binding proteins into mammalian cells or target tissues as described herein. Such methods can be used to administer nucleic acid encoding bispecific binding proteins to cells in vitro. In some embodiments, nucleic acid encoding bispecific binding proteins is administered for in vivo or ex vivo gene therapy use. In other embodiments, gene delivery techniques are used to study the activity of bispecific binding proteins in cell-based or animal models. Non-viral vector delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as liposomes. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to cells. Such methods are well known in the art.

[0335] Non-viral delivery methods of nucleic acid encoding bispecific binding proteins include lipofection, microinjection, gene gun, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and drug-enhanced uptake of DNA. Lipofection methods and lipofection reagents are well known in the art (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides include the lipids of Felgner, WO91 / 17424, WO91 / 16024. Delivery can be to cells (ex vivo administration) or target tissues (in vivo administration). Preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to those skilled in the art.

[0336] The use of RNA or DNA virus-based systems for delivery of nucleic acids encoding the bispecific binding proteins described herein takes advantage of the highly evolved process for targeting viruses to specific cells in the body and transporting the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo) or used to treat cells in vitro, and the modified cells are administered to patients (ex vivo). Conventional virus-based systems for delivery of the bispecific binding proteins of the present disclosure can include retroviral, lentiviral, adenoviral, adeno-associated viral, and herpes simplex viral vectors for gene transfer. Viral vectors are currently the most efficient and versatile method of gene transfer in target cells and tissues. Integration into the host genome is possible with retroviral, lentiviral, and adeno-associated viral gene transfer methods, often resulting in long-term expression of the inserted transgene. Furthermore, high transduction efficiency has been observed in many different cell types and target tissues.

[0337] In one method of treatment, the pharmaceutical composition comprising a bispecific binding protein that binds CD137 and a tumor-associated antigen may further comprise a therapeutic or toxic agent, conjugated or not, to the bispecific binding protein that binds CD137 and a tumor-associated antigen. In a specific embodiment, the bispecific binding protein that binds CD137 and a tumor-associated antigen is used to target ADCs with a cytotoxic payload to tumors that express and / or overexpress the tumor-associated antigen.

[0338] The broad scope of the present disclosure is best understood with reference to the following examples, which are not intended to limit the disclosure to the specific embodiments. The specific embodiments described herein are offered by way of example only, and the present disclosure is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. EXAMPLES

[0339] Common methods Methods for protein purification, including immunoprecipitation, chromatography, and electrophoresis, have been described. See, e.g., Coligan et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York. Chemical analysis, chemical modification, post-translational modification, production of fusion proteins, and glycosylation of proteins have been described. See, e.g., Coligan et al. (2000) Current Protocols in Protein Science, Vol. 2, John Wiley and Sons, Inc., New York; Ausubel et al. (2001) Current Protocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life Science Research, St. Louis, Mo.; pp. 45-89; Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, NJ, pp. 384-391. Production, purification, and fragmentation of polyclonal and monoclonal antibodies have been described. Coligan et al. (2001) Current Protocols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra.

[0340] Hybridoma or cell culture supernatants containing antibody proteins disclosed herein were purified via HiTrap Protein G columns (GE, Cat. No. 17040401) according to the manufacturer's procedure. Briefly, the supernatants were equilibrated with DPBS (Gibco, Cat. No. 14190-136) for 5 CV and loaded via a syringe / infusion pump (Legato 200, KD Scientific) at ambient temperature and 3 min residence time. The column was washed with 5 CV of DPBS and elution was performed with 4 CV of pH 2.8 elution buffer (Fisher Scientific, Cat. No. PI21004). The eluate was fractionated and the fractions were neutralized with 1M Tris-HCL, pH 8.5 (Fisher Scientific, Cat. No. 50-843-270) and assayed by A280 (Dropsensiti96, Trinean). Peak fractions were pooled and buffer exchanged into DPBS. Centrifugal filters (EMD Millipore, Cat. No. UFC803024) were equilibrated in DPBS at 4,000 x g for 2 minutes. Purified samples were loaded, DPBS was added, and samples were spun at 4,000 x g for 5-10 minutes until the total DPBS volume reached ≥6 DV. The final pool was analyzed by A280.

[0341] Standard methods in molecular biology are described. For example, see Maniatis et al. (1982) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, Calif. Standard methods are also described in Ausbel et al. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, NY, which describes cloning and DNA mutagenesis in bacterial cells (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4).

[0342] Stable cell lines expressing the target TSA / TAA disclosed herein were generated by transfecting pcDNA3.1-based plasmids expressing the TSA / TAA proteins disclosed herein into selected host cells (i.e., CHO-K1, HEK293T) using electroporation-based transfection. Geneticin was used to select integrated cells. After 7-10 days of Geneticin selection, stable clones were isolated by FACS. After expansion, stable clones were further confirmed by flow cytometry for expression of the TSA / TAA target.

[0343] An internal control anti-CD137 antibody based on the anti-CD137 antibody (Urelumab), referred to herein as "Urelumab-NR", was prepared based on the publicly available information published in U.S. Pat. No. 7,288,638 (VH of SEQ ID NO: 3 and VL of SEQ ID NO: 6 therein).

[0344] The sequences of the heavy and light chain variable regions of the hybridoma clones were determined as described below: 1–2 × 10 6 Total RNA was extracted from hybridoma cells. cDNA was generated by performing 5'RACE reaction using SMARTer RACE 5' / 3'Kit from Takara Bio Inc. (Mountain View, CA, USA). PCR was performed using Q5 High Fidelity DNA Polymerase from NEB Laboratories Inc. (Ipswich, MA, USA) to amplify variable regions from heavy and light chains using Takara Universal Primer Mix in combination with gene-specific primers for the 3' mouse constant region of the appropriate immunoglobulin. The amplified variable regions of heavy and light chains were run on a 2% agarose gel, the appropriate bands were excised, and then gel purified using Qiagen's Mini Elute Gel Extraction Kit. The purified PCR products were cloned using Invitrogen's Zero Blunt PCR Cloning Kit (Carlsbad, CA, USA), transformed into Takara Bio's Stellar Competent E.Coli Cells, and plated on LB agar + 50ug / ml kanamycin plates. Direct colony Sanger sequencing was performed by GeneWiz (South Plainfield, NJ, USA). The resulting nucleotide sequences were analyzed using IMGT V-QUEST to identify productive rearrangements and to analyze the translated protein sequences. CDR determination was based on Kabat numbering.

[0345] Recombinant monoclonal or bispecific binding proteins were expressed and purified as follows: the respective heavy or light chains were PCR amplified or synthesized and cloned into pcDNA3.4-based expression vectors carrying constant regions from human IgG1 (Uniprot P01857) or human kappa light chain (UniProt P01834) or human lambda light chain (UniProt P0DOY2). Expi293 cells (Thermo Fisher Scientific) were transfected with pairs of heavy and light chain expression plasmids according to the supplier's protocol for the Expi293 Expression System. Five days after transfection, culture supernatants were harvested by centrifugation. Antibodies were purified by one-step affinity purification using a protein A column and buffer exchanged into 20 mM sodium acetate pH 5.0 or PBS pH 7.2.

[0346] Methods for flow cytometry are available, including the Fluorescence Activated Cell Sorting Detection System (FACS®). See, for example, Owens et al. (1994) Flow Cytometry Principles for Clinical Laboratory Practice, John Wiley and Sons, Hoboken, NJ; Givan (2001) Flow Cytometry, 2nd ed.; Wiley-Liss, Hoboken, NJ; Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons, Hoboken, NJ. Fluorescent reagents suitable for modifying nucleic acids, including nucleic acid primers and probes, polypeptides, and antibodies, for use, for example, as diagnostic reagents, are available. Molecular Probes (2003) Catalogue, Molecular Probes, Inc., Eugene, Oreg.; Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.

[0347] Standard techniques are available for characterizing ligand / receptor interactions. See, e.g., Coligan et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York. Standard methods of antibody function characterization suitable for characterizing antibodies with a particular mechanism of action are also well known to those skilled in the art.

[0348] For example, software packages and databases are available for determining antigenic fragments, leader sequences, protein folding, functional domains, CDR annotations, glycosylation sites, and sequence alignments.

[0349] The reference sequences used herein are shown in Table 9. [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4]

[0350] Example 1: Generation of binding proteins that bind to CD137 and Claudin6, Claudin18.2, or Nectin-4 Fully human anti-human CD137, anti-human Claudin 6, and anti-human Claudin 18.2 antibodies were generated by immunizing Trianni mice, which are human Ig transgenic mice that express human antibody VH and VL genes (see, e.g., WO2013 / 063391, TRIANNI (registered trademark) mice).

