Binding molecules for cancer treatment

Abstract

The present invention relates to binding molecules that specifically bind to CD137 and FAP and to medicaments containing same, their use in pharmaceutical preparations and methods of using same as medicaments for the treatment and / or prevention of cancer.

Description

[Technical Field]

[0001] Sequence List This application includes a sequence listing submitted electronically in ASCII format, the entirety of which is incorporated herein by reference. The ASCII copy said to have been created on 22 April 2021 is named 09-0703-WO-1_SL.txt and has a size of 873,263 bytes.

[0002] Field of Invention The present invention relates to a binding molecule that binds to CD137 (4-1BB, TNFRSF9), a member of the TNFR family, and fibroblast-activating protein (FAP), as well as its use in pharmaceuticals, pharmaceutical compositions containing the same, and methods for using the same as an agent for the treatment and / or prevention of cancer.

[0003] Background of the Invention Cancer is generally a group of diseases characterized by abnormal cell proliferation and the potential for cancerous cells to invade or spread throughout the body. It is a serious disease worldwide and a leading cause of death.

[0004] Various treatment methods have been used in attempts to manage or, in some cases, treat cancer, including surgery, chemotherapy, radiation therapy, and hormone therapy. Recent advances in immunotherapy have changed the landscape of cancer treatment. Nevertheless, since most patients with locally advanced or metastatic tumors will die from the disease, the substantial need for new treatment strategies is justified.

[0005] Antibody biomolecules offer the potential of powerful therapeutic agents for treating cancer. Antibodies are designed to recognize and bind to specific proteins (their target antigens) on the surface of cells, and such proteins may be present only on the surface of certain cancer cells or on immune cells. This binding can trigger many different biological responses, depending on the function of those target antigen proteins and the structure of the antibody itself.

[0006] For example, some antibodies induce an immune response that attacks and kills cancer cells, either by attracting immune cells to cancer cells or by directly influencing the activity of the immune system itself. Further types of antibody-based therapies bind to cancer cells, stopping or reducing cell division, thus slowing and preventing abnormal cell proliferation. Other types of antibodies have drugs or radioactive particles attached to them, and therefore deliver these therapies directly to the cancer cells themselves.

[0007] FAP is a member of the dipeptidyl peptidase (DPP) family (also known as fibroblast-activating protein alpha, prolyl endopeptidase FAP, 170kDa melanoma membrane-bound gelatinase, or endometrial serine protease). FAP is transiently expressed in some fetal mesenchymal tissues and rarely expressed in healthy adult tissues. In this case, the presence of FAP is usually limited to endometrial cells. FAP is also expressed in diseases associated with activated stroma, including wound healing, rheumatoid arthritis, osteoarthritis, cirrhosis, and pulmonary fibrosis, and is often induced in activated fibroblasts after trauma or injury to tissue. FAP is also expressed in the tumor stromal tissue of all types of human epithelial tumors, as well as in malignant cells of various bone and soft tissue sarcomas. FAP is expressed in more than 90% of human epithelial malignancies, including colorectal, ovarian, breast, bladder, and lung tumors. FAPs are preferentially found in fibroblasts that develop in the vicinity of newly forming or established blood vessels, forming specific cellular compartments between tumor capillary endothelium and actual malignant epithelial cells and cell clusters.

[0008] Stromal fibroblasts play a crucial role in the development, proliferation, and metastasis of carcinomas. FAP expression profiles suggest that FAP is involved in tumor invasion into healthy tissues, as well as tumor formation and metastasis. FAP inhibitors, i.e., substances that can reduce or inhibit the proteolytic activity of FAP, are useful therapeutic agents for treating all types of tumor diseases. FAP inhibitors can preferably be used to treat epithelial tumors, such as breast tumors, non-small cell lung cancer, colorectal cancer, and soft tissue cancers.

[0009] FAP antibodies are well known in the art. For example, sibrotuzumab is a humanized mouse FAP, and after humanization, it bound to human FAP, but no detectable binding to mouse FAP was shown, and it did not show tumor death in the mouse model (WO No. 1993005804, Cheng, et al. Tumors and their microenvironments: tilling the soil. Commentary re: AM Scott et al., A Phase I dose-escalation study of sibrotuzumab in patients with advanced or metastatic fibroblast activation protein-positive cancer. Clin. Cancer Res., 9: 1639-1647). At least 15 antibodies targeting FAP are in commercial preclinical or clinical development (e.g., US No. 8999342, Ludwig Institute for Cancer Research (mAb / preclinical); WO No. 2016110598, US No. 20170369592, Mabimmune Diagnostics / Univ Zurich (mAb / preclinical); WO No. 2015118030, US No. 10137202, Oncomatryx (mAb-conjugate / preclinical); WO No. 1993005804, WO No. 1999057151, WO No. 2001068708, WO No. 2002083171, WO No. 2007077173, US No. 8568727, cibrotuzumab, Boehringer See Ingelheim (humanized mouse FAP / termination after PH1 in colorectal).

[0010] CD137 (4-1BB, TNFRSF9, CDw137, T cell antigen 4-1BB homology, T cell antigen ILA, CD antigen CD137) is also a member of the tumor necrosis factor receptor superfamily. CD137 is expressed on activated T lymphocytes, and its binding to a ligand enhances T cell function. CD137 activation is determined by receptor oligomerization. CD137 is expressed on activated CD4+ and CD8+ T cells, Tregs, DCs, monocytes, mast cells, and eosinophils. CD137 activation plays a crucial role in the activation and survival of CD8+ T cells. CD137 activation does not initiate effector function, but rather sustains and enhances it, preferentially supporting TH1 cytokine production. In CD4+ T cells, CD137 stimulation initially leads to activation, followed by activation-induced cell death. This suggests a mechanism by which CD137 agonist antibodies can have therapeutic effects in tumor immunity and autoimmunity.

[0011] Furthermore, CD137 is expressed on antigen-presenting cells, such as dendritic cells and macrophages, and stimulation of these cell types can induce immune activation that can lead to tumor-targeting immunity. CD137 plays an essential role in immune surveillance. CD137 deficiency leads to reduced T cell immune responses, such as cytokine production and cytolytic T cell activity, in knockout mice (Kwon, 2002, Narazaki, 2010). In humans, CD137 deficiency is associated with immune disorder, impaired lymphocyte response, and EBV-associated lymphoma development (Somekh, 2019).

[0012] Furthermore, CD137 agonist antibodies have been shown to activate endothelial cells in the tumor environment, leading to upregulation of ICAM-1 and VCAM-1 and improving T cell recruitment. Several studies have demonstrated the induction of tumor immunity by treatment with agonist CD137 antibodies, including pioneering research dating back to 1997 (Melero, 1997).

[0013] More than 25 drugs targeting CD137 are currently in preclinical or clinical development, but progress in this target has been slow. Urelumab (BMS-66513), a fully human IgG4 antibody developed by Bristol-Myers Squibb, was the first CD137 agonist to enter clinical practice in 2005. In combination with pembrolizumab, it showed promising efficacy in melanoma patients, but several trials were discontinued due to concerns about hepatotoxicity. The future of urelumab is unclear, and although many BMS-supported trials are still ongoing, it has been dropped from the BMS pipeline. Another major CD137 agonist, utomirumab (PF-05082566), a fully human IgG2 antibody from Pfizer, has shown only limited clinical efficacy in various combinations. Urelumab can stimulate T cells without further crosslinking, whereas utomirumab requires FcγR-mediated crosslinking to initiate its agonist effect (Fisher, 2012). In the latter case, FcγR-mediated crosslinking is not a predictable process, and this process limits the antitumor efficacy of utomirumab. In April 2018, Pfizer announced that it would not develop utomirumab as monotherapy or in combination with pembrolizumab, although it may proceed with other combination trials. Therefore, although preliminary data suggested that utomirumab was tolerable, it showed only minimal tumor activity in combination with avelumab and rituximab, in contrast to urelumab. Pfizer's Phase III clinical trials have recently decreased in number from 500 to 29 patients.

[0014] Newer CD137 agents may have enhanced activity without increased toxicity by simultaneously targeting tumor antigens (e.g., HER2), thereby developing focal activity in tumors. CD137 / Her2 bispecific PRS-343 has recently shown promising results in Phase I trials involving numerous pre-conditioned cancer patients across multiple tumor types. PRS-343 demonstrated tumor activity, increased tumor-infiltrating CD8+ T cells, and a favorable safety profile. FAP expression on tumor-associated fibroblasts in the stroma is more stable than HER2 expression on cancer cells; therefore, the combination of CD137 and HER2 as co-localization targets may limit efficacy.

[0015] Another bispecific CD137 / FAP DARPin molecule (MP-0310 from AMGEN / Molecular Partners) is another such tumor target molecule formatted as monovalent FAP and a monovalent CD137 binder. It is unclear whether this format allows for binding to the native CD137 ligand in vivo or whether the monovalent CD137 engager cross-links CD137 and thus can adequately activate T cells. DARPin (engineered ankyrin repeat protein) is not an antibody, but a genetically engineered antibody-mimicking protein, representing a new class of non-immunoglobulin proteins that has been used as an alternative to antibodies for target binding in drug discovery and development (e.g., WO 2002 / 020565, US 20130244940, and Link et al. "Preclinical pharmacology of MP0310: a 4-1BB / FAP bi-specific DARPin® drug candidate promoting tumor restricted T cell co-stimulation." Poster 3572 (https: / / investors.molecularpartners.com / ~ / media / Files / M / Molecular-Partners / documents / 201804-mp0310-pharmacology-poster-3752.pdf).

[0016] Furthermore, the CD137-L / FAP molecule (RG-7827 from Roche targeting) is a trimerized human CD137 ligand that is not antibody-arm bound. It is unclear whether this CD137 ligand enables the higher-order multimerization of CD137, which is thought to be necessary for improving agonist activity. Furthermore, RG-7827 is expected to compete with endogenous CD137 ligands (see, for example, WO Nos. 2019175125, 2017055398, 2017060144, US Nos. 20170114141, 20170247467, and J. Sam, C. Claus, C. Ferrara, S. Lang, V. Nicolini, S. Colombetti, V. Teichgraber, S. Evers, M. Bacac, P. Umana, C. Klein. (AACR 2018, Poster 5621, FAP-4-1BBL: A novel versatile tumor-stroma targeted 4-1BB agonist for combination immunotherapy with checkpoint inhibitors, T-cell bispecific antibodies, and ADCC-mediating antibodies)).

[0017] Thus, while systemic administration of agonist 4-1BB antibodies may have shown potential as an effective tumor targeting agent in preclinical models, clinical development has been hampered by either significant dose-limiting hepatotoxicity or limited clinical efficacy, possibly due to relatively low potency and / or dependence on Fcγ receptor-mediated hyperclustering, which has prevented successful progress in past Phase II trials. Therefore, although bispecific CD137 molecules and CD137 molecules with various combination partners are currently under development, the safety and clinical efficacy of this antigen have yet to be demonstrated.

[0018] In general, despite significant recent advances in cancer treatment, some solid tumors remain difficult to treat and do not respond to available therapies; they do not respond to either current standard treatments or newer, initially promising drugs currently in clinical trials. Even if patients initially respond, they often do not experience a long-term response and rarely achieve complete remission or cure. For example, in the case of non-small cell lung cancer, one such unmet need is that approximately 40-50% of all NSCLC patients do not respond to first-line treatment, while another 50% who do respond do not experience a long-term sustained response with current first-line treatments (including anti-PD-1 immunotherapy). Furthermore, fewer than 30-40% of patients maintain progression-free status after 12 months. Options for further treatment of patients in second / third-line treatment who have previously received anti-PD-L1 therapy are limited and not well-established, as chemotherapy options, such as docetaxel or platinum-based regimens (if not used as first-line), have limited benefits.

[0019] Given the available preclinical and clinical data, as well as the poor outlook for such cancer patients, there is a clear need to identify more effective therapies, particularly those that are better tolerable by patients and have targeted specificity for tumor types resistant to currently available treatments. Therefore, novel approaches, including immunologically active agents with different modes of action, are required.

[0020] Summary of the Invention This invention is based on the concept that tumor-restricted activation of tumor-specific TILs represents a promising mode for cancer treatment. Embodiments of this invention are bispecific, bivalent molecules that combine a binding site that specifically binds to fibroblast-activating protein (FAP) with a binding site that specifically binds to CD137 (4-1BB, TNFRSF9) within a single binding molecule and a low-affinity binding site. The bispecific molecules of this invention activate and engage with CD137 on T cells only in the cancer stromal context of the tumor microenvironment where FAP is expressed.

[0021] As will be discussed in more detail below, one advantage of the molecule of the present invention is the improved activation of tumor-responsive T cells and the expansion of the existing tumor response to the tumor microenvironment, resulting in tumor regression in patients with improved safety compared to conventional CD137 agonists. By conditioning CD137 engagement on proximal expression of FAP due to its low affinity, CD137 activity and T cell recruitment occur only in the FAP+ tumor environment of many types of cancer. This low-affinity engagement is an improvement over other monovalent CD137 approaches. This is because the bivalent molecule of the present invention significantly binds to and activates tumor-affiliated T cells only in the presence of FAP, causing them to proliferate and secrete IFNγ, thereby resulting in a more localized tumor-targeted immune response. Ultimately, the targeted activity of the CD137 / FAP antibody will result in tumor regression in patients with better tolerability and an improved safety profile compared to conventional CD137 agonists.

[0022] Therefore, a first aspect of the present invention provides an immunoglobulin-like binding molecule having at least one antigen-binding site that specifically binds to fibroblast-activating protein (FAP) and at least one antigen-binding site that specifically binds to CD137 (4-1BB, TNFRSF9).

[0023] In a preferred embodiment of the binding molecule of the present invention, the molecule is bispecific and tetravalent, being divalent with respect to CD137 and divalent with respect to FAP.

[0024] In preferred embodiments of the binding molecule of the present invention, the antigen-binding site that specifically binds to CD137(4-1BB,TNFRSF9) is a part of an immunoglobulin (Ig) molecule, and the antigen-binding site that specifically binds to fibroblast-activating protein (FAP) preferably includes one or more scFv fused to the N-terminus of the Fc domain via a peptide linker.

[0025] In a preferred embodiment of the binding molecule of the present invention, one or more scFv molecules have a VL-VH orientation from the N-terminus to the C-terminus.

[0026] In a preferred embodiment of the binding molecule of the present invention, one or more scFv molecules are fused to the C-terminus of the heavy chain of the Ig molecule.

[0027] In a preferred embodiment of the binding molecule of the present invention, the Ig molecule is IgG1KO.

[0028] In a preferred embodiment of the binding molecule of the present invention, one or more scFv molecules are fused to an Ig molecule by a peptide linker, preferably a peptide linker having a length of about 4 to 20 amino acids.

[0029] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site that specifically binds to CD137(4-1BB) is as follows: i) Heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 290 (CDR1), SEQ ID NO: 8 (CDR2), and SEQ ID NO: 9 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 12 (CDR1), SEQ ID NO: 13 (CDR2), and SEQ ID NO: 14 (CDR3) or ii) Heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 18 (CDR2), and SEQ ID NO: 9 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 22 (CDR1), SEQ ID NO: 23 (CDR2), and SEQ ID NO: 14 (CDR3) or iii) Heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 28 (CDR2), and SEQ ID NO: 9 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 32 (CDR1), SEQ ID NO: 33 (CDR2), and SEQ ID NO: 14 (CDR3) or iv) Heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 38 (CDR2), and SEQ ID NO: 9 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 42 (CDR1), SEQ ID NO: 43 (CDR2), and SEQ ID NO: 14 (CDR3) or v) Heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 48 (CDR2), and SEQ ID NO: 9 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 52 (CDR1), SEQ ID NO: 53 (CDR2), and SEQ ID NO: 14 (CDR3) or vi) Heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 58 (CDR2), and SEQ ID NO: 9 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 62 (CDR1), SEQ ID NO: 63 (CDR2), and SEQ ID NO: 14 (CDR3) or vii) Heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 308 (CDR1), SEQ ID NO: 68 (CDR2), and SEQ ID NO: 69 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 72 (CDR1), SEQ ID NO: 73 (CDR2), and SEQ ID NO: 74 (CDR3) or viii) Heavy chain CDRs containing SEQ ID NO: 308 (CDR1), SEQ ID NO: 78 (CDR2), and SEQ ID NO: 69 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 72 (CDR1), SEQ ID NO: 73 (CDR2), and SEQ ID NO: 74 (CDR3) or ix) Heavy chain CDRs containing SEQ ID NO: 308 (CDR1), SEQ ID NO: 88 (CDR2), and SEQ ID NO: 69 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 92 (CDR1), SEQ ID NO: 93 (CDR2), and SEQ ID NO: 74 (CDR3) or x) Heavy chain CDRs containing SEQ ID NO: 308 (CDR1), SEQ ID NO: 98 (CDR2), and SEQ ID NO: 69 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 93 (CDR1), SEQ ID NO: 93 (CDR2), and SEQ ID NO: 74 (CDR3) or xi) Heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 672 (CDR1) (where X1 is selected from the group consisting of A or S), SEQ ID NO: 335 (CDR2) (where X1 is selected from the group consisting of N or Q, X2 is selected from the group consisting of D or E, X3 is selected from the group consisting of G or A, X4 is selected from the group consisting of T or K, X5 is selected from the group consisting of L or V, X6 is selected from the group consisting of D or E, X7 is selected from the group consisting of L or V, and X8 is selected from the group consisting of S or G), and SEQ ID NO: 9 (CDR3), as well as SEQ ID NO: 336 A light chain CDR containing the amino acid sequences of (CDR1) (where X1 is selected from the group consisting of K or R, X2 is selected from the group consisting of D or S, X3 is selected from the group consisting of V or I, X4 is selected from the group consisting of S or T, and X5 is selected from the group consisting of V and L), SEQ ID NO: 337 (CDR2) (where X1 is selected from the group consisting of S or A, X2 is selected from the group consisting of Y or S, X3 is selected from the group consisting of R or L, X4 is selected from the group consisting of Y and Q, and X5 is selected from the group consisting of S or T), and SEQ ID NO: 14 (CDR3) or xii) A heavy chain CDR containing SEQ ID NO: 308 (CDR1), SEQ ID NO: 338 (CDR2) (where X1 is selected from the group consisting of Y or I) and SEQ ID NO: 69 (CDR3), and a light chain CDR containing the amino acid sequence of SEQ ID NO: 669 (CDR1) (where X1 is selected from the group consisting of N or Q), SEQ ID NO: 339 (CDR2) (where X1 is selected from the group consisting of L or G) and SEQ ID NO: 74 (CDR3) or xiii) CD137 antigen-binding molecules that bind to an epitope on CD137 with the extracellular domain CRD3 of amino acids 87-118 (SEQ ID NO: 352), blocking the binding of any of the above antigen-binding molecules (i)-(xii) and / or competing for binding with them. xvi) CD137 antigen-binding molecules that bind to epitopes on CD137 via the extracellular domains CRD2 / CRD3 of amino acids 46-117 (SEQ ID NO: 356), blocking the binding of any of the above antigen-binding molecules (i)-(xvi) and / or competing for binding with them. It is selected from the group that includes it.

[0030] In a preferred embodiment of the immunoglobulin-like binding molecule of the present invention, the antigen-binding site that specifically binds to CD137(4-1BB) is a part of an immunoglobulin molecule including a variable heavy chain region and a variable light chain region. Here, the VH and VL regions are as follows: i) A variable heavy chain containing the amino acid sequence of SEQ ID NO: 10 and a variable light chain containing the amino acid sequence of SEQ ID NO: 15 or ii) A variable heavy chain containing the amino acid sequence of SEQ ID NO: 20 and a variable light chain containing the amino acid sequence of SEQ ID NO: 25 or iii) A variable heavy chain containing the amino acid sequence of SEQ ID NO: 30 and a variable light chain containing the amino acid sequence of SEQ ID NO: 35 or iv) A variable heavy chain containing the amino acid sequence of SEQ ID NO: 40 and a variable light chain containing the amino acid sequence of SEQ ID NO: 45 or v) A variable heavy chain containing the amino acid sequence of SEQ ID NO: 50 and a variable light chain containing the amino acid sequence of SEQ ID NO: 55 or vi) A variable heavy chain containing the amino acid sequence of SEQ ID NO: 60 and a variable light chain containing the amino acid sequence of SEQ ID NO: 65 or vii) A variable heavy chain containing the amino acid sequence of SEQ ID NO: 70 and a variable light chain containing the amino acid sequence of SEQ ID NO: 75 or viii) A variable heavy chain containing the amino acid sequence of SEQ ID NO: 80 and a variable light chain containing the amino acid sequence of SEQ ID NO: 85 or ix) A variable heavy chain containing the amino acid sequence of SEQ ID NO: 90 and a variable light chain containing the amino acid sequence of SEQ ID NO: 95 or x) A variable heavy chain containing the amino acid sequence of SEQ ID NO: 100 and a variable light chain containing the amino acid sequence of SEQ ID NO: 105 or xi) A variable heavy chain containing the amino acid sequence of SEQ ID NO: 10, which has a different amino acid sequence from CDRH1 SEQ ID NO: 672 (where X1 is selected from the group consisting of A or S) by amino acid substitution of one or fewer amino acids, and / or has a different amino acid sequence from CDRH2 SEQ ID NO: 335 (where X1 is selected from the group consisting of N or Q, X2 is selected from the group consisting of D or E, X3 is selected from the group consisting of G or A, X4 is selected from the group consisting of T or K, X5 is selected from the group consisting of L or V, X6 is selected from the group consisting of D or E, X7 is selected from the group consisting of L or V, and X8 is selected from the group consisting of S or G) by amino acid substitution of four or fewer amino acids, and CDRL1 A variable light chain containing the amino acid sequence of SEQ ID NO: 15, which has a different amino acid sequence from SEQ ID NO: 336 (wherein X1 is selected from the group consisting of K or R, X2 is selected from the group consisting of D or S, X3 is selected from the group consisting of V or I, X4 is selected from the group consisting of S or T, and X5 is selected from the group consisting of V and L) and / or CDRL2 due to amino acid substitution of 5 or fewer amino acids; or SEQ ID NO: 337 (wherein X1 is selected from the group consisting of S or A, X2 is selected from the group consisting of Y or S, X3 is selected from the group consisting of R or L, X4 is selected from the group consisting of Y and Q, and X5 is selected from the group consisting of S or T); or xii) A variable heavy chain containing the amino acid sequence of SEQ ID NO: 70, which differs in amino acid sequence from CDRH2 SEQ ID NO: 338 (where X1 is selected from the group consisting of Y or I) by amino acid substitution of one or fewer amino acids, and a variable light chain containing the amino acid sequence of SEQ ID NO: 75, which differs in amino acid sequence from CDRL1 SEQ ID NO: 669 (where X1 is selected from the group consisting of N or Q) by amino acid substitution of one or fewer amino acids, and which differs in amino acid sequence from CDRL2 SEQ ID NO: 339 (where X1 is selected from the group consisting of L or G) by amino acid substitution of one or fewer amino acids, or xiii) Variable heavy chain region VH containing an amino acid sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 20, SEQ ID NO: 30, SEQ ID NO: 40, SEQ ID NO: 50, and SEQ ID NO: 60, and a light chain variable region VL containing an amino acid sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 25, SEQ ID NO: 35, SEQ ID NO: 45, SEQ ID NO: 55, and SEQ ID NO: 65, or xiv) Variable heavy chain region VH containing an amino acid sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 70, SEQ ID NO: 80, SEQ ID NO: 90, and SEQ ID NO: 100, and light chain variable region VL containing an amino acid sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 75, SEQ ID NO: 85, SEQ ID NO: 95, and SEQ ID NO: 105. xv) The extracellular domain CRD3 of amino acids 87-118 (SEQ ID NO: 352) binds to an epitope on CD137, blocking the binding of any of the above antigen-binding molecules (i)-(xiv) and / or competing for binding with them. xvi) CD137 antigen-binding molecules that bind to epitopes on CD137 with extracellular domains CRD2 / CRD3 of amino acids 46-117 (SEQ ID NO: 356), blocking the binding of any of the above antigen-binding molecules (i)-(xiv) and / or competing for binding with them. It is selected from the group that includes it.

[0031] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site that specifically binds to fibroblast-activating protein (FAP) is as follows: i) Heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 108 (CDR2), and SEQ ID NO: 109 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 111 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3) or ii) Heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 117 (CDR2), and SEQ ID NO: 109 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 120 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3) or iii) Heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 126 (CDR2), and SEQ ID NO: 109 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 129 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3) or iv) Heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 328 (CDR1), SEQ ID NO: 135 (CDR2), and SEQ ID NO: 136 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 138 (CDR1), SEQ ID NO: 139 (CDR2), and SEQ ID NO: 140 (CDR3) or v) Heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 333 (CDR1), SEQ ID NO: 144 (CDR2), and SEQ ID NO: 145 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 138 (CDR1), SEQ ID NO: 139 (CDR2), and SEQ ID NO: 140 (CDR3) or vi) A heavy chain CDR containing the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 341 (CDR2) (where X1 is selected from the group consisting of D or E) and SEQ ID NO: 109 (CDR3), and a light chain CDR containing the amino acid sequences of SEQ ID NO: 691 (CDR1) (where X1 is selected from the group consisting of N, R or S, and X2 is selected from the group consisting of N or S), SEQ ID NO: 112 (CDR2) and SEQ ID NO: 113 (CDR3) or vii) Heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 694 (CDR1) (where X1 is selected from the group consisting of S or N), SEQ ID NO: 342 (CDR2) (where X1 is selected from the group consisting of D or E), and SEQ ID NO: 343 (CDR3) (where X1 is selected from N or E), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 138 (CDR1), SEQ ID NO: 139 (CDR2), and SEQ ID NO: 140 (CDR3), or Selected from a group of scFvs that include [this].

[0032] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site that specifically binds to fibroblast-activating protein (FAP) is as follows: i) A variable heavy chain containing the amino acid sequence of SEQ ID NO: 106 and a variable light chain containing the amino acid sequence of SEQ ID NO: 110 or ii) A variable heavy chain containing the amino acid sequence of SEQ ID NO: 115 and a variable light chain containing the amino acid sequence of SEQ ID NO: 119 or iii) A variable heavy chain containing the amino acid sequence of SEQ ID NO: 124 and a variable light chain containing the amino acid sequence of SEQ ID NO: 128 or iv) A variable heavy chain containing the amino acid sequence of SEQ ID NO: 133 and a variable light chain containing the amino acid sequence of SEQ ID NO: 137 or v) A variable heavy chain containing the amino acid sequence of SEQ ID NO: 142 and a variable light chain containing the amino acid sequence of SEQ ID NO: 146 or vi) A variable heavy chain containing the amino acid sequence of SEQ ID NO: 106, which differs from the amino acid sequence of CDRH2 SEQ ID NO: 341 (where X1 is selected from D or E) by amino acid substitution of one or fewer amino acids, and a variable light chain containing the amino acid sequence of SEQ ID NO: 110, which differs from the amino acid sequence of CDRL1 SEQ ID NO: 691 (where X1 is selected from the group consisting of N, R or S, and X2 is selected from N or S) by amino acid substitution of two or fewer amino acids, or vii) A variable heavy chain containing the amino acid sequence of SEQ ID NO: 133 and a variable light chain containing the amino acid sequence of SEQ ID NO: 137, which differs in amino acid sequence from CDRH1 SEQ ID NO: 694 (where X1 is selected from the group consisting of S or N) by amino acid substitution of one or fewer amino acids and / or differs in amino acid sequence from CDRH2 SEQ ID NO: 342 (where X1 is selected from the group consisting of D or E) by amino acid substitution of one or fewer amino acids and / or differs in amino acid sequence from CDRH3 SEQ ID NO: 343 (where X1 is selected from the group consisting of N or E) by amino acid substitution of one or fewer amino acids or viii) Variable heavy chain region VH containing an amino acid sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 106, SEQ ID NO: 115, and SEQ ID NO: 124, and light chain variable region VL containing an amino acid sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 110, SEQ ID NO: 119, and SEQ ID NO: 128. viv) Variable heavy chain region VH containing an amino acid sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 133 and SEQ ID NO: 142, and light chain variable region VL containing an amino acid sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 137 and SEQ ID NO: 146. Selected from a group of scFvs that include [this].

