Combination therapy

Abstract

The invention provides inter alia a method of treating cancer in a subject comprising administering to the subject a first construct comprising an LTBR binding agent and a second construct comprising a topoisomerase inhibitor.

Description

[0001] COMBINATION THERAPY

[0002] Field of the invention

[0003] The invention relates to the treatment of cancer using (i) a first construct comprising an LTBR binding agent and (ii) a second construct comprising a topoisomerase inhibitor, and related aspects.

[0004] Background of the invention

[0005] Cancer can affect multiple cell types and tissues but the underlying cause is a breakdown in the control of cell division. This process is highly complex, requiring careful coordination of multiple pathways, many of which remain to be fully characterised.

[0006] Cancer immunotherapy involves the use of a subject's own immune system to treat or prevent cancer. These therapies are designed to provoke the body’s immune system to eliminate cancer cells and exploit the fact that cancers accumulate genetic mutations resulting in the expression of tumor antigens. These tumor antigens consist of tumor-associated antigens such as proteins and tumor-specific antigens or neoantigens, which are peptides that are presented by major histocompatibility molecules (Xie et al 2023). However, malignant tumors can evade detection by the immune system via tumor cell intrinsic mechanisms and mechanisms linked to the tumor microenvironment leading to therapy resistance (Sharma et al 2017). Durable responses to immunotherapy have only been observed in a minority of cancer patients and thus there is a need for the development of new therapies. Key challenges in mounting durable antitumor responses include limited infiltration of immune effector cells in the tumor, immune cell dysfunction and the lack of effective priming of anti-tumor immune cells, e.g. due to lacking tumor immunogenicity.

[0007] Another area of cancer therapy consists of antibody-drug conjugates (ADCs) which have emerged as promising class of cancer therapeutics. An ADC consists of a monoclonal antibody able to bind to and internalize into cancer cells, and a potent cytotoxic payload connected via a chemical linker intended to kill tumor cells. ADCs have been shown to improve patient outcomes in various tumor types and several ADCs have been approved. However, responses to these agents are often limited by drug resistance and safety-related adverse effects of the treatment (Tsuchikama et al 2024).

[0008] There remains a need for new approaches and therapies to treat cancer. Such approaches may demonstrate high response rates and durable responses, reduction in the dose required for effect, an improved safety profile / reduced side effects, or the like.Summary of the invention

[0009] The inventors have established that administration of an LTBR binding agent and a topoisomerase inhibitor is surprisingly effective in treating cancer. The effects of this combination therapy are demonstrated in particular in Example 5 below.

[0010] Accordingly, the invention provides in one aspect a composition comprising a first construct comprising an LTBR binding agent and a second construct comprising a topoisomerase inhibitor.

[0011] The invention also provides a first construct comprising an LTBR binding agent for use in the treatment of cancer in combination with a second construct comprising a topoisomerase inhibitor.

[0012] The invention also provides a second construct comprising a topoisomerase inhibitor for use in the treatment of cancer in combination with a first construct comprising an LTBR binding agent.

[0013] The invention also provides a method of treating cancer in a subject comprising administering to the subject a first construct comprising an LTBR binding agent and a second construct comprising a topoisomerase inhibitor.

[0014] The invention also provides the use of a first construct comprising an LTBR binding agent in the manufacture of a medicament for the treatment of cancer in combination with a second construct comprising a topoisomerase inhibitor.

[0015] The invention also provides the use of a second construct comprising a topoisomerase inhibitor in the manufacture of a medicament for the treatment of cancer in combination with a first construct comprising an LTBR binding agent.

[0016] Certain embodiments of the invention may be expected to benefit from one or more of the following advantages over the prior art:

[0017] (a) agonism of LTBR (e.g. activation of NFKB pathways, such as the classical or alternative NFKB pathways)

[0018] (b) clustering of LTBR

[0019] (c) reduced toxicity (e.g. liver toxicity)

[0020] (d) reduced doses (e.g. reduced doses of topoisomerase inhibitor relative to monotherapy)

[0021] (e) improved biodistribution

[0022] (f) improved therapeutic efficacy (e.g. improved therapeutic response rates, progression free survival and duration of response)

[0023] (g) localisation to, and conditional activation at the target site

[0024] (h) upregulation of intercellular adhesion molecules (e.g. ICAM-1)

[0025] (i) increased secretion of CCL5 (e.g. from RPMI-7951 cells)(j) increased yield and / or purity

[0026] (k) improved half-life and / or reduced rate of clearance.

[0027] Further aspects of the invention will become clear from the below.

[0028] Summary of sequences

[0029] SEQ ID NO: 1 Polypeptide sequence of BHA10 heavy chain variable region

[0030] SEQ ID NO: 2 Polypeptide sequence of BHA10 light chain variable region

[0031] SEQ ID NO: 3 Polypeptide sequence of a G4S peptide linker

[0032] SEQ ID NO: 4 Polypeptide sequence of a (648)4 peptide linker

[0033] SEQ ID NO: 5 Polypeptide sequence of 5G11 heavy chain variable region

[0034] SEQ ID NO: 6 Polypeptide sequence of 5G11 light chain variable region

[0035] SEQ ID NO: 7 Polypeptide sequence of 28H1 heavy chain variable region

[0036] SEQ ID NO: 8 Polypeptide sequence of 28H1 light chain variable region

[0037] SEQ ID NO: 9 Polypeptide sequence of full length human LTBR

[0038] SEQ ID NO: 10 Polypeptide sequence of human LTBR extracellular domain

[0039] SEQ ID NO: 11 Polypeptide sequence of full length Macaca fascicularis LTBR

[0040] SEQ ID NO: 12 Polypeptide sequence of Macaca fascicularis LTBR extracellular domain SEQ ID NO: 13 Polypeptide sequence of full length mouse LTBR

[0041] SEQ ID NO: 14 Polypeptide sequence of mouse LTBR extracellular domain

[0042] SEQ ID NO: 15 Polypeptide sequence of full length human FAP

[0043] SEQ ID NO: 16 Polypeptide sequence of human FAP extracellular domain

[0044] SEQ ID NO: 17 Polypeptide sequence of full length Macaca fascicularis FAP

[0045] SEQ ID NO: 18 Polypeptide sequence of Macaca fascicularis FAP extracellular domain SEQ ID NO: 19 Polypeptide sequence of full length mouse FAP

[0046] SEQ ID NO: 20 Polypeptide sequence of mouse FAP extracellular domain

[0047] SEQ ID NO: 21 Polypeptide sequence of human LIGHT (including ECD)

[0048] SEQ ID NO: 22 Polypeptide sequence of human LIGHT ECD

[0049] SEQ ID NO: 23 Polypeptide sequence of Clone 1.5 heavy chain variable region SEQ ID NO: 24 Polypeptide sequence of Clone 1.6 heavy chain variable region SEQ ID NO: 25 Polypeptide sequence of Clone 1.7 heavy chain variable region SEQ ID NO: 26 Polypeptide sequence of Clone 1.8 heavy chain variable region SEQ ID NO: 27 Polypeptide sequence of Clone 2.1 heavy chain variable region SEQ ID NO: 28 Polypeptide sequence of Clone 2.1 light chain variable region

[0050] SEQ ID NO: 29 Polypeptide sequence of Clone 2.2 heavy chain variable region SEQ ID NO: 30 Polypeptide sequence of Clone 2.2 light chain variable region

[0051] SEQ ID NO: 31 Polypeptide sequence of Clone 2.3 heavy chain variable regionSEQ ID NO: 32 Polypeptide sequence of Clone 2.3 light chain variable region SEQ ID NO: 33 Polypeptide sequence of Clone 2.4 heavy chain variable region SEQ ID NO: 34 Polypeptide sequence of Clone 2.4 light chain variable region SEQ ID NO: 35 Polypeptide sequence of Clone 2.5 heavy chain variable region SEQ ID NO: 36 Polypeptide sequence of Clone 2.5 light chain variable region SEQ ID NO: 37 Polypeptide sequence of Clone 2.6 heavy chain variable region SEQ ID NO: 38 Polypeptide sequence of Clone 2.6 light chain variable region SEQ ID NO: 39 Polypeptide sequence of Clone 2.7 heavy chain variable region SEQ ID NO: 40 Polypeptide sequence of Clone 2.7 light chain variable region SEQ ID NO: 41 Polypeptide sequence of Clone 2.8 heavy chain variable region SEQ ID NO: 42 Polypeptide sequence of Clone 2.8 light chain variable region SEQ ID NO: 43 Polypeptide sequence of Clone 3.5 heavy chain variable region SEQ ID NO: 44 Polypeptide sequence of Clone 3.5 light chain variable region SEQ ID NO: 45 Polypeptide sequence of Clone 3.6 heavy chain variable region SEQ ID NO: 46 Polypeptide sequence of Clone 3.6 light chain variable region SEQ ID NO: 47 Polypeptide sequence of Clone 3.7 heavy chain variable region SEQ ID NO: 48 Polypeptide sequence of Clone 3.7 light chain variable region SEQ ID NO: 49 Polypeptide sequence of Clone 3.4 heavy chain variable region SEQ ID NO: 50 Polypeptide sequence of Clone 3.4 light chain variable region SEQ ID NO: 51 Polypeptide sequence of Clone 3.1 heavy chain variable region SEQ ID NO: 52 Polypeptide sequence of Clone 3.1 light chain variable region SEQ ID NO: 53 Polypeptide sequence of Clone 3.2 heavy chain variable region SEQ ID NO: 54 Polypeptide sequence of Clone 3.2 light chain variable region SEQ ID NO: 55 Polypeptide sequence of Clone 3.3 heavy chain variable region SEQ ID NO: 56 Polypeptide sequence of Clone 3.3 light chain variable region SEQ ID NO: 57 Polypeptide sequence of Clone 1.5 HCDR1