[0351] The immunized TRIANNI mice described above were immunized by injecting either recombinant human protein, stable cell lines expressing the target protein, or DNA via intraperitoneal (IP), subcutaneous (SC), tail base, or footpad injection.

[0352] Mouse anti-Nectin-4 antibodies were generated by immunizing Balb / c mice with recombinant human Nectin-4 protein by injection either intraperitoneally (IP), subcutaneously (SC), or at the base of the tail or footpad.

[0353] Immune responses were monitored by retro-orbital bleeding. Plasma was screened by ELISA, flow cytometry (FACS), or imaging (as described below). Mice with sufficient anti-CD137, anti-Claudin6, anti-Claudin18.2, or anti-Nectin-4 titers were used for fusions. Mice were boosted intraperitoneally, at the base of the tail, in the footpad, or intravenously with immunogens before being sacrificed and the spleens and lymph nodes removed.

[0354] To select mice producing antibodies that bind to CD137, Claudin6, Claudin18.2, or Nectin-4, sera from immunized mice were screened by ELISA, FACS, or imaging for binding to human CD137, Claudin6, Claudin18.2, or Nectin-4 protein, respectively.

[0355] For ELISA, briefly, recombinant human CD137 or Nectin-4 coated ELISA plates were incubated with dilutions of serum from immune mice for 1 h at room temperature, and the assay plates were washed. Specific antibody binding was detected by incubation with HRP-conjugated anti-mouse IgG antibody (Jackson ImmunoResearch, Cat. No. 115-036-071) for 1 h at room temperature, washing, and then incubation with ABTS substrate (Moss, Cat. No. ABTS-1000) for 30 min at room temperature. Plates were read using an ELISA plate reader (Biotech).

[0356] For FACS, briefly, HEK293T or CHO-K1 cells expressing CD137, Claudin6, Claudin18.2, or Nectin-4, or parental HEK293T or CHO-K1 cells were incubated with dilutions of serum from immune mice at 4°C for 2 hours. Cells were fixed with 2% PFA (Alfa Aesar, Cat. No. J61899) for 15 minutes at 4°C and then washed. After one hour of incubation at 4°C, specific antibody binding was detected with Alexa 647-labeled goat anti-mouse IgG antibody (Thermo Fisher Scientific, Cat. No. A21235). Flow cytometry analysis was performed on a flow cytometry instrument (Intellicyte IQue plus, Sartorius).

[0357] Additionally, mouse sera were tested by imaging. Briefly, HEK293T cells or CHO-K1 cells expressing CD137, Claudin6, Claudin18.2, or Nectin-4 were incubated with dilutions of sera from immune mice. Cells were washed, fixed with paraformaldehyde, washed, and specific antibody binding was detected with secondary Alexa488 goat anti-mouse antibody and Hoechst (Invitrogen). Plates were scanned and analyzed on an imaging device (Cytation 5, Biotek).

[0358] To generate hybridomas producing human antibodies against CD137, Claudin6, Claudin18.2, or Nectin-4, splenocytes and lymph node cells were isolated from immunized mice and fused to appropriate immortalized cell lines, such as mouse myeloma cell lines. The resulting hybridomas were screened for production of antigen-specific antibodies. For example, single cell suspensions of splenocytes, lymph node cells from immunized mice were fused to an equal number of mouse IgG non-secreting myeloma cells Sp2 / 0 (ATCC, CRL 1581) by electrofusion. Cells were seeded into flat-bottom 96-well tissue culture plates, followed by approximately one week of incubation in selection medium (HAT medium) and then replaced with hybridoma culture medium. Approximately 10-14 days after cell seeding, supernatants from individual wells were screened by ELISA, imaging, or FACS as described above. Antibody-secreting hybridomas were transferred to 24-well plates and screened again, and if anti-CD137, anti-Claudin6, anti-Claudin18.2, or anti-Nectin-4 were still positive, the positive hybridomas were subcloned by sorting using a single cell sorter. The subclones were screened again by ELISA, imaging, or FACS as described above. Stable subclones were then cultured in vitro to generate small amounts of antibody for purification and characterization.

[0359] A mouse anti-Nectin-4 antibody was humanized by CDR grafting. Briefly, the VH and VL of 1925Ab4 were used as queries to search human antibody germline sequences for the most similar human framework regions, respectively. The mouse CDRs (based on Kabat numbering) were grafted into the identified human antibody frameworks. Multiple pairs of humanized VH and VL variants were expressed and purified, and one pair (1925Ab4 VH(Hz) and 1925Ab4 VL(Hz)) with the highest binding affinity to human Nectin-4 was used to construct a bispecific antibody.

[0360] Example 2: Molecular design and production of TAA / CD137 bispecifics (BsAb_A) As a representative example of a binding protein that binds to a TAA, we prepared a symmetric bispecific (Claudin 6 x CD137) BsAb_A characterized by the molecular format shown in Figure 2, containing the subunits / components summarized in Figures 3 and 4. 1912Ab3 1. Heavy chain SEQ ID NO: 12, which comprises the components anti-Claudin6 antibody heavy chain, linker, and anti-CD137 scFv (VH-VL with CC) (N→C), and 2. Light chain SEQ ID NO: 9 containing the anti-Claudin6 antibody light chain

[0361] DNA segment 1 having a polynucleotide sequence (sequence number 12) encoding the heavy chain component of 1912Ab3 was inserted into an expression vector, and DNA segment 2 having a polynucleotide sequence (sequence number 9) encoding the light chain of 1912Ab3 was inserted into the above expression vector. 1912Ab5 1. Heavy chain: SEQ ID NO: 72, which comprises the components of the heavy chain of an anti-Claudin6 antibody, a linker, and an anti-CD137 scFv (VH-VL with CC) (N→C), and 2. Light chain: SEQ ID NO: 9 comprising the anti-Claudin6 antibody light chain.

[0362] In an alternative example, DNA segment 1 having a polynucleotide sequence (sequence number 72) encoding the heavy chain component of 1912Ab5 was inserted into an expression vector, and DNA segment 2 having a polynucleotide sequence (sequence number 9) encoding the light chain of 1912Ab5 was inserted into the above expression vector.

[0363] The constructed expression vectors were transiently expressed in Expi293 cells (Scientific) and cultured in Expi293 expression medium for 5 days at 37°C in a CO2 incubator. The bispecific antibodies were purified from cell culture supernatants by recombinant Protein A affinity chromatography (Hitrap Mabselect SuRe, GE Healthcare) and, if necessary, by a second step of purification by ion exchange or gel filtration chromatography. SDS-PAGE (BiRad), size exclusion HPLC (Agilent Technologies, 1100 series) analysis using a SE-HPLC column (Tosoh, G3000SWXL), and CE-SDS (SCIEX, PA800 Plus) were performed to detect and confirm the size and purity of the bispecific antibodies. The purified protein was buffer exchanged into the desired buffer, concentrated by ultrafiltration using an Amicon Ultra 15 30K device, and protein concentration was estimated using DropSense (Unchained Labs). Transient transfection could be used in a two-vector system or with a one-vector system containing both heavy and light chain components in one single vector. Alternatively, bispecific antibodies could be purified from the supernatant of a CHO stable cell line.

[0364] Example 3: Molecular design and production of TAA / CD137 bispecifics (BsAb_B) As a representative example of a binding protein that binds to a TAA, we prepared a symmetric bispecific (Claudin 18.2 x CD137) BsAb_B characterized by the molecular format shown in Figure 2, containing the subunits / components summarized in Figures 3 and 4. 1901Ab3 1. Heavy chain SEQ ID NO: 4, which contains the heavy chain of the anti-Claudin18.2 antibody; and 2. Light chainSEQ ID NO: 5 comprising the light chain of the anti-Claudin 18.2 antibody, a linker, and an anti-CD137 scFv (VH-VL with CC) (N→C)

[0365] DNA segment 1 having a polynucleotide sequence (sequence number 4) encoding the heavy chain component of 1901Ab3 was inserted into an expression vector, and DNA segment 2 having a polynucleotide sequence (sequence number 5) encoding the light chain of 1901Ab3 was inserted into the above expression vector.