[0033] Preferred embodiments of the immunoglobulin-like binding molecule of the present invention are as follows: i)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 290 (CDR1), SEQ ID NO: 8 (CDR2), and SEQ ID NO: 9 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 12 (CDR1), SEQ ID NO: 13 (CDR2), and SEQ ID NO: 14 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 108 (CDR2), and SEQ ID NO: 109 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 111 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3) or ii) (a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 290 (CDR1), SEQ ID NO: 8 (CDR2), and SEQ ID NO: 9 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 12 (CDR1), SEQ ID NO: 13 (CDR2), and SEQ ID NO: 14 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 117 (CDR2), and SEQ ID NO: 109 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 120 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3) or iii)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 290 (CDR1), SEQ ID NO: 8 (CDR2), and SEQ ID NO: 9 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 12 (CDR1), SEQ ID NO: 13 (CDR2), and SEQ ID NO: 14 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 126 (CDR2), and SEQ ID NO: 109 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 129 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3) or iv) (a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 290 (CDR1), SEQ ID NO: 8 (CDR2), and SEQ ID NO: 9 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 12 (CDR1), SEQ ID NO: 13 (CDR2), and SEQ ID NO: 14 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 328 (CDR1), SEQ ID NO: 135 (CDR2), and SEQ ID NO: 136 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 138 (CDR1), SEQ ID NO: 139 (CDR2), and SEQ ID NO: 140 (CDR3) or v)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 290 (CDR1), SEQ ID NO: 8 (CDR2), and SEQ ID NO: 9 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 12 (CDR1), SEQ ID NO: 13 (CDR2), and SEQ ID NO: 14 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 333 (CDR1), SEQ ID NO: 144 (CDR2), and SEQ ID NO: 145 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 138 (CDR1), SEQ ID NO: 139 (CDR2), and SEQ ID NO: 140 (CDR3) or (vi)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 18 (CDR2), and SEQ ID NO: 9 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 22 (CDR1), SEQ ID NO: 23 (CDR2), and SEQ ID NO: 14 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 108 (CDR2), and SEQ ID NO: 109 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 111 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3) or (vii)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 18 (CDR2), and SEQ ID NO: 9 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 22 (CDR1), SEQ ID NO: 23 (CDR2), and SEQ ID NO: 14 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 117 (CDR2), and SEQ ID NO: 109 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 120 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3) or (viii)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 18 (CDR2), and SEQ ID NO: 9 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 22 (CDR1), SEQ ID NO: 23 (CDR2), and SEQ ID NO: 14 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 126 (CDR2), and SEQ ID NO: 109 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 129 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3) or (ix)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 18 (CDR2), and SEQ ID NO: 9 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 22 (CDR1), SEQ ID NO: 23 (CDR2), and SEQ ID NO: 14 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 328 (CDR1), SEQ ID NO: 135 (CDR2), and SEQ ID NO: 136 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 138 (CDR1), SEQ ID NO: 139 (CDR2), and SEQ ID NO: 140 (CDR3) or (x)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 18 (CDR2), and SEQ ID NO: 9 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 22 (CDR1), SEQ ID NO: 23 (CDR2), and SEQ ID NO: 14 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 333 (CDR1), SEQ ID NO: 144 (CDR2), and SEQ ID NO: 145 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 138 (CDR1), SEQ ID NO: 139 (CDR2), and SEQ ID NO: 140 (CDR3) or (xi)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 28 (CDR2), and SEQ ID NO: 9 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 32 (CDR1), SEQ ID NO: 33 (CDR2), and SEQ ID NO: 14 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 108 (CDR2), and SEQ ID NO: 109 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 111 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3) or (xii)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 28 (CDR2), and SEQ ID NO: 9 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 32 (CDR1), SEQ ID NO: 33 (CDR2), and SEQ ID NO: 14 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 117 (CDR2), and SEQ ID NO: 109 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 120 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3) or (xiii)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 28 (CDR2), and SEQ ID NO: 9 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 32 (CDR1), SEQ ID NO: 33 (CDR2), and SEQ ID NO: 14 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 126 (CDR2), and SEQ ID NO: 109 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 129 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3) or (xiv)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 28 (CDR2), and SEQ ID NO: 9 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 32 (CDR1), SEQ ID NO: 33 (CDR2), and SEQ ID NO: 14 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 328 (CDR1), SEQ ID NO: 135 (CDR2), and SEQ ID NO: 136 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 138 (CDR1), SEQ ID NO: 139 (CDR2), and SEQ ID NO: 140 (CDR3) or (xv)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 28 (CDR2), and SEQ ID NO: 9 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 32 (CDR1), SEQ ID NO: 33 (CDR2), and SEQ ID NO: 14 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 333 (CDR1), SEQ ID NO: 144 (CDR2), and SEQ ID NO: 145 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 138 (CDR1), SEQ ID NO: 139 (CDR2), and SEQ ID NO: 140 (CDR3) or (xvi)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 38 (CDR2), and SEQ ID NO: 9 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 42 (CDR1), SEQ ID NO: 43 (CDR2), and SEQ ID NO: 14 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 108 (CDR2), and SEQ ID NO: 109 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 111 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3) or (xvii)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 38 (CDR2), and SEQ ID NO: 9 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 42 (CDR1), SEQ ID NO: 43 (CDR2), and SEQ ID NO: 14 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 117 (CDR2), and SEQ ID NO: 109 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 120 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3) or (xviii)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 38 (CDR2), and SEQ ID NO: 9 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 42 (CDR1), SEQ ID NO: 43 (CDR2), and SEQ ID NO: 14 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 126 (CDR2), and SEQ ID NO: 109 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 129 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3) or (xix)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 38 (CDR2), and SEQ ID NO: 9 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 42 (CDR1), SEQ ID NO: 43 (CDR2), and SEQ ID NO: 14 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 328 (CDR1), SEQ ID NO: 135 (CDR2), and SEQ ID NO: 136 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 138 (CDR1), SEQ ID NO: 139 (CDR2), and SEQ ID NO: 140 (CDR3) or (xx)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 38 (CDR2), and SEQ ID NO: 9 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 42 (CDR1), SEQ ID NO: 43 (CDR2), and SEQ ID NO: 14 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 333 (CDR1), SEQ ID NO: 144 (CDR2), and SEQ ID NO: 145 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 138 (CDR1), SEQ ID NO: 139 (CDR2), and SEQ ID NO: 140 (CDR3) or (xxi)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 48 (CDR2), and SEQ ID NO: 9 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 52 (CDR1), SEQ ID NO: 53 (CDR2), and SEQ ID NO: 14 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 108 (CDR2), and SEQ ID NO: 109 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 111 (CDR1), SEQ ID NO: 111 (CDR2), and SEQ ID NO: 113 (CDR3) or (xxii)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 48 (CDR2), and SEQ ID NO: 9 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 52 (CDR1), SEQ ID NO: 53 (CDR2), and SEQ ID NO: 14 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 117 (CDR2), and SEQ ID NO: 109 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 120 (CDR1), SEQ ID NO: 121 (CDR2), and SEQ ID NO: 113 (CDR3) or (xxiii)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 48 (CDR2), and SEQ ID NO: 9 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 52 (CDR1), SEQ ID NO: 53 (CDR2), and SEQ ID NO: 14 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 126 (CDR2), and SEQ ID NO: 109 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 129 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3) or (xxiv)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 48 (CDR2), and SEQ ID NO: 9 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 52 (CDR1), SEQ ID NO: 53 (CDR2), and SEQ ID NO: 14 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 328 (CDR1), SEQ ID NO: 135 (CDR2), and SEQ ID NO: 136 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 138 (CDR1), SEQ ID NO: 139 (CDR2), and SEQ ID NO: 140 (CDR3) or (xxv)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 48 (CDR2), and SEQ ID NO: 9 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 52 (CDR1), SEQ ID NO: 53 (CDR2), and SEQ ID NO: 14 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 333 (CDR1), SEQ ID NO: 144 (CDR2), and SEQ ID NO: 145 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 138 (CDR1), SEQ ID NO: 139 (CDR2), and SEQ ID NO: 140 (CDR3) or (xxvi)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 58 (CDR2), and SEQ ID NO: 9 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 62 (CDR1), SEQ ID NO: 63 (CDR2), and SEQ ID NO: 14 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 108 (CDR2), and SEQ ID NO: 109 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 111 (CDR1), SEQ ID NO: 111 (CDR2), and SEQ ID NO: 113 (CDR3) or (xxvii)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 58 (CDR2), and SEQ ID NO: 9 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 62 (CDR1), SEQ ID NO: 63 (CDR2), and SEQ ID NO: 14 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 117 (CDR2), and SEQ ID NO: 109 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 120 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3) or (xxviii)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 58 (CDR2), and SEQ ID NO: 9 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 62 (CDR1), SEQ ID NO: 63 (CDR2), and SEQ ID NO: 14 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 126 (CDR2), and SEQ ID NO: 109 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 129 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3) or (xxix)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 58 (CDR2), and SEQ ID NO: 9 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 62 (CDR1), SEQ ID NO: 63 (CDR2), and SEQ ID NO: 14 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 328 (CDR1), SEQ ID NO: 135 (CDR2), and SEQ ID NO: 136 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 138 (CDR1), SEQ ID NO: 139 (CDR2), and SEQ ID NO: 140 (CDR3) or (xxx)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 58 (CDR2), and SEQ ID NO: 9 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 62 (CDR1), SEQ ID NO: 63 (CDR2), and SEQ ID NO: 14 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 333 (CDR1), SEQ ID NO: 144 (CDR2), and SEQ ID NO: 145 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 138 (CDR1), SEQ ID NO: 139 (CDR2), and SEQ ID NO: 140 (CDR3) or (xxxi)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 308 (CDR1), SEQ ID NO: 68 (CDR2), and SEQ ID NO: 69 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 72 (CDR1), SEQ ID NO: 73 (CDR2), and SEQ ID NO: 74 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 108 (CDR2), and SEQ ID NO: 109 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 111 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3) or (xxxii)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 308 (CDR1), SEQ ID NO: 68 (CDR2), and SEQ ID NO: 69 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 72 (CDR1), SEQ ID NO: 73 (CDR2), and SEQ ID NO: 74 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 117 (CDR2), and SEQ ID NO: 109 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 120 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3) or (xxxiii)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 308 (CDR1), SEQ ID NO: 68 (CDR2), and SEQ ID NO: 69 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 72 (CDR1), SEQ ID NO: 73 (CDR2), and SEQ ID NO: 74 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 126 (CDR2), and SEQ ID NO: 109 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 129 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3) or (xxxiv)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 308 (CDR1), SEQ ID NO: 68 (CDR2), and SEQ ID NO: 69 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 72 (CDR1), SEQ ID NO: 73 (CDR2), and SEQ ID NO: 74 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 328 (CDR1), SEQ ID NO: 135 (CDR2), and SEQ ID NO: 136 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 138 (CDR1), SEQ ID NO: 139 (CDR2), and SEQ ID NO: 140 (CDR3) or (xxxv)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 308 (CDR1), SEQ ID NO: 68 (CDR2), and SEQ ID NO: 69 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 72 (CDR1), SEQ ID NO: 73 (CDR2), and SEQ ID NO: 74 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 333 (CDR1), SEQ ID NO: 144 (CDR2), and SEQ ID NO: 145 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 138 (CDR1), SEQ ID NO: 139 (CDR2), and SEQ ID NO: 140 (CDR3) or (xxxvi)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 308 (CDR1), SEQ ID NO: 78 (CDR2), and SEQ ID NO: 69 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 72 (CDR1), SEQ ID NO: 73 (CDR2), and SEQ ID NO: 74 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 108 (CDR2), and SEQ ID NO: 109 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 111 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3) or (xxxvii)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 308 (CDR1), SEQ ID NO: 78 (CDR2), and SEQ ID NO: 69 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 72 (CDR1), SEQ ID NO: 73 (CDR2), and SEQ ID NO: 74 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 117 (CDR2), and SEQ ID NO: 109 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 120 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3) or (xxxviii)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 308 (CDR1), SEQ ID NO: 78 (CDR2), and SEQ ID NO: 69 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 72 (CDR1), SEQ ID NO: 73 (CDR2), and SEQ ID NO: 74 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 126 (CDR2), and SEQ ID NO: 109 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 129 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3) or (xxxix)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 308 (CDR1), SEQ ID NO: 78 (CDR2), and SEQ ID NO: 69 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 72 (CDR1), SEQ ID NO: 73 (CDR2), and SEQ ID NO: 74 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 328 (CDR1), SEQ ID NO: 135 (CDR2), and SEQ ID NO: 136 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 138 (CDR1), SEQ ID NO: 139 (CDR2), and SEQ ID NO: 140 (CDR3) or (xl)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 308 (CDR1), SEQ ID NO: 78 (CDR2), and SEQ ID NO: 69 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 72 (CDR1), SEQ ID NO: 73 (CDR2), and SEQ ID NO: 74 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 333 (CDR1), SEQ ID NO: 144 (CDR2), and SEQ ID NO: 145 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 138 (CDR1), SEQ ID NO: 139 (CDR2), and SEQ ID NO: 140 (CDR3) or (xli)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 308 (CDR1), SEQ ID NO: 88 (CDR2), and SEQ ID NO: 69 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 92 (CDR1), SEQ ID NO: 93 (CDR2), and SEQ ID NO: 74 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 108 (CDR2), and SEQ ID NO: 109 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 111 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3) or (xlii)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 308 (CDR1), SEQ ID NO: 88 (CDR2), and SEQ ID NO: 69 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 92 (CDR1), SEQ ID NO: 93 (CDR2), and SEQ ID NO: 74 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 117 (CDR2), and SEQ ID NO: 109 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 120 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3) or (xliii)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 308 (CDR1), SEQ ID NO: 88 (CDR2), and SEQ ID NO: 69 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 92 (CDR1), SEQ ID NO: 93 (CDR2), and SEQ ID NO: 74 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 126 (CDR2), and SEQ ID NO: 109 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 129 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3) or (xliv)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 308 (CDR1), SEQ ID NO: 88 (CDR2), and SEQ ID NO: 69 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 92 (CDR1), SEQ ID NO: 93 (CDR2), and SEQ ID NO: 74 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 328 (CDR1), SEQ ID NO: 135 (CDR2), and SEQ ID NO: 136 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 138 (CDR1), SEQ ID NO: 139 (CDR2), and SEQ ID NO: 140 (CDR3) or (xlv)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 308 (CDR1), SEQ ID NO: 88 (CDR2), and SEQ ID NO: 69 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 92 (CDR1), SEQ ID NO: 93 (CDR2), and SEQ ID NO: 74 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 333 (CDR1), SEQ ID NO: 144 (CDR2), and SEQ ID NO: 145 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 138 (CDR1), SEQ ID NO: 139 (CDR2), and SEQ ID NO: 140 (CDR3) or (xlvi)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 308 (CDR1), SEQ ID NO: 98 (CDR2), and SEQ ID NO: 69 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 92 (CDR1), SEQ ID NO: 93 (CDR2), and SEQ ID NO: 74 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 108 (CDR2), and SEQ ID NO: 109 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 111 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3) or (xlvii)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 308 (CDR1), SEQ ID NO: 98 (CDR2), and SEQ ID NO: 69 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 92 (CDR1), SEQ ID NO: 93 (CDR2), and SEQ ID NO: 74 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 117 (CDR2), and SEQ ID NO: 109 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 120 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3) or (xlviii)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 308 (CDR1), SEQ ID NO: 98 (CDR2), and SEQ ID NO: 69 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 92 (CDR1), SEQ ID NO: 93 (CDR2), and SEQ ID NO: 74 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 126 (CDR2), and SEQ ID NO: 109 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 129 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3) or (xlix)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 308 (CDR1), SEQ ID NO: 98 (CDR2), and SEQ ID NO: 69 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 92 (CDR1), SEQ ID NO: 93 (CDR2), and SEQ ID NO: 74 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 328 (CDR1), SEQ ID NO: 135 (CDR2), and SEQ ID NO: 136 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 138 (CDR1), SEQ ID NO: 139 (CDR2), and SEQ ID NO: 140 (CDR3) or (l)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 308 (CDR1), SEQ ID NO: 98 (CDR2), and SEQ ID NO: 69 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 92 (CDR1), SEQ ID NO: 93 (CDR2), and SEQ ID NO: 74 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 333 (CDR1), SEQ ID NO: 144 (CDR2), and SEQ ID NO: 145 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 138 (CDR1), SEQ ID NO: 139 (CDR2), and SEQ ID NO: 140 (CDR3) or (li)(a) Heavy chain CDRs containing the amino acid sequences of (i)(a)Sequence ID: 672(CDR1) (where X1 is A or S), Sequence ID: 335(CDR2) (where X1 is selected from the group consisting of N or Q, X2 is selected from the group consisting of D or E, X3 is selected from the group consisting of G or A, X4 is selected from the group consisting of T or K, X5 is selected from the group consisting of L or V, X6 is selected from the group consisting of D or E, X7 is selected from the group consisting of L or V, and X8 is selected from the group consisting of S or G) and Sequence ID: 9(CDR3), and Sequence ID: 336(CDR1) (here The CD137 binding site includes a light chain CDR containing the amino acid sequences of SEQ ID NO: 337 (CDR2) (where X1 is selected from the group consisting of K or R, X2 is selected from the group consisting of D or S, X3 is selected from the group consisting of V or I, X4 is selected from the group consisting of S or T, and X5 is selected from the group consisting of V and L), SEQ ID NO: 14 (CDR3), and the CD137 binding site including the light chain CDR. (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 341 (CDR2) (where X1 is selected from D or E) and SEQ ID NO: 109 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 691 (CDR1) (where X1 is selected from the group consisting of N, R or S, and X2 is selected from the group consisting of N or S), SEQ ID NO: 112 (CDR2) and SEQ ID NO: 113 (CDR3) or (lii)(a) Heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 672 (CDR1) (where X1 is selected from the group consisting of A or S), SEQ ID NO: 335 (CDR2) (where X1 is selected from the group consisting of N or Q, X2 is selected from the group consisting of D or E, X3 is selected from the group consisting of G or A, X4 is selected from the group consisting of T or K, X5 is selected from the group consisting of L or V, X6 is selected from the group consisting of D or E, X7 is selected from the group consisting of L or V, and X8 is selected from the group consisting of S or G), and SEQ ID NO: 9 (CDR3), as well as SEQ ID NO: 336 (CDR 1) CD137 binding site including a light chain CDR containing the amino acid sequences of 1) (wherein X1 is selected from the group consisting of K or R, X2 is selected from the group consisting of D or S, X3 is selected from the group consisting of V or I, X4 is selected from the group consisting of S or T, and X5 is selected from the group consisting of V and L), SEQ ID NO: 337 (CDR2) (wherein X1 is selected from the group consisting of S or A, X2 is selected from the group consisting of Y or S, X3 is selected from the group consisting of R or L, X4 is selected from the group consisting of Y and Q, and X5 is selected from the group consisting of S or T), and SEQ ID NO: 14 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 694 (CDR1) (wherein X1 is selected from the group consisting of S or N), SEQ ID NO: 342 (CDR2) (wherein X1 is selected from the group consisting of D or E), and SEQ ID NO: 343 (CDR3) (wherein X1 is selected from N or E), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 138 (CDR1), SEQ ID NO: 139 (CDR2), and SEQ ID NO: 140 (CDR3), or (liii)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 308 (CDR1), SEQ ID NO: 338 (CDR2) (where X1 is selected from the group consisting of Y or I) and SEQ ID NO: 69 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 669 (CDR1) (where X1 is selected from the group consisting of N or Q), SEQ ID NO: 339 (CDR2) (where X1 is selected from the group consisting of L or G) and SEQ ID NO: 74 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 341 (CDR2) (where X1 is selected from the group consisting of D or E) and SEQ ID NO: 109 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 691 (CDR1) (where X1 is selected from the group consisting of N, R or S, and X2 is selected from the group consisting of N or S), SEQ ID NO: 112 (CDR2) and SEQ ID NO: 113 (CDR3) or (lix)(a) CD137 binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 308 (CDR1), SEQ ID NO: 338 (CDR2) (where X1 is selected from the group consisting of Y or I) and SEQ ID NO: 69 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 669 (CDR1) (where X1 is selected from the group consisting of N or Q), SEQ ID NO: 339 (CDR2) (where X1 is selected from the group consisting of L or G) and SEQ ID NO: 74 (CDR3), and (b) FAP binding sites including heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 694 (CDR1) (where X1 is selected from the group consisting of S or N), SEQ ID NO: 342 (CDR2) (where X1 is selected from the group consisting of D or E), and SEQ ID NO: 343 (CDR3) (where X1 is selected from N or E), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 138 (CDR1), SEQ ID NO: 139 (CDR2), and SEQ ID NO: 140 (CDR3). It includes a first antigen-binding site that specifically binds to CD137(4-1BB) and a second antigen-binding site that specifically binds to fibroblast-activating protein (FAP), selected from the group including the above.

[0034] Further aspects of the present invention are described below: (i) an immunoglobulin heavy chain containing the amino acid sequence of SEQ ID NO: 151 and fused to scFv at its C-terminus, and a light chain containing the amino acid sequence of SEQ ID NO: 152 or (ii) an immunoglobulin heavy chain containing the amino acid sequence of SEQ ID NO: 153 and fused to scFv at its C-terminus, and a light chain containing the amino acid sequence of SEQ ID NO: 154 or (iii) an immunoglobulin heavy chain containing the amino acid sequence of SEQ ID NO: 155 and fused to scFv at its C-terminus, and a light chain containing the amino acid sequence of SEQ ID NO: 156 or (iv) an immunoglobulin heavy chain containing the amino acid sequence of SEQ ID NO: 157 and fused to scFv at its C-terminus, and a light chain containing the amino acid sequence of SEQ ID NO: 158 or (v) an immunoglobulin heavy chain containing the amino acid sequence of SEQ ID NO: 159 and fused to scFv at its C-terminus, and a light chain containing the amino acid sequence of SEQ ID NO: 160 or (vi) an immunoglobulin heavy chain containing the amino acid sequence of SEQ ID NO: 164 and fused to scFv at its C-terminus, and a light chain containing the amino acid sequence of SEQ ID NO: 165 or (vii) an immunoglobulin heavy chain containing the amino acid sequence of SEQ ID NO: 169 and fused to scFv at its C-terminus, and a light chain containing the amino acid sequence of SEQ ID NO: 170 or (viii) an immunoglobulin heavy chain containing the amino acid sequence of SEQ ID NO: 174 and fused to scFv at its C-terminus, and a light chain containing the amino acid sequence of SEQ ID NO: 175 or (ix) An immunoglobulin heavy chain containing the amino acid sequence of SEQ ID NO: 179 and fused to scFv at its C-terminus, and a light chain containing the amino acid sequence of SEQ ID NO: 180 or (x) an immunoglobulin heavy chain containing the amino acid sequence of SEQ ID NO: 184 and fused to scFv at its C-terminus, and a light chain containing the amino acid sequence of SEQ ID NO: 185 or (xi) an immunoglobulin heavy chain containing the amino acid sequence of SEQ ID NO: 189 and fused to scFv at its C-terminus, and a light chain containing the amino acid sequence of SEQ ID NO: 190 or (xii) An immunoglobulin heavy chain containing the amino acid sequence of SEQ ID NO: 194 and fused to scFv at its C-terminus, and a light chain containing the amino acid sequence of SEQ ID NO: 195 or (xiii) an immunoglobulin heavy chain containing the amino acid sequence of SEQ ID NO: 199 and fused to scFv at its C-terminus, and a light chain containing the amino acid sequence of SEQ ID NO: 200 or (xiv) An immunoglobulin heavy chain containing the amino acid sequence of SEQ ID NO: 204 and fused to scFv at its C-terminus, and a light chain containing the amino acid sequence of SEQ ID NO: 205 or (xv) An immunoglobulin heavy chain containing the amino acid sequence of SEQ ID NO: 209 and fused to scFv at its C-terminus, and a light chain containing the amino acid sequence of SEQ ID NO: 210 or (xvi) An immunoglobulin heavy chain containing the amino acid sequence of SEQ ID NO: 214 and fused to scFv at its C-terminus, and a light chain containing the amino acid sequence of SEQ ID NO: 215 or (xvii) An immunoglobulin heavy chain containing the amino acid sequence of SEQ ID NO: 219 and fused to scFv at its C-terminus, and a light chain containing the amino acid sequence of SEQ ID NO: 221 or (xviii) An immunoglobulin heavy chain containing the amino acid sequence of SEQ ID NO: 224 and fused to scFv at its C-terminus, and a light chain containing the amino acid sequence of SEQ ID NO: 225 or (xix) An immunoglobulin heavy chain containing the amino acid sequence of SEQ ID NO: 229 and fused to scFv at its C-terminus, and a light chain containing the amino acid sequence of SEQ ID NO: 230 or (xx) An immunoglobulin heavy chain containing the amino acid sequence of SEQ ID NO: 234 and fused to scFv at its C-terminus, and a light chain containing the amino acid sequence of SEQ ID NO: 235 or (xvii) An immunoglobulin heavy chain containing the amino acid sequence of SEQ ID NO: 239 and fused to scFv at its C-terminus, and a light chain containing the amino acid sequence of SEQ ID NO: 240 or (xviii) An immunoglobulin heavy chain containing the amino acid sequence of SEQ ID NO: 244 and fused to scFv at its C-terminus, and a light chain containing the amino acid sequence of SEQ ID NO: 245 or (xix) An immunoglobulin heavy chain containing the amino acid sequence of SEQ ID NO: 249, fused to scFv at its C-terminus, and a light chain containing the amino acid sequence of SEQ ID NO: 250 or (xx) An immunoglobulin heavy chain containing the amino acid sequence of SEQ ID NO: 254 and fused to scFv at its C-terminus, and a light chain containing the amino acid sequence of SEQ ID NO: 255 or (xxi) An immunoglobulin heavy chain containing the amino acid sequence of SEQ ID NO: 259 and fused to scFv at its C-terminus, and a light chain containing the amino acid sequence of SEQ ID NO: 260 or (xxii) An immunoglobulin heavy chain containing the amino acid sequence of SEQ ID NO: 264 and fused to scFv at its C-terminus, and a light chain containing the amino acid sequence of SEQ ID NO: 265 or (xxiii) An immunoglobulin heavy chain containing the amino acid sequence of SEQ ID NO: 269 and fused to scFv at its C-terminus, and a light chain containing the amino acid sequence of SEQ ID NO: 270 or (xxiv) An immunoglobulin heavy chain containing the amino acid sequence of SEQ ID NO: 274 and fused to scFv at its C-terminus, and a light chain containing the amino acid sequence of SEQ ID NO: 275 or (xxv)(a) A first polypeptide comprising an immunoglobulin heavy chain having the amino acid sequence of SEQ ID NO: 151 and fused to scFv at its C-terminus, wherein the variable heavy chain portion that specifically binds to CD137 is determined by amino acid substitution of one or fewer amino acids to form CDRH1, which has a different amino acid sequence from SEQ ID NO: 672 (where X1 is selected from the group consisting of A or S) and / or by amino acid substitution of four or fewer amino acids to form CDRH2 Sequence ID: 335 (where X1 is selected from the group consisting of N or Q, X2 is selected from the group consisting of D or E, X3 is selected from the group consisting of G or A, X4 is selected from the group consisting of T or K, X5 is selected from the group consisting of L or V, X6 is selected from the group consisting of D or E, X7 is selected from the group consisting of L or V, and X8 is selected from the group consisting of S or G) has a different amino acid sequence, where the scFv is an FAP-binding scFv containing a heavy chain domain (VH) and a light chain domain, where the FAP VH is CDRH2 by amino acid substitution of one or fewer amino acids Sequence ID: 341 (where X1 is selected from D or E) has a different amino acid sequence, where the FAP VL is CDRL1 by amino acid substitution of two or fewer amino acids A first polypeptide with an amino acid sequence different from Sequence ID: 691 (where X1 is selected from the group consisting of N, R, or S, and X2 is selected from the group consisting of N or S), and (b) A second polypeptide containing a light chain domain (VL) that specifically binds to CD137, having an amino acid sequence different from CDRL1 SEQ ID NO: 336 (wherein X1 is selected from the group consisting of K or R, X2 is selected from the group consisting of D or S, X3 is selected from the group consisting of V or I, X4 is selected from the group consisting of S or T, and X5 is selected from the group consisting of V and L) by amino acid substitution of 4 or fewer amino acids, and / or having an amino acid sequence different from CDRL2 SEQ ID NO: 337 (wherein X1 is selected from the group consisting of S or A, X2 is selected from the group consisting of Y or S, X3 is selected from the group consisting of R or L, X4 is selected from the group consisting of Y and Q, and X5 is selected from the group consisting of S or T) by amino acid substitution of 5 or fewer amino acids, or (xxvi)(a) A first polypeptide comprising an immunoglobulin heavy chain having the amino acid sequence of SEQ ID NO: 155 and fused to scFv at its C-terminus, wherein the variable heavy chain portion that specifically binds to CD137 is determined by amino acid substitution of one or fewer amino acids to form CDRH1, which has a different amino acid sequence from SEQ ID NO: 672 (where X1 is selected from the group consisting of A or S) and / or is determined by amino acid substitution of four or fewer amino acids to form CDRH2 Unlike Sequence ID: 335 (where X1 is selected from the group consisting of N or Q, X2 is selected from the group consisting of D or E, X3 is selected from the group consisting of G or A, X4 is selected from the group consisting of T or K, X5 is selected from the group consisting of L or V, X6 is selected from the group consisting of D or E, X7 is selected from the group consisting of L or V, and X8 is selected from the group consisting of S or G), here the scFv is an FAP-bound scFv containing a heavy chain domain (VH) and a light chain domain (VL), and here the FAP VH is CDRH1 by amino acid substitution of one or fewer amino acids, has a different amino acid sequence from Sequence ID: 694 (where X1 is selected from the group consisting of S or N) and / or CDRH2 by amino acid substitution of one or fewer amino acids, has a different amino acid sequence from Sequence ID: 342 (where X1 is selected from the group consisting of D or E) and / or CDRH3 by amino acid substitution of one or fewer amino acids A first polypeptide with an amino acid sequence different from Sequence ID: 343 (where X1 is selected from the group consisting of N or E), and (b) A second polypeptide containing a light chain that specifically binds to CD137, having an amino acid sequence different from CDRL1 SEQ ID NO: 336 (wherein X1 is selected from the group consisting of K or R, X2 is selected from the group consisting of D or S, X3 is selected from the group consisting of V or I, X4 is selected from the group consisting of S or T, and X5 is selected from the group consisting of V and L) by amino acid substitution of 4 or fewer amino acids, and / or having an amino acid sequence different from CDRL2 SEQ ID NO: 337 (wherein X1 is selected from the group consisting of S or A, X2 is selected from the group consisting of S or Y, X3 is selected from the group consisting of S or Y, X4 is selected from the group consisting of Y or Q, and X5 is selected from the group consisting of S or T) by amino acid substitution of 5 or fewer amino acids, or (xxvii)(a) A first polypeptide comprising an immunoglobulin heavy chain fused to an scFv at its C-terminus, wherein the variable heavy chain portion that specifically binds to CD137 has a different amino acid sequence from CDRH2 SEQ ID NO: 338 (where X1 is selected from the group consisting of Y or I) by amino acid substitution of one or fewer amino acids, wherein the scFv is an FAP-binding scFv comprising a heavy chain domain (VH) and a light chain domain, wherein the FAP VH has a different amino acid sequence from CDRH2 SEQ ID NO: 341 (where X1 is selected from D or E) by amino acid substitution of one or fewer amino acids, and the FAP VL has a different amino acid sequence from CDRL1 SEQ ID NO: 691 (where X1 is selected from the group consisting of N, R or S, and X2 is selected from the group consisting of N or S) by amino acid substitution of two or fewer amino acids, and (b) A second polypeptide containing a light chain that specifically binds to CD137, having an amino acid sequence different from CDRL1 SEQ ID NO: 669 (where X1 is selected from the group consisting of N or Q) by one or fewer amino acid substitutions, and / or having an amino acid sequence different from CDRL2 SEQ ID NO: 339 (where X1 is selected from the group consisting of L or G) by one or fewer amino acid substitutions, or (xxviii)(a) A first polypeptide comprising an immunoglobulin heavy chain fused to an scFv at its C-terminus, wherein the variable heavy chain portion that specifically binds to CD137 has a different amino acid sequence from CDRH2 SEQ ID NO: 338 (where X1 is selected from the group consisting of Y or I) by amino acid substitution of one or fewer amino acids, wherein the scFv is an FAP-binding scFv comprising a heavy chain domain (VH) and a light chain domain (VL), wherein the FAP VH has a different amino acid sequence from CDRH2 SEQ ID NO: 341 (where X1 is selected from D or E) by amino acid substitution of one or fewer amino acids, and the FAP VL has a different amino acid sequence from CDRL1 SEQ ID NO: 691 (where X1 is selected from the group consisting of N, R or S, and X2 is selected from the group consisting of N or S) by amino acid substitution of two or fewer amino acids, and (b) A second polypeptide containing a light chain that specifically binds to CD137, having an amino acid sequence different from CDRL1 SEQ ID NO: 669 (where X1 is selected from the group consisting of N or Q) by one or fewer amino acid substitutions, and / or having an amino acid sequence different from CDRL2 SEQ ID NO: 339 (where X1 is selected from the group consisting of L or G) by one or fewer amino acid substitutions, the amino acid sequence of SEQ ID NO: 154. This invention provides immunoglobulin-like binding molecules, including [specific component].

