binding construct

Abstract

The present invention provides a construct comprising an FAP binder and an LTBR binder.

Description

Technical Field

[0001] This invention relates to constructs comprising FAP binders and LTBR binders, particularly for the treatment of diseases such as cancer, and related aspects. This invention also relates to FAP binders and LTBR binders.

[0002] Existing technology

[0003] Cancer can affect a variety of cell types and tissues, but its root cause is the dysregulation of cell division control. This process is highly complex and requires the precise coordination of multiple pathways, many of which are not yet fully characterized.

[0004] Cancer immunotherapy involves using an individual's own immune system to treat or prevent cancer. These therapies aim to stimulate the body's immune system to eliminate cancer cells and utilize the expression of tumor antigens resulting from accumulated gene mutations in cancer. These tumor antigens include tumor-associated antigens such as proteins and tumor-specific antigens or neoantigens, which are presented by major tissue compatibility molecules (Xie et al., 2023). However, malignant tumors can evade the immune system's surveillance through intrinsic tumor cell mechanisms and mechanisms associated with the tumor microenvironment, leading to therapy resistance (Sharma et al., 2017).

[0005] Durable responses to immunotherapy have been observed only in a small number of cancer patients, necessitating the development of novel therapies. Key challenges in generating durable antitumor responses include limited infiltration of immune effector cells within the tumor, immune cell dysfunction, and a lack of initiation of antitumor immune cells. Recent studies have shown that the presence of high endothelial venules (HEVs) and tertiary lymphoid structures (TLSs) in cancer patients is associated with better survival and improved responses to therapy (Cabrita et al., 2020; Vanhersecke et al., 2021; Asrir et al., 2022; Zhang et al.; Front Immunol, 2022). HEVs are specialized structures within blood vessels that serve as gateways for immune cells to enter the tumor, while TLSs are aggregates of ectopic immune cells that provide a favorable local immune environment and promote local initiation of immune cells (Sautès-Fridman et al., 2019; Schumacher et al., 2022). Therefore, therapeutic strategies aimed at enhancing HEVs and TLSs in patients will contribute to improving antitumor immune responses.

[0006] Lymphotoxin β receptor (LTBR), also known as tumor necrosis factor receptor superfamily member 3 (TNFRSF3), is a lymphotoxin cell surface receptor involved in cytokine release and apoptosis. It is a member of the tumor necrosis factor receptor superfamily. LTBR regulates the expression of various homeostatic lymphoid tissue cytokines (such as CCL5, CCL19, CCL21, and CXCL13) and adhesion molecules (ICAM-1, VCAM-1, MAdCAM1) through the classical and alternative NFKB pathways, playing a central role in the development and homeostasis of secondary lymphoid organs (SLO), lymphoid tissue tracts (TLS), and high-density leukocyte extremities (HEVs) (Dejardin et al., 2002; Schneider et al., 2004; Fernandes et al., 2016). LTBR is activated by two different trimeric ligands: LIGHT (TNFSF14) and lymphotoxin α1β2 (LTα1β2). Although LTα1β2 is specific only for LTBR, ​​LIGHT also binds to and activates HVEM (TNFRSF14), a receptor expressed on immune cells and involved in their regulation (Pasero et al., 2012).

[0007] Studies have found that activation of LTBR by its ligands can lead to the formation of ectopic TLS and HEVs (Schrama et al., 2001; Tang et al., 2017; Allen et al., 2017; Asrir et al., 2022). The presence of TLS and HEVs in the tumor microenvironment is often associated with immune cell infiltration and better prognosis, suggesting that TLS and HEVs are involved in antitumor immune responses (Dieu-Nosjean et al., 2008; Weinstein and Storkus, 2015; Vanhersecke et al., 2021; Asrir et al., 2022). Therefore, LTBR activation has the potential to promote the formation of HEVs and TLSs in the tumor microenvironment, induce antitumor responses, and improve current cancer therapies.

[0008] Recent evidence suggests that activation of the LTBR (lymphotoxin beta receptor) can stimulate resident fibroblasts to mature into immune-functional fibroblast reticulum cells (FRCs) (Sautès-Fridman et al., 2019). FRCs are a specialized subset of stromal cells that highly express the fibroblast activation protein alpha (FAP) in lymphoid organs such as TLS. They play a crucial role in the regulation of immune responses and the migration, retention, and activation of immune cells through close interaction with various immune cells, including T cells, B cells, and dendritic cells.

[0009] Because LTBR is widely expressed in organisms, pro-effect LTBR-binding constructs that can induce TLS formation and create an immune-activating environment pose a significant risk of systemic immune-related adverse events. Johansson-Percival et al. reported weight loss in mice after systemic administration of the LTBR activator VTP-LIGHT (Johansson-Percival et al., 2017), while overexpression of lymphotoxin α and lymphotoxin β induced hepatotoxicity in mice (Haybaeck et al., 2009). US20120014947 indicates that lymphotoxin signaling may lead to hepatotoxicity, particularly in hepatitis models; knockout of LTBR or inhibition of LTBR signaling can reduce the increase in liver enzymes (such as AST and ALT), which are associated with hepatotoxicity. A phase 1 open-label, dose-escalation study of humanized LTBR activating antibody (hCBE11) in patients with advanced solid tumors was intended to determine its safety and tolerability, but was terminated before enrollment was completed (ClinicalTrials.gov, NCT00105170).

[0010] Therefore, as the prior art suggests, there is a need for a therapeutic approach that can specifically activate LTBR in tumors rather than other tissues to reduce the risk of toxicity and generate well-tolerated drugs that can be used in combination therapies (Tang et al., 2017).

[0011] US2021 / 0188990 discloses multispecific binding molecules, such as bispecific antibodies, with a first specificity against LTBR and a second specificity against the outer domain B (EDB) of fibronectin (US2021 / 0188990, paragraph 17), particularly for the purpose of killing cancer cells (US2021 / 0188990, paragraph 2). This disclosure indicates that using a bispecific antibody having one arm binding to LTBR and another arm binding to a tumor-associated antigen (TAA) (the “targeting” arm) enables tumor antigen-dependent activation of LTBR, ​​and that using the targeting arm, which binds to an extracellular matrix antigen, is more effective than binding to a cell surface antigen co-expressed on the same cell as LTBR. Conversely, the inventors of this application have demonstrated that bispecific antibodies having a first specificity against LTBR and a second specificity against FAP can activate LTBR in a tumor antigen-dependent manner.

[0012] WO2023117834 discloses activating LTBR antibodies and bispecific FAP / LTBR antibodies comprising these antibodies. Comparative data on the FAP / LTBR bispecific antibodies disclosed in WO2023117834 are provided in Example 4 below.

[0013] When providing bispecific agents targeting LTBR, ​​it may be advantageous for the LTBR-binding component to have relatively low affinity while still achieving high activity after incorporating a bispecific form. In other words, the ratio of bispecific activity to monospecific affinity may need to be high. Lower target binding affinity may be beneficial for agonists, as literature reports that low-affinity antibodies exert higher activity than high-affinity antibodies by increasing aggregation (Yu et al., Nature 2023). Furthermore, lower binding affinity for the target may be advantageous for agonists to reduce off-tumor binding.

[0014] New methods and therapies are still needed in cancer treatment. These methods can demonstrate high response rates and durable responses, reduce the dosage required to achieve efficacy, improve safety profiles, and reduce side effects. Summary of the Invention

[0015] This invention relates to several aspects involving targeting FAP and / or LTBR. The inventors have demonstrated that certain FAP / LTBR bispecific constructs can achieve conditional activation of LTBR in the presence of FAP, providing advantages including potentially reduced toxicity. Certain FAP / LTBR bispecific constructs can activate LTBR on cells that are double-positive for both FAP and LTBR (e.g., CAFs and FRCs). The inventors have also demonstrated that certain FAP / LTBR bispecific constructs can target each of two or more cell types that are single-positive for either FAP or LTBR and activate LTBR. Furthermore, certain FAP / LTBR bispecific constructs can achieve upregulation of adhesion molecules and / or induction of chemokines (as shown in Examples 4.1 to 4.10). These and other advantageous properties of certain constructs have been shown to translate into in vivo effects, such as the formation of HEVs and TLS, an increase in immune cells, and inhibition / regression of tumor growth (as shown in Examples 4.11 to 4.13).

[0016] The inventors have also identified anti-FAP binding peptides and anti-LTBR binding peptides that (a) benefit from advantageous properties when in monovalent, monospecific form (as shown in Examples 1 to 3) and (b) represent components that are useful in the manufacture of the aforementioned advantageous bispecific constructs.

[0017] One aspect of the present invention provides a construct comprising an FAP binder and an LTBR binder. Another aspect of the present invention provides a construct according to the invention for cancer treatment. Another aspect of the present invention also provides a polynucleotide encoding the construct of the present invention. Furthermore, the present invention provides (i) certain anti-LTBR binding peptides and (ii) certain anti-FAP binding peptides.

[0018] Certain embodiments of the present invention are expected to benefit from one or more of the following advantages over the prior art: (a) Enhanced affinity, (b) Enhanced avidity (c) The facilitating effect of LTBR (e.g., activation of NFKB pathways, including classic or alternative NFKB pathways), (d) Aggregation of LTBR, (e) Reduce toxicity (e.g., hepatotoxicity), (f) Reduce the dose, (g) Improve biological distribution, (h) Increase the ratio of monomer affinity to bispecific potency. (i) Improve treatment efficacy, (j) Combining novel and advantageous epitopes, (k) Localization and conditional activation at the target site (i.e., in a FAP-dependent manner), (l) Upregulation of intercellular adhesion molecules (e.g., ICAM-1), (m) Simultaneously binds to LTBR and FAP on the same cell and activates LTBR on that cell. (n) Increase the secretion of CCL5 (e.g., from RPMI-7951 cells), (o) Increased yield and / or purity, (p) Easier to purify, (q) Reduce immunogenicity, (r) Increase biological distribution, (s) Improved half-life and / or reduced clearance, (t) Reduces the binding of Fc receptors.

[0019] Other aspects of the invention will become clear in the following.

[0020] Sequence Summary

[0021] SEQ ID NO: 1 Peptide sequence of the BHA10 heavy chain variable region

[0022] SEQ ID NO: 2 Peptide sequence of the variable region of the BHA10 light chain

[0023] SEQ ID NO: 3 Peptide sequence of linker G4S

[0024] SEQ ID NO: 4 Peptide sequence of peptide linker (G4S)4

[0025] SEQ ID NO: 5 5G11 Heavy Chain Variable Region Peptide Sequence

[0026] SEQ ID NO: 6 5G11 light chain variable region polypeptide sequence

[0027] SEQ ID NO: 7 28H1 heavy chain variable region polypeptide sequence

[0028] SEQ ID NO: 8 28H1 Peptide sequence of the light chain variable region

[0029] SEQ ID NO: 9 Full-length human LTBR polypeptide sequence

[0030] SEQ ID NO: 10 Peptide sequence of the extracellular domain of human LTBR

[0031] SEQ ID NO: 11 Peptide sequence of full-length cynomolgus monkey LTBR

[0032] SEQ ID NO: 12 Peptide sequence of the extracellular domain of the LTBR in cynomolgus monkeys

[0033] SEQ ID NO: 13 Full-length mouse LTBR polypeptide sequence

[0034] SEQ ID NO: 14 Peptide sequence of the extracellular domain of mouse LTBR

[0035] SEQ ID NO: 15 Full-length human FAP polypeptide sequence

[0036] SEQ ID NO: 16 Peptide sequence of the extracellular domain of human FAP

[0037] SEQ ID NO: 17 Full-length cynomolgus monkey FAP polypeptide sequence

[0038] SEQ ID NO: 18 Peptide sequence of the extracellular domain of FAP in cynomolgus monkeys

[0039] SEQ ID NO: 19 Full-length mouse FAP polypeptide sequence

[0040] SEQ ID NO: 20 Peptide sequence of the extracellular domain of mouse FAP cells

[0041] SEQ ID NO: 21 Peptide sequence of human light (including ECD)

[0042] SEQ ID NO: 22 Peptide sequence of human LIGHT ECD

[0043] SEQ ID NO: 23 Peptide sequence of the heavy chain variable region of clone 1.5

[0044] SEQ ID NO: 24 Peptide sequence of the heavy chain variable region of clone 1.6

[0045] SEQ ID NO: 25 Peptide sequence of the heavy chain variable region of clone 1.7

[0046] SEQ ID NO: 26 Peptide sequence of the heavy chain variable region of clone 1.8

[0047] SEQ ID NO: 27 Peptide sequence of the heavy chain variable region of clone 2.1

[0048] SEQ ID NO: 28 Peptide sequence of the light chain variable region of clone 2.1

[0049] SEQ ID NO: 29 Peptide sequence of the heavy chain variable region of clone 2.2

[0050] SEQ ID NO: 30 Peptide sequence of the light chain variable region of clone 2.2

[0051] SEQ ID NO: 31 Peptide sequence of the heavy chain variable region of clone 2.3

[0052] SEQ ID NO: 32 Peptide sequence of the light chain variable region of clone 2.3

[0053] SEQ ID NO: 33 Peptide sequence of the heavy chain variable region of clone 2.4

[0054] SEQ ID NO: 34 Peptide sequence of the light chain variable region of clone 2.4

[0055] SEQ ID NO: 35 Peptide sequence of the heavy chain variable region of clone 2.5

[0056] SEQ ID NO: 36 Peptide sequence of clone 2.5 light chain variable region

[0057] SEQ ID NO: 37 Peptide sequence of the heavy chain variable region of clone 2.6

[0058] SEQ ID NO: 38 Peptide sequence of clone 2.6 light chain variable region

[0059] SEQ ID NO: 39 Peptide sequence of the heavy chain variable region of clone 2.7

[0060] SEQ ID NO: 40 Peptide sequence of the light chain variable region of clone 2.7

[0061] SEQ ID NO: 41 Peptide sequence of the heavy chain variable region of clone 2.8

[0062] SEQ ID NO: 42 Peptide sequence of the light chain variable region of clone 2.8

[0063] SEQ ID NO: 43 Peptide sequence of the heavy chain variable region of clone 3.5

[0064] SEQ ID NO: 44 Peptide sequence of the light chain variable region of clone 3.5

[0065] SEQ ID NO: 45 Peptide sequence of the heavy chain variable region of clone 3.6

[0066] SEQ ID NO: 46 Peptide sequence of the light chain variable region of clone 3.6

[0067] SEQ ID NO: 47 Peptide sequence of the heavy chain variable region of clone 3.7

[0068] SEQ ID NO: 48 Peptide sequence of the light chain variable region of clone 3.7

[0069] SEQ ID NO: 49 Peptide sequence of the heavy chain variable region of clone 3.4

[0070] SEQ ID NO: 50 Peptide sequence of the light chain variable region of clone 3.4

[0071] SEQ ID NO: 51 Peptide sequence of the heavy chain variable region of clone 3.1

[0072] SEQ ID NO: 52 Peptide sequence of the light chain variable region of clone 3.1

[0073] SEQ ID NO: 53 Peptide sequence of the heavy chain variable region of clone 3.2

[0074] SEQ ID NO: 54 Peptide sequence of the light chain variable region of clone 3.2

[0075] SEQ ID NO: 55 Peptide sequence of the heavy chain variable region of clone 3.3

[0076] SEQ ID NO: 56 Peptide sequence of the light chain variable region of clone 3.3

[0077] SEQ ID NO: 57 Peptide sequence of clone 1.5 HCDR1

[0078] SEQ ID NO: 58 Peptide sequence of clone 1.5 HCDR2

[0079] SEQ ID NO: 59 Peptide sequence of clone 1.5 HCDR3

[0080] SEQ ID NO: 60 Peptide sequence of clone 1.6 HCDR1

[0081] SEQ ID NO: 61 Peptide sequence of clone 1.6 HCDR2

[0082] SEQ ID NO: 62 Peptide sequence of clone 1.6 HCDR3

[0083] SEQ ID NO: 63 Peptide sequence of clone 1.7 HCDR1

[0084] SEQ ID NO: 64 Peptide sequence of clone 1.7 HCDR2

[0085] SEQ ID NO: 65 Peptide sequence of clone 1.7 HCDR3

[0086] SEQ ID NO: 66 Peptide sequence of clone 1.8 HCDR1

[0087] SEQ ID NO: 67 Peptide sequence of clone 1.8 HCDR2

[0088] SEQ ID NO: 68 Peptide sequence of clone 1.8 HCDR3

[0089] SEQ ID NO: 69 Peptide sequence of clone 2.1 HCDR1

[0090] SEQ ID NO: 70 Peptide sequence of clone 2.1 HCDR2

[0091] SEQ ID NO: 71 Peptide sequence of clone 2.1 HCDR3

[0092] SEQ ID NO: 72 Peptide sequence of clone 2.1 LCDR1

[0093] SEQ ID NO: 73 Peptide sequence of clone 2.1 LCDR2

[0094] SEQ ID NO: 74 Peptide sequence of clone 2.1 LCDR3

[0095] SEQ ID NO: 75 Peptide sequence of clone 2.2 HCDR1

[0096] SEQ ID NO: 76 Peptide sequence of clone 2.2 HCDR2

[0097] SEQ ID NO: 77 Peptide sequence of clone 2.2 HCDR3

[0098] SEQ ID NO: 78 Peptide sequence of clone 2.2 LCDR1

[0099] SEQ ID NO: 79 Peptide sequence of clone 2.2 LCDR2

[0100] SEQ ID NO: 80 Peptide sequence of clone 2.2 LCDR3

[0101] SEQ ID NO: 81 Peptide sequence of clone 2.3 HCDR1

[0102] SEQ ID NO: 82 Peptide sequence of clone 2.3 HCDR2

[0103] SEQ ID NO: 83 Peptide sequence of clone 2.3 HCDR3

[0104] SEQ ID NO: 84 Peptide sequence of clone 2.3 LCDR1

[0105] SEQ ID NO: 85 Peptide sequence of clone 2.3 LCDR2

[0106] SEQ ID NO: 86 Peptide sequence of clone 2.3 LCDR3

[0107] SEQ ID NO: 87 Peptide sequence of clone 2.4 HCDR1

[0108] SEQ ID NO: 88 Peptide sequence of clone 2.4 HCDR2

[0109] SEQ ID NO: 89 Peptide sequence of clone 2.4 HCDR3

[0110] SEQ ID NO: 90 Peptide sequence of clone 2.4 LCDR1

[0111] SEQ ID NO: 91 Peptide sequence of clone 2.4 LCDR2

[0112] SEQ ID NO: 92 Peptide sequence of clone 2.4 LCDR3

[0113] SEQ ID NO: 93 Peptide sequence of clone 2.5 HCDR1

[0114] SEQ ID NO: 94 Peptide sequence of clone 2.5 HCDR2

[0115] SEQ ID NO: 95 Peptide sequence of clone 2.5 HCDR3

[0116] SEQ ID NO: 96 Peptide sequence of clone 2.5 LCDR1

[0117] SEQ ID NO: 97 Peptide sequence of clone 2.5 LCDR2

[0118] SEQ ID NO: 98 Peptide sequence of clone 2.5 LCDR3

[0119] SEQ ID NO: 99 Peptide sequence of clone 2.6 HCDR1

[0120] SEQ ID NO: 100 Peptide sequence of clone 2.6 HCDR2

[0121] SEQ ID NO: 101 Peptide sequence of clone 2.6 HCDR3

[0122] SEQ ID NO: 102 Peptide sequence of clone 2.6 LCDR1

[0123] SEQ ID NO: 103 Peptide sequence of clone 2.6 LCDR2

[0124] SEQ ID NO: 104 Peptide sequence of clone 2.6 LCDR3

[0125] SEQ ID NO: 105 Peptide sequence of clone 2.7 HCDR1

[0126] SEQ ID NO: 106 Peptide sequence of clone 2.7 HCDR2

[0127] SEQ ID NO: 107 Peptide sequence of clone 2.7 HCDR3

[0128] SEQ ID NO: 108 Peptide sequence of clone 2.7 LCDR1

[0129] SEQ ID NO: 109 Peptide sequence of clone 2.7 LCDR2

[0130] SEQ ID NO: 110 Peptide sequence of clone 2.7 LCDR3

[0131] SEQ ID NO: 111 Peptide sequence of clone 2.8 HCDR1

[0132] SEQ ID NO: 112 Peptide sequence of clone 2.8 HCDR2

[0133] SEQ ID NO: 113 Peptide sequence of clone 2.8 HCDR3

[0134] SEQ ID NO: 114 Peptide sequence of clone 2.8 LCDR1

[0135] SEQ ID NO: 115 Peptide sequence of clone 2.8 LCDR2

[0136] SEQ ID NO: 116 Peptide sequence of clone 2.8 LCDR3

[0137] SEQ ID NO: 117 Peptide sequence of clone 3.5 HCDR1

[0138] SEQ ID NO: 118 Peptide sequence of clone 3.5 HCDR2

[0139] SEQ ID NO: 119 Peptide sequence of clone 3.5 HCDR3

[0140] SEQ ID NO: 120 Peptide sequence of clone 3.5 LCDR1

[0141] SEQ ID NO: 121 Peptide sequence of clone 3.5 LCDR2

[0142] SEQ ID NO: 122 Peptide sequence of clone 3.5 LCDR3

[0143] SEQ ID NO: 123 Peptide sequence of clone 3.6 HCDR1

[0144] SEQ ID NO: 124 Peptide sequence of clone 3.6 HCDR2

[0145] SEQ ID NO: 125 Peptide sequence of clone 3.6 HCDR3

[0146] SEQ ID NO: 126 Peptide sequence of clone 3.6 LCDR1

[0147] SEQ ID NO: 127 Peptide sequence of clone 3.6 LCDR2

[0148] SEQ ID NO: 128 Peptide sequence of clone 3.6 LCDR3

[0149] SEQ ID NO: 129 Peptide sequence of clone 3.7 HCDR1

[0150] SEQ ID NO: 130 Peptide sequence of clone 3.7 HCDR2

[0151] SEQ ID NO: 131 Peptide sequence of clone 3.7 HCDR3

[0152] SEQ ID NO: 132 Peptide sequence of clone 3.7 LCDR1

[0153] SEQ ID NO: 133 Peptide sequence of clone 3.7 LCDR2

[0154] SEQ ID NO: 134 Peptide sequence of clone 3.7 LCDR3

[0155] SEQ ID NO: 135 Peptide sequence of clone 3.4 HCDR1

[0156] SEQ ID NO: 136 Peptide sequence of clone 3.4 HCDR2

[0157] SEQ ID NO: 137 Peptide sequence of clone 3.4 HCDR3

[0158] SEQ ID NO: 138 Peptide sequence of clone 3.4 LCDR1

[0159] SEQ ID NO: 139 Peptide sequence of clone 3.4 LCDR2

[0160] SEQ ID NO: 140 Peptide sequence of clone 3.4 LCDR3

[0161] SEQ ID NO: 141 Peptide sequence of clone 3.1 HCDR1

[0162] SEQ ID NO: 142 Peptide sequence of clone 3.1 HCDR2

[0163] SEQ ID NO: 143 Peptide sequence of clone 3.1 HCDR3

[0164] SEQ ID NO: 144 Peptide sequence of clone 3.1 LCDR1

[0165] SEQ ID NO: 145 Peptide sequence of clone 3.1 LCDR2

[0166] SEQ ID NO: 146 Peptide sequence of clone 3.1 LCDR3

[0167] SEQ ID NO: 147 Peptide sequence of clone 3.2 HCDR1

[0168] SEQ ID NO: 148 Peptide sequence of clone 3.2 HCDR2

[0169] SEQ ID NO: 149 Peptide sequence of clone 3.2 HCDR3

[0170] SEQ ID NO: 150 Peptide sequence of clone 3.2 LCDR1

[0171] SEQ ID NO: 151 Peptide sequence of clone 3.2 LCDR2

[0172] SEQ ID NO: 152 Peptide sequence of clone 3.2 LCDR3

[0173] SEQ ID NO: 153 Peptide sequence of clone 3.3 HCDR1

[0174] SEQ ID NO: 154 Peptide sequence of clone 3.3 HCDR2

[0175] SEQ ID NO: 155 Peptide sequence of clone 3.3 HCDR3

[0176] SEQ ID NO: 156 Peptide sequence of clone 3.3 LCDR1

[0177] SEQ ID NO: 157 Peptide sequence of clone 3.3 LCDR2

[0178] SEQ ID NO: 158 Peptide sequence of clone 3.3 LCDR3

[0179] The polypeptide sequence of clone 1.1 of SEQ ID NO: 159

[0180] The polypeptide sequence of clone 1.2 of SEQ ID NO: 160

[0181] The polypeptide sequence of clone 1.3 of SEQ ID NO: 161

[0182] The polypeptide sequence of clone 1.4 of SEQ ID NO: 162

[0183] SEQ ID NO: 163 Peptide sequence of clone 1.1 HCDR1

[0184] SEQ ID NO: 164 Peptide sequence of clone 1.1 HCDR2

[0185] SEQ ID NO: 165 Peptide sequence of clone 1.1 HCDR3

[0186] SEQ ID NO: 166 Peptide sequence of clone 1.2 HCDR1

[0187] SEQ ID NO: 167 Peptide sequence of clone 1.2 HCDR2

[0188] SEQ ID NO: 168 Peptide sequence of clone 1.2 HCDR3

[0189] SEQ ID NO: 169 Peptide sequence of clone 1.3 HCDR1

[0190] SEQ ID NO: 170 Peptide sequence of clone 1.3 HCDR2

[0191] SEQ ID NO: 171 Peptide sequence of clone 1.3 HCDR3

[0192] SEQ ID NO: 172 Peptide sequence of clone 1.4 HCDR1

[0193] SEQ ID NO: 173 Peptide sequence of clone 1.4 HCDR2

[0194] SEQ ID NO: 174 Peptide sequence of clone 1.4 HCDR3

[0195] SEQ ID NO: 175 Peptide sequence of clone 4.1 HC1

[0196] SEQ ID NO: 176 Peptide sequence of clone 4.1 HC2

[0197] SEQ ID NO: 177 4.1 LC polypeptide sequence

[0198] SEQ ID NO: 178 Peptide sequence of clone 4.2 HC1

[0199] SEQ ID NO: 179 Peptide sequence of clone 4.2 HC2

[0200] SEQ ID NO: 180 4.2 LC polypeptide sequence

[0201] SEQ ID NO: 181 Peptide sequence of clone 4.3 HC1

[0202] SEQ ID NO: 182 Peptide sequence of clone 4.3 HC2

[0203] SEQ ID NO: 183 Peptide sequence of clone 4.3 LC

[0204] SEQ ID NO: 184 Peptide sequence of clone 4.4 HC1

[0205] SEQ ID NO: 185 Peptide sequence of clone 4.4 HC2

[0206] SEQ ID NO: 186 Peptide sequence of clone 4.4 LC

[0207] SEQ ID NO: 187 Peptide sequence of clone 4.5 HC1

[0208] SEQ ID NO: 188 Peptide sequence of clone 4.5 HC2

[0209] SEQ ID NO: 189 4.5 LC polypeptide sequence

[0210] SEQ ID NO: 190 Peptide sequence of clone 4.6 HC1

[0211] SEQ ID NO: 191 Peptide sequence of clone 4.6 HC2

[0212] SEQ ID NO: 192 4.6 LC polypeptide sequence

[0213] SEQ ID NO: 193 Peptide sequence of clone 4.7 HC1

[0214] SEQ ID NO: 194 Peptide sequence of clone 4.7 HC2

[0215] SEQ ID NO: 195 Peptide sequence of clone 4.7 LC

[0216] SEQ ID NO: 196 Peptide sequence of clone 4.8 HC

[0217] SEQ ID NO: 197 Peptide sequence of clone 4.8 LC

[0218] SEQ ID NO: 198 Peptide sequence of clone 4.9 HC

[0219] SEQ ID NO: 199 Peptide sequence of clone 4.9 LC

[0220] SEQ ID NO: 200 Peptide sequence of clone 4.10 HC1

[0221] SEQ ID NO: 201 Peptide sequence of clone 4.10 HC2

[0222] SEQ ID NO: 202 Peptide sequence of clone 4.10 LC

[0223] SEQ ID NO: 203 Truncation of the hinge region polypeptide sequence

[0224] SEQ ID NO: 204 Polypeptide sequence of the natural hinge region

[0225] SEQ ID NO: 205 is a combination of the polypeptide sequences of SEQ ID NO: 58 and 167.

[0226] SEQ ID NO: 206 is a combination of the polypeptide sequences of SEQ ID NO: 67 and 170.

[0227] SEQ ID NO: 207 is a combination of the polypeptide sequences of SEQ ID NO: 65 and 165.

[0228] SEQ ID NO: 208 is a combination of the polypeptide sequences of SEQ ID NO: 68 and 171.

[0229] The polypeptide sequence of peptide SEQ ID NO: 209 E22 Attached Figure Description

[0230] Figure 1a Anti-LTBR VHH screening: HepG2 reporter gene detection in the absence of anti-His Tag crosslinking antibody.

[0231] Figure 1b Anti-LTBR VHH screening: HepG2 reporter gene detection in the presence of anti-His Tag crosslinking antibody.

[0232] Figure 2. Detection of the activity of VHH Fc fusion protein in vitro using chemokine induction from the tumor cell line HCC1187.

[0233] Figure 3a Ligand receptor inhibition assay: binding of anti-LTBR VHH-Fc fusion protein to A549 cell line.

[0234] Figure 3b Ligand receptor inhibition assay: Ligand receptor inhibition.

[0235] Figure 4 FACS binding assay: binding of anti-LTBR antibody to LTBR-positive cell line A375.

[0236] Figure 5a NFKB activation in HepG2 reporter cells expressing human LTBR: HepG2 reporter gene assay.

[0237] Figure 5b NFKB activation in HepG2 reporter cells expressing human LTBR: HepG2 reporter gene assay in the presence of crosslinking antibody.

[0238] Figure 6 FACS binding assay: binding of anti-FAP antibody to HEK293 cell line overexpressing FAP.

[0239] Figure 7 Production of human and mouse-specific FAP / LTBR bispecific antibodies: a schematic diagram of a specific 1:1 form.

[0240] Figure 7 b. Production of human and mouse-specific FAP / LTBR bispecific antibodies: a schematic diagram of a specific 2:1 formulation.

[0241] Figure 7 c. Production of human and mouse-specific FAP / LTBR bispecific antibodies: a schematic diagram of a specific 2:2 formulation.

[0242] Figure 7 d Production of human and mouse-specific FAP / LTBR bispecific antibodies: a schematic diagram of a specific 2:2 formulation.

[0243] Figure 8a The activity of the in vitro FAP / LTBR bispecific antibody was compared with the activity of HCC1187 monoculture in a chemokine induction assay using FAP-positive primary human mammary CAFs co-cultured with tumor cell line HCC1187.

[0244] Figure 8b The activity of the in vitro FAP / LTBR bispecific antibody was measured in a chemokine induction assay using FAP-positive primary human mammary CAFs co-cultured with tumor cell line HCC1187: the activity of HCC1187:CHO-FAP co-cultured.

[0245] Figure 8c The activity of the in vitro FAP / LTBR bispecific antibody was compared with the activity of HCC1187 monoculture in a chemokine induction assay using FAP-positive primary human mammary CAFs co-cultured with tumor cell line HCC1187.

[0246] Figure 8d The activity of the in vitro FAP / LTBR bispecific antibody was measured in a chemokine induction assay using FAP-positive primary human mammary CAFs co-cultured with tumor cell line HCC1187: the activity of HCC1187:CHO-FAP co-cultured.

[0247] Figure 8e The activity of the in vitro FAP / LTBR bispecific antibody was compared with the activity of HCC1187 monoculture in a chemokine induction assay using FAP-positive primary human mammary CAFs co-cultured with tumor cell line HCC1187.

[0248] Figure 8f The activity of the in vitro FAP / LTBR bispecific antibody was measured in a chemokine induction assay using FAP-positive primary human mammary CAFs co-cultured with tumor cell line HCC1187: the activity of HCC1187:CHO-FAP co-cultured.

[0249] Figure 9a In vitro FAP / LTBR bispecific activity assay in human mammary fibroblast CAFs: ICAM-1 upregulation in primary human mammary fibroblast CAFs after FAP / LTBR bsAb stimulation.

[0250] Figure 9b In vitro FAP / LTBR bispecific activity assay in human mammary fibroblast CAFs: ICAM-1 upregulation in primary human mammary fibroblast CAFs after FAP / LTBR bsAb stimulation.

[0251] Figure 10a Chemokinetic induction assay using tumor cell line RPMI-7951: CCL5 secretion in RPMI-7951 cells after FAP / LTBR bsAb stimulation.

[0252] Figure 10b Chemokinetic induction assay using tumor cell line RPMI-7951: CCL5 secretion in RPMI-7951 cells after FAP / LTBR bsAb stimulation.

[0253] Figure 11a HCC-1187:RPMI-7951 CCL-19 Readout: Transactivation in the presence of low FAP expression cell lines.

[0254] Figure 11b HCC-1187: RPMI-7951 CCL-5 readout: cis-activation in the presence of double-positive cells.

[0255] Figure 12a In a chemokine induction assay using tumor cell line 4T1 co-cultured with a cell line overexpressing FAP-positive CHO (single culture), the in vitro FAP / LTBR bispecific activity was replaced.

[0256] Figure 12b In a chemokine induction assay using tumor cell line 4T1 co-cultured with a cell line overexpressing FAP-positive CHO, the in vitro FAP / LTBR bispecific activity was replaced.

[0257] Figure 12c In a chemokine induction assay using tumor cell line 4T1 co-cultured with a cell line overexpressing FAP-positive CHO, the in vitro FAP / LTBR bispecific activity was replaced.