[0366] The constructed expression vectors were transiently expressed in Expi293 cells (Scientific) and cultured in Expi293 expression medium for 5 days at 37°C in a CO2 incubator. The bispecific antibodies were purified from cell culture supernatants by recombinant Protein A affinity chromatography (Hitrap Mabselect SuRe, GE Healthcare) and, if necessary, by a second step of purification by ion exchange or gel filtration chromatography. SDS-PAGE (BiRad), size exclusion HPLC (Agilent Technologies, 1100 series) analysis using a SE-HPLC column (Tosoh, G3000SWXL), and CE-SDS (SCIEX, PA800 Plus) were performed to detect and confirm the size and purity of the bispecific antibodies. The purified protein was buffer exchanged into the desired buffer, concentrated by ultrafiltration using an Amicon Ultra 15 30K device, and protein concentration was estimated using DropSense (Unchained Labs). Transient transfection could be used in a two-vector system or with a one-vector system containing both heavy and light chain components in one single vector. Alternatively, bispecific antibodies could be purified from the supernatant of a CHO stable cell line.

[0367] Example 4: Molecular design and production of TAA / CD137 bispecifics (BsAb_C) As a representative example of a binding protein that binds to a TAA, we prepared a symmetric bispecific (Nectin-4 x CD137) BsAb_C characterized by the molecular format shown in Figure 2, containing the subunits / components summarized in Figures 3 and 4. 1925Ab3 1. Heavy chain SEQ ID NO: 18, which comprises the components anti-CD137 scFv (VH-VL with CC) (N→C), a linker, and the heavy chain of a humanized anti-Nectin-4 antibody; and 2. Light chain SEQ ID NO: 15 containing the humanized Nectin-4 antibody light chain

[0368] DNA segment 1 having a polynucleotide sequence (sequence number 18) encoding the heavy chain component of 1925Ab3 was inserted into an expression vector, and DNA segment 2 having a polynucleotide sequence (sequence number 15) encoding the light chain of 1925Ab3 was inserted into the above expression vector.

[0369] The constructed expression vectors were transiently expressed in Expi293 cells (Scientific) and cultured in Expi293 expression medium for 5 days at 37°C in a CO2 incubator. The bispecific antibodies were purified from cell culture supernatants by recombinant Protein A affinity chromatography (Hitrap Mabselect SuRe, GE Healthcare) and, if necessary, by a second step of purification by ion exchange or gel filtration chromatography. SDS-PAGE (BiRad), size exclusion HPLC (Agilent Technologies, 1100 series) analysis using a SE-HPLC column (Tosoh, G3000SWXL), and CE-SDS (SCIEX, PA800 Plus) were performed to detect and confirm the size and purity of the bispecific antibodies. The purified protein was buffer exchanged into the desired buffer, concentrated by ultrafiltration using an Amicon Ultra 15 30K device, and protein concentration was estimated using DropSense (Unchained Labs). Transient transfection could be used in a two-vector system or with a one-vector system containing both heavy and light chain components in one single vector. Alternatively, bispecific antibodies could be purified from the supernatant of a CHO stable cell line.

[0370] Example 5: Binding of CLDN6 / CD137 BsAb to Claudin6 on the cell surface The bispecific CLDN6 / CD137 binding protein was generated, produced, and purified as described in Example 4. To examine the binding activity of BsAbs 1912Ab3 and 1912Ab4 to Claudin6, immunofluorescence binding assays were performed using NEC8 WT cells or Claudin6 KO NEC8 cells expressing endogenous human Claudin6 on the cell surface. Claudin6 KO NEC8 cells were generated by CRISPR gene editing technology. These cells were cultured in RPMI containing 10% FBS. On the day of the experiment, the cells were harvested, washed, and stained with BsAbs 1912Ab3 and 1912Ab4, and mAbs 1912Ab1 and 1912Ab2 at 4°C for 2 hours, and then fixed at room temperature for 15 minutes. The fixed cells were washed three times with PBS and then stained with Alexa Fluor® 488 goat anti-human IgG antibody (Invitrogen, Cat. No. A-11013) for detection at room temperature for 1 h. The binding signal was evaluated by quantifying the fluorescence intensity using iQue Screener PLUS (Sartorius, MI).

[0371] As shown in FIG. 6A, at a concentration of 10 μg / ml, the disclosed bispecific binding proteins including 1912Ab3 and 1912Ab4 bound similarly to human Claudin6 on the cell surface of NEC8 cells compared to the monospecific control antibodies 1912Ab1 and 1912Ab2. No binding was detected when the Claudin 6 gene was deleted by CRISPR gene editing technology. This result confirmed that the binding of the bispecific binding proteins on NEC8 WT cells was specific through Claudin6 expressed on the cell surface of NEC8 cells. The concentration-dependent binding curve of 1912Ab5 to Claudin6 is shown in FIG. 6B. 1912Ab5 has a binding EC of 1.5 nM. 50 Binds to NEC8 cells at 100 ng / ml.

[0372] Given the high homology between Claudin6 and Claudin9, and the expression profile of Claudin9 in normal cells, a highly selective Claudin6 antibody is desirable for treating cancer and minimizing safety issues. To evaluate the binding selectivity of Claudin6 over Claudin9, two CHO cell lines overexpressing either Claudin6 or Claudin9 were used in an image-based cell binding assay. As shown in Figure 6C, 1912Ab5 binds to CHO-Claudin6 cells but not to CHO-Claudin9 cells.

[0373] Example 6: Binding of CLDN6 / CD137 BsAb to CD137 The binding of Claudin6-CD137 BsAb to CD137 was measured by SPR assay and immunofluorescence imaging assay. As shown in Figure 7A, 1912Ab5 has the desired fast on and fast off kinetics when binding to human CD137. From three experiments, 1912Ab5 had an average ka value of 1.33E+06 (1 / Ms) and an average kd value of 4.62E-02 (1 / s). The average KD was 3.46E+08M. Intermittent binding may reduce the risk of overstimulating T cells and causing T cell exhaustion.

[0374] HEK293T cells stably transfected with human CD137 expression constructs were used in cell-based binding assays to evaluate CD137 binding affinity. Cells were seeded in complete medium containing DMEM with 10% FBS and then incubated overnight at 37°C. After staining cells with test antibodies for 2 hours at 4°C, cells were fixed for 15 minutes at room temperature. Fixed cells were washed three times with PBS and then stained with Alexa Fluor® 488 goat anti-human IgG (H+L) secondary antibody (Invitrogen, Cat. No. A-11013) for 1 hour at room temperature for detection. Binding signals were evaluated by imaging cells and quantifying fluorescence intensity using a Cytation Imager (Biotech, VT).

[0375] The results shown in Figure 7B indicate that the bispecific antibodies 1912Ab3 and 1912Ab4, as well as the monospecific control antibody 1923Ab4, bound to human CD137 at similar levels at a concentration of 10 μg / ml. The concentration-dependent binding curve of 1912Ab5 is shown in Figure 7C. 1912Ab5 had a binding EC 50 EC values ​​for binding to HEK293-Claudin6 cells and the benchmark control, urelumab-NR 50 was 0.22 nM.

[0376] Example 7: Claudin6-dependent activation of CD137 signaling CLDN6 / CD137 BsAbs were evaluated for their ability to induce Claudin6-dependent CD137 agonism. Briefly, a Jurkat T reporter cell line stably expressing CD137 and containing an NFkB-Luc reporter was used to quantify CD137 signaling, and NEC8 WT cells expressing endogenous Claudin6 on the cell surface were used as target cells to provide Claudin6. Claudin6 KO NEC8 cells were used as a negative control to demonstrate Claudin6 dependency. The disclosed antibodies 1912Ab3 and 1912Ab4 are bispecific antibodies that bind both Claudin6 and CD137. The monospecific antibody Urelumab-NR binds only to CD137 and is used as a control antibody. Jurkat T reporter cells were co-cultured with either NEC8 WT or Claudin6 KO cells and stimulated with the disclosed binding proteins at 37°C, 5% CO2 for 16 hours. ONE-Glo™ Luciferase Reagent (Promega, Catalog No. E6130) was added and the plate was incubated at room temperature for 10 minutes. Luminescence signals were measured by Synergy Neo2 plate reader (Biotech) and data were analyzed by GraphPad Prism. Figure 8A shows that only Urelumab-NR activated CD137 signaling in both NEC8 WT and Claudin6 KO target cells. 1912Ab3 and 1912Ab4 induced stronger CD137 signaling than Urelumab-NR in the presence of NEC8 WT cells. Only background activity was detected in Claudin6 knockout NEC8 cells.

[0377] The dose-response curves of 1912Ab3, 1912Ab4, and urelumab-NR to induce CD137 signaling in the presence of NEC8 WT cells are shown in Figure 8B. The EC 50 The values ​​(potency) were 0.20 nM, 0.18 nM, and 0.31 nM, respectively. Both 1912Ab3 and 1912Ab4 showed better efficacy (higher Emax) than urelumab-NR.