[0052] SEQ ID NO: 58 Polypeptide sequence of Clone 1.5 HCDR2

[0053] SEQ ID NO: 59 Polypeptide sequence of Clone 1.5 HCDR3

[0054] SEQ ID NO: 60 Polypeptide sequence of Clone 1.6 HCDR1

[0055] SEQ ID NO: 61 Polypeptide sequence of Clone 1.6 HCDR2

[0056] SEQ ID NO: 62 Polypeptide sequence of Clone 1.6 HCDR3

[0057] SEQ ID NO: 63 Polypeptide sequence of Clone 1.7 HCDR1

[0058] SEQ ID NO: 64 Polypeptide sequence of Clone 1.7 HCDR2

[0059] SEQ ID NO: 65 Polypeptide sequence of Clone 1.7 HCDR3

[0060] SEQ ID NO: 66 Polypeptide sequence of Clone 1.8 HCDR1

[0061] SEQ ID NO: 67 Polypeptide sequence of Clone 1.8 HCDR2SEQ ID NO: 68 Polypeptide sequence of Clone 1.8 HCDR3 SEQ ID NO: 69 Polypeptide sequence of Clone 2.1 HCDR1 SEQ ID NO: 70 Polypeptide sequence of Clone 2.1 HCDR2 SEQ ID NO: 71 Polypeptide sequence of Clone 2.1 HCDR3 SEQ ID NO: 72 Polypeptide sequence of Clone 2.1 LCDR1 SEQ ID NO: 73 Polypeptide sequence of Clone 2.1 LCDR2 SEQ ID NO: 74 Polypeptide sequence of Clone 2.1 LCDR3 SEQ ID NO: 75 Polypeptide sequence of Clone 2.2 HCDR1 SEQ ID NO: 76 Polypeptide sequence of Clone 2.2 HCDR2 SEQ ID NO: 77 Polypeptide sequence of Clone 2.2 HCDR3 SEQ ID NO: 78 Polypeptide sequence of Clone 2.2 LCDR1 SEQ ID NO: 79 Polypeptide sequence of Clone 2.2 LCDR2 SEQ ID NO: 80 Polypeptide sequence of Clone 2.2 LCDR3 SEQ ID NO: 81 Polypeptide sequence of Clone 2.3 HCDR1 SEQ ID NO: 82 Polypeptide sequence of Clone 2.3 HCDR2 SEQ ID NO: 83 Polypeptide sequence of Clone 2.3 HCDR3 SEQ ID NO: 84 Polypeptide sequence of Clone 2.3 LCDR1 SEQ ID NO: 85 Polypeptide sequence of Clone 2.3 LCDR2 SEQ ID NO: 86 Polypeptide sequence of Clone 2.3 LCDR3 SEQ ID NO: 87 Polypeptide sequence of Clone 2.4 HCDR1 SEQ ID NO: 88 Polypeptide sequence of Clone 2.4 HCDR2 SEQ ID NO: 89 Polypeptide sequence of Clone 2.4 HCDR3 SEQ ID NO: 90 Polypeptide sequence of Clone 2.4 LCDR1 SEQ ID NO: 91 Polypeptide sequence of Clone 2.4 LCDR2 SEQ ID NO: 92 Polypeptide sequence of Clone 2.4 LCDR3 SEQ ID NO: 93 Polypeptide sequence of Clone 2.5 HCDR1 SEQ ID NO: 94 Polypeptide sequence of Clone 2.5 HCDR2 SEQ ID NO: 95 Polypeptide sequence of Clone 2.5 HCDR3 SEQ ID NO: 96 Polypeptide sequence of Clone 2.5 LCDR1 SEQ ID NO: 97 Polypeptide sequence of Clone 2.5 LCDR2 SEQ ID NO: 98 Polypeptide sequence of Clone 2.5 LCDR3 SEQ ID NO: 99 Polypeptide sequence of Clone 2.6 HCDR1 SEQ ID NO: 100 Polypeptide sequence of Clone 2.6 HCDR2 SEQ ID NO: 101 Polypeptide sequence of Clone 2.6 HCDR3 SEQ ID NO: 102 Polypeptide sequence of Clone 2.6 LCDR1 SEQ ID NO: 103 Polypeptide sequence of Clone 2.6 LCDR2SEQ ID NO: 104 Polypeptide sequence of Clone 2.6 LCDR3 SEQ ID NO: 105 Polypeptide sequence of Clone 2.7 HCDR1 SEQ ID NO: 106 Polypeptide sequence of Clone 2.7 HCDR2 SEQ ID NO: 107 Polypeptide sequence of Clone 2.7 HCDR3 SEQ ID NO: 108 Polypeptide sequence of Clone 2.7 LCDR1 SEQ ID NO: 109 Polypeptide sequence of Clone 2.7 LCDR2 SEQ ID NO: 110 Polypeptide sequence of Clone 2.7 LCDR3 SEQ ID NO: 111 Polypeptide sequence of Clone 2.8 HCDR1 SEQ ID NO: 112 Polypeptide sequence of Clone 2.8 HCDR2 SEQ ID NO: 113 Polypeptide sequence of Clone 2.8 HCDR3 SEQ ID NO: 114 Polypeptide sequence of Clone 2.8 LCDR1 SEQ ID NO: 115 Polypeptide sequence of Clone 2.8 LCDR2 SEQ ID NO: 116 Polypeptide sequence of Clone 2.8 LCDR3 SEQ ID NO: 117 Polypeptide sequence of Clone 3.5 HCDR1 SEQ ID NO: 118 Polypeptide sequence of Clone 3.5 HCDR2 SEQ ID NO: 119 Polypeptide sequence of Clone 3.5 HCDR3 SEQ ID NO: 120 Polypeptide sequence of Clone 3.5 LCDR1 SEQ ID NO: 121 Polypeptide sequence of Clone 3.5 LCDR2 SEQ ID NO: 122 Polypeptide sequence of Clone 3.5 LCDR3 SEQ ID NO: 123 Polypeptide sequence of Clone 3.6 HCDR1 SEQ ID NO: 124 Polypeptide sequence of Clone 3.6 HCDR2 SEQ ID NO: 125 Polypeptide sequence of Clone 3.6 HCDR3 SEQ ID NO: 126 Polypeptide sequence of Clone 3.6 LCDR1 SEQ ID NO: 127 Polypeptide sequence of Clone 3.6 LCDR2 SEQ ID NO: 128 Polypeptide sequence of Clone 3.6 LCDR3 SEQ ID NO: 129 Polypeptide sequence of Clone 3.7 HCDR1 SEQ ID NO: 130 Polypeptide sequence of Clone 3.7 HCDR2 SEQ ID NO: 131 Polypeptide sequence of Clone 3.7 HCDR3 SEQ ID NO: 132 Polypeptide sequence of Clone 3.7 LCDR1 SEQ ID NO: 133 Polypeptide sequence of Clone 3.7 LCDR2 SEQ ID NO: 134 Polypeptide sequence of Clone 3.7 LCDR3 SEQ ID NO: 135 Polypeptide sequence of Clone 3.4 HCDR1 SEQ ID NO: 136 Polypeptide sequence of Clone 3.4 HCDR2 SEQ ID NO: 137 Polypeptide sequence of Clone 3.4 HCDR3 SEQ ID NO: 138 Polypeptide sequence of Clone 3.4 LCDR1 SEQ ID NO: 139 Polypeptide sequence of Clone 3.4 LCDR2SEQ ID NO: 140 Polypeptide sequence of Clone 3.4 LCDR3 SEQ ID NO: 141 Polypeptide sequence of Clone 3.1 HCDR1 SEQ ID NO: 142 Polypeptide sequence of Clone 3.1 HCDR2 SEQ ID NO: 143 Polypeptide sequence of Clone 3.1 HCDR3 SEQ ID NO: 144 Polypeptide sequence of Clone 3.1 LCDR1 SEQ ID NO: 145 Polypeptide sequence of Clone 3.1 LCDR2 SEQ ID NO: 146 Polypeptide sequence of Clone 3.1 LCDR3 SEQ ID NO: 147 Polypeptide sequence of Clone 3.2 HCDR1 SEQ ID NO: 148 Polypeptide sequence of Clone 3.2 HCDR2 SEQ ID NO: 149 Polypeptide sequence of Clone 3.2 HCDR3 SEQ ID NO: 150 Polypeptide sequence of Clone 3.2 LCDR1 SEQ ID NO: 151 Polypeptide sequence of Clone 3.2 LCDR2 SEQ ID NO: 152 Polypeptide sequence of Clone 3.2 LCDR3 SEQ ID NO: 153 Polypeptide sequence of Clone 3.3 HCDR1 SEQ ID NO: 154 Polypeptide sequence of Clone 3.3 HCDR2 SEQ ID NO: 155 Polypeptide sequence of Clone 3.3 HCDR3 SEQ ID NO: 156 Polypeptide sequence of Clone 3.3 LCDR1 SEQ ID NO: 157 Polypeptide sequence of Clone 3.3 LCDR2 SEQ ID NO: 158 Polypeptide sequence of Clone 3.3 LCDR3 SEQ ID NO: 159 Polypeptide sequence of Clone 1.1 SEQ ID NO: 160 Polypeptide sequence of Clone 1.2 SEQ ID NO: 161 Polypeptide sequence of Clone 1.3 SEQ ID NO: 162 Polypeptide sequence of Clone 1.4 SEQ ID NO: 163 Polypeptide sequence of Clone 1.1 HCDR1 SEQ ID NO: 164 Polypeptide sequence of Clone 1.1 HCDR2 SEQ ID NO: 165 Polypeptide sequence of Clone 1.1 HCDR3 SEQ ID NO: 166 Polypeptide sequence of Clone 1.2 HCDR1 SEQ ID NO: 167 Polypeptide sequence of Clone 1.2 HCDR2 SEQ ID NO: 168 Polypeptide sequence of Clone 1.2 HCDR3 SEQ ID NO: 169 Polypeptide sequence of Clone 1.3 HCDR1 SEQ ID NO: 170 Polypeptide sequence of Clone 1.3 HCDR2 SEQ ID NO: 171 Polypeptide sequence of Clone 1.3 HCDR3 SEQ ID NO: 172 Polypeptide sequence of Clone 1.4 HCDR1 SEQ ID NO: 173 Polypeptide sequence of Clone 1.4 HCDR2 SEQ ID NO: 174 Polypeptide sequence of Clone 1.4 HCDR3 SEQ ID NO: 175 Polypeptide sequence of Clone 4.1 HC1SEQ ID NO: 176 Polypeptide sequence of Clone 4.1 HC2

[0062] SEQ ID NO: 177 Polypeptide sequence of Clone 4.1 LC

[0063] SEQ ID NO: 178 Polypeptide sequence of Clone 4.2 HC1

[0064] SEQ ID NO: 179 Polypeptide sequence of Clone 4.2 HC2

[0065] SEQ ID NO: 180 Polypeptide sequence of Clone 4.2 LC

[0066] SEQ ID NO: 181 Polypeptide sequence of Clone 4.3 HC1

[0067] SEQ ID NO: 182 Polypeptide sequence of Clone 4.3 HC2

[0068] SEQ ID NO: 183 Polypeptide sequence of Clone 4.3 LC

[0069] SEQ ID NO: 184 Polypeptide sequence of Clone 4.4 HC1

[0070] SEQ ID NO: 185 Polypeptide sequence of Clone 4.4 HC2

[0071] SEQ ID NO: 186 Polypeptide sequence of Clone 4.4 LC

[0072] SEQ ID NO: 187 Polypeptide sequence of Clone 4.5 HC1

[0073] SEQ ID NO: 188 Polypeptide sequence of Clone 4.5 HC2

[0074] SEQ ID NO: 189 Polypeptide sequence of Clone 4.5 LC

[0075] SEQ ID NO: 190 Polypeptide sequence of Clone 4.6 HC1

[0076] SEQ ID NO: 191 Polypeptide sequence of Clone 4.6 HC2

[0077] SEQ ID NO: 192 Polypeptide sequence of Clone 4.6 LC

[0078] SEQ ID NO: 193 Polypeptide sequence of Clone 4.7 HC1

[0079] SEQ ID NO: 194 Polypeptide sequence of Clone 4.7 HC2

[0080] SEQ ID NO: 195 Polypeptide sequence of Clone 4.7 LC

[0081] SEQ ID NO: 196 Polypeptide sequence of Clone 4.8 HC

[0082] SEQ ID NO: 197 Polypeptide sequence of Clone 4.8 LC

[0083] SEQ ID NO: 198 Polypeptide sequence of Clone 4.9 HC

[0084] SEQ ID NO: 199 Polypeptide sequence of Clone 4.9 LC

[0085] SEQ ID NO: 200 Polypeptide sequence of Clone 4.10 HC1

[0086] SEQ ID NO: 201 Polypeptide sequence of Clone 4.10 HC2

[0087] SEQ ID NO: 202 Polypeptide sequence of Clone 4.10 LC

[0088] SEQ ID NO: 203 Polypeptide sequence of truncated hinge region

[0089] SEQ ID NO: 204 Polypeptide sequence of native hinge region

[0090] SEQ ID NO: 205 Polypeptide sequence combining SEQ ID NOs 58 and 167 SEQ ID NO: 206 Polypeptide sequence combining SEQ ID NOs 67 and 170 SEQ ID NO: 207 Polypeptide sequence combining SEQ ID NOs 65 and 165 SEQ ID NO: 208 Polypeptide sequence combining SEQ ID NOs 68 and 171 SEQ ID NO: 209 Example second construct linker

[0091] SEQ ID NO: 210 Heavy chain polypeptide sequence of trastuzumab SEQ ID NO: 211 Light chain polypeptide sequence of trastuzumabSEQ ID NO: 212 Polypeptide sequence of HER2

[0092] Summary of the figures

[0093] Figure 1a Screening of anti-LTBR VHHs: HepG2 reporter assay in the absence of anti-His Tag cross-linking antibody

[0094] Figure 1 b Screening of anti-LTBR VHHs: HepG2 reporter assay in the presence of anti-His Tag cross-linking antibody

[0095] Figure 2 In vitro VHH Fc fusion protein activity in chemokine induction assay using tumour cell line HCC1187

[0096] Figure 3a Ligand receptor inhibition assay: Anti-LTBR VHH-Fc fusion proteins binding to A549 cell line

[0097] Figure 3b Ligand receptor inhibition assay: Ligand Receptor Inhibition

[0098] Figure 4 FACS Binding assay: Binding of anti-LTBR Abs to LTBR positive cell line A375 Figure 5a NFKB activation of HepG2 reporter cells expressing human LTBR: HepG2

[0099] reporter assay

[0100] Figure 5b NFKB activation of HepG2 reporter cells expressing human LTBR: HepG2

[0101] reporter assay in the presence of cross-linking Ab

[0102] Figure 6 FACS Binding assay: Binding of anti-FAP Abs to FAP-overexpressing HEK293 cell line

[0103] Figure 7a Production of human and mouse-specific FAP / LTBR bispecific antibodies:

[0104] schematic diagram of a specific 1:1 format

[0105] Figure 7b Production of human and mouse-specific FAP / LTBR bispecific antibodies:

[0106] schematic diagram of a specific 2:1 format

[0107] Figure 7c Production of human and mouse-specific FAP / LTBR bispecific antibodies:

[0108] schematic diagram of a specific 2:2 format

[0109] Figure 7d Production of human and mouse-specific FAP / LTBR bispecific antibodies:

[0110] schematic diagram of a specific 2:2 format

[0111] Figure 8a HepG2 NFKB activation-induced luciferase assay results illustrating activation of LTBR by FAP / LTBR bispecific antibodies in the presence of FAP (monoculture)