[0258] Figure 12d In a chemokine induction assay using tumor cell line 4T1 co-cultured with a cell line overexpressing FAP-positive CHO, the in vitro FAP / LTBR bispecific activity was replaced.

[0259] Figure 12e In a chemokine induction assay using tumor cell line 4T1 co-cultured with a cell line overexpressing FAP-positive CHO (single culture), the in vitro FAP / LTBR bispecific activity was replaced.

[0260] Figure 13In vitro activity of mouse FAP / LTBR activating antibody in primary mouse lung fibroblasts (Figure I, Repeat Experiment I).

[0261] Figure 14 In vitro activity of mouse FAP / LTBR activating antibody in primary mouse lung fibroblasts (Figure II, repeat experiment II).

[0262] Figure 15 In vivo efficacy of FAP / LTBR bispecific molecules in an orthotopic breast cancer model: tumor growth inhibition after treatment with FAP / LTBR bispecific molecules.

[0263] Figure 16 Treatment schedule for radiotherapy research on the MMTV-PyMT mBR9071 tumor model.

[0264] Figure 17 In vivo efficacy of FAP / LTBR bispecific replacement molecules in an orthotopic breast cancer model: HEV induction after treatment.

[0265] Figure 18 In vivo efficacy of FAP / LTBR bispecific replacement molecules in an in situ breast cancer model: treatment response in the in situ EMT6 model.

[0266] Figure 19a In vivo efficacy of FAP / LTBR bispecific replacement molecules in an orthotopic breast cancer model: increased B cells after treatment.

[0267] Figure 19b In vivo efficacy of FAP / LTBR bispecific replacement molecules in an orthotopic breast cancer model: HEV induction after treatment.

[0268] Figure 20 In vivo efficacy of FAP / LTBR bispecific alternative molecules in a subcutaneous lung cancer model: treatment response in a subcutaneous lung cancer model.

[0269] Figure 21a In vivo efficacy of FAP / LTBR bispecific replacement molecules in a subcutaneous lung cancer model: HEV induction after treatment.

[0270] Figure 21b In vivo efficacy of FAP / LTBR bispecific replacement molecules in a subcutaneous lung cancer model: CD4+ cell count increased after treatment.

[0271] Figure 21c In vivo efficacy of FAP / LTBR bispecific replacement molecules in a subcutaneous lung cancer model: CD8+ cell count increased after treatment.

[0272] Figure 22aDevelopment of TLS structure in a subcutaneous lung cancer model after combination therapy with PD-L1 and alternative FAP / LTBR BsAb. Representative images of T cell aggregates (CD8 staining).

[0273] Figure 22b Development of TLS structure in a subcutaneous lung cancer model after combination therapy with PD-L1 and alternative FAP / LTBR BsAb. Representative images of B-cell aggregates (CD19 staining).

[0274] Figure 23a Histological quantification of changes in tumor microenvironment (increased CD8) after combination therapy with PD-L1 and alternative FAP / LTBR BsAb.

[0275] Figure 23b Histological quantification of changes in tumor microenvironment (increased CD19) after combination therapy with PD-L1 and alternative FAP / LTBR BsAb.

[0276] Figure 24 In vivo efficacy of LTBR monospecific replacement molecule combined with radiotherapy in a subcutaneous colorectal cancer model: tumor growth inhibition of primary (irradiated) tumors after treatment.

[0277] Figure 25 In a subcutaneous colorectal cancer model, the in vivo efficacy of LTBR monospecific replacement molecule combined with radiotherapy: tumor growth inhibition of remote (unirradiated) tumors after treatment.

[0278] Figure 26a In vivo efficacy of LTBR monospecific replacement molecule combined with radiotherapy in a subcutaneous colorectal cancer model: changes in the tumor microenvironment (increased CD4) after fractionated radiotherapy and FAP / LTBR BsAb replacement therapy.

[0279] Figure 26b In vivo efficacy of LTBR monospecific replacement molecule combined with radiotherapy in a subcutaneous colorectal cancer model: changes in the tumor microenvironment (increase in CD8) after fractionated radiotherapy and replacement FAP / LTBR BsAb.

[0280] Figure 27a HepG2 NFKB activation-induced luciferase assay results showed that LTBR was activated by FAP / LTBR bispecific antibody in the presence of FAP (single culture).

[0281] Figure 27b HepG2 NFKB activation-induced luciferase assay results showed that LTBR was activated by FAP / LTBR bispecific antibody in the presence of FAP (co-culture).

[0282] Figure 27c HepG2 NFKB activation-induced luciferase assay results showed that LTBR was activated by FAP / LTBR bispecific antibody in the presence of FAP (single culture).

[0283] Figure 27d HepG2 NFKB activation-induced luciferase assay results showed that LTBR was activated by FAP / LTBR bispecific antibody in the presence of FAP (co-culture).

[0284] Figure 28a In vivo efficacy of FAP / LTBR bispecific alternative molecules in an orthotopic breast cancer model: therapeutic response in an orthotopic EMT6 model (tumor volume) combined with a cancer vaccine (peptide antigen + adjuvant).

[0285] Figure 28b In vivo efficacy of FAP / LTBR bispecific replacement molecule in an orthotopic breast cancer model: CD4+ increased after treatment.

[0286] Figure 28c In vivo efficacy of FAP / LTBR bispecific replacement molecule in an orthotopic breast cancer model: CD8+ increased after treatment.

[0287] Figure 29 In vivo efficacy of FAP / LTBR bispecific alternative molecules in an orthotopic breast cancer model: therapeutic response in combination with an orthotopic EMT6 model (survival probability) and a cancer vaccine (peptide antigen + adjuvant).

[0288] Figure 30 In vivo efficacy of FAP / LTBR bispecific alternative molecules in an orthotopic breast cancer model: re-excitation response of an orthotopic EMT6 model (tumor volume) combined with a cancer vaccine (peptide antigen + adjuvant). Invention Details

[0290] Binding agents

[0291] The binder can bind with affinity (appropriately expressed by KD value, Ka value, and binding rate (k)). on ) and / or dissociation rate (k offThe term "binding peptide" (as further described herein) indicates that the peptide binds to a target. Suitablely, the binding agent can enhance, reverse enhance, block, or neutralize a specific target. In one embodiment, the binding agent is a small molecule. Suitablely, the binding agent comprises or consists of a binding peptide (also referred to as a "binding domain"). A peptide is called a binding peptide when it contains a domain comprising one or more amino acid residues that form an antigen-binding site and is capable of binding to an epitope on a target with affinity. Hereinafter, "binding peptide" and "antigen-binding peptide" are used synonymously. A peptide binding LTBR is synonymous with an anti-LTBR peptide, and a peptide binding FAP is synonymous with an anti-FAP peptide. Binding peptides may include antibodies and fragments thereof, such as variable domains (all of which will be further described below), antibodies modified to include additional binding regions, and antibody mimics. Other binding peptides may also include DARPin (Binz et al., 2003), Affimer™, Fynomer™, Centyrin, Nanofitin®, and cyclic peptides.

[0292] The following examples describe various binding agents (including single-specific binding domains), which are referred to as “clones” and assigned specific clone numbers or identifiers.

[0293] Antibodies and their fragments

[0294] The binder is preferably an antibody or a binding fragment thereof. Such fragments can be included in unrelated antibodies (or their antigen-binding fragments), making the construct containing the binder a chimeric antibody (or its chimeric fragment). A conventional antibody or immunoglobulin (Ig) is a protein consisting of four polypeptide chains: two heavy chains (H or HC) and two light chains (L or LC), each divided into constant and variable regions. The variable region contains the heavy chain variable region paired with the light chain variable region. In this document, the heavy (H) chain variable region is referred to simply as the VH region, and the light (L) chain variable region is referred to simply as the VL region. These domains, their associated domains, and the domains derived from them are referred to herein as variable domains. The VH and VL regions (or “domains”) can be further subdivided into highly variable regions, referred to as “complementarity-determining regions” (“CDRs”), and regions scattered between the more conserved regions, referred to as “frame regions” (“FRs”). Frame regions and complementarity-determining regions have been precisely defined (Kabat et al., 1991). Since this invention relates to binders or peptides comprising complementarity-determining regions (CDRs) and selective frame regions (FRs), the determination of these CDRs / FRs is explained first. The determination of CDR regions within variable region sequences typically depends on the algorithm / method applied, such as Kabat, Chothia, Martin (enhanced Chothia), IMGT (ImMunoGeneTics Information System), or the AbM numbering scheme. See, for example, Kabat et al., 1991; Chothia and Lesk, 1987; the AbM definition is a compromise between Kabat and Chothia used in Oxford Molecular's AbM antibody modeling software, see also Martin and Allen 2007; Strohl and Strohl 2012, Chapter 3: Antibody Structure-Function Relationships, pp. 37-56; www.imgt.org / IMGTScientificChart / Numbering / IMGTnumbering.html.

[0295] Applying different methods to the same variable region sequence may produce different CDR amino acid sequences. These differences may lie in the length and / or delineation of the CDR sequence within the variable region sequence; see WO2022136649A1. Figure 14Figure 8 of WO2022136647A1 illustrates the parallel application of the IMGT and Kabat systems on the same sequence, and Figure 64 of WO2022175532A1 illustrates the parallel application of the Kabat, MacCallum, IMGT, AbM, and Chothia systems on the same sequence. Therefore, the CDRs contained in the binders or peptides of the present invention can be described as CDR sequences present in the variable region sequences characterized herein (more particularly, the anti-LTBR or anti-FAP antibody / immunoglobulin / VHH sequences characterized herein), or determined or depicted according to well-known methods, such as according to the Kabat, Chothia, Martin (enhanced Chothia), IMGT, or AbM numbering schemes or methods. The depiction of CDRs determines the depiction of frame regions (FRs). In the claims and clauses of this invention, as stated in the claims and clauses herein, the Kabat CDR definition system should be used unless otherwise specified.

[0296] In conventional antibodies, each VH and VL region consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDRs and FRs in the VH region are denoted as HCDR1, HCDR2, HCDR3, HFR1, HFR2, HFR3, and HFR4, respectively. The CDRs and FRs in the VL region are denoted as LCDR1, LCDR2, LCDR3, LFR1, LFR2, LFR3, and LFR4, respectively. A conventional antibody tetramer consisting of two heavy immunoglobulin chains and two light immunoglobulin chains is formed by the heavy and light immunoglobulin chains being linked together by, for example, disulfide bonds, and the heavy chains are similarly linked. The constant region of the heavy chain includes three domains CH1, CH2, and CH3, while the constant region of the light chain consists of one domain CL. The variable domains of the heavy and light chains are binding domains that interact with the antigen. Antibody constant regions typically mediate the binding of antibodies to host tissues or factors, including various cells in the immune system (such as effector cells) and the first component (C1q) of the classical complement system.

[0297] Conventional antibodies are homodimers consisting of two identical heavy chains (HC) and two identical light chains (LC). Figure 7 (As shown in c and 7d). Antibodies containing two different heavy chains (HC1 and HC2) and two identical light chains (LC) can also be produced (e.g. Figure 7 (as shown in a and 7b).

[0298] Another exception to the structure of conventional antibodies has been found in the serum of camels. In addition to regular antibodies, these sera contain specialized IgG antibodies. These IgG antibodies, known as heavy chain antibodies (HCAbs), lack the light (L) chain polypeptide and the first constant region (CH1). The N-terminal region of the heavy chain (H chain) of the homodimeric protein contains a specialized immunoglobulin chain variable domain, called VHH, for binding to its homologous antigens (Muyldermans 2013; Hamers-Casterman et al 1993).

[0299] The term "antibody" includes any substantially intact antibody protein containing at least one antibody variable domain (containing at least one antigen-binding site (ABS)). Antibodies include, but are not limited to, immunoglobulins of the types IgA, IgG, IgE, IgD, and IgM (and their subtypes, such as IgG1). The overall structure of immunoglobulin G (IgG) antibodies, assembled from two identical heavy chain (H) and two identical light chain (L) polypeptides, has been well established and is highly conserved in mammals (Padlan 1994).

[0300] As used herein, antibody fragments (also referred to as “antibody fragments,” “antibody-binding fragments,” “immunoglobulin fragments,” “antigen-binding fragments,” or “antigen-binding polypeptides”) refer to the portion of an antibody that binds to a target (e.g., a molecule in which one or more immunoglobulin chains are not full-length but can bind to a target). Examples of binding fragments covered by the term “antibody fragment” include: Fab fragment (a monovalent fragment composed of the VL, VH, CL, and CH1 domains); F(ab')2 fragment (a divalent fragment formed by two Fab fragments connected by disulfide bonds in the hinge region); Fd fragment (composed of VH and CH1 domains); Fv fragment (variable domain or variable region, consisting of the VL and VH domains of the antibody arm, where the VL and VH domains together form the antigen binding site); Single-chain variable fragments scFv (composed of VL and VH domains, which may be linked by synthetic linkers (e.g., peptides) using recombination methods, enabling them to be made into a single protein chain in which the VL and VH regions pair to form a monovalent molecule); A VH (heavy-chain variable field); A VL (light chain variable domain); A single-domain antibody (dAb, composed of VH or VL domains); A minibody (consisting of a pair of scFv fragments linked by a CH3 domain); A diabody (composed of a non-covalent dimer of scFv fragments, which are formed by linking the VH domain of one antibody to the VL domain of another antibody via a peptide linker); A VHH (a heavy chain variable domain as described in detail above).

[0301] The antibody or a fragment thereof may be a human antibody or derived from a human antibody. "Human antibody" refers to an antibody having variable and constant regions derived from human germline immunoglobulin sequences. Administration of such human antibodies to a human individual does not produce a cross-species antibody response against the major amino acids contained in the body (e.g., a reaction known as HAMA – human anti-mouse antibody). Such human antibodies may include amino acid residues encoded by non-human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutations, or mutations introduced by somatic mutations), such as in the CDR, and especially CDR3. However, this term does not include antibodies that graft germline CDR sequences derived from other mammals (such as mice) onto human framework sequences. Human antibodies prepared, expressed, established, or isolated through recombinant methods, such as antibodies expressed by transfection into host cells using a recombinant expression vector, antibodies isolated from a recombinant human antibody library, antibodies isolated from transgenic animals (e.g., mice) containing human immunoglobulin genes, or antibodies prepared, expressed, established, or isolated by any other method involving splicing human immunoglobulin gene sequences with other DNA sequences, can also be referred to as "recombinant human antibodies".

[0302] The process of replacing at least one amino acid residue in the variable domain framework region of a non-human immunoglobulin with a corresponding residue from a human variable domain is called "humanization." Humanization of the variable domain may reduce its immunogenicity in humans.

[0303] Antibodies contain a hinge region, and certain constructs containing antibody domains and antibody fragments also contain a hinge region. A naturally occurring hinge region is a flexible amino acid sequence of the central portion of the heavy chain of IgG and IgA immunoglobulins, which connects the two chains via disulfide bonds. As used herein, a hinge region includes naturally occurring hinge regions and variants thereof, wherein these variants appropriately maintain sufficient flexibility and rigidity to maintain the integrity of the antibody, fragment, or construct while maintaining its binding capacity. A naturally occurring hinge region may, for example, contain or consist of the polypeptide sequence of SEQ ID NO: 204. A variant may be a truncated version of a naturally occurring hinge region (“truncated hinge region”), for example, a hinge region truncated by 5 amino acids relative to a naturally occurring hinge region, or most preferably a truncated hinge region containing or consisting of SEQ ID NO: 203. In one embodiment, the hinge region consists of 10 to 80 amino acids, for example 20 to 70 amino acids, for example 30 to 60 amino acids, for example 40 to 50 amino acids.

[0304] The construct suitably comprises an antibody or antibody fragment. In one embodiment, the antibody fragment comprises scFv, Fv, Fab, Fab', F(ab')2, a variable domain (e.g., VH, VL, VNAR, or VHH), a biantibody, or a microantibody. If the construct is an antibody or antibody fragment, the binding region of that antibody or antibody fragment (e.g., the Fab region of the antibody or antibody fragment, or other binding domains introduced synthetically, such as VHH) provides the FAP and LTBR binding agents in the construct of the present invention.

[0305] Most suitably, the construct is an antibody (i.e., a full-length antibody). The antibody of the present invention can be of any class, such as IgG, IgA, IgM, IgE, IgD, or a subclass thereof, and may contain a κ or λ light chain. In one embodiment, the antibody is an IgG antibody, for example, at least one subclass of IgG1, IgG2, IgG3, or IgG4. In one embodiment, the antibody is IgG1, most preferably human IgG1. In another embodiment, the antibody may be a modified form to impart desired properties, such as an IgG form, where its Fc region is mutated to reduce effector function, prolong half-life, alter ADCC, or improve hinge stability. Such modifications are well known in the art, and exemplary embodiments are described herein. Suitably, the antibody contains PGLALA substitutions in the Fc region (i.e., Pro329Gly, Leu234Ala, and Leu235Ala substitutions, Schlothauer et al., 2016). Suitably, the Fc region of the antibody contains 329Gly, 234Ala, and 235Ala. Alternatively, the Fc region of the antibody may contain LALA substitutions (i.e., Leu234Ala and Leu235Ala substitutions, see Schlothauer et al., 2016). Suitably, the Fc region of the antibody may contain 234Ala and 235Ala. In another embodiment, the antibody may contain a STR-silenced Fc region (i.e., G236R binds to the aforementioned LALA substitutions, see Wilkinson et al., 2021). Thus, in one embodiment, the Fc region may contain 234Ala, 235Ala, and 236Arg. In another embodiment, the Fc region may be non-glycosylated. These same Fc region modifications can be applied to any construct containing an Fc.

[0306] In one embodiment, the antibody is multispecific, such as bispecific, and multivalent, such as bivalent. Suitable constructs of these and other forms are further detailed below.

[0307] In one particular embodiment, the construct comprises, consists primarily of, or consists only of an antibody or a pair of scFvs. Most preferably, the construct comprises an antibody.

[0308] In another embodiment, the construct comprises or consists only of an antibody and VHH, wherein the VHH replaces one of the Fab regions (e.g., Figure 7 (as shown in a), the VHH is a binding polypeptide that binds to LTBR, ​​and the paired VH and VL in the remaining Fab region are binding polypeptides that bind to FAP.

[0309] In another embodiment, the construct comprises an antibody and a VHH or thereof, wherein the VHH is fused to the Fc region (e.g., the CH3 domain) of the antibody (e.g., as shown in the image). Figure 7(As shown in b), VHH is a binding polypeptide that binds to LTBR, ​​while the paired VH and VL in each of the two Fab regions are binding polypeptides that bind to FAP. Suitablely, another VHH is fused to the Fc region (e.g., the second CH3 domain), where the other VHH is a binding polypeptide that binds to LTBR (e.g., ...). Figure 7 (as shown in c).

[0310] In one particular embodiment, the construct comprises or consists of an antibody and an scFv, wherein the scFv lineage is fused to the Fc region (e.g., the CH3 domain) of the antibody, the scFv is a binding polypeptide that binds to the LTBR, ​​and the paired VH and VL of each of the two Fab regions are binding polypeptides that bind to the FAP. Suitablely, another scFv lineage is fused to the Fc region (e.g., a second CH3 domain), and the other scFv is a binding polypeptide that binds to the LTBR (e.g., ...). Figure 7 (as shown in d).

[0311] In one embodiment, the scFv may fuse to the C-terminus of the Fc. Suitablely, a peptide linker may be used, such as a linker containing glycine and serine residues, for example, a G4S linker (SEQ ID NO: 3) or a (G4S)4 linker (SEQ ID NO: 4). Suitablely, the disulfide bond may be located at the VH44:VL100 position (Weatherill et al., 2012). In one embodiment, the linker does not contain an Ig domain. In one embodiment, the linker is a linear peptide. In one embodiment, the linker is a hinge region. Most suitablely, the scFv fuses to the CH3 domain via a linear peptide linker.

[0312] Specificity, affinity, and potency

[0313] "Specificity" refers to the number of different types of antigens or antigenic determinants that a particular antibody or its fragment can bind to. Antibody specificity refers to the antibody's ability to recognize a specific antigen as a unique molecular entity and distinguish it from other antigens. The term "specific binding" of an antibody to an antigen or epitope is widely understood in the field. A molecule is said to exhibit "specific binding" when the frequency, rate, duration, and / or affinity of its reaction with a specific target antigen or epitope is significantly higher than its reaction with other targets. If the binding affinity, affinity, ease of binding, and / or longer binding time of an antibody (or its fragment) to a target antigen or epitope is significantly higher than its binding to other substances, it is called "specific binding." Specific binding can also be considered if the binding of an antibody (or its fragment) to a target is statistically significantly higher than its binding to unrelated binders. The specific binding of an antibody or fragment thereof to an antigen or antigenic determinant can be determined by any known suitable method, including but not limited to Scatchard analysis and / or competitive binding assays such as radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competitive assays, as well as methods such as equilibrium dialysis, equilibrium binding, gel filtration, ELISA, or spectroscopy (e.g., using fluorescence assays) and variations thereof known in the art.

[0314] In one embodiment, "specific binding" refers to a binding affinity constant (KD) of 10 between the target and the substance. -8 M or lower, such as 10 -9 M or lower, while the binding affinity constant (KD) of any other solid target is 10. -7 M or higher, such as 10 -6 M or higher. More appropriately, the combination of any other entity cannot be measured.

[0315] Affinity is expressed as the equilibrium constant (KD) for the dissociation of the antigen and the antigen-binding polypeptide. It measures the strength of the binding between the antigenic determinant and the antigen-binding site on the antibody (or its fragments): the smaller the KD value, the stronger the binding between the antigenic determinant and the antigen-binding polypeptide. Alternatively, affinity can also be expressed as the affinity constant (KA), i.e., 1 / KD. The method for determining affinity depends on the specific antigen; any KD value below 10... -6 M is considered to be bonded. A suitable configuration should be able to bond at 10 -6 A target with a dissociation constant (M) or lower (KD) is more suitable, especially one with a dissociation constant of 10. -7 M or lower, 10 is more suitable -8 M or lower, 10 is the most suitable. -9 M or lower.

[0316] Suitablely, the KD value of the peptides of the present invention can be determined using surface plasmon resonance (SPR) or biolayer interferometry (“BLI”, for example, using an Octet instrument, or, for example, the method described in Example 4).

[0317] Affinity is a measure of the strength of binding between a peptide, antibody, or fragment thereof and a related antigen. Affinity is related to the affinity between the antigenic determinant and the antigen-binding site on the antibody, as well as the number of related binding sites on the antibody.

[0318] "Potential" refers to the amount of a therapeutic agent required to produce a specific strength of effect, used to measure its activity. Highly potent therapeutic agents elicit a stronger response at low concentrations, while less potent agents elicit a weaker response at low concentrations. Potential is a function of affinity and efficacy. Efficacy refers to the ability of a therapeutic agent to produce a biological response after binding to a target and the quantitative strength of that response. The half-maximal effective concentration (EC50) is the concentration at which a therapeutic agent elicits a response between baseline and maximum at a given exposure time. Therapeutic agents may cause inhibitory or stimulatory effects. This metric is often used to measure potency.

[0319] LTBR

[0320] Lymphotoxin beta receptor (LTBR), also known as tumor necrosis factor receptor superfamily member 3 (TNFRSF3), is a cell surface receptor involved in apoptosis and cytokine release. It is a member of the tumor necrosis factor receptor superfamily.

[0321] The construct of the present invention comprises an LTBR binder. LTBR binders have been disclosed in the prior art, such as anti-LTBR binding peptides BHA10 (WO2004002431, comprising the heavy and light chain variable regions of SEQ ID NO: 1 and 2, respectively) and huCBE11 (WO2004002431).

[0322] Suitablely, one or more LTBR binders contain or consist of a variable domain that binds to the LTBR, ​​such as VH, VL, VNAR, or VHH, and more suitably a heavy chain variable domain, such as VHH.

[0323] LTBRs are suitably located on the surface of stromal cells, fibroblasts, endothelial cells, and / or cancer cells. Suitably, LTBRs are located on the surface of a cell that also contains FAPs.

[0324] LTBR binders appropriately bind to the extracellular domain of LTBR.

[0325] In one embodiment, LTBR is a polypeptide that has at least 50% sequence identity with SEQ ID NO: 9 (full-length human LTBR), for example, at least 60% identity, for example, at least 70% identity, for example, at least 80% identity, for example, at least 90% identity, for example, at least 95% identity. Suitably, LTBR comprises or is composed of SEQ ID NO: 9 (full-length human LTBR).

[0326] Suitably, LTBR is the extracellular domain of human LTBR, ​​i.e., an LTBR binder binds to the extracellular domain of human LTBR. In one embodiment, LTBR is a polypeptide that has at least 50% sequence identity with SEQ ID NO: 10 (extracellular domain of human LTBR), for example, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity. Suitably, LTBR is a polypeptide comprising or composed of SEQ ID NO: 10 (extracellular domain of human LTBR).

[0327] In one embodiment, LTBR is a polypeptide having at least 50% sequence identity, such as at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity, with SEQ ID NO: 11 (full-length cynomolgus monkey LTBR). Suitably, LTBR comprises or is composed of SEQ ID NO: 11 (full-length cynomolgus monkey LTBR).

[0328] Suitably, the LTBR is the extracellular domain of the cynomolgus monkey LTBR, ​​i.e., the LTBR binder binds to the extracellular domain of the cynomolgus monkey LTBR. In one embodiment, the LTBR is a polypeptide having at least 50% sequence identity, such as at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity, with SEQ ID NO: 12 (cynomolgus monkey LTBR extracellular domain). Suitably, the LTBR is a polypeptide comprising or composed of SEQ ID NO: 12 (cynomolgus monkey LTBR extracellular domain).

[0329] In one embodiment, LTBR is a polypeptide having at least 50% sequence identity, such as at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity, with SEQ ID NO: 13 (full-length mouse LTBR). Suitably, LTBR comprises or is composed of SEQ ID NO: 13 (full-length mouse LTBR).

[0330] Suitably, the LTBR is the extracellular domain of a mouse LTBR, ​​meaning the LTBR binder binds to the extracellular domain of a mouse LTBR. In one embodiment, the LTBR is a polypeptide having at least 50% sequence identity, such as at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity, with SEQ ID NO: 14 (extracellular domain of mouse LTBR). In one embodiment, the LTBR is a polypeptide comprising or composed of SEQ ID NO: 14 (extracellular region of mouse LTBR).

[0331] Suitablely, one or all LTBR binders have an EC50 value of 9.0E-09 M or lower, such as 8.0E-09 M or lower, such as 7.0E-09 M or lower, such as 6.0E-09 M or lower, such as 5.0E-09 M or lower, such as 4.0E-09 M or lower, such as 3.0E-09 M or lower, such as 2.0E-09 M or lower, such as 1.0E-09 M or lower, such as 9.0E-10 M or lower, such as 8.0E-10 M or lower, such as 7.0E-10 M or lower, such as 6.0E-10 M or lower, such as 5.0E-10 M or lower, such as 4.0E-10 M or lower, such as 3.0E-10 M or lower, such as 2.0E-10 M or lower, such as 1.0E-10 M or lower, such as 9.0E-11 M or lower, for example, 8.0E-11 M or lower, for example, 7.0E-11 M or lower, for example, 6.0E-11 M or lower, for example, 5.0E-11 M or lower, for example, 4.0E-11 M or lower, for example, 3.0E-11 M or lower, for example, 2.0E-11 M or lower, for example, 1.0E-11 M or lower, for example, 9.0E-12 M or lower, for example, 8.0E-12 M or lower, for example, 7.0E-12 M or lower, for example, 6.0E-12 M or lower, for example, 5.0E-12 M or lower, for example, 4.0E-12 M or lower, for example, 3.0E-12 M or lower, for example, 2.0E-12 M or lower, for example, 1.0E-12 M or lower. Appropriately, the EC50 value is determined by chemokine induction assay, for example, using an ELISA. More appropriately, the EC50 value was determined by the experiment shown in any of the examples.

[0332] Appropriately, one or all LTBR binders have an EC50 value or lower as described for any LTBR binder listed in the example table.

[0333] Appropriately, the LTBR binder specifically binds to LTBR, ​​meaning that the binder does not bind significantly to targets other than human LTBR, ​​such as targets other than human, cynomolgus monkey, or mouse LTBR.

[0334] In one embodiment, the LTBR binder is an LTBR ligand, namely LIGHT, more appropriately human LIGHT.

[0335] In one embodiment, the LTBR binder is not an LTBR ligand, human light, or more appropriately, the LTBR binder is not light.

[0336] Suitablely, LIGHT is a polypeptide comprising, substantially consisting of, or consisting of a sequence having at least 70%, for example at least 80%, for example at least 90%, for example at least 99%, for example at least 100% identity with SEQ ID NO: 21 or SEQ ID NO: 22.

[0337] The constructs of the present invention can be coupled with LTBR with the following affinities: 1.0E-08 M or lower, such as 9.0E-09 M or lower, such as 7.0E-09 M or lower, such as 6.7E-09 M or lower, such as 5.0E-09 M or lower, such as 3.0E-09 M or lower, such as 2.0E-09 M or lower, such as 1.0E-09 M or lower.

[0338] Suitable, one or all LTBR binders are bound to LTBR with the following affinity (KD): 20 nM or lower, for example 15 nM or lower, for example 10 nM or lower, for example 9 nM or lower, for example 8 nM or lower, for example 7 nM or lower, for example 6 nM or lower, for example 5 nM or lower, for example 4 nM or lower, for example 3 nM or lower, for example 2 nM or lower, for example 1 nM or lower. Suitable, the KD value is determined using the determination shown in Example 4.

[0339] Suitablely, the LTBR binder is a promoter (i.e., an activator), a de-activator, or a neutralizer of LTBR. In one embodiment, the LTBR binder is an agonist of LTBR. Suitablely, the agonist activates such NFKB signaling pathways. If the LTBR binder is an agonist of LTBR, ​​then the binder (a) suitably induces LTBR aggregation, and more suitably (b) activates classical NFKB pathways (suitably leading to the expression of adhesion molecules such as ICAMs, e.g., ICAM-1, VCAM-1, and / or MAdCAM-1) and / or activates alternative NFKB pathways (suitably leading to the expression of chemokines, e.g., CCL5, CCL19, CCL21, and / or CXCL13).

[0340] Suitablely, the LTBR binder is an LTBR agonist, wherein the LTBR binder has an EC50 value of 9.0E-09 M or lower, for example 1.0E-08 M or lower, for example 8.0E-09 M or lower, for example 7.0E-09 M or lower, for example 6.0E-09 M or lower, for example 5.0E-09 M or lower, for example 4.0E-09 M or lower, for example 3.0E-09 M or lower, for example 2.0E-09 M or lower, for example 1.0E-09 M or lower, for example 9.0E-10 M or lower, for example 8.0E-10 M or lower, for example 7.0E-10 M or lower, for example 6.0E-10 M or lower, for example 5.0E-10 M or lower, for example 4.0E-10 M or lower, for example 3.0E-10 M or lower, for example 2.0E-10 M or lower, such as 1.0E-10 M or lower, such as 9.0E-11 M or lower, such as 8.0E-11 M or lower, such as 7.0E-11 M or lower, such as 6.0E-11 M or lower, such as 5.0E-11 M or lower, such as 4.0E-11 M or lower, such as 3.0E-11 M or lower, such as 2.0E-11 M or lower, such as 1.0E-11 M or lower, such as 9.0E-12 M or lower, such as 8.0E-12 M or lower, such as 7.0E-12 M or lower, such as 6.0E-12 M or lower, such as 5.0E-12 M or lower, such as 4.0E-12 M or lower, such as 3.0E-12 M or lower, such as 2.0E-12 M or lower, such as 1.0E-12 M or lower. Alternatively, the LTBR binder may have an EC50 value that is the same as or lower than that of any LTBR binder listed in the example table. Appropriately, the EC50 value is established by a chemically induced assay, such as using an ELISA. More appropriately, the EC50 value is established using any of the assay methods listed in the examples.

[0341] In one embodiment, the construct binds to mouse LTBR. In another embodiment, the construct binds to cynomolgus monkey LTBR. In yet another embodiment, the construct binds to human LTBR.

[0342] One, more, or all LTBR binders may be variable regions (e.g., heavy chain variable regions, such as VHH) that bind LTBR, ​​as appropriately defined below.

[0343] In one embodiment, the LTBR binder or peptide comprises at least one CDR, wherein the CDR is HCDR1, HCDR2, or HCDR3 contained in the heavy chain variable region of SEQ ID NO: 23-26 or 159-162. Suitably, such binders or peptides comprise at least one of these HCDR1 and HCDR2, one of these HCDR1 and HCDR3, or one of these HCDR2 and HCDR3. More suitably, such binders or peptides comprise one of these HCDR1, HCDR2, and HCDR3. More suitably, HCDR1, HCDR2, and HCDR3 are described according to the Kabat, Chothia, Martin, IMGT, or AbM methods. More appropriately, HCDR1 is selected from SEQ ID NO: 57, 60, 63, 66, 163, 166, 169 or 172 as defined by the Kabat method; HCDR2 is selected from SEQ ID NO: 58, 61, 64, 67, 164, 167, 170, 173, 205 or 206 as defined by the Kabat method; and / or HCDR3 is selected from SEQ ID NO: 59, 62, 65, 68, 165, 168, 171, 174, 207 or 208 as defined by the Kabat method.