[0378] Similarly, 1912Ab5 and Urelumab-NR were evaluated in a CD137 signaling assay using either NEC8 cells (Figure 8C) or OV90 cells (Figure 8D). As shown in the figures, 1912Ab5 had ECs of 0.066 nM and 0.064 nM, respectively. 50 The control antibody, urelumab-NR, induced dose-dependent CD137 signaling with EC values ​​(potency) of 0.28 nM and 0.62 nM, respectively. 50 1912Ab5 showed stronger agonism in CD137 signaling in T cells than urelumab-NR.

[0379] Example 8: Claudin6-dependent activation of CD8 T cells Co-culture experiments were used to measure T cell activation by Claudin6-CD137 BsAb. Healthy donor-derived CD8 T cells and NEC8 cells were used as effector and target cells, respectively. These two cells were co-cultured in RPMI1640 medium supplemented with 10% FBS and 0.5ug / ml mouse anti-hCD3 clone OKT3 (Biolegend, Cat. No. 317325). The disclosed binding proteins were added to stimulate the T cells. The disclosed antibodies 1912Ab3 and 1912Ab4 are bispecific antibodies that bind both Claudin6 and CD137. The monospecific antibody Urelumab-NR binds only to CD137 and is used as a control antibody. The plates were incubated at 37°C with 5% CO2 for 3 days. After 72 hours of incubation, supernatants were collected and used to measure secreted IFNγ by AlphaLISA (PerkinElmer, Cat. No. AL217C / F) using the manufacturer's suggested protocol. The amount of IFNγ represents T cell activation.

[0380] Figure 9A shows that Urelumab-NR stimulates T cell activation independent of Claudin6 expression. Similar levels of IFNγ were detected when CD8 T cells were co-cultured with NEC8 WT or Claudin6 KO NEC8 cells. However, 1912Ab3 and 1912Ab4 stimulate CD8 T cell activation only in the presence of NEC8 WT cells, but not in the presence of Claudin6 KO NEC8 cells. This result confirms the Claudin6-dependent T cell activation activity of the disclosed bispecific binding proteins.

[0381] The dose-response curves of 1912Ab3, 1912Ab4, and urelumab-NR to induce CD8 T cell activation in the presence of NEC8 WT cells are shown in Figure 9B. The EC 50 The values ​​(potency) were 0.042 nM, 0.15 nM, and 0.9 nM, respectively. Both 1912Ab3 and 1912Ab4 demonstrated greater potency and efficacy (higher Emax) for inducing IFNγ production, a hallmark of T cell activation, than urelumab-NR.

[0382] Similarly, 1912Ab5 and Urelumab-NR were evaluated in a T cell activation assay using either NEC8 cells (Figure 9C) or Claudin6 KO NEC8 cells (Figure 9D). As shown in the figure, 1912Ab5 had an EC of 0.17 nM only in the presence of NEC8 wild-type cells. 50 In contrast, the control antibody, urelumab-NR, showed activity in both NEC8 wild-type and Claudin6 KO NEC8 cells, with EC 50 The values ​​were 0.82 nM and 0.99 nM, respectively, indicating that its activity was independent of Claudin6 expression. 1912Ab5 showed higher agonism levels than urelumab-NR in T cell activation assays only when the target tumor antigen was available.

[0383] Example 9: Tumor-killing activity of CD8 T cells induced by BsAb CLDN6 / CD137 Co-culture experiments were performed to evaluate the T cell derived tumor killing activity (TDCC) mediated by BsAbs 1912Ab3 and 1912Ab4. Briefly, CD8 T cells from healthy donors were pre-activated with ImmunoCult™ human CD3 / CD28 T cell activator (StemCell, Inc., Cat. No. 10971) for 2 days. The activated cells were washed to remove the CD3 / CD28 activator. The activated CD8 T cells were then co-cultured with NEC8 tumor cells stably transfected with a GFP expression construct and treated with the disclosed bispecific binding proteins for 108 hours. The disclosed antibodies 1912Ab3 and 1912Ab4 are bispecific antibodies that bind both Claudin6 and CD137. Cell numbers were determined by the area of ​​green fluorescent cells measured using Cytation (Biotech, VT). The percentage of killing was calculated by the following formula: % killing = (area of ​​GFP cells from wells without binding protein treatment - area of ​​GFP cells from wells treated with binding protein) / area of ​​GFP cells from wells without binding protein treatment * 100%

[0384] Figure 10A shows that 1912Ab3 and 1912Ab4 induced potent T cell-mediated cytotoxicity. Approximately 80% of tumor cells were killed by CD8 T cells upon treatment with BsAb for 108 hours. The EC 50 The values ​​were 0.11 nM and 0.16 nM, respectively.

[0385] Similar co-culture experiments were performed to evaluate the TDCC effect of BsAb 1912Ab5. Briefly, CD8 T cells from healthy donors were co-cultured with ovarian cancer cell line OV90 cells stably transfected with GFP in the presence of mouse anti-hCD3 clone OKT3 (Biolegend, Cat. No. 317325). Co-cultured cells were treated with BsAb 1912Ab5 or control for 144 hours. The disclosed antibody 1912Ab5 is a bispecific antibody that binds both Claudin6 and CD137. Viable cell counts were measured using Cytation (Biotech, VT). Percentage killing was calculated by the following formula: % killing = (area of ​​GFP cells from wells without binding protein treatment - area of ​​GFP cells from wells treated with binding protein) / area of ​​GFP cells from wells without binding protein treatment * 100%

[0386] Figure 10B shows that 1912Ab5 induced potent T cell-mediated cytotoxicity. Approximately 70% of tumor cells were killed by CD8 T cells upon 144 h treatment with BsAb. The EC 50 The value was 0.036 nM.

[0387] Example 10: Effect of CLDN6 / CD137 BsAb on tumor growth in a subcutaneous syngeneic MC38-hClaudin 6 mouse tumor model in humanized B-h4-1BB mice Female B-h4-1BB mice (Biocytogen), 6-8 weeks old and weighing 16-20 g, were acclimated for 7 days prior to study enrollment. The MC38 mouse colon cancer cell line was genetically modified to overexpress human Claudin6. Cells were maintained in vitro as monolayer cultures in DMEM supplemented with 10% heat-inactivated FBS at 37°C in a 5% CO atmosphere. Cells were harvested and cultured at 5 × 10 in 100 μl of PBS. 5 Cells were implanted subcutaneously into the right front flank for tumor development. On day 7, tumor-bearing mice were cultured at approximately 100–150 mm 3Mice were randomly enrolled into three study groups with a mean tumor size of 100 mm. Each group consisted of six mice. Tumor size was measured twice weekly in two dimensions using calipers, and volume was calculated using the following formula: 3 It is expressed as V=0.5a×b 2 where a and b are the long and short dimensions of the tumor, respectively. On days 7, 11, 14, and 18, mice were treated with 5 mg / kg of 1912Ab3, 1912Ab4, or PBS as a negative control by intraperitoneal injection. The study was terminated on day 28.

[0388] Figure 11A shows the tumor growth curves for the three treatment groups. Both 1912Ab3 and 1912Ab4 significantly inhibited tumor growth compared to the vehicle control. All mice injected with 1912Ab3 and 5 of 6 mice injected with 1912Ab4 showed complete tumor remission at day 28.

[0389] Hepatotoxicity is monitored by measuring the activity of ALT and AST in mouse serum from serum samples on day 21. As shown in Figure 11B, ALT level does not increase significantly between treated and control groups. Similarly, as shown in Figure 11C, AST level does not increase significantly from treated groups, indicating low risk of antibody-induced hepatotoxicity.

[0390] To evaluate whether bsAb Claudin6 / CD137 can induce tumor immunity in mice with complete tumor remission. Four mice were pre-treated with 1912Ab3, and four mice treated with 1912Ab were re-challenged with MC38-Claudin6 tumors 45 days after the last dose of pre-treatment. Four naive mice were used as a control group in this study. As shown in Figure 11D, all naive mice developed tumors, but none of the pre-treated mice with complete tumor remission developed tumors after re-challenge.

[0391] To find the effective dose of antibody 1912Ab5, a dose titration study was performed. Female B-h4-1BB mice (Biocytogen, Boston, MA) were administered 5 × 10 5 Viable MC38 cells were inoculated subcutaneously. The tumor size was approximately 100 mm 3 When the mice reached 1912Ab5 antibody titers, they were randomized into three groups and treatment was initiated by intraperitoneal injection: Group 1 received vehicle control, Group 2 received 0.3 mpk of 1912Ab5 antibody, Group 3 received 1 mpk of 1912Ab5 antibody, and Group 4 received 3 mpk of 1912Ab5 antibody. Treatment was administered twice weekly for two weeks.