[0112] Figure 8b HepG2 NFKB activation-induced luciferase assay results illustrating activation of LTBR by FAP / LTBR bispecific antibodies in the presence of FAP (co-culture) Figure 8c HepG2 NFKB activation-induced luciferase assay results illustrating activation of LTBR by FAP / LTBR bispecific antibodies in the presence of FAP (monoculture)Figure 8d HepG2 NFKB activation-induced luciferase assay results illustrating activation of LTBR by FAP / LTBR bispecific antibodies in the presence of FAP (co-culture) Figure 9a In vitro FAP / LTBR bispecific activity in chemokine induction assay using tumour cell line HCC1187 cultured with FAP positive primary human breast CAFs: Activity in HCC1187 monoculture

[0113] Figure 9b In vitro FAP / LTBR bispecific activity in chemokine induction assay using tumour cell line HCC1187 cultured with FAP positive primary human breast CAFs: Activity in HCC1187:CHO-FAP co-culture

[0114] Figure 9c In vitro FAP / LTBR bispecific activity in chemokine induction assay using tumour cell line HCC1187 cultured with FAP positive primary human breast CAFs: Activity in HCC1187 monoculture

[0115] Figure 9d In vitro FAP / LTBR bispecific activity in chemokine induction assay using tumour cell line HCC1187 cultured with FAP positive primary human breast CAFs: Activity in HCC1187:CHO-FAP co-culture

[0116] Figure 9e In vitro FAP / LTBR bispecific activity in chemokine induction assay using tumour cell line HCC1187 cultured with FAP positive primary human breast CAFs: Activity in HCC1187:CHO-FAP monoculture

[0117] Figure 9f In vitro FAP / LTBR bispecific activity in chemokine induction assay using tumour cell line HCC1187 cultured with FAP positive primary human breast CAFs: Activity in HCC1187:CHO-FAP co-culture

[0118] Figure 10a In vitro FAP / LTBR bispecific activity in ICAM-1 upregulation assay on human breast CAFs: ICAM-1 upregulation on primary human breast fibroblast CAFs post-FAP / LTBR bsAb stimulation

[0119] Figure 10b In vitro FAP / LTBR bispecific activity in ICAM-1 upregulation assay on human breast CAFs: ICAM-1 upregulation on primary human breast fibroblast CAFs post-FAP / LTBR bsAb stimulation

[0120] Figure 11a In vitro FAP / LTBR bispecific activity in chemokine induction assay using tumour cell line RPMI-7951: CCL5 secretion post-FAP / LTBR bsAb stimulation in RPMI- 7951 cells

[0121] Figure 11b In vitro FAP / LTBR bispecific activity in chemokine induction assay using tumour cell line RPMI-7951: CCL5 secretion post-FAP / LTBR bsAb stimulation in RPMI- 7951 cells

[0122] Figure 12a HCC-1187:RPMI-7951 CCL-19 read out: Trans-activation in the presence of low FAP expressing line

[0123] Figure 12b HCC-1187:RPMI-7951 CCL-5 read out: Cis-activation in the presence of double-positive cellsFigure 13a In vitro surrogate FAP / LTBR bispecific activity in chemokine induction assay using tumour cell line 4T1 cultured with FAP positive CHO-overexpressing line (monoculture)

[0124] Figure 13b In vitro surrogate FAP / LTBR bispecific activity in chemokine induction assay using tumour cell line 4T1 cultured with FAP positive CHO-overexpressing line (co-culture)

[0125] Figure 13c In vitro surrogate FAP / LTBR bispecific activity in chemokine induction assay using tumour cell line 4T1 cultured with FAP positive CHO-overexpressing line Figure 13d In vitro surrogate FAP / LTBR bispecific activity in chemokine induction assay using tumour cell line 4T1 cultured with FAP positive CHO-overexpressing line Figure 13e In vitro surrogate FAP / LTBR bispecific activity in chemokine induction assay using tumour cell line 4T1 cultured with FAP positive CHO-overexpressing line Figure 14 In vitro activity of mouse FAP / LTBR agonist antibody in primary mouse lung fibroblasts (graph I, repeat experiment I)

[0126] Figure 15 In vitro activity of mouse FAP / LTBR agonist antibody in primary mouse lung fibroblasts (graph II, repeat experiment II)

[0127] Figure 16 In vivo efficacy with FAP / LTBR bispecific surrogate molecules in orthotopic breast cancer models: Treatment response in orthotopic EMT6 human HER2 syngeneic mouse model in combination with trastuzumab deruxtecan (tumor volume)

[0128] Figure 17 In vivo efficacy with FAP / LTBR bispecific surrogate molecules in orthotopic breast cancer models: Treatment response in orthotopic EMT6 human HER2 syngeneic mouse model in combination with trastuzumab deruxtecan (tumor weight)

[0129] Detailed description of the invention

[0130] The invention utilises a first construct comprising a lymphotoxin beta receptor (LTBR) binding agent and a second construct comprising a topoisomerase inhibitor.

[0131] First construct

[0132] LTBR binding agent

[0133] LTBR is a cell surface receptor for lymphotoxin involved in apoptosis and cytokine release. LTBR is a member of the tumour necrosis factor receptor superfamily and is also known as tumour necrosis factor receptor superfamily member 3 (TNFRSF3).

[0134] The first construct comprises an LTBR binding agent. LTBR binding agents are disclosed in the prior art, for example anti-LTBR binding polypeptide BHA10 (W02004002431,comprising heavy and light chain variable regions of SEQ ID NO: 1 and 2, respectively) and huCBE11 (W02004002431).

[0135] Suitably one or all of the LTBR binding agents are LTBR binding polypeptides, such as an LTBR binding antibody or a LTBR binding fragment thereof. Suitably one or all of the LTBR binding agents comprise or consist of a variable domain which binds to LTBR, such as a VH, VL, VNAR or VHH, and more suitably a heavy chain variable domain such as a VHH.

[0136] The LTBR is suitably situated on the surface of stromal cells, fibroblasts, endothelial cells and / or cancer cells. In one embodiment the LTBR is situated on the surface of a cell wherein the cell also comprises FAP on its surface. The LTBR binding agent suitably binds to the extracellular domain of LTBR.

[0137] In one embodiment, LTBR is a polypeptide sharing 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, such as at least 95% identity with SEQ ID NO: 9 (full length human LTBR).

[0138] Suitably, LTBR comprises such as consists of SEQ ID NO: 9 (full length human LTBR).

[0139] Suitably, the LTBR is the extracellular domain of human LTBR, i.e. the LTBR binding agent binds to the extracellular domain of human LTBR. In one embodiment, LTBR is a polypeptide sharing 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, such as at least 95% identity with SEQ ID NO: 10 (human LTBR extracellular domain). Suitably, LTBR is a polypeptide comprising or consisting of SEQ ID NO: 10 (human LTBR extracellular domain).

[0140] In one embodiment, LTBR is a polypeptide sharing 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, such as at least 95% identity with SEQ ID NO: 11 (full length Macaca fascicularis LTBR). Suitably, LTBR comprises such as consists of SEQ ID NO: 11 (full length Macaca fascicularis LTBR).

[0141] Suitably, the LTBR is the extracellular domain of Macaca fascicularis LTBR, i.e. the LTBR binding agent binds to the extracellular domain of Macaca fascicularis LTBR. In one embodiment, LTBR is a polypeptide sharing 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, such as at least 95% identity with SEQ ID NO: 12 (Macaca fascicularis LTBR extracellular domain). Suitably, LTBR is a polypeptide comprising or consisting of SEQ ID NO: 12 (Macaca fascicularis LTBR extracellular domain).

[0142] In one embodiment, LTBR is a polypeptide sharing 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, such as at least 95% identity with SEQ ID NO: 13 (full length mouse LTBR). Suitably, LTBR comprises such as consists of SEQ ID NO: 13 (full length mouse LTBR).Suitably, the LTBR is the extracellular domain of mouse LTBR, i.e. the LTBR binding agent binds to the extracellular domain of mouse LTBR. In one embodiment, LTBR is a polypeptide sharing 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, such as at least 95% identity with SEQ ID NO: 14 (mouse LTBR extracellular domain). In one embodiment, LTBR is a polypeptide comprising or consisting of SEQ ID NO: 14 (mouse LTBR extracellular domain).

[0143] Suitably one or all of the LTBR binding agents have an EC50 of 9.0E-09 M or less, such as 8.0E-09 M or less, such as 7.0E-09 M or less, such as 6.0E-09 M or less, such as 5.0E-09 M or less, such as 4.0E-09 M or less, such as 3.0E-09 M or less, such as 2.0E-09 M or less, such as 1.0E-09 M or less, such as 9.0E-10 M or less, such as 8.0E-10 M or less, such as 7.0E-10 M or less, such as 6.0E-10 M or less, such as 5.0E-10 M or less, such as 4.0E-10 M or less, such as 3.0E-10 M or less, such as 2.0E-10 M or less, such as 1.0E-10 M or less, such as 9.0E-11 M or less, such as 8.0E-11 M or less, such as 7.0E-11 M or less, such as 6.0E-11 M or less, such as 5.0E-11 M or less, such as 4.0E-11 M or less, such as 3.0E-11 M or less, such as 2.0E-11 M or less, such as 1.0E-11 M or less, such as 9.0E-12 M or less, such as 8.0E-12 M or less, such as 7.0E-12 M or less, such as 6.0E-12 M or less, such as 5.0E-12 M or less, such as 4.0E-12 M or less, such as 3.0E-12 M or less, such as 2.0E-12 M or less, such as 1.0E-12 M or less. Suitably the EC50 is established using a chemokine induction assay, for example assayed using an ELISA. More suitably the EC50 is established using an assay set out in any one of the Examples.

[0144] Suitably one or all of the LTBR binding agents have an EC50 as recited for any of the LTBR binding agents specified in the tables of the Examples or less.

[0145] Suitably the LTBR binding agent binds to LTBR specifically, i.e. the binding agent does not bind significantly to targets other than human LTBR, such as does not bind significantly to targets other than human, Macaca fascicularis or mouse LTBR, such as does not bind significantly to targets other than LTBR.

[0146] In one embodiment, the LTBR binding agent is the LTBR ligand, LIGHT, more suitably human LIGHT.

[0147] In one embodiment, the LTBR binding agent is not the LTBR ligand, human LIGHT, or more suitably the LTBR binding agent is not LIGHT.

[0148] Suitably LIGHT is a polypeptide comprising, consisting essentially of, or consisting of a sequence sharing at least 70%, such as at least 80%, such as at least 90%, such as at least 99%, such as 100% identity, with SEQ ID NO: 21 or SEQ ID NO: 22.

[0149] The first construct of the invention may bind to LTBR with an avidity of 1.0E-08 M or less, such as 9.0E-09 M or less, such as 7.0E-09 M or less, such as 6.7E-09 M or less, suchas 5.0E-09 M or less, such as 3.0E-09 M or less, such as 2.0E-09 M or less, such as 1.0E-09 M or less.

[0150] Suitably one or all of the LTBR binding agents bind to LTBR with an affinity (KD) of 20 nM or less, such as 15 nM or less, such as 10 nM or less, such as 9 nM or less, such as 8 nM or less, such as 7 nM or less, such as 6 nM or less, such as 5 nM or less, such as 4 nM or less, such as 3 nM or less, such as 2 nM or less, such as 1 nM or less. Suitably the KD is established using the assay set out in Example 4.

[0151] Suitably the LTBR binding agent agonises (i.e. activates), inversely agonises, antagonises or neutralises LTBR. In one embodiment the LTBR binding agent is an LTBR agonist. Suitably the agonist activates such NFKB signalling pathways. If the LTBR binding agent is an LTBR agonist, then suitably the LTBR binding agent (a) induces clustering of LTBR and more suitably (b) activates the classical NFKB pathway (suitably leading to expression of adhesion molecules such as ICAMs, such as ICAM-1, VCAM-1 and / or MAdCAM-1) and / or activates the alternative NFKB pathway (suitably leading to expression of chemokines, such as CCL5, CCL19, CCL21 and / or CXCL13).

[0152] Suitably the LTBR binding agent agonises LTBR in that the LTBR binding agent has an EC50 of 9.0E-09 M or less, such as 1.0E-08 M or less, such as 8.0E-09 M or less, such as 7.0E-09 M or less, such as 6.0E-09 M or less, such as 5.0E-09 M or less, such as 4.0E-09 M or less, such as 3.0E-09 M or less, such as 2.0E-09 M or less, such as 1.0E-09 M or less, such as 9.0E-10 M or less, such as 8.0E-10 M or less, such as 7.0E-10 M or less, such as 6.0E-10 M or less, such as 5.0E-10 M or less, such as 4.0E-10 M or less, such as 3.0E-10 M or less, such as 2.0E-10 M or less, such as 1.0E-10 M or less, such as 9.0E-11 M or less, such as 8.0E-11 M or less, such as 7.0E-11 M or less, such as 6.0E-11 M or less, such as 5.0E-11 M or less, such as 4.0E-11 M or less, such as 3.0E-11 M or less, such as 2.0E-11 M or less, such as 1.0E-11 M or less, such as 9.0E-12 M or less, such as 8.0E-12 M or less, such as 7.0E-12 M or less, such as 6.0E-12 M or less, such as 5.0E-12 M or less, such as 4.0E-12 M or less, such as 3.0E-12 M or less, such as 2.0E-12 M or less, such as 1.0E-12 M or less. Alternatiely, the LTBR binding agent may have an EC50 as recited for any of the LTBR binding agents specified in the tables of the Examples or less. Suitably the EC50 is established using a chemokine induction assay, for example assayed using an ELISA. More suitably the EC50 is established using an assay set out in any one of the Examples.