[0344] In one embodiment, the variable domain binding to the LTBR comprises or consists of three heavy chain complementarity-determining regions (HCDR1-HCDR3) and four frame regions (FR1-FR4), wherein HCDR1 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 57, 60, 63, 66, 163, 166, 169, or 172 (e.g., 57, 60, 63, or 66), or wherein HCDR1 comprises or consists of a polypeptide sequence having 3, 2, or 1 amino acid sequence differences from SEQ ID NO: 57, 60, 63, 66, 163, 166, 169, or 172 (e.g., 57, 60, 63, or 66); HCDR2 comprises or consists of a polypeptide sequence having 3, 2, or 1 amino acid sequence differences from SEQ ID NO: 58, 61, 64, 67, 164, 167, 170, 173, 205, or 206 (e.g., 58, 61, 64, or 67) having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity, or HCDR2 comprising or consisting of a polypeptide sequence differing from SEQ ID NO: 58, 61, 64, 67, 164, 167, 170, 173, 205, or 206 (e.g., 58, 61, 64, or 67) by 3, 2, or 1 amino acid; and HCDR3 comprising a polypeptide sequence differing from SEQ ID NO: 59, 62, 65, 68, 165, 168, 171, 174, 207, or 208 (e.g., 59, 62, 65, or 68) having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity, or HCDR3 comprising or consisting of a polypeptide sequence that differs from SEQ ID NO: 59, 62, 65, 68, 165, 168, 171, 174, 207, or 208 (e.g., 59, 62, 65, or 68) by 3, 2, or 1 amino acid. More appropriately, HCDR1 comprises or consists of the polypeptide sequence of SEQ ID NO: 57, 60, 63, 66, 163, 166, 169, or 172 (e.g., 57, 60, 63, or 66), HCDR2 comprises or consists of the polypeptide sequence of SEQ ID NO: 58, 61, 64, 67, 164, 167, 170, 173, 205, or 206 (e.g., 58, 61, 64, or 67), and HCDR3 comprises or consists of the polypeptide sequence of SEQ ID NO: 59, 62, 65, 68, 165, 168, 171, 174, 207, or 208 (e.g., 59, 62, 65, or 68). In particular, these CDR polypeptide sequences are determined according to or by the Kabat method.

[0345] In one embodiment, the variable domain bound to the LTBR comprises the three heavy chain complementarity-determining region amino acid sequences (HCDR1-HCDR3) of the variable domain amino acid sequence of SEQ ID NO: 23, 24, 25, 26, 159, 160, 161 or 162 (e.g., 23, 24, 25 or 26).

[0346] In one embodiment, the variable domain binding to the LTBR comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher sequence identity with SEQ ID NO: ID NO: 23, 24, 25, 26, 159, 160, 161, or 162 (e.g., 23, 24, 25, or 26). More suitably, the variable domain binding to the LTBR comprises or is composed of a polypeptide sequence of SEQ ID NO: ID NO: 23, 24, 25, 26, 159, 160, 161, or 162 (e.g., 23, 24, 25, or 26). In a further embodiment, the variation of the polypeptide sequence is located in one or more of the frame regions FR1 to FR4 contained in SEQ ID NO: ID NO: 23, 24, 25, 26, 159, 160, 161 or 162 (e.g., 23, 24, 25 or 26). Alternatively, the variation of the polypeptide sequence is located in one or more of the frame regions FR1 to FR4 contained in SEQ ID NO: ID NO: 23, 24, 25, 26, 159, 160, 161 or 162 (e.g., 23, 24, 25 or 26), and the CDR polypeptide sequence contained in SEQ ID NO: ID NO: 23, 24, 25, 26, 159, 160, 161 or 162 (e.g., 23, 24, 25 or 26) is the HCDR1 to HCDR3 polypeptide sequences as described above. In one embodiment, one, more, or all of the LTBR binders comprise or consist of paired heavy chain variable domains and light chain variable domains bound to the LTBR (e.g., scFv), further defined as appropriate.

[0347] In one embodiment, the heavy chain variable domain binding to the LTBR comprises or consists of three complementarity-determining regions (HCDR1-HCDR3) and four frame regions (HFR1-HFR4), wherein HCDR1 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 69, 75, 81, 87, 93, 99, 105, or 111; HCDR2 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 70, 76, 82, 88, 94, 100, 106, or 112; and HCDR3 comprises a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 71, 77, 83, 89, 95, 101, or 107. Or 113, a polypeptide sequence having or consisting of 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity, and the light chain variable domain binding to LTBR comprising or consisting of three complementarity-determining regions (LCDR1-LCDR3) and four frame regions (LFR1-LFR4), wherein LCDR1 comprises or consists of a polypeptide sequence having or consisting of 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 72, 78, 84, 90, 96, 102, 108, or 114, and LCDR2 comprises a polypeptide sequence having or consisting of SEQ ID NO: 73, 79, 85, 91, 97, 103, or 109. Or 115 has a polypeptide sequence or is composed of a sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity, and LCDR3 contains or is composed of a polypeptide sequence or is composed of a sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 74, 80, 86, 92, 98, 104, 110, or 116.More appropriately, HCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 69, 75, 81, 87, 93, 99, 105, or 111; HCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 70, 76, 82, 88, 94, 100, 106, or 112; and HCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 71, 77, 83, 89, 95, 101, 107, or 113; LCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 72, 78, 84, 90, 96, 102, 108, or 114; LCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 73, 79, 85, 91, 97, 103, 109, or 115; and LCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 69, 75, 81, 87, 93, 99, 105, or 115. The polypeptide sequence of 74, 80, 86, 92, 98, 104, 110 or 116 or composed of such sequences.

[0348] In one embodiment, the heavy chain variable domain bound to the LTBR comprises the three heavy chain complementarity-determining region amino acid sequences (HCDR1 to HCDR3) of the heavy chain variable domain amino acid sequence of SEQ ID NO: 27, 29, 31, 33, 35, 37, 39 or 41, while the light chain variable domain bound to the LTBR comprises the three light chain complementarity-determining region amino acid sequences (LCDR1 to LCDR3) of the light chain variable domain amino acid sequence of SEQ ID NO: 28, 30, 32, 34, 36, 38, 40 or 42.

[0349] In one embodiment, the heavy chain variable domain bound to the LTBR comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher sequence identity with SEQ ID NO: 27, 29, 31, 33, 35, 37, 39, or 41, while the light chain variable domain bound to the LTBR comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher sequence identity with SEQ ID NO: 28, 30, 32, 34, 36, 38, 40, or 42, respectively. More appropriately, the heavy chain variable domain bound to the LTBR contains or is composed of the amino acid sequence of SEQ ID NO: 27, 29, 31, 33, 35, 37, 39 or 41, while the light chain variable domain bound to the LTBR contains or is composed of the amino acid sequence of SEQ ID NO: 28, 30, 32, 34, 36, 38, 40 or 42.

[0350] Preferably, HCDR (and LCDR) are provided in the heavy chain variable domain (and the light chain variable domain combined with the LTBR) in the same combination as shown in Tables 17A / B. For example, it is preferred to use the combination of HCDR1-3 of SEQ ID NO:: 57, 58 and 59 in VHH (as listed in clone 1.5), and it is preferred to use the combination of (a) HCDR1-3 of SEQ ID NO:: 69, 70 and 71 and (b) LCDR1-3 of SEQ ID NO:: 72, 73 and 74 (as listed in clone 2.1) in, for example, scFv. The same principle also applies to the combination of heavy chain and light chain variable regions, as shown in Table 16.

[0351] When multiple LTBR binders are used in the constructs of this invention, these binders may be (a) the same or (b) different LTBR binders. If not simultaneously, these LTBR binders are suitably and independently selected from the LTBR binders described herein, such as those detailed above.

[0352] If the construct is an antibody, then in one embodiment, one or all LTBR binders are binding domains (e.g., VHH or scFv, more preferably VHH) linked to a constant region (e.g., CH3 region).

[0353] FAP

[0354] Fibroblast activation protein α (FAP-α, FAP), also known as proline endopeptidase FAP, is an enzyme encoded by the FAP gene in humans. FAP is a membrane-bound gelatinase with a molecular weight of 170 kDa. FAP is highly expressed in cancer tissues of various cancer types, especially on the surface of cancer-associated fibroblasts (CAFs) in the cancer stroma and on the surface of cancer cells (Zboralski et al., 2022; Zhao et al., 2022).

[0355] The construct of the present invention comprises an FAP binder. FAP binders have been disclosed in the prior art, for example, anti-FAP binding peptide 28H1 (US10577429B2, which comprises heavy chain and light chain variable regions, respectively, SEQ ID NO: 7 and 8).

[0356] FAP binders can achieve localization (e.g., targeting tumors) and co-localize with LTBR binders or accumulate LTBR in bispecific constructs; i.e., conditional activation. This conditional activation should activate LTBR-positive cells in the presence of FAP in the tumor, thereby reducing toxicity (while not activating in tissues where FAP is not expressed). FAP is suitably located on the surface of the stroma and / or cancer cells. FAP binders are suitably localized on the cell surface, wherein the cell also contains LTBR on its surface.

[0357] FAP binders appropriately bind to the extracellular domain of FAP.

[0358] In one embodiment, FAP is a polypeptide having at least 50% identity, such as at least 60% identity, such as at least 70% identity, such as at least 80% identity, such as at least 90% identity, or such as at least 95% identity with SEQ ID NO: 15 (full-length human FAP). Suitably, FAP comprises or is composed of SEQ ID NO: 15 (full-length human FAP).

[0359] Suitablely, FAP is the extracellular domain of human FAP, i.e., FAP binder binds to the extracellular domain of human FAP. In one embodiment, FAP is a polypeptide having at least 50% identity with SEQ ID NO: 16 (extracellular domain of human FAP), for example, at least 60% identity, for example, at least 70% identity, for example, at least 80% identity, for example, at least 90% identity, for example, at least 95% identity. Suitablely, FAP comprises or consists of SEQ ID NO: 16.

[0360] In one embodiment, FAP is a polypeptide that shares at least 50% identity with SEQ ID NO: 17 (full-length cynomolgus monkey FAP), for example, at least 60% identity, for example, at least 70% identity, for example, at least 80% identity, for example, at least 90% identity, for example, at least 95% identity. Suitably, FAP comprises or is composed of SEQ ID NO: 17 (full-length cynomolgus monkey FAP).

[0361] Suitablely, FAP is the extracellular domain of cynomolgus monkey FAP, i.e., the FAP binder binds to the extracellular domain of cynomolgus monkey FAP. In one embodiment, FAP is a polypeptide that has at least 50% identity with SEQ ID NO: 18 (cynomolgus monkey FAP extracellular domain), for example, at least 60% identity, for example, at least 70% identity, for example, at least 80% identity, for example, at least 90% identity, for example, at least 95% identity. Suitablely, FAP comprises or is composed of SEQ ID NO: 18.

[0362] In one embodiment, FAP is a polypeptide that shares at least 50% identity with SEQ ID NO: 19 (the full-length sequence of mouse FAP), for example, at least 60% identity, for example, at least 70% identity, for example, at least 80% identity, for example, at least 90% identity, for example, at least 95% identity. Suitably, FAP comprises or is composed of SEQ ID NO: 19.

[0363] Suitablely, FAP is the extracellular domain of mouse FAP, i.e., the FAP binder binds to the extracellular domain of mouse FAP. In one embodiment, FAP is a polypeptide that has at least 50% identity with SEQ ID NO: 20 (mouse FAP extracellular domain), for example, at least 60% identity, for example, at least 70% identity, for example, at least 80% identity, for example, at least 90% identity, for example, at least 95% identity. Suitablely, FAP is a polypeptide comprising or composed of SEQ ID NO: 20 (mouse FAP extracellular domain).

[0364] Suitablely, one or all FAP binders have an EC50 value of 9.0E-09 M or lower, such as 8.0E-09 M or lower, such as 7.0E-09 M or lower, such as 6.0E-09 M or lower, such as 5.0E-09 M or lower, such as 4.0E-09 M or lower, such as 3.0E-09 M or lower, such as 2.0E-09 M or lower, such as 1.0E-09 M or lower, such as 9.0E-10 M or lower, such as 8.0E-10 M or lower, such as 7.0E-10 M or lower, such as 6.0E-10 M or lower, such as 5.0E-10 M or lower, such as 4.0E-10 M or lower, such as 3.0E-10 M or lower, such as 2.0E-10 M or lower, such as 1.0E-10 M or lower, such as 9.0E-11 M. M or lower, such as 8.0E-11 M or lower, such as 7.0E-11 M or lower, such as 6.0E-11 M or lower, such as 5.0E-11 M or lower, such as 4.0E-11 M or lower, such as 3.0E-11 M or lower, such as 2.0E-11 M or lower, such as 1.0E-11 M or lower, such as 9.0E-12 M or lower, such as 8.0E-12 M or lower, such as 7.0E-12 M or lower, such as 6.0E-12 M or lower, such as 5.0E-12 M or lower, such as 4.0E-12 M or lower, such as 3.0E-12 M or lower, such as 2.0E-12 M or lower, such as 1.0E-12 M or lower.

[0365] Appropriately, EC50 is determined using one of the assays listed in the Examples. Appropriately, the EC50 value of one or all FAP binders should be equal to or lower than the EC50 value of any clone specified in the Examples table.

[0366] Appropriately, the FAP binder specifically binds to FAP, meaning that the binder does not significantly bind to targets other than human FAP, such as targets other than human, cynomolgus monkey, or mouse FAP.

[0367] The constructs of the present invention bind to FAP with an affinity of 9.0E-10 M or lower, such as 8.0E-10 M, 7.2E-10 M, 5.0E-10 M or lower, 1.0E-10 M or lower, 9.0E-11 M or lower, 7.0E-11 M or lower, 5.0E-11 M or lower, or 1.0E-11 M or lower.

[0368] Appropriately, one or all FAP binders have an affinity of 400 pM or lower (K). D Combined with FAP, such as 300 pM or lower, such as 200 pM or lower, such as 150 pM or lower, such as 130 pM or lower, such as 110 pM or lower, such as 100 pM or lower, such as 50 pM or lower.

[0369] Appropriately, KD was established using the determination shown in Example 4.

[0370] In one embodiment, the construct binds to mouse FAP. In another embodiment, the construct binds to cynomolgus monkey FAP. In yet another embodiment, the construct binds to human FAP.

[0371] In one embodiment, one, more, or all FAP binders comprise or consist of paired heavy chain variable domains and light chain variable domains bound to the FAP (e.g., a Fab segment), as appropriately further defined below.

[0372] In one embodiment, the heavy chain variable domain binding to FAP comprises or consists of three complementarity-determining regions (HCDR1 to HCDR3) and four frame regions (HFR1 to HFR4), wherein HCDR1 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 117, 123, 129, 135, 141, 147, or 153; HCDR2 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 118, 124, 130, 136, 142, 148, or 154; and HCDR3 ... SEQ ID NO: 119, 125, 131, 137, 143, 149, or 155 have a polypeptide sequence or constitutes thereof with 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity, and the light chain variable domain binding to FAP comprises or constitutes thereof three complementarity-determining regions (LCDR1 to LCDR3) and four frame regions (LFR1 to LFR4), wherein LCDR1 comprises or constitutes thereof a polypeptide sequence or constitutes thereof with 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity, and LCDR2 comprises or constitutes thereof a polypeptide sequence or constitutes thereof with SEQ ID NO: 120, 126, 132, 138, 144, 150, or 156, and LCDR2 comprises or constitutes thereof a polypeptide sequence or constitutes thereof with 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity, and LCDR2 comprises thereof a polypeptide sequence or constitutes thereof with sequence identity, and LCDR3 comprises thereof a polypeptide sequence or constitutes thereof with sequence identity, and LCDR4 comprises thereof a polypeptide sequence or constitutes thereof with sequence identity, and LCDR5 comprises thereof a polypeptide sequence or constitutes thereof with sequence identity, and LCDR6 comprises thereof a polypeptide sequence or constitutes thereof with sequence identity, and LCDR7 comprises thereof a polypeptide sequence or constitutes thereof with sequence identity, and LCDR8 comprises thereof a polypeptide sequence or constitutes thereof with sequence identity, and LCDR9 comprises thereof a polypeptide sequence or constitutes thereof with sequence identity, and LCDR1 comprises thereof a polypeptide sequence or constitutes thereof with sequence identity, and LCDR2 comprises thereof a polypeptide sequence or constitutes thereof with sequence identity, and LCDR3 comprises thereof a polypeptide sequence or constitutes thereof with sequence identity, and 121, 127, 133, 139, 145, 151 or 157 having a sequence identity of 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher or 95% or higher, and LCDR3 containing or consisting of a polypeptide sequence having a sequence identity of 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher or 95% or higher with SEQ ID NO: 122, 128, 134, 140, 146, 152 or 158.More appropriately, HCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 117, 123, 129, 135, 141, 147, or 153; HCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 118, 124, 130, 136, 142, 148, or 154; and HCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 119, 125, 131, 137, 143, 149, or 155; and LCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 120, 126, 132, 138, 144, 150, or 156; LCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 121, 127, 133, 139, 145, 151, or 157; and LCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 118, 124, 130, 136, 142, 148, or 154. The polypeptide sequence of 122, 128, 134, 140, 146, 152 or 158 or composed of such sequences.

[0373] In one embodiment, the heavy chain variable domain binding to FAP comprises the three heavy chain complementarity-determining region amino acid sequences (HCDR1-HCDR3) of the heavy chain variable domain amino acid sequence of SEQ ID NO: 43, 45, 47, 49, 51, 53 or 55, while the light chain variable domain binding to FAP comprises the three light chain complementarity-determining region amino acid sequences (LCDR1-LCDR3) of the light chain variable domain amino acid sequence of SEQ ID NO: 44, 46, 48, 50, 52, 54 or 56.

[0374] In another embodiment, the heavy chain variable domain binding to FAP comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher sequence identity with the sequence of SEQ ID NO: 43, 45, 47, 49, 51, 53, or 55, while the light chain variable domain binding to FAP comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher sequence identity with the amino acid sequence of SEQ ID NO: 44, 46, 48, 50, 52, 54, or 56, 97% or higher, 98% or higher, or 99% or higher sequence identity with the sequence of SEQ ID NO: 44, 46, 48, 50, 52, 54, or 56, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher ... More preferably, the heavy chain variable domain contains or is composed of the amino acid sequence of SEQ ID NO: 43, 45, 47, 49, 51, 53 or 55, while the light chain variable domain contains or is composed of the amino acid sequence of SEQ ID NO: 44, 46, 48, 50, 52, 54 or 56.

[0375] Preferably, HCDR and LCDR are provided as heavy chain variable domains combined with FAP and light chain variable domains combined with FAP in the same combination as listed in Table 17A. For example, it is preferred to use HCDR1-3 of (a) SEQ ID NO: 117, 118 and 119 in combination with LCDR1-3 of (b) SEQ ID NO: 120, 121 and 122 (as listed in clone 3.5) for example in a Fab. The same principle applies to the combinations of variable regions listed in Table 16.

[0376] When multiple FAP binders are used in the constructs of this invention, these FAP binders may be (a) the same or (b) different FAP binders. If not simultaneously, these FAP binders are suitably and independently selected from the FAP binders described herein, such as those detailed above.

[0377] If the construct is an antibody, then in one embodiment, one or all of the FAP binders are Fab (linked to the CH2 region).

[0378] polypeptide and polynucleotide sequences

[0379] To compare two closely related peptide sequences, NCBI BLAST v2.0 can be used with the standard peptide sequence setting (BLASTP) to calculate the "percentage of sequence identity (%)" between the first and second peptide sequences. Similarly, to compare two closely related polynucleotide sequences, NCBI BLAST v2.0 can be used with the standard nucleotide sequence setting (BLASTN) to calculate the "percentage of sequence identity (%)" between the first and second nucleotide sequences.

[0380] A polypeptide or polynucleotide sequence is considered identical or "identical" if it has 100% sequence identity across its entire length. Residues in the sequence are numbered from left to right: for polypeptides, from the N-terminus to the C-terminus; for polynucleotides, from the 5' end to the 3' end.

[0381] The "difference" between polypeptide sequences refers to the insertion, deletion, or substitution of a single amino acid residue at a position in the second sequence compared to the first sequence. Two polypeptide sequences may contain one, two, or more such amino acid differences. Insertion, deletion, or substitution in a second sequence that is identical to the first sequence (100% sequence identity) results in a decrease in the percentage of sequence identity. For example, if the identical sequence is 9 amino acid residues long, a substitution in the second sequence will reduce the sequence identity to 88.9%. If the first and second polypeptide sequences are both 9 amino acid residues long and have 6 identical residues, then the first and second polypeptide sequences have greater than 66% identity (the first and second polypeptide sequences have 66.7% identity).

[0382] In the clauses and requests herein, the numbering of polypeptide sequences and the definitions of CDRs and FRs (i.e., HCDR1, HCDR2, HCDR3, HFR1, HFR2, HFR3, HFR4, LCDR1, LCDR2, LCDR3, LFR1, LFR2, LFR3, and LFR4) are defined according to the Kabat system (Kabat et al., 1991, incorporated herein by reference for the purposes of defining CDRs and FRs) unless otherwise stated. In addition to the Kabat system, any of the numbering schemes of Chothia, Martin (enhanced Chothia), IMGT (ImMunoGeneTics information system), or AbM may be applied. The “corresponding” amino acid residue between the first and second polypeptide sequences refers to an amino acid residue in the first sequence that, according to the Kabat system, has the same position as an amino acid residue in the second sequence, while the amino acid residue in the second sequence may differ in identity from that in the first sequence. Appropriately, if the frame and CDR have the same length according to the Kabat definition (or according to the definition or description of Chothia, Martin (enhanced Chothia), IMGT (immunogenetic information system), or AbM), the corresponding residues will have the same numbering (and lettering). Alignment can be performed manually or using known sequence alignment calculator algorithms, such as NCBI BLAST v2.0 (BLASTP or BLASTN) with standard settings.

[0383] The residues described in the polypeptide sequences listed herein may be unmodified or modified. If modified, they may be, for example, isomerized, deaminated, or oxidized.

[0384] Construct

[0385] In at least some embodiments, the constructs of the present invention benefit from certain properties, such as the ability to aggregate LTBR, ​​bind LTBR and FAP on the same cell, potently promote LTBR (e.g., LTBR NFKB pathway, such as classical and alternative NFKB pathways), bind LTBR and FAP with desired affinity, and achieve favorable biodistribution (e.g., tumor targeting) upon administration due to FAP-dependent conditional activation. The properties specified regarding the composition of monospecific LTBR and FAP binders may be suitably equivalently applied to the constructs of the present invention.

[0386] The constructs of this invention are suitably conditionally activated at a target site. Conditional activation means that the construct accumulates LTBR at the target site in a FAP-dependent manner (due to the construct's specificity for FAP), thereby activating the LTBR and leading to activation of the classical and / or alternative NFKB pathways (as described further in detail herein). The target site is suitably a tumor.

[0387] Suitablely, the LTBR binder can bind to LTBR on the cell, and the FAP binder can simultaneously bind to FAP on the same cell, and / or the LTBR binder can bind to LTBR on the first cell, and the FAP binder can simultaneously bind to FAP on the second cell. Suitablely, the LTBR binder can activate LTBR on cells that simultaneously express LTBR and FAP.

[0388] The construct according to the invention may comprise multiple binders, and is therefore multivalent (e.g., bivalent). These multiple binders bind to different targets, thus the construct is multispecific (e.g., bispecific). Such a construct may comprise (a) no more than one LTBR binder and no more than one FAP binder, (b) no more than one LTBR binder and more than one FAP binder, (c) more than one LTBR binder and no more than one FAP binder, or (d) more than one LTBR binder and more than one FAP binder. If the construct comprises more than one LTBR binder and / or FAP binder, the LTBR binders may be the same or different, and the FAP binders may be the same or different. Suitablely, the LTBR binders are the same. Suitablely, the FAP binders are the same. Most suitablely, the LTBR binders are the same and the FAP binders are the same. If the LTBR binders are different, then suitablely they are independently selected from LTBR binders as defined herein. If the FAP binders are different, they are appropriately selected independently from the FAP binders defined herein.

[0389] In one embodiment, the construct contains only one FAP binder. Alternatively, the construct contains more than one FAP binder. In one embodiment, the construct contains only two FAP binders.

[0390] In one embodiment, the construct contains only one LTBR binder. Alternatively, the construct contains more than one LTBR binder. In one embodiment, the construct contains only two LTBR binders.

[0391] The potency and specificity of the constructs of this invention can be expressed as the ratio of FAP binding valence to LTBR binding valence, for example, 1:1, 2:1, etc. A bispecific construct that is monovalent to both FAP and LTBR can be described as 1:1. A bispecific construct that is bivalent to both FAP and LTBR can be described as 2:1. A bispecific construct that is bivalent to both FAP and LTBR can be described as 2:2, and so on.

[0392] A FAP monovalent construct is a construct with a single anti-FAP valence. A FAP bivalent construct is a construct with two anti-FAP valences, and so on. An LTBR monovalent construct is a construct with a single anti-LTBR valence. An LTBR bivalent construct is a construct with two anti-LTBR valences, and so on.

[0393] In one embodiment, the construct comprises or consists of an antibody, such as a bispecific antibody and / or a bivalent antibody.

[0394] The constructs according to the invention may contain at least one other binding agent (e.g., an antibody or its antigen-binding fragment), which is neither an LTBR binding agent nor a FAP binding agent. Alternatively, the constructs according to the invention may not contain any other binding agents.

[0395] A suitable construct may contain or consist of antibodies, wherein in one embodiment, a first variable region binds to LTBR and a second variable region binds to FAP.

[0396] Another suitable construct could contain multiple antibodies.

[0397] Other suitable constructs may comprise or consist of one or more antibody fragments as described above, wherein each antibody fragment provides one or more binding domains. Suitable antibody fragments include, for example, scFv, Fv, Fab, Fab', F(ab')2, variable regions (e.g., VH, VL, VNAR, or VHH), biantibodies, or microantibodies. In one embodiment, the construct comprises or consists of two scFvs.

[0398] Binders can be linked (or “fused”) together directly (i.e., without a linker) or indirectly via a linker or other components (such as immunoglobulin domains). Binders can also be linked (or “fused”) to components of the construct (such as constant domains) directly (i.e., without a linker) or indirectly via a linker. Suitablely, the linker is a peptide. Linkers are appropriately selected to ensure that the binder can bind to its target while maintaining structural integrity. If the construct is to be administered to an individual, the linker should be appropriately non-immunogenic in the individual.

[0399] Appropriately, the length of the peptide linker is no more than 50 amino acids, for example, no more than 40 amino acids, for example, no more than 30 amino acids, for example, no more than 20 amino acids, for example, no more than 10 amino acids, for example, no more than 9 amino acids, for example, no more than 8 amino acids, for example, no more than 7 amino acids, for example, no more than 6 amino acids, for example, no more than 5 amino acids.

[0400] Appropriately, the length of the peptide linker is greater than 6 amino acids, for example greater than 7 amino acids, for example greater than 8 amino acids, for example greater than 9 amino acids, for example greater than 10 amino acids, for example greater than 20 amino acids, for example greater than 30 amino acids, for example greater than 40 amino acids, for example greater than 50 amino acids.

[0401] Suitable, the peptide linker comprises or is composed of glycine and serine residues, for example, comprising or is composed of a G4S linker (SEQ ID NO: 3).

[0402] In one embodiment, the binders are linked together in the construct in the form of bispecific, multivalent (e.g., bivalent) antibodies. Such bispecific antibodies may be referred to as "bsAb".

[0403] A particularly suitable form of bispecific antibody is generated via a "knobs-into-holes" method (Merchant et al., 1998). Alternatively, one or more (ideally all) Golay mutations can be introduced into the CH1 / CL region to obtain efficient light chain pairing (Golay et al., 2016).

[0404] In one embodiment, the bispecific construct of the present invention having multiple valences for LTBR and / or FAP may include multiple scFvs, wherein each scFv is fused to the C-terminus of Fc via a peptide linker (e.g., a linker containing glycine and serine residues, such as a G4S linker (SEQ ID NO: 3)).

[0405] If the Fc is present in the construct (e.g., if the construct is an antibody), an appropriate LTBR binder may be directly or indirectly linked to the Fc (e.g., linked to one of the CH3 domains). If two LTBR binders are present, each LTBR binder may be linked to one of the CH3 domains of the Fc, i.e., the first LTBR binder is linked to the first CH3 domain and the second LTBR binder is linked to the second CH3 domain.

[0406] In one embodiment, the linker does not contain an Ig domain. In one embodiment, the linker is a linear peptide. In another embodiment, the linker is a hinge region. Most preferably, the LTBR binder is indirectly linked to the CH3 domain via a linear peptide linker.

[0407] The various constructs described in the following examples (including bispecific constructs) are referred to as “clones” and are assigned a specific clone number or ID.

[0408] cancer

[0409] In one embodiment, the construct of the present invention is used for cancer treatment. In one embodiment, the cancer may be a tumor. In another embodiment, the cancer may be a non-hematologic cancer. In this case, it is most ideal that the LTBR binder is an LTBR agonist (as discussed in detail above).

[0410] Drug administration

[0411] The constructs and peptides of the present invention are generally intended for use in mammalian individuals, particularly human individuals. These constructs and peptides are typically administered to individuals in need of them, particularly mammalian individuals in need of them, and even more particularly human individuals in need of them.

[0412] The construct or peptide can be administered via any suitable route, depending on the nature of the specific agent. Exemplary routes include oral, parenteral, buccal, sublingual, nasal, or rectal administration. Appropriately, the construct may be administered via parenteral administration, such as intravenous (iv or IV) or intraperitoneal (ip or IP).

[0413] If a cancer vaccine and / or adjuvant are available, they can be administered via any suitable route, depending on the nature of the specific agent. Exemplary routes include oral, parenteral, buccal, sublingual, nasal, or rectal administration. Appropriately, the vaccine and / or adjuvant may be administered parenterally, such as intraperitoneally.

[0414] The construct or peptide may be provided in the form of a pharmaceutical composition comprising the construct or peptide and a pharmaceutically acceptable carrier or excipient.

[0415] If delivered orally, the construct or peptide may be administered as a solid pharmaceutical composition (such as a tablet, capsule, or lozenge) or a liquid pharmaceutical composition (such as a suspension, emulsion, or solution).

[0416] Liquid formulations typically consist of a suspension or solution of the construct in a suitable liquid carrier, such as an aqueous solvent like water, ethanol, or glycerol, or a non-aqueous solvent like polyethylene glycol or oil. The formulation may also contain suspending agents, preservatives, flavoring agents, and / or coloring agents.

[0417] Tablet formulations can be prepared using any suitable pharmaceutical carrier commonly used in the preparation of solid dosage forms, such as magnesium stearate, starch, lactose, sucrose, and cellulose.

[0418] Where appropriate, the pharmaceutical composition is presented in unit dose form, such as tablets, capsules, ampoules, or pre-filled syringes. Where appropriate, the unit dose form is intended for intravenous administration.

[0419] Pharmaceutical compositions may contain, for example, 0.1% to 99.99% of the active ingredient by weight, or, for example, 10% to 60% by weight, depending on the route of administration. Pharmaceutical compositions may contain 0.01% to 99% of a carrier by weight, or, for example, 40% to 90% by weight, depending on the route of administration. Pharmaceutical compositions may contain 0.05 mg to 2000 mg of the active ingredient, or, for example, 1.0 mg to 500 mg, depending on the route of administration. Pharmaceutical compositions may contain 50 mg to 1000 mg of a carrier, or, for example, 100 mg to 400 mg, depending on the route of administration.

[0420] The dosage of the pharmaceutical ingredients used will vary in a conventional manner based on the severity of the cancer, patient weight, and other similar factors. However, as a general guideline, suitable unit doses can range from 0.05 mg to 1000 mg, with 1.0 mg to 500 mg being more appropriate, and these unit doses can be administered multiple times daily, such as twice or three times daily. This therapy can continue for weeks, months, or longer.

[0421] The dose provided to an individual is typically a safe and effective dose, meaning an amount that achieves an acceptable balance between providing the intended benefit and avoiding unwanted side effects. A “safe and effective dose” refers to the amount of compound that effectively achieves the desired effect in treating a disease state. The desired effect is typically clinically significant and measurable, for example, in cases of: (a) inhibiting the disease state, i.e., slowing or halting its progression; and / or (b) alleviating the disease state, i.e., causing disease regression or a reduction in associated symptoms. A safe and effective dose is an amount sufficient to achieve these desired effects.

[0422] For the avoidance of doubt, the "safe and effective amount" described herein can be achieved by any suitable dosing regimen. Therefore, for example, the safe and effective amount of a component mentioned herein (e.g., via a specific route of administration) includes achieving a safe and effective amount via a single dose or multiple doses (e.g., via a specific route of administration). For example, a safe and effective amount administered intravenously includes both a single intravenous dose and multiple intravenous doses, as long as a safe and effective amount is achieved via intravenous administration.

[0423] Polynucleotides, carriers and cells

[0424] In one embodiment, this document provides one or more polynucleotides encoding constructs of the present invention. One or more expression vectors are also provided, which collectively contain these polynucleotides. Additionally, a cell containing these expression vectors or polynucleotides is provided. Further, a method for producing constructs of the present invention is provided, the method comprising culturing cells under suitable conditions to express the polynucleotides, thereby producing the construct.

[0425] combination

[0426] In some embodiments, the constructs or peptides of the present invention may be used in combination with other agents. For example, in one embodiment, a composition is provided comprising the construct or protein of the present invention and a checkpoint inhibitor (such as an anti-CTLA-4 antibody, an anti-PD-L1 antibody, and / or an anti-PD-1 antibody). These compositions are suitable for the treatment of cancer (e.g., tumors).