[0035] Therefore, in a further embodiment, the present invention is (i) polypeptides that can specifically bind to CD137, (a) Heavy chain variable region VH containing an amino acid sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 20, SEQ ID NO: 30, SEQ ID NO: 40, SEQ ID NO: 50, and SEQ ID NO: 60, and light chain variable region VL containing an amino acid sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 25, SEQ ID NO: 35, SEQ ID NO: 45, SEQ ID NO: 55, and SEQ ID NO: 65, or (b) Variable heavy chain region VH containing an amino acid sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 70, SEQ ID NO: 80, SEQ ID NO: 90, and SEQ ID NO: 100, and a light chain variable region VL containing an amino acid sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 75, SEQ ID NO: 85, SEQ ID NO: 95, and SEQ ID NO: 105 Includes, (ii) polypeptides that can specifically bind to FAP, (a) Variable heavy chain region VH containing an amino acid sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 106, SEQ ID NO: 115, and SEQ ID NO: 124, and light chain variable region VL containing an amino acid sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 110, SEQ ID NO: 119, and SEQ ID NO: 128 (b) Variable heavy chain region VH containing an amino acid sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 133 and SEQ ID NO: 142, and light chain variable region VL containing an amino acid sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 137 and SEQ ID NO: 146. This invention provides a bispecific antigen-binding molecule that includes [the specified component].

[0036] Further aspects of the present invention provide one or more nucleic acid molecules encoding the immunoglobulin-like binding molecules of the present invention, or one or more expression vectors containing such nucleic acid molecules. The terms “vector” or “expression vector” are synonymous with “expression construct” and refer to a DNA molecule used to introduce a specific gene of relevant manipulative properties into a target cell and to direct its expression. The term includes vectors as self-replicating nucleic acid constructs and vectors incorporated into the genome of a host cell into which they are introduced. The expression vectors of the present invention include an expression cassette. The term “expression cassette” refers to a polynucleotide that is recombinant or synthetically produced with a set of specific nucleic acid elements that enable the transcription of a particular nucleic acid in a target cell. Recombinant expression cassettes can be incorporated into plasmids, chromosomes, mitochondrial DNA, plasmid DNA, viruses, or nucleic acid fragments. Typically, the recombinant expression cassette portion of an expression vector includes the nucleic acid sequence to be transcribed and a promoter, among other sequences. In certain embodiments, the expression cassette of the present invention comprises a polynucleotide sequence encoding the bispecific antigen-binding molecule or a fragment thereof of the present invention, and typically, multiple expression vectors encoding different parts of the immunoglobulin-like binding molecule of the present invention (e.g., the heavy and light chains are encoded by different expression vectors) are simultaneously transfected into the same host cell. The expression vectors enable the transcription of large amounts of stable mRNA. Once the expression vectors enter the target cell, the ribonucleic acid molecules or proteins encoded by the genes are produced by the cell's transcription and / or translation machinery. In one embodiment, the expression vector or vector of the present invention comprises an expression cassette or cassette containing a polynucleotide sequence encoding the bispecific antigen-binding molecule or a fragment thereof of the present invention.

[0037] Further aspects of the present invention provide host cells containing the nucleic acid molecule of the present invention functionally associated with an expression regulatory sequence. The terms “host cell,” “host cell lineage,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced (including offspring of such cells). Host cells include “transformed” and “transformed cells,” which include transformed primary cells and their offspring, regardless of the number of passages. Offspring may not be exactly identical to parent cells in nucleic acid content and may contain mutations. Mutant offspring having the same function or biological activity as those screened or selected in the initially transformed cells are included herein. Host cells are any type of cell lineage that can be used to generate the bispecific antigen-binding molecule of the present invention. Host cells include cultured cells, e.g., mammalian cultured cells, e.g., CHO cells or hybridoma cells, yeast cells, insect cells, and plant cells, and, to name just a few, cells contained within transgenic animals or animal tissues.

[0038] A further aspect of the present invention is a method for producing the binding molecule of the present invention as described herein. (a) A step of culturing the host cells of the present invention under conditions that enable the expression of the molecule, (b) A method is provided which includes the step of recovering the molecule.

[0039] A further aspect of the present invention provides an immunoglobulin-like binding molecule for use in pharmaceuticals.

[0040] As used herein, the term "cancer" refers to proliferative diseases such as lymphoma, lymphocytic leukemia, lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, gastric cancer. This includes cancers such as colon cancer, breast cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvis cancer, mesothelioma, hepatocellular carcinoma, biliary tract cancer, neoplasms of the central nervous system (CNS), spinal axial tumors, brainstem gliomas, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, and Ewing's sarcoma (including refractory versions of any of the above cancers) or a combination of one or more of the above cancers. Further aspects of the present invention provide immunoglobulin-like binding molecules for use in the treatment of cancer, preferably colorectal cancer (CRC) (e.g., colorectal adenocarcinoma), gastric cancer (GC) (e.g., gastric adenocarcinoma), pancreatic cancer (PAC) (e.g., pancreatic adenocarcinoma), and lung cancer (LC) (e.g., squamous cell carcinoma of the lung, lung adenocarcinoma).

[0041] Further aspects of the present invention provide a pharmaceutical composition comprising an immunoglobulin-like binding molecule described in any one embodiment of the present invention, together with a pharmaceutically acceptable carrier and optionally one or more further active ingredients, such as chemotherapeutic agents, radiotherapy agents and / or other agents for use in cancer immunotherapy.

[0042] In one embodiment, a pharmaceutical composition is provided comprising a bispecific immunoglobulin-like conjugate molecule according to any one of the described embodiments or the immunoglobulin-like conjugate molecule of the present invention for use in stimulating a T cell response, supporting the survival and / or recruitment of activated T cells, suppressing and / or preventing anergy of immune cells; treating infections, treating cancer, delaying the progression of cancer, and / or extending the survival of patients with cancer.

[0043] A further aspect of the present invention provides a method for treating cancer, comprising administering an effective amount of the immunoglobulin-like binding molecule of the present invention to a patient in need. [Brief explanation of the drawing]