[0153] In one embodiment, the first construct binds to mouse LTBR. In an alternative embodiment, the first construct binds to Macaca fascicularis LTBR. In an alternative embodiment, the first construct binds to human LTBR.One, a plurality, or all of the LTBR binding agents may be a variable domain (e.g. a heavy chain variable domain, e.g. a VHH) which binds to LTBR, suitably as defined further below.

[0154] In one embodiment, the LTBR-binding agent or polypeptide comprises at least one CDR, wherein the CDR is a HCDR1, HCDR2 or HCDR3 comprised in a heavy chain variable domain of SEQ ID NOs: 23-26 or 159-162. Suitably, such agent or polypeptide comprises at least such HCDR1 and HCDR2, such HCDR1 and HCDR3, or such HCDR2 and HCDR3. More suitably, such agent or polypeptide comprises such HCDR1, HCDR2 and HCDR3. More suitably, the HCDR1, HCDR2 and HCDR3 are delineated according to the Kabat-, Chothia-, Martin-, IMGT-, or AbM-method. More suitably the HCDR1 is chosen from SEQ ID NOs: 57, 60, 63, 66, 163, 166, 169 or 172 as defined by the Kabat-method; the HCDR2 is chosen from SEQ ID NOs: 58, 61, 64, 67, 164, 167, 170, 173, 205 or 206 as defined by the Kabat-method; and / or the HCDR3 is chosen from SEQ ID NOs: 59, 62, 65, 68, 165, 168, 171, 174, 207 or 208 as defined by the Kabat-method.

[0155] In one embodiment the variable domain which binds 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 sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater 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 difference with 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 sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 58, 61, 64, 67, 164, 167, 170, 173, 205 or 206 (e.g. 58, 61, 64 or 67) or wherein HCDR2 comprises or consists of a polypeptide sequence having 3, 2 or 1 amino acid sequence difference with 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 sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 59, 62, 65, 68, 165, 168, 171, 174, 207 or 208 (e.g. 59, 62, 65 or 68) or wherein HCDR3 comprises or consists of a polypeptide sequence having 3, 2 or 1 amino acid sequence difference with SEQ ID NO: 59, 62, 65, 68, 165, 168, 171, 174, 207 or 208 (e.g. 59, 62, 65 or 68). More suitably HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 57, 60, 63, 66, 163, 166, 169 or 172 (e.g.

[0156] 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 or208 (e.g. 59, 62, 65 or 68). In particular, these CDR polypeptide sequences were determined according to or with the Kabat-method.

[0157] In one embodiment the variable domain which binds to 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).

[0158] In one embodiment the variable domain which binds to LTBR comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 23, 24, 25, 26, 159, 160, 161 or 162 (e.g.

[0159] 23, 24, 25 or 26). More suitably the variable domain which binds to LTBR comprises or consists of a polypeptide sequence of SEQ ID NO: 23, 24, 25, 26, 159, 160, 161 or 162 (e.g.

[0160] 23, 24, 25 or 26). In one further embodiment thereto, the polypeptide sequence variation is in one or more the framework regions FR1-FR4 comprised in SEQ ID NO: 23, 24, 25, 26, 159, 160, 161 or 162 (e.g. 23, 24, 25 or 26). Alternatively, the polypeptide sequence variation is one or more of the framework regions FR1-FR4 comprised in SEQ ID NO: 23, 24, 25, 26, 159, 160, 161 or 162 (e.g. 23, 24, 25 or 26) and the CDR polypeptide sequences comprised within SEQ ID NO: 23, 24, 25, 26, 159, 160, 161 or 162 (e.g. 23, 24, 25 or 26) are the HCDR1-HCDR3 polypeptide sequences as outlined above. In one embodiment one, a plurality, or all of the LTBR binding agents comprise or consist of a paired heavy chain variable domain and light chain variable domain which bind to LTBR (e.g. an scFv), suitably as defined further below.

[0161] In one embodiment the heavy chain variable domain which binds 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 sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 69, 75, 81, 87, 93, 99, 105 or 111, HCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 70, 76, 82, 88, 94, 100, 106 or 112 and HCDR3 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 71, 77, 83, 89, 95, 101, 107 or 113 and the light chain variable domain which binds to LTBR comprises or consists of three complementarity determining regions (LCDR1-LCDR3) and four framework regions (LFR1-LFR4), wherein LCDR1 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 72, 78, 84, 90, 96, 102, 108 or 114, LCDR2 comprises orconsists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 73, 79, 85, 91, 97, 103, 109 or 115 and LCDR3 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 74, 80, 86, 92, 98, 104, 110 or 116. More suitably HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 69, 75, 81, 87, 93, 99, 105 or 111 , HCDR2 comprises or consists of a polypeptide sequence of SEQ I D NO: 70, 76, 82, 88, 94, 100, 106 or 112 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 71, 77, 83, 89, 95, 101, 107 or 113 and LCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 72, 78, 84, 90, 96, 102, 108 or 114, LCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 73, 79, 85, 91, 97, 103, 109 or 115 and LCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 74, 80, 86, 92, 98, 104, 110 or 116.

[0162] In one embodiment the heavy chain variable domain which binds to LTBR comprises the three heavy chain complementarity determining region amino acid sequences (HCDR1-HCDR3) of the heavy variable domain amino acid sequence of SEQ ID NO: 27, 29, 31, 33, 35, 37, 39 or 41 and the light chain variable domain which binds to LTBR comprises the three light chain complementarity determining region amino acid sequences (LCDR1-LCDR3) of the light variable domain amino acid sequence of SEQ ID NO: 28, 30, 32, 34, 36, 38, 40 or 42.

[0163] In one embodiment the heavy chain variable domain which binds to LTBR comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 27, 29, 31 , 33, 35, 37, 39 or 41 and the light chain variable domain which binds to LTBR comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 28, 30, 32, 34, 36, 38, 40 or 42. More suitably the heavy chain variable domain which binds to LTBR comprises or consists of a polypeptide sequence of SEQ ID NO: 27, 29, 31 , 33, 35, 37, 39 or 41 and the light chain variable domain which binds to LTBR comprises or consists of a polypeptide sequence of SEQ ID NO: 28, 30, 32, 34, 36, 38, 40 or 42.

[0164] Preferably the HCDRs (and LCDRs) are provided in the heavy chain variable domain which binds to LTBR (and light chain variable domain which binds to LTBR) in the same combinations as set out in Table 17A / B. For example, it is preferable that HCDRs1-3 of SEQ ID NOs: 57, 58 and 59 are deployed in combination in e.g. a VHH (as set out for Clone 1.5), and it is preferable that (a) HCDRs1-3 of SEQ ID NOs: 69, 70 and 71 and (b) LCDRs1-3 ofSEQ ID NOs: 72, 73 and 74 are deployed in combination (as set out for Clone 2.1) e.g. in an scFv. The same principle applies for combinations of heavy and light chain variable regions as set out in Table 16.

[0165] In one embodiment, the first construct comprises only one LTBR binding agent. In a further embodiment, the first construct comprises multiple LTBR binding agents. If multiple LTBR binding agents are comprised in the first construct, the LTBR binding agents may be (a) identical or (b) different LTBR binding agents. If different, the LTBR binding agents are suitably independently selected from the LTBR binding agents described herein, such as those detailed above and below.

[0166] If the first construct is an antibody, then in one embodiment one or all of the LTBR binding agents are a binding domain (e.g. a VHH or scFv, more suitably a VHH), most suitably linked to the constant region (e.g. the CH3 region).

[0167] Monospecific polypeptides (“clones”) which bind to LTBR used in the examples are summarised as follows. Such binding polypeptides may be incorporated into the first construct as an LTBR binding agent.

[0168]

[0169]

[0170] The LTBR binding polypeptide may comprise or consist of a variable domain which binds to LTBR (e.g. a heavy chain variable domain, such as a VHH), suitably as defined further below.

[0171] In one embodiment, the LTBR-binding polypeptide comprises at least one CDR, wherein the CDR is a HCDR1, HCDR2 or HCDR3 comprised in a heavy chain variable domain of SEQ ID NOs: 23-26 or 159-162. Suitably, such polypeptide comprises at least such HCDR1 and HCDR2, such HCDR1 and HCDR3, or such HCDR2 and HCDR3. More suitably, such polypeptide comprises such HCDR1, HCDR2 and HCDR3. More suitably, the HCDR1, HCDR2 and HCDR3 are delineated according to the Kabat-, Chothia-, Martin-, IMGT-, or AbM-method. More suitably the HCDR1 is chosen from SEQ ID NOs: 57, 60, 63, 66, 163, 166, 169 or 172 as defined by the Kabat-method; the HCDR2 is chosen from SEQ ID NOs: 58, 61, 64, 67, 164, 167, 170, 173, 205 or 206 as defined by the Kabat-method; and / or the HCDR3 is chosen from SEQ ID NOs: 59, 62, 65, 68, 165, 168, 171, 174, 207 or 208 as defined by the Kabat-method.

[0172] In one embodiment the variable domain which binds 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 sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater 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 difference with 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 sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 58, 61, 64, 67, 164, 167, 170, 173, 205 or 206 (e.g. 58, 61, 64 or 67) or wherein HCDR2 comprises or consists of a polypeptide sequence having 3, 2 or 1 amino acid sequence difference with 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 sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 59, 62, 65, 68, 165, 168, 171, 174, 207 or 208 (e.g. 59, 62, 65 or 68) or wherein HCDR3 comprises or consists of a polypeptidesequence having 3, 2 or 1 amino acid sequence difference with SEQ ID NO: 59, 62, 65, 68, 165, 168, 171, 174, 207 or 208 (e.g. 59, 62, 65 or 68). More suitably HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 57, 60, 63, 66, 163, 166, 169 or 172 (e.g.

[0173] 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 were determined or delineated according to or with the Kabat-method.

[0174] In one embodiment the variable domain which binds to LTBR comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 23, 24, 25, 26, 159, 160, 161 or 162 (e.g.

[0175] 23, 24, 25 or 26). More suitably the variable domain which binds to LTBR comprises or consists of a polypeptide sequence of SEQ ID NO: 23, 24, 25, 26, 159, 160, 161 or 162 (e.g.

[0176] 23, 24, 25 or 26). In one further embodiment thereto, the polypeptide sequence variation is in one or more the framework regions FR1-FR4 comprised in SEQ ID NO: 23, 24, 25, 26, 159, 160, 161 or 162 (e.g. 23, 24, 25 or 26). Alternatively, the polypeptide sequence variation is one or more of the framework regions FR1-FR4 comprised in SEQ ID NO: 23, 24, 25, 26, 159, 160, 161 or 162 (e.g. 23, 24, 25 or 26) and the CDR polypeptide sequences comprised within SEQ ID NO: 23, 24, 25, 26, 159, 160, 161 or 162 (e.g. 23, 24, 25 or 26) are the HCDR1-HCDR3 polypeptide sequences as outlined above.

[0177] In one embodiment the LTBR binding polypeptide comprises or consists of a paired heavy chain variable domain and light chain variable domain which bind to LTBR (e.g. an scFv), suitably as defined further below.

[0178] In one embodiment the heavy chain variable domain which binds 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 sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 69, 75, 81, 87, 93, 99, 105 or 111, HCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 70, 76, 82, 88, 94, 100, 106 or 112 and HCDR3 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 71, 77, 83, 89, 95, 101, 107 or 113 and the light chain variable domain which binds to LTBR comprises or consists of three complementarity determining regions (LCDR1-LCDR3) and four framework regions (LFR1-LFR4), wherein LCDR1 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 72, 78, 84, 90, 96, 102, 108 or 114, LCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 73, 79, 85, 91, 97, 103, 109 or 115 and LCDR3 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 74, 80, 86, 92, 98, 104, 110 or 116. More suitably HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 69, 75, 81, 87, 93, 99, 105 or 111 , HCDR2 comprises or consists of a polypeptide sequence of SEQ I D NO: 70, 76, 82, 88, 94, 100, 106 or 112 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 71, 77, 83, 89, 95, 101, 107 or 113 and LCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 72, 78, 84, 90, 96, 102, 108 or 114, LCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 73, 79, 85, 91, 97, 103, 109 or 115 and LCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 74, 80, 86, 92, 98, 104, 110 or 116.