[0427] The constructs, compositions, or peptides of the present invention can be used to treat cancer (e.g., tumors), optionally in combination with radiotherapy (e.g., low-fractionation radiotherapy), chemotherapy, or antibody-drug conjugates (ADCs). The constructs of the present invention can be used to treat cancer in conjunction with one of the aforementioned agents, or one of the aforementioned agents can be used to treat cancer in conjunction with the constructs of the present invention. (a) The administration of the constructs or peptides of the present invention with (b) checkpoint inhibitors and / or radiotherapy and / or chemotherapy or ADCs can be sequential, separate, or simultaneous.

[0428] Other specific embodiments

[0429] Specific constructs of the present invention are characterized in the examples.

[0430] In one embodiment, the construct of the present invention comprises, is primarily composed of, or is composed of the forms of clones 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 4.10, 4.11, or 4.12 described in Table 15.

[0431] The constructs of this invention may comprise, consist primarily of, or be composed of Figure 7The constructs of the present invention comprise, are primarily composed of, or are composed of, the polypeptide sequences of clones 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 4.10, 4.11, or 4.12.

[0432] In some embodiments, the constructs of the present invention may be provided in the following specific forms.

[0433] The construct may comprise, substantially comprise, or consist of: a bispecific, FAP-monovalent, LTBR-monovalent antibody comprising a heterodimer of two polypeptide chains, the first chain comprising, or consisting of, a Fab region, a hinge region, a CH2 domain, and a CH3 domain from the N-terminus to the C-terminus; and the second chain comprising, or consisting of, a VHH domain, a hinge region, a CH2 domain, and a CH3 domain from the N-terminus to the C-terminus, wherein the hinge regions are linked by disulfide bonds, the CH2 and CH3 domains are linked to form an Fc region, the Fab region comprises a paired VH and VL domain at its N-terminus forming an FAP binding site, and the VHH forming an LTBR binding site (e.g., as shown in the image). Figure 7 (as shown in a). This is an example of a 1:1 form construct.

[0434] The construct may comprise, substantially consist of, or be composed of: a first heavy chain, a light chain, and a second heavy chain, wherein the first heavy chain contains VH, CH1, CH2, and CH3 from the N-terminus to the C-terminus, the second heavy chain contains VHH, CH2, and CH3 from the N-terminus to the C-terminus, the light chain contains VL and CL from the N-terminus to the C-terminus, and binds with VH and CH1 of the first heavy chain to form a Fab region, and the CH2 and CH3 domains of the first and second heavy chains bind to form an Fc region, wherein VH and VL bind to the FAP, and VHH binds to the LTBR (e.g., as shown in the figure). Figure 7 (as shown in a). This is an example of a 1:1 form construct.

[0435] The construct may comprise, substantially comprise, or consist of: a bispecific, FAP-bivalent, LTBR-monovalent antibody comprising a heterodimer of two polypeptide chains. The first polypeptide chain, from its N-terminus to its C-terminus, comprises or consists of: a Fab region, a hinge region, a CH2 domain, a CH3 domain, and a VHH. The second polypeptide chain, from its N-terminus to its C-terminus, comprises or consists of: a Fab region, a hinge region, a CH2 domain, and a CH3 domain. The hinge regions are linked by disulfide bonds, the CH2 and CH3 domains combine to form an Fc region, each Fab region contains a paired VH and VL at its N-terminus, each forming an FAP binding site, while the VHH forms an LTBR binding site at its C-terminus (e.g., substantially as shown in the diagram). Figure 7 (as shown in b). This is an example of a 2:1 form construct.

[0436] The construct may comprise, and is primarily composed of, the following: a first heavy chain, a first light chain, a second heavy chain, and a second light chain. The first heavy chain, from its N-terminus to its C-terminus, sequentially contains VH, CH1, CH2, CH3, and VHH. The second heavy chain, from its N-terminus to its C-terminus, sequentially contains VH, CH1, CH2, and CH3. The first light chain, from its N-terminus to its C-terminus, sequentially contains VL and CL, and combines with VH and CH1 of the first heavy chain to form a Fab region. The second light chain, from its N-terminus to its C-terminus, sequentially contains VL and CL, and combines with VH and CH1 of the second heavy chain to form a Fab region. The CH2 and CH3 domains of the first and second heavy chains combine to form an Fc region. The VH and VL of each Fab region are bound to the FAP, while the VHH is bound to the LTBR (e.g., essentially as shown). Figure 7 (as shown in b). This is an example of a 2:1 form construct.

[0437] The construct may include, consist primarily of, or be composed of: a bispecific, FAP-bivalent, LTBR-bivalent antibody comprising two homodimers of polypeptide chains. The first chain, from N-terminus to C-terminus, comprises a Fab region, a hinge region, a CH2 domain, a CH3 domain, and a VHH; the second chain, from N-terminus to C-terminus, comprises a Fab region, a hinge region, a CH2 domain, a CH3 domain, and a VHH, wherein the hinge regions are linked by disulfide bonds, the CH2 and CH3 domains bind to form an Fc region, each Fab region contains a paired VH and VL at its N-terminus, forming an FAP binding site, while the VHH forms an LTBR binding site at its C-terminus (e.g., essentially as shown). Figure 7 (As shown in c). This is an example of a 2:2 form construct.

[0438] The construct may comprise, substantially consist of, or be composed of: a first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein the first heavy chain contains VH, CH1, CH2, CH3, and VHH from the N-terminus to the C-terminus; the second heavy chain contains VH, CH1, CH2, CH3, and VHH from the N-terminus to the C-terminus; the first light chain contains VL and CL from the N-terminus to the C-terminus and combines with VH and CH1 of the first heavy chain to form a Fab region; the second light chain contains VL and CL from the N-terminus to the C-terminus and combines with VH and CH1 of the second heavy chain to form a Fab region; and the CH2 and CH3 regions of the first and second heavy chains combine to form an Fc region, wherein the VH and VL of each Fab region are combined with FAP, and the VHH is combined with LTBR (e.g., substantially as shown). Figure 7 (As shown in c). This is an example of a 2:2 form construct.

[0439] The construct may comprise, substantially comprise, or consist of: a bispecific, FAP-bivalent, LTBR-bivalent antibody comprising a homodimer of two polypeptide chains, the first chain comprising a Fab region, a hinge region, a CH2 region, a CH3 region, and an scFv from the N-terminus to the C-terminus; and the second chain comprising a Fab region, a hinge region, a CH2 region, a CH3 region, and an scFv from the N-terminus to the C-terminus, wherein the hinge regions are linked by disulfide bonds, the CH2 and CH3 regions form an Fc region, each Fab region comprising a paired VH and VL at its N-terminus, each forming an FAP binding site, and each scFv forming an LTBR binding site at its C-terminus (e.g., substantially as shown). Figure 7 (As shown in d). This is an example of a 2:2 form construct.

[0440] The construct may comprise, substantially consist of, or be composed of: a first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein the first heavy chain, from its N-terminus to its C-terminus, sequentially comprises VH, CH1, CH2, CH3, and paired VH and VL; the second heavy chain, from its N-terminus to its C-terminus, sequentially comprises VH, CH1, CH2, CH3, and paired VH and VL; the first light chain, from its N-terminus to its C-terminus, sequentially comprises VL and CL, and combines with the VH and CH1 of the first heavy chain to form a Fab region; the second light chain, from its N-terminus to its C-terminus, sequentially comprises VL and CL, and combines with the VH and CH1 of the second heavy chain to form a Fab region; and the CH2 and CH3 domains of the first and second heavy chains combine to form an Fc region, wherein the VH and VL of each Fab region are combined with FAP, and the paired VH and VL are combined with LTBR (e.g., substantially as shown). Figure 7 (As shown in d). This is an example of a 2:2 form construct.

[0441] The present invention also provides: (a) an antibody that binds to an epitope bound to the polypeptide or construct of the present invention, (b) an antibody that competes with the construct of the present invention for binding LTBR, ​​and (c) an antibody that competes with the construct of the present invention for binding FAP.

[0442] The present invention also provides an embodiment of a construct, which is an antibody, comprising: (a) One or more Fab regions, each containing a heavy chain variable domain and a light chain variable domain paired with one of the FAPs, wherein: (i) The heavy chain variable domain comprises or is composed of the polypeptide sequence of SEQ ID NO: 43, and the light chain variable domain comprises or is composed of the polypeptide sequence of SEQ ID NO: 44; (ii) The heavy chain variable domain comprises or is composed of the polypeptide sequence of SEQ ID NO: 45, and the light chain variable domain comprises or is composed of the polypeptide sequence of SEQ ID NO: 46; or (iii) The heavy chain variable domain comprises or is composed of the polypeptide sequence of SEQ ID NO: 47, and the light chain variable domain comprises or is composed of the polypeptide sequence of SEQ ID NO: 48; as well as (b) One or more paired heavy chain variable domains and light chain variable domains combined with LTBR; The heavy chain variable domain comprises or is composed of the polypeptide sequence of SEQ ID NO: 31, and the light chain variable domain comprises or is composed of the polypeptide sequence of SEQ ID NO: 32; Or one or more variable domains combined with LTBR; where (i) wherein the heavy chain variable domain comprises or is composed of the polypeptide sequence of SEQ ID NO: 24; or (ii) wherein the heavy chain variable domain comprises or is composed of the polypeptide sequence of SEQ ID NO: 25.

[0443] In a further embodiment, the construct is an antibody comprising: (i) A heterodimer of two different heavy chains (HC1 and HC2) and two identical light chains (LC), wherein: (a) The HC1 polypeptide sequence comprises or is composed of SEQ ID NO: 175, the HC2 polypeptide sequence comprises or is composed of SEQ ID NO: 176, and the LC polypeptide sequence comprises or is composed of SEQ ID NO: 177; (b) The HC1 polypeptide sequence comprises or is composed of SEQ ID NO: 178, the HC2 polypeptide sequence comprises or is composed of SEQ ID NO: 179, and the LC polypeptide sequence comprises or is composed of SEQ ID NO: 180; (c) The HC1 polypeptide sequence comprises or is composed of SEQ ID NO: 181, the HC2 polypeptide sequence comprises or is composed of SEQ ID NO: 182, and the LC polypeptide sequence comprises or is composed of SEQ ID NO: 183; (d) The HC1 polypeptide sequence comprises or is composed of SEQ ID NO: 184, the HC2 polypeptide sequence comprises or is composed of SEQ ID NO: 185, and the LC polypeptide sequence comprises or is composed of SEQ ID NO: 186; (e) The HC1 polypeptide sequence comprises or is composed of SEQ ID NO: 187, the HC2 polypeptide sequence comprises or is composed of SEQ ID NO: 188, and the LC polypeptide sequence comprises or is composed of SEQ ID NO: 189; (f) The HC1 polypeptide sequence comprises or is composed of SEQ ID NO: 190, the HC2 polypeptide sequence comprises or is composed of SEQ ID NO: 191, and the LC polypeptide sequence comprises or is composed of SEQ ID NO: 192; (g) The HC1 polypeptide sequence comprises or is composed of SEQ ID NO: 193, the HC2 polypeptide sequence comprises or is composed of SEQ ID NO: 194, and the LC polypeptide sequence comprises or is composed of SEQ ID NO: 195; or (h) The HC1 polypeptide sequence comprises or is composed of SEQ ID NO: 200, the HC2 polypeptide sequence comprises or is composed of SEQ ID NO: 201, and the LC polypeptide sequence comprises or is composed of SEQ ID NO: 202, or (ii) Homodimers of two identical heavy chains (HC) and two identical light chains (LC), wherein: (a) The HC polypeptide sequence comprises or is composed of SEQ ID NO: 196, and the LC polypeptide sequence comprises or is composed of SEQ ID NO: 197, or (b) The HC polypeptide sequence comprises or is composed of SEQ ID NO: 198, and the LC polypeptide sequence comprises or is composed of SEQ ID NO: 199.

[0444] The present invention also provides a construct comprising an antibody or antibody fragment, wherein the antibody or antibody fragment comprises an FAP binding domain and an LTBR binding domain, wherein: (i) The LTBR binding domain is a variable domain bound to the LTBR, ​​which contains or consists of three heavy chain complementarity-determining regions (HCDR1-HCDR3) and four frame regions (FR1-FR4), wherein: (a) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 60 or 172, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 61 or 173, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 62 or 174, or (b) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 57 or 166, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 58 or 167, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 59 or 168, or (c) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 63 or 163, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 64 or 164, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 65 or 165, or (d) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 66 or 169, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 67 or 170, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 68 or 171. or (ii) The LTBR binding domain is a pair of heavy chain variable domains and light chain variable domains, wherein the heavy chain variable domain contains or consists of three complementary determinant regions (HCDR1-HCDR3) and four frame regions (HFR1-HFR4), and the light chain variable domain contains or consists of three complementary determinant regions (LCDR1-LCDR3) and four frame regions (LFR1-LFR4), wherein: (a) HCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 69, HCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 70, and HCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 71, wherein LCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 72, LCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 73, and LCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 74, or (b) HCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 75, HCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 76, and HCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 77, wherein LCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 78, LCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 79, and LCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 80, or (c) HCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 81, HCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 82, and HCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 83, wherein LCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 84, LCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 85, and LCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 86, or (d) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 87, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 88, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 89, wherein LCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 90, LCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 91, and LCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 92, or (e) HCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 93, HCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 94, and HCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 95, wherein LCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 96, LCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 97, and LCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 98, or (f) HCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 99, HCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 100, and HCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 101, wherein LCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 102, LCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 103, and LCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 104, or (g) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 105, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 106, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 107; LCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 108, LCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 109, and LCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 110, or (h) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 111, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 112, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 113; LCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 114, LCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 115, and LCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 116. and The FAP binding domain is a pair of heavy chain variable domains and light chain variable domains, wherein the heavy chain variable domain contains three complementary determinant regions (HCDR1-HCDR3) and four frame regions (HFR1-HFR4) or composed thereof, and the light chain variable domain contains three complementary determinant regions (LCDR1-LCDR3) and four frame regions (LFR1-LFR4) or composed thereof, wherein: (a) HCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 117, HCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 118, and HCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 119, wherein LCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 120, LCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 121, and LCDR3 comprises or is composed of the polypeptide sequence shown in SEQ ID NO: 122, or (b) HCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 123, HCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 124, and HCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 125, wherein LCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 126, LCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 127, and LCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 128, or (c) HCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 129, HCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 130, HCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 131, and wherein LCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 132, LCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 133, and LCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 134, or (d) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 135, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 136, HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 137, and wherein LCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 138, LCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 139, and LCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 140, or (e) HCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 141, HCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 142, and HCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 143, wherein LCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 144, LCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 145, and LCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 146, or (f) HCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 147, HCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 148, and HCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 149, wherein LCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 150, LCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 151, and LCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 152, or (g) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 153, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 154, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 155, wherein LCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 156, LCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 157, and LCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 158.

[0445] polypeptides with LTBR or FAP junction

[0446] In addition to constructs comprising both FAP and LTBR binders, the present invention also provides (a) constructs that bind to FAP and (b) constructs that bind to LTBR. These constructs can be used, for example, in the bispecific constructs of the present invention. The monospecific polypeptides (“clones”) used in the examples of the present invention are summarized below.

[0447]

[0448] Peptides that bind to LTBR

[0449] LTBR-binding peptides may contain or be composed of a variable domain that binds to LTBR (e.g., a heavy chain variable domain, such as VHH), as defined below.

[0450] In one embodiment, the LTBR-binding peptide comprises at least one CDR, wherein the CDR is HCDR1, HCDR2, or HCDR3, and these CDRs are contained within the heavy chain variable domain of SEQ ID NO:: 23-26 or 159-162. Suitably, the peptide comprises at least one such HCDR1 and HCDR2, one such HCDR1 and HCDR3, or one such HCDR2 and HCDR3. More suitably, the peptide comprises one such HCDR1, HCDR2, and HCDR3. More suitably, HCDR1, HCDR2, and HCDR3 are characterized according to the Kabat, Chothia, Martin, IMGT, or AbM methods. More appropriately, HCDR1 as defined by the Kabat method is selected from SEQ ID NO:: 57, 60, 63, 66, 163, 166, 169 or 172; HCDR2 as defined by the Kabat method is selected from SEQ ID NO:: 58, 61, 64, 67, 164, 167, 170, 173, 205 or 206; and / or HCDR3 as defined by the Kabat method is selected from SEQ ID NO:: 59, 62, 65, 68, 165, 168, 171, 174, 207 or 208.

[0451] In one embodiment, the variable domain binding to the LTBR comprises or consists of three heavy chain complementarity-determining regions (HCDR1 to HCDR3) and four frame regions (FR1 to FR4), wherein HCDR1 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 57, 60, 63, 66, 163, 166, 169, or 172 (e.g., 57, 60, 63, or 66), or wherein HCDR1 comprises or consists of a polypeptide sequence having 3, 2, or 1 amino acid differences from SEQ ID NO: 57, 60, 63, 66, 163, 166, 169, or 172 (e.g., 57, 60, 63, or 66); HCDR2 comprises or consists of a polypeptide sequence having 3, 2, or 1 amino acid differences from SEQ ID NO: 57, 60, 63, 66, 163, 166, 169, or 172 (e.g., 57, 60, 63, or 66); 58, 61, 64, 67, 164, 167, 170, 173, 205, or 206 (e.g., 58, 61, 64, or 67) having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity, or wherein HCDR2 comprises or consists of a polypeptide sequence differing from SEQ ID NO: 58, 61, 64, 67, 164, 167, 170, 173, 205, or 206 (e.g., 58, 61, 64, or 67) by 3, 2, or 1 amino acids; and HCDR3 comprises a polypeptide sequence differing from SEQ ID NO: 59, 62, 65, 68, 165, 168, 171, 174, 207, or 208 (e.g., 59, 62, 65, or 68) having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity, or wherein HCDR3 comprises or consists of a polypeptide sequence that differs from SEQ ID NO: 59, 62, 65, 68, 165, 168, 171, 174, 207, or 208 (e.g., 59, 62, 65, or 68) by 3, 2, or 1 amino acid. More appropriately, HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 57, 60, 63, 66, 163, 166, 169, or 172 (e.g., 57, 60, 63, or 66), HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 58, 61, 64, 67, 164, 167, 170, 173, 205, or 206 (e.g., 58, 61, 64, or 67), and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 59, 62, 65, 68, 165, 168, 171, 174, 207, or 208 (e.g., 59, 62, 65, or 68). In particular, these CDR polypeptide sequences are determined or described according to the Kabat method.

[0452] In one embodiment, the variable domain binding to the LTBR comprises a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher sequence identity with SEQ ID NO: 23, 24, 25, 26, 159, 160, 161, or 162 (e.g., 23, 24, 25, or 26). More suitably, the variable domain binding to the LTBR comprises or comprises a polypeptide sequence of or constitutes thereof with SEQ ID NO: 23, 24, 25, 26, 159, 160, 161, or 162 (e.g., 23, 24, 25, or 26). In another embodiment, the variation of the polypeptide sequence is located in one or more frame regions FR1 to FR4 contained in SEQ ID NO: 23, 24, 25, 26, 159, 160, 161, or 162 (e.g., 23, 24, 25, or 26). Alternatively, the variation of the polypeptide sequence is located in one or more frame regions FR1 to FR4 contained in SEQ ID NO: 23, 24, 25, 26, 159, 160, 161, or 162 (e.g., 23, 24, 25, or 26), and the CDR polypeptide sequence is the HCDR1 to HCDR3 polypeptide sequences contained in SEQ ID NO: 23, 24, 25, 26, 159, 160, 161, or 162 (e.g., 23, 24, 25, or 26), as listed above.

[0453] In one embodiment, the LTBR-binding polypeptide comprises or consists of a pair of heavy chain variable domains and light chain variable domains, which are capable of binding to an LTBR (e.g., an scFv), as further defined appropriately below.

[0454] In one embodiment, the heavy chain variable domain binding to the LTBR comprises or consists of three complementarity-determining regions (HCDR1 to HCDR3) and four frame regions (HFR1 to HFR4), wherein HCDR1 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 69, 75, 81, 87, 93, 99, 105, or 111; HCDR2 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 70, 76, 82, 88, 94, 100, 106, or 112; and HCDR3 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: SEQ ID NOs 71, 77, 83, 89, 95, 101, 107, or 113 contain polypeptide sequences or are composed thereof with 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity, and the light chain variable domain bound to the LTBR comprises or is composed of three complementarity-determining regions (LCDR1 to LCDR3) and four frame regions (LFR1 to LFR4), wherein LCDR1 contains or is composed of a polypeptide sequence or is composed thereof with 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity, and LCDR2 contains or is composed of a polypeptide sequence ... Or 115 is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity, and LCDR3 comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 74, 80, 86, 92, 98, 104, 110, or 116.More appropriately, HCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 69, 75, 81, 87, 93, 99, 105, or 111; HCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 70, 76, 82, 88, 94, 100, 106, or 112; and HCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 71, 77, 83, 89, 95, 101, 107, or 113. LCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 72, 78, 84, 90, 96, 102, 108, or 114; LCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 73, 79, 85, 91, 97, 103, 109, or 115; and LCDR3 comprises the polypeptide sequence of SEQ ID NO: 69, 75, 81, 87, 93, 99, 105, or 115. The polypeptide sequence of 74, 80, 86, 92, 98, 104, 110 or 116 or composed of such sequences.

[0455] In one embodiment, the heavy chain variable domain binding to the LTBR comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher sequence identity with SEQ ID NO: 27, 29, 31, 33, 35, 37, 39, or 41, and the light chain variable domain binding to the LTBR comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher sequence identity with SEQ ID NO: 28, 30, 32, 34, 36, 38, 40, or 42, and the sequence identity is ...97% or higher, 98% or higher, or 99% or higher sequence identity with SEQ ID NO: 28, 30, 32, 34, 36, 38, 40, or 42, and the sequence identity is 99% or higher, 70% or higher, 80% or higher, 85% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, More appropriately, the heavy chain variable domain that binds to the LTBR comprises or is composed of a polypeptide sequence of SEQ ID NO: 27, 29, 31, 33, 35, 37, 39 or 41, and the light chain variable domain that binds to the LTBR comprises or is composed of a polypeptide sequence of SEQ ID NO: 28, 30, 32, 34, 36, 38, 40 or 42.

[0456] In another embodiment, the variable domain binding to the LTBR comprises or consists of three heavy chain complementarity-determining regions (HCDR1-HCDR3) and four frame regions (FR1-FR4), wherein HCDR1 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 163, 166, 169, or 172; HCDR2 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 164, 167, 170, or 173; and HCDR3 comprises a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher sequence identity with SEQ ID NO: 165, 168, 171, or 174. The polypeptide sequence or constitutes thereof has 85% or higher, 90% or higher, or 95% or higher sequence identity. More preferably, HCDR1 contains or constitutes thereof a polypeptide sequence of SEQ ID NO: 163, 166, 169, or 172, HCDR2 contains or constitutes thereof a polypeptide sequence of SEQ ID NO: 164, 167, 170, or 173, and HCDR3 contains or constitutes thereof a polypeptide sequence of SEQ ID NO: 165, 168, 171, or 174.

[0457] In one embodiment, the variable domain binding to the LTBR comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher amino acid sequence identity with the sequence of SEQ ID NO: 159, 160, 161, or 162. More suitably, the variable domain binding to the LTBR comprises or consists of a polypeptide sequence of sequence listing 159, 160, 161, or 162.

[0458] Preferably, HCDRs (and LCDRs) are provided in the same combinations listed in Tables 17A / B in heavy chain variable domains (and paired light chain variable domains, which are also combined with LTBR) linked to LTBR. For example, it is preferred to use HCDRs 1-3 of SEQ ID NO: 57, 58 and 59 in combination (as listed in Clone 1.5) for example in VHH, and it is preferred to use HCDRs 1-3 of (a) SEQ ID NO: 69, 70 and 71 and LCDRs 1-3 of (b) SEQ ID NO: 72, 73 and 74 in combination (as listed in Clone 2.1) for example in scFv. The same principle also applies to the combinations of heavy chain variable domains and light chain variable domains listed in Table 16.

[0459] The present invention also provides a polypeptide that binds to LTBR, ​​comprising or consisting of a polypeptide sequence of clones 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 1.10, 1.11, 1.12, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7 or 2.8; or an antibody that competes with the anti-LTBR polypeptide of the present invention for binding to LTBR.

[0460] Appropriately, the polypeptide binds specifically to the LTBR, ​​for example, by its dissociation constant (Ki) when binding to any other entity. D ) is 10 -7 M or higher, such as 10 -6 M or higher. Appropriately, the peptide specifically binds to the extracellular domain of the LTBR. In some embodiments, the LTBR is located on the surface of stromal cells. In some embodiments, the LTBR is located on the surface of endothelial cells. In some embodiments, the LTBR is located on the surface of fibroblasts. In other embodiments, the LTBR is located on the surface of cancer cells, myeloid cells, and / or macrophages (e.g., present in the tumor microenvironment (TME)).

[0461] In some embodiments, the LTBR-binding peptide is an LTBR agonist such that its binding to the LTBR leads to activation of LTBR NFKB pathways, such as classical and / or non-classical NFKB pathways. If the LTBR binder is an LTBR agonist, then suitably the LTBR binder (a) induces LTBR aggregation and more suitably (b) activates classical NFKB pathways (suitably leading to the expression of adhesion molecules such as ICAMs (e.g., ICAM-1, VCAM-1, and / or MAdCAM-1)) and / or activates alternative NFKB pathways (suitably leading to the expression of chemokines such as CCL5, CCL19, CCL21, and / or CXCL13).

[0462] LTBR-binding peptides can have an affinity of 20 nM or lower (K). D The LTBR-binding peptide can bind to LTBR at a concentration of, for example, 15 nM or less, 10 nM or less, 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, or 1 nM or less. In some embodiments, the LTBR-binding peptide can have the affinity (K0) described in Table 9. D (or lower affinity) binds to LTBR. Affinity can be determined as shown in Example 2.

[0463] Suitable, the EC50 value of the LTBR-binding peptide is 9.0E-09 M or lower, for example, 8.0E-09 M or lower, for example, 7.0E-09 M or lower, for example, 6.0E-09 M or lower, for example, 5.0E-09 M or lower, for example, 4.0E-09 M or lower, for example, 3.0E-09 M or lower, for example, 2.0E-09 M or lower, for example, 1.0E-09 M or lower, for example, 9.0E-10 M or lower, for example, 8.0E-10 M or lower, for example, 7.0E-10 M or lower, for example, 6.0E-10 M or lower, for example, 5.0E-10 M or lower, for example, 4.0E-10 M or lower, for example, 3.0E-10 M or lower, for example, 2.0E-10 M or lower, for example, 1.0E-10 M or lower, for example, 9.0E-11 M. M or lower, such as 8.0E-11 M or lower, such as 7.0E-11 M or lower, such as 6.0E-11 M or lower, such as 5.0E-11 M or lower, such as 4.0E-11 M or lower, such as 3.0E-11 M or lower, such as 2.0E-11 M or lower, such as 1.0E-11 M or lower, such as 9.0E-12 M or lower, such as 8.0E-12 M or lower, such as 7.0E-12 M or lower, such as 6.0E-12 M or lower, such as 5.0E-12 M or lower, such as 4.0E-12 M or lower, such as 3.0E-12 M or lower, such as 2.0E-12 M or lower, such as 1.0E-12 M or lower.

[0464] Alternatively, the LTBR-binding polypeptide may have an EC50 value or lower as described in any of the clones specified in the example table.

[0465] The above-described features of the multispecific constructs of the present invention are also applicable to certain embodiments of the polypeptides bound to LTBR in the present invention.

[0466] The present invention also provides an anti-LTBR polypeptide or construct, wherein the polypeptide or construct is used in combination with a cancer vaccine to treat cancer. Suitably, the polypeptide or construct may be used in combination with an adjuvant along with the cancer vaccine. Suitably, the polypeptide, construct, cancer vaccine, and / or adjuvant may be administered sequentially, alone, or simultaneously. In some embodiments, the cancer vaccine may be an antigenic peptide or a polynucleotide (e.g., RNA (such as mRNA) or DNA) encoding an antigen, optionally contained in a pharmaceutically acceptable excipient. The cancer vaccine may be a tumor-associated antigen or encode a tumor-associated antigen. Suitably, the cancer vaccine comprises or consists of the polypeptide sequence KCLQDNNWDYTRYAQAFTLLKAKG (SEQ ID NO: 209). In one embodiment, the adjuvant is a TLR receptor agonist, such as a TLR-9 agonist, like a CpG oligonucleotide.

[0467] Peptides that bind to FAP

[0468] In one embodiment, the polypeptide that binds to FAP comprises or consists of a pair of heavy chain variable domains and light chain variable domains (e.g., a Fab fragment), as appropriately further defined below.

[0469] In one embodiment, the heavy chain variable domain binding to FAP comprises or consists of three complementarity-determining regions (HCDR1 to HCDR3) and four frame regions (HFR1 to HFR4), wherein HCDR1 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 117, 123, 129, 135, 141, 147, or 153; HCDR2 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 118, 124, 130, 136, 142, 148, or 154; and HCDR3 ... SEQ ID NO: 119, 125, 131, 137, 143, 149, or 155 have a polypeptide sequence or constitutes thereof with 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity, and the light chain variable domain binding to FAP comprises or constitutes thereof three complementarity-determining regions (LCDR1 to LCDR3) and four frame regions (LFR1 to LFR4), wherein LCDR1 comprises or constitutes thereof a polypeptide sequence or constitutes thereof with 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity, and LCDR2 comprises or constitutes thereof a polypeptide sequence or constitutes thereof with SEQ ID NO: 120, 126, 132, 138, 144, 150, or 156, and LCDR2 comprises or constitutes thereof a polypeptide sequence or constitutes thereof with 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity, and LCDR2 comprises thereof a polypeptide sequence or constitutes thereof with sequence identity, and LCDR3 comprises thereof a polypeptide sequence or constitutes thereof with sequence identity, and LCDR4 comprises thereof a polypeptide sequence or constitutes thereof with sequence identity, and LCDR5 comprises thereof a polypeptide sequence or constitutes thereof with sequence identity, and LCDR6 comprises thereof a polypeptide sequence or constitutes thereof with sequence identity, and LCDR7 comprises thereof a polypeptide sequence or constitutes thereof with sequence identity, and LCDR8 comprises thereof a polypeptide sequence or constitutes thereof with sequence identity, and LCDR9 comprises thereof a polypeptide sequence or constitutes thereof with sequence identity, and LCDR1 comprises thereof a polypeptide sequence or constitutes thereof with sequence identity, and LCDR2 comprises thereof a polypeptide sequence or constitutes thereof with sequence identity, and LCDR3 comprises thereof a polypeptide sequence or constitutes thereof with sequence identity, and 121, 127, 133, 139, 145, 151 or 157 having a sequence identity of 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher or 95% or higher, and LCDR3 containing or consisting of a polypeptide sequence having a sequence identity of 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher or 95% or higher with SEQ ID NO: 122, 128, 134, 140, 146, 152 or 158.More appropriately, HCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 117, 123, 129, 135, 141, 147, or 153; HCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 118, 124, 130, 136, 142, 148, or 154; and HCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 119, 125, 131, 137, 143, 149, or 155; LCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 120, 126, 132, 138, 144, 150, or 156; LCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 121, 127, 133, 139, 145, 151, or 157; and LCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 118, 124, 130, 136, 142, 148, or 154. The polypeptide sequence of 122, 128, 134, 140, 146, 152 or 158 or composed of such sequences.

[0470] In one embodiment, the heavy chain variable domain binding to FAP comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher sequence identity with the sequence of SEQ ID NO: 43, 45, 47, 49, 51, 53, or 55, and the light chain variable domain binding to FAP comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher sequence identity with the sequence of SEQ ID NO: 44, 46, 48, 50, 52, 54, or 56, and ...4, or 56, and the sequence of SEQ ID NO: 44, 46, 48, 50, 52, 54, or 56, and the sequence of SEQ ID NO: 45, 97% or higher, 98% or higher, or 99% or higher sequence identity with the sequence of SEQ ID NO: 46, 97% or higher, or 98% or higher, or 99% or higher sequence identity with the sequence of SEQ ID NO: 46, 97% or higher, or 98% or higher sequence identity with the sequence of SEQ ID NO: 46, 97% or higher, or 98% or higher sequence identity with the sequence of SEQ ID NO: More appropriately, the heavy chain variable domain comprises or is composed of the polypeptide sequence of SEQ ID NO: 43, 45, 47, 49, 51, 53 or 55, while the light chain variable domain comprises or is composed of the polypeptide sequence of SEQ ID NO: 44, 46, 48, 50, 52, 54 or 56.

[0471] Preferably, the HCDR and LCDR are provided in the same combination as the heavy chain variable domains and light chain variable domains combined with FAP listed in Table 17A. For example, it is preferred to use a combination of (a) HCDR 1-3 of SEQ ID NO: 117, 118 and 119 and (b) LCDR 1-3 of SEQ ID NO: 120, 121 and 122 (as listed in clone 3.5), for example in a Fab. The same principle also applies to the combinations of variable regions listed in Table 16.

[0472] The present invention also provides a polypeptide that binds to FAP, comprising or consisting of a polypeptide sequence of clone 3.1, 3.2, 3.3, 3.4, 3.5, 3.6 or 3.7; or an antibody that competes with the anti-FAP polypeptide of the present invention for binding to FAP.

[0473] Appropriately, the FAP-binding polypeptide specifically binds to FAP, for example, its binding dissociation constant (KD) with any other entity is 10. -7 M or higher, such as 10 -6 M or higher. In some embodiments, the FAP is located on the surface of mesenchymal cells (e.g., CAFs) and / or cancer cells. Appropriately, the FAP-binding peptide specifically binds to the extracellular domain of the FAP.