[0392] As shown in Figure 12, single agent 1912Ab5 demonstrated strong efficacy: at a dose of 0.3mpk, 1912Ab5 exhibited 97.7% tumor growth inhibition (TGI) at 32 days after tumor inoculation, 1912Ab5 at 1mpk exhibited 106.2% tumor growth inhibition (TGI) at 32 days after tumor inoculation, and 1912Ab5 at 3mpk exhibited 106.4% tumor growth inhibition (TGI) at 32 days after tumor inoculation.

[0393] A follow-up study was performed to compare the efficacy of Ab 1912Ab5 with the benchmark CD137 Ab, urelumab-NR. Female B-h4-1BB mice (Biocytogen, Boston, MA) were injected with 5 × 10 5 Viable MC38 cells were inoculated subcutaneously. The tumor size was approximately 100 mm 3 When the mice reached 100% CI, they were randomized into three groups and treatment was initiated by intraperitoneal injection: Group 1 received vehicle control, Group 2 received 0.1 mpk of 1912Ab5 antibody, and Group 3 received 0.1 mpk of urelumab-NR. Treatment was administered twice weekly for two weeks.

[0394] As shown in Figure 13, single agent 1912Ab5 demonstrated superior efficacy compared to the benchmark antibody Urelumab-NR. At a dose of 0.1 mpk, 1912Ab5 exhibited 77.2% tumor growth inhibition (TGI) at day 27 post-tumor inoculation, whereas Urelumab-NR at 0.1 mpk exhibited only 36.6% tumor growth inhibition (TGI).

[0395] To determine whether the Claudin6 / CD137 bispecific Ab could be used to treat large established tumors, 5 × 10 5 Viable MC38 cells were inoculated subcutaneously. The tumor size was approximately 400 mm 3 When tumor growth rate reached 100%, mice were randomized into two groups and treated twice weekly for one week. Group 1 received vehicle control and group 2 received two doses of 2mpk 1912Ab5 antibody. As shown in Figure 14, 1912Ab5 showed strong efficacy, i.e., 62.7% tumor growth inhibition (TGI), 35 days after tumor inoculation.

[0396] Example 11: Immune composition analysis of tumors treated with Claudin6-CD137 antibody Multiplex fluorescent immunohistochemistry (IHC) testing and tumor infiltrating lymphocyte (TIL) analysis were performed to evaluate immune cell content in tumors after Claudin6 / CD137 bsAb treatment.

[0397] Female B-h4-1BB mice (Biocytogen, Boston, MA) were treated with 5 × 10 5 Viable MC38 cells were inoculated subcutaneously. The tumor size was approximately 100 mm 3 When tumor tissue size reached 100%, mice were randomized into two groups, 8 mice per group, and treated twice on days 15 and 19. Group 1 was treated with vehicle control and group 2 was treated with 1mpk 1912Ab5. On day 26 after tumor inoculation, mice were euthanized and fresh tumors were harvested for IHC and Til testing.

[0398] Two tumors from each treatment group were formalin fixed and paraffin embedded. Fluorescent IHC was performed on 5 mm FFPE tissue sections. After deparaffinization, slides were stained with primary antibodies detecting CD45, CD3, CD4, and CD8 for multiplex immune cell profiling. Representative images are shown in Figure 15. 1912Ab5-treated tumors had significantly increased lymphocyte infiltration, as well as CD4 and CD8 T cell infiltration (Figure 15B) compared to vehicle controls (Figure 15A).

[0399] Tumor-infiltrating lymphocyte analysis was performed using six fresh tumors from each treatment group. An enzyme-based method was used to dissociate tumors. Cells from digested tumors were filtered, washed, and used for multiplex flow cytometry. T cell populations were gated by active CD45+CD3+, CD4 T cells were gated by active CD45+CD3+CD4+CD8-, CD8 T cells were gated by active CD45+CD3+CD4-CD8+, exhausted T cells were gated by active CD45+CD3+Tim-3+, central memory T cells were gated by CD45+CD3+CD8+highCD44highCD62L, resident memory T cells were gated by active CD45+CD3+CD8+CD69+CD103+, and M2-like macrophage cells were gated by active CD45+CD11b+F4 / 80++CD206+.

[0400] As shown in Figure 16, both CD4 (Figure 16A) and CD8 (Figure 16B) cells were increased in all 1912Ab5-treated tumors, suggesting enhanced T cell-derived tumor killing. Furthermore, memory T cells, central memory T cells (Figure 16C) and resident memory T cells (Figure 16D), were significantly increased along with the tumor immunity observed in 1912Ab5-treated animals (Figure 11D), suggesting that treatment with Claudin6-CD137 bsAb may promote the formation of anti-tumor memory. Furthermore, the reduction of exhausted T cells (Figure 16E) and reduction of M2-like macrophages (Figure 16F) indicate the tumor microenvironment modulating effect of Claudin6-CD137 bsAb.

[0401] Based on the data disclosed herein, it is expected that the antitumor effect of CLDN6 / CD137 BsAb treatment leads to modulation of the TME, with increased lymphocyte infiltration converting the suppressive TME into an inflammatory TME. Tumor cell-dependent CD137 activation specifically enhances T cell activation through tumors and promotes T memory formation, and the fast off kinetics of bsAb may help reduce T cell exhaustion. Reduced M2 macrophages reduce inhibitory cytokine release in the TME, thus improving T cell activation.

[0402] Example 12: Evaluation of Claudin6-CD137 antibodies in the PD1-resistant model B16F10 To predict the therapeutic potential of Claudin6 / CD137 antibody treatment in PD1-resistant patients, we evaluated the efficacy of antibody 1912Ab5 using the B16F10 melanoma model, a PD1-resistant tumor model. Six- to seven-week-old female homozygous B-h4-1BB mice (Biocytogen, Boston, MA) were injected with 1 × 10 5 Viable B16-F10 cells were injected subcutaneously into the right flank. 3When the mice reached 100% CI, they were randomized into two groups and treatment was initiated by intraperitoneal injection. Group 1 received the vehicle control and group 2 received the 3mpk 1912Ab5 antibody. Treatment was administered twice weekly for two weeks.

[0403] Body weight was measured twice a week. Tumor volumes were determined at different time points using the formula V=1 / 2×L×W×W, where L is the long dimension and W is the short dimension of the xenograft. 3 Any mice with tumors exceeding 100 were sacrificed. Mice survival was monitored up to 27 days after tumor implantation.

[0404] As shown in FIG. 17, mice from the 3mpk 1912Ab5 antibody treatment group had a TGI value of 67.1% 20 days after tumor implantation, indicating significant efficacy.

[0405] Example 13: CLDN18.2 / CD137 to Claudin18.2 on the cell surface BsAb binding Bispecific CLDN18.2 / CD137 antibodies were generated, produced, and purified as described in Example 3. To examine the binding activity of these binding proteins to Claudin18.2, NUGC4 cells that endogenously express human Claudin18.2 were used in immunofluorescence binding assays. Cells were cultured in RPMI medium containing 10% FBS. On the day of the experiment, cells were harvested, washed, and stained with BsAbs 1901Ab2 and 1901Ab3, as well as monospecific anti-CLDN18.2 control antibody 1901Ab1, for 2 hours at 4°C, followed by fixing the cells with paraformaldehyde for 15 minutes at room temperature. Monoclonal antibody 1901Ab1 specifically binds to Claudin18.2. The fixed cells were then washed three times with PBS, after which the cells were stained with Alexa Fluor® 488 goat anti-human IgG antibody (Invitrogen, Cat. No. A-11013) for 1 hour at room temperature for detection. Binding signals were assessed by quantifying fluorescence intensity using iQue Screener PLUS (Sartorius, MI).

[0406] As shown in FIG. 18, at a concentration of 10 μg / ml, the disclosed bispecific binding proteins including 1901Ab2 and 1901Ab3 bound similarly to human Claudin 18.2 on NUGC4 cells compared to the control monoclonal antibody 1901Ab1.

[0407] Example 14: Binding of CLDN18 / CD137 BsAb to cell surface CD137 The binding affinity of CLDN18.2 / CD137 BsAbs 1901Ab2 and 1901Ab3 was evaluated using an immunofluorescence imaging assay. Briefly, HEK293T-huCD137 cells stably expressing human CD137 were seeded in complete medium containing DMEM with 10% FBS and then incubated overnight at 37°C. After binding of the cells with the BsAbs for 2 hours at 4°C, the cells were fixed in paraformaldehyde for 15 minutes at room temperature. The monospecific antibody 1923Ab4 binds only to CD137 and is used as a control antibody. The fixed cells were washed three times with PBS and then stained with Alexa Fluor® 488 goat anti-human IgG antibody (Invitrogen, Cat. No. A-11013) for 1 hour at room temperature for detection. The binding signal was evaluated by imaging the cells and quantifying the fluorescence intensity using a Cytation Imager (Biotech, VT).