[0179] In one embodiment the heavy chain variable domain which binds to LTBR comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 27, 29, 31, 33, 35, 37, 39 or 41 and the light chain variable domain which binds to LTBR comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 28, 30, 32, 34, 36, 38, 40 or 42. More suitably the heavy chain variable domain which binds to LTBR comprises or consists of a polypeptide sequence of SEQ ID NO: 27, 29, 31 , 33, 35, 37, 39 or 41 and the light chain variable domain which binds to LTBR comprises or consists of a polypeptide sequence of SEQ ID NO: 28, 30, 32, 34, 36, 38, 40 or 42.

[0180] In one embodiment the variable domain which binds 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 sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 163, 166, 169 or 172, HCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 164, 167, 170 or 173 and HCDR3 comprises or consists of a polypeptide sequence sharing 70% orgreater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 165, 168, 171 or 174. More suitably HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 163, 166, 169 or 172, HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 164, 167, 170 or 173 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 165, 168, 171 or 174.

[0181] In one embodiment the variable domain which binds to LTBR comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 159, 160, 161 or 162. More suitably the variable domain which binds to LTBR comprises or consists of a polypeptide sequence of SEQ ID NO: 159, 160, 161 or 162.

[0182] Preferably the HCDRs (and LCDRs) are provided in the heavy chain variable domain which binds to LTBR (and paired light chain variable domain which binds to LTBR) in the same combinations as set out in Table 17A / B. For example, it is preferable that HCDRs1-3 of SEQ ID Nos: 57, 58 and 59 are deployed in combination (as set out for Clone 1.5) e.g. in a VHH, and it is preferable that (a) HCDRs1-3 of SEQ ID Nos: 69, 70 and 71 and (b) LCDRs1-3 of SEQ ID Nos: 72, 73 and 74 are deployed in combination (as set out for Clone 2.1) e.g. in an scFv. The same principle applies for combinations of heavy chain variable regions and light chain variable regions as set out in Table 16.

[0183] The polypeptide which binds to LTBR may comprise or consist of the polypeptide sequence(s) of clone 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 may comprise or consist of an antibody which competes for binding to LTBR with an anti-LTBR polypeptide disclosed herein.

[0184] Suitably the polypeptide which binds to LTBR specifically binds to LTBR, for example in that any other entity is bound to with a KD of 10'7M or more, such as 10'6M or more. Suitably the polypeptide specifically binds to the extracellular domain of LTBR. In some embodiments, the LTBR is on the surface of stromal cells. In some embodiments, the LTBR is on the surface of endothelial cells. In some embodiments, the LTBR is on the surface of fibroblasts. In further embodiments, the LTBR is situated on the surface of cancer cells, myeloid cells and / or macrophages (e.g. present in the TME).

[0185] The LTBR binding polypeptide may bind to LTBR with an affinity (KD) of 20 nM or less, such as 15 nM or less, such as 10 nM or less, such as 9 nM or less, such as 8 nM or less, such as 7 nM or less, such as 6 nM or less, such as 5 nM or less, such as 4 nM or less, such as 3 nM or less, such as 2 nM or less, such as 1 nM or less. In some embodiments, the LTBR binding polypeptide may bind to LTBR with an affinity (KD) as recited in Table 9 or less. The affinity may be established as set out in Example 2.Suitably the LTBR binding polypeptide has an EC50 of 9.0E-09 M or less, such as 8.0E-09 M or less, such as 7.0E-09 M or less, such as 6.0E-09 M or less, such as 5.0E-09 M or less, such as 4.0E-09 M or less, such as 3.0E-09 M or less, such as 2.0E-09 M or less, such as 1.0E-09 M or less, such as 9.0E-10 M or less, such as 8.0E-10 M or less, such as 7.0E-10 M or less, such as 6.0E-10 M or less, such as 5.0E-10 M or less, such as 4.0E-10 M or less, such as 3.0E-10 M or less, such as 2.0E-10 M or less, such as 1.0E-10 M or less, such as 9.0E-11 M or less, such as 8.0E-11 M or less, such as 7.0E-11 M or less, such as 6.0E-11 M or less, such as 5.0E-11 M or less, such as 4.0E-11 M or less, such as 3.0E-11 M or less, such as 2.0E-11 M or less, such as 1.0E-11 M or less, such as 9.0E-12 M or less, such as 8.0E-12 M or less, such as 7.0E-12 M or less, such as 6.0E-12 M or less, such as 5.0E-12 M or less, such as 4.0E-12 M or less, such as 3.0E-12 M or less, such as 2.0E-12 M or less, such as 1.0E-12 M or less.

[0186] Alternatively, the LTBR binding polypeptide may have an EC50 as recited for any of the clones specified in the tables of the Examples or less.

[0187] FAP binding agent

[0188] In addition to an LTBR binding agent, in one embodiment the first construct may comprise a FAP (fibroblast activation protein alpha) binding agent. FAP, also known as prolyl endopeptidase, is an enzyme that in humans is encoded by the FAP gene. FAP is a 170 kDa membrane-bound gelatinase. FAP is highly expressed in cancer tissue across a wide range of cancer types and is particularly expressed on the surface of cancer associated fibroblasts (CAFs) present in the cancer stroma and on cancer cells (Zboralski et al 2022, Zhao et al 2022).

[0189] FAP binding agents are disclosed in the prior art, for example anti-FAP binding polypeptide 28H1 (US10577429B2, comprising heavy and light chain variable regions of SEQ ID NO: 7 and 8, respectively).

[0190] Suitably one or all of the FAP binding agents are FAP binding polypeptides, such as an FAP binding antibody or a FAP binding fragment thereof. Suitably one or all of the FAP binding agents comprise or consist of a variable domain which binds to FAP, such as a VH, VL, VNAR orVHH.

[0191] Suitably the FAP binding agent achieves localisation (e.g. targeting of a tumour), colocation with the LTBR binding agent or clustering of LTBR; i.e. conditional activation. This conditional activation phenomenon should allow for reduced toxicity due to activation of LTBR-positive cells in the presence of FAP in the tumour (and not in tissues where FAP is not expressed). The FAP is suitably situated on the surface of stromal and / or cancer cells.Suitably the FAP is situated on the surface of a cell wherein the cell also comprises LTBR on its surface.

[0192] The FAP binding agent suitably binds to the extracellular domain of FAP.

[0193] In one embodiment, FAP is a polypeptide sharing 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, such as at least 95% identity with SEQ ID NO: 15 (full length human FAP). Suitably, FAP comprises such as consists of SEQ ID NO: 15 (full length human FAP).

[0194] Suitably, the FAP is the extracellular domain of human FAP, i.e. the FAP binding agent binds to the extracellular domain of human FAP. In one embodiment, FAP is a polypeptide sharing 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, such as at least 95% identity with SEQ ID NO: 16 (human FAP extracellular domain). Suitably, FAP is a polypeptide comprising or consisting of SEQ ID NO: 16.

[0195] In one embodiment, FAP is a polypeptide sharing 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, such as at least 95% identity with SEQ ID NO: 17 (full length Macaca fascicularis FAP). Suitably, FAP comprises such as consists of SEQ ID NO: 17 (full length Macaca fascicularis FAP).

[0196] Suitably, the FAP is the extracellular domain of Macaca fascicularis FAP, i.e. the FAP binding agent binds to the extracellular domain of Macaca fascicularis FAP. In one embodiment, FAP is a polypeptide sharing 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, such as at least 95% identity with SEQ ID NO: 18 (Macaca fascicularis FAP extracellular domain). Suitably, FAP is a polypeptide comprising or consisting of SEQ ID NO: 18.

[0197] In one embodiment, FAP is a polypeptide sharing 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, such as at least 95% identity with SEQ ID NO: 19 (full length mouse FAP). Suitably, FAP comprises such as consists of SEQ ID NO: 19 (full length mouse FAP).

[0198] Suitably, the FAP is the extracellular domain of mouse FAP, i.e. the FAP binding agent binds to the extracellular domain of mouse FAP. In one embodiment, FAP is a polypeptide sharing 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, such as at least 95% identity with SEQ ID NO: 20 (mouse FAP extracellular domain). Suitably, FAP is a polypeptide comprising or consisting of SEQ ID NO: 20 (mouse FAP extracellular domain).

[0199] Suitably one or all of the FAP binding agents have an EC50 of 9.0E-09 M or less, such as 8.0E-09 M or less, such as 7.0E-09 M or less, such as 6.0E-09 M or less, such as 5.0E-09M or less, such as 4.0E-09 M or less, such as 3.0E-09 M or less, such as 2.0E-09 M or less, such as 1.0E-09 M or less, such as 9.0E-10 M or less, such as 8.0E-10 M or less, such as 7.0E-10 M or less, such as 6.0E-10 M or less, such as 5.0E-10 M or less, such as 4.0E-10 M or less, such as 3.0E-10 M or less, such as 2.0E-10 M or less, such as 1.0E-10 M or less, such as 9.0E-11 M or less, such as 8.0E-11 M or less, such as 7.0E-11 M or less, such as 6.0E-11 M or less, such as 5.0E-11 M or less, such as 4.0E-11 M or less, such as 3.0E-11 M or less, such as 2.0E-11 M or less, such as 1.0E-11 M or less, such as 9.0E-12 M or less, such as 8.0E-12 M or less, such as 7.0E-12 M or less, such as 6.0E-12 M or less, such as 5.0E-12 M or less, such as 4.0E-12 M or less, such as 3.0E-12 M or less, such as 2.0E-12 M or less, such as 1.0E-12 M or less.

[0200] Suitably the EC50 is established using one of the assays set out in the examples. Suitably one or all of the FAP binding agents have an EC50 as recited for any of the clones specified in the tables of the Examples or less.

[0201] Suitably the FAP binding agent binds to FAP specifically, i.e. the binding agent does not bind significantly to targets other than human FAP, such as does not bind significantly to targets other than human, Macaca fascicularis or mouse FAP, such as does not bind significantly to targets other than FAP.

[0202] The first construct of the invention may bind to FAP with an avidity of 9.0E-10 M or less, such as 8.0E-10, such as 7.2E-10, such as 5.0E-10 M or less, such as 1.0E-10 M or less, such as 9.0E-11 M or less, such as 7.0E-11 M or less, such as 5.0E-11 M or less, such as 1.0E-11 M or less.

[0203] Suitably one or all of the FAP binding agents bind to FAP with an affinity (KD) of 400 pM or less, such as 300 pM or less, such as 200 pM or less, such as 150 pM or less, such as 130 pM or less, such as 110 pM or less, such as 100 pM or less, such as 50 pM or less.

[0204] Suitably the KD is established using the assay set out in Example 4.

[0205] In one embodiment, the first construct binds to mouse FAP. In an alternative embodiment, the first construct binds to Macaca fascicularis FAP. In an alternative embodiment, the first construct binds to human FAP.

[0206] In one embodiment one, a plurality, or all of the FAP binding agents comprise or consist of a paired heavy chain variable domain and light chain variable domain which bind to FAP (e.g. a Fab), suitably as defined further below.

[0207] In one embodiment the heavy chain variable domain which binds to FAP 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 sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 117, 123, 129, 135, 141, 147 or 153,HCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 118, 124, 130, 136, 142, 148 or 154 and HCDR3 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 119, 125, 131, 137, 143, 149 or 155 and the light chain variable domain which binds to FAP comprises or consists of three complementarity determining regions (LCDR1-LCDR3) and four framework regions (LFR1-LFR4), wherein LCDR1 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 120, 126, 132, 138, 144, 150 or 156, LCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 121, 127, 133, 139, 145, 151 or 157 and LCDR3 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 122, 128, 134, 140, 146, 152 or 158. More suitably HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 117, 123, 129, 135, 141, 147 or 153, HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 118, 124, 130, 136, 142, 148 or 154 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 119, 125, 131, 137, 143, 149 or 155 and LCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 120, 126, 132, 138, 144, 150 or 156, LCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 121, 127, 133, 139, 145, 151 or 157 and LCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 122, 128, 134, 140, 146, 152 or 158.

[0208] In one embodiment the heavy chain variable domain which binds to FAP comprises the three heavy chain complementarity determining region amino acid sequences (HCDR1-HCDR3) of the heavy variable domain amino acid sequence of SEQ ID NO: 43, 45, 47, 49, 51, 53 or 55 and the light chain variable domain which binds to FAP comprises the three light chain complementarity determining region amino acid sequences (LCDR1-LCDR3) of the light variable domain amino acid sequence of SEQ ID NO: 44, 46, 48, 50, 52, 54 or 56.