[0044] [Figure 1A] Figures 1A-1B: A. Schematic diagram of the Doppelmab immunoglobulin-like binding molecule of the present invention. B. Schematic diagram of the mechanism of action of the CD137 / FAP binding molecule in tumor stroma. In this case, the CD137 agonist activity will be limited to the microenvironment that expresses FAP+ stromal cells. [Figure 1B] Figures 1A-1B: A. Schematic diagram of the Doppelmab immunoglobulin-like binding molecule of the present invention. B. Schematic diagram of the mechanism of action of the CD137 / FAP binding molecule in tumor stroma. In this case, the CD137 agonist activity will be limited to the microenvironment that expresses FAP+ stromal cells. [Figure 2A] Figures 2A-2B: A. Binding of anti-human CD137 antibodies to surface-expressed cynomolgus monkey CD137: Representative clones of CD137 #B13, B17, B19, B20, B21, B27, B28, B30 and B31 expressed as average fluorescence intensity against increasing Ab concentration (nM); B. Binding of anti-human CD137 antibodies to surface-expressed human CD137: Representative clones of CD137 #B13, B17, B19, B20, B21, B27, B28, B30 and B31 expressed as average fluorescence intensity against increasing Ab concentration (nM). [Figure 2B] Figures 2A-2B: A. Binding of anti-human CD137 antibodies to surface-expressed cynomolgus monkey CD137: Representative clones of CD137 #B13, B17, B19, B20, B21, B27, B28, B30 and B31 expressed as average fluorescence intensity against increasing Ab concentration (nM); B. Binding of anti-human CD137 antibodies to surface-expressed human CD137: Representative clones of CD137 #B13, B17, B19, B20, B21, B27, B28, B30 and B31 expressed as average fluorescence intensity against increasing Ab concentration (nM). [Figure 3A] Figures 3A-3D: Crosslinking-dependent activity of the CD137 molecule. A. EC50 graphs of anti-CD137 purified chimeric IgG agonist activation with and without secondary antibody crosslinking (CL+). Representative clones of CD137 #A16, A17, A18, A19, A20, A21, A49, A51, A50, A53, A54, and A57. B. Bar graphs of anti-CD137 purified chimeric IgG agonist activation with and without secondary antibody. (C) Activity in the Jurkat NFκB assay, measured as RLU, which shows agonist activity only with secondary antibody crosslinking. Positive controls were urelumab chimeric IgG1-KO and utomirumab as IgG1-KO. Representative anti-CD137 clones include #B2, B5, B7, B9, B10, B12, B13, B17, B19, B27, B30, B31, A1, A13, A2, A25, A3, A30, A39, A4, A47, B3, B4, B21, A12, A16, A17, A19, A26, A27, A41, A44, A45, A46, A49, A57, A8, A34, and A35. C. Activity in the Jurkat NFκB assay, measured as RLU, which shows agonist activity only without secondary antibody crosslinking. [Figure 3B]Figures 3A-3D: Crosslinking-dependent activity of the CD137 molecule. A. EC50 graphs of anti-CD137 purified chimeric IgG agonist activation with and without secondary antibody crosslinking (CL+). Representative clones of CD137 #A16, A17, A18, A19, A20, A21, A49, A51, A50, A53, A54, and A57. B. Bar graphs of anti-CD137 purified chimeric IgG agonist activation with and without secondary antibody. (C) Activity in the Jurkat NFκB assay, measured as RLU, which shows agonist activity only with secondary antibody crosslinking. Positive controls were urelumab chimeric IgG1-KO and utomirumab as IgG1-KO. Representative anti-CD137 clones include #B2, B5, B7, B9, B10, B12, B13, B17, B19, B27, B30, B31, A1, A13, A2, A25, A3, A30, A39, A4, A47, B3, B4, B21, A12, A16, A17, A19, A26, A27, A41, A44, A45, A46, A49, A57, A8, A34, and A35. C. Activity in the Jurkat NFκB assay, measured as RLU, which shows agonist activity only without secondary antibody crosslinking. [Figure 3C]Figures 3A-3D: Crosslinking-dependent activity of the CD137 molecule. A. EC50 graphs of anti-CD137 purified chimeric IgG agonist activation with and without secondary antibody crosslinking (CL+). Representative clones of CD137 #A16, A17, A18, A19, A20, A21, A49, A51, A50, A53, A54, and A57. B. Bar graphs of anti-CD137 purified chimeric IgG agonist activation with and without secondary antibody. (C) Activity in the Jurkat NFκB assay, measured as RLU, which shows agonist activity only with secondary antibody crosslinking. Positive controls were urelumab chimeric IgG1-KO and utomirumab as IgG1-KO. Representative anti-CD137 clones include #B2, B5, B7, B9, B10, B12, B13, B17, B19, B27, B30, B31, A1, A13, A2, A25, A3, A30, A39, A4, A47, B3, B4, B21, A12, A16, A17, A19, A26, A27, A41, A44, A45, A46, A49, A57, A8, A34, and A35. C. Activity in the Jurkat NFκB assay, measured as RLU, which shows agonist activity only without secondary antibody crosslinking. [Figure 3D]Figures 3A-3D: Crosslinking-dependent activity of the CD137 molecule. A. EC50 graphs of anti-CD137 purified chimeric IgG agonist activation with and without secondary antibody crosslinking (CL+). Representative clones of CD137 #A16, A17, A18, A19, A20, A21, A49, A51, A50, A53, A54, and A57. B. Bar graphs of anti-CD137 purified chimeric IgG agonist activation with and without secondary antibody. (C) Activity in the Jurkat NFκB assay, measured as RLU, which shows agonist activity only with secondary antibody crosslinking. Positive controls were urelumab chimeric IgG1-KO and utomirumab as IgG1-KO. Representative anti-CD137 clones include #B2, B5, B7, B9, B10, B12, B13, B17, B19, B27, B30, B31, A1, A13, A2, A25, A3, A30, A39, A4, A47, B3, B4, B21, A12, A16, A17, A19, A26, A27, A41, A44, A45, A46, A49, A57, A8, A34, and A35. C. Activity in the Jurkat NFκB assay, measured as RLU, which shows agonist activity only without secondary antibody crosslinking. [Figure 4A] Figures 4A-4B: A. Schematic diagram of a 2D assay in which bound CD137 Fab fragments are incubated in the presence of biotinylated huCD137 antigen, and the bound biotinylated antigen is detected with streptavidin-labeled flurochrome. B. ELISA titration of graft Fab against chimeric B21 Fab. Various graft preparations were evaluated by ELISA binding, and chimeric Fab showed better binding compared to graft Fab. 2H11a-unrelated Fab was retained as a negative control. [Figure 4B]Figures 4A-4B: A. Schematic diagram of a 2D assay in which bound CD137 Fab fragments are incubated in the presence of biotinylated huCD137 antigen, and the bound biotinylated antigen is detected with streptavidin-labeled flurochrome. B. ELISA titration of graft Fab against chimeric B21 Fab. Various graft preparations were evaluated by ELISA binding, and chimeric Fab showed better binding compared to graft Fab. 2H11a-unrelated Fab was retained as a negative control. [Figure 5] Figure 5: ELISA of top-framework optimized Fabs of CD137 clones. Fabs identified based on binding in the ELISA assay were evaluated for binding to chimeric CD137 B21 clone Fabs. The binding analysis of Fabs to human CD137 is shown for 22 candidate molecules from a VK / VH combination library (including B21.V55, V49, V51, V69, V47, V61, V72, V48, V56, V75, V54, V50, V71, V53, V70, V63, V74, V52, V62, and V61) arranged compared to the parent B21 parent molecule. [Figure 6A] Figures 6A-6B: A. Binding analysis of anti-CD137 candidates after mutational modification of Vk-CDR. Each bar represents the position of amino acid modification within the variable light chain. B. Binding analysis of anti-CD137 candidates after mutational modification of VH-CDR. Each bar represents the position of amino acid modification within the variable heavy chain and the corresponding OD. [Figure 6B] Figures 6A-6B: A. Binding analysis of anti-CD137 candidates after mutational modification of Vk-CDR. Each bar represents the position of amino acid modification within the variable light chain. B. Binding analysis of anti-CD137 candidates after mutational modification of VH-CDR. Each bar represents the position of amino acid modification within the variable heavy chain and the corresponding OD. [Figure 7]Figure 7. ELISA results comparing the binding of anti-CD137 Fabs incorporating Vk and VH amino acid modification combinations. Subsequently, the Fabs identified based on the primary binding ELISA assay were evaluated for binding to chimeric B21-chimeric parent Fabs. Binding analysis of Fabs to human CD137 is shown for 15 positive binders from a VK / VH combination library, including V68, V73, V74, V14, V18, V64, V15, V17, V16, V67, V75, V66, V62, V71, and V72, compared to the parent B21 chimeric Fab parent. [Figure 8A] Figures 8A-8B. A. Crosslink-dependent activity of optimized anti-CD137 candidates after Vk and VH optimization. A and B: Agonist activation with and without (A) secondary antibody crosslinking, as measured by NFκB activity in the Jurkat assay, expressed as RLU. Representative CD137 clones B21, V1-V40 and A49, V41-V48 compared to parental clones. [Figure 8B] Figures 8A-8B. A. Crosslink-dependent activity of optimized anti-CD137 candidates after Vk and VH optimization. A and B: Agonist activation with and without (A) secondary antibody crosslinking, as measured by NFκB activity in the Jurkat assay, expressed as RLU. Representative CD137 clones B21, V1-V40 and A49, V41-V48 compared to parental clones. [Figure 9] Figure 9. Epitope mapping data of anti-CD137 binding candidates. CD137 antibodies were able to bind to full-length human CD137-expressing Jurkat cell lines, but binding was lost when various human CRDs (huCD137) were systematically replaced with mouse CRDs (mCRD1, mCRD2, mCRD3, and mCRD4). Each CD137 clonal variant is labeled in the left panel. Epitope binding is illustrated as a gradient scale (as shown on the right). Here, "black" = +binding and "white" = no binding. [Figure 10A]Figures 10A-10B. Effects of CD137-binding molecules on inhibiting the activation of human CD137-Jurkat cells induced by CD137 ligand-expressing cells. (A) Various CD137-binding molecules differentially affect the activation of human CD137-expressing Jurkat cells in the presence of 293 cells expressing human CD137 ligand. BMSAB, PfizerAb, and the Roche split-trimeric-4-1BBL(71-248) / FAP(28H1) molecule (see "Construction 2.11", WO 2017 / 194438) inhibit the activation of CD137 ligand-induced Jurkat cells, while the CD137 B21 mutant enables the binding of CD137 ligand and the activation of productive T cells induced by CD137 ligand. (B) Extensive dose titration of the CD137 B21 variant and construct 2.11 molecule in the assay system described in (A). With the CD137 B21 variant, the functional interaction between human CD137 and human CD137 ligand is not inhibited when tested up to 100 nM. [Figure 10B] Figures 10A-10B. Effects of CD137-binding molecules on inhibiting the activation of human CD137-Jurkat cells induced by CD137 ligand-expressing cells. (A) Various CD137-binding molecules differentially affect the activation of human CD137-expressing Jurkat cells in the presence of 293 cells expressing human CD137 ligand. BMSAB, PfizerAb, and the Roche split-trimeric-4-1BBL(71-248) / FAP(28H1) molecule (see "Construction 2.11", WO 2017 / 194438) inhibit the activation of CD137 ligand-induced Jurkat cells, while the CD137 B21 mutant enables the binding of CD137 ligand and the activation of productive T cells induced by CD137 ligand. (B) Extensive dose titration of the CD137 B21 variant and construct 2.11 molecule in the assay system described in (A). With the CD137 B21 variant, the functional interaction between human CD137 and human CD137 ligand is not inhibited when tested up to 100 nM. [Figure 11A]Figures 11A-11C. A. Serum titers from various OminChicken®-derived anti-human FAP antibodies showing cross-reactivity to human, mouse, and cynomolgus monkey FAPs as measured by Elisa. B. MFI of anti-FAP antibody binding to stable cell lines expressing human FAP (HT1080-FAP) compared to wild-type HT1080 wild-type (WT) cells. Representative clones #D6, E11, G3, H12, H3, A9, C12, C3, C8, D2, E1, E11.2, and H9. C. MFI of anti-FAP antibody binding to stable cell lines expressing mouse FAP (B16-FAP) compared to wild-type B16 wild-type cells. Representative clones #D6, E11, G3, H12, H3, A9, C12, C3, C8, D2, E1, E11.2, and H9. [Figure 11B] Figures 11A-11C. A. Serum titers from various OminChicken®-derived anti-human FAP antibodies showing cross-reactivity to human, mouse, and cynomolgus monkey FAPs as measured by Elisa. B. MFI of anti-FAP antibody binding to stable cell lines expressing human FAP (HT1080-FAP) compared to wild-type HT1080 wild-type (WT) cells. Representative clones #D6, E11, G3, H12, H3, A9, C12, C3, C8, D2, E1, E11.2, and H9. C. MFI of anti-FAP antibody binding to stable cell lines expressing mouse FAP (B16-FAP) compared to wild-type B16 wild-type cells. Representative clones #D6, E11, G3, H12, H3, A9, C12, C3, C8, D2, E1, E11.2, and H9. [Figure 11C]Figures 11A-11C. A. Serum titers from various OminChicken®-derived anti-human FAP antibodies showing cross-reactivity to human, mouse, and cynomolgus monkey FAPs as measured by Elisa. B. MFI of anti-FAP antibody binding to stable cell lines expressing human FAP (HT1080-FAP) compared to wild-type HT1080 wild-type (WT) cells. Representative clones #D6, E11, G3, H12, H3, A9, C12, C3, C8, D2, E1, E11.2, and H9. C. MFI of anti-FAP antibody binding to stable cell lines expressing mouse FAP (B16-FAP) compared to wild-type B16 wild-type cells. Representative clones #D6, E11, G3, H12, H3, A9, C12, C3, C8, D2, E1, E11.2, and H9. [Figure 12A] Figures 12A-12C. Effects of the bispecific CD137(4-1BB,TNFRSF9) / fibroblast-activating protein (FAP) molecule on cell activation. A. FAP+HT1080 cells and FAP-ve HT1080 cells were incubated with the CD137 / FAP bispecific molecule for 24 hours. B. Comparison with urelumab, monovalent CD137(4-1BB,TNFRSF9) antibody alone, or C. utomirumab, monovalent CD137(4-1BB,TNFRSF9) antibody alone. [Figure 12B] Figures 12A-12C. Effects of the bispecific CD137(4-1BB,TNFRSF9) / fibroblast-activating protein (FAP) molecule on cell activation. A. FAP+HT1080 cells and FAP-ve HT1080 cells were incubated with the CD137 / FAP bispecific molecule for 24 hours. B. Comparison with urelumab, monovalent CD137(4-1BB,TNFRSF9) antibody alone, or C. utomirumab, monovalent CD137(4-1BB,TNFRSF9) antibody alone. [Figure 12C]Figures 12A-12C. Effects of the bispecific CD137(4-1BB,TNFRSF9) / fibroblast-activating protein (FAP) molecule on cell activation. A. FAP+HT1080 cells and FAP-ve HT1080 cells were incubated with the CD137 / FAP bispecific molecule for 24 hours. B. Comparison with urelumab, monovalent CD137(4-1BB,TNFRSF9) antibody alone, or C. utomirumab, monovalent CD137(4-1BB,TNFRSF9) antibody alone. [Figure 13A]Figures 13A-13D: A. CD137 B21 mutant co-cultured with HT1080-FAP-expressing cells. NF-κB-dependent induction of the luciferase reporter gene in Jurkat-CD137 cells when co-cultured with HT1080-FAP (A, C) or HT1080-wild-type cells (B, D). The levels of reporter gene activity are B21.V16(CD137 #2) / FAP#5, B21.V22(CD137 #3) / FAP #3, B21.V37(CD137 #6) / FAP #3, B21.V16(CD137 #2) / FAP #2, B21.V22(CD137 #4) / FAP #3, B21.V25(CD135 #5) / FAP #3, B21.V22(CD137 #4) / FAP #2, B21.V25(CD137 #5) / FAP #2, B21.V22 CD137 #3 / FAP #2, B21.V25(CD137 #4) / FAP #5, B21.V22(CD137 #3) / FAP Correlated with representative clonal mutants including #5, B21.V37(CD137 #6) / FAP #2, B21.V29(CD137 #5) / FAP #5, and B21.V37(CD137 #6) / FAP#5. B. CD137 B21 mutant shown in (A) with HT1080-wild-type cells. C. CD137 A49 mutant co-cultured with HT1080-FAP-expressing cells. Representative clonal mutants include A49.V48(CD137 #7) / FAP #3, A49.V47(CD137 #9) / FAP #3, A49.V43(CD137 #8) / FAP #3, A49.V48(CD137 #7) / FAP #5, A49.V48(CD137 #7) / FAP #2, A49.V47(CD137 #9) / FAP #5, A49.V47(CD135 #9) / FAP #2, A49.V43(CD137 #8) / FAP #2, and A49.V43(CD137 #8) / FAP #5. CD137 A49 mutants shown in (C) with D.HT1080 wild-type cells. [Figure 13B]Figures 13A-13D: A. CD137 B21 mutant co-cultured with HT1080-FAP-expressing cells. NF-κB-dependent induction of the luciferase reporter gene in Jurkat-CD137 cells when co-cultured with HT1080-FAP (A, C) or HT1080-wild-type cells (B, D). The levels of reporter gene activity are B21.V16(CD137 #2) / FAP#5, B21.V22(CD137 #3) / FAP #3, B21.V37(CD137 #6) / FAP #3, B21.V16(CD137 #2) / FAP #2, B21.V22(CD137 #4) / FAP #3, B21.V25(CD135 #5) / FAP #3, B21.V22(CD137 #4) / FAP #2, B21.V25(CD137 #5) / FAP #2, B21.V22 CD137 #3 / FAP #2, B21.V25(CD137 #4) / FAP #5, B21.V22(CD137 #3) / FAP Correlated with representative clonal mutants including #5, B21.V37(CD137 #6) / FAP #2, B21.V29(CD137 #5) / FAP #5, and B21.V37(CD137 #6) / FAP#5. B. CD137 B21 mutant shown in (A) with HT1080-wild-type cells. C. CD137 A49 mutant co-cultured with HT1080-FAP-expressing cells. Representative clonal mutants include A49.V48(CD137 #7) / FAP #3, A49.V47(CD137 #9) / FAP #3, A49.V43(CD137 #8) / FAP #3, A49.V48(CD137 #7) / FAP #5, A49.V48(CD137 #7) / FAP #2, A49.V47(CD137 #9) / FAP #5, A49.V47(CD135 #9) / FAP #2, A49.V43(CD137 #8) / FAP #2, and A49.V43(CD137 #8) / FAP #5. CD137 A49 mutants shown in (C) with D.HT1080 wild-type cells. [Figure 13C]Figures 13A-13D: A. CD137 B21 mutant co-cultured with HT1080-FAP-expressing cells. NF-κB-dependent induction of the luciferase reporter gene in Jurkat-CD137 cells when co-cultured with HT1080-FAP (A, C) or HT1080-wild-type cells (B, D). The levels of reporter gene activity are B21.V16(CD137 #2) / FAP#5, B21.V22(CD137 #3) / FAP #3, B21.V37(CD137 #6) / FAP #3, B21.V16(CD137 #2) / FAP #2, B21.V22(CD137 #4) / FAP #3, B21.V25(CD135 #5) / FAP #3, B21.V22(CD137 #4) / FAP #2, B21.V25(CD137 #5) / FAP #2, B21.V22 CD137 #3 / FAP #2, B21.V25(CD137 #4) / FAP #5, B21.V22(CD137 #3) / FAP Correlated with representative clonal mutants including #5, B21.V37(CD137 #6) / FAP #2, B21.V29(CD137 #5) / FAP #5, and B21.V37(CD137 #6) / FAP#5. B. CD137 B21 mutant shown in (A) with HT1080-wild-type cells. C. CD137 A49 mutant co-cultured with HT1080-FAP-expressing cells. Representative clonal mutants include A49.V48(CD137 #7) / FAP #3, A49.V47(CD137 #9) / FAP #3, A49.V43(CD137 #8) / FAP #3, A49.V48(CD137 #7) / FAP #5, A49.V48(CD137 #7) / FAP #2, A49.V47(CD137 #9) / FAP #5, A49.V47(CD135 #9) / FAP #2, A49.V43(CD137 #8) / FAP #2, and A49.V43(CD137 #8) / FAP #5. CD137 A49 mutants shown in (C) with D.HT1080 wild-type cells. [Figure 13D]Figures 13A-13D: A. CD137 B21 mutant co-cultured with HT1080-FAP-expressing cells. NF-κB-dependent induction of the luciferase reporter gene in Jurkat-CD137 cells when co-cultured with HT1080-FAP (A, C) or HT1080-wild-type cells (B, D). The levels of reporter gene activity are B21.V16(CD137 #2) / FAP#5, B21.V22(CD137 #3) / FAP #3, B21.V37(CD137 #6) / FAP #3, B21.V16(CD137 #2) / FAP #2, B21.V22(CD137 #4) / FAP #3, B21.V25(CD135 #5) / FAP #3, B21.V22(CD137 #4) / FAP #2, B21.V25(CD137 #5) / FAP #2, B21.V22 CD137 #3 / FAP #2, B21.V25(CD137 #4) / FAP #5, B21.V22(CD137 #3) / FAP Correlated with representative clonal mutants including #5, B21.V37(CD137 #6) / FAP #2, B21.V29(CD137 #5) / FAP #5, and B21.V37(CD137 #6) / FAP#5. B. CD137 B21 mutant shown in (A) with HT1080-wild-type cells. C. CD137 A49 mutant co-cultured with HT1080-FAP-expressing cells. Representative clonal mutants include A49.V48(CD137 #7) / FAP #3, A49.V47(CD137 #9) / FAP #3, A49.V43(CD137 #8) / FAP #3, A49.V48(CD137 #7) / FAP #5, A49.V48(CD137 #7) / FAP #2, A49.V47(CD137 #9) / FAP #5, A49.V47(CD135 #9) / FAP #2, A49.V43(CD137 #8) / FAP #2, and A49.V43(CD137 #8) / FAP #5. CD137 A49 mutants shown in (C) with D.HT1080 wild-type cells. [Figure 14A]Figures 14A-14H. IFN-γ production in PBMCs in the presence of CD137 / FAP mutants: A. Anti-CD3 stimulated PBMCs co-cultured with FAP-expressing HT1080 cells produced increased IFN-γ through CD137 co-stimulation. Representative clonal mutants include B21.V16(CD137 #2) / FAP #5, B21.V16(CD137 #2) / FAP #2, and B21.V16(CD137 #2) / FAP #3. B. Representative clonal mutants include B21.V22(CD137 #3) / FAP #5, B21.V22 CD137 #3 / FAP #2, and B21.V22(CD137 #3) / FAP #3. C. Representative clonal mutants include B21.V25(CD137 #4) / FAP #5, B21.V22(CD137 #4) / FAP #2, and B21.V22(CD137 #4) / FAP #3. D. Representative mutants include B21.V29(CD137 #5) / FAP #5, B21.V25(CD137 #5) / FAP #2, and B21.V25(CD135 #5) / FAP #3. E. Representative mutants include B21.V37(CD137 #6) / FAP #5, B21.V37(CD137 #6) / FAP #2, and B21.V37(CD137 #6) / FAP #3. F. Representative mutants A49.V43(CD137 #8) / FAP #5, A49.V43(CD137 #8) / FAP #2, A49.V43(CD137 #8) / FAP #3. G. Representative mutants A49.V47(CD137 #9) / FAP #5, A49.V47(CD135 #9) / FAP #2, A49.V47(CD137 #9) / FAP #3. H. Representative mutants A49.V48(CD137 #7) / FAP #5, A49.V48(CD137 #7) / FAP #2, A49.V48(CD137 #7) / FAP #3. [Figure 14B]Figures 14A-14H. IFN-γ production in PBMCs in the presence of CD137 / FAP mutants: A. Anti-CD3 stimulated PBMCs co-cultured with FAP-expressing HT1080 cells produced increased IFN-γ through CD137 co-stimulation. Representative clonal mutants include B21.V16(CD137 #2) / FAP #5, B21.V16(CD137 #2) / FAP #2, and B21.V16(CD137 #2) / FAP #3. B. Representative clonal mutants include B21.V22(CD137 #3) / FAP #5, B21.V22 CD137 #3 / FAP #2, and B21.V22(CD137 #3) / FAP #3. C. Representative clonal mutants include B21.V25(CD137 #4) / FAP #5, B21.V22(CD137 #4) / FAP #2, and B21.V22(CD137 #4) / FAP #3. D. Representative mutants include B21.V29(CD137 #5) / FAP #5, B21.V25(CD137 #5) / FAP #2, and B21.V25(CD135 #5) / FAP #3. E. Representative mutants include B21.V37(CD137 #6) / FAP #5, B21.V37(CD137 #6) / FAP #2, and B21.V37(CD137 #6) / FAP #3. F. Representative mutants A49.V43(CD137 #8) / FAP #5, A49.V43(CD137 #8) / FAP #2, A49.V43(CD137 #8) / FAP #3. G. Representative mutants A49.V47(CD137 #9) / FAP #5, A49.V47(CD135 #9) / FAP #2, A49.V47(CD137 #9) / FAP #3. H. Representative mutants A49.V48(CD137 #7) / FAP #5, A49.V48(CD137 #7) / FAP #2, A49.V48(CD137 #7) / FAP #3. [Figure 14C]Figures 14A-14H. IFN-γ production in PBMCs in the presence of CD137 / FAP mutants: A. Anti-CD3 stimulated PBMCs co-cultured with FAP-expressing HT1080 cells produced increased IFN-γ through CD137 co-stimulation. Representative clonal mutants include B21.V16(CD137 #2) / FAP #5, B21.V16(CD137 #2) / FAP #2, and B21.V16(CD137 #2) / FAP #3. B. Representative clonal mutants include B21.V22(CD137 #3) / FAP #5, B21.V22 CD137 #3 / FAP #2, and B21.V22(CD137 #3) / FAP #3. C. Representative clonal mutants include B21.V25(CD137 #4) / FAP #5, B21.V22(CD137 #4) / FAP #2, and B21.V22(CD137 #4) / FAP #3. D. Representative mutants include B21.V29(CD137 #5) / FAP #5, B21.V25(CD137 #5) / FAP #2, and B21.V25(CD135 #5) / FAP #3. E. Representative mutants include B21.V37(CD137 #6) / FAP #5, B21.V37(CD137 #6) / FAP #2, and B21.V37(CD137 #6) / FAP #3. F. Representative mutants A49.V43(CD137 #8) / FAP #5, A49.V43(CD137 #8) / FAP #2, A49.V43(CD137 #8) / FAP #3. G. Representative mutants A49.V47(CD137 #9) / FAP #5, A49.V47(CD135 #9) / FAP #2, A49.V47(CD137 #9) / FAP #3. H. Representative mutants A49.V48(CD137 #7) / FAP #5, A49.V48(CD137 #7) / FAP #2, A49.V48(CD137 #7) / FAP #3. [Figure 14D]Figures 14A-14H. IFN-γ production in PBMCs in the presence of CD137 / FAP mutants: A. Anti-CD3 stimulated PBMCs co-cultured with FAP-expressing HT1080 cells produced increased IFN-γ through CD137 co-stimulation. Representative clonal mutants include B21.V16(CD137 #2) / FAP #5, B21.V16(CD137 #2) / FAP #2, and B21.V16(CD137 #2) / FAP #3. B. Representative clonal mutants include B21.V22(CD137 #3) / FAP #5, B21.V22 CD137 #3 / FAP #2, and B21.V22(CD137 #3) / FAP #3. C. Representative clonal mutants include B21.V25(CD137 #4) / FAP #5, B21.V22(CD137 #4) / FAP #2, and B21.V22(CD137 #4) / FAP #3. D. Representative mutants include B21.V29(CD137 #5) / FAP #5, B21.V25(CD137 #5) / FAP #2, and B21.V25(CD135 #5) / FAP #3. E. Representative mutants include B21.V37(CD137 #6) / FAP #5, B21.V37(CD137 #6) / FAP #2, and B21.V37(CD137 #6) / FAP #3. F. Representative mutants A49.V43(CD137 #8) / FAP #5, A49.V43(CD137 #8) / FAP #2, A49.V43(CD137 #8) / FAP #3. G. Representative mutants A49.V47(CD137 #9) / FAP #5, A49.V47(CD135 #9) / FAP #2, A49.V47(CD137 #9) / FAP #3. H. Representative mutants A49.V48(CD137 #7) / FAP #5, A49.V48(CD137 #7) / FAP #2, A49.V48(CD137 #7) / FAP #3. [Figure 14E]Figures 14A-14H. IFN-γ production in PBMCs in the presence of CD137 / FAP mutants: A. Anti-CD3 stimulated PBMCs co-cultured with FAP-expressing HT1080 cells produced increased IFN-γ through CD137 co-stimulation. Representative clonal mutants include B21.V16(CD137 #2) / FAP #5, B21.V16(CD137 #2) / FAP #2, and B21.V16(CD137 #2) / FAP #3. B. Representative clonal mutants include B21.V22(CD137 #3) / FAP #5, B21.V22 CD137 #3 / FAP #2, and B21.V22(CD137 #3) / FAP #3. C. Representative clonal mutants include B21.V25(CD137 #4) / FAP #5, B21.V22(CD137 #4) / FAP #2, and B21.V22(CD137 #4) / FAP #3. D. Representative mutants include B21.V29(CD137 #5) / FAP #5, B21.V25(CD137 #5) / FAP #2, and B21.V25(CD135 #5) / FAP #3. E. Representative mutants include B21.V37(CD137 #6) / FAP #5, B21.V37(CD137 #6) / FAP #2, and B21.V37(CD137 #6) / FAP #3. F. Representative mutants A49.V43(CD137 #8) / FAP #5, A49.V43(CD137 #8) / FAP #2, A49.V43(CD137 #8) / FAP #3. G. Representative mutants A49.V47(CD137 #9) / FAP #5, A49.V47(CD135 #9) / FAP #2, A49.V47(CD137 #9) / FAP #3. H. Representative mutants A49.V48(CD137 #7) / FAP #5, A49.V48(CD137 #7) / FAP #2, A49.V48(CD137 #7) / FAP #3. [Figure 14F]Figures 14A-14H. IFN-γ production in PBMCs in the presence of CD137 / FAP mutants: A. Anti-CD3 stimulated PBMCs co-cultured with FAP-expressing HT1080 cells produced increased IFN-γ through CD137 co-stimulation. Representative clonal mutants include B21.V16(CD137 #2) / FAP #5, B21.V16(CD137 #2) / FAP #2, and B21.V16(CD137 #2) / FAP #3. B. Representative clonal mutants include B21.V22(CD137 #3) / FAP #5, B21.V22 CD137 #3 / FAP #2, and B21.V22(CD137 #3) / FAP #3. C. Representative clonal mutants include B21.V25(CD137 #4) / FAP #5, B21.V22(CD137 #4) / FAP #2, and B21.V22(CD137 #4) / FAP #3. D. Representative mutants include B21.V29(CD137 #5) / FAP #5, B21.V25(CD137 #5) / FAP #2, and B21.V25(CD135 #5) / FAP #3. E. Representative mutants include B21.V37(CD137 #6) / FAP #5, B21.V37(CD137 #6) / FAP #2, and B21.V37(CD137 #6) / FAP #3. F. Representative mutants A49.V43(CD137 #8) / FAP #5, A49.V43(CD137 #8) / FAP #2, A49.V43(CD137 #8) / FAP #3. G. Representative mutants A49.V47(CD137 #9) / FAP #5, A49.V47(CD135 #9) / FAP #2, A49.V47(CD137 #9) / FAP #3. H. Representative mutants A49.V48(CD137 #7) / FAP #5, A49.V48(CD137 #7) / FAP #2, A49.V48(CD137 #7) / FAP #3. [Figure 14G]Figures 14A-14H. IFN-γ production in PBMCs in the presence of CD137 / FAP mutants: A. Anti-CD3 stimulated PBMCs co-cultured with FAP-expressing HT1080 cells produced increased IFN-γ through CD137 co-stimulation. Representative clonal mutants include B21.V16(CD137 #2) / FAP #5, B21.V16(CD137 #2) / FAP #2, and B21.V16(CD137 #2) / FAP #3. B. Representative clonal mutants include B21.V22(CD137 #3) / FAP #5, B21.V22 CD137 #3 / FAP #2, and B21.V22(CD137 #3) / FAP #3. C. Representative clonal mutants include B21.V25(CD137 #4) / FAP #5, B21.V22(CD137 #4) / FAP #2, and B21.V22(CD137 #4) / FAP #3. D. Representative mutants include B21.V29(CD137 #5) / FAP #5, B21.V25(CD137 #5) / FAP #2, and B21.V25(CD135 #5) / FAP #3. E. Representative mutants include B21.V37(CD137 #6) / FAP #5, B21.V37(CD137 #6) / FAP #2, and B21.V37(CD137 #6) / FAP #3. F. Representative mutants A49.V43(CD137 #8) / FAP #5, A49.V43(CD137 #8) / FAP #2, A49.V43(CD137 #8) / FAP #3. G. Representative mutants A49.V47(CD137 #9) / FAP #5, A49.V47(CD135 #9) / FAP #2, A49.V47(CD137 #9) / FAP #3. H. Representative mutants A49.V48(CD137 #7) / FAP #5, A49.V48(CD137 #7) / FAP #2, A49.V48(CD137 #7) / FAP #3. [Figure 14H]Figures 14A-14H. IFN-γ production in PBMCs in the presence of CD137 / FAP mutants: A. Anti-CD3 stimulated PBMCs co-cultured with FAP-expressing HT1080 cells produced increased IFN-γ through CD137 co-stimulation. Representative clonal mutants include B21.V16(CD137 #2) / FAP #5, B21.V16(CD137 #2) / FAP #2, and B21.V16(CD137 #2) / FAP #3. B. Representative clonal mutants include B21.V22(CD137 #3) / FAP #5, B21.V22 CD137 #3 / FAP #2, and B21.V22(CD137 #3) / FAP #3. C. Representative clonal mutants include B21.V25(CD137 #4) / FAP #5, B21.V22(CD137 #4) / FAP #2, and B21.V22(CD137 #4) / FAP #3. D. Representative mutants include B21.V29(CD137 #5) / FAP #5, B21.V25(CD137 #5) / FAP #2, and B21.V25(CD135 #5) / FAP #3. E. Representative mutants include B21.V37(CD137 #6) / FAP #5, B21.V37(CD137 #6) / FAP #2, and B21.V37(CD137 #6) / FAP #3. F. Representative mutants A49.V43(CD137 #8) / FAP #5, A49.V43(CD137 #8) / FAP #2, A49.V43(CD137 #8) / FAP #3. G. Representative mutants A49.V47(CD137 #9) / FAP #5, A49.V47(CD135 #9) / FAP #2, A49.V47(CD137 #9) / FAP #3. H. Representative mutants A49.V48(CD137 #7) / FAP #5, A49.V48(CD137 #7) / FAP #2, A49.V48(CD137 #7) / FAP #3. [Figure 15A] Figures 15A-15B. IFN-γ secretion from individual donor PBMCs incubated in the presence of HT1080 wild-type (A) or HT1080-FAP expressing cells (B) while increasing the concentration of exemplary CD137 B21 / FAP bispecific molecules. [Figure 15B]Figures 15A-15B. IFN-γ secretion from individual donor PBMCs incubated in the presence of HT1080 wild-type (A) or HT1080-FAP expressing cells (B) while increasing the concentration of exemplary CD137 B21 / FAP bispecific molecules. [Figure 16A] Figures 16A-16B. A. FAP Enzyme Assay: CD137-B21 / FAP-C3 and mouse CD137 B21 / FAP-A11, which are exemplary molecules of the present invention, do not interfere with the enzymatic activity of the FAP protein. B. Talabostat Mesylate Inhibition Assay: A positive control for FAP inhibition. [Figure 16B] Figures 16A-16B. A. FAP Enzyme Assay: CD137-B21 / FAP-C3 and mouse CD137 B21 / FAP-A11, which are exemplary molecules of the present invention, do not interfere with the enzymatic activity of the FAP protein. B. Talabostat Mesylate Inhibition Assay: A positive control for FAP inhibition. [Figure 17] Figure 17. Schematic diagram of the administration schedule of the molecule in a subcutaneous syngeneic MC38 colorectal cancer model in CD137 KI HuGEMM® mice. [Figure 18A] Figures 18A-18D. In vivo efficacy of human CD137 B21 / FAP C3 and mouse CD137 B21 / FAP-A11 monotherapy in a subcutaneous syngeneic MC38 colorectal cancer model in CD137 KI HuGEMM mice. (A) Mean tumor growth curve after treatment with human CD137 B21 / FAP C3, (B) Individual tumor growth curve after treatment with human CD137 B21 / FAP C3, (C) Mean tumor growth curve after treatment with mouse CD137 B21 / FAP A11, and (D) Individual tumor growth curve after treatment with mouse CD137 B21 / FAP A11. [Figure 18B]Figures 18A-18D. In vivo efficacy of human CD137 B21 / FAP C3 and mouse CD137 B21 / FAP-A11 monotherapy in a subcutaneous syngeneic MC38 colorectal cancer model in CD137 KI HuGEMM mice. (A) Mean tumor growth curve after treatment with human CD137 B21 / FAP C3, (B) Individual tumor growth curve after treatment with human CD137 B21 / FAP C3, (C) Mean tumor growth curve after treatment with mouse CD137 B21 / FAP A11, and (D) Individual tumor growth curve after treatment with mouse CD137 B21 / FAP A11. [Figure 18C] Figures 18A-18D. In vivo efficacy of human CD137 B21 / FAP C3 and mouse CD137 B21 / FAP-A11 monotherapy in a subcutaneous syngeneic MC38 colorectal cancer model in CD137 KI HuGEMM mice. (A) Mean tumor growth curve after treatment with human CD137 B21 / FAP C3, (B) Individual tumor growth curve after treatment with human CD137 B21 / FAP C3, (C) Mean tumor growth curve after treatment with mouse CD137 B21 / FAP A11, and (D) Individual tumor growth curve after treatment with mouse CD137 B21 / FAP A11. [Figure 18D] Figures 18A-18D. In vivo efficacy of human CD137 B21 / FAP C3 and mouse CD137 B21 / FAP-A11 monotherapy in a subcutaneous syngeneic MC38 colorectal cancer model in CD137 KI HuGEMM mice. (A) Mean tumor growth curve after treatment with human CD137 B21 / FAP C3, (B) Individual tumor growth curve after treatment with human CD137 B21 / FAP C3, (C) Mean tumor growth curve after treatment with mouse CD137 B21 / FAP A11, and (D) Individual tumor growth curve after treatment with mouse CD137 B21 / FAP A11. [Figure 19A] Figures 19A-19B. TIL recruitment by CD137 / FAP molecules: The human CD137 B21 / FAP C3 and mouse CD137 B21 / FAPA11 binding molecules of the present invention can induce increased CD3+(A) or CD8+(B) T cell infiltration into the tumor microenvironment compared to the control group. [Figure 19B] Figures 19A-19B. TIL recruitment by CD137 / FAP molecules: The human CD137 B21 / FAP C3 and mouse CD137 B21 / FAPA11 binding molecules of the present invention can induce increased CD3+(A) or CD8+(B) T cell infiltration into the tumor microenvironment compared to the control group. [Figure 20A] Figures 20A-20E. Combined Therapy PD-1 and CD137 / FAP. A. Schematic diagram of the mouse model and the administration schedule of CD137 / FAP and PD-1. B. The conjugating molecule of the present invention, when used in combination with a PD-1 antagonist mAb, can induce a reduction in tumor volume compared to the control group, and the effect is dose-dependent. (B) and (D) show combination therapy with mouse PD-1 antibody and human CD137 B21 / FAP C3, or (C) and (E) show combination therapy with mouse PD-1 and mouse CD137 B21 / FAP A11. Both resulted in potent and statistically significant (p<0.0001) inhibition of tumor growth, as demonstrated by the reduction in tumor volume. [Figure 20B] Figures 20A-20E. Combined Therapy PD-1 and CD137 / FAP. A. Schematic diagram of the mouse model and the administration schedule of CD137 / FAP and PD-1. B. The conjugating molecule of the present invention, when used in combination with a PD-1 antagonist mAb, can induce a reduction in tumor volume compared to the control group, and the effect is dose-dependent. (B) and (D) show combination therapy with mouse PD-1 antibody and human CD137 B21 / FAP C3, or (C) and (E) show combination therapy with mouse PD-1 and mouse CD137 B21 / FAP A11. Both resulted in potent and statistically significant (p<0.0001) inhibition of tumor growth, as demonstrated by the reduction in tumor volume. [Figure 20C]Figures 20A-20E. Combined Therapy PD-1 and CD137 / FAP. A. Schematic diagram of the mouse model and the administration schedule of CD137 / FAP and PD-1. B. The conjugating molecule of the present invention, when used in combination with a PD-1 antagonist mAb, can induce a reduction in tumor volume compared to the control group, and the effect is dose-dependent. (B) and (D) show combination therapy with mouse PD-1 antibody and human CD137 B21 / FAP C3, or (C) and (E) show combination therapy with mouse PD-1 and mouse CD137 B21 / FAP A11. Both resulted in potent and statistically significant (p<0.0001) inhibition of tumor growth, as demonstrated by the reduction in tumor volume. [Figure 20D] Figures 20A-20E. Combined Therapy PD-1 and CD137 / FAP. A. Schematic diagram of the mouse model and the administration schedule of CD137 / FAP and PD-1. B. The conjugating molecule of the present invention, when used in combination with a PD-1 antagonist mAb, can induce a reduction in tumor volume compared to the control group, and the effect is dose-dependent. (B) and (D) show combination therapy with mouse PD-1 antibody and human CD137 B21 / FAP C3, or (C) and (E) show combination therapy with mouse PD-1 and mouse CD137 B21 / FAP A11. Both resulted in potent and statistically significant (p<0.0001) inhibition of tumor growth, as demonstrated by the reduction in tumor volume. [Figure 20E] Figures 20A-20E. Combined Therapy PD-1 and CD137 / FAP. A. Schematic diagram of the mouse model and the administration schedule of CD137 / FAP and PD-1. B. The conjugating molecule of the present invention, when used in combination with a PD-1 antagonist mAb, can induce a reduction in tumor volume compared to the control group, and the effect is dose-dependent. (B) and (D) show combination therapy with mouse PD-1 antibody and human CD137 B21 / FAP C3, or (C) and (E) show combination therapy with mouse PD-1 and mouse CD137 B21 / FAP A11. Both resulted in potent and statistically significant (p<0.0001) inhibition of tumor growth, as demonstrated by the reduction in tumor volume. [Figure 21A]Figures 21A to 21G. The binding molecule of the present invention, when used in combination with a PD-1 agonist, can induce both CD4 and CD8 T cell infiltration compared to the control group. (A) Surviving tumor area measured after treatment with the media alone, anti-PD-1 alone, huCD137 B21 / FAP-C3 alone, or huCD137 B21 / FAP-C3 + PD-1 combination. (B) CD4+ cell density measured in the surviving tumor area after treatment with the media alone, anti-PD-1 alone, huCD137 B21 / FAP-C3 alone, or huCD137 B21 / FAP-C3 + PD-1 combination. (C) CD8+ cell density measured in the surviving tumor area after treatment with the media alone, anti-PD-1 alone, huCD137 B21 / FAP-C3 alone, or huCD137 B21 / FAP-C3 + PD-1 combination. (D) Plot tumor survival area as a function of CD8+ T cell density with treatment by the medium alone. (E) Plot tumor survival area as a function of CD8+ T cell density with treatment by anti-PD-1 alone. (F) Plot tumor survival area as a function of CD8+ T cell density with treatment by huCD137-B21 / FAP-C3 alone. (G) Plot tumor survival area as a function of CD8+ T cell density with treatment by huCD137-B21 / FAP-C3 in combination with anti-PD-1. Furthermore, the data demonstrate that increased CD8+ T cell infiltration is positively correlated with the disappearance of tumor survival area. [Figure 21B]Figures 21A to 21G. The binding molecule of the present invention, when used in combination with a PD-1 agonist, can induce both CD4 and CD8 T cell infiltration compared to the control group. (A) Surviving tumor area measured after treatment with the media alone, anti-PD-1 alone, huCD137 B21 / FAP-C3 alone, or huCD137 B21 / FAP-C3 + PD-1 combination. (B) CD4+ cell density measured in the surviving tumor area after treatment with the media alone, anti-PD-1 alone, huCD137 B21 / FAP-C3 alone, or huCD137 B21 / FAP-C3 + PD-1 combination. (C) CD8+ cell density measured in the surviving tumor area after treatment with the media alone, anti-PD-1 alone, huCD137 B21 / FAP-C3 alone, or huCD137 B21 / FAP-C3 + PD-1 combination. (D) Plot tumor survival area as a function of CD8+ T cell density with treatment by the medium alone. (E) Plot tumor survival area as a function of CD8+ T cell density with treatment by anti-PD-1 alone. (F) Plot tumor survival area as a function of CD8+ T cell density with treatment by huCD137-B21 / FAP-C3 alone. (G) Plot tumor survival area as a function of CD8+ T cell density with treatment by huCD137-B21 / FAP-C3 in combination with anti-PD-1. Furthermore, the data demonstrate that increased CD8+ T cell infiltration is positively correlated with the disappearance of tumor survival area. [Figure 21C]Figures 21A to 21G. The binding molecule of the present invention, when used in combination with a PD-1 agonist, can induce both CD4 and CD8 T cell infiltration compared to the control group. (A) Surviving tumor area measured after treatment with the media alone, anti-PD-1 alone, huCD137 B21 / FAP-C3 alone, or huCD137 B21 / FAP-C3 + PD-1 combination. (B) CD4+ cell density measured in the surviving tumor area after treatment with the media alone, anti-PD-1 alone, huCD137 B21 / FAP-C3 alone, or huCD137 B21 / FAP-C3 + PD-1 combination. (C) CD8+ cell density measured in the surviving tumor area after treatment with the media alone, anti-PD-1 alone, huCD137 B21 / FAP-C3 alone, or huCD137 B21 / FAP-C3 + PD-1 combination. (D) Plot tumor survival area as a function of CD8+ T cell density with treatment by the medium alone. (E) Plot tumor survival area as a function of CD8+ T cell density with treatment by anti-PD-1 alone. (F) Plot tumor survival area as a function of CD8+ T cell density with treatment by huCD137-B21 / FAP-C3 alone. (G) Plot tumor survival area as a function of CD8+ T cell density with treatment by huCD137-B21 / FAP-C3 in combination with anti-PD-1. Furthermore, the data demonstrate that increased CD8+ T cell infiltration is positively correlated with the disappearance of tumor survival area. [Figure 21D]Figures 21A to 21G. The binding molecule of the present invention, when used in combination with a PD-1 agonist, can induce both CD4 and CD8 T cell infiltration compared to the control group. (A) Surviving tumor area measured after treatment with the media alone, anti-PD-1 alone, huCD137 B21 / FAP-C3 alone, or huCD137 B21 / FAP-C3 + PD-1 combination. (B) CD4+ cell density measured in the surviving tumor area after treatment with the media alone, anti-PD-1 alone, huCD137 B21 / FAP-C3 alone, or huCD137 B21 / FAP-C3 + PD-1 combination. (C) CD8+ cell density measured in the surviving tumor area after treatment with the media alone, anti-PD-1 alone, huCD137 B21 / FAP-C3 alone, or huCD137 B21 / FAP-C3 + PD-1 combination. (D) Plot tumor survival area as a function of CD8+ T cell density with treatment by the medium alone. (E) Plot tumor survival area as a function of CD8+ T cell density with treatment by anti-PD-1 alone. (F) Plot tumor survival area as a function of CD8+ T cell density with treatment by huCD137-B21 / FAP-C3 alone. (G) Plot tumor survival area as a function of CD8+ T cell density with treatment by huCD137-B21 / FAP-C3 in combination with anti-PD-1. Furthermore, the data demonstrate that increased CD8+ T cell infiltration is positively correlated with the disappearance of tumor survival area. [Figure 21E]Figures 21A to 21G. The binding molecule of the present invention, when used in combination with a PD-1 agonist, can induce both CD4 and CD8 T cell infiltration compared to the control group. (A) Surviving tumor area measured after treatment with the media alone, anti-PD-1 alone, huCD137 B21 / FAP-C3 alone, or huCD137 B21 / FAP-C3 + PD-1 combination. (B) CD4+ cell density measured in the surviving tumor area after treatment with the media alone, anti-PD-1 alone, huCD137 B21 / FAP-C3 alone, or huCD137 B21 / FAP-C3 + PD-1 combination. (C) CD8+ cell density measured in the surviving tumor area after treatment with the media alone, anti-PD-1 alone, huCD137 B21 / FAP-C3 alone, or huCD137 B21 / FAP-C3 + PD-1 combination. (D) Plot tumor survival area as a function of CD8+ T cell density with treatment by the medium alone. (E) Plot tumor survival area as a function of CD8+ T cell density with treatment by anti-PD-1 alone. (F) Plot tumor survival area as a function of CD8+ T cell density with treatment by huCD137-B21 / FAP-C3 alone. (G) Plot tumor survival area as a function of CD8+ T cell density with treatment by huCD137-B21 / FAP-C3 in combination with anti-PD-1. Furthermore, the data demonstrate that increased CD8+ T cell infiltration is positively correlated with the disappearance of tumor survival area. [Figure 21F]Figures 21A to 21G. The binding molecule of the present invention, when used in combination with a PD-1 agonist, can induce both CD4 and CD8 T cell infiltration compared to the control group. (A) Surviving tumor area measured after treatment with the media alone, anti-PD-1 alone, huCD137 B21 / FAP-C3 alone, or huCD137 B21 / FAP-C3 + PD-1 combination. (B) CD4+ cell density measured in the surviving tumor area after treatment with the media alone, anti-PD-1 alone, huCD137 B21 / FAP-C3 alone, or huCD137 B21 / FAP-C3 + PD-1 combination. (C) CD8+ cell density measured in the surviving tumor area after treatment with the media alone, anti-PD-1 alone, huCD137 B21 / FAP-C3 alone, or huCD137 B21 / FAP-C3 + PD-1 combination. (D) Plot tumor survival area as a function of CD8+ T cell density with treatment by the medium alone. (E) Plot tumor survival area as a function of CD8+ T cell density with treatment by anti-PD-1 alone. (F) Plot tumor survival area as a function of CD8+ T cell density with treatment by huCD137-B21 / FAP-C3 alone. (G) Plot tumor survival area as a function of CD8+ T cell density with treatment by huCD137-B21 / FAP-C3 in combination with anti-PD-1. Furthermore, the data demonstrate that increased CD8+ T cell infiltration is positively correlated with the disappearance of tumor survival area. [Figure 21G]Figures 21A to 21G. The binding molecule of the present invention, when used in combination with a PD-1 agonist, can induce both CD4 and CD8 T cell infiltration compared to the control group. (A) Surviving tumor area measured after treatment with the media alone, anti-PD-1 alone, huCD137 B21 / FAP-C3 alone, or huCD137 B21 / FAP-C3 + PD-1 combination. (B) CD4+ cell density measured in the surviving tumor area after treatment with the media alone, anti-PD-1 alone, huCD137 B21 / FAP-C3 alone, or huCD137 B21 / FAP-C3 + PD-1 combination. (C) CD8+ cell density measured in the surviving tumor area after treatment with the media alone, anti-PD-1 alone, huCD137 B21 / FAP-C3 alone, or huCD137 B21 / FAP-C3 + PD-1 combination. (D) Plot tumor survival area as a function of CD8+ T cell density with treatment by the medium alone. (E) Plot tumor survival area as a function of CD8+ T cell density with treatment by anti-PD-1 alone. (F) Plot tumor survival area as a function of CD8+ T cell density with treatment by huCD137-B21 / FAP-C3 alone. (G) Plot tumor survival area as a function of CD8+ T cell density with treatment by huCD137-B21 / FAP-C3 in combination with anti-PD-1. Furthermore, the data demonstrate that increased CD8+ T cell infiltration is positively correlated with the disappearance of tumor survival area.

[0045] Detailed description of the invention CD137 is widely expressed throughout the hematopoietic and non-hematopoietic compartments: activated T cells, regulatory T cells, NK cells, dendritic cells, activated monocytes, neutrophils, eosinophils, mast cells, activated B cells, Reed-Sternberg cells, and vascular walls (on endothelium and vascular smooth muscle cells). As demonstrated in preclinical studies, clinical trials of urelumab, a potent monospecific CD137 agonist antibody, have reported severe hepatotoxicity, including death. Both high CD137 affinity and high clustering via FcγR on hepatic resident T cells are thought to contribute to urelumab-mediated hepatotoxicity.

[0046] The CD137 / FAP molecule of the present invention has been engineered to have lower CD137 binding affinity, LALA mutations for reduced FcγR binding, and the need for co-binding to CD137 and tumor stroma-specific FAP for activity. FAP (fibroblast-activating protein) is the anchor target. FAP is expressed only in activated fibroblasts located within the tumor stroma. Therefore, the FAP bispecific molecule will function only to promote T cell activation and the killing of cancer cells that are in close physical contact with activated fibroblasts. Tumor cells that are not in direct contact with activated fibroblasts will not be affected by this treatment and will continue to proliferate. Therefore, there is a clear advantage to using FAP as an anchor target to mediate CD137 receptor-induced activation and T cell infiltration only at the tumor site.