[0408] As shown in FIG. 19, at a concentration of 10 μg / ml, the disclosed bispecific binding proteins 1901Ab2 and 1901Ab3 bound to cell surface human CD137 similarly to their monoclonal antibody control 1923Ab4.

[0409] Example 15: Target cell-dependent activation of CD137 signaling by CLDN18.2 / CD137 BsAb Anti-CLDN18.2 / CD137 BsAbs 1901Ab2 and 1901Ab3 were also evaluated for their ability to induce target cell-dependent CD137 agonism. Briefly, a Jurkat T reporter cell line stably expressing CD137 and containing an NFkB-luc reporter was used to quantify CD137 signaling. NUGC4 cells, which expressed endogenous Claudin18.2 on the cell surface, were used as target cells. Jurkat T reporter cells were co-cultured with or without NUGC4 target cells and stimulated with the disclosed binding proteins for 16 hours at 37°C and 5% CO2. ONE-Glo™ Luciferase Reagent (Promega, Cat. No. E6130) was then added and the plates were incubated at room temperature for 10 minutes. The monospecific antibody Urelumab-NR (produced by NovaRock Biotherapeutics based on publicly available sequence information) binds only to CD137 and is used as a control antibody. Luminescence signals were measured by a Synergy Neo2 plate reader (Biotech) and data were analyzed by GraphPad Prism.

[0410] Figure 20A shows that, as expected, Urelumab-NR activates CD137 signaling independently of the presence of NUGC4 target cells. In contrast, 1901Ab2 induces CD137 signaling only in the presence of NUGC4 target cells. Moreover, 1901Ab2 induces stronger CD137 signaling than Urelumab-NR in the presence of NUGC4 target cells. This result confirms that 1901Ab2 exhibits CD137 agonism only when it engages with Claudin18.2 expressed on the cell surface of NUGC4 cells. In the absence of NUGC4 cells, the agonist activity of 1901Ab2 is not detected.

[0411] The dose-response curves of 1901Ab2, 1901Ab3, and urelumab-NR to induce CD137 signaling in the presence of NUGC4 cells are shown in Figure 20B. The EC 50 The potency values ​​were 0.047 nM, 0.10 nM, and 0.21 nM, respectively. Both 1901Ab2 and 1901Ab3 had better EC values ​​for inducing CD137 signaling than urelumab-NR. 50 value and high signaling intensity (E max ) was shown.

[0412] Example 16: Activation of CD8 T cells by CLDN18.2 / CD137 BsAb Co-culture experiments were used to measure T cell activation by BsAbs 1901Ab2 and 1901Ab3 in the presence of TCR signaling. Healthy donor-derived CD8 T cells and NUGC4 cells were used as effector and target cells. These two cells were co-cultured in RPMI1640 medium supplemented with 10% FBS and 0.5ug / ml mouse anti-hCD3 clone OKT3 (Biolegend, Cat. No. 317325) to provide TCR signaling. The disclosed binding proteins were added to stimulate T cells. Plates were incubated at 37°C with 5% CO2 for 3 days. After 72 hours of incubation, supernatants were collected and used to measure secreted IFNγ by AlphaLISA (PerkinElmer, Cat. No. AL217C / F) using the protocol suggested by the manufacturer. The amount of IFNγ represents T cell activation.

[0413] The dose-response curves of 1901Ab2, 1901Ab3, and urelumab-NR to induce CD8 T cell activation in the presence of NUGC4 cells are shown in Figure 21. EC 50The values ​​were 0.081 nM, 0.12 nM, and 0.51 nM, respectively. Both 1901Ab2 and 1901Ab3 showed better potency and higher Emax for inducing IFNγ production, a hallmark of T cell activation, than urelumab-NR.

[0414] Example 17: Tumor killing activity from CD8 T cells induced by CLDN18.2 / CD137 BsAb Co-culture experiments were performed to measure the tumor killing activity of CD8 T cells treated with CLDN18.2 / CD137 BsAbs 1901Ab2 and 1901Ab3. Briefly, CD8 T cells from healthy donors were pre-activated with ImmunoCult™ Human CD3 / CD28 T Cell Activator (StemCell, Catalog No. 10971) for 2 days. The activated cells were then washed to remove the CD3 / CD28 activator. The activated CD8 T cells were then co-cultured with NUGC4 tumor cells stably transfected with GFP and treated with the disclosed bispecific binding proteins for 96 hours. Cell numbers were measured by green fluorescence intensity using Cytation (Biotech, VT). The percentage of killing was calculated by the following formula: % killing = (GFP signal from wells without binding protein treatment - GFP signal from wells treated with antibody) / GFP signal from wells without binding protein treatment * 100%

[0415] Figure 22 shows that 1901Ab2 and 1901Ab3 induced potent T cell-mediated cytotoxicity. Approximately 75% of tumor cells were killed by CD8 T cells after 96 hours of treatment with the disclosed bispecific binding proteins. The EC 50 The values ​​are 0.043 nM and 0.033 nM, respectively.

[0416] Example 18: Effect of CLDN18.2 / CD137 BsAb 1901Ab2 on tumor growth in a subcutaneous syngeneic MC38-hClaudin18.2 mouse tumor model in humanized B-h4-1BB mice Female B-h4-1BB mice (Biocytogen), 6-8 weeks old and weighing 16-20 g, were acclimated for 7 days prior to study enrollment. The MC38 mouse colon cancer cell line was genetically modified to overexpress human Claudin18.2. Cells are maintained in vitro as monolayer cultures in DMEM supplemented with 10% heat-inactivated FBS at 37°C in a 5% CO atmosphere. Cells were harvested and cultured at 5 × 10 in 100 μl of PBS. 5 Cells were implanted subcutaneously into the right front flank for tumor development. On day 7, tumor-bearing mice were cultured at approximately 100–150 mm 3 Mice were randomly enrolled into three study groups (each group containing six mice) with a mean tumor size of 100 mm. Tumor size was measured twice weekly in two dimensions using calipers, and volume was calculated using the following formula: 3 It is expressed as V=0.5a×b 2 where a and b are the major and minor dimensions of the tumor, respectively. Mice were treated with 5 mg / kg 1901Ab2 or PBS control by intraperitoneal injection on days 7, 10, 14, and 17. The study was terminated on day 34.

[0417] Figure 23 shows the tumor growth curves for the two treatment groups. 1901Ab2 significantly inhibited tumor growth compared to PBS-treated controls. Four of six mice injected with 1901Ab2 showed complete tumor remission at day 34.

[0418] Example 19: Binding of Nectin-4 / CD137 BsAb to Cell Surface Nectin4 Bispecific Nectin-4 / CD137 binding proteins were generated, produced, and purified as described in Example 4. To examine the binding activity of BsAbs 1925Ab1, 1925Ab2, and 1925Ab3 to Nectin4, CHO cells expressing human Nectin4 were used in immunofluorescence binding assays. Cells were cultured in F12K medium containing 10% FBS. On the day of the experiment, cells were harvested, washed, and stained with binding proteins at 4°C for 2 hours, and then fixed with paraformaldehyde at room temperature for 15 minutes. The disclosed antibodies 1925Ab1, 1925Ab2, and 1925Ab3 are bispecific antibodies that bind to both Nectin-4 and CD137. Monospecific antibody 1925Ab4 (parent mouse antibody) binds only to Nectin-4 and is used as a control antibody. The fixed cells were then washed three times with PBS, after which the cells were stained with Alexa Fluor® 488 goat anti-human IgG antibody (Invitrogen, Cat. No. A-11013) for detection at room temperature for 1 h. The binding signal was assessed by quantifying the fluorescence intensity using iQue Screener PLUS (Sartorius, MI).

[0419] As shown in FIG. 24, at a concentration of 10 μg / ml, the disclosed bispecific binding proteins including 1925Ab1, 1925Ab2, and 1925Ab3 bound similarly to human Nectin4 on CHO cells compared to the control monoclonal antibody 1925Ab4.