[0209] In one embodiment the heavy chain variable domain which binds to FAP comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 43, 45, 47, 49, 51 , 53 or 55 and the light chain variable domain which binds to FAP comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greatersequence identity with SEQ ID NO: 44, 46, 48, 50, 52, 54 or 56. More suitably the heavy chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 43, 45, 47, 49, 51 , 53 or 55 and the light chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 44, 46, 48, 50, 52, 54 or 56.

[0210] Preferably the HCDRs and LCDRs are provided in the heavy chain variable domain which binds to FAP and light chain variable domain which binds to FAP in the same combinations as set out in Table 17A. For example, it is preferable that (a) HCDRs1-3 of SEQ ID NOs: 117, 118 and 119 and (b) LCDRs1-3 of SEQ ID NOs: 120, 121 and 122 are deployed in combination (as set out for Clone 3.5) e.g. in an Fab. The same principle applies for combinations of variable regions as set out in Table 16.

[0211] If multiple FAP binding agents are deployed in the first construct, the FAP binding agents may be (a) identical or (b) different FAP binding agents. If different, the FAP binding agents are suitably independently selected from the FAP binding agents described herein, such as those detailed above.

[0212] If the first construct is an antibody, then in one embodiment one or all of the FAP binding agents are a Fab (linked to the CH2 region).

[0213] Monospecific polypeptides (“clones”) which bind to FAP used in the examples of the invention are summarised as follows. Such binding polypeptides may, for example, be incorporated into the first construct.

[0214]

[0215] In one embodiment the FAP binding polypeptide comprises or consists of a paired heavy chain variable domain and light chain variable domain which bind to FAP (e.g. a Fab), suitably as defined further below.

[0216] In one embodiment the heavy chain variable domain which binds to FAP 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 sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or95% or greater sequence identity with SEQ ID NO: 117, 123, 129, 135, 141, 147 or 153, HCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 118, 124, 130, 136, 142, 148 or 154 and HCDR3 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 119, 125, 131, 137, 143, 149 or 155 and the light chain variable domain which binds to FAP comprises or consists of three complementarity determining regions (LCDR1-LCDR3) and four framework regions (LFR1-LFR4), wherein LCDR1 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 120, 126, 132, 138, 144, 150 or 156, LCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 121, 127, 133, 139, 145, 151 or 157 and LCDR3 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 122, 128, 134, 140, 146, 152 or 158. More suitably HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 117, 123, 129, 135, 141, 147 or 153, HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 118, 124, 130, 136, 142, 148 or 154 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 119, 125, 131, 137, 143, 149 or 155 and LCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 120, 126, 132, 138, 144, 150 or 156, LCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 121, 127, 133, 139, 145, 151 or 157 and LCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 122, 128, 134, 140, 146, 152 or 158.

[0217] In one embodiment the heavy chain variable domain which binds to FAP comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 43, 45, 47, 49, 51 , 53 or 55 and the light chain variable domain which binds to FAP comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 44, 46, 48, 50, 52, 54 or 56. More suitably the heavy chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 43, 45, 47, 49, 51 , 53 or 55 and the light chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 44, 46, 48, 50, 52, 54 or 56.Preferably the HCDRs and LCDRs are provided in the heavy chain variable domain which binds to FAP and light chain variable domain which binds to FAP in the same combinations as set out in Table 17A. For example, it is preferable that (a) HCDRs1-3 of SEQ ID Nos: 117, 118 and 119 and (b) LCDRs1-3 of SEQ ID Nos: 120, 121 and 122 are deployed in combination (as set out for Clone 3.5) e.g. in a Fab. The same principle applies for combinations of variable regions as set out in Table 16.

[0218] The polypeptide which binds to FAP may comprise or consist of the polypeptide sequence(s) of clone 3.1, 3.2, 3.3, 3.4, 3.5, 3.6 or 3.7; or, an antibody which competes for binding to FAP with an anti-FAP polypeptide disclosed herein.

[0219] Suitably the FAP binding polypeptide specifically binds to FAP, for example such that any other entity is bound to with a KD of 10'7M or more, such as 10'6M or more. In some embodiments, the FAP is on the surface of stromal cells (e.g. CAFs) and / or on the surface of cancer cells. Suitably the FAP binding polypeptide specifically binds to the extracellular domain of FAP.

[0220] Suitably the FAP binding polypeptide binds to FAP with an affinity (KD) of 400 pM or less, such as 300 pM or less, such as 200 pM or less, such as 150 pM or less, such as 130 pM or less, such as 110 pM or less, such as 100 pM or less, such as 50 pM or less. Alternatively the FAP binding polypeptide binds to FAP with an affinity (KD) as recited in Table 13 or less. Suitably the affinity is established as set out in Example 3.

[0221] Suitably the FAP binding polypeptide has an EC50 of 9.0E-09 M or less, such as 8.0E-09 M or less, such as 7.0E-09 M or less, such as 6.0E-09 M or less, such as 5.0E-09 M or less, such as 4.0E-09 M or less, such as 3.0E-09 M or less, such as 2.0E-09 M or less, such as 1.0E-09 M or less, such as 9.0E-10 M or less, such as 8.0E-10 M or less, such as 7.0E-10 M or less, such as 6.0E-10 M or less, such as 5.0E-10 M or less, such as 4.0E-10 M or less, such as 3.0E- 10 M or less, such as 2.0E-10 M or less, such as 1.0E-10 M or less, such as 9.0E-11 M or less, such as 8.0E-11 M or less, such as 7.0E-11 M or less, such as 6.0E-11 M or less, such as 5.0E- 11 M or less, such as 4.0E-11 M or less, such as 3.0E-11 M or less, such as 2.0E-11 M or less, such as 1.0E-11 M or less, such as 9.0E-12 M or less, such as 8.0E-12 M or less, such as 7.0E- 12 M or less, such as 6.0E-12 M or less, such as 5.0E-12 M or less, such as 4.0E-12 M or less, such as 3.0E-12 M or less, such as 2.0E-12 M or less, such as 1.0E-12 M or less. Alternatively the FAP binding polypeptide has an EC50 as recited for any of the clones specified in the tables of the Examples or less.

[0222] The polypeptides described above which bind to LTBR or FAP may comprise an antibody, such as consist of an antibody, or may comprise or consist of one or more antibody fragments. A suitable fragment may include for example an scFv, Fv, Fab, Fab’, F(ab’)2, variable domain (e.g. VH, VL, VNAR or VHH), diabody or minibody.Structure and properties of first construct

[0223] Suitably the first construct comprises an antibody, or an antibody fragment. In one embodiment, the antibody fragment comprises an scFv, Fv, Fab, Fab’, F(ab')2, variable domain (e.g. VH, VL, VNAR or VHH), diabody or minibody. If the first construct is an antibody or antibody fragment, then the binding regions of the antibody or antibody fragment (e.g. the Fab regions of the antibody or antibody fragment, or synthetically introduced further binding domains such as VHHs) provide the LTBR binding agent(s) of the first construct (and the FAP binding agent(s) of the first construct, if present).

[0224] Most suitably the first construct is an antibody (i.e. a full length antibody). Antibodies used in the invention can be of any class, e.g. IgG, IgA, IgM, IgE, IgD, or subclass thereof, and can comprise a kappa or lambda light chain. In one embodiment, the antibody is an IgG antibody, for example, at least one of subclasses, I gG 1 , I gG2 , lgG3 or lgG4. In one embodiment, the antibody is an lgG1, most suitably human lgG1. The antibody may comprise further variable domains (in addition to the conventional two Fab regions) which bind to a target (such as LTBR or FAP), and the further variable domains may for example be VHHs. In a further embodiment, the antibody may be in a format, such as an IgG format, that has been modified to confer desired properties, such as having the Fc mutated to reduce effector function, extend 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 comprises PGLALA substitutions (substitutions Pro329Gly, Leu234Ala, and Leu235Ala, Schlothauer et al. 2016) in the Fc region. Suitably the antibody Fc region comprises 329Gly, 234Ala and 235Ala. Alternatively, the antibody comprises LALA substitutions (substitutions Leu234Ala, and Leu235Ala, Schlothauer et al. 2016) in the Fc region. Suitably the antibody Fc region comprises 234Ala and 235Ala. In a further embodiment the antibody comprises an STR-silenced Fc region (i.e. G236R in combination with the LALA substitutions described above, see Wilkinson et al. 2021). Therefore, in one embodiment, the Fc region comprises 234Ala, 235Ala and 236Arg. In a yet further embodiment, the Fc region is aglycosylated. The same Fc modifications may be applied to any construct comprising an Fc.

[0225] The antibody may be monospecific or multispecific, such as bispecific. The antibody may be monovalent or multivalent, such as bivalent. In one embodiment the antibody is multispecific, such as bispecific and multivalent, such as bivalent. Suitable constructs of these formats and other formats are detailed further below.

[0226] In a particular embodiment, the first construct comprises, essentially consists of or consists of an antibody or a pair of scFvs. Most suitably the construct comprises an antibody.

[0227] In a further embodiment, the first construct comprises or consists of an antibody and a VHH, wherein the VHH replaces one of the Fab regions (such as illustrated in Figure 7a), theVHH is a binding polypeptide which binds to LTBR and the paired VH and VL of the remaining Fab region is a binding polypeptide which binds to FAP.

[0228] In an alternative embodiment, the first construct comprises or consists of an antibody and a VHH, wherein the VHH is fused to the Fc region (e.g. a CH3 domain) of the antibody (such as illustrated in Figure 7b), the VHH is a binding polypeptide which binds to LTBR and the paired VH and VL of each of the two Fab regions are binding polypeptides which bind to FAP. Suitably a further VHH is fused to the Fc region (e.g. the second CH3 domain), wherein the further VHH is a binding polypeptide which binds to LTBR (such as illustrated in Figure 7c).

[0229] In a particular embodiment, the first construct comprises or consists of an antibody and an scFv, wherein the scFv is fused to the Fc region (e.g. a CH3 domain) of the antibody, the scFv is a binding polypeptide which binds to LTBR and the paired VH and VL of each of the two Fab regions are binding polypeptides which bind to FAP. Suitably a further scFv is fused to the Fc region (e.g. the second CH3 domain), wherein the further scFv is a binding polypeptide which binds to LTBR (such as illustrated in Figure 7d).

[0230] In one embodiment, the first construct may comprise or consist of an scFv fused to the C-terminus of an Fc. Suitably, a peptide linker such as a linker comprising Glycine and Serine residues, such as a G4S linker (SEQ ID NO: 3) or a (648)4 linker (SEQ ID NO: 4) may be used to link the LTBR binding agent(s) to the Fc region, for example to the CH3 region. Suitably a disulfide bond may be situated in the VH44:VL100 position (Weatherill et al. 2012).

[0231] The first construct in at least some embodiments benefits from certain properties such as the ability to cluster LTBR, bind to LTBR and FAP on the same cell, potently agonise LTBR (e.g. LTBR NFKB pathways, such as classical and alternative NFKB pathways), bind to LTBR (and optionally FAP) with desired affinity, and achieve favourable biodistribution when administered to a subject (e.g. tumour-targeting), due to FAP-dependent conditional activation.

[0232] Suitably the first construct is conditionally activated at a target site. By conditionally activated it is meant that the first construct clusters LTBR at a target site in a FAP-dependent manner (due to the anti-FAP specificity of the first construct, if present) such that the construct activates LTBR, resulting in activation of the classical and / or alternative NFKB pathway (as detailed further herein). The target site is suitably a tumour.

[0233] If a FAP binding polypeptide is present in the first construct, then suitably the LTBR binding agent is capable of binding LTBR on a cell and the FAP binding agent is capable of binding FAP on the same cell simultaneously and / or the LTBR binding agent is capable of binding LTBR on a first cell and the FAP binding agent is capable of binding FAP on a second cell simultaneously. Suitably the LTBR binding agent is capable of activating LTBR on cells which express both LTBR and FAP.The first construct may comprise a single binding agent and is therefore monovalent. Such a first construct may comprise no more than one LTBR binding agent. The first construct may be monospecific, i.e. does not have specificity to any further targets.

[0234] The first construct may comprise multiple binding agents and is therefore multivalent (e.g. bivalent). These multiple binding agents may bind to different targets and therefore the first construct is multispecific (e.g. bispecific). A further specificity may be anti-FAP. Such a first construct may comprise (a) no more than one LTBR binding agent and no more than one FAP binding agent, (b) no more than one LTBR binding agent and more than one FAP binding agent (c) more than one LTBR binding agent and no more than one FAP binding agent or (d) more than one LTBR binding agent and more than one FAP binding agent. If the construct comprises more than one LTBR binding agent and / or FAP binding agent, then the LTBR binding agents may be the same or different and the FAP binding agents may be the same or different. Suitably the LTBR binding agents are the same. Suitably the FAP binding agents are the same. Most suitably, the LTBR binding agents are the same and the FAP binding agents are the same. If the LTBR binding agents are different, suitably they are independently selected from the LTBR binding agents defined herein. If the FAP binding agents are different, suitably they are independently selected from the FAP binding agents defined herein.