[0474] Suitable, the FAP-binding peptide binds to FAP with the following affinity (KD): 400 pM or lower, for example, 300 pM or lower, for example, 200 pM or lower, for example, 150 pM or lower, for example, 130 pM or lower, for example, 110 pM or lower, for example, 100 pM or lower, for example, 50 pM or lower. Alternatively, the binding affinity (KD) of the FAP-binding peptide to FAP is as listed in Table 13 or lower. Suitable, this affinity is determined as described in Example 3.

[0475] Suitable, the FAP-binding peptide has an EC50 value of 9.0E-09 M or lower, such as 8.0E-09 M or lower, such as 7.0E-09 M or lower, such as 6.0E-09 M or lower, such as 5.0E-09 M or lower, such as 4.0E-09 M or lower, such as 3.0E-09 M or lower, such as 2.0E-09 M or lower, such as 1.0E-09 M or lower, such as 9.0E-10 M or lower, such as 8.0E-10 M or lower, such as 7.0E-10 M or lower, such as 6.0E-10 M or lower, such as 5.0E-10 M or lower, such as 4.0E-10 M or lower, such as 3.0E-10 M or lower, such as 2.0E-10 M or lower, such as 1.0E-10 M or lower, such as 9.0E-11 M. M or lower, such as 8.0E-11 M or lower, such as 7.0E-11 M or lower, such as 6.0E-11 M or lower, such as 5.0E-11 M or lower, such as 4.0E-11 M or lower, such as 3.0E-11 M or lower, such as 2.0E-11 M or lower, such as 1.0E-11 M or lower, such as 9.0E-12 M or lower, such as 8.0E-12 M or lower, such as 7.0E-12 M or lower, such as 6.0E-12 M or lower, such as 5.0E-12 M or lower, such as 4.0E-12 M or lower, such as 3.0E-12 M or lower, such as 2.0E-12 M or lower, such as 1.0E-12 M or lower. Alternatively, the FAP-binding peptide has an EC50 value that is the same as or lower than the EC50 value of any clone listed in the table in the examples.

[0476] The above-described features of the multispecific constructs of the present invention can also be applied accordingly to certain peptide embodiments bound to FAP in the present invention.

[0477] Peptides that bind to LTBR or FAP

[0478] The polypeptides that bind to LTBR or FAP may contain an antibody, for example, consist of a single antibody, or may contain or consist of one or more antibody fragments. Suitable fragments may include, for example, scFv, Fv, Fab, Fab', F(ab')2, variable domains (e.g., VH, VL, VNAR, or VHH), biantibodies, or microantibodies.

[0479] These peptides may also contain an additional binding agent that is neither an LTBR binding agent nor an FAP binding agent. In some embodiments, these peptides may not contain any additional binding agent.

[0480] The present invention also provides an antibody that binds to an epitope of either the anti-LTBR or anti-FAP polypeptide of the present invention.

[0481] The anti-LTBR or anti-FAP peptides of the present invention can be used as pharmaceuticals, for example, for treating cancer; or can be used to manufacture pharmaceuticals, for example, for treating cancer. The present invention provides a method of treating cancer, comprising administering the anti-FAP or anti-LTBR peptides of the present invention to an individual requiring the treatment. The cancer can be a tumor or a non-hematologic cancer.

[0482] The present invention also provides one or more polynucleotides encoding the anti-FAP or anti-LTBR polypeptide of the present invention, one or more expression vectors comprising such polynucleotides in a set, and cells comprising such expression vectors or polynucleotides. The present invention also provides a method for producing the anti-FAP or anti-LTBR polypeptide of the present invention, the method comprising culturing cells under suitable conditions to induce polynucleotide expression and produce the polypeptide.

[0483] The present invention is further illustrated by the following non-limiting examples.

[0484] 1. Example 1: Generation of anti-LTBR nanobody (VHH)

[0485] 1.1 Immunization and Library Construction

[0486] VHH was generated by immunizing llamas and alpacas with recombinant proteins, essentially using the same method described in the literature (Pardon et al., 2014) (Henry & MacKenzie, 2018). In short, the animals were immunized with human LTBR human IgG1 Fc chimera (Acro Biosystems, product number # LTR-H5251).

[0487] The preparation and use of phage display libraries derived from peripheral blood mononuclear cells (PBMCs) were as previously described (Pardon et al., 2014) (Henry & MacKenzie, 2018). The VHH fragment was cloned into an M13 phagemid vector containing Myc and His6 tags. The library was rescued by superinfection with *E. coli* TG1 cells grown at the infection index and using VCSM13 helper phages.

[0488] The phage library used for human LTBR immunization underwent two rounds of successive selection for human LTBR human IgG1 Fc chimeric protein (SinoBiological, product number #10581-H02H).

[0489] 1.2 Screening for anti-LTBR VHH

[0490] In the combined ELISA, 830 VHH clones from immunization and selection candidates were screened using crude periplasmic extract. Plates were coated with 5 µg / ml NeutrAvidin in PBS (incubated overnight at 4°C) and then blocked with 1% casein in PBS (at room temperature for 2 hours). Subsequently, 10 nM biotinylated hLTBR (OriGene, ID # AR51717PU-S) was captured in 1% casein in PBS (at room temperature for 1 hour). Detection was performed using 5 µg / ml anti-c-myc antibody 9E10 (Merck, ID # 11667203001) and 0.16 µg / ml anti-mouse IgG-HRP (Jackson Immuno Research, ID # 715-035-150), both in 1% casein in PBS (at room temperature for 1 hour). Sequence analysis of 808 positive clones revealed sequences belonging to 149 distinct related sequence clusters. Then, dissociation rate analysis was used to confirm the binding affinity of 270 unique sequences to human and cynomolgus monkey LTBRs (as described below).

[0491] To confirm species cross-reactivity, recombinant human or cynomolgus monkey LTBRs fused to human Fc (SinoBiological, numbers #10581-H02H and 90101-C02H) were immobilized on a CM5 sensor chip (number #CFJB334). A 1:5 dilution of crude periplasmic extracts from clones expressing VHHs was injected at 30 µl / min into HBS-EP pH 7.4 buffer for 2 min to allow binding to the chip-bound antigen. Then, HBS-EP buffer (pH 7.4) without VHHs was injected at the same flow rate for 5 min to allow spontaneous dissociation of the bound VHHs. Surface regeneration was performed between samples using 1 mM glycine (pH 1.5) / 1 M NaCl, with 10 µl injected twice. Sensor spectra obtained using a Biacore T200 instrument were fitted with a 1:1 interaction model (Langmuir model) using Insight Evaluation Software (Biacore) to calculate koff values. Based on this analysis, four clones were selected for further evaluation, and the results are shown in Table 1. The results confirmed that the selected clones could distinguish between humans and cynomolgus monkey LTBRs with comparable binding strength.

[0492] Table 1. Dissociation rate analysis of selected leads in humans and cynomolgus monkeys using Biacore.

[0493]

[0494] The synthetic DNA fragment encoding VHH was cloned into an *E. coli* expression vector, expressed under the control of an IPTG-induced lac promoter, and contained within a frame of reference for the N-terminal PelB signal peptide (which directs the recombinant protein into the periplasmic space) and the C-terminal FLAG3 and His6 tags. The VHH protein was purified from these clones using IMAC chromatography and desalted according to an established procedure (Pardon et al., 2014).

[0495] The purified protein was tested for its binding affinity to endogenously expressed human LTBR in HepG2 cells. Expression levels were confirmed using the anti-LTBR clone CBE-11 (“huCBE11” as described in WO2004002431). HepG2 cells were resuspended in FACS buffer to a final concentration of 1.0 × 10⁻⁶. 6 Cells / mL. A series of diluted purified VHH was incubated with mouse anti-FLAG biotinylated antibody (Sigma Aldrich, product number: F9291-1MG) at a concentration of 5 µg / mL in FACS buffer at room temperature with shaking for 30 min. The cell suspension was aliquoted into 96-well V plates and incubated with the above VHH / antibody mixture on ice with shaking for 1 h. VHH binding to cells was detected using streptavidin R-PE (Invitrogen, product number: SA10044) at a dilution of 1:400 (0.18 µg / mL) in FACS buffer, with shaking on ice for 30 min and protection from light. Cells were obtained by flow cytometry, and data were analyzed using a FloJo V10 and GraphPad Prism. EC50 values ​​are shown in Table 2.

[0496] Table 2. EC50 values ​​of HepG2 cell line binding curves

[0497] LTBR activation occurs via the aggregation of multiple LTBR receptors, leading to activation of an intracellular signaling cascade, through the ligands LTα1β2 or LIGHT. Therefore, monovalent molecules are not expected to activate the receptors. Human LTBR activation was assessed using NFKB luciferase reporter gene HepG2 cells (endogenous LTBR expression, Signosis, product number #SL-0017) with or without cross-linked anti-His antibodies.

[0498] For reporter gene assays, cells are prepared at a density of 3 × 10⁻⁶. 4Cells were seeded at a density of 100 cells / well in 96-well plates (Greiner) and cultured overnight at 37°C and 5% CO2 in Eagle Minimum Essential Medium (EMEM, Gibco) supplemented with 10% heat-inactivated FBS and 100 U / mL penicillin and streptomycin (Gibco). Cells were then cultured for 6 hours with monovalent His-tagged VHH crosslinked with an anti-His-tagged monoclonal antibody (Jackson, product number #300-005-240). After 6 hours, ONE-Glo Ex solution (Promega, product number E8120) was added to the wells and incubated for 10 minutes. Readings were then performed using a BMG Pherastar FSX plate reader, and background-corrected RLU values ​​relative to the stimulus (antibody or antibody fragment) concentration were plotted in GraphPad Prism for all test conditions. EC50 values ​​are shown in Table 3. Figure 1a and 1b The results show that, as expected, monovalent VHH does not lead to LTBR activation when not crosslinked with an anti-His tag antibody.

[0499] Table 3. EC50 values ​​from HepG2 reporter gene assay

[0500] Sequence optimization of unit price VHH

[0501] Four selected VHH proteins were sequenced to enhance their humanization of the human IGHV3 and JH germline common sequences and improve their chemical and physical stability while minimizing their impact on target binding. To this end, different mutant variants were generated for each lead and their ability to competitively bind to endogenously expressed human LTBR and its respective FLAG3-tagged parental sequences in HepG2 cells was compared using competitive flow cytometry. The melting point (Tm) of the isovariates was determined. These variants were also subjected to temperature stress testing (1 week at 40°C), followed by analysis of size exclusion chromatography combined with multi-angle laser scattering (aSEC-MALLS) to assess their oligomerization tendency. Furthermore, VHHs underwent long-term temperature stress testing (4 weeks at 40°C) and forced oxidative stress testing (10 mM H2O2 at 37°C for 3 hours), and their amino acid stability was assessed by detailed peptide mapping mass spectrometry. Amino acid residues with poor chemical stability (modification rate >5% after relevant stress tests) were replaced with suitable substitutes. Sequence-optimized mutants with the desired properties were selected for further evaluation.

[0502] 1.4 Construction and functional testing of the VHH-Fc fusion protein

[0503] The VHH-Fc fusion protein was generated by binding a sequence-optimized anti-LTBR VHH to the Fc region of human IgG1, separated by a flexible glycine-serine linker. These constructs were cloned into expression vectors and expressed via transient CHO suspension cell transfection. The VHH-Fc fusion protein was purified from cell culture supernatant using protein A via affinity chromatography, followed by a single purification chromatography step: preparative size exclusion chromatography (SEC).

[0504] The purity and heterogeneity of the samples were assessed by SEC-HPLC and mass spectrometry. All samples were confirmed to contain ≥95% monomer content and <10% impurities before functional readings. A list of constructs is available in Table 4.

[0505] Table 4. List of VHH-Fc Fusion Builds

[0506] NFKB activation in HepG2 reporter cells expressing human LTBR

[0507] The ability of the anti-LTBR VHH-Fc fusion protein to activate LTBR was tested in the NFKB luciferase reporter gene HepG2 (Signosis, catalog number SL0017-FP). Accumulation of endogenous LTBR in HepG2 cells led to activation of endogenous NFKB, causing it to translocate from the cytoplasm and nucleus, bind to the promoter region, and induce luciferase expression. Subsequently, a luciferase matrix / lysis reagent mixture (ONE-GLO EX Promega) was added to the cells, and the enzyme activity was quantified in a bioluminescent reaction, where the enzyme activity was directly proportional to luciferase expression.

[0508] A monospecific anti-LTBR agonist (BHA10, as disclosed in WO2004002431) was used as a positive control. Briefly, HepG2 NFKB luciferase (Lc) cells (30,000 cells / well) were seeded, and serial 1:10 dilutions of the VHH-Fc fusion protein and the control were added to the cells. The cells were incubated at 37°C, 5% CO2 for 6 hours, followed by the addition of the assay reagent. After 6 hours, ONE-Glo Ex solution (Promega, catalog number E8120) was added to the wells and incubated for 10 minutes, followed by reading using a BMG Pherastar FSX plate reader. Background-corrected RLU values ​​relative to stimulus concentrations were plotted in GraphPad Prism for all test conditions, and EC50 values ​​are shown in Table 5. The results confirmed that constructing the monomeric VHH as a bivalent molecular line resulted in LTBR activation with potency comparable to or higher than the control monoclonal antibody (BHA10).

[0509] Table 5. EC50 values ​​of luciferase activity induced by HepG2 NFKB activation

[0510] 1.4.2 Chemokinetic assay of VHH Fc fusion protein activity in vitro using tumor cell line HCC1187

[0511] An activation assay for measuring chemokine secretion was used to investigate the ability of the anti-LTBR VHH-Fc fusion protein to activate endogenously expressed LTBR in tumor cells (HCC1187). In short, HCC1187 cells (20,000 cells per well) were seeded in growth medium. The antibody and control were serially diluted 7 times at a 1:10 ratio (double-diluted) to prepare a stock solution at twice the concentration in the assay medium. After adding the stock solution to the cells, the cells were incubated at 37°C and 5% CO2 for 24 hours. After incubation, the supernatant was collected, clarified by centrifugation, and chemokine levels were measured using a CCL19 DuoSet ELISA (R&D system, DY361) according to the manufacturer's instructions. Absorbance was read using a BMG Pherastar FSX plate reader. The OD correction values ​​(450–540 nm) of the standard curve were analyzed in Prism according to the manufacturer's recommendations, and the cytokine concentrations in the test wells were calculated by interpolation. Dose-response curves were plotted using GraphPad Prism Version 9 with a nonlinear fit (log(agonist) vs response(variable slope - four parameters)). EC50 values ​​are shown in Table 6.

[0512] Table 6. EC50 values ​​of CCL19 upregulation in HCC1187 chemokine induction assay

[0513] Figure 2 The anti-LTBR VHH-Fc fusion protein induced dose-dependent CCL19 production in HCC1887 cells.

[0514] Ligand receptor inhibition assay

[0515] The ability of the VHH-Fc fusion protein to bind to endogenously expressed LTBR on A549 tumor cells and to inhibit the binding of human LIGHT (Acro, product number LIT-H5242) to A549 cells was evaluated. BHA10 was used as a positive control in the experiment. Recombinant human LIGHT was labeled with PE fluorescein using the PE / R-phycoerythrin (R-PE) conjugation kit – Lightning-Link® (Abcam, product number ab102918) – according to the manufacturer's instructions. 1x10 6 Cells were suspended in FACS buffer (1% BSA, 0.1% sodium azide in PBS), and then 50 µl (5 x 10) was added to each well of a 96-well U-shaped plate. 5 Cells were washed and resuspended in 25 µl of antibody dilution (2-fold final concentration) and incubated at 4°C for 0.5 h. Subsequently, 25 µl of PE-labeled LIGHT at 48 nM (2-fold final concentration) in FACS buffer was added to the cells, and incubation continued at 4°C for 1 h. After washing, the cells were further stained with a secondary detection antibody, namely Alexa Fluor® 647 conjugated AffiniPure Fab fragment goat anti-human IgG (H+L) (Stratech, product number 109-607-003), and incubated at 4°C for 1 h. The cells were then thoroughly washed and fixed for 20 min with 4% paraformaldehyde solution in PBS (Thermo Scientific, product number J19943.K2), following the manufacturer's instructions. The cells were then washed, resuspended in PBS, and data were read on a Beckman Coulter CytoFLEX instrument. Data were analyzed using FloJo V10 and GraphPad Prism v9. The IC50 and EC50 values ​​are shown in Table 7.

[0516] Table 7. IC50 values ​​of ligand-receptor inhibition and EC50 values ​​of binding curves with LTBR-positive cell line (A549)

[0517] Figure 3a All tested antibodies were confirmed to bind strongly to LTBR (tumor necrosis factor receptor superfamily member 14) endogenously expressed in the A549 tumor cell line.

[0518] Figure 3b All tested clones were confirmed to exhibit the ability to inhibit LTBR-LIGHT interaction, with IC50 values ​​ranging from 0.16 nM to 0.09 nM. The positive control antibody BHA10 showed partial inhibitory activity.

[0519] Epitope Competition Test on Octet

[0520] Epitope classification was performed using the Octet Red96 assay to confirm whether selected anti-LTBR VHHs competed for the same epitope. In short, 5 μg / ml of recombinant human LTBR was ligated to the AR2G biosensor at pH 5.0 via amine coupling, following the manufacturer's instructions. First, a first VHH was loaded onto the pre-coated LTBR biosensor until saturation, and then the sensor was exposed to a competitor / second VHH. If the second antibody detected additional binding on the biosensor, this indicated the presence of an unoccupied epitope (non-competitor). Conversely, if no binding of the second antibody was detected on the sensor, this indicated that the epitope was blocked (competitor). Results showed that only clone 1.10 bound to a distinct epitope, while all other clones bound to an overlapping epitope.

[0521] 2. Example 2: Generation of anti-LTBR monoclonal antibody

[0522] 2.1. Immunization

[0523] Five transgenic humanized mice (ATX-GK Cross) from each group were immunized with either recombinant human LTBR protein or plastid DNA encoding human LTBR cDNA. The standard 5-week RIMMS protocol for protein was used (10 μg of antigen emulsified in complete Freund's adjuvant subcutaneously, followed by 5 weeks of subcutaneous administration of antigen emulsified in incomplete Freund's adjuvant), or the protocol for DNA elongation was used (25 μg co-injected with pBoost4 adjuvant vector, intradermal (ID) once a week for six weeks, followed by intraperitoneal (IP) injection of 10 μg of recombinant protein before fusion).

[0524] Bleeding samples were collected in the fourth week, and the titers of antigen-positive and purified label-negative serum were tested by ELISA. ELISA plates were coated with the following materials: 1 μg / ml of human LTBR / human Fc fusion protein (AcroH5259), cynomolgus monkey LTBR / human Fc fusion protein (R&D 10759-LR), human LTBR / mouse Fc fusion protein (M-300-15, in-house produced), mouse LTBR / mouse Fc fusion protein (R&D 1008-LR), or an in-house mouse Fc control group (P-110). Antigen-coated plates were incubated with 8 spots of 10-fold serial dilution of serum at an initial concentration of 1:100. Antigen-binding antibodies were detected using anti-mouse IgG HRP secondary antibody and a one-step TMB solution. The absorbance signal at 450 nm was measured using an ELISA microplate reader.

[0525] 2.2. Fusion tumor

[0526] Immunological tissues were collected and stored from high-titer mice for antibody development. Fusion tumor cell lines producing LTBR antibodies were generated by fusing single B cells from the spleen and lymph nodes of titer-positive mice with myeloma cells. Twenty 96-well plates of fusion tumor cells were generated and expanded. Antibodies specifically binding to LTBR in the fusion tumor cell supernatant were detected by ELISA antigen binding. Antibody affinity in the fusion tumor supernatant was measured using the Octet instrument via biolayer interferometry (BLI). The LTBR antibody in the fusion tumor supernatant was loaded onto a biosensor, and the reaction was measured as nanoscale displacement of the interference pattern, which was proportional to the amount of antibody bound to the biosensor surface. The binding interaction between LTBR and the immobilized antibody was measured as binding force (kon). After analyte binding, the biosensor was immersed in LTBR-free PBS, causing the bound antigen to dissociate from the antibody (kdis), i.e., KD(M), or the antibody affinity to LTBR was measured as kdis / kon.

[0527] The heavy and light chains of validated fusionomas were sequenced. RNA was isolated from fusionomas secreting LTBR antibodies, and the variable regions of the heavy and light chains were cloned by reverse transcription using gene-specific primers followed by PCR amplification using variable region gene-specific primers. The PCR products were sequenced using standard Sanger sequencing methods.

[0528] 2.3. Phage Display

[0529] Variable heavy and light chains were amplified from the spleens of high-titer immunized mice using reverse transcription with gene-specific primers followed by PCR amplification with variable region gene-specific primers. These two variable regions were cloned into a phage display vector designed to express Fabs on the phage g3p protein, thus creating diverse libraries. For common light chain libraries, as before, only the heavy chain variable region was cloned into the phage display vector. Phage libraries expressing unique Fabs were amplified and purified. Phages were then bound to biotinylated LTBR antigens (human, mouse, or cynomolgus monkey) captured on magnetic streptavidin beads. After several rigorous washes, the phage system still bound to the antigen beads was eluted with alkaline triethylamine solution and neutralized with Tris buffer at pH 8.0. The eluted phages were re-infected with TG1 bacterial cells, amplified by co-infection with M13 helper phages, and purified by PEG precipitation. Purified phages expressing Fabs were screened for antigen binding according to the method described. The second round of selected phages were diluted and used to infect TG1 cells. After two rounds of selection, polycolonal pools of phages were tested by ELISA to confirm the presence of LTBR-specific phages. Variable heavy and light chain regions from single-infected bacterial colonies were sequenced using rolling circle amplification and standard Sanger sequencing.

[0530] 2.4. Antibody Expression

[0531] All unique variable heavy and light chain pairs obtained from the discovery techniques were cloned into vectors designed for the expression of full-length antibodies as IgG containing the LALA silencing mutation in HEK293 cells under the control of a CMV activator. The antibody expression vectors were conjugated with polyethyleneimine and transfected into HEK293 cell cultures. After 5 days of agitated culture in HEK293 cell medium at 37°C, the antibodies were captured on agarose-based protein A resin. After several rigorous washes, the antibodies were eluted with glycine solution at pH 3, neutralized with HEPES solution at pH 9, and the buffer was transferred to PBS. The purity of the purified proteins was confirmed by SEC-HPLC and CE-SDS analysis. Each antibody was assigned a unique clone number.

[0532] 2.5 Binding of anti-LTBR antibody to A375 tumor cell line

[0533] To facilitate binding to LTBR-positive cells, the A375 tumor cell line was used. LTBR-negative HEK293 cells were also tested to confirm specificity. Briefly, cells were resuspended in FACS binding buffer, and live / dead efluro450 viability dye was added to the cell suspension at a 1:1000 dilution, followed by incubation in the dark at room temperature for 30 minutes. The cell concentration was then adjusted to 2 × 10⁶ cells / year. 5 Cells / mL, with 100 μL added to the wells of a 96-well U-plate as needed. To generate EC50, a 100 nM stock solution was prepared and then serially diluted 3-fold at 12 spots. Cells were collected by centrifugation, and the supernatant was discarded. A series of diluted anti-LTBR antibody was added, and the cells were incubated at 4°C in the dark for 45 min. Cells were washed twice with PBS containing 3% (w / v) BSA, and then 60 μL of fluorescently labeled secondary antibody (goat anti-human IgG) diluted 1:200 in PBS was added. Incubation continued at 4°C for at least 30 min, followed by two washes with PBS at pH 7.4. Cell lines were fixed with 2% PFA (30 min at 4°C), washed, and stored in 100 μL of PBS, or analyzed immediately on a flow cytometer. Data were analyzed using a FloJo V10 and a GraphPad Prism. No binding to the HEK cell line was detected in any of the test clones, confirming the specific interaction between the anti-LTBR antibody and the LTBR receptor. The results are summarized in Table 8. Figure 4 shows the dose-dependent binding to the naturally occurring form of LTBR expressed on the A375 tumor cell line.

[0534] Table 8. EC50 values ​​of anti-LTBR antibody binding to A375 cell line

[0535] NP has not reached its plateau, so the EC50 cannot be evaluated.

[0536] 2.6. Binding kinetics of anti-LTBR antibody to human and cynomolgus monkey LTBR

[0537] The binding of anti-LTBR antibodies to human and cynomolgus monkey LTBR was investigated using surface plasmon resonance (SPR) assays on a Carterra LSA. Antibodies were captured using an EC30M chip. Chips were prepared for amine conjugation using EDC and sNHS activation at MES pH 5.5. Goat anti-human IgG Fc antibodies were then captured onto the chip via a 10-minute incubation. Excess binding sites were quenched with 1M ethanolamine at pH 8. To measure monovalent binding kinetics, antibodies were printed / spotted onto the chip and allowed to bind to the goat antibody, followed by washing. After preparation, the spotted chips were exposed to multiple binding and dissociation cycles with gradually increasing analyte concentrations. In this study, recombinant proteins (human LTBR (Acro) or cynomolgus monkey LTBR His (R&D)) were serially diluted 3-fold starting at 1500 nM for a total of 10 concentration points. Curve fitting was performed using a global fit of a 1:1 binding model to obtain KD values. Table 9 The binding affinity for both species was reported. The antibodies tested showed comparable affinity for both humans and cynomolgus monkey LTBR.

[0538] Table 9. KD values ​​of LTBR binding kinetics in humans and cynomolgus monkeys

[0539] 2.7 Epitope Competition Analysis of Carterra LSA

[0540] High-throughput epitope classification experiments were conducted using a real-time label-free biosensor (Carterra LSA) to classify a group of mAbs based on their mutual blocking abilities. In pairwise epitope classification analysis, antigen and antibody 2 (analyte antibody) were sequentially applied to a sensor chip (HC200M) pre-covalently preloaded with antibody 1 (ligand antibody). Upon exposure to the assay antibody, an increase in the reaction signal indicated no competition between the two antibodies; no change in the signal indicated competition. Antibodies with the same blocking properties in the test set were grouped into the same bin. Community network graphs were used to explore the clustering of monoclonal antibodies with similar but not necessarily identical competitive properties. Unlike sandwich / blocking clustering based solely on heatmaps, community clustering graphs employ a hierarchical clustering method, analyzing the sorted heatmaps to generate a tree diagram, progressively grouping the mAbs. The community clustering results for the tested anti-LTBR antibodies are listed in Table 10.

[0541] Table 10. Anti-LTBR antibody community grouping defined by epitope competition analysis on the Carterra platform

[0542] 2.8. NFKB activation in HepG2 reporter cells expressing human LTBR

[0543] To investigate the prokinetic function of anti-LTBR antibodies, the HepG2 cell line (Signosis, product number SL0017-FP) was used. In HepG2 cells, the accumulation of endogenous LTBR leads to the activation of endogenous NfKb, which translocates from the cytoplasm to the nucleus, binds to the promoter region, and induces luciferase expression. Subsequently, a luciferase matrix / lysis reagent mixture (ONE-GLO EX Promega) was added to the cells to quantify the enzyme activity in a bioluminescent reaction, where the activity was directly proportional to luciferase expression. The antibodies were tested at a 2:1 ratio with and without cross-linked antibodies (anti-human Fc, Jackson, product number 109-005-098). BHA10 was used as a positive control for this assay.

[0544] HepG2 NF-Kb Lc (30,000 cells / well) were individually seeded in their growth medium. Serial 1:10 dilutions of the monospecific antibody were added to the cells with or without a cross-linking agent, and the cells were incubated at 37°C, 5% CO2 for 6 hours, followed by the addition of the assay reagent. After 6 hours, ONE-Glo Ex solution (Promega, product number E8120) was added, and the cells were incubated for 10 minutes. Data were then read using a BMG Pherastar FSX plate reader. Background-corrected RLU values ​​for all test conditions were plotted against stimulus concentration in a GraphPad Prism and displayed separately. Figure 5a (without cross-linking antibodies) and Figure 5b (With cross-linked antibodies) The EC50 values ​​are shown in Table 11.

[0545] Table 11. EC50 values ​​of the activating activity of anti-LTBR antibodies in the HepG2 reporter gene assay (with or without cross-linking antibodies).

[0546]

[0547] A series of anti-LTBR antibodies with different activating activities were screened. In most cases, the presence of cross-linked antibodies showed higher potency.

[0548] 3. Example 3: Generation of anti-FAP antibodies

[0549] 3.1. Immunization

[0550] Two groups of transgenic humanized mice (n=5 per group) were immunized with either recombinant human FAP (Acro Bioscience, FAP-H5244) protein or plasso DNA encoding human FAP cDNA. For the protein, a standard 5-week RIMMS protocol was used (10 μg of antigen subcutaneously administered in a fully Freund's adjuvant-emulsified antigen, followed by five weekly subcutaneous administrations of antigen in a partially Freund's adjuvant-emulsified antigen), or an extension protocol for the DNA (25 μg co-injected with pBoost4 adjuvant vector, weekly intradermal injections for six weeks, followed by a final intraperitoneal booster injection of 10 μg of recombinant protein before fusion). Samples were collected at week 4 for ELISA testing of antigen-positive and purified tag-negative serum titers. ELISA plates were coated with 1 μg / ml of human FAP-His (Acro) or an internal His-tag control (CD22-His). Antigen-coated plates were incubated with serum at 10-fold serial dilutions starting at 1:100, in 8 spots. Antibodies binding to the antigen were detected using anti-mouse IgG HRP secondary antibody and a one-step TMB solution. Absorbance signal was measured at 450 nm using an ELISA microplate reader. Strong titers were observed in mice administered either the recombinant protein or cDNA human FAP on day 21, with no response to the His tag.

[0551] 3.2. Fusion tumor

[0552] Immune tissues were collected from high-titer mice and stored for antibody development. A fusion tumor cell line producing FAP antibodies was generated by fusing single B cells from the spleen and lymph nodes of titer-positive mice with myeloma cells. Twenty 96-well plates of fusion tumor cells were generated and expanded. Fusion tumors expressing FAP-specific antibodies were identified by antigen binding assay using ELISA. Antibody affinity in the fusion tumor supernatant was measured using an Octet instrument via biolayer interference (BLI). FAP antibodies from the fusion tumor supernatant were loaded onto a biosensor, and the reaction was measured as nanodisplacement of the interference pattern, proportional to the amount of antibody bound to the biosensor surface. The binding interaction between FAP and the immobilized antibody was measured as binding force (kon). After analyte binding, the biosensor was immersed in FAP-free PBS solution, causing the bound antigen to dissociate from the antibody (kdis), i.e., KD(M), or the antibody affinity for FAP was measured as kdis / kon.

[0553] The heavy and light chains of validated fusionomas were sequenced. RNA was isolated from FAP-secreting fusionomas, and the variable regions of the heavy and light chains were cloned by reverse transcription using gene-specific primers followed by PCR amplification using variable region gene-specific primers. The PCR products were sequenced using standard Sanger sequencing methods.

[0554] 3.3. Phage Display

[0555] Reverse transcription was performed on the spleens of mice immunized with high titers using gene-specific primers, followed by PCR amplification using variable chain gene-specific primers to amplify the variable heavy and light chains. Both variable regions were cloned into a phage display vector designed to express Fabs on the phage g3p protein to construct diverse libraries. For the common light chain library, as before, only the heavy chain variable region was cloned into the phage display vector. Phage libraries expressing unique Fabs were amplified and purified. Phages were then bound to biotinylated FAP antigens (human, mouse, or cynomolgus monkey) captured on magnetic streptavidin beads. After several rigorous washes, the phage system still bound to the antigen beads was eluted with alkaline triethylamine solution and neutralized with Tris buffer at pH 8.0. The eluted phages were re-infected with TG1 bacterial cells, amplified by co-infection with M13 helper phages, and purified by PEG precipitation. The purified phages expressing Fabs were screened for antigen binding as described above. The second round of selected phages were diluted and used to infect TG1 cells. The resulting phage polyclonal pool was then tested by ELISA to confirm the presence of FAP-specific phages. Variable heavy and light chain regions from single-infected bacterial colonies were sequenced using rolling circle amplification and standard Sanger sequencing.

[0556] 3.4. Antibody Expression

[0557] Unique variable heavy and light chain pairs from all discovered technologies were cloned into vectors designed to express full-length antibodies as IgG containing the LALA silencing mutation in HEK293 cells under the control of a CMV activator. The antibody expression vectors were conjugated with polyethyleneimine and transfected into HEK293 cell cultures. After 5 days of agitated culture in HEK293 cell medium at 37°C, the antibodies were captured on agarose-based protein A resin. After several rigorous washes, the antibodies were eluted with glycine solution at pH 3, neutralized with HEPES solution at pH 9, and the buffer was transferred to PBS. The purity of the purified proteins was confirmed by SEC-HPLC and CE-SDS analysis. Each antibody was assigned a unique clone number.

[0558] 3.5. Binding of anti-FAP antibody to the FAP-overexpressing HEK293 cell line

[0559] The binding of the antibody to human FAP expressed on transfected HEK293 cells was measured using FACS. Additionally, to confirm the antibody's specificity, binding to parental HEK293 cells was also tested. The specific procedure was as follows: the cell suspension was resuspended in FACS binding buffer, and live / dead efluro450 viability dye was added at a 1:1000 dilution. The cells were then incubated at room temperature in the dark for 30 minutes. Subsequently, the cells were diluted to 2 × 10⁶ cells / mL. 5 Cells / mL were collected, with 100 μL added to the wells of a 96-well U-shaped plate as needed. To generate EC50 values, a 100 nM antibody stock solution was prepared and 3-fold diluted at 12 spots. Cells were collected by centrifugation, and after removing the supernatant, a series of diluted anti-FAP antibody solutions were added, followed by incubation at 4°C in the dark for 45 min. Cells were washed twice with PBS containing 3% (w / v) BSA, followed by 60 μL of fluorescently labeled secondary antibody (goat anti-human IgG), diluted 1:200 in PBS. Incubation continued at 4°C for at least 30 min, followed by two washes with PBS at pH 7.4. Cells could be fixed with 2% PFA (30 min at 4°C), washed, and stored in 100 μL of PBS, or immediately analyzed by flow cytometry. No binding to the HEK parental cell line was detected in any of the tested clones, confirming the specific interaction between the anti-FAP antibody and the FAP receptor. Results are summarized in Table 12. Figure 6 The antibody was shown to bind in a dose-dependent manner to the HEK293 cell line overexpressing FAP.