[0047] The inventors discovered co-localization of CD137 and FAP in tumors. They investigated the prevalence of co-localization in large subsets of colorectal cancer (CRC), gastric cancer (GC), and pancreatic cancer (PAC). High prevalence was consistently observed in CRC for both CD137 expression (88-100%) and FAP expression (87-100%). In GC, CD137 expression was also demonstrated in 56-90% of all cases, and was significantly more frequent in the intestinal type compared to the diffuse type. On the other hand, FAP has been shown to be constitutively expressed at high levels in primary and metastatic gastric cancer. In PAC, CD137 expression was observed in 50-82% of all cases, and FAP in 81%.

[0048] Therefore, CD137 and FAP exhibit co-localization in various tumors, while showing little to no co-expression in non-cancerous cells. In particular, FAP could not be detected in normal liver tissue or hepatocytes that have been reported to be sensitive to CD137 activation.

[0049] Terms not specifically defined herein should be given the meanings that a person skilled in the art would assign to them in consideration of this disclosure and the context. However, as used herein, unless otherwise specified, the following terms shall have the meanings indicated and the following conventions shall be observed.

[0050] A first aspect of the present invention provides an immunoglobulin-like binding molecule having at least one antigen-binding site that specifically binds to CD137(4-1BB,TNFRSF9) (wherein the antigen-binding site that specifically binds to CD137(4-1BB ligand receptor) is a part of an immunoglobulin (Ig) molecule) and at least one antigen-binding site that specifically binds to fibroblast-activating protein (FAP) (wherein the antigen-binding site that specifically binds to fibroblast-activating protein (FAP) includes two scFv). Each protein and the genes associated with them are known in the art and are well represented in biological databases.

[0051] "Human CD137 (4-1BB, TNFRSF9)" is defined as the protein (SEQ ID NO: 1) and the nucleic acid sequence encoding that protein, provided under NCBI:NP_001552 and Uniprot:Q07011. The amino acids that define the various CRD regions of the extracellular domain (ECD) are as defined in Table 1 below. The amino acid sequences of the human-Fc-His proteins tagged for human CD137 and cynomolgus monkey CD137 used as immunogens in immunization campaigns correspond to SEQ ID NOs: 335 and 343 and are included in Table 1. "Cynomolgus monkey CD137 (4-1BB, TNFRSF9)" is defined as the protein (SEQ ID NO: 2) and the nucleic acid sequence encoding that protein, provided under Uniprot accession number F6W5G6. "Mouse CD137 (4-1BB, TNFRSF9)" is defined as the protein (SEQ ID NO: 3) and the nucleic acid sequence encoding that protein, provided under Uniprot accession P20334.

[0052] [Table 1] TIFF0007911965000002.tif254169 TIFF0007911965000003.tif254169 TIFF0007911965000004.tif254169

[0053] "Human fibroblast-activating protein (FAP)" is defined as the protein (SEQ ID NO: 4) provided in NCBI:NP_004451 and Uniprot:Q12884, and the nucleic acid sequence encoding that protein. The term "fibroblast-activating protein (FAP)" is also known as prolyl endopeptidase FAP or seplacase (EC 3.4.21). In one embodiment, the antigen-binding molecule of the present invention is specifically bindable to human, mouse, and / or cynomolgus monkey FAP. The extracellular domain (ECD) of human FAP extends from amino acids 26 to 760. The amino acid sequence of mouse FAP is shown in UniProt accession number P97321 (SEQ ID NO: 4) or NCBI RefSeq NP_032012.1. The extracellular domain (ECD) of mouse FAP extends from amino acids 26 to 761. Preferably, the anti-FAP binding molecule of the present invention binds to the extracellular domain of FAP.

[0054] This invention relates to a binding molecule having binding specificity for at least two different sites. In this invention, the immunoglobulin-like binding molecule is derived from an antibody. Techniques for preparing the binding molecule include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983), WO 93 / 08829 and Traunecker et al., EMBO J. 10: 3655 (1991)) and the "knob-in-hole" operation (see, for example, US No. 5,731,168). Furthermore, the immunoglobulin-like binding molecule of the present invention can be used to manipulate the electrostatic steering effect for preparing antibody Fc-heterodimer molecules (WO No. 2009 / 089004), to crosslink two or more antibodies or fragments (see, e.g., US No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)), to use a leucine zipper for producing bispecific antibodies (see, e.g., Kostelny et al., Immunol., 148(5): 1547-1553 (1992)), to use the "diabody" technique for preparing bispecific antibody fragments (see, e.g., Hollinger et al., PNAS. USA, 90:6444-6448 (1993)), and to use single-stranded Fv(sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368). (See 1994) and can also be prepared by preparing a trispecific antibody, such as the one described in Tutt et al. J. Immunol. 147: 60 (1991).

[0055] As used herein, the term "antigen-binding site" refers to the heavy chain variable domain (V) derived from the antibody. H ) and light chain variable domain (V L) includes. The term “variable region” or “variable domain” refers to a domain of the antibody heavy or light chain involved in the binding of an antigen-binding molecule to an antigen. The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) generally have similar structures, and each domain contains four conserved framework regions (FRs) and three hypervariable regions. In such cases, each variable domain of VH and VL contains three complementarity-determining regions (CDRs) that constitute a hypervariable region or loop. Generally, a native quadruple-chain antibody contains six hypervariable regions: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). In some cases, one VH or VL domain may be sufficient to confer antigen-binding specificity. In one embodiment, the antigen-binding site or particular portion of a protein of the present invention generally originates from the antibody. Generalized structures of antibody or immunoglobulin molecules are well known to those skilled in the art.

[0056] The term "antigen-binding molecule" or "antigen-binding polypeptide," in its broadest sense, refers to a molecule that specifically binds to an antigenic determinant. Examples of antigen-binding molecules or polypeptides are antibodies and antibody fragments.

[0057] Antibodies, or immunoglobulin molecules (also known as immunoglobulins, abbreviated as Ig), are gamma globulin proteins found in the blood or other bodily fluids of vertebrates, used by the immune system to identify and neutralize foreign substances, such as bacteria and viruses. They are typically composed of basic structural units to form monomers (one unit), dimers (two units), or pentamers (five units), each having two large heavy chains and two small light chains. Antibodies can bind to other molecules or structures known as antigens through non-covalent interactions. This binding is specific in the sense that the antibody will bind to only certain structures with high affinity. The specific portion of an antigen recognized by an antibody is called an epitope or antigenic determinant. The portion of the antibody that binds to an epitope is sometimes called a paratope and is located in the so-called variable domain or variable region (Fv) of the antibody. The variable domain contains three so-called complementarity-determining regions (CDRs) separated by a framework region (FR). "Immunoglobulin-like binding molecules" possess antigen-binding characteristics similar to those of immunoglobulin molecules and utilize the same functional strategies as antibody molecules; that is, they are capable of specifically binding to antigens and possess at least one antigen-binding region. However, immunoglobulin-like molecules are not limited to structures and sequences found in nature.

[0058] The term "bispecificity" means that an antigen-binding molecule is capable of specifically binding to at least two distinguishable antigenic determinants. The term "valence" refers to the presence of a specific number of binding sites in the antigen-binding molecule that are specific to one distinguishable antigenic determinant for one distinguishable antigenic determinant. Therefore, the term "divalent" indicates the presence of two binding sites in the antigen-binding molecule that are specific to each particular antigenic determinant. In certain embodiments of the present invention, the bispecific antigen-binding molecules are also divalent for each antigenic determinant, meaning that in that context they have two binding sites specific to CD137 and two binding sites specific to FAP.

[0059] It is well known in the art that the terms "complementarity-determining region" and "CDR" refer to discontinuous sequences of amino acids within the antibody variable region that confer antigen specificity and binding affinity. Generally, there are three CDRs (CDR-H1, CDR-H2, CDR-H3) in each heavy chain variable region and three CDRs (CDR-L1, CDR-L2, CDR-L3) in each light chain variable region.

[0060] The amino acid sequence boundaries of the specified CDRs are defined using the following methods: Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. ("Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme), MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745 ("Contact" numbering scheme), and Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol, 2003. The numbering scheme can be determined using one of many known schemes, including January; 27(1):55-77 ("IMGT" or "CCG" numbering scheme) and Honegger A and Pltickthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, 2001 Jun. 8; 309(3):657-70.

[0061] The boundaries of a given CDR may vary depending on the naming convention. The amino acid positions assigned to CDR and FR can be defined, for example, according to a numbering system. Here, specific amino acids in the variable region of the antibody of the present invention are numbered in a sequence starting from the N-terminus as amino acid "1" of the molecule and ending at the C-terminus of the molecule, and the boundaries of the CDR are defined by the following amino acid numbering.

[0062] [Table 2]

[0063] [Table 3]

[0064] [Table 4]

[0065] [Table 5]

[0066] For example, according to the Kabat rules for CD137 #1-6, the VH CDRs are arranged as follows: residues 31-35 (CDR1), 50-66 (CDR2), and 99-108 (CDR3). Also, according to the Kabat numbering system for CD137 #1-6, the VL CDRs are arranged as follows: residues 24-34 (CDR1), 50-56 (CDR2), and 89-97 (CDR3). According to the Kabat rules for CD137 VH #7-10, the VH CDRs are arranged as follows: residues 31-35 (CDR1), 50-65 (CDR2), and 98-107 (CDR3). Furthermore, according to the Kabat numbering system for CD137 #7~10, the VL CDRs are arranged as follows: residues 24~39 (CDR1), 55~61 (CDR2), and 94~102 (CDR3).

[0067] In the context of this invention, references to CDRs are based on the definition of the Kabat Rules, but this disclosure is not limited to FRs and CDRs defined by any one numbering system, but includes all numbering systems, including those considered above.

[0068] Therefore, unless otherwise specified, the terms “CDR” and “complementarity-determining region” of a given antibody or region, for example, the variable region, and individual CDRs (e.g., CDR-H1, CDR-H2), as well as the framework region (FR) of the antibody or region, should be understood to encompass each region (e.g., complementarity-determining region) defined by any of the known schemes described herein. In some cases, a scheme is specified for identifying one or more specific CDRs, for example, CDRs defined by Kabat, Chothia, CCG, IMGT, or other methods known in the art. In other cases, a specific amino acid sequence of the CDR is given. Alternative naming conventions for each CDR region of the present invention are given in Tables 3A to 3H below.

[0069] [Table 6] TIFF0007911965000010.tif254169 TIFF0007911965000011.tif105169

[0070] [Table 7] TIFF0007911965000013.tif254169 TIFF0007911965000014.tif105169

[0071] [Table 8] TIFF0007911965000016.tif242169 TIFF0007911965000017.tif249169 TIFF0007911965000018.tif96169

[0072] Table 9 TIFF0007911965000020.tif235169 TIFF0007911965000021.tif232169 TIFF0007911965000022.tif94169

[0073] Table 10 TIFF0007911965000024.tif101169

[0074] Table 11 TIFF0007911965000026.tif111169

[0075] Table 12 TIFF0007911965000028.tif100169

[0076] Table 13 TIFF0007911965000030.tif106169

[0077] The amino acid sequence modification of antibodies is intended. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. The term “amino acid sequence variant” includes substantial variants in which amino acid substitutions exist in one or more hypervariable region residues of a parent antigen-binding molecule (e.g., a humanized antibody or a human antibody). Generally, the obtained variants selected for further study will have modifications in specific biological properties suitable for the intended function. These modifications may include, but are not limited to, “improvements” in specific biological properties of the parent antigen-binding molecule, such as increased affinity or decreased immunogenicity, and / or will substantially retain the specific biological properties of the parent antigen-binding molecule. Amino acid sequence variants can be prepared by introducing appropriate nucleotide changes into the nucleic acid encoding the antibody variant or by peptide synthesis. Such modifications include, for example, deletions of residues in the amino acid sequence of the antibody variant and / or insertions and / or substitutions of residues into the same sequence. Any combination of deletions, insertions, and substitutions is made so as to arrive at a final construct, provided that the final construct possesses the desired characteristics. Furthermore, amino acid changes can alter the post-translational processing of antibody variants (for example, changes in the number or location of glycosylation sites).

[0078] For example, one, two, three, four, five, or six amino acids can be inserted, substituted, or deleted in each CDR (depending on their length, of course), while one, two, three, four, five, six, seven, eight, nine, ten, one, two, three, four, five, six, seven, eight, nine, ten, one, two, three, three, four, five, six, seven, eight, nine, five, six, seven, eight, nine, or ten residues can be inserted, substituted, or deleted in each FR. Preferably, amino acid sequence insertion into an antibody construct includes amino-terminal fusions and / or carboxyl-terminal fusions ranging in length from one, two, three, four, five, six, seven, eight, nine, or ten residues to polypeptides containing 100 or more residues, as well as intrasequence insertions of one or more amino acid residues. Furthermore, amino acid sequence deletions from antibody constructs in the amino-terminal and / or carboxy-terminal regions, ranging in length from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues, are intended to maximize and / or modify desired characteristics in the antibody construct, in addition to its ability to bind to the target antigen.

[0079] The most interesting sites for substitutional mutagenesis are the CDRs of the heavy and / or light chains, particularly the hypervariable regions (CDRs), but FR modifications in the heavy and / or light chains are also intended. Substitutions are preferably conservative substitutions as described herein. Preferably, depending on the length of the CDR or FR, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids can be substituted in the CDR, while 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25 amino acids can be substituted in the framework region (FR). For example, if the CDR sequence contains 6 amino acids, it is conceivable that 1, 2, or 3 of these amino acids will be substituted. Similarly, if the CDR sequence contains 15 amino acids, it is conceivable that 1, 2, 3, 4, 5, or 6 of these amino acids will be substituted.

[0080] Generally, when amino acids are substituted in one or more or all of the heavy chain and / or light chain CDRs, the resulting “substituted” sequence is preferably at least 60% or 65%, more preferably 70% or 75%, even more preferably 80% or 85%, and most preferably 90% or 95% identical to the “original” CDR sequence. This means that the degree to which a CDR is identical to the “substituted” sequence depends on the length of the CDR. For example, a CDR with five amino acids is preferably 80% identical to its substituted sequence because it has at least one substituted amino acid. Therefore, the CDRs of an antibody construct may have varying degrees of identity with respect to their substituted sequences; for example, CDRL1 may have 80% identity, while CDRL3 may have 90% identity.

[0081] The "Fc portion" of an antibody does not directly participate in the binding of the antibody to the antigen, but exhibits various effector functions. The term "Fc portion of an antibody" is well known to those skilled in the art and is defined based on the papain cleavage of the antibody. Depending on the amino acid sequence of the constant region of their heavy chain, antibodies or immunoglobulins are classified into the classes IgA, IgD, IgE, IgG, and IgM. According to the heavy chain constant region, the various classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. Some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, and IgG4, IgAl, and IgA2. The Fc portion of an antibody is directly involved in ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement-dependent cell-mediated cytotoxicity) based on complement activation, Clq binding, and Fc receptor binding. Complement activation (CDC) is initiated by the binding of the complement factor Clq to the Fc portion of most IgG antibody subclasses. The effect of antibodies on the complement system depends on specific conditions, but binding to Clq is triggered by a defined binding site on the Fc portion. Such binding sites are known in the art, for example, Boakle et al., Nature 282 (1975) 742-743, Lukas et al., J. Immunol. 127 (1981) 2555-2560, Brunhouse and Cebra, Mol. Immunol. 16 (1979) 907-917, Burton et al., Nature 288 (1980) 338-344, Thommesen et al., Mol. Immunol. 37 (2000) 995-1004, Idusogie et al., J. Immunol. 164 (2000) 4178-4184, Hezareh et al., J. Virology 75 (2001) 12161-12168, Morgan et al., Immunology This is described in 86 (1995) 319-324, EP No. 0307434. Such binding sites are, for example, L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numbered according to the Kabat EU index).Of these residues, L234 and L235 are the most important for mediating C1q and Fc gamma receptor binding in IgG1 (Hezareh et al., J. Virology 75 (2001) 12161-12168). Antibodies of subclasses IgG1 and IgG3 typically exhibit complement activation and Clq and C3 binding, while IgG2 and IgG4 do not activate the complement system and do not bind to Clq and C3.

[0082] In embodiments of the present invention, the binding of the binding molecule to the complement product C1q or Fc gamma receptor is removed by utilizing the constant region of IgG1, which involves L-to-A mutagenesis at positions 234 and 235 (corresponding to amino acids 117 and 118 of human IgG1 SEQ ID NO: 283 and human IgG1 KO SEQ ID NO: 284).

[0083] In the art, antibodies have been further developed and have become a versatile tool for use in medicine and technology. Therefore, in the context of this invention, the terms “antibody molecule” or “antibody” (as used synonymously herein) encompass not only naturally occurring antibodies, such as those containing, for example, two light chains and two heavy chains or just two heavy chains, as in camel species, but also all molecules containing at least one paratope having binding specificity to an antigen and structural similarity to the variable domain of immunoglobulins.

[0084] Therefore, antibodies may include monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, antibody fragments, particularly Fv, Fab, Fab', or F(ab')2 fragments, single-chain antibodies, particularly single-chain variable fragments (scFv), small module immunotherapy drugs (SMIPs), domain antibodies, nanobodies, and diabodies.

[0085] Monoclonal antibodies (mAbs) are monospecific antibodies with identical amino acid sequences. They can be produced using hybridoma technology from hybrid cell lines (called hybridomas) that represent clones of fusion products between specific antibody-producing B cells and melanoma (B-cell cancer) cells (Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975; 256:495-7.). Alternatively, monoclonal antibodies can be produced by recombinant expression in host cells (Norderhaug L, Olafsen T, Michaelsen TE, Sandlie I. (May 1997). "Versatile vectors for transient and stable expression of recombinant antibody molecules in mammalian cells." J Immunol Methods 204 (1): 77-87, see also below).

[0086] For human application, it is often desirable to reduce the immunogenicity of antibodies originally derived from other species, such as mice. This can be achieved by constructing chimeric antibodies or through a process called "humanization." In this context, a "chimeric antibody" is understood to be an antibody containing a sequence portion (e.g., a variable domain) derived from one species (e.g., mouse) fused with a sequence portion (e.g., a constant domain) derived from a different species (e.g., human). A "humanized antibody" is an antibody containing a variable domain originally derived from a non-human species, where specific amino acids have been mutated to make the overall sequence of the variable domain more closely resemble the sequence of a human variable domain. Methods for the chimerization and humanization of antibodies are well known in this art (Billetta R, Lobuglio AF. "Chimeric antibodies". Int Rev Immunol. 1993;10(2-3):165-76; Riechmann L, Clark M, Waldmann H, Winter G (1988). "Reshaping human antibodies for therapy". Nature: 332:323).

[0087] Furthermore, techniques for producing antibodies based on sequences derived from the human genome have been developed, for example, through phage display or the use of transgenic animals (WO No. 90 / 05144, D. Marks, HR Hoogenboom, TP Bonnert, J. McCafferty, AD Griffiths and G. Winter (1991) "By-passing immunisation. Human antibodies from V-gene libraries displayed on phage." J.Mol.Biol., 222, 581-597, Knappik et al., J. Mol. Biol. 296: 57-86, 2000, S. Carmen and L. Jermutus, "Concepts in antibody phage display." Briefings in Functional Genomics and Proteomics 2002 1(2):189-203, Lonberg N, Huszar D. "Human antibodies from transgenic mice." Int Rev Immunol. 1995;13(1):65-93., Bruggemann M, Taussig MJ. "Production of human antibody repertoires in transgenic mice." Curr Opin Biotechnol. 1997 Aug;8(4):455-8.). Such antibodies are referred to as "human antibodies" in the context of this invention.

[0088] Furthermore, antibodies may also include immunoglobulin fragments that retain antigen-binding properties, such as Fab, Fab', or F(ab')2 fragments. Such fragments can be obtained, for example, by fragmentation of immunoglobulins by proteolytic digestion or by recombinant expression of such fragments. For example, immunoglobulin digestion can be achieved by routine techniques, for example, using papain or pepsin (WO 94 / 29348). Papain digestion of antibodies typically produces two identical antigen-binding fragments, so-called Fab fragments, each having one antigen-binding site and a residual Fc fragment. Pepsin treatment generates F(ab')2. In Fab molecules, the variable domains are preferably fused to the constant domains of human immunoglobulins. Thus, the heavy chain variable domain can be fused to the CH1 domain (so-called Fd fragment), and the light chain variable domain can be fused to the CL domain. Fab molecules can be produced by recombinant expression of each nucleic acid in host cells. See below.

[0089] Numerous techniques have been developed to place the variable domains of immunoglobulins or molecules derived from such variable domains into different molecular contexts. These are the “immunoglobulin-like” molecules of the present invention. In some cases, these immunoglobulin-like molecules can be smaller in size compared to naturally occurring immunoglobulins and may, for example, contain one or more amino acid chains. For example, a single-stranded variable fragment (scFv) is a fusion of the variable regions of the heavy and light chains of an immunoglobulin, linked together with a short linker, usually serine (S) or glycine (G) (WO Nos. 88 / 01649, 91 / 17271, Huston et al; International Reviews of Immunology, Volume 10, 1993, 195-217). A "single-domain antibody" or "nanobody" has an antigen-binding site in one Ig-like domain (WO No. 94 / 04678, No. 03 / 050531, Ward et al., Nature. 1989 Oct 12;341 (6242):544-6, Revets et al., Expert Opin Biol Ther. 5(1):111-24, 2005). One or more single-domain antibodies with binding specificity for the same or different antigens can be linked together. A nanobody is a bivalent antibody molecule consisting of two amino acid chains containing two variable domains (WO No. 94 / 13804, Holliger et al., Proc Natl Acad Sci US A. 1993 Jul 15;90(14):6444-8). Other examples of antibody-like molecules are immunoglobulin superfamily antibodies (IgSF, Srinivasan and Roeske, Current Protein Pept. Sci. 2005, 6(2): 185-96). From a different concept, so-called small module immunotherapies (SMIPs) arise, which contain an Fv domain linked to a single-strand hinge and an effector domain lacking the constant domain CH1 (WO No. 02 / 056910).

[0090] With respect to the present invention, a first aspect of the present invention provides an immunoglobulin-like binding molecule having at least one antigen-binding site that specifically binds to CD137 (4-1BB, TNFRSF9) and at least one antigen-binding site that specifically binds to fibroblast-activating protein (FAP).

[0091] In one embodiment, the immunoglobulin-like binding molecule of the present invention binds to CD137 (4-1BB, TNFRSF9) or fibroblast-activating protein (FAP) target antigens with affinity (1 pM to 100 μM, preferably with a KD value in the range of 1 pM to 1 μM) as measured by surface plasmon resonance analysis (Malmqvist M., "Surface plasmon resonance for detection and measurement of antibody-antigen affinity and kinetics.", Curr Opin Immunol. 1993 Apr;5(2):282-6.). Antibody affinity can also be measured using kinetic exclusion assay (KinExA) technology (Darling, RJ, and Brault PA., "Kinetic exclusion assay technology: Characterization of Molecular Interactions." ASSAY and Drug Development Technologies. 2004, Dec 2(6): 647-657).

[0092] As used herein, the terms “binding” or “specifically binding” refer to the binding of an antibody and / or immunoglobulin-like molecule to an antigen epitope in an in vitro assay, preferably a surface plasmon resonance assay (SPR, BIAcore, GE-Healthcare Uppsala, Sweden). The binding affinity is k on (Rate constant for antibody association from antibody / antigen complex), k off (Dissociation constant) and KD(k off / k onis defined by the term "specifically binds." Specifically binding generally refers to the formation of a complex between a receptor molecule and its ligand. In the context of antibody-antigen binding, high-affinity antibodies typically bind to their target antigens with an affinity of 10 -9 M or less.

[0093] An "antibody that specifically binds to CD137" or an "immunoglobulin-like binding molecule that specifically binds to CD137" or an "antibody that specifically binds to FAP" or an "immunoglobulin-like binding molecule that specifically binds to FAP" refers to a molecule that can bind with sufficient affinity such that the antibody and / or immunoglobulin-like binding molecule is useful as a diagnostic agent and / or therapeutic agent in targeting CD137 or FAP, respectively. In one embodiment, the degree of binding of an anti-CD137 binding molecule to an unrelated non-CD137 protein is less than about 10% of the binding of the antibody and / or immunoglobulin-like binding molecule to CD137, as measured, for example, by radioimmunoassay (RIA) or flow cytometry (FAC). In certain embodiments, the antibody and / or immunoglobulin-like binding molecule that binds to CD1 have a dissociation constant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, ≦0.001 nM. Similarly, in one embodiment, the degree of binding of an anti-FAP antibody to an unrelated non-FAP protein is less than about 10% of the binding of the antibody and / or immunoglobulin-like binding molecule to FAP, as measured, for example, by radioimmunoassay (RIA) or flow cytometry (FAC). In certain embodiments, the antibody and / or immunoglobulin-like binding molecule that binds to FAP have a dissociation constant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, ≦0.001 nM (e.g., 10 -6 M or less, 10 -50 M to 10 -13 M, e.g., 10 -8 M to 10 -10 M).

[0094] The binding affinity of antibody molecules can be improved by known processes known as affinity maturation (Marks et al., 1992, Biotechnology 10:779-783, Barbas, et al., 1994, Proc. Nat. Acad. Sci, USA 91:3809-3813, Shier et al., 1995, Gene 169:147-155). Therefore, affinity-matured antibodies and / or immunoglobulin-like binding molecules are also included in the present invention.

[0095] In a more preferred embodiment, the antigen-binding site that specifically binds to CD137(4-1BB,TNFRSF9) is part of an immunoglobulin (Ig) molecule, and the antigen-binding site that specifically binds to fibroblast-activating protein (FAP) includes one or more scFv, scFab, Fab, or Fv binding elements. Preferably, the antigen-binding site that specifically binds to fibroblast-activating protein (FAP) includes two scFvs.

[0096] A "single-stranded Fv fragment" (scFv) is a polypeptide comprising an antibody heavy chain variable domain (VH), a linker, and an antibody light chain variable domain (VL), wherein the antibody domain and the linker have one of the following sequences from the N-terminus to the C-terminus: a) VH-linker-VL, or b) VL-linker-VH, where the linker is a polypeptide having lengths of 15 and 25 amino acids, preferably 20 amino acids.

[0097] In addition, these single-chain Fab molecules can be further stabilized by incorporating disulfide bonds between the VH domain and the VL domain, or within the VH domain or within the VL domain, via the incorporation of cysteine ​​residues. The term N-terminus refers to the first amino acid of the polypeptide chain, while the term C-terminus refers to the last amino acid of the C-terminus of the polypeptide chain. Accordingly, embodiments of the present invention include one or more scFv molecules containing additional cysteine ​​residues for forming disulfide bonds.

[0098] As demonstrated in the attached examples, the inventors have shown that FAP scFv having a VL-VH orientation from the N-terminus to the C-terminus can function in the binding molecule of the present invention to induce CD137 crosslinking in target cells. FAP scFv having a VH-VL orientation from the N-terminus to the C-terminus can also function, but its activity may be reduced in this orientation. Therefore, a preferred embodiment of the present invention is when the order is VL-VH from the N-terminus to the C-terminus.

[0099] A more preferred embodiment of the present invention is in which one or more scFvs are fused to an Ig molecule by a peptide linker, preferably a peptide linker having a length of about 4 to 20 amino acids. Preferably, the scFv is fused to the C-terminus of the heavy chain of the Ig molecule. Preferably, the Ig molecule is IgG.

[0100] Methods for linking scFv molecules to the C-terminus of the heavy chain of an IgG molecule are well known in the art. Typically, small linker sequences of glycine and serine amino acids (called GS minilinkers) are used. The number of amino acids in the linker can be varied to 4 (GGGS) (SEQ ID NO: 279), 6 (GGSGGS) (SEQ ID NO: 280), 10 (GGGGSGGGGS) (SEQ ID NO: 281), 20 (GGGGSGGGGSGGGGSGGGGS) (SEQ ID NO: 282) or more. In practice, the linker is usually formed by combining a nucleic acid molecule encoding the IgG of interest (which in this case would include a nucleic acid encoding the variable domain of the heavy chain for the CD137 (4-1BB, TNFRSF9) binding site) with a nucleic acid encoding the desired scFv (which in this case would include a nucleic acid encoding the variable domain of the heavy chain for the fibroblast-activating protein (FAP) binding site), spaced apart by a nucleic acid molecule encoding the linker sequence. Next, as will be further explained below, this complete HC-scFv coding nucleic acid molecule is placed in an expression vector and introduced into a suitable host cell so that a complete IgG heavy chain-scFv single polypeptide is formed.

[0101] Preferably, the GS linker is GGGGSGGGGSGGGGSGGGGS (Sequence ID: 282).

[0102] Immunoglobulin-like binding molecules can be fused (as fusion proteins) to other molecular entities that have a desired effect on the properties of antibody molecules, or they can be linked in some way (by covalent or non-covalent bonds). For example, it may be desirable to improve the pharmacokinetic properties of antibody molecules, such as their stability in body fluids, such as blood, particularly in the case of single-chain antibodies or domain antibodies. In this regard, many techniques have been developed, in particular, to extend the half-life of such antibody molecules in circulation, such as pegylation (WO Nos. 98 / 25971, 98 / 48837, and 2004081026), fusing or covalently attaching antibody molecules to other antibody molecules that have affinity for serum proteins, such as albumin (WO Nos. 2004041865 and 2004003019), or expressing antibody molecules as fusion proteins with all or part of serum proteins, such as albumin or transferrin (WO No. 2001079258).

[0103] The Fc region of natural antibodies interacts with many Fc receptors, resulting in many important functional capabilities (referred to as "effector functions"). The immunoglobulin-like binding molecules of the present invention contain a portion of the Fc region that has been engineered to avoid unintended crosslinking by soluble Fc gamma receptors or complement C1q. In one embodiment, such an antibody variant has a significantly lower affinity for Fc gamma receptors and complement C1q than the parent antibody. Therefore, embodiments of the present invention include an Fc variant in which the Ig molecule has reduced affinity for Fc gamma receptors or complement receptors, or both, compared to the wild-type Fc region.

[0104] Further embodiments of the present invention include an Fc region or related portion thereof that has been manipulated to modify serum levels (half-life) by optimizing its interaction with neonatal Fc receptors (FcRn).

[0105] Methods for preparing binding sites that bind to specific target antigens are well known in the art. Those skilled in the art can readily use these methods to devise binding sites with the specificity required for CD137 (4-1BB, TNFRSF9) or fibroblast-activating protein (FAP) target antigens.