[0235] In one embodiment, the first construct comprises no FAP binding agent. In a further embodiment the first construct comprises only one FAP binding agent. Alternatively, the first construct comprises more than one FAP binding agent. In one embodiment, the first construct comprises only two FAP binding agents.

[0236] In one embodiment, the first construct comprises only one LTBR binding agent.

[0237] Alternatively, the first construct comprises more than one LTBR binding agent. In one embodiment, the first construct comprises only two LTBR binding agents.

[0238] The valency and specificity of a first construct may be expressed as a ratio of FAP-binding to LTBR-binding valencies e.g. 1:1, 2:1, etc. A bispecific construct which is monovalent for FAP and monovalent for LTBR may be described as 1 : 1. A bispecific first construct which is bivalent for FAP and monovalent for LTBR may be described as 2:1. A bispecific first construct which is bivalent for FAP and bivalent for LTBR may be described as 2:2, and so on.

[0239] A FAP-monovalent first construct is a construct with a single anti-FAP valency. A FAP-bivalent construct is a first construct with two anti-FAP valencies, and so on. An LTBR-monovalent first construct is a first construct with a single anti-LTBR valency. An LTBR-bivalent first construct is a first construct with two anti-LTBR valencies, and so on.

[0240] In one embodiment the first construct comprises or consists of an antibody, such as a bispecific antibody and / or bivalent antibody.A first construct may comprise at least one further binding agent (such as an antibody or antigen-binding fragment thereof), which is (a) not an LTBR binding agent, and / or (b) not a FAP binding agent. Alternatively, a first construct may comprise no further binding agents.

[0241] A suitable first construct may comprise or consist of an antibody, wherein in one embodiment the first variable region binds to LTBR and the second variable region binds to FAP.

[0242] A further suitable first construct may comprise multiple antibodies.

[0243] Other suitable first constructs may comprise or consist of one or a plurality of antibody fragments as described above, wherein each antibody fragment may contribute one or more binding domains. Suitable antibody fragments include for example an scFv, Fv, Fab, Fab’, F(ab')2, variable domain (e.g. VH, VL, VNAR or VHH), diabody or minibody. In one embodiment, the first construct comprises or consists of an antibody, or two scFvs.

[0244] The binding agents can be linked (or ‘fused’) to each other directly (i.e. without use of a linker) or indirectly via a linker or via further components such as immunoglobulin domains. The binding agents can be linked (or ‘fused’) to components of the first construct, such as constant domains, directly (i.e. without use of a linker) or indirectly via a linker. If the first construct comprises an Fc region, then optionally one or all of the LTBR binding agents are linked by a peptide linker to the Fc region, for example to the CH3 region.

[0245] A linker comprised in the first construct is referred to herein as a ‘first construct linker’. Suitably, the linker is a peptide. Suitably the linker is selected so as to allow binding of the binding agents to their targets, while maintaining structural integrity. If intended for administration to a subject, the linker is suitably non-immunogenic in the subject to which the binding agents are administered.

[0246] Suitably the peptide linker is no longer than 50 amino acids, such as no longer than 40 amino acids, such as no longer than 30 amino acids, such as no longer than 20 amino acids, such as no longer than 10 amino acids, such as no longer than 9 amino acids, such as no longer than 8 amino acids, such as no longer than 7 amino acids, such as no longer than 6 amino acids, such as no longer than 5 amino acids.

[0247] Suitably the peptide linker is shorter than 6 amino acids, such as shorter than 7 amino acids, such as shorter than 8 amino acids, such as shorter than 9 amino acids, such as shorter than 10 amino acids, such as shorter than 20 amino acids, such as shorter than 30 amino acids, such as shorter than 40 amino acids, such as shorter than 50 amino acids.

[0248] Suitably the peptide linker comprises or consists of glycine and serine residues, such as comprising or consisting of a G4S linker (SEQ ID NO: 3).In one embodiment the binding agents are linked to each other in a first construct in the form of a bispecific, multivalent (e.g. bivalent) antibody. A bispecific antibody may be referred to as a “bsAb”.

[0249] A particularly suitable bispecific antibody format is that generated via the knobs-into-holes approach (Merchant et al. 1998). Alternatively, or in addition, one or more (most suitably, all) Golay mutations in the CH1 / CL regions may be introduced to obtain efficient light chain pairing (Golay et al. 2016).

[0250] In one embodiment, bispecific first constructs having more than one valency for LTBR and / or FAP may comprise multiple scFvs, wherein each scFv is fused to the C-terminal of an Fc by a peptide linker, such as a linker comprising glycine and serine residues, such as a G4S linker (SEQ ID NO: 3).

[0251] If an Fc is present in the first construct (e.g. if the first construct is an antibody), then suitably the LTBR binding agent is linked directly or indirectly to the Fc (e.g. to one of the CH3 domains). If two or more LTBR binding agents are present, each LTBR binding agent may be linked to one of the CH3 domains of the Fc.

[0252] A number of first constructs (including bispecific constructs) described in the examples below are referred to as ‘clones’ and are assigned specific clone numbers or IDs.

[0253] Specific first constructs are characterised in the examples.

[0254] In one embodiment the first construct comprises, essentially consists of or consists of a format as described in Table 15 for clone 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.

[0255] The first construct may comprise, essentially consist of or consist of a format as depicted in Figures 7a-7d. The first construct may comprise or consist of of the polypeptide sequence(s) of clone 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.

[0256] The first construct may in some embodiments be provided in the following particular formats.

[0257] The first construct may comprise, essentially consist or consist of a bispecific, FAP-monovalent, LTBR-monovalent antibody comprising a heterodimer of two polypeptide chains, the first chain comprising or consisting of from N to C terminus a Fab region, a hinge region, a CH2 domain and a CH3 domain; and the second chain comprising or consisting of from N to C terminus a VHH, a hinge region and a CH2 domain and a CH3 domain, wherein the hinge regions are linked via a disulphide bond, the CH2 and CH3 domains are associated to form an Fc region, the Fab region comprises a paired VH and VL at its N-terminus which form a FAP binding site and the VHH forms an LTBR binding site (e.g. essentially as set out in Figure 7a). This is one embodiment of a 1:1 format first construct.The first construct may comprise, essentially consist or consist of a bispecific, FAP-bivalent, LTBR-monovalent antibody comprising a heterodimer of two polypeptide chains, the first chain comprising or consisting of from N to C terminus a Fab region, a hinge region, a CH2 domain, a CH3 domain and a VHH; and the second chain comprising or consisting of from N to C terminus a Fab region, a hinge region, a CH2 domain and a CH3 domain, wherein the hinge regions are linked via a disulphide bond, the CH2 and CH3 domains are associated to form an Fc region, the Fab regions each comprise a paired VH and VL at their N-terminus which each form a FAP binding site and the VHH forms an LTBR binding site at the C-terminus (e.g. essentially as set out in Figure 7b). This is one embodiment of a 2:1 format first construct.

[0258] The first construct may comprise, essentially consist or consist of a bispecific, FAP-bivalent, LTBR-bivalent antibody comprising a homodimer of two polypeptide chains, the first chain comprising or consisting of from N to C terminus a Fab region, a hinge region, a CH2 domain, a CH3 domain and a VHH; and the second chain comprising or consisting of from N to C terminus a Fab region, a hinge region, a CH2 domain, a CH3 domain and a VHH wherein the hinge regions are linked via a disulphide bond, the CH2 and CH3 domains are associated to form an Fc region, the Fab regions each comprise a paired VH and VL at their N-terminus which each form a FAP binding site and the VHHs each form an LTBR binding site at the C-terminus (e.g. essentially as set out in Figure 7c). This is one embodiment of a 2:2 format first construct.

[0259] The first construct may comprise, essentially consist or consist of a bispecific, FAP-bivalent, LTBR-bivalent antibody comprising a homodimer of two polypeptide chains, the first chain comprising or consisting of from N to C terminus a Fab region, a hinge region, a CH2 domain, a CH3 domain and an scFv; and the second chain comprising or consisting of from N to C terminus a Fab region, a hinge region, a CH2 domain, a CH3 domain and an scFv wherein the hinge regions are linked via a disulphide bond, the CH2 and CH3 domains are associated to form an Fc region, the Fab regions each comprise a paired VH and VL at their N-terminus which each form a FAP binding site and the scFvs each form an LTBR binding site at the C-terminus (e.g. essentially as set out in Figure 7d). This is one embodiment of a 2:2 format first construct.

[0260] In one embodiment the first construct is an antibody comprising

[0261] (a) one or more Fab domains, each comprising a paired heavy chain variable domain and light chain variable domain which bind to FAP; wherein

[0262] (i) the heavy chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 43 and the light chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 44;(ii) the heavy chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 45 and the light chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 46; or

[0263] (iii) the heavy chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 47 and the light chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 48,

[0264] and

[0265] (b) one or more paired heavy chain variable domain and light chain variable domain which bind to LTBR;

[0266] wherein the heavy chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 31 and the light chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 32

[0267] or one or more variable domains which bind to LTBR; wherein

[0268] (i) wherein the heavy chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 24; or

[0269] (ii) wherein the heavy chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 25.

[0270] In a further embodiment the first construct is an antibody comprising:

[0271] (i) a heterodimer of two different heavy chains (HC1 and HC2) and two identical light chains (LC) wherein:

[0272] (a) the HC1 polypeptide sequence comprises or consists of SEQ ID NO: 175, the HC2 polypeptide sequence comprises or consists of SEQ ID NO: 176 and the LC polypeptide sequence comprises or consists of SEQ ID NO: 177;

[0273] (b) the HC1 polypeptide sequence comprises or consists of SEQ ID NO: 178, the HC2 polypeptide sequence comprises or consists of SEQ ID NO: 179 and the LC polypeptide sequence comprises or consists of SEQ ID NO: 180;

[0274] (c) the HC1 polypeptide sequence comprises or consists of SEQ ID NO: 181, the HC2 polypeptide sequence comprises or consists of SEQ ID NO: 182 and the LC polypeptide sequence comprises or consists of SEQ ID NO: 183;

[0275] (d) the HC1 polypeptide sequence comprises or consists of SEQ ID NO: 184, the HC2 polypeptide sequence comprises or consists of SEQ ID NO: 185 and the LC polypeptide sequence comprises or consists of SEQ ID NO: 186;

[0276] (e) the HC1 polypeptide sequence comprises or consists of SEQ ID NO: 187, the HC2 polypeptide sequence comprises or consists of SEQ ID NO: 188 and the LC polypeptide sequence comprises or consists of SEQ ID NO: 189;(f) the HC1 polypeptide sequence comprises or consists of SEQ ID NO: 190, the HC2 polypeptide sequence comprises or consists of SEQ ID NO: 191 and the LC polypeptide sequence comprises or consists of SEQ ID NO: 192;

[0277] (g) the HC1 polypeptide sequence comprises or consists of SEQ ID NO: 193, the HC2 polypeptide sequence comprises or consists of SEQ ID NO: 194 and the LC polypeptide sequence comprises or consists of SEQ ID NO: 195; or (h) the HC1 polypeptide sequence comprises or consists of SEQ ID NO: 200, the HC2 polypeptide sequence comprises or consists of SEQ ID NO: 201 and the LC polypeptide sequence comprises or consists of SEQ ID NO: 202 or (ii) a homodimer of two identical heavy chains (HC) and two identical light chains (LC) wherein:

[0278] (a) the HC polypeptide sequence comprises or consists of SEQ ID NO: 196 and the LC polypeptide sequence comprises or consists of SEQ ID NO: 197 or (b) the HC polypeptide sequence comprises or consists of SEQ ID NO: 198 and the LC polypeptide sequence comprises or consists of SEQ ID NO: 199.