[0560] Table 12. EC50 values ​​of anti-FAP antibody binding to FAP-HEK293 cell line

[0561] 3.6. Binding kinetics of anti-FAP antibodies to human and cynomolgus monkey FAP

[0562] The binding of anti-FAP antibodies to human and cynomolgus FAP was investigated using surface plasmon resonance (SPR) technology on a Carterra LSA. Antibodies were captured using an EC30M chip. The chip was prepared for amino coupling by bioconjugation activation with EDC and sNHS at MES pH 5.5. Goat anti-human IgG Fc antibodies were then captured onto the chip after a 10-minute incubation. Excess conjugated sites were quenched with 1M ethanolamine at pH 8. To measure monovalent binding kinetics, antibodies were printed / spotted onto the chip and allowed to bind to the goat antibody, followed by washing. Once prepared, the printed chip was exposed to multiple binding and dissociation cycles with analyte concentrations progressively increased. In this study, the concentrations of recombinant proteins (human FAP (Acro) or cynomolgus FAP His (Acro)) were started at 1500 nM and diluted 3-fold for a total of 10 concentration spots. Curves were globally fitted using a 1:1 binding model to obtain KD values. The binding affinity of anti-FAP antibodies to human and cynomolgus monkey FAP is reported in Table 13. The antibodies tested showed comparable affinity for human and cynomolgus monkey FAP.

[0563] Table 13. Binding affinity of anti-FAP antibodies to human and cynomolgus monkey FAP.

[0564] 3.7. Epitope Competition Analysis

[0565] High-throughput epitope clustering experiments were performed on a real-time label-free biosensor (Carterra LSA) to cluster a group of monoclonal antibodies (mAbs) based on their ability to mutually block antigen binding. In pairwise epitope clustering analysis, antigen and antibody 2 (analyte antibody) were sequentially applied to a sensor chip (HC200M) pre-covalently loaded with antibody 1 (ligand antibody). An increased response upon exposure to the assay antibody indicated no competition between the two antibodies; no change in signal indicated competition. Antibodies with the same blocking properties in the test set were grouped together. Community network graphs were used to explore mAbs sharing similar but not identical competitive properties. Unlike sandwich / blocking assignments that rely solely on heatmaps, community network graphs use hierarchical clustering to analyze sorted heatmaps to generate a dendrogram, progressively clustering mAbs. The community clustering results for the tested anti-FAP antibodies are shown in Table 14.

[0566] The results identified a diverse group of anti-FAP antibodies, which were selected for further evaluation.

[0567] Table 14. Community groupings defined by epitope competition analysis on Carterra

[0568] 3.8. Sequence optimization of anti-FAP antibody

[0569] Antibody variable regions that have undergone somatic mutations may contain framework residues that differ from the germline sequence derived from the antibody. Therefore, to reduce the immunogenicity of the antibody variable regions (VH and VL), the framework residues are restored to the corresponding human germline sequences. In short, different mutant variants are generated for each clone, and their ability to bind to recombinant FAP on the Octet is compared. In each case, the clone closest to the human germline sequence is selected for further evaluation.

[0570] 4. Example 4: Generating bispecific antibodies targeting FAP and LTBR

[0571] 4.1. Production of human and mouse-specific FAP / LTBR bispecific antibodies

[0572] Based on the results of Example 1, the identified anti-LTBR VHHs, after cross-linking with an external cross-linking agent (e.g., an anti-His antibody or when present as a bivalent molecule in the human IgG1 Fc region), exhibited activation activity in the NFKB reporter gene assay. These clones exhibited the most favorable functional properties, making them suitable for the evaluation of bispecific antibodies.

[0573] In previous examples, highly diverse panels of anti-FAP antibodies with multiple affinities and binding to different huFAP regions have been identified.

[0574] Humanized cynomolgus monkeys cross-reacting anti-LTBR agonist VHH (as described in Example 1) or anti-LTBR agonist antibody (as described in Example 2), and anti-FAP antibody (as described in Example 3) were used to generate constructs containing both FAP and LTBR binders. These constructs were FAP / LTBR bispecific agonist antibodies. Different forms of bispecific antibodies were prepared, binding bivalently or monovalently to LTBR and monovalently or bivalently to FAP, i.e., FAP:LTBR 1:1, 2:1, or 2:2 formulations. Figure 7 A through 7d show schematic diagrams of the various bispecific forms produced. These FAP / LTBR bispecific activating antibodies contain one or more FAP binders and one or more LTBR binders, specifically in the form of: (a) one or two anti-FAP Fab regions, and (b) one or two anti-LTBR VHHs or one or two anti-LTBR scFvs. Figure 7 a contains a VHH that is anti-LTBR, ​​replacing a Fab. Figure 7 b includes a VHH that resists -LTBR, ​​which is connected to the CH3 domain via a synthetic linker. Figure 7c contains a VHH that resists -LTBR and is connected to two CH3 domains via a synthetic linker, while Figure 7 d contains an anti-LTBR scFv that is connected to each CH3 domain via a synthetic linker. Figure 7 a contains a Fab that is anti-FAP, while Figure 7 b through 7d contain two anti-FAP Fabs. Each construct is assigned a unique clone number.

[0575] The variable region of the human-specific construct was cloned into an expression vector containing the constant region of human IgG1. In some cases, to generate a 2:1 or 2:2 form, the scFv or VHH containing the variable region sequence of the anti-LTBR antibody (Examples 1 and 2) was fused to the C-terminus of the Fc via a G4S linker (SEQ ID NO: 3). Due to potential stability issues with the scFv fragment, an additional disulfide bond was introduced at the VH44:VL100 position (Weatherill et al., 2012). To ensure proper assembly of the heavy chain, the well-known "knob-into-holes" strategy was employed (Merchant et al., 1998). Furthermore, to reduce binding to FcyRs, LALA (Leu234Ala and Leu235Ala) or PGLALA (Pro329Gly, Leu234Ala and Leu235Ala) mutations were introduced into the Fc region (Schlothauer et al., 2016).

[0576] To generate mouse alternative antibodies, the variable region of the mouse-specific construct was cloned into an expression vector containing the constant region of mouse IgG2a or mouse IgG1. To ensure proper assembly of the light and heavy chains, a publicly disclosed mutation in CH1 / CL was introduced to promote sufficient light chain pairing, and a charge-directing mutation was introduced in the CH3 domain to ensure specific heavy chain pairing (Wang et al., 2020). Similar to the human construct, to prevent binding to FcyRs, the Fc region was mutated with PGLALA (Pro329Gly, Leu234Ala, and Leu235Ala) mutations (Schlothauer et al., 2016). In some cases, to generate a 2:1 or 2:2 form, the scFv containing the 5G11 sequence of the variable region was fused to the C-terminus of the Fc via a G4S linker (SEQ ID NO: 3). Due to potential stability issues with the scFv fragment, an additional disulfide bond was introduced at the VH44:VL100 position (Weatherill et al., 2012).

[0577] Simultaneously, (i) monospecific control antibodies, (ii) anti-human LTBR control clones BHA10 and CBE-11 (as revealed in WO2004002431), (iii) anti-FAP clone 28H1 (as revealed in US10577429B2), and (iv) anti-mouse LTBR control clone (clone 5G11) were cloned into expression vectors containing the constant regions of human IgG1, rat IgG2a, or mouse IgG1. PGLALA mutations (Pro329Gly, Leu234Ala, and Leu235Ala, (Schlothauer et al., 2016)) were introduced into the constant regions of the human IgG1 / mouse IgG2a heavy chains to eliminate binding to FcyR.

[0578] In addition, FAP / LTBR bispecific comparative antibodies were produced, clones: P1AH5886, P1AG5459 and P1AG5461 (as disclosed in WO2023 / 117834A1).

[0579] Bispecific and monospecific control groups were expressed by transient transfection of CHO suspension cells. Antibodies were purified from cell culture supernatants using protein A affinity chromatography, followed by one or two-step chromatography: preparative size exclusion chromatography (SEC) and / or ion exchange chromatography to enrich heterodimers. Sample purity and diversity were assessed by SEC-HPLC and mass spectrometry. All samples were confirmed to have monomer content ≥95% and impurity content <10% prior to functional evaluation.

[0580] The generated bispecific and monospecific antibodies are listed in Table 15, and the clonal variable region sequences listed in the table are shown in Table 16.

[0581] Table 15. List of bispecific antibodies and monospecific control groups

[0582] All 1:1 type constructs listed in the table contain a truncated hinge region in HC1 (DKTHTCPPCP, sequence number: 203), while HC2 contains a native hinge region (EPKSCDKTHTCP, sequence number: 204). Constructs of other types contain a native hinge region in HC (EPKSCDKTHTCP, SEQ ID NO: 204).

[0583] Table 16. Variable region amino acid sequences of FAP and LTBR clones

[0584] Table 17A. CDR amino acid sequences of FAP and LTBR clones. For VHH, the HCDR sequence is provided. For paired VH / VL clones, the HCDR sequence is provided in the first row of each clone, followed by the LCDR sequence of the same clone in the second row.

[0585]

[0586] The heavy and light chain polypeptide sequences of clones 4.1 to 4.10 are as follows, where for homodimers, HC = heavy chain and LC = light chain. In the case of heterodimers, in addition to the polypeptide sequence of the light chain, polypeptide sequences of the first (HC1) and second (HC2) heavy chains are also provided.

[0587] Clones 4.1 HC1: SEQ ID NO: 175, HC2: SEQ ID NO: 176, LC: SEQ ID NO: 177

[0588] Clones 4.2 HC1: SEQ ID NO: 178, HC2: SEQ ID NO: 179, LC: SEQ ID NO: 180

[0589] Clones 4.3 HC1: SEQ ID NO: 181, HC2: SEQ ID NO: 182, LC: SEQ ID NO: 183

[0590] Clones 4.4 HC1: SEQ ID NO: 184, HC2: SEQ ID NO: 185, LC: SEQ ID NO: 186

[0591] Clones 4.5 HC1: SEQ ID NO: 187, HC2: SEQ ID NO: 188, LC: SEQ ID NO: 189

[0592] Clones 4.6 HC1: SEQ ID NO: 190, HC2: SEQ ID NO: 191, LC: SEQ ID NO: 192

[0593] Clones 4.7 HC1: SEQ ID NO: 193, HC2: SEQ ID NO: 194, LC: SEQ ID NO: 195

[0594] Clones 4.8 HC: SEQ ID NO: 196, LC: SEQ ID NO: 197

[0595] Clones 4.10 HC1: SEQ ID NO: 200, HC2: SEQ ID NO: 201, LC: SEQ ID NO: 202

[0596] Table 17B. Alternative CDR or LTBR VHH amino acid sequences. Alternative CDR sequences formed by combining nearly identical individual CDRs are provided, along with their amino acid sequences and SEQ ID NO:.

[0597]

[0598] 4.2. Binding of FAP / LTBR bispecific antibody to cells expressing human FAP and LTBR

[0599] To test the binding of the FAP / LTBR bispecific antibody to cells expressing human FAP, the CHO-K1 recombinant cell line overexpressing FAP (BPS, product number 79947) or the transient HEK293 cell line overexpressing FAP were used. To test the binding of the FAP / LTBR bispecific antibody to cells expressing human LTBR, ​​the A549 (ATCC, product number CCL-185) or A375 tumor cell lines endogenously expressing LTBR were used. In this experiment, the LTBR monospecific antibody (BHA10) was used as a control group.

[0600] In some cases, secondary antibodies can interfere with detection; therefore, according to the manufacturer's instructions, the bispecific antibody is directly labeled using the AlexaFluor® 647 Conjugation Kit (Fast) - Lightning-Link® (Abcam, product number ab269823). In this case, in addition to adding the secondary antibody, the staining protocol is as described below.

[0601] Specifically, 1 × 10 6The cells were resuspended in FACS buffer (1% BSA, 0.1% sodium azide in PBS), and 50 µl was added to each well of a 96-well U-plate (5 × 10⁶ cells per well). 4 Cells were washed once and resuspended in 50 µl / well of FACS buffer containing 1:3, 11-spot serial dilutions of constructs 4.1–4.6 and clones 4.8–4.9, and then incubated at 4°C for 1 hour. After thorough washing, cells were further stained with a secondary antibody, namely R-phycoerythrin-conjugated AffiniPure goat anti-human IgG, Fcγ fragment-specific antibody (Stratech, product number 109-115-098-JIR), and incubated at 4°C for 1 hour. Finally, cells were washed and resuspended with FACS buffer, and data were read using Beckman Coulter CytoFLEX. Data were analyzed using FloJo V10 and GraphPad Prismv9. As described in Examples 2.4 and 3.5, the binding affinity of clone 4.7 to A375 (LTBR-positive cell line) and HEK293-FAP-overexpressing cell line was tested. EC50 values ​​are shown in Table 18.

[0602] Table 18. EC50 values ​​of FAP / LTBR bispecific antibody binding to LTBR and FAP positive cell lines

[0603] NP = The plateau period has not been reached, and the EC50 cannot be evaluated.

[0604] As shown in the table above, all FAP / LTBR bispecific antibodies exhibited lower binding potency to LTBR-positive cells than the control group BHA10, whose EC50 values ​​were within the pM range. As mentioned above, lower binding affinity may be more advantageous in the context of activating antibodies.

[0605] 4.3. Activation of NFKB expressed by human LTBR in HepG2 reporter cells, co-cultured with CHO cell lines overexpressing FAP.

[0606] For FAP-mediated crosslinking, the FAP-CHO-K1 recombinant cell line (BPS, number 79947) was used.

[0607] To evaluate the conditional activation of LTBR by the FAP / LTBR bispecific antibody, these molecules were tested in the NFKB luciferase reporter gene HepG2 assay (Signosis, SL0017-FP) in CHO cell lines with or without FAP overexpression (FAP-CHO-K1). Accumulation of endogenous LTBR in HepG2 cells leads to activation of endogenous NFKB, which translocates from the cytoplasm to the nucleus, binds to the promoter region, and induces luciferase expression. Subsequently, a luciferase matrix / lysis reagent mixture (ONE-GLO EX, Promega) was added to the cells to quantify the enzyme activity in a bioluminescent reaction, where enzyme activity was directly proportional to luciferase expression.

[0608] The monospecific anti-LTBR agonist CBE-11 was used as a positive control group.

[0609] HepG2 NFKB Lc (30,000 cells / well) was co-seeded with or alone with FAP-overexpressing CHO cells (15,000 cells / well) in their growth medium. Bispecific antibodies and control groups were added to the cells at serial dilutions of 1:10 and incubated at 37°C, 5% CO2 for 6 hours, followed by the addition of assay reagents. After 6 hours, ONE-Glo Ex solution (Promega, product number E8120) was added to the wells and incubated for 10 minutes. Data were then read using a BMG Pherastar FSX plate reader, and background-corrected RLU values ​​relative to stimulus concentrations were plotted in GraphPad Prism for all test conditions. EC50 values ​​are shown in Table 19.

[0610] Table 19. EC50 values ​​of luciferase activity induced by HepG2 NFKB activation

[0611] The results confirmed that FAP / LTBR bispecific antibodies (1:1 and 2:1 forms) containing a monovalent LTBR arm activate LTBR in a FAP-dependent manner, and their activity is minimal or nonexistent in the absence of FAP cells, as shown in the HEPG2 reporter gene assay. Figure 27a c (single culture) and Figure 27b This was confirmed by representative data from co-culture (d). Compared to the control group monospecific antibody CBE-11, it activated HepG2 cells to a similar degree in the presence or absence of FAP-positive cells.

[0612] FAP / LTBR bispecific antibodies (clones 4.8 and 4.9) and the comparative antibody clone P1AH5886 (WO2023117834), containing two LTBR binding sites, retained greater LTBR activity in the absence of FAP. Compared to the monospecific control group, activity was further increased in the presence of FAP-positive cells. As stated above, this conditional activation should reduce the toxicity caused by the activation of LTBR-positive cells in the presence of FAP in tumors (rather than in tissues that do not express FAP).

[0613] Due to the complexity of bispecific antibodies, the lowest activity in single cultures may be due to the presence of some residual impurities in the sample.

[0614] 4.4. Chemokine induction assay of FAP / LTBR bispecific antibody using FAP-positive primary human mammary cancer cells co-cultured with tumor cell line HCC1187 to determine in vitro activity.

[0615] An activation assay measuring chemokine secretion was used to evaluate the ability of the LTBR / FAP bispecific antibody to conditionally accumulate on cells (tumor cell line HCC1187) and activate endogenously expressed LTBR.

[0616] Primary human mammary cancer cells (CAFs) were used as a source of FAP to determine the bioactivity of LTBR / FAP bispecific antibodies in physiologically relevant environments.

[0617] Flow cytometry was used to confirm LTBR expression in HCC1187 and primary human mammary CAFs.

[0618] HCC1187 cells (20,000 cells / well) were co-seeded with or seeded alone with primary human mammary cell-associated antigens (CAFs) isolated from cancer patients in their growth media. Bispecific antibodies and controls were prepared at a 1:10 ratio using seven-spot serial dilutions, with each concentration replicated twice. Stock solutions of twice the concentration were prepared in assay medium, and after cell addition, cells were incubated at 37°C and 5% CO2 for 24 hours.

[0619] After incubation, the supernatant was collected and clarified by centrifugation. Chemokine levels were then determined using a CCL19DuoSet ELISA (R&D system, product number: DY361) according to the manufacturer's instructions. Absorbance values ​​were read on a BMG Pherastar FSX plate reader. The OD correction values ​​(450-540 nm) of the standard curve were analyzed in Prism software as recommended by the manufacturer, and the cytokine concentrations in the test wells were calculated by extrapolation. Dose-response curves were plotted using GraphPad Prism Version 9, and a nonlinear fit (log(agonist) vs. response (variable slope - four parameters)) was applied.

[0620] FAP / LTBR bispecific antibody induces CCL19 secretion in HCC1187 cells in a dose-dependent manner. Figures 8a to 8f These data show that for molecules with a single LTBR binding site, LTBR activation is only achieved through LTBR aggregation in the presence of FAP-positive cells (primary human mammary CAFs in this example). Similarly, as shown in Example 4.3, bispecific constructs with two LTBR binding sites retain some function in single cultures. EC50 values ​​are shown in Table 20.

[0621] Surprisingly, the FAP / LTBR bispecific antibodies (clones 4.1 to 4.6) exhibited significantly higher potency in co-culture than the control antibody CBE-11, which can be explained by their lower affinity for binding to LTBR-positive cells. As reported in the literature, low-affinity anti-4-1BB and anti-CD40 monoclonal antibodies mediate higher agonistic activity, suggesting that low-affinity antibody-induced agonistic activity is a conserved feature of TNFR (Yu et al., Nature 2023).

[0622] Table 20. EC50 values ​​of CCL19 upregulation on HCC1187 in the presence or absence of FAP-positive primary human mammary cancer cells (CAFs)

[0623] 4.5. Activity of in vitro FAP / LTBR bispecific antibody on ICAM-1 of human mammary CAFs (catheter arterial fibrils) in a test.

[0624] It has been reported that stimulation of LTBR leads to activation of the classical NF-KB1-RelA and alternative NF-KB2-RelB pathways (Bénézech et al., 2012), which in turn leads to increased expression of chemokines CXCL13, CCl21 and CCL19, as well as adhesion molecules ICAM1 and VCAM1.

[0625] Single-culture assays were performed using primary human mammary gland CAFs to verify whether the FAP / LTBR bispecific antibody could upregulate adhesion molecules by activating LTBR. ICAM-1 expression was assessed by immunofluorescence staining.

[0626] The expression of two targets on primary human mammary carcinoma cells (CAFs) was confirmed by flow cytometry using a control antibody.

[0627] Human breast cancer-associated fibroblasts (CAFs) co-expressing two receptors (FAP and LTBR) were seeded in 96-well plates at a density of 20,000 cells / well and allowed to attach for 24 hours. Subsequently, a 7-point, 10-fold stock solution was prepared in culture medium to prepare a 7-point, 10-fold dilution series of bispecific antibodies (bsAb) and a control LTBR agonist, which was added to the cells as a 10-fold concentration stock solution and incubated at 37°C, 5% CO2 for 24 hours. After stimulation, cells were fixed by adding 100 µL of 8% formaldehyde (final concentration 4%) for 20 minutes, followed by washing with PBS. Cells were incubated at room temperature for 1 hour with blocking buffer (3% BSA and 0.3% Triton X-100 (w / v) in PBS) to prevent nonspecific binding. For immunostaining, cells were incubated overnight at 4°C with an affinity-purified rabbit monoclonal antibody against ICAM-1 (Abcam, catalog number ab282575, dilution 1 / 500). Cells were then washed three times with 100 µL of wash buffer (10 mM sodium phosphate, 0.15 M sodium chloride, 0.05% Tween-20 buffer) and incubated for 1 hour at room temperature with a polyclonal antibody against rabbit IgG (H+L) conjugated to secondary antibody AF546 (Invitrogen, catalog number A10040, 1 / 500 dilution, 3% BSA (w / v) in PBS). Simultaneously, cell nuclei were counterstained using Hoechst 33342 (Invitrogen, H3570, 1 / 10000 dilution). Cells were washed three more times with wash buffer and then transferred to PBS for imaging. Fluorescent images were acquired on a Cell Insight CX7 HCS platform. Image acquisition and analysis were performed using Thermo Scientific HCS Studio: Cellomics Scan V6.6.2 software and Spot DetectorBioapplication V4. This bioapplication measures fluorescence intensity at the single-cell level, allowing for rapid protein expression analysis. Data are presented as the average fluorescence intensity per cell (ICAM-1 expression) minus the intensity under untreated conditions (baseline correction), and plotted using GraphPad Prism Version 9.

[0628] Figure 9a and 9bThe results showed that, except for comparative example P1AH5886 (WO2023117834) and clone 4.10, the FAP / LTBR bispecific antibody achieved potent LTBR activation, which was measured by the upregulation of ICAM-1 in primary fibroblasts. Comparative example P1AH5886 failed to achieve potent LTBR activation in this experiment.

[0629] Therefore, we can conclude that anti-FAP / LTBR bispecific antibodies can act in both cis and trans directions, targeting LTBR expressed on one cell (e.g., the matrix) and FAP expressed on adjacent cells. Figures 8a-8f Zhongwei confirmed that the data is summarized in Table 21.

[0630] The 2:2 form of FAP / LTBR bispecific antibodies (clones 4.8 and 4.9) had the lowest EC50 values, indicating that their potency was higher than that of the 2:1 or 1:1 forms. However, as the previous examples show, the activity of the 2:2 form may be FAP-dependent or FAP-independent, as the bivalent LTBR form still retains some of its activating properties (examples 4.3 and 4.4).

[0631] Surprisingly, FAP / LTBR bispecific antibody clone 4.10 failed to upregulate ICAM-1 when FAP and LTBR were expressed in the same cells, even though it contained the same functional LTBR binder as clones 4.1, 4.3, 4.4, and 4.5. However, clone 4.10 contained a different FAP binder (binding to a different epitope). This unique epitope proved crucial in achieving ICAM-1 upregulation with the FAP / LTBR bispecific antibody.

[0632] Without being bound by theory, this observation may suggest that ICAM-1 upregulation can be achieved by simultaneously binding to FAP and LTBR (“cis”) on the same cell (while simultaneously binding to FAP on the first cell and LTBR on the second cell (“trans”) may not induce ICAM-1 upregulation). This FAP epitope may facilitate the simultaneous binding of FAP and LTBR on the same cell.

[0633] Table 21. EC50 values ​​of ICAM1 upregulation in CAF single-cell culture assays

[0634] 4.6. Activity of in vitro FAP / LTBR bispecific antibody against tumor cell line RPMI-7951 in chemokine induction assays

[0635] To investigate whether the FAP / LTBR bispecific antibody could effectively activate LTBR in cis-double-positive cells, such as fibroblast reticulocytes, which are crucial for the formation and maintenance of TLS, a monoculture assay was performed using the tumor cell line RPMI-7951. The expression of FAP and LTBR was confirmed by flow cytometry. Briefly, RPMI-7951 cells were seeded at a concentration of 50,000 cells per well in 96-well V-bottom plates and cultured in medium. A series of 7-point 10-fold dilutions of the FAP / LTBR bispecific antibody and the control LTBR agonist antibody CBE11 were prepared as 10-fold stock solutions in medium, and after adding the cells, the plates were cultured at 37°C and 5% CO2 for 24 hours. After 24 hours, the plates were centrifuged, and the supernatant was collected. The CCL5 level in the supernatant was determined using a Human CCL5 DuoSet ELISA kit (R&D system, product number DY278) according to the manufacturer's instructions. Absorbance values ​​were read using a BMG Pherastar FSX plate reader. Following the manufacturer's recommendations, corrected absorbance values ​​(450–540 nm) of the standard curves were analyzed in GraphPad Prism software to interpolate cytokine concentrations in the test wells. Dose-response curves were plotted using GraphPad Prism version 9, with a nonlinear fit applied (log(agonist) vs. response(variable slope – four parameters)). Data are summarized in Table 22.

[0636] Figure 10a and 10b The results showed that the FAP / LTBR bispecific antibody and the LTBR agonist control antibody potently activated LTBR, ​​leading to upregulation of the pro-inflammatory CCL5 chemokine. This contrasts with the comparative bispecific antibody P1AH5886, which did not induce CCL5 secretion in RPMI-7951 cells. The results suggest that the FAP / LTBR bispecific antibody can adequately aggregate LTBR even with co-expression of both receptors. Similarly, as described in Example 4.5, clones 4.8 and 4.9 induced higher levels of CCL5 compared to the 1:1 or 2:1 formulations of the FAP / LTBR bispecific antibody.

[0637] In summary, these data confirm that the FAP / LTBR bispecific antibody can fully activate LTBR in cis (when both receptors are co-expressed) and in a FAP-dependent manner, activate trans-LTBR in neighboring cells, and activate LTBR in a FAP-dependent manner (i.e., trans-activation) in neighboring cells, thereby inducing the production of chemokines (such as CCL19 and CCL5) and upregulating the expression of adhesion molecules (such as ICAM-1).

[0638] Table 22. Chemokines-induced EC50 values ​​in RPMI-7951 cells

[0639] 4.7. Transactivation of FAP / LTBR bispecific antibody in chemokine induction assays when low FAP-expressing cells (RPMI-7951) were co-cultured with HCC-1187.

[0640] The potency of the FAP / LTBR bispecific antibody was determined in the presence of a low-FAP-expressing cell line (RPMI-7951). Expression levels were confirmed by flow cytometry, and FAP expression was approximately 4-fold lower than that of previously used human breast cancer-associated fibroblasts (CAFs) (Example 4.4). Because RPMI-7951 and HCC1187 secrete unique chemokines upon LTBR stimulation, as described in Examples 4.4 and 4.6, both cis and trans activation could be assessed simultaneously.

[0641] Since HCC1187 cells are single-positive cells (containing LTBR on their surface but lacking FAP), for FAP / LTBR bispecific antibodies to aggregate LTBR and subsequently activate downstream signaling pathways, these bispecific antibodies need to bind simultaneously to both FAP expressed on RPMI-7951 cells and LTBR expressed on HCC1187 cells. If CCL19 secretion is observed after the addition of FAP / LTBR bispecific antibodies in this assay, it indicates trans-activation of LTBR. Cis-activation is characterized by RPMI-7951 cells simultaneously expressing FAP and LTBR, ​​and secreting CCL5 upon LTBR stimulation.

[0642] In summary, RPMI-7951 cells (20,000 cells / well) were seeded in 96-well plates for both monoculture and coculture, while culture plates containing only culture medium were used for monoculture of HCC1187 cells. These culture plates were incubated at 37°C and 5% CO2 for 24 hours before the addition of HCC1187 cells and the compound. The following day, HCC1187 cells (20,000 cells / well) were seeded in culture plates containing only culture medium, and RPMI-7951 coculture plates were prepared on day 1. A 10-fold stock solution was prepared using a 7-point, 10-fold dilution series of FAP / LTBR bispecific antibody and a control LTBR agonist antibody (CBE-11), and added to the cell culture plates. After 24 hours, the culture plates were centrifuged, and the supernatant was collected. Following the manufacturer's instructions, CCL5 and CCL19 chemokine levels in the supernatant were determined using the Human CCL5 DuoSet ELISA (R&D system, product number DY278) and the Human CCL19 DuoSet ELISA (R&D system, product number DY361). Absorbance was read using a BMG Pherastar FSX plate reader. As recommended by the manufacturer, the OD correction values ​​(450–540 nm) of the standard curve were analyzed using GraphPad Prism software, and cytokine concentrations in the test wells were calculated by interpolation. Dose-response curves were plotted using GraphPad Prism Version 9, and a nonlinear fit (log(agonist) versus response (variable slope – four parameters)) was applied. Data are presented in Table 23.

[0643] Table 23. EC50 values ​​of cis and trans activation in HCC1187:RPMI-7951 co-culture assay.

[0644] Figure 11a shows that the 1:1 form of the FAP / LTBR bispecific antibody (see Bispecific Antibodies 4.1, 4.2 and 4.3) can induce CCL19 secretion through FAP-mediated LTBR activation even at low FAP expression levels.

[0645] Figure 11b confirms the previous results described in Example 4.6, that the FAP / LTBR bispecific antibody can sufficiently accumulate LTBR on double-positive cells (FAP and LTBR positive) to lead to dose-dependent secretion of CCL5.

[0646] Combining these data with the results of Example 4.4 shows that the potency of the FAP / LTBR bispecific antibody is correlated with the FAP expression density on cells.

[0647] 4.8. The binding affinity of the LTBR / FAP bispecific antibody to recombinant LTBR and FAP in humans and cynomolgus monkeys was determined by Octet.

[0648] To confirm species cross-reactivity, the binding affinity of the dual-target antibody molecules to recombinant proteins in humans and cynomolgus monkeys was determined using Octet. This cross-reactivity is advantageous because it allows for the evaluation of antibody molecule dosage and safety in preclinical testing in cynomolgus monkeys.

[0649] In summary, biotinylated human LTBR-His-Avitag (Acro, product number LTR-H82E9-25 μg) was loaded into the SA streptavidin biosensor (Fortebio) and then serially diluted 2-fold in kinetic buffer (ForteBio) with an initial concentration of 50 nM FAP / LTBR bispecific antibody. Binding kinetics were studied in 1x kinetic buffer with a binding time of 100 seconds followed by a dissociation time of 300 seconds. Inter-step correlations were assessed by subtracting parallel buffer blanks, baseline alignment (y-axis), and dissociation alignment, and data smoothing was performed using a Savitzky-Gola filter in the data analysis software. The processed data were fitted using a 1:1 Langmuir binding model, with R² values ​​used as a measure of fitting accuracy. KD and R² values ​​are shown in Table 24.

[0650] Cynomolgus recombinant LTBR, ​​serially diluted twofold starting at 50 nM, was immobilized on the surface of an ARG biosensor (Fortebio), followed by the addition of 25 nM of FAP / LTBR bispecific antibody. Binding kinetics were studied in a 1-fold kinetic buffer with a binding phase of 180 seconds and a dissociation phase of 100 seconds. The sensor tip was regenerated using ethylene glycol. The generated data were referenced by subtracting parallel buffer blanks, baseline aligned to the y-axis, and inter-step correlation corrected by dissociation alignment. The data were smoothed using a Savitzky-Golay filter in the data analysis software. A 1:1 Langmuir binding model was used to fit the processed data, with R² values ​​used as a measure of fit accuracy. KD values ​​are shown in Table 24.

[0651] The binding kinetics of the two targets in clone 4.7 were studied as described in Examples 2.6 and 3.6, and their KD values ​​are shown in Table 24.

[0652] Binding studies of the recombinant FAP protein were performed using an Octet instrument via biolayer interference assay (BLI). Anti-Fc antibodies were immobilized on the surface of an ARG biosensor (Fortebio) using standard amino-coating chemistry. FAP / LTBR bispecific antibodies were loaded onto the pre-coated anti-Fc capture antibody surface, followed by the addition of 2-fold serial dilutions of cynomolgus monkey FAP (Acro, product number FAP-C82Q5) or human FAP (Acro, product number FAP-H82Q6) starting at 50 nM. Binding kinetics were studied in a 1-fold kinetic buffer with a binding phase of 200 seconds and a dissociation phase of 400 seconds. The sensor tip was regenerated using 10 mM glycine at pH 2.0. The generated data were referenced by subtracting parallel buffer blanks, baseline-aligned to the y-axis, and corrected for inter-step correlations by dissociation alignment. The processed data were smoothed using a Savitzky-Golay filter in the data analysis software. The processed data were fitted using a 1:1 Langmuir binding model, with R² value used as a measure of fitting accuracy. The results are summarized in Table 24. Since FAP is a natural dimeric antigen, affinity was observed, and the detection capability of these molecules on Octet was at its limit. Cross-species cross-reactivity of all tested samples was confirmed.

[0653] Table 24. Kinetic parameters of FAP / LTBR bispecific antibody binding to human and cynomolgus monkey LTBR and FAP as measured by Octet. Values ​​outside the instrument specification range

[0654] 4.9. Using tumor cell line 4T1 and FAP-positive CHO overexpressing cell line for culture, the activity of the in vitro alternative FAP / LTBR bispecific antibody was detected in a chemokine induction assay.