[0106] Methods for generating antibodies and antibody fragments are well known in the art. For example, antibodies can be generated by one of several methods that utilize the induction of in vivo production of antibody molecules, screening of immunoglobulin libraries (Orlandi et al, 1989. Proc. Natl. Acad. Sci. USA 86:3833-3837; Winter et al 1991, Nature 349:293-299), or the generation of monoclonal antibody molecules by cell lines in culture. These include, but are not limited to, hybridoma technology, human B-cell hybridoma technology, and Epstein-Barr virus (EBV) hybridoma technology (Kohler et al 1975. Nature 256:4950497; Kozbor et al 1985. J. Immunol. Methods 81:31-42; Cote et al 1983. Proc. Natl. Acad. Sci. USA 80:2026-2030; Cole et al 1984. Mol. Cell. Biol. 62:109-120).

[0107] Using these methods, it would be routine for those skilled in the art to prepare antibodies having binding sites with the specificity required for CD137 (4-1BB, TNFRSF9) or fibroblast-activating protein (FAP) target antigens. Isolation of binding domains from such antibodies is a routine practice, and further information on methods that can be used in practice is provided in the accompanying examples.

[0108] The inventors prepared specific CD137(4-1BB,TNFRSF9) / fibroblast-activating protein (FAP) immunoglobulin-like binding molecules using exemplary antigen-binding sites for CD137 and FAP listed below. These are examined in the attached examples.

[0109] As a non-limiting example, bispecific molecules were prepared using exemplary antigen-binding sites specific to CD137 (4-1BB, TNFRSF9), and are CD137 #1, CD137 #2, CD137 #3, CD137 #4, CD137 #5, CD137 #6, CD137 #7, CD137 #8, CD137 #9, and CD137 #10.

[0110] As a non-limiting example, bispecific molecules were prepared using exemplary antigen-binding sites specific to fibroblast-activating proteins (FAPs), and these were referred to as FAP #1, FAP #2, FAP #3, FAP #4, and FAP #5.

[0111] The amino acid sequence of the specific antigen-binding site is provided in this description and the sequence listing.

[0112] The following details of preferred embodiments of the present invention, including specific binding sites for CD137 (4-1BB, TNFRSF9) or fibroblast-activating protein (FAP), are provided below.

[0113] To avoid any doubt, each of the specific embodiments listed below for the first aspect of the present invention can also be considered an independent aspect of the present invention.

[0114] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific to CD137 includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 290 (CDR1), SEQ ID NO: 8 (CDR2), and SEQ ID NO: 9 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 12 (CDR1), SEQ ID NO: 13 (CDR2), and SEQ ID NO: 14 (CDR3). In this embodiment, the antigen-binding site specific to CD137 is CD137 #1.

[0115] Preferably, the antigen-binding site specific to CD137 includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 10 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 15. In this embodiment, the antigen-binding site specific to CD137 is CD137 #1.

[0116] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific to CD137 includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 18 (CDR2), and SEQ ID NO: 9 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 22 (CDR1), SEQ ID NO: 23 (CDR2), and SEQ ID NO: 14 (CDR3). In this embodiment, the antigen-binding site specific to CD137 is CD137 #2.

[0117] Preferably, the antigen-binding site specific to CD137 includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 20 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 25. In this embodiment, the antigen-binding site specific to CD137 is CD137 #2.

[0118] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific to CD137 includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 28 (CDR2), and SEQ ID NO: 9 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 32 (CDR1), SEQ ID NO: 33 (CDR2), and SEQ ID NO: 14 (CDR3). In this embodiment, the antigen-binding site specific to CD137 is CD137 #3.

[0119] Preferably, the antigen-binding site specific to CD137 includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 30 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 35. In this embodiment, the antigen-binding site specific to CD137 is CD137 #3.

[0120] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific to CD137 includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 38 (CDR2), and SEQ ID NO: 9 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 42 (CDR1), SEQ ID NO: 43 (CDR2), and SEQ ID NO: 14 (CDR3). In this embodiment, the antigen-binding site specific to CD137 is CD137 #4.

[0121] Preferably, the antigen-binding site specific to CD137 includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 40 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 45. In this embodiment, the antigen-binding site specific to CD137 is CD137 #4.

[0122] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific to CD137 includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 48 (CDR2), and SEQ ID NO: 9 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 52 (CDR1), SEQ ID NO: 53 (CDR2), and SEQ ID NO: 14 (CDR3). In this embodiment, the antigen-binding site specific to CD137 is CD137 #5.

[0123] Preferably, the antigen-binding site specific to CD137 includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 50 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 55. In this embodiment, the antigen-binding site specific to CD137 is CD137 #5.

[0124] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific to CD137 includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 58 (CDR2), and SEQ ID NO: 9 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 62 (CDR1), SEQ ID NO: 63 (CDR2), and SEQ ID NO: 14 (CDR3). In this embodiment, the antigen-binding site specific to CD137 is CD137 #6.

[0125] Preferably, the antigen-binding site specific to CD137 includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 60 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 65. In this embodiment, the antigen-binding site specific to CD137 is CD137 #6.

[0126] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific to CD137 includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 308 (CDR1), SEQ ID NO: 68 (CDR2), and SEQ ID NO: 69 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 72 (CDR1), SEQ ID NO: 73 (CDR2), and SEQ ID NO: 74 (CDR3). In this embodiment, the antigen-binding site specific to CD137 is CD137 #7.

[0127] Preferably, the antigen-binding site specific to CD137 includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 70 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 75. In this embodiment, the antigen-binding site specific to CD137 is CD137 #7.

[0128] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific to CD137 includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 308 (CDR1), SEQ ID NO: 78 (CDR2), and SEQ ID NO: 69 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 72 (CDR1), SEQ ID NO: 73 (CDR2), and SEQ ID NO: 74 (CDR3). In this embodiment, the antigen-binding site specific to CD137 is CD137 #8.

[0129] Preferably, the antigen-binding site specific to CD137 includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 80 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 85. In this embodiment, the antigen-binding site specific to CD137 is CD137 #8.

[0130] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific for CD137 comprises a variable heavy chain region and a variable light chain region, wherein the heavy chain CDRs comprise the amino acid sequences of SEQ ID NO: 308 (CDR1), SEQ ID NO: 88 (CDR2) and SEQ ID NO: 69 (CDR3), and the light chain CDRs comprise the amino acid sequences of SEQ ID NO: 92 (CDR1), SEQ ID NO: 93 (CDR2) and SEQ ID NO: 74 (CDR3). In this embodiment, the antigen-binding site specific for CD137 is CD137 #9. Preferably, the antigen-binding site specific for CD137 comprises a variable heavy chain region comprising the amino acid sequence of SEQ ID NO: 90 and a variable light chain region comprising the amino acid sequence of SEQ ID NO: 95. In this embodiment, the antigen-binding site specific for CD137 is CD137 #9. In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific for CD137 comprises a variable heavy chain region and a variable light chain region, wherein the heavy chain CDRs comprise the amino acid sequences of SEQ ID NO: 308 (CDR1), SEQ ID NO: 98 (CDR2) and SEQ ID NO: 69 (CDR3), and the light chain CDRs comprise the amino acid sequences of SEQ ID NO: 92 (CDR1), SEQ ID NO: 93 (CDR2) and SEQ ID NO: 74 (CDR3). In this embodiment, the antigen-binding site specific for CD137 is CD137 #10.

[0131] Preferably, the antigen-binding site specific for CD137 comprises a variable heavy chain region comprising the amino acid sequence of SEQ ID NO: 100 and a variable light chain region comprising the amino acid sequence of SEQ ID NO: 105. In this embodiment, the antigen-binding site specific for CD137 is CD137 #10.

[0132] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific for FAP comprises a variable heavy chain region and a variable light chain region, wherein the heavy chain CDRs comprise the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 108 (CDR2) and SEQ ID NO: 109 (CDR3), and the light chain CDRs comprise the amino acid sequences of SEQ ID NO: 111 (CDR1), SEQ ID NO: 112 (CDR2) and SEQ ID NO: 113 (CDR3). In this embodiment, the antigen-binding site specific for FAP is FAP #1.

[0133] Preferably, the antigen-binding site specific for FAP comprises a variable heavy chain region comprising the amino acid sequence of SEQ ID NO: 106 and a variable light chain region comprising the amino acid sequence of SEQ ID NO: 110. In this embodiment, the antigen-binding site specific for FAP is FAP #1.

[0134] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific for FAP comprises a variable heavy chain region and a variable light chain region, wherein the heavy chain CDRs comprise the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 117 (CDR2) and SEQ ID NO: 109 (CDR3), and the light chain CDRs comprise the amino acid sequences of SEQ ID NO: 120 (CDR1), SEQ ID NO: 112 (CDR2) and SEQ ID NO: 113 (CDR3). In this embodiment, the antigen-binding site specific for FAP is FAP #2.

[0135] Preferably, the antigen-binding site specific for FAP comprises a variable heavy chain region comprising the amino acid sequence of SEQ ID NO: 115 and a variable light chain region comprising the amino acid sequence of SEQ ID NO: 119. In this embodiment, the antigen-binding site specific for FAP is FAP #2.

[0136] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific for FAP comprises a variable heavy chain region and a variable light chain region, wherein the heavy chain CDRs comprise the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 126 (CDR2) and SEQ ID NO: 109 (CDR3), and the light chain CDRs comprise the amino acid sequences of SEQ ID NO: 129 (CDR1), SEQ ID NO: 112 (CDR2) and SEQ ID NO: 113 (CDR3). In this embodiment, the antigen-binding site specific for FAP is FAP #3.

[0137] Preferably, the antigen-binding site specific for FAP comprises a variable heavy chain region comprising the amino acid sequence of SEQ ID NO: 124 and a variable light chain region comprising the amino acid sequence of SEQ ID NO: 128. In this embodiment, the antigen-binding site specific for FAP is FAP #3.

[0138] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific to FAP includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 328 (CDR1), SEQ ID NO: 135 (CDR2), and SEQ ID NO: 136 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 138 (CDR1), SEQ ID NO: 139 (CDR2), and SEQ ID NO: 140 (CDR3). In this embodiment, the antigen-binding site specific to FAP is FAP #4.

[0139] Preferably, the antigen-binding site specific to FAP includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 133 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 137. In this embodiment, the antigen-binding site specific to FAP is FAP #4.

[0140] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific to FAP includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 333 (CDR1), SEQ ID NO: 144 (CDR2), and SEQ ID NO: 145 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 138 (CDR1), SEQ ID NO: 139 (CDR2), and SEQ ID NO: 140 (CDR3). In this embodiment, the antigen-binding site specific to FAP is FAP #5.

[0141] Preferably, the antigen-binding site specific to FAP includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 142 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 146. In this embodiment, the antigen-binding site specific to FAP is FAP #5.

[0142] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific to CD137 includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 290 (CDR1), SEQ ID NO: 8 (CDR2), and SEQ ID NO: 9 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 12 (CDR1), SEQ ID NO: 13 (CDR2), and SEQ ID NO: 14 (CDR3). The antigen-binding site specific to FAP includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 108 (CDR2), and SEQ ID NO: 109 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 111 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3). In this embodiment, the antigen-binding site specific to CD137 is CD137 #1, and the antigen-binding site specific to FAP is FAP #1.

[0143] Preferably, the antigen-binding site specific to CD137 includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 10 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 15, and the antigen-binding site specific to FAP includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 106 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 110. In this embodiment, the antigen-binding site specific to CD137 is CD137 #1, and the antigen-binding site specific to FAP is FAP #1.

[0144] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific to CD137 includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 308 (CDR1), SEQ ID NO: 68 (CDR2), and SEQ ID NO: 69 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 72 (CDR1), SEQ ID NO: 73 (CDR2), and SEQ ID NO: 74 (CDR3). The antigen-binding site specific to FAP includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 108 (CDR2), and SEQ ID NO: 109 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 111 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3). In this embodiment, the antigen-binding site specific to CD137 is CD137 #7, and the antigen-binding site specific to FAP is FAP #1.

[0145] Preferably, the antigen-binding site specific to CD137 includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 70 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 75, and the antigen-binding site specific to FAP includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 106 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 110. In this embodiment, the antigen-binding site specific to CD137 is CD137 #7, and the antigen-binding site specific to FAP is FAP #1.

[0146] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific to CD137 includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 290 (CDR1), SEQ ID NO: 8 (CDR2), and SEQ ID NO: 9 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 12 (CDR1), SEQ ID NO: 13 (CDR2), and SEQ ID NO: 14 (CDR3). The antigen-binding site specific to FAP includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 328 (CDR1), SEQ ID NO: 135 (CDR2), and SEQ ID NO: 136 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 138 (CDR1), SEQ ID NO: 139 (CDR2), and SEQ ID NO: 140 (CDR3). In this embodiment, the antigen-binding site specific to CD137 is CD137 #1, and the antigen-binding site specific to FAP is FAP #4.

[0147] Preferably, the antigen-binding site specific to CD137 includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 10 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 15, and the antigen-binding site specific to FAP includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 133 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 137. In this embodiment, the antigen-binding site specific to CD137 is CD137 #1, and the antigen-binding site specific to FAP is FAP #4.

[0148] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific to CD137 includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 308 (CDR1), SEQ ID NO: 68 (CDR2), and SEQ ID NO: 69 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 72 (CDR1), SEQ ID NO: 73 (CDR2), and SEQ ID NO: 74 (CDR3). The antigen-binding site specific to FAP includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 328 (CDR1), SEQ ID NO: 135 (CDR2), and SEQ ID NO: 136 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 138 (CDR1), SEQ ID NO: 139 (CDR2), and SEQ ID NO: 140 (CDR3). In this embodiment, the antigen-binding site specific to CD137 is CD137 #7, and the antigen-binding site specific to FAP is FAP #4.

[0149] Preferably, the antigen-binding site specific to CD137 includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 70 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 75, and the antigen-binding site specific to FAP includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 133 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 137. In this embodiment, the antigen-binding site specific to CD137 is CD137 #7, and the antigen-binding site specific to FAP is FAP #4.

[0150] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific to CD137 includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 18 (CDR2), and SEQ ID NO: 9 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 22 (CDR1), SEQ ID NO: 23 (CDR2), and SEQ ID NO: 14 (CDR3). The antigen-binding site specific to FAP includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 333 (CDR1), SEQ ID NO: 144 (CDR2), and SEQ ID NO: 145 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 138 (CDR1), SEQ ID NO: 139 (CDR2), and SEQ ID NO: 140 (CDR3). In this embodiment, the antigen-binding site specific to CD137 is CD137 #2, and the antigen-binding site specific to FAP is FAP #5.

[0151] Preferably, the antigen-binding site specific to CD137 includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 20 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 25, and the antigen-binding site specific to FAP includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 142 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 146. In this embodiment, the antigen-binding site specific to CD137 is CD137 #2, and the antigen-binding site specific to FAP is FAP #5.

[0152] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific to CD137 includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 18 (CDR2), and SEQ ID NO: 19 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 22 (CDR1), SEQ ID NO: 23 (CDR2), and SEQ ID NO: 14 (CDR3). The antigen-binding site specific to FAP includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 117 (CDR2), and SEQ ID NO: 109 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 120 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3). In this embodiment, the antigen-binding site specific to CD137 is CD137 #2, and the antigen-binding site specific to FAP is FAP #2.

[0153] Preferably, the antigen-binding site specific to CD137 includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 20 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 25, and the antigen-binding site specific to FAP includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 115 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 119. In this embodiment, the antigen-binding site specific to CD137 is CD137 #2, and the antigen-binding site specific to FAP is FAP #2.

[0154] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific for CD137 comprises a variable heavy chain region and a variable light chain region, wherein the heavy chain CDRs comprise the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 18 (CDR2), and SEQ ID NO: 9 (CDR3), and the light chain CDRs comprise the amino acid sequences of SEQ ID NO: 22 (CDR1), SEQ ID NO: 23 (CDR2), and SEQ ID NO: 14 (CDR3); the antigen-binding site specific for FAP comprises a variable heavy chain region and a variable light chain region, wherein the heavy chain CDRs comprise the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 126 (CDR2), and SEQ ID NO: 109 (CDR3), and the light chain CDRs comprise the amino acid sequences of SEQ ID NO: 129 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3). In this embodiment, the antigen-binding site specific for CD137 is CD137 #2, and the antigen-binding site specific for FAP is FAP #3.

[0155] Preferably, the antigen-binding site specific for CD137 comprises a variable heavy chain region comprising the amino acid sequence of SEQ ID NO: 30 and a variable light chain region comprising the amino acid sequence of SEQ ID NO: 35, and the antigen-binding site specific for FAP comprises a variable heavy chain region comprising the amino acid sequence of SEQ ID NO: 124 and a variable light chain region comprising the amino acid sequence of SEQ ID NO: 128. In this embodiment, the antigen-binding site specific for CD137 is CD137 #2, and the antigen-binding site specific for FAP is FAP #3.

[0156] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific to CD137 includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 28 (CDR2), and SEQ ID NO: 9 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 32 (CDR1), SEQ ID NO: 33 (CDR2), and SEQ ID NO: 14 (CDR3). The antigen-binding site specific to FAP includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 333 (CDR1), SEQ ID NO: 144 (CDR2), and SEQ ID NO: 145 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 138 (CDR1), SEQ ID NO: 139 (CDR2), and SEQ ID NO: 140 (CDR3). In this embodiment, the antigen-binding site specific to CD137 is CD137 #3, and the antigen-binding site specific to FAP is FAP #5.

[0157] Preferably, the antigen-binding site specific to CD137 includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 30 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 35, and the antigen-binding site specific to FAP includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 142 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 146. In this embodiment, the antigen-binding site specific to CD137 is CD137 #3, and the antigen-binding site specific to FAP is FAP #5.

[0158] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific to CD137 includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 28 (CDR2), and SEQ ID NO: 9 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 32 (CDR1), SEQ ID NO: 33 (CDR2), and SEQ ID NO: 14 (CDR3). The antigen-binding site specific to FAP includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 117 (CDR2), and SEQ ID NO: 109 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 120 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3). In this embodiment, the antigen-binding site specific to CD137 is CD137 #3, and the antigen-binding site specific to FAP is FAP #2.

[0159] Preferably, the antigen-binding site specific to CD137 includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 30 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 35, and the antigen-binding site specific to FAP includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 115 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 119. In this embodiment, the antigen-binding site specific to CD137 is CD137 #3, and the antigen-binding site specific to FAP is FAP #2.

[0160] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific to CD137 includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 28 (CDR2), and SEQ ID NO: 9 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 32 (CDR1), SEQ ID NO: 33 (CDR2), and SEQ ID NO: 14 (CDR3). The antigen-binding site specific to FAP includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 126 (CDR2), and SEQ ID NO: 109 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 129 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3). In this embodiment, the antigen-binding site specific to CD137 is CD137 #3, and the antigen-binding site specific to FAP is FAP #3.

[0161] Preferably, the antigen-binding site specific to CD137 includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 30 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 35, and the antigen-binding site specific to FAP includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 124 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 128. In this embodiment, the antigen-binding site specific to CD137 is CD137 #3, and the antigen-binding site specific to FAP is FAP #3.

[0162] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific to CD137 includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 38 (CDR2), and SEQ ID NO: 9 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 42 (CDR1), SEQ ID NO: 43 (CDR2), and SEQ ID NO: 14 (CDR3). The antigen-binding site specific to FAP includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 333 (CDR1), SEQ ID NO: 144 (CDR2), and SEQ ID NO: 145 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 138 (CDR1), SEQ ID NO: 139 (CDR2), and SEQ ID NO: 140 (CDR3). In this embodiment, the antigen-binding site specific to CD137 is CD137 #4, and the antigen-binding site specific to FAP is FAP #5.

[0163] Preferably, the antigen-binding site specific to CD137 includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 40 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 45, and the antigen-binding site specific to FAP includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 142 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 146. In this embodiment, the antigen-binding site specific to CD137 is CD137 #4, and the antigen-binding site specific to FAP is FAP #5.

[0164] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific to CD137 includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 38 (CDR2), and SEQ ID NO: 9 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 42 (CDR1), SEQ ID NO: 43 (CDR2), and SEQ ID NO: 14 (CDR3). The antigen-binding site specific to FAP includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 117 (CDR2), and SEQ ID NO: 109 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 120 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3). In this embodiment, the antigen-binding site specific to CD137 is CD137 #4, and the antigen-binding site specific to FAP is FAP #2.

[0165] Preferably, the antigen-binding site specific to CD137 includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 40 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 45, and the antigen-binding site specific to FAP includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 115 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 119. In this embodiment, the antigen-binding site specific to CD137 is CD137 #4, and the antigen-binding site specific to FAP is FAP #2.

[0166] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific to CD137 includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 38 (CDR2), and SEQ ID NO: 9 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 42 (CDR1), SEQ ID NO: 43 (CDR2), and SEQ ID NO: 14 (CDR3). The antigen-binding site specific to FAP includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 126 (CDR2), and SEQ ID NO: 109 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 129 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3). In this embodiment, the antigen-binding site specific to CD137 is CD137 #4, and the antigen-binding site specific to FAP is FAP #3.

[0167] Preferably, the antigen-binding site specific to CD137 includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 40 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 45, and the antigen-binding site specific to FAP includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 124 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 128. In this embodiment, the antigen-binding site specific to CD137 is CD137 #4, and the antigen-binding site specific to FAP is FAP #3.

[0168] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific to CD137 includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 48 (CDR2), and SEQ ID NO: 9 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 52 (CDR1), SEQ ID NO: 53 (CDR2), and SEQ ID NO: 14 (CDR3). The antigen-binding site specific to FAP includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 333 (CDR1), SEQ ID NO: 144 (CDR2), and SEQ ID NO: 145 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 138 (CDR1), SEQ ID NO: 139 (CDR2), and SEQ ID NO: 140 (CDR3). In this embodiment, the antigen-binding site specific to CD137 is CD137 #5, and the antigen-binding site specific to FAP is FAP #5.

[0169] Preferably, the antigen-binding site specific to CD137 includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 50 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 55, and the antigen-binding site specific to FAP includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 142 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 146. In this embodiment, the antigen-binding site specific to CD137 is CD137 #5, and the antigen-binding site specific to FAP is FAP #5.

[0170] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific to CD137 includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 48 (CDR2), and SEQ ID NO: 9 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 52 (CDR1), SEQ ID NO: 53 (CDR2), and SEQ ID NO: 14 (CDR3). The antigen-binding site specific to FAP includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 117 (CDR2), and SEQ ID NO: 109 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 120 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3). In this embodiment, the antigen-binding site specific to CD137 is CD137 #5, and the antigen-binding site specific to FAP is FAP #2.

[0171] Preferably, the antigen-binding site specific to CD137 includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 50 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 55, and the antigen-binding site specific to FAP includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 115 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 119. In this embodiment, the antigen-binding site specific to CD137 is CD137 #5, and the antigen-binding site specific to FAP is FAP #2.

[0172] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific to CD137 includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 48 (CDR2), and SEQ ID NO: 9 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 52 (CDR1), SEQ ID NO: 53 (CDR2), and SEQ ID NO: 14 (CDR3). The antigen-binding site specific to FAP includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 126 (CDR2), and SEQ ID NO: 109 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 129 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3). In this embodiment, the antigen-binding site specific to CD137 is CD137 #5, and the antigen-binding site specific to FAP is FAP #3.

[0173] Preferably, the antigen-binding site specific to CD137 includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 50 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 55, and the antigen-binding site specific to FAP includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 124 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 128. In this embodiment, the antigen-binding site specific to CD137 is CD137 #5, and the antigen-binding site specific to FAP is FAP #3.

[0174] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific to CD137 includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 58 (CDR2), and SEQ ID NO: 9 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 62 (CDR1), SEQ ID NO: 63 (CDR2), and SEQ ID NO: 14 (CDR3). The antigen-binding site specific to FAP includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 333 (CDR1), SEQ ID NO: 144 (CDR2), and SEQ ID NO: 145 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 138 (CDR1), SEQ ID NO: 139 (CDR2), and SEQ ID NO: 140 (CDR3). In this embodiment, the antigen-binding site specific to CD137 is CD137 #6, and the antigen-binding site specific to FAP is FAP #5.

[0175] Preferably, the antigen-binding site specific to CD137 includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 60 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 65, and the antigen-binding site specific to FAP includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 142 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 146. In this embodiment, the antigen-binding site specific to CD137 is CD137 #6, and the antigen-binding site specific to FAP is FAP #5.

[0176] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific to CD137 includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 58 (CDR2), and SEQ ID NO: 9 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 62 (CDR1), SEQ ID NO: 63 (CDR2), and SEQ ID NO: 14 (CDR3). The antigen-binding site specific to FAP includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 117 (CDR2), and SEQ ID NO: 109 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 120 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3). In this embodiment, the antigen-binding site specific to CD137 is CD137 #6, and the antigen-binding site specific to FAP is FAP #2.

[0177] Preferably, the antigen-binding site specific to CD137 includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 60 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 65, and the antigen-binding site specific to FAP includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 115 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 119. In this embodiment, the antigen-binding site specific to CD137 is CD137 #6, and the antigen-binding site specific to FAP is FAP #2.

[0178] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific to CD137 includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 58 (CDR2), and SEQ ID NO: 9 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 62 (CDR1), SEQ ID NO: 63 (CDR2), and SEQ ID NO: 14 (CDR3). The antigen-binding site specific to FAP includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 126 (CDR2), and SEQ ID NO: 109 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 129 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3). In this embodiment, the antigen-binding site specific to CD137 is CD137 #6, and the antigen-binding site specific to FAP is FAP #3.

[0179] Preferably, the antigen-binding site specific to CD137 includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 60 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 64, and the antigen-binding site specific to FAP includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 124 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 128. In this embodiment, the antigen-binding site specific to CD137 is CD137 #6, and the antigen-binding site specific to FAP is FAP #3.

[0180] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific to CD137 includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 308 (CDR1), SEQ ID NO: 78 (CDR2), and SEQ ID NO: 69 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 72 (CDR1), SEQ ID NO: 73 (CDR2), and SEQ ID NO: 74 (CDR3). The antigen-binding site specific to FAP includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 333 (CDR1), SEQ ID NO: 144 (CDR2), and SEQ ID NO: 145 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 138 (CDR1), SEQ ID NO: 139 (CDR2), and SEQ ID NO: 140 (CDR3). In this embodiment, the antigen-binding site specific to CD137 is CD137 #8, and the antigen-binding site specific to FAP is FAP #5.

[0181] Preferably, the antigen-binding site specific to CD137 includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 80 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 85, and the antigen-binding site specific to FAP includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 142 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 146. In this embodiment, the antigen-binding site specific to CD137 is CD137 #8, and the antigen-binding site specific to FAP is FAP #5.

[0182] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific to CD137 includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 308 (CDR1), SEQ ID NO: 78 (CDR2), and SEQ ID NO: 69 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 72 (CDR1), SEQ ID NO: 73 (CDR2), and SEQ ID NO: 74 (CDR3). The antigen-binding site specific to FAP includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 117 (CDR2), and SEQ ID NO: 109 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 120 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3). In this embodiment, the antigen-binding site specific to CD137 is CD137 #8, and the antigen-binding site specific to FAP is FAP #2.

[0183] Preferably, the antigen-binding site specific to CD137 includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 80 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 85, and the antigen-binding site specific to FAP includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 115 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 119. In this embodiment, the antigen-binding site specific to CD137 is CD137 #8, and the antigen-binding site specific to FAP is FAP #2.

[0184] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific to CD137 includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 308 (CDR1), SEQ ID NO: 78 (CDR2), and SEQ ID NO: 69 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 72 (CDR1), SEQ ID NO: 73 (CDR2), and SEQ ID NO: 74 (CDR3). The antigen-binding site specific to FAP includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 126 (CDR2), and SEQ ID NO: 109 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 129 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3). In this embodiment, the antigen-binding site specific to CD137 is CD137 #8, and the antigen-binding site specific to FAP is FAP #3.

[0185] Preferably, the antigen-binding site specific to CD137 includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 80 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 85, and the antigen-binding site specific to FAP includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 124 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 128. In this embodiment, the antigen-binding site specific to CD137 is CD137 #8, and the antigen-binding site specific to FAP is FAP #3.

[0186] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific to CD137 includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 308 (CDR1), SEQ ID NO: 88 (CDR2), and SEQ ID NO: 69 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 92 (CDR1), SEQ ID NO: 93 (CDR2), and SEQ ID NO: 74 (CDR3). The antigen-binding site specific to FAP includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 333 (CDR1), SEQ ID NO: 144 (CDR2), and SEQ ID NO: 145 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 138 (CDR1), SEQ ID NO: 139 (CDR2), and SEQ ID NO: 140 (CDR3). In this embodiment, the antigen-binding site specific to CD137 is CD137 #9, and the antigen-binding site specific to FAP is FAP #5.

[0187] Preferably, the antigen-binding site specific to CD137 includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 90 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 95, and the antigen-binding site specific to FAP includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 142 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 146. In this embodiment, the antigen-binding site specific to CD137 is CD137 #9, and the antigen-binding site specific to FAP is FAP #5.

[0188] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific to CD137 includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 308 (CDR1), SEQ ID NO: 88 (CDR2), and SEQ ID NO: 69 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 92 (CDR1), SEQ ID NO: 93 (CDR2), and SEQ ID NO: 74 (CDR3). The antigen-binding site specific to FAP includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 117 (CDR2), and SEQ ID NO: 109 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 120 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3). In this embodiment, the antigen-binding site specific to CD137 is CD137 #9, and the antigen-binding site specific to FAP is FAP #2.

[0189] Preferably, the antigen-binding site specific to CD137 includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 90 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 95, and the antigen-binding site specific to FAP includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 115 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 119. In this embodiment, the antigen-binding site specific to CD137 is CD137 #9, and the antigen-binding site specific to FAP is FAP #2.

[0190] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific to CD137 includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 308 (CDR1), SEQ ID NO: 88 (CDR2), and SEQ ID NO: 69 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 92 (CDR1), SEQ ID NO: 93 (CDR2), and SEQ ID NO: 74 (CDR3). The antigen-binding site specific to FAP includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 126 (CDR2), and SEQ ID NO: 109 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 129 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3). In this embodiment, the antigen-binding site specific to CD137 is CD137 #9, and the antigen-binding site specific to FAP is FAP #3.