[0420] Example 20: Binding of Nectin-4 / CD137 BsAb to cell surface CD137 The binding affinity of BsAbs 1925Ab1, 1925Ab2, and 1925Ab3 was evaluated using an immunofluorescence imaging assay. Briefly, HEK293T-huCD137 cells stably expressing human CD137 were seeded in complete medium containing DMEM with 10% FBS and then incubated overnight at 37°C. The cells were allowed to bind with the disclosed binding proteins for 2 hours at 4°C, after which the cells were fixed with paraformaldehyde for 15 minutes at room temperature. The disclosed antibodies 1925Ab1, 1925Ab2, and 1925Ab3 are bispecific antibodies that bind to both Nectin-4 and CD137. The monospecific anti-CD137 antibody 1923Ab4 binds only to CD137 and is used as a control antibody. Fixed cells were washed three times with PBS and then stained with Alexa Fluor® 488 goat anti-human IgG antibody (Invitrogen, Cat. No. A-11013) for detection at room temperature for 1 h. Binding signals were assessed by imaging cells and quantifying the fluorescence intensity using a Cytation Imager (BioTek, VT).

[0421] As shown in FIG. 25, at a concentration of 10 μg / ml, the disclosed bispecific binding proteins, including 1925Ab1, 1925Ab2 and 1925Ab3, bound to cell surface human CD137 similarly to their monoclonal antibody control 1923Ab4.

[0422] Example 21: Target cell-dependent activation of CD137 signaling by Nectin-4 / CD137 BsAb Nectin-4 / CD137 BsAbs 1925Ab1, 1925Ab2, 1925Ab3 were evaluated for their ability to induce target cell-dependent CD137 agonism. Briefly, a Jurkat T reporter cell line stably expressing CD137 and containing an NFkB-luc reporter was used to quantify CD137 signaling. CHO cells stably transfected with human Nectin4 (CHO-Nectin4) were used as target cells. Jurkat T reporter cells were co-cultured with or without CHO-Nectin4 target cells and stimulated with the disclosed binding proteins for 16 hours at 37°C, 5% CO2. ONE-Glo™ Luciferase Reagent (Promega, Cat. No. E6130) was then added and the plates were incubated at room temperature for 10 minutes. The disclosed antibodies 1925Ab1, 1925Ab2, and 1925Ab3 are bispecific antibodies that bind to both Nectin-4 and CD137. The monospecific antibody Urelumab-NR binds only to CD137 and is used as a control antibody. The luminescence signal was measured by Synergy Neo2 plate reader (Biotech), and the data was analyzed by GraphPad Prism.

[0423] Urelumab-NR was produced by Novaloc Biotherapeutics, Inc. based on the publicly available sequence of Urelumab. Figure 26A shows that Urelumab-NR activated CD137 signaling independent of the presence of CHO-Nectin4 target cells, as expected. In contrast, 1925Ab1, 1925Ab2, and 1925Ab3 induced CD137 signaling only in the presence of CHO-Nectin4 target cells. Furthermore, 1925Ab1, 1925Ab2, and 1925Ab3 induced stronger CD137 signaling than Urelumab-NR in the presence of NUGC4 target cells. This result confirms that the disclosed bispecific binding protein exhibited CD137 agonism only when it engaged with Nectin4 expressed on the cell surface of CHO cells. No agonist activity of these disclosed bispecific binding proteins was detected in the absence of CHO-Nectin4 cells.

[0424] The dose-response curves of 1925Ab1, 1925Ab2, 1925Ab3, and Urelumab-NR to induce CD137 signaling in the presence of NUGC4 cells are shown in Figure 26B. The EC 50 The potency values ​​were 0.027 nM, 0.080 nM, 0.049 nM, and 0.26 nM, respectively. The disclosed bispecific binding proteins, including 1925Ab1, 1925Ab2, and 1925Ab3, had better EC values ​​for inducing CD137 signaling than urelumab-NR. 50 value and high signaling intensity (E max ) was shown.

[0425] Example 22: Evaluation of antibody-induced immune cell infiltration in mouse liver Hepatotoxicity has been a known side effect of some early CD137 agonist antibody therapeutics. Urelumab has been reported to induce inflammatory hepatotoxicity at doses of 0.3 mg / kg or higher, with a maximum tolerated dose (MTD) of 0.1 mg / kg, limiting its therapeutic window (Segal NH, et al. Clin Cancer Res. 2017;23(8):1929-1936). Studies have shown that urelumab-induced hepatotoxicity is due to hepatic inflammation marked by immune cell infiltration in the liver and a significant increase in serum ALT levels (Zhang H, et al. Journal for ImmunoTherapy of Cancer 2020;8).

[0426] Bispecific antibodies that bind CD137 and tumor-associated antigens (TAAs) offer the advantage of potent costimulation targeting the tumor microenvironment (TME) and reduced risk of hepatic inflammation / hepatotoxicity, which may widen the therapeutic window.

[0427] To demonstrate that the disclosed bispecific TAA-CD137 antibody has lower hepatotoxicity, 6-8 week old female B-h4-1BB mice (Biocytogen) weighing 16-20 g were acclimated for 7 days prior to study enrollment. B-h4-1BB mice were randomly enrolled into three study groups. Each group consisted of six mice. On days 0, 3, 7, and 10, mice were treated by intraperitoneal injection of 10 mg / kg urelumab-NR, 1912Ab5, or PBS as a negative control. The study was terminated on day 13. Livers from each treatment group were formalin fixed and paraffin embedded. Fluorescent IHC was performed using 5 mm FFPE tissue sections. After deparaffinization, slides were stained with primary antibodies detecting CD4, CD8 T cells, and macrophages for immune cell identification.

[0428] In FIG. 27, infiltration of mouse CD4 T cells (A), CD8 T cells (B), and mouse macrophages (C) was significantly increased only in urelumab-NR-treated mice, but not in 1912Ab5-treated mice.

[0429] Consistent with Example 10, Figures 11B and 11C, the lack of immune cell infiltration induced by 1912Ab5 indicates a low risk of hepatotoxicity from CLDN6 / CD137 bsAb.

[0430] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weights, reaction conditions, and the like used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of at least the number of reported significant digits and by applying ordinary rounding techniques.

[0431] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0432] The terms "a", "an", "the", and similar references used in the context of describing this disclosure (particularly in the context of the claims below) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand way of individually referring to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated herein as if set forth individually herein. All methods described herein can be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to better illuminate the disclosure and does not create a limitation on the scope of the disclosure as otherwise described in the claims. No language in this specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

[0433] Grouping of alternative elements or embodiments of the disclosure disclosed herein should not be construed as limiting. Each group member may be referenced and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in or deleted from a group for reasons of convenience and / or patentability. When such inclusion or deletion occurs, the specification is considered to include the group as modified, and therefore fulfills the written description of all Markush groups used in the appended claims.

[0434] Certain embodiments of the present disclosure are described herein, including the best mode known to the inventors for carrying out the present disclosure. Needless to say, variations of these described embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors expect that those skilled in the art will adopt such variations as necessary, and the inventors intend for the present disclosure to be practiced in ways other than as specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the present disclosure unless otherwise indicated herein or clearly contradicted by context.

[0435] Certain embodiments disclosed herein may be further limited in the claims using the language "consisting of" or "consisting essentially of." When used in a claim, whether at the time of filing or when added per amendment, the transitional term "consisting of" excludes any element, step, or ingredient not specified in the claim. The transitional term "consisting essentially of" limits the claim to the specified materials or steps, as well as materials or steps that do not materially affect the basic and novel characteristics. The embodiments of the present disclosure so claimed are essentially or explicitly described and enabled herein.

[0436] It should be understood that the embodiments of the present disclosure disclosed herein are illustrative of the principles of the present disclosure. Other modifications that can be employed are within the scope of the present disclosure. Thus, by way of example, but not of limitation, alternative configurations of the present disclosure can be utilized in accordance with the teachings herein. Thus, the present disclosure is not limited to that precisely as shown and described.

[0437] Although the present disclosure is described and illustrated herein by reference to various specific materials, procedures, and examples, it should be understood that the present disclosure is not limited to the specific combination of materials and procedures selected for that purpose. Multiple variations of such details may be implied, as would be understood by those skilled in the art. It is intended that the present specification and examples be considered as merely illustrative, with the true scope and spirit of the present disclosure being indicated by the following claims. All references, patents, and patent applications mentioned in this application are incorporated herein by reference in their entirety.

Claims

1. 1. A bispecific binding protein that binds to a tumor-associated antigen and CD137, (a) an antibody scaffold module comprising a first antigen-binding site that binds to the tumor-associated antigen and a second antigen-binding site that binds to the tumor-associated antigen; and (b) at least one first binding module comprising a third antigen-binding site that binds to CD137; The bispecific binding protein, wherein the tumor-associated antigen is Claudin6, Claudin18.2, or Nectin-4.