[0655] Because the anti-FAP antibody (clone 28H1) selectively binds to human and mouse antigens, the CHO-human FAP-K1 cell line can be used as a source of FAP in the following tests.

[0656] To evaluate the ability of alternative mouse FAP / LTBR bispecific antibodies to conditionally activate LTBR, ​​the FAP / LTBR bispecific antibody and a monoclonal LTBR-promoting antibody were tested in 4T1 (ATCC, CRL-2539) cells with or without the FAP-CHO-K1 cell line. Furthermore, a monospecific anti-LTBR antibody was tested with or without a cross-linking agent to induce sufficient LTBR aggregation. An anti-Fc antibody was used as the cross-linking agent.

[0657] In short, 4T1 cells (20,000 cells / well) were co-seeded with FAP-CHO-K1 cells (20,000 cells / well) or seeded separately in their growth medium. A stock solution of the bispecific antibody and control group was prepared in the experimental medium at a 1:10 seven-point dilution series (repeated twice) to a 10-fold concentration, added to the cells, and incubated at 37°C and 5% CO2 for 24 hours.

[0658] After incubation, the supernatant was collected and clarified by centrifugation. Chemokine levels were then determined using the CCL5 DuoSet ELISA (DY478-05) according to the manufacturer's instructions. Absorbance values ​​were read on a BMG Pherastar FSX plate reader. The corrected OD values ​​(450-540 nm) of the standard curve were analyzed in Prism software as recommended by the manufacturer to interpolate cytokine concentrations in the wells. Dose-response curves were plotted using GraphPad Prism Version 9, and a nonlinear fit (log(agonist) vs. response (variable slope - four parameters)) was applied. EC50 values ​​are shown in Table 25.

[0659] Table 25. Chemokines-induced EC50 values ​​in 4T1 cells

[0660] Figures 12a and 12b show that, compared to the non-targeted LTBR bispecific antibody (clone 4.13), the alternative FAP / LTBR bispecific antibody system conditionally activates LTBR, ​​exhibiting limited activity in the absence of FAP. The results indicate that clone 4.11 exhibits stronger FAP-dependent activity, a lower EC50 value, and higher maximum activity compared to the cross-linked monospecific LTBR agonist antibody (clone 4.14).

[0661] Figure 12c shows that, regardless of the Fc backbone, the monospecific LTBR agonist antibody 5G11 exhibited the same potency in 4T1 cell single culture assays, leading to dose-dependent secretion of CCL5 (results from clones 4.14 and 4.15).

[0662] Using monospecific LTBR agonist antibodies to achieve LTBR activation in the absence of FAP may potentially lead to toxicity due to the widespread expression of LTBR in normal tissues. Higher LTBR activation levels are achieved when the monospecific antibody is cross-linked with an anti-Fc antibody.

[0663] Compared to alternatives to FAP / LTBR bispecific antibodies (clone 4.12), Figure 12d The results showed that, in a 4T1 single-culture assay, the alternative FAP / LTBR bispecific molecules (FAP-targeting P1AG5459 and non-targeting P1AG55461) achieved the same level of LTBR activation as the monospecific LTBR agonist control antibody (clone 4.14) without cross-linking. Compared to the non-targeting control group, the FAP-targeting comparative molecules showed significantly higher LTBR activation levels when co-cultured with FAP-positive cell lines. Figure 12e The efficacy of the target was enhanced, while the non-targeted control group showed similar activity in single culture and co-culture.

[0664] 4.10. In vitro activity of mouse LTBR / FAP bispecific agonist antibody in primary mouse lung fibroblasts

[0665] To understand whether stimulation of fibroblasts with the mouse FAP / LTBR bispecific antibody agonist leads to the upregulation of adhesion molecules (e.g., ICAM-1), a single culture assay of primary mouse lung fibroblasts (PMLF) was performed.

[0666] Primary mouse lung fibroblasts (PMLF) were derived from naive (untreated) mice. PMLF was seeded (20,000 cells / well) in 96-well plates and allowed to attach within 24 hours. Subsequently, stock solutions of seven concentrations of FAP / LTBR bispecific antibody (bsAb) and a control LTBR agonist antibody, diluted 10-fold, were prepared in culture medium to create 10-fold concentrations. These stock solutions were added to the cells and incubated at 37°C and 5% CO2 for 48 hours. After stimulation, cells were fixed for 20 minutes with 100 µL of 8% formaldehyde (final concentration 4%) and washed with PBS. Cells were then incubated at room temperature for 1 hour with blocking buffer (PBS solution containing 3% BSA and 0.3% Triton X-100, w / v / v) to prevent nonspecific binding. For immunostaining, cells were incubated overnight at 4°C with a purified ICAM-1 rat monoclonal affinity antibody (Biolegend, catalog number 116102) diluted 1 / 100 in 3% BSA in PBS (w / v). Cells were then washed three times with 100 µL of wash buffer (10 mM sodium phosphate, 0.15 M sodium chloride, 0.05% Tween-20) and incubated for 1 hour at room temperature with a secondary antibody, anti-rat IgG (H+L) AF488 pAb (from donkey IgG, Invitrogen, catalog number A-21208, diluted 1 / 500 in 3% BSA in PBS (w / v)). Simultaneously, the nuclei were counterstained with Hoechst 33342 nuclear staining agent (Invitrogen, H3570, 1 / 10000 dilution). Cells were washed three more times with wash buffer and then transferred to PBS for image acquisition. Fluorescent images were acquired using a CellInsight CX7 HCS platform. Image acquisition and analysis were performed using Thermo Scientific HCS Studio: Cellomics Scan V6.6.2 software and Spot Detector Bioapplication V4. This bioapplication enables rapid protein expression analysis by measuring fluorescence intensity at the single-cell level. Data are presented as mean fluorescence intensity per cell (ICAM1 expression) minus the intensity under untreated conditions (baseline correction), and graphs were plotted using GraphPad PrismVersion 9. Stimulation with an LTBR / FAP bispecific agonist antibody resulted in a concentration-dependent upregulation of adhesion molecule expression, such as... Figure 13 and 14 As shown.

[0667] These data show that the alternative FAP / LTBR bispecific antibody can induce LTBR aggregation and signaling in an FAP-dependent manner (due to FAP binding), leading to the induction of chemokines and the upregulation of adhesion molecules on the cell surface, similar to the human construct. In contrast, the control group with no target (clone 4.13, not bound to FAP) showed lower upregulation of adhesion molecules (Figs. 13 and 14) and no induction of chemokines (Figs. 12a and 12b).

[0668] Table 26. EC50 values ​​of ICAM1 adjusted upwards in PMLF

[0669] 4.11. In vivo efficacy of FAP / LTBR alternative bispecific antibodies in an orthotopic breast cancer model

[0670] Alternative FAP / LTBR bispecific antibodies have been shown to activate the LTBR receptor in a FAP-dependent manner, leading to dose-dependent induction of chemokines and upregulation of adhesion molecules. Therefore, it is desirable to test the in vivo function of FAP / LTBR bispecific antibodies in tumor models with homologous immune activity.

[0671] In a mouse breast cancer MMTV-PyMT mBR9071 tumor model (Crown Biosciences), the efficacy of a combination of an alternative FAP / LTBR bispecific antibody and an anti-CTLA-4 checkpoint inhibitor was tested in mice. Briefly, the mBR9071 tumor model was derived from tumor tissue fragments collected from MMTV-PyMT mice with a C57BL / 6 background. These tumor fragments were passaged in stored C57BL / 6 mice, and the fragments were collected and inoculated into the final recipient C57BL / 6 mice. Each recipient mouse received a 2–3 mm diameter tumor tissue fragment orthotopically inoculated into the mammary fat pad. When the average tumor size reached approximately 80–120 mm³, mice were randomly assigned to treatment groups. Mice received twice weekly an isotype control antibody, an anti-CTLA-4 antibody, an anti-CTLA-4 antibody + LTBR antibody (clone 4.14), or an anti-CTLA-4 + FAP / LTBR bispecific antibody (clone 4.11) (Table 27). All antibodies were administered intraperitoneally (IP) at a dose of 10 mg / kg. The anti-CTLA-4 antibody was clone 9D9, product number BP0164 (BioXell). The study was terminated when the control group mice reached the humane endpoint. On the termination day, tumor tissues from all groups were dissociated and enzymatically digested for flow cytometry analysis. Cells were stained to detect HEVs (CD31+, MECA 79+), T cells (CD3+, CD4+, CD8+), and B cells (CD19+). Cells were fixed and analyzed on a BD LSR Fortessa flow cytometer, and data were analyzed using Kaluza software.

[0672] Table 27. Treatment timeline for MMTV-PyMT in situ breast cancer

[0673] Compared with the isotype control group, there was no significant difference in the effect of anti-CTLA-4 treatment alone on tumor growth. Mice receiving anti-CTLA-4 in combination with clonal 4.14 or 4.11 showed highly significant inhibition of tumor growth compared with the isotype control group or mice treated with anti-CTLA-4 alone. Figure 15 No significant weight loss or changes in liver enzymes ALT and AST were observed in any treatment group (data not shown). Statistical analysis was performed using two-way ANOVA.

[0674] Compared to the isotype control group or the anti-CTLA-4 treatment alone, the combination therapy also resulted in an increase in the number of CD8+ T cells in the tumor, as measured by flow cytometry at the end of the study (data not shown). Induction of high endothelial microveins (HEVs-CD31+MECA-79+ cells) was significantly increased only in mice treated with the anti-CTLA-4 plus FAPxLTBR bispecific antibody (clone 4.11), compared to all other treatment groups, as measured by flow cytometry (data not shown). Figure 17 Tumor growth curves and flow cytometry results were plotted using GraphPad Prism Version 9. Statistical analysis was performed using one-way ANOVA. p<0.05, p<0.01, p<0.001, p<0.0001).

[0675] The efficacy of the combination of clone 4.11 and an anti-PD-L1 checkpoint inhibitor was also measured in an syngeneic EMT6 breast cancer model. In short, EMT6 tumor cells (5 × 10⁻⁶) showed efficacy in this combination. 5 (Number of cells) were orthotopically inoculated into the mammary glands of BALB / c mice to develop tumors. When the average tumor size reached approximately 100 mm³, the mice were randomly assigned to treatment groups. Mice were treated with isotype control, anti-PD-L1 (clone 10F.9G2, product number BP0101, BioXell), FAP / LTBR bispecific antibody clone 4.11, or a combination of anti-PD-L1 and FAP / LTBR bispecific antibody clone 4.11. All antibodies were administered intraperitoneally twice weekly at a dose of 10 mg / kg (Table 28).

[0676] Table 28. Treatment schedule for the orthotopic EMT6 mouse model

[0677] Compared with the isotype control, combination therapy with the anti-PD-L1 + FAP / LTBR bispecific antibody 4.11 clone showed highly significant tumor growth inhibition (Fig. 18), resulting in tumor regression in 4 out of 10 mice. No significant weight loss was observed in any treatment group. Compared with the isotype control or anti-PD-L1 treatment alone, treatment with clone 4.11 monotherapy and combination therapy with anti-PD-L1 + clone 4.11 also significantly increased the proportion of B cells in the tumor (Fig. 19a). Flow cytometry measurements showed that monotherapy with clone 4.11 and combination therapy with anti-PD-L1 + clone 4.11 significantly increased the formation of high endothelial venules (HEVs) (Fig. 19b).

[0678] 4.12. In vivo efficacy of FAP / LTBR bispecific alternative antibodies in a subcutaneous lung cancer model.

[0679] The in vivo efficacy of FAP / LTBR bispecific antibody clone 4.11 against anti-PD-L1 was tested in the mLU6054 mBR9071 mouse model (Crown Biosciences). Tumor fragments from the mLU6054 mouse lung tumor model were collected from stored C57BL / 6 mice and inoculated into the final recipient mice. One tumor fragment (2-3 mm in diameter) was subcutaneously inoculated into the right anterior side of each mouse. When the average tumor volume reached approximately 80-120 mm³, the recipient mice were randomly assigned to treatment groups. Mice received isotype control, anti-PD-L1, or anti-PD-L1 + FAP / LTBR bispecific antibody clone 4.11 twice a week at a dose of 10 mg / kg via intraperitoneal injection (see Figure 16 and Table 29 for details). The study was terminated when the control mice reached the human termination point. On the termination day, the tumors in each group were isolated and enzymatically digested for flow cytometry analysis. Cells were stained to detect HEVs (CD31+, MECA 79+) and T cells (CD3+, CD4+, CD8+). Cells were collected after fixation on a BD LSR Fortessa and data analysis was performed using Kaluza software. For IHC analysis, tumors were fixed in 10% neutral buffered formalin for 24 hours, then transferred to 70% ethanol, and finally embedded in paraffin.

[0680] Table 22. Treatment schedule for mLU6054 subcutaneous mouse model

[0681] Combination therapy with the anti-PD-L1 + FAP / LTBR bispecific antibody clone 4.11 showed a significant inhibitory effect on tumor growth compared with the isotype control (Figure 20). No significant weight loss was observed in any treatment group (data not shown). Tumor growth curves and flow cytometry results were plotted using GraphPad Prism Version 9. Statistical analysis of tumor growth curves was performed using a two-factor ANOVA (…). p<0.05, p<0.01, p<0.0001).

[0682] Compared with isotype control or anti-PD-L1 monotherapy, the combination therapy of anti-PD-L1 + clonal 4.11 significantly increased the formation of high endothelial venules (HEVs) (CD31+ MECA79+ cells) in tumors and increased CD4+ and CD8+ T cells (Fig. 21a, b, and c). Statistical analysis was performed using a single-factor ANOVA (…). p<0.05, p<0.01, p<0.001, p<0.0001).

[0683] For immunohistochemical (IHC) analysis, tumors were fixed in 10% neutral buffered formalin for 24 hours, then transferred to 70% ethanol and embedded in paraffin. FFPE tumor samples were subjected to IHC staining for CD8 and CD19 to observe the location of CD8+ T cells and CD19+ B cells. mLU6054 tumors treated with anti-PD-L1 + clone 4.11 combination therapy showed histologically infiltrated CD8+ T cells (Fig. 22a) and CD19+ B cells (Fig. 22b), a hallmark of tertiary lymphoid structure (TLS) development. These structures were not observed in isotype control mice or mice treated with anti-PD-L1 monotherapy.

[0684] Furthermore, digital quantification of CD8+ and CD19+ cell counts in tumors using IHC showed that, compared to allotype control or anti-PD-L1 monotherapy alone, combination therapy increased the number of CD8+ T cells and significantly increased the number of CD19+ B cells in the tumor microenvironment. Figure 23a and 23b ).

[0685] In summary, combination therapy with anti-PD-L1 and FAP / LTBR bispecific antibodies led to changes in the tumor microenvironment, manifested as an increase in immune cells (T cells and B cells) and the formation of high endothelial venules (HEVs), specialized vessels mediating the migration of lymphocytes to lymph nodes (LNs) and other secondary lymphoid organs. Furthermore, highly organized aggregates of immune cells were detected, containing clearly defined T-cell and B-cell regions forming lymphoid tissues (TLSs).

[0686] 4.13. In vivo efficacy of anti-LTBR alternative clonal therapy combined with radiotherapy in a subcutaneous colorectal cancer model.

[0687] Radiation therapy (RT) is a highly effective cancer treatment tool designed to control or eliminate solid tumors for clinical benefit. Furthermore, evidence in the literature suggests that certain RT doses and fractionation regimens can induce immune cell death, thereby generating a systemic anti-cancer response, known as the abscopal effect. To investigate whether low-fractionation RT can be combined with checkpoint inhibitors or LTBR agonists to induce a systemic anti-tumor immune response and act on remotely unirradiated tumors (the abscopal effect), the MC38 tumor model was used.

[0688] MC38 tumor fragments were obtained from subcutaneously implanted C56bl / 6 mice. The tumor fragments (20–30 mg) were subcutaneously implanted into both sides of C57bl / 6 recipient mice. Mice were randomly assigned to groups based on the tumor volume on the right side of each mouse. Randomization occurred when the right-side tumor volume reached approximately 80–100 mm³. Mice receiving radiotherapy only to the right-side tumor received 5 Gy of radiation once daily for three consecutive days. The left-side tumor was not radiotreated during the study. All antibody treatments were administered intraperitoneally at a dose of 10 mg / kg. Treatments received by mice included: allotype control, anti-PD-L1, radiotherapy alone, radiotherapy + anti-PD-L1, radiotherapy + LTBR agonist clone 4.15, or LTBR agonist clone 4.15 alone (Table 30). The study was terminated when the primary and secondary tumors in the control mice reached the humane termination point. On the day of termination, primary tumors in all groups were dissected and prepared for flow cytometry by enzymatic digestion. Cells were stained to detect the presence of T cells (CD3+, CD4+, CD8+). Cells were fixed and acquired on a BD LSR Fortessa, and data were analyzed using Kaluza software. Tumor growth curves and flow cytometry results were plotted using GraphPad Prism Version 9. Statistical analysis of tumor growth curves was performed using two-factor ANOVA analyses. p<0.05, p<0.01, p<0.0001).

[0689] Table 30. Treatment plan for the MC38 mouse in vivo model

[0690] In the tumors on the right side (receiving radiation therapy), the combination of anti-LTBR clone 4.15 and radiation therapy significantly inhibited tumor growth compared to antibody therapy alone (Figure 24). In the tumors on the left side (not receiving radiation therapy), mice treated with radiation therapy plus anti-LTBR clone 4.15 showed significantly inhibited tumor growth compared to radiation therapy alone or anti-LTBR therapy alone, indicating that the combination of anti-LTBR and radiation therapy produces a systemic anti-tumor response. Figure 25 Anti-PD-L1 therapy did not produce a long-range effect in tumors that had not received radiation therapy (data not shown). Tumors in the isotype control group grew rapidly, and the animals quickly reached the humane endpoint and were removed from the study; therefore, no relevant data were shown.

[0691] In the right-sided (radiation-treated) tumor, flow cytometry analysis showed that the combination of anti-LTBR and radiation therapy significantly increased CD4+ and CD8+ T cells compared to single therapy (Figures 26a and 26b), indicating a change in the tumor immune microenvironment and a synergistic effect between the treatments. Statistical analysis was performed using a single-factor ANOVA (Analog-Nutritional Scale). p<0.05, p<0.01, p<0.001, p<0.0001).

[0692] 4.14. In vivo efficacy of FAP-LTBR replacement molecule and E22 peptide antigen in an orthotopic breast cancer model

[0693] Cancer vaccines typically consist of tumor-specific peptide components administered in conjunction with an adjuvant (e.g., a TLR agonist). Literature reports that EMT6 tumors express tumor-associated antigens, including the E22 peptide KCLQDNNWDYTRYAQAFTLLKAKG (Castiglioni et al., 2023, SEQ ID NO: 209). To demonstrate the benefits of combining an LTBR agonist with a cancer vaccine, a combination study was conducted using a model cancer vaccine consisting of an E22 peptide and CpG-ODN 1826 (a well-known TLR9 agonist). The activity of this model cancer vaccine was tested both alone with the adjuvant and in combination with a FAP / LTBR agonist bispecific antibody. Results showed that, when used in combination with the model cancer vaccine, the FAP / LTBR agonist bispecific antibody induced a strong anti-tumor immune response that inhibited tumor growth in an EMT6 breast cancer model.

[0694] The potency of the combination of clone 4.11 and the E22 peptide antigen was measured in an endogenic EMT6 breast cancer model. In short, the potency of the combination of EMT6 tumor cells (5 × 10⁻⁶) was measured in a endogenic EMT6 breast cancer model. 5 (Number of cells) were injected into the mammary fat pads of BALB / c mice to develop tumors. When the average tumor size reached approximately 100 mm... 3 Mice were randomly assigned to treatment groups. Mice received either an isotype control, an adjuvant (TLR-9 agonist, InvivoGen, product number: vac-1826-1) + E22 peptide as a control, FAP / LTBR bispecific antibody clone 4.11, or a combination of adjuvant + E22 peptide and FAP / LTBR bispecific antibody clone 4.11. The FAP / LTBR bispecific antibody was administered intraperitoneally three times weekly at a dose of 10 mg / kg (Table 31). A control group receiving only adjuvant was also included, but its activity increased in the absence of the peptide, potentially indicating that the adjuvant has different mechanisms of action with or without the peptide. Therefore, we compared the FAP / LTBR bsAb + peptide + adjuvant group with the control group receiving only peptide + adjuvant. The study was terminated when mice in the control group reached the humane endpoint. On the day of termination, tumors in all groups were dissected and enzymatically digested for flow cytometry analysis. If a tumor in a group was too small to be treated (approximately 100 mm), the tumor was removed. 3 Multiple tumors were then combined for flow cytometry analysis. Cells were stained with T-cell markers (CD45+, CD3+, CD4+, CD8+). After fixation, cells were sampled using a BD LSR Fortessa, and data were analyzed using Kaluza software.

[0695] Table 31. Treatment schedule for the orthotopic EMT6 mouse model

[0696] Compared with the control group (adjuvant + peptide), the combination of FAP / LTBR bispecific antibody (clone 4.11) + adjuvant + peptide significantly inhibited tumor growth, resulting in tumor regression in 8 out of 10 mice, compared with 3 out of 10 mice in the peptide + adjuvant group and 1 out of 10 mice in the clone 4.11 control group. Figure 28a Tumor growth curves and flow cytometry results were plotted using GraphPad Prism Version 9. Statistical analysis was performed using one-way ANOVA. p<0.05, p<0.01, p<0.001, p<0.0001).

[0697] Compared with the isotype control or adjuvant + peptide group, the combination of FAP / LTBR bispecific antibody (clone 4.11) + adjuvant + peptide significantly increased CD4+ and CD8+ T cells in tumors. Figure 28b and 28c Statistical analysis was performed using a one-way ANOVA. p<0.05, p<0.01, p<0.001, p<0.0001).

[0698] 4.15. In vivo efficacy of FAP-LTBR alternative molecules and E22 peptide antigen in an orthotopic breast cancer model (re-stimulation study)

[0699] Successful cancer immunotherapy requires inducing a durable anti-tumor immune response, including eliminating the primary tumor and preventing recurrence. This rechallenge study evaluates the resilience of the immune response when tumor cells are reintroduced after complete tumor regression following initial treatment.

[0700] To elucidate the perpetuity of response in combination with clone 4.11 and E22 peptide (Castiglioni et al., Nature 2023), additional in vivo studies were conducted using an syngeneic EMT6 breast cancer model. Mice were randomly assigned to treatment groups when the mean tumor volume reached approximately 100 mm³. The treatment groups consisted of: (1) an allotype control group, (2) an adjuvant (TLR-9 agonist, InvivoGen, product number vac-1826-1) + E22 peptide as a control group, (3) the FAP / LTBR bispecific antibody clone 4.11, and (4) a combination treatment group of adjuvant + E22 peptide + FAP / LTBR bispecific antibody clone 4.11. The FAP / LTBR bispecific antibody was administered intraperitoneally three times weekly at a dose of 10 mg / kg. Mice were euthanized when the tumor volume reached a predetermined threshold. Details of the treatment regimen are provided in Table 32.

[0701] Combination therapy with FAP-LTBR bispecific antibody (clone 4.11) and peptide vaccine (E22 peptide) achieved durable complete remission (CR) in 28% of individuals, such as Figure 29 As shown. Survival curves were generated using the Kaplan-Meier method, and mouse survival data were analyzed using the Log-rank test of GraphPad Prism Version 9. Statistical analysis was performed using univariate ANOVA (...). p<0.05, p<0.01, p<0.001, (p<0.0001). Subsequently, on day 91 after the end of treatment, two mice from the vaccine monotherapy group showing complete regression and seven mice from the vaccine + clone 4.11 group underwent rechallenge. The rechallenge consisted of implanting EMT6 cells into the contralateral (left) mammary fat pad, and age-matched untreated (naive) mice that had not been exposed to primary tumor implantation received the same number of rechallenge cells as a control group.

[0702] Notably, compared to age-matched, treatment-naïve controls, all re-triggered mice rejected the tumor, indicating the establishment of a strong and durable immune memory, such as... Figure 30 As shown. The tumor growth curve was plotted using GraphPadPrism Version 9.

[0703] Table 32. Treatment schedule for the in situ EMT6-huHER2 mouse model

[0704] The higher frequency of complete regression observed in the combination therapy group may indicate a synergistic effect between the peptide vaccine and the bispecific antibody.

[0705] Successful rejection of re-engaged tumors may indicate the generation of functional memory T cells capable of recognizing and eliminating tumor cells. This result supports the hypothesis that combination therapy can induce a robust and durable adaptive immune response to tumor-associated antigens.

[0706] In this specification and subsequent requests, unless the context otherwise requires, the word “comprise” and its variations such as “comprises” and “comprising” shall be understood to include the specified integer, step, group of integers or set of steps, but not to exclude any other integer, step, group of integers or set of steps.

[0707] Unless otherwise stated, all concentration percentages mentioned herein are w / v. The unit prefixes µ and u are used interchangeably in this document to mean "micro".

[0708] The patent application to which this specification and claims pertain may serve as the basis for priority in any subsequent application. Claims in a subsequent application may target any feature or combination of features described herein. These claims may be product, composition, method, or use claims, and may include, but are not limited to, the claims exemplified below.

[0709] Terms of this invention: The provisions of this invention include, but are not limited to, the following. These provisions relate primarily to constructs comprising FAP binders and LTBR binders.

[0710] Item 1. A construct comprising an FAP binder and an LTBR binder.

[0711] Item 2. The construct according to Item 1, wherein the construct contains only one FAP binder.

[0712] Item 3. The construct according to Item 1, wherein the construct contains more than one FAP binder.

[0713] Item 4. The construct according to Item 1, wherein the construct contains only two FAP binders.

[0714] Item 5. The construct according to any one of items 1 to 4, wherein the construct contains only one LTBR binder.

[0715] Item 6. The construct according to any one of items 1 to 4, wherein the construct comprises more than one LTBR binder.

[0716] Item 7. The construct according to Item 6, wherein the construct contains only two LTBR binders.

[0717] Item 8. The construct according to any one of items 1 to 7, wherein one or more LTBR binders comprise or are composed of a binding polypeptide that binds to LTBR.

[0718] Item 9. The construct according to any one of items 1 to 8, wherein one or more FAP binders comprise or are composed of a binding polypeptide that binds to FAP.

[0719] Item 10. A construct according to any one of items 1 to 9, wherein the construct contains an antibody.

[0720] Item 11. A construct according to any one of items 1 to 9, wherein the construct is composed of an antibody.

[0721] Item 12. The construct according to Item 10 or Item 11, wherein the antibody is a bispecific antibody.

[0722] Item 13. The construct according to any one of items 10 to 12, wherein the antibody is a bivalent antibody.

[0723] Item 14. The construct according to Item 13, wherein the antibody is mainly composed of bispecific, bivalent antibodies.

[0724] Item 15. The construct according to Item 14, wherein the antibody is composed of a bispecific, bivalent antibody.

[0725] Item 16. The construct according to any one of items 10 to 15, wherein the antibody is an IgG1 antibody.

[0726] Item 17. A construct according to any one of items 1 to 10, wherein the construct comprises one or more antibody fragments.

[0727] Item 18. A construct according to any one of items 1 to 9, wherein the construct comprises one or more antibody fragments.

[0728] Item 19. The construct according to Item 17 or Item 18, wherein the one or more antibody fragments are each independently selected from scFv, Fv, Fab, Fab', F(ab')2, variable domains (e.g., VH, VL, VNAR or VHH), biantibodies or microantibodies.

[0729] Item 20. The construct according to Item 19, wherein the construct comprises or consists of two scFvs.

[0730] Item 21. According to the construct of Item 19 or Item 20, wherein the scFvs contains a disulfide bond at the VH44:VL100 position.

[0731] Item 22. A construct according to any one of Items 1, 10, 11 or 16 to 19, wherein the construct has a 1:1 form.

[0732] Item 23. A construct according to any one of Items 1, 10, 11 or 16 to 19, wherein the construct has a 2:1 form.

[0733] Item 24. A construct according to any one of Items 1, 10, 11 or 16 to 19, wherein the construct has a 2:2 form.

[0734] Item 25. The construct according to any one of items 1 to 24, wherein one or all of the LTBR binders comprises or consists of a variable domain bound to or formed thereto the LTBR.

[0735] Item 26. Based on the construct of Item 25, wherein the variable domain associated with LTBR is selected from VH, VL, VNAR or VHH.

[0736] Item 27. Based on the construct of Item 26, the variable field associated with LTBR is a heavy-chain variable field.

[0737] Item 28. Based on the construct of Item 27, the heavy chain variable domain that binds to LTBR is VHH.

[0738] Item 29. A construct according to any one of items 1 to 28, wherein the construct further comprises another binder that is neither an LTBR binder nor a FAP binder.

[0739] Item 30. A construct according to any one of items 1 to 28, wherein the construct does not contain any other binder.

[0740] Item 31. A construct according to any one of items 25 to 30, wherein the variable domain binding to LTBR comprises or consists of three heavy chain complementarity-determining regions (HCDR1-HCDR3) and four framework regions (FR1-FR4), wherein HCDR1 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 57 or 166, or wherein HCDR1 comprises or consists of a polypeptide sequence having 3, 2, or 1 amino acid sequence difference from SEQ ID NO: 57 or 166, and HCDR2 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 205, 58, or 167, or wherein ... 205, 58, or 167 are polypeptide sequences having a sequence difference of 3, 2, or 1 amino acid, or are composed thereof, and HCDR3 comprises a polypeptide sequence having a sequence identity of 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher with respect to the sequence of SEQ ID NO: 59 or 168, or wherein HCDR3 comprises a polypeptide sequence having a sequence difference of 3, 2, or 1 amino acid, or is composed thereof.

[0741] Item 32 is based on the construct of Item 31, wherein HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 57 or 166, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 205, 58 or 167, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 59 or 168.

[0742] Item 33. A construct of any one of items 25 to 30, wherein the variable domain binding to LTBR comprises or consists of three heavy chain complementarity-determining regions (HCDR1-HCDR3) and four frame regions (FR1-FR4), wherein HCDR1 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 60 or 172, or wherein HCDR1 comprises or consists of a polypeptide sequence having 3, 2, or 1 amino acid sequence differences from SEQ ID NO: 60 or 172, and HCDR2 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 61 or 173, or wherein ... 61 or 173 has a polypeptide sequence that differs from or is composed of a polypeptide sequence with 3, 2 or 1 amino acid sequence differences; and HCDR3 contains or is composed of a polypeptide sequence that has 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher or 95% or higher sequence identity with or is composed of a polypeptide sequence that differs from ...

[0743] The 34th embodiment is the construct according to the 33rd embodiment, wherein HCDR1 comprises or is composed of SEQ ID NO: 60 or 172, HCDR2 comprises or is composed of SEQ ID NO: 61 or 173, and HCDR3 comprises or is composed of SEQ ID NO: 62 or 174.

[0744] Item 35. A construct according to any one of items 25 to 30, wherein the variable domain binding to LTBR comprises or consists of three heavy chain complementarity-determining regions (HCDR1-HCDR3) and four framework regions (FR1-FR4), wherein HCDR1 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 63 or 163, or wherein HCDR1 comprises or consists of a polypeptide sequence having 3, 2, or 1 amino acid sequence differences from SEQ ID NO: 63 or 163, and HCDR2 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 64 or 164, or wherein HCDR2 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 64 or 164 contains or is composed of a polypeptide sequence that differs from SEQ ID NO: 207, 65 or 165 by 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher or 95% or higher sequence identity, or HCDR3 contains or is composed of a polypeptide sequence that differs from SEQ ID NO: 207, 65 or 165 by 3, 2 or 1 amino acid sequence.

[0745] Item 36: The construct according to Item 35, wherein HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 63 or 163, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 64 or 164, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 207, 65 or 165.

[0746] Item 37: A construct according to any one of items 25 to 30, wherein the variable domain binding to LTBR comprises or consists of three heavy chain complementarity-determining regions (HCDR1-HCDR3) and four framework regions (FR1-FR4), wherein HCDR1 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 66 or 169, or wherein HCDR1 comprises or consists of a polypeptide sequence having 3, 2, or 1 amino acid sequence differences from SEQ ID NO: 66 or 169, and HCDR2 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 206, 67, or 170, or wherein HCDR2 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 206, 67, or 170 are polypeptide sequences having a sequence difference of 3, 2, or 1 amino acid, or are composed thereof, and HCDR3 comprises or is composed of a polypeptide sequence having a sequence identity of 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher with respect to the sequence of SEQ ID NO: 208, 68, or 171, or wherein HCDR3 comprises or is composed of a polypeptide sequence having a sequence difference of 3, 2, or 1 amino acid, or is composed thereof with respect to the sequence of SEQ ID NO: 208, 68, or 171.

[0747] Item 38. The construct as described in Item 37, wherein HCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 66 or 169, HCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 206, 67 or 170, and HCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 208, 68 or 171.

[0748] Item 39. The construct as described in any one of items 25 to 30, wherein the variable domain binding to LTBR comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher sequence identity with SEQ ID NO: 23 or 160.

[0749] Item 40. The construct as described in Item 39, wherein the variable domain that binds to LTBR comprises or is composed of the polypeptide sequence of SEQ ID NO: 23 or 160.

[0750] Item 41. The construct as described in any one of items 25 to 30, wherein the variable domain bound to the LTBR comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher sequence identity with SEQ ID NO: 24 or 162.

[0751] Item 42. The construct as described in Item 41, wherein the variable domain that binds to LTBR comprises or is composed of the polypeptide sequence of SEQ ID NO: 24 or 162.

[0752] Item 43. An construct according to any one of items 25 to 30, wherein the variable domain binding to LTBR comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher sequence identity with SEQ ID NO: 25 or 159.