[0191] Preferably, the antigen-binding site specific to CD137 includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 90 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 95, and the antigen-binding site specific to FAP includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 124 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 128. In this embodiment, the antigen-binding site specific to CD137 is CD137 #9, and the antigen-binding site specific to FAP is FAP #3.

[0192] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific to CD137 includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 308 (CDR1), SEQ ID NO: 98 (CDR2), and SEQ ID NO: 69 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 92 (CDR1), SEQ ID NO: 93 (CDR2), and SEQ ID NO: 74 (CDR3). The antigen-binding site specific to FAP includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 333 (CDR1), SEQ ID NO: 144 (CDR2), and SEQ ID NO: 145 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 138 (CDR1), SEQ ID NO: 139 (CDR2), and SEQ ID NO: 140 (CDR3). In this embodiment, the antigen-binding site specific to CD137 is CD137 #10, and the antigen-binding site specific to FAP is FAP #5.

[0193] Preferably, the antigen-binding site specific to CD137 includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 100 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 105, and the antigen-binding site specific to FAP includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 142 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 146. In this embodiment, the antigen-binding site specific to CD137 is CD137 #10, and the antigen-binding site specific to FAP is FAP #5.

[0194] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific to CD137 includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 308 (CDR1), SEQ ID NO: 98 (CDR2), and SEQ ID NO: 69 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 92 (CDR1), SEQ ID NO: 93 (CDR2), and SEQ ID NO: 74 (CDR3). The antigen-binding site specific to FAP includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 117 (CDR2), and SEQ ID NO: 109 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 120 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3). In this embodiment, the antigen-binding site specific to CD137 is CD137 #10, and the antigen-binding site specific to FAP is FAP #2.

[0195] Preferably, the antigen-binding site specific to CD137 includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 100 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 105, and the antigen-binding site specific to FAP includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 115 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 119. In this embodiment, the antigen-binding site specific to CD137 is CD137 #10, and the antigen-binding site specific to FAP is FAP #2.

[0196] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site specific to CD137 includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 308 (CDR1), SEQ ID NO: 98 (CDR2), and SEQ ID NO: 69 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 92 (CDR1), SEQ ID NO: 93 (CDR2), and SEQ ID NO: 74 (CDR3). The antigen-binding site specific to FAP includes a variable heavy chain region and a variable light chain region, where the heavy chain CDR includes the amino acid sequences of SEQ ID NO: 319 (CDR1), SEQ ID NO: 126 (CDR2), and SEQ ID NO: 109 (CDR3), and the light chain CDR includes the amino acid sequences of SEQ ID NO: 129 (CDR1), SEQ ID NO: 112 (CDR2), and SEQ ID NO: 113 (CDR3). In this embodiment, the antigen-binding site specific to CD137 is CD137 #10, and the antigen-binding site specific to FAP is FAP #3.

[0197] Preferably, the antigen-binding site specific to CD137 includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 100 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 105, and the antigen-binding site specific to FAP includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 124 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 128. In this embodiment, the antigen-binding site specific to CD137 is CD137 #10, and the antigen-binding site specific to FAP is FAP #3. The above are specific combinations of antigen-binding sites specific to CD137 and FAP that can be used in the binding molecule of the present invention.

[0198] In each of these embodiments, the variable heavy chain containing the antigen-binding site CD137 is fused to a human heavy chain constant region, such as IgG, IgG2, IgG3, IgG4, IgA, IgE, or IgM. Preferably, the heavy chain constant region of human IgG1 is used.

[0199] A further embodiment of the present invention is when a variable light chain containing the antigen-binding site CD137 is fused to a human light chain constant region, either kappa or lambda. Preferably, the human kappa light chain constant region is used.

[0200] An exemplary sequence for the heavy chain constant region of wild-type human IgG1 is provided in SEQ ID NO: 283, and an IgG1 KO is provided in SEQ ID NO: 284.

[0201] An exemplary sequence for the light chain constant region of *Hitokappa* is provided in SEQ ID NO: 285.

[0202] The following provides the binding molecules of the present invention. Each specific molecule of the present invention includes a modified immunoglobulin molecule comprising an immunoglobulin heavy chain containing an amino acid sequence of a variable heavy chain domain that specifically binds to CD137, an scFv that specifically binds to FAP, and an antibody light chain containing an amino acid sequence of a variable light chain domain that specifically binds to CD137.

[0203] A further aspect of the present invention provides an immunoglobulin-like binding molecule comprising an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 151 and fused to scFv at its C-terminus, and a light chain comprising the amino acid sequence of SEQ ID NO: 152. In this aspect, the antigen-binding site specific to CD137 is CD137 #1, and the antigen-binding site specific to FAP is FAP #1.

[0204] A further aspect of the present invention provides an immunoglobulin-like binding molecule comprising an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 153 and fused to scFv at its C-terminus, and a light chain comprising the amino acid sequence of SEQ ID NO: 154. In this aspect, the antigen-binding site specific to CD137 is CD137 #7, and the antigen-binding site specific to FAP is FAP #1.

[0205] A further aspect of the present invention provides an immunoglobulin-like binding molecule comprising an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 155 and fused to scFv at its C-terminus, and a light chain comprising the amino acid sequence of SEQ ID NO: 156. In this aspect, the antigen-binding site specific to CD137 is CD137 #1, and the antigen-binding site specific to FAP is FAP #4.

[0206] A further aspect of the present invention provides an immunoglobulin-like binding molecule comprising an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 157 and fused to scFv at its C-terminus, and a light chain comprising the amino acid sequence of SEQ ID NO: 158. In this aspect, the antigen-binding site specific to CD137 is CD137 #7, and the antigen-binding site specific to FAP is FAP #4.

[0207] A further aspect of the present invention provides an immunoglobulin-like binding molecule comprising an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 159 and fused to scFv at its C-terminus, and a light chain comprising the amino acid sequence of SEQ ID NO: 160. In this aspect, the antigen-binding site specific to CD137 is CD137 #2, and the antigen-binding site specific to FAP is FAP #5.

[0208] A further aspect of the present invention provides an immunoglobulin-like binding molecule comprising an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 164 and fused to scFv at its C-terminus, and a light chain comprising the amino acid sequence of SEQ ID NO: 165. In this aspect, the antigen-binding site specific to CD137 is CD137 #2, and the antigen-binding site specific to FAP is FAP #2.

[0209] A further aspect of the present invention provides an immunoglobulin-like binding molecule comprising an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 169 and fused to scFv at its C-terminus, and a light chain comprising the amino acid sequence of SEQ ID NO: 170. In this aspect, the antigen-binding site specific to CD137 is CD137 #2, and the antigen-binding site specific to FAP is FAP #3.

[0210] A further aspect of the present invention provides an immunoglobulin-like binding molecule comprising an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 174 and fused to scFv at its C-terminus, and a light chain comprising the amino acid sequence of SEQ ID NO: 175. In this aspect, the antigen-binding site specific to CD137 is CD137 #3, and the antigen-binding site specific to FAP is FAP #5.

[0211] A further aspect of the present invention provides an immunoglobulin-like binding molecule comprising an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 179 and fused to scFv at its C-terminus, and a light chain comprising the amino acid sequence of SEQ ID NO: 180. In this aspect, the antigen-binding site specific to CD137 is CD137 #3, and the antigen-binding site specific to FAP is FAP #2.

[0212] A further aspect of the present invention provides an immunoglobulin-like binding molecule comprising an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 184 and fused to scFv at its C-terminus, and a light chain comprising the amino acid sequence of SEQ ID NO: 185. In this aspect, the antigen-binding site specific to CD137 is CD137 #3, and the antigen-binding site specific to FAP is FAP #3.

[0213] A further aspect of the present invention provides an immunoglobulin-like binding molecule comprising an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 189 and fused to scFv at its C-terminus, and a light chain comprising the amino acid sequence of SEQ ID NO: 190. In this aspect, the antigen-binding site specific to CD137 is CD137 #4, and the antigen-binding site specific to FAP is FAP #5.

[0214] A further aspect of the present invention provides an immunoglobulin-like binding molecule comprising an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 194 and fused to scFv at its C-terminus, and a light chain comprising the amino acid sequence of SEQ ID NO: 195. In this aspect, the antigen-binding site specific to CD137 is CD137 #4, and the antigen-binding site specific to FAP is FAP #2.

[0215] A further aspect of the present invention provides an immunoglobulin-like binding molecule comprising an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 199 and fused to scFv at its C-terminus, and a light chain comprising the amino acid sequence of SEQ ID NO: 200. In this aspect, the antigen-binding site specific to CD137 is CD137 #4, and the antigen-binding site specific to FAP is FAP #3.

[0216] A further aspect of the present invention provides an immunoglobulin-like binding molecule comprising an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 204 and fused to scFv at its C-terminus, and a light chain comprising the amino acid sequence of SEQ ID NO: 205. In this aspect, the antigen-binding site specific to CD137 is CD137 #5, and the antigen-binding site specific to FAP is FAP #5.

[0217] A further aspect of the present invention provides an immunoglobulin-like binding molecule comprising an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 209 and fused to scFv at its C-terminus, and a light chain comprising the amino acid sequence of SEQ ID NO: 210. In this aspect, the antigen-binding site specific to CD137 is CD137 #5, and the antigen-binding site specific to FAP is FAP #2.

[0218] A further aspect of the present invention provides an immunoglobulin-like binding molecule comprising an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 214 and fused to scFv at its C-terminus, and a light chain comprising the amino acid sequence of SEQ ID NO: 215. In this aspect, the antigen-binding site specific to CD137 is CD137 #5, and the antigen-binding site specific to FAP is FAP #3.

[0219] A further aspect of the present invention provides an immunoglobulin-like binding molecule comprising an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 219 and fused to scFv at its C-terminus, and a light chain comprising the amino acid sequence of SEQ ID NO: 220. In this aspect, the antigen-binding site specific to CD137 is CD137 #6, and the antigen-binding site specific to FAP is FAP #5.

[0220] A further aspect of the present invention provides an immunoglobulin-like binding molecule comprising an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 224 and fused to scFv at its C-terminus, and a light chain comprising the amino acid sequence of SEQ ID NO: 225. In this aspect, the antigen-binding site specific to CD137 is CD137 #6, and the antigen-binding site specific to FAP is FAP #2.

[0221] A further aspect of the present invention provides an immunoglobulin-like binding molecule comprising an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 229 and fused to scFv at its C-terminus, and a light chain comprising the amino acid sequence of SEQ ID NO: 230. In this aspect, the antigen-binding site specific to CD137 is CD137 #6, and the antigen-binding site specific to FAP is FAP #3.

[0222] A further aspect of the present invention provides an immunoglobulin-like binding molecule comprising an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 234 and fused to scFv at its C-terminus, and a light chain comprising the amino acid sequence of SEQ ID NO: 235. In this aspect, the antigen-binding site specific to CD137 is CD137 #8, and the antigen-binding site specific to FAP is FAP #5.

[0223] A further aspect of the present invention provides an immunoglobulin-like binding molecule comprising an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 239 and fused to scFv at its C-terminus, and a light chain comprising the amino acid sequence of SEQ ID NO: 240. In this aspect, the antigen-binding site specific to CD137 is CD137 #8, and the antigen-binding site specific to FAP is FAP #2.

[0224] A further aspect of the present invention provides an immunoglobulin-like binding molecule comprising an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 244 and fused to scFv at its C-terminus, and a light chain comprising the amino acid sequence of SEQ ID NO: 245. In this aspect, the antigen-binding site specific to CD137 is CD137 #8, and the antigen-binding site specific to FAP is FAP #3.

[0225] A further aspect of the present invention provides an immunoglobulin-like binding molecule comprising an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 249 and fused to scFv at its C-terminus, and a light chain comprising the amino acid sequence of SEQ ID NO: 250. In this aspect, the antigen-binding site specific to CD137 is CD137 #9, and the antigen-binding site specific to FAP is #5.

[0226] A further aspect of the present invention provides an immunoglobulin-like binding molecule comprising an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 254 and fused to scFv at its C-terminus, and a light chain comprising the amino acid sequence of SEQ ID NO: 255. In this aspect, the antigen-binding site specific to CD137 is CD137 #9, and the antigen-binding site specific to FAP is FAP #2.

[0227] A further aspect of the present invention provides an immunoglobulin-like binding molecule comprising an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 259 and fused to scFv at its C-terminus, and a light chain comprising the amino acid sequence of SEQ ID NO: 260. In this aspect, the antigen-binding site specific to CD137 is CD137 #9, and the antigen-binding site specific to FAP is FAP #3.

[0228] A further aspect of the present invention provides an immunoglobulin-like binding molecule comprising an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 264 and fused to scFv at its C-terminus, and a light chain comprising the amino acid sequence of SEQ ID NO: 265. In this aspect, the antigen-binding site specific to CD137 is CD137 #10, and the antigen-binding site specific to FAP is FAP #5.

[0229] A further aspect of the present invention provides an immunoglobulin-like binding molecule comprising an immunoglobulin heavy chain having the amino acid sequence of SEQ ID NO: 269 and fused to scFv at its C-terminus, and a light chain having the amino acid sequence of SEQ ID NO: 270. In this aspect, the antigen-binding site specific to CD137 is CD137 #10, and the antigen-binding site specific to FAP is FAP #2.

[0230] A further aspect of the present invention provides an immunoglobulin-like binding molecule comprising an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 274 and fused to scFv at its C-terminus, and a light chain comprising the amino acid sequence of SEQ ID NO: 275. In this aspect, the antigen-binding site specific to CD137 is CD137 #10, and the antigen-binding site specific to FAP is FAP #3.

[0231] A further aspect of the present invention provides a nucleic acid molecule encoding the binding molecule of the present invention or an expression vector containing such a nucleic acid molecule.

[0232] In some embodiments, the binding molecule of the present invention comprises heavy chain and light chain polypeptides. As those skilled in the art will understand, nucleic acid molecules encoding heavy chain polypeptides, light chain polypeptides, or heavy chain polypeptides and light chain polypeptides can be readily prepared.

[0233] Nucleic acid molecules encoding light and heavy chains can be synthesized chemically and enzymatically by polymerase chain reaction (PCR) using standard methods. First, suitable oligonucleotides can be synthesized by methods known in the art that can be used to produce synthetic genes (e.g., Gait, 1984). Methods for generating synthetic genes from oligonucleotides are known in the art (e.g., Stemmer et al., 1995; Ye et al., 1992; Hayden and Mandecki, 1988; Frank et al., 1987).

[0234] The nucleic acid molecules of the present invention include, but are not limited to, DNA molecules encoding polypeptide sequences shown in sequence listings. The present invention also relates to nucleic acid molecules that hybridize to DNA molecules encoding polypeptide sequences shown in sequence listings under high stringency binding and washing conditions, as defined in WO 2007 / 042309. Preferred molecules (in terms of mRNA) have at least 75% or 80% (preferably at least 85%, more preferably at least 85%, most preferably at least 95%) homology or sequence identity with one of the DNA molecules described herein. For example, considering antibody expression in eukaryotic cells, the DNA sequences shown in sequence listings are designed to match codon utilization in eukaryotic cells. If antibody expression in E. coli is desired, these sequences can be modified to match codon utilization in E. coli. Variants of the DNA molecules of the present invention can be constructed in several ways, for example, as described in WO 2007 / 042309.

[0235] As used herein, the terms “identical” or “% identity” refer to two or more sequences or subsequences that, in the context of two or more nucleic acid or polypeptide sequences, are identical or have a specific proportion of identical nucleotide or amino acid residues when compared and aligned for the greatest correspondence. To determine % identity, the sequences are aligned for the best possible comparison (for example, a gap may be introduced into the sequence of the first amino acid or nucleic acid sequence for the best possible alignment with the second amino acid or nucleic acid sequence). Then, the amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The % identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions (e.g., duplicate positions) × 100). In some embodiments, the two sequences being compared are of the same length after a gap has been appropriately introduced within the sequences (e.g., excluding any further sequences that extend beyond the sequences being compared). For example, when variable-region sequences are being compared, the leader and / or stationary-domain sequences are not considered. For sequence comparisons between two sequences, the "corresponding" CDR refers to the CDR at the same position in both sequences (e.g., CDR-H1 of each sequence).

[0236] The determination of % identity or % similarity between two sequences can be achieved using mathematical algorithms. A preferred non-restrictive example of a mathematical algorithm used for comparing two sequences is the algorithm from Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such algorithms are incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403-410. A BLAST nucleotide search can be performed using the NBLAST program with a score of 100 and a word length of 12 to obtain nucleotide sequences homologous to the nucleic acid encoding the target protein. BLAST protein searches can be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the target protein. For comparison purposes, gap alignment can be obtained using Gapped BLAST, as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used for iterative searches to detect distant relationships between molecules (ibid.). When using the BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. Another preferred non-restrictive example of a mathematical algorithm used for sequence comparison is the algorithm by Myers and Miller, CABIOS (1989). Such algorithms are incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.Further algorithms for sequence analysis are publicly known in the art, including ADVANCE and ADAM described in Torellis and Robotti, 1994, Comput. Appl. Biosci. 10:3-5, and FASTA described in Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444-8. Within FASTA, ktup is a control option that sets the sensitivity and speed of the search. When ktup=2, similar regions in the two sequences being compared are found by looking at aligned residue pairs, and when ktup=1, a single aligned amino acid is examined. ktup can be set to 2 or 1 for protein sequences or 1-6 for DNA sequences. The default value when ktup is not specified is 2 for proteins and 6 for DNA. Alternatively, protein sequence alignment can be performed using the CLUSTAL W algorithm described in Higgins et al., 1996, Methods Enzymol. 266:383-402.

[0237] A further aspect of the present invention is a method for producing a bound molecule according to any one of the prior claims, (a) Culturing the host cells of the present invention under conditions that enable the expression of the molecule, (b) A method is provided which includes recovering the molecule.

[0238] Embodiments of this aspect of the present invention are (c) a method of production further comprising the step of further purifying and / or modifying and / or formulating the binding molecule of the present invention.

[0239] To produce the binding molecule of the present invention, a DNA molecule encoding the full-length light chain and / or heavy chain or a fragment thereof is inserted into an expression vector such that the sequence is manipulably linked to a transcription and translation control sequence.

[0240] To produce the antibody of the present invention, those skilled in the art can select from a wide variety of expression systems well known in the art, such as those outlined in Kipriyanov and Le Gall, 2004.

[0241] Expression vectors include plasmids, retroviruses, cosmids, EBV-derived episomes, etc. The expression vector and expression regulatory sequence are selected to be compatible with host cells. Antibody light chain genes and antibody heavy chain genes can be inserted into separate vectors. In certain embodiments, both DNA sequences are inserted into the same expression vector. A convenient vector is one that encodes a functionally complete human CH or CL immunoglobulin sequence and has appropriate restriction sites engineered to allow easy insertion and expression of any VH or VL sequence as described above. The constant chain is typically kappa or lambda for the antibody light chain, and can be, but is not limited to, any IgG isotype (IgG1, IgG2, IgG3, IgG4) or other immunoglobulins including allele variants for the antibody heavy chain.

[0242] Furthermore, recombinant expression vectors can encode signal peptides that facilitate the secretion of antibody chains from host cells. The DNA encoding the antibody chain can be cloned into the vector so that the signal peptide is in-frame ligated to the amino terminus of the mature antibody chain DNA. The signal peptide can be an immunoglobulin signal peptide or a heterologous peptide derived from a non-immunoglobulin protein. Alternatively, the DNA sequence encoding the antibody chain may already contain the signal peptide sequence.

[0243] Recombinant expression vectors have a regulatory sequence including promoters, enhancers, terminal and polyadenylation signals, in addition to the DNA sequence encoding the antibody chain, as well as other expression regulatory elements that control the expression of the antibody chain in host cells. Examples of promoter sequences (exemplified for expression in mammalian cells) include (CMV) (e.g., CMV Simian Virus 40 (SV40) (e.g., SV40 promoter / enhancer)), adenoviruses (e.g., adenovirus major late promoter (AdMLP)), promoters and / or enhancers derived from polyomas, and potent mammalian promoters, such as native immunoglobulin and actin promoters. Examples of polyadenylation signals include BGH polyA, SV40 late or early polyA. Alternatively, the 3'UTR of an immunoglobulin gene, etc., can be used.

[0244] Furthermore, recombinant expression vectors may also have sequences (e.g., origins of replication) and selection marker genes that regulate vector replication in host cells. A vector comprising nucleic acid molecules encoding the heavy chain or its antigen-binding portion and / or light chain or its antigen-binding portion of the molecule of the present invention, and these DNA molecules, can be introduced into host cells, such as bacterial cells or higher eukaryotic cells, such as mammalian cells, according to transfection methods known in the art, including liposome-mediated transfection, polycation-mediated transfection, protoplast fusion, microinjection, calcium phosphate precipitation, electroporation, or transfer by a viral vector.

[0245] Preferably, the nucleic acid molecules encoding the heavy chain and light chain are located on two vectors that are simultaneously transfected into host cells, preferably mammalian cells.

[0246] Therefore, a further aspect of the present invention provides a host cell comprising an expression vector containing a nucleic acid molecule encoding a heavy chain and an expression vector containing a nucleic acid molecule encoding a light chain.

[0247] Mammalian cell lines available as hosts for expression are well known in the art, and in particular include Chinese hamster ovary (CHO, CHO-DG44) cells, NSO, SP2 / 0 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human cancer cells (e.g., Hep G2), A549 cells, 3T3 cells, or derivatives / offspring of any such cell lines. Other mammalian cells, including (but not limited to) human, mouse, rat, monkey, and rodent cell lines, or other eukaryotic or prokaryotic cells, including (but not limited to) yeast, insect, and plant cells, such as bacteria, can be used. The binding molecule of the present invention is produced by culturing the host cells for a period of time sufficient to allow the expression of the binding molecule in the host cells.

[0248] Antibody molecules are preferably recovered from the culture medium as secreted polypeptides, or, for example, from host cell lysates if expressed without a secretion signal. To obtain a substantially homogeneous preparation of the antibody, the antibody molecules must be purified using standard protein purification methods used for recombinant proteins and host cell proteins. For example, a state-of-the-art purification method useful for obtaining the conjugated molecules of the present invention includes, as a first step, the removal of cells and / or particulate cell debris from the culture medium or lysate. The antibody is then purified from hybrid soluble proteins, polypeptides, and nucleic acids by, for example, fractionation on an immunoaffinity column or ion-exchange column, ethanol precipitation, reverse-phase HPLC, Sephadex chromatography, or chromatography on silica or cation-exchange resin. As a final step in the method for obtaining CD137 and FAP conjugated molecules, the purified antibody molecules can be dried, for example, lyophilized, as described below for therapeutic applications.

[0249] A further aspect of the present invention provides a binding molecule for use in pharmaceuticals.

[0250] In one embodiment, the present invention relates to a CD137 / FAP binding molecule, wherein the binding molecule has the following characteristics: (a) K less than or equal to 1 × 10⁻⁸M D It binds to human CD137. (b) Binds to human CD137 and cynomolgus monkey CD137, (c) A CRD3 epitope or CRD2 / 3 epitope that binds to human and cynomolgus monkey CD137, (d) Blocking and / or competing with the binding of any of the antigen-binding molecules described herein to human and cynomolgus monkey CD137 at the CRD3 epitope or CRD2 / 3 epitope, (d) In the absence of FAP binding, CD137 cluster formation and T cell activation are not substantially mediated. (e) Increases T cell proliferation (in a mixed lymphocyte reaction (MLR) assay) (f) Increase interferon-gamma production in MLR assays. (g) Increase IL-2 secretion in MLR assay, (h) Does not inhibit the binding of CD137 to CD137L. (i) Stimulate antigen-specific memory responses, (j) Stimulates the antibody response, (k)in inhibits tumor cell growth in vivo, and (l) Does not show hepatotoxicity in vivo. Show at least one of them.

[0251] Preferably, the binding molecule is 1 × 10 -8 M~1×10 -10 M KD for humans and cynomolgus monkeys CD137 and 1×10 -8 M and 1×10 -10 M binds more weakly to human, cynomolgus monkey, and mouse FAP proteins in the KD stage.

[0252] In yet another embodiment, the present invention provides a CD137 / FAP binding molecule, wherein the CD137 binding region comprises a heavy chain variable region and a light chain variable region, (a) The heavy chain variable region contains an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100. (b) The light chain variable region contains an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 25, 35, 45, 55, 65, 75, 85, 95, and 105. (c) Antibodies should have a K content of 1 × 10⁻⁸ M or less. D It then binds to human CD137, (d) The antibody does not substantially mediate CD137 activation in the absence of FAP.

[0253] A preferred embodiment of the present invention is 10 -10 M's K D It binds to human CD137, and 10 -10 M's K D It binds to cynomolgus macaque CD137. A preferred embodiment of the present invention is 10 -10 M's K D Human FAP, 10 -10 M's K D Crab-eating macaque FAP and 10 -9 M's K D It then binds to the mouse FAP.

[0254] In yet another aspect, the present invention provides a CD137-binding molecule in which the CD137 heavy chain variable region is human IGHV3-7 *01 The molecule contains an amino acid sequence derived from a germ cell lineage sequence, preferably, the CD137-binding molecule contains a heavy chain variable region which contains an amino acid sequence that is at least 78%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 20, 30, 40, 50, and 60.

[0255] In yet another embodiment, the present invention provides a CD137-binding molecule in which the CD137 light chain variable region is human IGKV1-NL1 * 01 The amino acid sequence comprises an amino acid sequence derived from a germ cell lineage sequence, preferably where the light chain variable region comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 25, 35, 45, 55, and 65.

[0256] In yet another embodiment, the present invention provides an FAP-binding molecule in which the FAP heavy chain variable region is human IGHV3-23 * 04 The FAP-binding molecule comprises an amino acid sequence derived from a germ cell lineage sequence, preferably, the FAP-binding molecule comprises a heavy chain variable region containing an amino acid sequence that is at least 78%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 106, 115, 124, 133, or 142.

[0257] In yet another embodiment, the present invention provides an FAP-binding molecule in which the FAP light chain variable region is human IGKV3-11 * 01 The FAP-binding molecule comprises an amino acid sequence derived from a germ cell lineage sequence, preferably, the FAP-binding molecule comprises a light chain variable region containing an amino acid sequence that is at least 78%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 110, 119, 128, 137, or 146.

[0258] In yet another embodiment, the present invention provides a CD137 / FAP binding molecule, wherein the FAP binding region comprises a heavy chain variable region and a light chain variable region, (a) The heavy chain variable region contains an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 106, 115, 124, 133, and 142. (b) The light chain variable region contains an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 110, 119, 128, 137, and 146. (c) The antibodies in (a) and (b) have a K content of 1 × 10⁻⁷ M or less. D It then binds to human CD137, (d) The antibodies in (a) to (c) do not substantially bind to human CD137L or CD137 in the absence of FAP crosslinking.

[0259] In yet another embodiment, the present invention provides a CD137 / FAP binding molecule, wherein the FAP binding region comprises a heavy chain variable region and a light chain variable region, (a) The heavy chain variable region contains an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 106, 115, 124, 133, and 142. (b) The light chain variable region contains an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 110, 119, 128, 137, and 146. (c) The antibodies of (a) and (b) bind to an epitope on human CD137 in the extracellular domain CRD3 between amino acids 87-118 (SEQ ID NO: 352) or (d) The antibodies of (a) and (b) bind to an epitope on human CD137 in the extracellular domain CRD2-3 between amino acids 46-117 (SEQ ID NO: 356), (e) The antibodies (a) to (c) do not substantially mediate CD137 cluster formation and T cell activation in the absence of FAP binding.

[0260] In a preferred embodiment, the above-described CD137 / FAP binding molecule has the following properties: (a) Antibodies increase T cell proliferation (in MLR assays), (b) The antibody increases interferon-gamma production (in the MLR assay), or (c) Antibodies increase IL-2 secretion (in MLR assays). It further includes at least one of the following.

[0261] In addition, or alternatively, the CD137 / FAP binding molecule may include one or more of the other features listed above.

[0262] Further embodiments of the present invention provide conjugated molecules of the present invention for use in the treatment or therapy of cancer. As used herein, “treatment” or “therapy” (and its variations, e.g., “to treat” or “to treat,” “therapeutic”) refers to a clinical intervention in an attempt to alter the natural course of the treated individual and can be carried out during the course of clinicopathology. Desired effects of treatment include, but are not limited to, limiting the onset or recurrence of disease, alleviating symptoms and reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the condition, and achieving remission or improving prognosis. In some embodiments, the molecules of the present invention are used to delay the onset of disease or slow the progression of disease. In one embodiment, the present invention is used for the treatment of T-cell infiltrating / FAP-positive tumors. The cancers are preferably colorectal cancer (CRC) (e.g., colorectal adenocarcinoma), gastric cancer (GC) (e.g., gastric adenocarcinoma), pancreatic cancer (PAC) (e.g., pancreatic adenocarcinoma), lung cancer (LC) (e.g., squamous cell carcinoma, lung adenocarcinoma, non-small cell lung cancer (NSCLC)), head and neck cancer, urothelial carcinoma, or melanoma, but other solid malignancies, which constitute 90% of all cancers, are also considered. This is due to the unmet clinical need for novel treatments for these types of less invasive tumors.

[0263] As described above, the inventors have identified that the binding molecule of the present invention is highly useful for T-cell FAP+ tissue-restricted CD137 activation and tumor killing, and therefore can be used to treat cancers having co-localization expression of both CD137(4-1BB,TNFRSF9) and fibroblast-activating protein (FAP). Methods for determining whether a particular tumor has co-localization expression of CD137(4-1BB,TNFRSF9) and fibroblast-activating protein (FAP) are well known in the art. For example, immunohistochemistry can be used to determine whether tumor tissue expresses CD137(4-1BB,TNFRSF9) and fibroblast-activating protein (FAP), and therefore would be suitable for treatment with the binding molecule of the present invention.

[0264] CRC is a distinct malignant tumor disease listed in ICD-10 and is one of the leading causes of cancer morbidity and mortality worldwide. Approximately 25% of CRC patients present with overt metastasis, and metastatic disease occurs in 40-50% of newly diagnosed patients. Recent improvements in chemotherapy have extended survival for metastatic CRC, but most patients will still die from the disease. Therefore, further therapeutic agents to treat this disease are greatly needed.

[0265] In a further embodiment, the present invention relates to a method for the treatment or prevention of cancer, comprising administering an effective amount of any of the above-described CD137 / FAP binding molecules to a human being.