2. the first antigen-binding site and the second antigen-binding site both bind to Claudin6; (i) a heavy chain variable region sequence comprising CDR1: SEQ ID NO: 45, CDR2: SEQ ID NO: 46, and CDR3: SEQ ID NO: 47, and a light chain variable region sequence comprising CDR1: SEQ ID NO: 48, CDR2: SEQ ID NO: 49, and CDR3: SEQ ID NO: 50; or (ii) a heavy chain variable region sequence comprising CDR1: SEQ ID NO:51, CDR2: SEQ ID NO:52, and CDR3: SEQ ID NO:53, and a light chain variable region sequence comprising CDR1: SEQ ID NO:54, CDR2: SEQ ID NO:55, and CDR3: SEQ ID NO:

56.

3. the first antigen-binding site and the second antigen-binding site both bind to Claudin6; (i) a heavy chain variable region sequence set forth in SEQ ID NO: 25 and a light chain variable region sequence set forth in SEQ ID NO: 26; or (ii) the heavy chain variable region sequence set forth in SEQ ID NO: 27, and the light chain variable region sequence set forth in SEQ ID NO:

28.

4. the first antigen-binding site and the second antigen-binding site both bind to Claudin18.2; 2. The bispecific binding protein of claim 1, comprising a heavy chain variable region sequence comprising CDR1: SEQ ID NO: 33, CDR2: SEQ ID NO: 34, and CDR3: SEQ ID NO: 35, and a light chain variable region sequence comprising CDR1: SEQ ID NO: 36, CDR2: SEQ ID NO: 37, and CDR3: SEQ ID NO:

38.

5. 5. The bispecific binding protein of claim 4, wherein the first antigen-binding site and the second antigen-binding site both bind to Claudin18.2 and comprise a heavy chain variable region sequence set forth in SEQ ID NO:21 and a light chain variable region sequence set forth in SEQ ID NO:

22.

6. the first antigen-binding site and the second antigen-binding site both bind to Nectin-4; 2. The bispecific binding protein of claim 1, comprising a heavy chain variable region sequence comprising CDR1: SEQ ID NO:57, CDR2: SEQ ID NO:58, and CDR3: SEQ ID NO:59, and a light chain variable region sequence comprising CDR1: SEQ ID NO:60, CDR2: SEQ ID NO:61, and CDR3: SEQ ID NO:

62.

7. the first antigen-binding site and the second antigen-binding site both bind to Nectin-4; (i) a heavy chain variable region sequence set forth in SEQ ID NO: 29 and a light chain variable region sequence set forth in SEQ ID NO: 30; or (ii) the heavy chain variable region sequence set forth in SEQ ID NO: 31, and the light chain variable region sequence set forth in SEQ ID NO:

32.

8. 2. The bispecific binding protein of claim 1 , wherein the first binding module is an antibody fragment.

9. The bispecific binding protein of claim 1 , wherein the first link module is an scFV that binds to CD137.

10. 2. The bispecific binding protein of claim 1, wherein the first link module and the antibody scaffold module are covalently linked to each other via an interlinker having the sequence set forth in SEQ ID NO:64 or SEQ ID NO:

65.

11. the first link module binds to CD137; 2. The bispecific binding protein of claim 1, comprising a heavy chain variable region sequence comprising CDR1: SEQ ID NO: 39, CDR2: SEQ ID NO: 40, and CDR3: SEQ ID NO: 41, and a light chain variable region sequence comprising CDR1: SEQ ID NO: 42, CDR2: SEQ ID NO: 43, and CDR3: SEQ ID NO:

44.

12. 12. The bispecific binding protein of claim 11 , wherein the first link module binds to CD137 and comprises a heavy chain variable region sequence set forth in SEQ ID NO: 23 and a light chain variable region sequence set forth in SEQ ID NO:

24.

13. the bispecific binding protein comprises two first binding modules that bind to CD137; the first antigen-binding site and the second antigen-binding site both bind to Claudin18.2 and comprise a heavy chain variable region sequence set forth in SEQ ID NO: 21 and a light chain variable region sequence set forth in SEQ ID NO: 22; each of the first link modules comprises a heavy chain variable region sequence set forth in SEQ ID NO: 23 and a light chain variable region sequence set forth in SEQ ID NO: 24; 2. The bispecific binding protein of claim 1, wherein the first link module is separately attached to the C-terminus of each heavy chain in the antibody scaffold module by a glycine-serine linker.

14. the bispecific binding protein comprises two first binding modules that bind to CD137; the first antigen-binding site and the second antigen-binding site both bind to Claudin18.2 and comprise a heavy chain variable region sequence set forth in SEQ ID NO: 21 and a light chain variable region sequence set forth in SEQ ID NO: 22; each of the first link modules comprises a heavy chain variable region sequence set forth in SEQ ID NO: 23 and a light chain variable region sequence set forth in SEQ ID NO: 24; 2. The bispecific binding protein of claim 1, wherein the first link module is separately attached to the C-terminus of each light chain in the antibody scaffold module by a glycine-serine linker.

15. the bispecific binding protein comprises two first binding modules that bind to CD137; the first antigen-binding site and the second antigen-binding site both bind to Claudin6 and comprise a heavy chain variable region sequence set forth in SEQ ID NO: 25 or 27 and a light chain variable region sequence set forth in SEQ ID NO: 26 or 28; each of the first link modules comprises a heavy chain variable region sequence set forth in SEQ ID NO: 23 and a light chain variable region sequence set forth in SEQ ID NO: 24; 2. The bispecific binding protein of claim 1, wherein the first link module is separately attached to the C-terminus of each heavy chain in the antibody scaffold module by a glycine-serine linker.

16. the bispecific binding protein comprises two first binding modules that bind to CD137; the first antigen-binding site and the second antigen-binding site both bind to Nectin-4 and comprise a heavy chain variable region sequence set forth in SEQ ID NO: 29 or 31 and a light chain variable region sequence set forth in SEQ ID NO: 30 or 32; each of the first link modules comprises a heavy chain variable region sequence set forth in SEQ ID NO: 23 and a light chain variable region sequence set forth in SEQ ID NO: 24; 2. The bispecific binding protein of claim 1, wherein the first link module is separately attached to the C-terminus of each light chain in the antibody scaffold module by a glycine-serine linker.

17. the bispecific binding protein comprises two first binding modules that bind to CD137; the first antigen-binding site and the second antigen-binding site both bind to Nectin-4 and comprise a heavy chain variable region sequence set forth in SEQ ID NO: 29 or 31 and a light chain variable region sequence set forth in SEQ ID NO: 30 or 32; each of the first link modules comprises a heavy chain variable region sequence set forth in SEQ ID NO: 23 and a light chain variable region sequence set forth in SEQ ID NO: 24; 2. The bispecific binding protein of claim 1, wherein the first link module is separately attached to the N-terminus of each heavy chain in the antibody scaffold module by a glycine-serine linker.

18. the bispecific binding protein comprises two first binding modules that bind to CD137; the first antigen-binding site and the second antigen-binding site both bind to Nectin-4 and comprise a heavy chain variable region sequence set forth in SEQ ID NO: 29 or 31 and a light chain variable region sequence set forth in SEQ ID NO: 30 or 32; each of the first link modules comprises a heavy chain variable region sequence set forth in SEQ ID NO: 23 and a light chain variable region sequence set forth in SEQ ID NO: 24; 2. The bispecific binding protein of claim 1, wherein the first link module is separately attached to the C-terminus of each heavy chain in the antibody scaffold module by a glycine-serine linker.

19. 10. The bispecific binding protein of claim 1, wherein the antibody scaffold module further comprises a constant region.

20. 20. The binding protein of claim 19, wherein the constant region comprises SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, or SEQ ID NO:

73.

21. 21. A pharmaceutical composition comprising the bispecific binding protein of any one of claims 1 to 20 and a pharmaceutically acceptable carrier.

22. 21. Use of a bispecific binding protein according to any one of claims 1 to 20 in the manufacture of a medicament for treating or preventing cancer.

23. 21. An isolated polynucleotide comprising a sequence encoding the bispecific binding protein of any one of claims 1 to 20.

24. A vector comprising the polynucleotide of claim 23.

25. A cell comprising the polynucleotide of claim 23.

26. A cell comprising the vector described in claim 24.

27. 26. A method for producing a bispecific binding protein according to any one of claims 1 to 20, comprising culturing the cell of claim 25.

28. A method for producing a bispecific binding protein according to any one of claims 1 to 20, comprising culturing a cell according to claim 26.

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