[0753] Item 44. The construct according to Item 43, wherein the variable domain that binds to LTBR comprises or is composed of the polypeptide sequence of SEQ ID NO: 25 or 159.

[0754] Item 45. The construct according to any one of items 25 to 30, wherein the variable domain binding to LTBR comprises or consists of a polypeptide sequence that is 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher in sequence identity with SEQ ID NO: 26 or 161.

[0755] Item 46. The construct according to Item 45, wherein the variable domain that binds to LTBR comprises or is composed of the polypeptide sequence of SEQ ID NO: 26 or 161.

[0756] Item 47. The construct according to any one of items 1 to 24, wherein one or all of the LTBR binders comprises or consists of paired heavy chain variable domains and light chain variable domains bound to the LTBR.

[0757] Item 48. According to the construct of Item 47, one or all of the LTBR binding agents are independently selected from scFv, Fv, Fab, Fab', F(ab')2, biantibody or microantibody.

[0758] Item 49: The construct according to Item 47 or 48, wherein the heavy chain variable domain binding to LTBR comprises or consists of three complementarity-determining regions (HCDR1-HCDR3) and four framework regions (HFR1-HFR4), wherein HCDR1 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 69, HCDR2 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 70, and HCDR3 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 70, and HCDR3 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 70. 71. A polypeptide sequence having or consisting of 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 72; the light chain variable domain binding to LTBR comprises or consists of three complementarity-determining regions (LCDR1-LCDR3) and four frame regions (LFR1-LFR4), wherein LCDR1 comprises or consists of a polypeptide sequence having or consisting of 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 72; LCDR2 comprises or consists of a polypeptide sequence having or consisting of 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 73; and LCDR3 comprises or consists of a polypeptide sequence having or consisting of 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 73; and LCDR3 comprises or consists of a polypeptide sequence having or consisting of 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 73. 74. A polypeptide sequence or a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity.

[0759] Item 50. The construct according to Item 49, wherein HCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 69, HCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 70, and HCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 71, and the light chain variable region comprises or is composed of three complementarity-determining regions (LCDR1 to LCDR3) and four frame regions (LFR1 to LFR4), wherein LCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 72, LCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 73, and LCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 74.

[0760] Item 51. The construct according to Item 47 or 48, wherein the heavy chain variable domain binding to LTBR comprises or consists of three complementarity-determining regions (HCDR1-HCDR3) and four framework regions (HFR1-HFR4), wherein HCDR1 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 75, HCDR2 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 76, and HCDR3 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 76, and HCDR3 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 76. 77. A polypeptide sequence having or consisting of 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 78; and the light chain variable domain binding to LTBR comprises or consists of three complementarity-determining regions (LCDR1-LCDR3) and four frame regions (LFR1-LFR4), wherein LCDR1 comprises or consists of a polypeptide sequence having or consisting of 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 78; LCDR2 comprises or consists of a polypeptide sequence having or consisting of 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 79; and LCDR3 comprises or consists of a polypeptide sequence having or consisting of 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 79; and LCDR3 comprises or consists of a polypeptide sequence having or consisting of 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 79. 80. A polypeptide sequence having or consisting of a sequence identity of 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher.

[0761] Item 52. The construct according to Item 51, wherein HCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO:75, HCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO:76, and HCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO:77, and the light chain variable domain comprises or is composed of three complementarity-determining regions (LCDR1-LCDR3) and four frame regions (LFR1-LFR4), wherein LCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO:78, LCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO:79, and LCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO:80.

[0762] Item 53. The construct according to Item 47 or 48, wherein the heavy chain variable domain binding to LTBR comprises or consists of three complementarity-determining regions (HCDR1-HCDR3) and four framework regions (HFR1-HFR4), wherein HCDR1 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 81, HCDR2 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 82, and HCDR3 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 82, and HCDR3 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 82. 83 has a polypeptide sequence or is composed of 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 84, and the light chain variable domain binding to LTBR comprises or is composed of three complementarity-determining regions (LCDR1-LCDR3) and four frame regions (LFR1-LFR4), wherein LCDR1 comprises or is composed of a polypeptide sequence or is composed of SEQ ID NO: 84 having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 85, and LCDR3 comprises or is composed of a polypeptide sequence or is composed of SEQ ID NO: 85 having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 85. 86. A polypeptide sequence having or consisting of 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity.

[0763] Item 54. The construct according to Item 53, wherein HCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 81, HCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 82, and HCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 83, and the light chain variable domain comprises or is composed of three complementarity-determining regions (LCDR1-LCDR3) and four frame regions (LFR1-LFR4), wherein LCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 84, LCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 85, and LCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 86.

[0764] Item 55. The construct according to Item 47 or 48, wherein the heavy chain variable domain binding to LTBR comprises or is composed of three complementarity-determining regions (HCDR1 to HCDR3) and four framework regions (HFR1 to HFR4), wherein HCDR1 comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 87, HCDR2 comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 88, and HCDR3 comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 88, and HCDR3 comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 88. 89 contains a polypeptide sequence or composed thereof having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity, and the light chain variable domain binding to LTBR comprises or is composed of three complementarity-determining regions (LCDR1-LCDR3) and four frame regions (LFR1-LFR4), wherein LCDR1 contains or is composed of a polypeptide sequence or composed thereof having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 90; LCDR2 contains or is composed of a polypeptide sequence or composed thereof having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 91; and LCDR3 contains or is composed of a polypeptide sequence or composed thereof having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 91; and LCDR3 contains or is composed of a polypeptide sequence or composed thereof having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 91. 92. A polypeptide sequence having or consisting of 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity.

[0765] Item 56. The construct according to Item 55, wherein HCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 87, HCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 88, and HCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 89, and the light chain variable region comprises or is composed of three complementarity-determining regions (LCDR1 to LCDR3) and four frame regions (LFR1 to LFR4), wherein LCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 90, LCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 91, and LCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 92.

[0766] Item 57. The construct according to Item 47 or 48, wherein the heavy chain variable domain binding to LTBR comprises or is composed of three complementarity-determining regions (HCDR1-HCDR3) and four framework regions (HFR1-HFR4), wherein HCDR1 comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 93, HCDR2 comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 94, and HCDR3 comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 94, and HCDR3 comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 94. 95 contains a polypeptide sequence or is composed of 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 96, and the light chain variable domain binding to LTBR comprises or is composed of three complementarity-determining regions (LCDR1-LCDR3) and four frame regions (LFR1-LFR4), wherein LCDR1 contains or is composed of a polypeptide sequence or is composed of SEQ ID NO: 96 containing 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 97, LCDR2 contains or is composed of a polypeptide sequence or is composed of SEQ ID NO: 97 containing 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 97, and LCDR3 contains or is composed of a polypeptide sequence or is composed of SEQ ID NO: 97 containing 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 97. 98. A polypeptide sequence having or consisting of 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity.

[0767] Item 58. The construct according to Item 57, wherein HCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 93, HCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 94, and HCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 95, and the light chain variable domain comprises or is composed of three complementarity-determining regions (LCDR1 to LCDR3) and four frame regions (LFR1 to LFR4), wherein LCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 96, LCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 97, and LCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 98.

[0768] Item 59. The construct according to Item 47 or 48, wherein the heavy chain variable domain binding to LTBR comprises or consists of three complementarity-determining regions (HCDR1-HCDR3) and four framework regions (HFR1-HFR4), wherein HCDR1 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 99, HCDR2 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 100, and HCDR3 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: SEQ ID NO: 101 contains or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity, and the light chain variable domain binding to LTBR comprises or is composed of three complementarity-determining regions (LCDR1-LCDR3) and four frame regions (LFR1-LFR4), wherein LCDR1 contains or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 102, LCDR2 contains or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 103, and LCDR3 contains or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 103, and LCDR3 contains or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 103. 104 consists of polypeptide sequences with 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity.

[0769] Item 60. The construct according to Item 59, wherein HCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 99, HCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 100, and HCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 101, and the light chain variable domain comprises or is composed of three complementarity-determining regions (LCDR1-LCDR3) and four frame regions (LFR1-LFR4), wherein LCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 102, LCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 103, and LCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 104.

[0770] Item 61. An construct according to any one of items 47 or 48, wherein the heavy chain variable domain binding to LTBR comprises or consists of three complementarity-determining regions (HCDR1-HCDR3) and four framework regions (HFR1-HFR4), wherein HCDR1 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 105, HCDR2 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 106, and HCDR3 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 106, and HCDR3 comprises or consists of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 106. 107 contains or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 108, and the light chain variable domain binding to LTBR comprises or is composed of three complementarity-determining regions (LCDR1-LCDR3) and four frame regions (LFR1-LFR4), wherein LCDR1 contains or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 109, LCDR2 contains or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 109, and LCDR3 contains or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 109. 110 is a polypeptide sequence or a polypeptide sequence having or consisting of 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity.

[0771] Item 62. The construct according to Item 61, wherein HCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 105, HCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 106, and HCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 107, and the light chain variable domain comprises or is composed of three complementarity-determining regions (LCDR1-LCDR3) and four frame regions (LFR1-LFR4), wherein LCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 108, LCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 109, and LCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 110.

[0772] Item 63. The construct according to Item 47 or 48, wherein the heavy chain variable domain binding to LTBR comprises or is composed of three complementarity-determining regions (HCDR1 to HCDR3) and four framework regions (HFR1 to HFR4), wherein HCDR1 comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 111, HCDR2 comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 112, and HCDR3 comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 112, and HCDR3 comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 112. 113 contains a polypeptide sequence having or being composed of 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 114, and the light chain variable domain binding to LTBR comprises or is composed of three complementarity-determining regions (LCDR1 to LCDR3) and four frame regions (LFR1 to LFR4), wherein LCDR1 contains or is composed of a polypeptide sequence having or being composed of 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 114, LCDR2 contains or is composed of a polypeptide sequence having or being composed of 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 115, and LCDR3 contains or is composed of a polypeptide sequence having or being composed of SEQ ID NO: 115. 116 is a polypeptide sequence or a polypeptide sequence having or consisting of 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity.

[0773] Item 64. The construct according to Item 63, wherein HCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 111, HCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 112, and HCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 113, and the light chain variable domain comprises or is composed of three complementarity-determining regions (LCDR1-LCDR3) and four frame regions (LFR1-LFR4), wherein LCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 114, LCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 115, and LCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 116.

[0774] Item 65. The construct according to Item 47 or 48, wherein the heavy chain variable domain binding to LTBR comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher sequence identity with SEQ ID NO: 27, and the light chain variable domain binding to LTBR comprises a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher sequence identity with SEQ ID NO: 28.

[0775] Item 66. The construct according to Item 65, wherein the heavy chain variable domain comprises or is composed of the polypeptide sequence of SEQ ID NO: 27, and the light chain variable domain comprises or is composed of the polypeptide sequence of SEQ ID NO: 28.

[0776] Item 67. The construct according to Item 47 or Item 48, wherein the heavy chain variable domain binding to LTBR comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher sequence identity with SEQ ID NO: 29, and the light chain variable domain binding to LTBR comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher sequence identity with SEQ ID NO: 30.

[0777] Item 68. The construct according to Item 67, wherein the heavy chain variable domain comprises or is composed of the polypeptide sequence of SEQ ID NO: 29, and the light chain variable domain comprises or is composed of the polypeptide sequence of SEQ ID NO: 30.

[0778] Item 69. The construct according to Item 47 or 48, wherein the heavy chain variable domain binding to LTBR comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher sequence identity with SEQ ID NO: 31, and the light chain variable domain binding to LTBR comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher sequence identity with SEQ ID NO: 32.

[0779] Item 70. The construct according to Item 69, wherein the heavy chain variable domain comprises or is composed of the polypeptide sequence of SEQ ID NO: 31, and the light chain variable domain comprises or is composed of the polypeptide sequence of SEQ ID NO: 32.

[0780] Item 71. The construct according to Item 47 or 48, wherein the heavy chain variable domain binding to LTBR comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher sequence identity with the sequence of SEQ ID NO: 33, and the light chain variable domain binding to LTBR comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher sequence identity with the sequence of SEQ ID NO: 34.

[0781] Item 72. The construct according to Item 71, wherein the heavy chain variable domain comprises or is composed of the polypeptide sequence of SEQ ID NO: 33, and the light chain variable domain comprises or is composed of the polypeptide sequence of SEQ ID NO: 34.

[0782] Item 73. The construct according to Item 47 or 48, wherein the heavy chain variable domain binding to LTBR comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher sequence identity with the sequence of SEQ ID NO: 35, and the light chain variable domain binding to LTBR comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher sequence identity with the sequence of SEQ ID NO: 36.

[0783] Item 74. The construct according to Item 73, wherein the heavy chain variable domain comprises or is composed of the polypeptide sequence of SEQ ID NO: 35, and the light chain variable domain comprises or is composed of the polypeptide sequence of SEQ ID NO: 36.

[0784] Item 75. The construct according to Item 47 or 48, wherein the heavy chain variable domain binding to LTBR comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher sequence identity with SEQ ID NO: 37, and the light chain variable domain binding to LTBR comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher sequence identity with SEQ ID NO: 38.

[0785] Item 76. The construct according to Item 75, wherein the heavy chain variable domain comprises or is composed of the polypeptide sequence of SEQ ID NO: 37, and the light chain variable region comprises or is composed of the polypeptide sequence of SEQ ID NO: 38.

[0786] Item 77. The construct according to Item 47 or 48, wherein the heavy chain variable domain bound to the LTBR comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher sequence identity with SEQ ID NO: 39, and the light chain variable domain bound to the LTBR comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher sequence identity with SEQ ID NO: 40.

[0787] Item 78. The construct according to Item 77, wherein the heavy chain variable domain comprises or is composed of the polypeptide sequence of SEQ ID NO: 39, and the light chain variable domain comprises or is composed of the polypeptide sequence of SEQ ID NO: 40.

[0788] Item 79. The construct according to Item 47 or 48, wherein the heavy chain variable domain binding to LTBR comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher sequence identity with SEQ ID NO: 41, and the light chain variable domain binding to LTBR comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher sequence identity with SEQ ID NO: 42.

[0789] Item 80. The construct according to Item 79, wherein the heavy chain variable domain comprises or is composed of the polypeptide sequence of SEQ ID NO: 41, and the light chain variable domain comprises or is composed of the polypeptide sequence of SEQ ID NO: 42.

[0790] Item 81. The construct according to any one of items 1 to 80, wherein one or all of the FAP binders comprises or consists of a heavy chain variable domain and a light chain variable domain bound to the FAP.

[0791] Item 82. According to the construct described in Item 81, one or all of the FAP binders are independently selected from scFv, Fv, Fab, Fab', bivalent antibody fragment (F(ab')2), biantibody or microantibody.

[0792] Item 83. The construct according to Item 81 or 82, wherein the heavy chain variable domain binding FAP comprises or is composed of three complementarity-determining regions (HCDR1-HCDR3) and four frame regions (HFR1-HFR4), wherein HCDR1 comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 117, HCDR2 comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 118, and HCDR3 comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 118, and HCDR3 comprises or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 118. SEQ ID NO: 119 contains or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity, and the light chain variable domain binding to FAP comprises or is composed of three complementarity-determining regions (LCDR1-LCDR3) and four frame regions (LFR1-LFR4), wherein LCDR1 contains or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 120, LCDR2 contains or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 121, and LCDR3 contains or is composed of a polypeptide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher sequence identity with SEQ ID NO: 120. 122. A polypeptide sequence having or consisting of a sequence identity of 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher.

[0793] Item 84. The construct according to Item 83, wherein HCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 117, HCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 118, and HCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 119, and the light chain variable domain comprises or is composed of three complementarity-determining regions (LCDR1 to LCDR3) and four frame regions (LFR1 to LFR4), wherein LCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 120, LCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 121, and LCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 122.

[0794] Item 85. The ...

Claims

1. A construct comprising an FAP binder and an LTBR binder.

2. The construct of claim 1, wherein the construct comprises more than one FAP binder.

3. The construct of claim 1 or 2, wherein the construct comprises more than one LTBR binder.

4. The construct according to any one of claims 1 to 3, wherein the construct comprises an antibody or antibody fragment, one or all of the FAP binders being FAP-binding domains, and one or all of the LTBR binders being LTBR-binding domains.

5. The construct of claim 4, wherein the antibody fragment comprises an Fc region, and one or all of the LTBR binders are directly or via a linker to the Fc region of the antibody or antibody fragment, and wherein the linker is no more than 20 amino acids in length.

6. The construct of claim 5, wherein one or all of the LTBR binders are connected to the CH3 domain of the Fc region.

7. The construct of claim 5 or 6, wherein one or all of the LTBR binders are connected to the Fc region via a linker of no more than 10 amino acid lengths consisting of glycine and serine residues.

8. The construct according to any one of claims 1 to 7, wherein the LTBR binder is an LTBR agonist.

9. The construct of claim 8, wherein the LTBR binder is an LTBR agonist, and wherein the EC50 of the LTBR binder as determined in a chemokine induction assay is 1.0E-08 M or lower.

10. The construct of claim 8 or 9, wherein the LTBR agonist activates the classical NFKB pathway.

11. The construct according to any one of claims 8 to 10, wherein the LTBR binder is capable of activating LTBR on cells expressing both LTBR and FAP.

12. The construct according to any one of claims 1 to 11, wherein one or all of the LTBR binders is a variable domain (e.g., VHH) that binds LTBR, ​​wherein the variable domain that binds LTBR comprises or consists of three heavy chain complementarity-determining regions (HCDR1-HCDR3) and four framework regions (FR1-FR4), wherein: (a) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 60 or 172, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 61 or 173, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 62 or 174, or (b) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 57 or 166, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 58 or 167, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 59 or 168, or (c) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 63 or 163, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 64 or 164, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 65 or 165, or (d)HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 66 or 169, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 67 or 170, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 68 or 171.

13. The construct of claim 12, wherein one or all of the LTBR binders are variable domains (e.g., VHH) that bind LTBR, ​​wherein the variable domains that bind LTBR comprise or consist of three heavy chain complementarity-determining regions (HCDR1-HCDR3) and four framework regions (FR1-FR4), wherein: (a) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 60, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 61, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 62, or (b) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 57, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 58, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 59, or (c) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 63, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 64, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 65, or (d) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 66, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 67, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO:

68.

14. The construct of claim 12, wherein one or all of the LTBR binders are variable domains (e.g., VHH) that bind LTBR, ​​wherein the variable domains that bind LTBR comprise or consist of three heavy chain complementarity-determining regions (HCDR1-HCDR3) and four framework regions (FR1-FR4), wherein: (a) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 172, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 173, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 174, or (b) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 166, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 167, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 168, or (c) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 163, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 164, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 165, or (d) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 169, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 170, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO:

171.

15. The construct of any one of claims 1 to 11, wherein one or all of the LTBR binders are paired heavy chain variable domains and light chain variable domains, wherein the heavy chain variable domains binding the LTBR comprise or consist of three complementary determinant regions (HCDR1-HCDR3) and four framework regions (HFR1-HFR4), and the light chain variable domains binding the LTBR comprise or consist of three complementary determinant regions (LCDR1-LCDR3) and four framework regions (LFR1-LFR4), wherein: (a) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 69, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 70, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 71, wherein LCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 72, LCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 73, and LCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 74, or (b) HCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 75, HCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 76, and HCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 77, wherein LCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 78, LCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 79, and LCDR3 comprises or is composed of the polypeptide sequence shown in SEQ ID NO: 80, or (c) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 81, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 82, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 83, wherein LCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 84, LCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 8, and LCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 86, or (d) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 87, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 88, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 89, wherein LCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 90, LCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 91, and LCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 92, or (e) HCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 93, HCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 94, and HCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 95, wherein LCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 96, LCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 97, and LCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 98, or (f) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 99, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 100, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 101, wherein LCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 102, LCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 103, and LCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 104, or (g) HCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 105, HCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 106, and HCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 107; and wherein LCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 108, LCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 109, and LCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 110, or (h)HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 111, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 112, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 113, wherein LCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 114, LCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 115, and LCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO:

116.

16. The construct according to any one of claims 1 to 15, wherein one or all of the FAP binders are paired heavy chain variable domains and light chain variable domains, wherein the heavy chain variable domains binding the FAP comprise or consist of three complementary determinant regions (HCDR1-HCDR3) and four framework regions (HFR1-HFR4), and The light chain variable domain combined with FAP comprises or consists of three complementary determinant regions (LCDR1-LCDR3) and four frame regions (LFR1-LFR4), wherein: (a) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 117, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 118, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 119, wherein LCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 120, LCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 121, and LCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 122, or (b) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 123, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 124, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 125, wherein LCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 126, LCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 127, and LCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 128, or (c) HCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 129, HCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 130, and HCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 131, wherein LCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 132, LCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 133, and LCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 134, or (d) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 135, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 136, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 137, wherein LCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 138, LCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 139, and LCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 140, or (e) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 141, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 142, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 143, wherein LCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 144, LCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 145, and LCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 146, or (f) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 147, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 148, HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 149, and wherein LCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 150, LCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 151, and LCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 152, or (g)HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 153, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 154, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 155, and wherein LCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 156, LCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 157, and LCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO:

158.

17. The construct of any one of claims 1 to 16, wherein one or all of the LTBR binders are VHH.

18. The construct of any one of claims 1 to 16, wherein the construct comprises: (a) A first heavy chain, a light chain, and a second heavy chain, wherein the first heavy chain contains VH, CH1, CH2, and CH3 from the N-terminus to the C-terminus, the second heavy chain contains VHH, CH2, and CH3 from the N-terminus to the C-terminus, and the light chain contains VL and CL from the N-terminus to the C-terminus, and combines with VH and CH1 of the first heavy chain to form the Fab region, while the CH2 and CH3 regions of the first and second heavy chains combine to form the Fc region, wherein the VH and VL regions combine to the FAP, and the VHH combines to the LTBR (e.g., essentially as shown in Figure 7a). (b) A first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein the first heavy chain contains VH, CH1, CH2, CH3, and VHH from the N-terminus to the C-terminus, the second heavy chain contains VH, CH1, CH2, and CH3 from the N-terminus to the C-terminus, the first light chain contains VL and CL from the N-terminus to the C-terminus and combines with VH and CH1 of the first heavy chain to form a Fab region, the second light chain contains VL and CL from the N-terminus to the C-terminus and combines with VH and CH1 of the second heavy chain to form a Fab region, and the CH2 and CH3 domains of the first and second heavy chains combine to form an Fc region, wherein the VH and VL of each Fab region are bound to the FAP, and the VHH is bound to the LTBR (e.g., essentially as shown in Figure 7b). (c) A first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein the first heavy chain contains VH, CH1, CH2, CH3, and VHH from the N-terminus to the C-terminus; the second heavy chain contains VH, CH1, CH2, CH3, and VHH from the N-terminus to the C-terminus; the first light chain contains VL and CL from the N-terminus to the C-terminus and combines with VH and CH1 of the first heavy chain to form a Fab region; the second light chain contains VL and CL from the N-terminus to the C-terminus and combines with VH and CH1 of the second heavy chain to form a Fab region; and the CH2 and CH3 domains of the first and second heavy chains combine to form an Fc region, wherein the VH and VL of each Fab region are bound to the FAP, and the VHH is bound to the LTBR (e.g., essentially as shown in Figure 7c), or (d) A first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein the first heavy chain contains VH, CH1, CH2, CH3 and paired VH and VL from the N-end to the C-end, the second heavy chain contains VH, CH1, CH2, CH3 and paired VH and VL from the N-end to the C-end, the first light chain contains VL and CL from the N-end to the C-end and combines with VH and CH1 of the first heavy chain to form a Fab region, the second light chain contains VL and CL from the N-end to the C-end and combines with VH and CH1 of the second heavy chain to form another Fab region, and the CH2 and CH3 domains of the first and second heavy chains combine to form an Fc region, wherein the VH and VL of each Fab region are bound to FAP, while the paired VH and VL are bound to LTBR (e.g., essentially as shown in Figure 7d).

19. The construct according to any one of claims 1 to 18, wherein the construct is an antibody, comprising: (a) One or more Fab regions, each Fab region containing a paired heavy chain variable domain and a light chain variable domain bound to the FAP; wherein (i) The heavy chain variable domain contains or is composed of the polypeptide sequence of SEQ ID NO: 43, while the light chain variable domain contains or is composed of the polypeptide sequence of SEQ ID NO: 44; (ii) The heavy chain variable domain contains or is composed of the polypeptide sequence of SEQ ID NO: 45, and the light chain variable domain contains or is composed of the polypeptide sequence of SEQ ID NO: 46; or (iii) The heavy chain variable domain contains or is composed of the polypeptide sequence of SEQ ID NO: 47, and the light chain variable domain contains or is composed of the polypeptide sequence of SEQ ID NO:

48. and (b) One or more paired heavy chain variable domains and light chain variable domains bound to the LTBR; The heavy chain variable domain contains or is composed of the polypeptide sequence of SEQ ID NO: 31, and the light chain variable domain contains or is composed of the polypeptide sequence of SEQ ID NO:

32. Or one or more variable domains that are combined into the LTBR; where (i) The heavy chain variable domain contains or is composed of the polypeptide sequence of SEQ ID NO: 24; or (ii) The heavy chain variable domain contains or is composed of or consists of the polypeptide sequence of SEQ ID NO:

25.

20. The construct as described in any one of claims 1 to 18, characterized in that... This construct is an antibody that contains: (i) A heterodimer of two different heavy chains (HC1 and HC2) and two identical light chains (LC), wherein: (a) The polypeptide sequence of HC1 contains or is composed of SEQ ID NO: 175, the polypeptide sequence of HC2 contains or is composed of SEQ ID NO: 176, and the polypeptide sequence of LC contains or is composed of SEQ ID NO:

177. (b) The HC1 polypeptide sequence contains or is composed of SEQ ID NO: 178, the HC2 polypeptide sequence contains or is composed of SEQ ID NO: 179, and the LC polypeptide sequence contains or is composed of SEQ ID NO:

180. (c) The HC1 polypeptide sequence contains or is composed of SEQ ID NO: 181, the HC2 polypeptide sequence contains or is composed of SEQ ID NO: 182, and the LC polypeptide sequence contains or is composed of SEQ ID NO:

183. (d) The HC1 polypeptide sequence contains or is composed of SEQ ID NO: 184, the HC2 polypeptide sequence contains or is composed of SEQ ID NO: 185, and the LC polypeptide sequence contains or is composed of SEQ ID NO:

186. (e) The HC1 polypeptide sequence contains or is composed of SEQ ID NO: 187, the HC2 polypeptide sequence contains or is composed of SEQ ID NO: 188, and the LC polypeptide sequence contains or is composed of SEQ ID NO:

189. (f) The HC1 polypeptide sequence contains or consists of SEQ ID NO: 190, the HC2 polypeptide sequence contains or consists of SEQ ID NO: 191, and the LC polypeptide sequence contains or consists of SEQ ID NO:

192. (g) The HC1 polypeptide sequence comprises or is composed of SEQ ID NO: 193, the HC2 polypeptide sequence comprises or is composed of SEQ ID NO: 194, and the LC polypeptide sequence comprises or is composed of SEQ ID NO: 195; or (h) The HC1 polypeptide sequence contains or is composed of SEQ ID NO: 200, the HC2 polypeptide sequence contains or is composed of SEQ ID NO: 201, and the LC polypeptide sequence contains or is composed of SEQ ID NO: 202; or (ii) A homodimer consisting of two identical heavy chains (HC) and two identical light chains (LC), wherein: (a) The HC polypeptide sequence contains or is composed of SEQ ID NO: 196, and the LC polypeptide sequence contains or is composed of SEQ ID NO: 197; or (b) The HC polypeptide sequence contains or is composed of SEQ ID NO: 198, and the LC polypeptide sequence contains or is composed of SEQ ID NO:

199.

21. The construct according to any one of claims 1 to 20, for use in tumor treatment.

22. A binding polypeptide comprising or consisting of a heavy chain variable domain (e.g., VHH) that binds to LTBR, ​​wherein the heavy chain variable domain comprises or consists of three heavy chain complementarity-determining regions (HCDR1-HCDR3) and four framework regions (FR1-FR4), and (a) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 60 or 172; HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 61 or 173; HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 62 or 174. (b) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 57 or 166, and HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 58 or 167, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 59 or 168; (c)HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 63 or 163, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 64 or 164, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 65 or 165. (d)HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 66 or 169, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 67 or 170, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 68 or 171. (e)HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 163, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 164, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO:

165. (f) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 166, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 167, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO:

168. (g) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 169, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 170, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 171; or (h)HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 172, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 173, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO:

174.

23. A binding polypeptide comprising, or consisting of, paired heavy chain variable domains and light chain variable domains that bind LTBR, ​​wherein the heavy chain variable domains comprise, or consist of, three complementarity-determining regions (HCDR1-HCDR3) and four framework regions (HFR1-HFR4), and (a) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 69, and HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 70, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 71, and the light chain variable domain contains or is composed of three complementarity-determining regions (LCDR1-LCDR3) and four frame regions (LFR1-LFR4), wherein LCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 72, LCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 73, and LCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO:

74. (b) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 75, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 76, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 77, and the light chain variable domain contains or is composed of three complementarity-determining regions (LCDR1-LCDR3) and four frame regions (LFR1-LFR4), wherein LCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 78, LCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 79, and LCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 80; (c) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 81, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 82, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 83, and the light chain variable domain contains or is composed of three complementarity-determining regions (LCDR1-LCDR3) and four frame regions (LFR1-LFR4), wherein LCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 84, LCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 85, and LCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 86; (d) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 87, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 88, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 89, and the light chain variable domain contains or is composed of three complementarity-determining regions (LCDR1-LCDR3) and four frame regions (LFR1-LFR4), wherein LCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 90, LCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 91, and LCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 92; (e) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 93, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 94, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 95, and the light chain variable domain contains or is composed of three complementarity-determining regions (LCDR1-LCDR3) and four frame regions (LFR1-LFR4), wherein LCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 96, LCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 97, and LCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 98; (f) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 99, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 100, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 101, and the light chain variable domain contains or is composed of three complementarity-determining regions (LCDR1-LCDR3) and four frame regions (LFR1-LFR4), wherein LCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 102, LCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 103, and LCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 104; (g) HCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 105, HCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 106, and HCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 107, and the light chain variable domain comprises or is composed of three complementarity-determining regions (LCDR1-LCDR3) and four frame regions (LFR1-LFR4), wherein LCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 108, LCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 109, and LCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 110; or (h)HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 111, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 112, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 113, and the light chain variable domain contains or is composed of three complementarity-determining regions (LCDR1-LCDR3) and four frame regions (LFR1-LFR4), wherein LCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 114, LCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 115, and LCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO:

116.

24. A binding polypeptide comprising, or consisting of, paired heavy chain variable domains and light chain variable domains that bind FAP, wherein the heavy chain variable domains comprise, or consist of, three complementarity-determining regions (HCDR1-HCDR3) and four framework regions (HFR1-HFR4), and (a) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 117, and HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 118, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 119, and the light chain variable domain contains or is composed of three complementarity-determining regions (LCDR1-LCDR3) and four frame regions (LFR1-LFR4), wherein LCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 120, LCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 121, and LCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO:

122. (b) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 123, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 124, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 125, and the light chain variable domain contains or is composed of three complementarity-determining regions (LCDR1-LCDR3) and four frame regions (LFR1-LFR4), wherein LCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 126, LCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 127, and LCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 128; (c) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 129, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 130, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 131, and the light chain variable domain contains or is composed of three complementarity-determining regions (LCDR1-LCDR3) and four frame regions (LFR1-LFR4), wherein LCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 132, LCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 133, and LCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 134; (d) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 135, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 136, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 137, and the light chain variable domain contains or is composed of three complementarity-determining regions (LCDR1-LCDR3) and four frame regions (LFR1-LFR4), wherein LCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 138, LCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 139, and LCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 140; (e) HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 141, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 142, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 143, and the light chain variable domain contains or is composed of three complementarity-determining regions (LCDR1-LCDR3) and four frame regions (LFR1-LFR4), wherein LCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 144, LCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 145, and LCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 146; (f) HCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 147, HCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 148, and HCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 149, and the light chain variable domain comprises or is composed of three complementarity-determining regions (LCDR1-LCDR3) and four frame regions (LFR1-LFR4), wherein LCDR1 comprises or is composed of the polypeptide sequence of SEQ ID NO: 150, LCDR2 comprises or is composed of the polypeptide sequence of SEQ ID NO: 151, and LCDR3 comprises or is composed of the polypeptide sequence of SEQ ID NO: 152; or (g)HCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 153, HCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 154, and HCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO: 155, and the light chain variable domain contains or is composed of three complementarity-determining regions (LCDR1-LCDR3) and four frame regions (LFR1-LFR4), wherein LCDR1 contains or is composed of the polypeptide sequence of SEQ ID NO: 156, LCDR2 contains or is composed of the polypeptide sequence of SEQ ID NO: 157, and LCDR3 contains or is composed of the polypeptide sequence of SEQ ID NO:

158.

25. The construct or polypeptide of any one of claims 1 to 24, for use in combination with radiotherapy, cancer vaccines or ADCs to treat cancer (e.g., tumors).

26. The construct or polypeptide of any one of claims 1 to 24, for use in combination with an anti-CTLA-4 antibody, an anti-PD-L1 antibody or an anti-PD-1 antibody for the treatment of cancer (e.g., tumors).

27. A binding polypeptide that binds to LTBR, ​​for use in combination with radiotherapy, cancer vaccines, or ADCs to treat cancer (e.g., tumors).

28. The binding polypeptide or construct of any one of claims 22 to 27, wherein the binding polypeptide is an antibody or a fragment thereof.

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