[0266] The preferred mode of application is parenteral administration by infusion or injection (intravenous, intramuscular, subcutaneous, intraperitoneal, or intradermal), but other modes of application, such as inhalation, percutaneous, intranasal, buccal, or oral, may also be applicable.

[0267] The "therapeutically effective dose" of the molecule administered is the minimum amount necessary to prevent, improve or treat the clinical symptoms of cancer, in particular, the minimum amount effective against these disorders.

[0268] The applicable daily dose range of the antibody of the present invention is typically 1 μg / kg to 100 mg / kg, preferably 0.1 mg / kg to 20 mg / kg.

[0269] The actual pharmacokinetically effective dose or therapeutic dose will naturally be determined by factors known to those skilled in the art, such as the patient's age and weight, the route of administration, and the severity of the disease. In any case, the combination will be administered in a dose and manner that allows the pharmacokinetically effective dose to be delivered based on the patient's specific condition.

[0270] The conjugated molecule of the present invention can be used alone or in combination with other pharmacologically active ingredients, such as cutting-edge or standard therapeutic compounds, such as cell proliferation inhibitors or cytotoxic substances, cell proliferation inhibitors, angiogenic substances, steroids, immunomodulators / checkpoint inhibitors, etc.

[0271] Accordingly, further embodiments of the present invention provide a pharmaceutical composition comprising any one of the embodiments of the present invention together with a pharmaceutically acceptable carrier and optionally one or more further active ingredients.

[0272] A further aspect of the present invention provides a conjugated molecule for use in the treatment of cancer, wherein the treatment comprises one or more pharmacologically active substances.

[0273] In further embodiments, the present invention provides the use of an anti-CD137 / FAP antibody or antigen-binding fragment for use in any one of the above-described embodiments of anti-CD137 / FAP for use in a pharmaceutical composition or for the treatment of a disease, wherein this use is for the treatment of cancer and / or tumors.

[0274] A further aspect of the present invention provides the use of one or more active ingredients in the manufacture of a pharmaceutical for the treatment of cancer and / or tumors, wherein the pharmaceutical comprises any one of the embodiments of the present invention.

[0275] Cell proliferation inhibitory and / or cytotoxic active substances that can be administered in combination with the binding molecule of the present invention include hormones, hormone analogs and antihormones, aromatase inhibitors, LHRH agonists and antagonists, growth factors (e.g., platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), insulin-like growth factor (IGF), human epidermal growth factor (HER, e.g., HER2, HER3, HER4), and hepatocyte growth factors. Inhibitors of growth factors, etc. (these inhibitors are, for example, growth factor antibodies, growth factor receptor antibodies, and tyrosine kinase inhibitors), such as cetuximab, gefitinib, afatinib, nintedanib, imatinib, lapatinib, bosutinib, and trastuzumab; antimetabolites (for example, folate antagonists, such as methotrexate, larcitrexed, pyrimidine analogs, such as 5-fluorouracil (5-FU), gemcitabine, irinotecan, doxorubicin, TAS-102, capecitabine, and gemcitabine, purines, and adenosine analogs, for example) , mercaptopurine, thioguanine, cladribine and pentostatin, cytarabine (araC), fludarabine), antitumor antibiotics (e.g., anthracyclines), platinum derivatives (e.g., cisplatin, oxaliplatin, carboplatin), alkylating agents (e.g., estramustine, mechloretamine, melphalan, chlorambucil, busulfan, dacarbazine, cyclophosphamide, ifosfamide, temozolomide, nitrosourea, e.g., carmustine and lomustine, thiotepa, etc.), antimitotic agents (e.g., vinca alkaloids, e.g., For example, angiogenesis inhibitors including vinblastine, vindesine, vinorelbine and vincristine, as well as taxanes (e.g., paclitaxel, docetaxel), bevacizumab, ramucirumab and aflibercept, tubulin inhibitors, DNA synthesis inhibitors, PARP inhibitors, topoisomerase inhibitors (e.g., epipodophyllotoxin, e.g., etoposide and etopophos, teniposide, amsacrin, topotecan, irinotecan, mitoxantrone), serine / threonine kinase inhibitors (e.g., PDK1 inhibitors, Raf inhibitors, A-Raf inhibitors,B-Raf inhibitors, C-Raf inhibitors, mTOR inhibitors, mTORC1 / 2 inhibitors, PI3K inhibitors, PI3Kα inhibitors, dual mTOR / PI3K inhibitors, STKT33 inhibitors, AKT inhibitors, PLK1 inhibitors (e.g., volasertib), CDK inhibitors including CDK9 inhibitors, Aurora kinase inhibitors), tyrosine kinase inhibitors (e.g., PTK2 / FAK inhibitors), protein-protein interaction inhibitors, MEK inhibitors, ERK inhibitors, FLT3 inhibitors, BRD4 inhibitors, IGF-1R inhibitors, Bcl-xL inhibitors, Bcl-2 inhibitors, Bcl-2 / Bcl-xL inhibitors, ErbB receptor inhibitors, BCR-ABL inhibitors, ABL inhibitors, Src inhibitors, rapamycin analogs (e.g., everolimus, tersilosimus, ridalolimus, sirolimus), androgen synthesis inhibitors, androgen receptor inhibitors, DNMT inhibitors, HDA This includes, but is not limited to, C inhibitors, ANG1 / 2 inhibitors, CYP17 inhibitors, radiopharmaceuticals, immunotherapeutic agents, such as immune checkpoint inhibitors (e.g., CTLA4, PD1, PD-L1, LAG3, and TIM3 binding molecules / immunoglobulins, e.g., ipilimumab, nivolumab, pembrolizumab) and various chemotherapeutic agents, such as amifostine, anagrelid, clodronat, filgrastine, interferon, interferon alfa, leucovorin, rituximab, procarbazine, levamisole, mesna, mitotane, pamidronate, and porfimer, proteasome inhibitors (e.g., bortezomib), Smac and BH3 mimetics, agents that restore p53 function including mdm2-p53 antagonists, Wnt / beta-catenin signaling pathway inhibitors, and / or cyclin-dependent kinase 9 inhibitors.

[0276] Growing evidence suggests that PD-1 signaling is a crucial mechanism utilized by tumors to evade anti-tumor immune responses (Dong, et al, 2002; Iwai et al, 2002; Shin and Ribas, 2015). Recent clinical trials with anti-PD-1 and PD-L1 monoclonal antibodies have shown clinical responsiveness in some patients with various cancers. These immune checkpoint inhibitors block the interaction between PD-1 and PD-L1, restoring stimulating signaling and promoting T cell activation. These T cells are often tumor-infiltrating T cells (TILs), which can trigger a robust immune response to eliminate cancer cells. In this context, the CD137 / FAP molecule of the present invention can enhance the elimination of cancer cells by activating and / or recruiting and / or maintaining TILs in the tumor microenvironment. Unfortunately, only a small fraction of treated patients respond to current immunotherapy treatments. For example, Valentini et al. reported that colorectal cancer (CRC) cells express little PD-L1, and that PD-L1 expression in microsatellite-stabilized (MSS) tumors (MSS CRCs) is mainly limited to tumor-infiltrating immune cells (Oncotarget, 2018). This could explain why MSS CRC patients did not respond to anti-PD-1 / PD-L1 therapy. Thus, developing therapies that can potentially increase patient response rates has become a top priority.

[0277] Studies have shown that the presence or absence of tumor-infiltrating lymphocytes (TILs) (i.e., "hot tumors") or tumor-infiltrating lymphocytes (TILs) (i.e., "cold tumors") is important in predicting the response to PD-1 therapy, and that the presence of TILs correlates with better patient outcomes during various antitumor therapies (Galon et al., 2006, Hwang et al., 2012, Mahmoud et al. 2011). Therefore, one aspect of the present invention is the combination of a CD137 / FAP bispecific molecule with a therapy that makes "non-inflammatory" tumors "inflammatory." For example, an embodiment of the present invention combines an immunotherapy that can attenuate PDL-1 inhibition (and / or overcome checkpoint blockade resistance in some way) with a CD137 / FAP bispecific molecule that specifically targets FAP+ expressing tumors. The combination of these therapies leads to the release of inhibition on the one hand, while simultaneously increasing new T cell infiltration into the tumor site and / or promoting T cell retention and activation in the tumor microenvironment (two-prong approach). The combination of therapies results in better tumor control than either treatment alone.

[0278] With the above in mind, the following: (i) For example, immunotherapies containing PD-1 and PD-L1 agents for the treatment of CRC patients, such as pembrolizumab and nivolumab (to be discussed below), (ii) SIRPα antibody (for example, any SIRP antagonist, in particular an antibody, preferably, for example, those disclosed in WO 2017 / 178653 incorporated herein by reference, other examples disclosed in WO 20200068752 and WO 2019023347), (iii) TcEngager (for example, preferably those disclosed in WO 2019234220 and EP 19201200.3), (iv) KISIMA vaccines (for example, preferably those disclosed in WO 2016 / 146260 and 2018 / 055060, which are incorporated herein by reference); (v) Oncolytic viruses (for example, preferably vesicular stomatitis viruses with or without specific gene cargo, e.g., VSV-GP and VSV-CCL21 as disclosed in WO 2010 / 040526 and PCT / EP 2020 / 051701, respectively, incorporated herein by reference), (vi) STING agonists (for example, preferably those disclosed in WO 2018060323 and US 10,537,590 incorporated herein by reference) and (vii) Chemotherapy agents used to treat CRC (including 5-fluorouracil, irinotecan, doxorubicin, and TAS-102) Treatment with the CD137 / FAP binding molecule of the present invention in combination with a drug selected from the above is particularly preferred.

[0279] In all senses of the present invention and its embodiments, a PD-1 pathway inhibitor is a compound that inhibits the interaction between PD-1 and its receptor. Preferably, a PD-1 pathway inhibitor can impair PD-1 pathway signaling mediated by the PD-1 receptor. A PD-1 inhibitor can be any inhibitor directed against any member of the PD-1 pathway capable of antagonizing PD-1 pathway signaling. Preferably, the inhibitor can be an antagonist antibody targeting any member of the PD-1 pathway directed against the PD-1 receptor, PD-L1, or PD-L2. Alternatively, a PD-1 pathway inhibitor can be a fragment of the PD-1 receptor or the PD-1 receptor itself, which blocks the activity of the PD-1 ligand.

[0280] PD-1 antagonists are well known in the art and are outlined, for example, in Li et al., Int. J. Mol. Sci. 2016, 17, 1151 (incorporated herein by reference). Any PD-1 antagonist, in particular antibodies, such as those disclosed in Li et al. and further antibodies disclosed herein, can be used in accordance with the present invention. Preferably, the PD-1 antagonist of the present invention and all embodiments thereof is selected from the group consisting of the following antibodies: pembrolizumab (anti-PD-1 antibody), nivolumab (anti-PD-1 antibody), pizilizumab (anti-PD-1 antibody), PDR-001 (anti-PD-1 antibody), PD1-1, PD1-2, PD1-3, PD1-4, and PD1-5, preferably BI-754019 (anti-PD-1 antibody), atezolizumab (anti-PD-L1 antibody), avelumab (anti-PD-L1 antibody), and durvalumab (anti-PD-L1 antibody), which are disclosed below in this specification.

[0281] For example, pembrolizumab (formerly known as lambrolizumab, trade name Keytruda, also known as MK-3475), disclosed in Hamid, O. et al. (2013) New England Journal of Medicine 369(2):134-44, is a humanized IgG4 monoclonal antibody that binds to PD-1 and contains a C228P mutation designed to inhibit Fc-mediated cytotoxicity. Pembrolizumab is disclosed, for example, in US No. 8,354,509 and WO No. 2009 / 114335. It is approved by the FDA for the treatment of patients with unresectable or metastatic melanoma and patients with metastatic NSCLC.

[0282] Nivolumab (CAS Registry Number: 946414-94-4, BMS-936558, or MDX1106b) is a fully human IgG4 monoclonal antibody that specifically blocks PD-1 and lacks detectable antibody-dependent cell cytogenesis (ADCC). Nivolumab is disclosed, for example, in US No. 8,008,449 and WO No. 2006 / 121168. It is approved by the FDA for the treatment of patients with unresectable or metastatic melanoma, metastatic NSCLC, and advanced renal cell carcinoma.

[0283] Pizilizumab (CT-011, Cure Tech) is a humanized IgG1k monoclonal antibody that binds to PD-1. Pizilizumab is disclosed, for example, in WO 2009 / 101611.

[0284] PDR-001 or PDR001 is a high-affinity ligand-blocking humanized anti-PD-1 IgG4 antibody that blocks the binding of PD-L1 and PD-L2 to PD-1. PDR-001 is disclosed in WO 2015 / 112900 and WO 2017 / 019896.

[0285] Antibodies PD1-1 to PD1-5 are antibody molecules defined by the sequences shown in Table 4. Here, HC represents the (full-length) heavy chain, and LC represents the (full-length) light chain.

[0286] [Table 14] TIFF0007911965000032.tif254169

[0287] Specifically, the anti-PD-1 antibody molecule described herein is (PD1-1) Heavy chain containing the amino acid sequence of SEQ ID NO: 652 and light chain containing the amino acid sequence of SEQ ID NO: 653 or (PD1-2) Heavy chain containing the amino acid sequence of SEQ ID NO: 654 and light chain containing the amino acid sequence of SEQ ID NO: 655 or (PD1-3) Heavy chain containing the amino acid sequence of SEQ ID NO: 656 and light chain containing the amino acid sequence of SEQ ID NO: 657 or (PD1-4) Heavy chain containing the amino acid sequence of SEQ ID NO: 658 and light chain containing the amino acid sequence of SEQ ID NO: 659 or (PD1-5) Heavy chain containing the amino acid sequence of SEQ ID NO: 660 and light chain containing the amino acid sequence of SEQ ID NO: 661 It has.

[0288] Atezolizumab (also known as Tecentriq, MPDL3280A) is a phage-derived human IgG1k monoclonal antibody that targets PD-L1, as described, for example, in Deng et al. mAbs 2016;8:593-603. It is approved by the FDA for the treatment of patients with urothelial carcinoma.

[0289] Avelumab is a fully human anti-PD-L1 IgG1 monoclonal antibody, as described, for example, in Boyerinas et al. Cancer Immunol. Res. 2015; 3:1148-1157.

[0290] Durvalumab (MEDI4736) is a human IgG1k monoclonal antibody with high specificity for PD-L1, and is described, for example, in Stewart et al. Cancer Immunol. Res. 2015;3:1052-1062 or Ibrahim et al. Semin. Oncol. 2015; 42:474-483.

[0291] Further PD-1 antagonists disclosed in Li et al. (above) or known to be in clinical trials, such as AMP-224, MEDI0680 (AMP-514), REGN2810, BMS-936559, JS001-PD-1, SHR-1210, BMS-936559, TSR-042, JNJ-63723283, MEDI4736, MPDL3280A and MSB0010718C, may be used instead of or in addition to the antagonists mentioned above.

[0292] As used herein, INN also means all biosimilar antibodies having the same or substantially the same amino acid sequence as the originator antibody, including, but not limited to, biosimilars authorized in the United States under 42 USC §262 subsection(k) and equivalent rules in other jurisdictions.

[0293] The PD-1 antagonists listed above are known in the art, along with their respective manufacturing processes, therapeutic uses, and properties.

[0294] In one embodiment, the PD-1 antagonist is pembrolizumab.

[0295] In another embodiment, the PD-1 antagonist is nivolumab.

[0296] In another embodiment, the PD-1 antagonist is pizilizumab.

[0297] In another embodiment, the PD-1 antagonist is atezolizumab.

[0298] In another embodiment, the PD-1 antagonist is avelumab.

[0299] In another embodiment, the PD-1 antagonist is durvalumab.

[0300] In another embodiment, the PD-1 antagonist is PDR-001.

[0301] In preferred embodiments, the proteins of the present invention, for example, CD137 #1 / FAP #1, CD137 #7 / FAP #1, CD137 #1 / FAP #4, CD137 #7 / FAP #4, CD137 #2 / FAP #5, CD137 #2 / FAP #2, CD137 #2 / FAP #3, CD137 #3 / FAP #5, CD137 #3 / FAP #2, CD137 #3 / FAP #3, CD137 #4 / FAP #5, CD137 #4 / FAP #2, CD137 #4 / FAP #3, CD137 #5 / FAP #5, CD137 #5 / FAP #2, CD137 #5 / FAP #3, CD137 #6 / FAP #5, CD137 #6 / FAP #2, CD137 #6 / FAP #3, CD137 CD137 #8 / FAP #5, CD137 #8 / FAP #2, CD137 #8 / FAP #3, CD137 #9 / FAP #5, CD137 #9 / FAP #2, and CD137 #9 / FAP #3, CD137 #10 / FAP #5, CD137 #10 / FAP #2, and CD137 #10 / FAP #3 are used in combination with PD-1 antagonists selected from the group consisting of PD1-1, PD1-2, PD1-3, PD1-4, and PD1-5 to treat cancer.

[0302] For therapeutic use, the conjugated molecules of the present invention are formulated into pharmaceutical compositions suitable for easy administration to animals or humans. Typical formulations of antibody molecules can be prepared by mixing the antibody molecules with physiologically acceptable carriers, excipients, or stabilizers in the form of lyophilized or dried formulations, aqueous solutions, or aqueous or non-aqueous suspensions. The carriers, excipients, modifiers, or stabilizers are nontoxic at the doses and concentrations used. These include buffer systems, e.g., phosphates, citrates, acetates, and other inorganic or organic acids and their salts; antioxidants, including ascorbic acid and methionine; preservatives, e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride, benzalkonium chloride, benzethonium chloride; phenols, butyl or benzyl alcohols; alkylparabens, e.g., methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol; proteins, e.g., serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, e.g., polyvinylpyrrolidone or polyethylene glycosides. This includes PEG (protein oxide); amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, oligosaccharides, or polysaccharides and other dehydrates, including glucose, mannose, sucrose, trehalose, dextrin, or dextran; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or ionic or nonionic surfactants, such as TWEEN® (polysorbate), PLURONICS®, or fatty acid esters, fatty acid ethers, or sugar esters. Organic solvents, such as ethanol or isopropanol, may also be included in the antibody preparation. Furthermore, excipients may have release-modifying or absorption-modifying functions.

[0303] The present invention will now be described by the following non-limiting embodiments.

[0304] Example 1: Design of binding molecules that recognize human CD137 (4-1BB, TNFRSF9) and fibroblast-activating protein (FAP) in relation to the tumor microenvironment. The inventors have developed a binding molecule that binds to CD137 (4-1BB, TNFRSF9) and fibroblast-activating protein (FAP), targeting CD137 T cell activation and recruitment of FAP-expressing fibroblasts in tumor stroma. The molecular design used has an IgG antibody (called a "master antibody") with specificity for one target antigen, and scFvs with different specificities are linked to the C-terminus of the heavy chain. A schematic of the design is shown in Figure 1A. A schematic of the mode of action is shown in Figure 1B.

[0305] Preferably, the binding molecule is bispecific and tetravalent.

[0306] The bispecific molecule contains a flexible peptide sequence between the variable heavy chain (VH) domain and the variable light chain (VL) domain of scFv, and the scFv domain is linked to the master IgG antibody via a further series of linkers. In one configuration, the scFv is oriented so that the VL domain forms the "N-terminus" of the scFv and is therefore fused to the C-terminus of the heavy chain of the master antibody, while the VH domain forms the C-terminus of the scFv, in fact, the entire heavy chain polypeptide. However, it can be understood that this "N-VL-VH-C" structure can be reversed, i.e., it can be "N-VH-VL-C".

[0307] The following examples illustrate methods used to generate bispecific molecules that bind to CD137 (4-1BB, TNFRSF9) and fibroblast-activating protein (FAP), as well as variations in the format and biological activity of these molecules.

[0308] Example 2: Preparation of a binding domain that recognizes CD137 (4-1BB, TNFRSF9) To understand how to prepare bispecific molecules that bind to human CD137 (4-1BB, TNFRSF9) and fibroblast-activating protein (FAP), it is necessary to obtain variable domains that bind to individual target antigens.

[0309] a. Immunization campaign regarding CD137 To prepare bispecific molecules that bind to human CD137(4-1BB,TNFRSF9), clonal hybridomas or single B cells derived from CD137(4-1BB,TNFRSF9)-immunized mice were cultured in vitro. The supernatant was screened for reactivity to human CD137(4-1BB,TNFRSF9). Then, immunoglobulin (Ig) VH and VL genes were amplified from the identified positive clones.

[0310] In short, wild-type CD1 mice were immunized with human and cynomolgus monkey CD137-Fc_His proteins (SEQ ID NOs: 349 and 359, respectively), and full Freund's adjuvant was used at various time points to enhance the antibody response. Serological testing was evaluated by ELISA using human and cynomolgus monkey CD137-Fc_His proteins as antigens. Serologically positive mice were given a final boost one week before spleen B cell isolation. Mouse spleens were harvested and processed to collect whole splenocytes. All procedures were performed according to protocols approved by IACUC.

[0311] To identify the binder for CD137, six selected mouse spleens were isolated, and the recovered splenocytes were stained according to a standard SBC antibody production method. First, the recovered splenocytes were T-cell depleted using mouse pan-T Dynabeads (Invitrogen 114.43D) according to the manufacturer's instructions. Then, the depleted cell preparation was incubated with 1 nM human CD137_huTNFRSF9_Hu-His cleavage-tagged biotin (SEQ ID NO: 335) and 1 nM MuGIPR-hu.FC-AF647, along with a fluorescent dye conjugated antibody against Sytox blue, which was included in the staining to aid in the identification of CD3, CD19, IgD, IgM, B220, and living memory B cells. Streptavidin-PE was added for the detection of B cells binding to biotinylated human CD137. Next, human CD137 antigen-positive memory B cells were individually sorted into 384-well plates and cultured at 37°C and 5% CO2 for 7 days. The B cell supernatant was screened for binding to biotinylated human and cynomolgus monkey CD137 proteins using AlphaLISA, according to the manufacturer's instructions.

[0312] To identify CD137-specific binders, antibodies that reacted only with human Fc were removed using the counter Fc bait MuGIPR-hu.FC-AF647 and the huCD137 bait huTNFRSF9_His-biotin. Both showed good binding independently to CD1 mouse B cell splenocytes, FMO1, and FMO2, respectively. Double-positive MuGIPR-hu.FC / huCD137 B cells were completely isolated from Hu CD137-positive B cells in fully stained samples. Thirty 384-well plates (10,560 wells) containing CD137-positive mouse B cells were collected and passaged for primary screening.

[0313] In the primary screening, 554 B cell supernatants were assayed for specific binding to both human CD137 with S / B > 2 and cynomolgus monkey CD137 with S / B > 2, with IgG > 10 ng / ml positivity (Figure 1). 554 positive B cell clones were isolated into six 96-well plates for DNA recovery and isolation and amplification of mouse VH and VL gene segments. Of the 554 clones, 376 B cell clones expressing anti-CD137 at titers ranging from 0.6 to 75 ug / ml were assayed for single-point binding to recombinant CHO cells (CHO-K1) expressing human CD137. Of the 376 B cell clones, 168 expressed anti-CD137 antibody at levels >2× relative to the background, ranging from 2 to 57× S / B (data not shown).

[0314] b. Identification of agonist CD137 antibodies To identify the agonist binder, the NF-κB-Luc2 / 4-1BB Jurkat assay system (Promega) was used. The NFκB activity assay uses a Jurkat reporter cell line that expresses human CD137 on its cell surface.

[0315] In short, the binding of HTP supernatant containing recombinant IgG1 (KO) antibody to the NF-kB-Luc2 / 4-1BB Jurkat cell line was assayed by flow cytometry. The CD137 antibody control was diluted in a staining buffer containing urelumab (BMS), a CD137-positive control antibody. Supernatant (50 μl) (as described above) collected from 168 anti-CD137 B cell clones was incubated with Jurkat cells in a 96-well plate at 4°C for 30 minutes. These cells were washed three times with staining buffer. Goat F(ab')2 anti-human IgG-Fc PE secondary antibody was added to these cells, and the plate was incubated at 4°C for 30 minutes. These cells were washed three times with staining buffer. The cells were suspended in 100 μl of cold staining buffer and analyzed by flow cytometry. The anti-CD137 antibody supernatant was analyzed for more than 2x background binding to the cell line. Of the 168 screened anti-CD137 antibody-containing supernatants, 10 showed 1.5-fold (1.64–5.21-fold) anti-CD137 agonist activity relative to the background, with antibody titers ranging from 0.76–31 μg / ml. The signal from clone CL-186330 was 5.61 S / B (data not shown).

[0316] c. V gene recovery for CD137 binding molecules Ten B cell clones were identified as having agonist activity exceeding a specific threshold selected for further analysis. First, cell lysates were collected, rearranged in 96-well plates, and stored at -20°C, after which the V gene was recovered. Genes encoding the mouse heavy chain variable region and light chain variable region were recovered by RT-PCR (see primers in Table 4) and then cloned in-frame into pTT5 expression vectors encoding human IgG1 (KO) and κ constant region, respectively.

[0317] B cell lysates were subjected to cDNA synthesis using the Smarter cDNA synthesis kit (Clontech, Mount View, CA). To facilitate cDNA synthesis, reverse transcription of all messenger RNAs was initiated using oligo(dT), followed by "5' capping" with Smarter IIA oligonucleotides. Subsequent amplification of VH and VL fragments was performed using two-step PCR amplification with a 5' primer targeting the Smarter IIA cap and a 3' primer targeting the consensus region at CH1. Briefly, each 50 μl PCR reaction consisted of 1 μl of each 20 μM primer (forward and reverse, final concentration 1 μM), 25 μl of PrimeStar® Max DNA polymerase premix (Clontech), 2 μl of unpurified cDNA, and 20 μl of double-distilled H2O. The cycle program is started and then performed for 35 cycles of 15 seconds at 94°C, 30 seconds at 50°C, and 50 seconds at 68°C, followed by completion at 68°C for 7 minutes. Second round PCR was performed using VL and VH second round primers containing 15 bp complementary extensions that "overlap" with each region in each pTT5 mother vector (VH and VL). Second round PCR was performed using the following program: 35 cycles (45 seconds at 94°C, 30 seconds at 50°C, and 50 seconds at 68°C), followed by completion at 68°C for 7 minutes.

[0318] To produce chimeric CD137, mouse VH and VL regions were fused to the heavy and light chain constant regions of human IgG1, and the In-Fusion® HD cloning kit (Clontech, USA) was used for directional cloning of ...

Claims

1. An immunoglobulin molecule having two antigen-binding sites that specifically bind to CD137 (4-1BB ligand receptor, 4-1BB), and two antigen-binding sites that specifically bind to fibroblast-activating protein (FAP), Here, both of the two antigen-binding sites that specifically bind to CD137 (4-1BB ligand receptor) are part of the immunoglobulin molecule, each of the two antigen-binding sites that specifically bind to fibroblast-activating protein (FAP) is scFv, and both of the two antigen-binding sites that specifically bind to CD137 (4-1BB) are as follows: Heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 295 (CDR1), SEQ ID NO: 18 (CDR2), and SEQ ID NO: 9 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 22 (CDR1), SEQ ID NO: 23 (CDR2), and SEQ ID NO: 14 (CDR3). Includes, Both antigen-binding sites that specifically bind to fibroblast-activating protein (FAP) Includes heavy chain CDRs containing the amino acid sequences of SEQ ID NO: 333 (CDR1), SEQ ID NO: 144 (CDR2), and SEQ ID NO: 145 (CDR3), and light chain CDRs containing the amino acid sequences of SEQ ID NO: 138 (CDR1), SEQ ID NO: 139 (CDR2), and SEQ ID NO: 140 (CDR3), A bispecific and tetravalent binding molecule.

2. The binding molecule according to claim 1, wherein the two scFv molecules have a VL-VH orientation from the N-terminus to the C-terminus.

3. The binding molecule according to claim 1 or 2, wherein two scFv molecules are each fused to the C-terminus of the heavy chain of an immunoglobulin molecule.

4. The binding molecule according to any one of claims 1 to 3, wherein the immunoglobulin molecule is IgG.

5. The binding molecule according to any one of claims 1 to 4, wherein two scFv molecules are fused to an immunoglobulin molecule by a peptide linker having a length of 4 to 20 amino acids.

6. The antigen-binding site that specifically binds to CD137 (4-1BB) is as follows: Variable heavy chain containing the amino acid sequence of SEQ ID NO: 20 and variable light chain containing the amino acid sequence of SEQ ID NO: 25 A binding molecule according to any one of claims 1 to 5, which is part of an immunoglobulin (Ig) molecule containing [the specified substance].

7. The antigen-binding site that specifically binds to fibroblast-activating protein (FAP) is as follows: Variable heavy chain containing the amino acid sequence of SEQ ID NO: 142 and variable light chain containing the amino acid sequence of SEQ ID NO: 146 A binding molecule according to any one of claims 1 to 6, comprising:

8. An immunoglobulin heavy chain containing the amino acid sequence of SEQ ID NO: 159, fused to scFv at the C-terminus, and an immunoglobulin light chain containing the amino acid sequence of SEQ ID NO:

160. including, The binding molecule according to any one of claims 1 to 7.

9. A nucleic acid molecule encoding the binding molecule according to any one of claims 1 to 8.

10. An expression vector containing a nucleic acid molecule encoding the binding molecule described in any one of claims 1 to 8.

11. A host cell containing the nucleic acid molecule described in claim 9.

12. A method for producing a bound molecule according to any one of claims 1 to 8, (i) Culturing the host cells according to claim 11 under conditions that enable the expression of the molecule, (ii) recovering the molecule, method.

13. A binding molecule according to any one of claims 1 to 8, for use in pharmaceuticals.

14. A binding molecule according to any one of claims 1 to 8, for use in the treatment of cancer.

15. The binding molecule according to claim 14, wherein the cancer is colorectal cancer (CRC) including colorectal adenocarcinoma, gastric cancer (GC) including gastric adenocarcinoma, pancreatic cancer (PAC) including pancreatic adenocarcinoma, or lung cancer (LC) including lung squamous cell carcinoma or lung adenocarcinoma.

16. A pharmaceutical composition comprising a binding molecule according to any one of claims 1 to 8 together with a pharmaceutically acceptable carrier.

17. The pharmaceutical composition according to claim 16, comprising one or more further active ingredients.

18. The binding molecule according to claim 13, wherein the binding molecule is administered to a patient in need in combination with a PD-1 antibody.

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