[0279] In a further embodiment the first construct comprises an antibody or antibody fragment comprising a FAP binding domain and an LTBR binding domain, wherein

[0280] (i) the LTBR binding domain is a variable domain which binds to LTBR which comprises or consists of three heavy chain complementarity determining regions (HCDR1-HCDR3) and four framework regions (FR1-FR4), wherein:

[0281] (a) HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 60 or 172, HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 61 or 173 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 62 or 174 or (b) HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 57 or 166, HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 58 or 167 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 59 or 168 or (c) HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 63 or 163, HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 64 or 164 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 65 or 165 or (d) HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 66 or 169, HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 67 or 170 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 68 or 171;

[0282] or

[0283] (ii) the LTBR binding domain is a paired heavy chain variable domain and light chain variable domain wherein the heavy chain variable domain comprises or consists of three complementarity determining regions (HCDR1-HCDR3) and four framework regions (HFR1-HFR4) and the light chain variable domain comprises or consists of three complementarity determining regions (LCDR1-LCDR3) and four framework regions (LFR1-LFR4), wherein: (a) HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 69, HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 70 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 71 and wherein LCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 72, LCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 73 and LCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 74 or

[0284] (b) HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 75, HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 76 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 77 and wherein LCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 78, LCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 79 and LCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 80 or

[0285] (c) HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 81 , HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 82 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 83 and wherein LCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 84, LCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 85 and LCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 86 or

[0286] (d) HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 87, HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 88 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 89 and wherein LCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 90, LCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 91 and LCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 92 or

[0287] (e) HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 93, HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 94 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 95 and wherein LCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 96, LCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 97 and LCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 98 or

[0288] (f) HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 99, HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 100 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 101 and wherein LCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 102, LCDR2 comprises or consists of apolypeptide sequence of SEQ ID NO: 103 and LCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 104 or

[0289] (g) HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 105, HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 106 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 107 and wherein LCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 108, LCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 109 and LCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 110 or

[0290] (h) HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 111, HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 112 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 113 and wherein LCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 114, LCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 115 and LCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 116;

[0291] and

[0292] the FAP binding domain is a paired heavy chain variable domain and light chain variable domain wherein the heavy chain variable domain comprises or consists of three complementarity determining regions (HCDR1-HCDR3) and four framework regions (HFR1-HFR4) and the light chain variable domain comprises or consists of three complementarity determining regions (LCDR1-LCDR3) and four framework regions (LFR1-LFR4), wherein: (a) HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 117, HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 118 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 119 and wherein LCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 120, LCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 121 and LCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 122 or

[0293] (b) HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 123, HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 124 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 125 and wherein LCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 126, LCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 127 and LCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 128 or

[0294] (c) HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 129, HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 130 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 131 and wherein LCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 132, LCDR2 comprises or consists of apolypeptide sequence of SEQ ID NO: 133 and LCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 134 or

[0295] (d) HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 135, HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 136 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 137 and wherein LCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 138, LCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 139 and LCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 140 or

[0296] (e) HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 141, HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 142 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 143 and wherein LCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 144, LCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 145 and LCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 146 or

[0297] (f) HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 147, HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 148 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 149 and wherein LCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 150, LCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 151 and LCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 152 or

[0298] (g) HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 153, HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 154 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 155 and wherein LCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 156, LCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 157 and LCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 158.

[0299] Second construct

[0300] Topoisomerase inhibitor

[0301] The second construct comprises a topoisomerase inhibitor. Suitably the topoisomerase inhibitor is a topoisomerase I inhibitor. Suitably the topoisomerase inhibitor is a topoisomerase II inhibitor. Alternatively, the topoisomerase inhibitor is a topoisomerase I inhibitor but not a topoisomerase II inhibitor. In a further embodiment, the topoisomerase inhibitor only inhibits topoisomerase I. Most suitably, the topoisomerase inhibitor is a topoisomerase I inhibitor.More suitably, the topoisomerase inhibitor may be selected from irinotecan, topotecan, camptothecin, rubitecan, MLN576, exatecan, belotecan and SN-38; or a functional derivative thereof and salts and / or solvates thereof; more suitably irinotecan, topotecan, camptothecin, rubitecan, MLN576, exatecan, belotecan and SN-38, more suitably exatecan ora derivative thereof and more suitably deruxtecan.

[0302] The structures of these topoisomerase inhibitors are as follows.

[0303] Irinotecan Topotecan

[0304]

[0305] Further second construct binding agents

[0306] In addition to a topoisomerase inhibitor, the second construct may comprise a further component. A suitable further component may be one or more further binding agents. A further binding agent may be a binding polypeptide, such as an antibody (e.g. a human or humanised antibody) or one or more antibody fragments. Suitable fragments may include for example an scFv, Fv, Fab, Fab’, F(ab’)2, variable domain (e.g. VH, VL, VNAR or VHH), diabody or minibody. The binding agent may bind to a tumour-associated antigen (TAA). In one embodiment, the TAA is selected from the list consisting of HER2, HER3, Nectin-4, TF (tissue factor), FRa (folate receptor alpha), EGFR, Claudin 18.2, a CEA (e.g. CEACAM5), mesothelin, B7-H3, B7-H4, NaPi-2b, EPCAM, ROR1, Trop2, cMET, CDH6, CD70, PSMA, SEZ6, Integrin B6 and combinations thereof. More suitably, the TAA is selected from HER2 or HER3. Most suitably the TAA is HER2. In one embodiment, the polypeptide seguence of HER2 is that provided in Uniprot entry P04626 (SEQ ID NO: 212).

[0307] The second construct may comprise for example (a) only a topoisomerase inhibitor, (b) a topoisomerase inhibitor and a further component, (c) a topoisomerase inhibitor and no more than one binding agent or (d) a topoisomerase inhibitor and more than one binding agent, such as two binding agents. The second construct may be monospecific, i.e. does not have specificity to any further targets.

[0308] The second construct may comprise multiple binding agents and is therefore multivalent (e.g. bivalent). These multiple binding agents may bind to different targets and therefore the first construct is multispecific (e.g. bispecific). If the second construct comprises more than one binding agent, then the binding agents may be the same or different. Suitably the binding agents are the same.

[0309] In one embodiment, the second construct comprises only one binding agent.

[0310] Alternatively, the second construct comprises more than one binding agent. In one embodiment, the second construct comprises only two binding agents.

[0311] In some embodiments the second construct comprises a HER2 (e.g. human HER2) binding agent. Receptor tyrosine-protein kinase erbB-2 is a protein that normally resides in the membranes of cells and is encoded by the ERBB2 gene. ERBB is abbreviated from erythroblastic oncogene B, a gene originally isolated from the avian genome. The human protein is also frequently referred to as “HER2” (human epidermal growth factor receptor 2) or CD340.

[0312] The HER2 binding agent may be a binding polypeptide, such as an antibody (e.g. a human or humanised antibody) or one or more antibody fragments. Suitable fragments may include for example an scFv, Fv, Fab, Fab’, F(ab’)2, variable domain (e.g. VH, VL, VNAR orVHH), diabody or minibody. Suitably the HER2 binding polypeptide comprises an antibody and more suitably the antibody is an IgG 1 antibody.

[0313] Suitably the HER2 binding agent has a minimum affinity to HER2 and / or binds specifically to HER2, for example binds to HER2 with a KD of 10'8M or less, such as 10'9M or less whereas suitably any other entity target is bound to with a KD of 10'7M or more such as 10'6M or more. More suitably, binding to any other entity cannot be measured.

[0314] Suitable HER2 binding antibodies include trastuzumab, pertuzumab and margetuximab. In one embodiment, the HER2 binding antibody comprises the CDRs of trastuzumab. In one embodiment, the CDRs of the HER2 binding antibody are those present in the heavy and light chain polypeptide sequences recited in SEQ ID NO: 210 and SEQ ID NO: 211, respectively. In a further embodiment, the VH and VL of the HER2 binding antibody are those present in the heavy and light chain polypeptide sequences recited in SEQ ID NO: 210 and SEQ ID NO: 211, respectively. In a further embodiment, the heavy and light chain polypeptide sequences of the HER2 binding antibody are the heavy and light chain polypeptide sequences recited in SEQ ID NO: 210 and SEQ ID NO: 211 , respectively. Most suitably the HER2 binding antibody is trastuzumab.

[0315] In one embodiment, the second construct comprises, such as consist essentially of, such as consists of, an antibody-drug conjugate (ADC).

[0316] A derivative of exatecan, deruxtecan, is used in the antibody-drug conjugate (ADC) trastuzumab deruxtecan (also known as “DS-8201a”). Trastuzumab deruxtecan consists of the humanized monoclonal anti-HER2 antibody trastuzumab covalently linked to the topoisomerase I inhibitor deruxtecan. Most suitably the second construct comprises (such as consists essentially of, such as consists of) trastuzumab deruxtecan. Trastuzumab deruxtecan (Iwata et al. 2018) is licensed for the treatment of certain cancers.

[0317] The structure of trastuzumab deruxtecan is as follows.

[0318]

[0319] Structure of second construct

[0320] Suitably the second construct comprises an antibody, or an antibody fragment. In one embodiment, the antibody fragment comprises an scFv, Fv, Fab, Fab’, F(ab')2, variable domain (e.g. VH, VL, VNAR or VHH), diabody or minibody.

[0321] Most suitably the second construct comprises an antibody (i.e. a full length antibody). The antibody may for example b an lgG1, an lgG2, an lgG3 or an lgG4. In one embodiment, the antibody is an I gG 1 , most suitably human I gG 1. In a further embodiment, the antibody may be in a format, such as an IgG format, that has been modified to confer desired properties, such as having the Fc mutated to reduce effector function, extend half-life, alter ADCC, or improve hinge stability. In one embodiment, the second construct is an ADC.

[0322] The second construct may for example comprise (a) a topoisomerase inhibitor and no further components, (b) one or more topoisomerase inhibitors and one or more HER2 binding agents, (c) no more than one topoisomerase inhibitor and one or more HER2 binding agents, (d) one or more topoisomerase inhibitors and no more than one HER2 binding agents or (e) one or more topoisomerase inhibitors and one or more HER2 binding agents.

[0323] If the construct comprises more than one topoisomerase inhibitors and / or HER2 binding agents, then the topoisomerase inhibitors may be the same or different and the HER2 binding agents may be the same or different.

[0324] The binding agents can be linked (or ‘fused’) to each other directly (i.e. without use of a linker) or indirectly via a linker or via further components such as immunoglobulin domains. The binding agents can be linked (or ‘fused’) to components of the first construct, such as constant domains, directly (i.e. without use of a linker) or indirectly via a linker. If the first construct comprises an Fc region, then optionally one or all of the LTBR binding agents are linked by a peptide linker to the Fc region, for example to the CH3 region. In someembodiments the HER2 binding agent (if present) and the topoisomerase inhibitor are connected by a linker, such as a cleavable linker, such as a cleavable linker wherein the linker is cleavable by a cathepsin, such as cathepsin B, cathepsin L, cathepsin D or cathepsin K (in particular, cathepsin B or cathepsin L). Alternatively the cleavable linker may be cleavable by glucuronidase. A linker comprised in the second construct is referred to herein as a ‘second construct linker’.

[0325] The linker may be a peptide linker, suitably consisting of 1 to 6 amino acids, such as 3 to 5 amino acids, such as 4 amino acids (i.e. a tetrapeptide linker). In one embodiment the linker consists of the amino acid sequence GGFG (SEQ ID NO: 209).

[0326] Suitably the linker is selected so as to allow binding of the binding agents to their targets, while maintaining structural integrity. If intended for administration to a subject, the linker is suitably non-immunogenic in the subject to which the binding agents are administered.

[0327] Treatment and administration

[0328] Certain embodiments of the invention relate to the treatment of cancer. In one embodiment, the cancer may be a tumour. In one embodiment, the cancer may be a non-haematological cancer. In one embodiment, the cancer may be a breast cancer. In the context of cancer treatment, the LTBR binding agent is most suitably an LTBR agonist (as discussed in more detail above).

[0329] The first and second constructs and compositions of the invention are typically intended for use with mammalian subjects, in particular human subjects. The first constructs and second constructs will typically be administered to a subject in need thereof, in particular a mammalian subject in need thereof, in particular a human subject in need thereof.

[0330] Administration of the first construct and the second construct may be sequential, separate or simultaneous. Suitably a safe and effective amount of the first construct and the second construct is administered.

[0331] Administration may be by any suitable route, which may depend on the nature of the specific agents. Exemplary routes include oral, parenteral, buccal, sublingual, nasal or rectal administration. Suitably, administration is performed parenterally, such as intravenously (i.v. or IV), intraperitoneally (i.p. or IP) or subcutaneously (s.c. or SC). In one embodiment the first construct is administered i.p.. In one embodiment the second construct is administered i.v.. In one embodiment the first construct is administered i.p. and the second construct is administered i.v..

[0332] Constructs and compositions may be provided in the form of a pharmaceutical composition comprising the constructs or composition and a pharmaceutically acceptable carrier or excipient.If delivered orally, the constructs or composition may suitably be delivered in a solid pharmaceutical composition (such as a tablet, capsule or lozenge) or in a liquid pharmaceutical composition (such as a suspension, emulsion or solution).

[0333] A liquid formulation will generally consist of a suspension or solution of the constructs or composition in a suitable liquid carrier e.g. an aqueous solvent such as water, ethanol or glycerine, or a non-aqueous solvent, such as polyethylene glycol or an oil. The formulation may also contain a suspending agent, preservative, flavouring and / or colouring agent.

[0334] A tablet formulation can be prepared using any suitable pharmaceutical carrier(s) routinely used for preparing solid formulations, such as magnesium stearate, starch, lactose, sucrose and cellulose.

[0335] Suitably, the pharmaceutical composition is in unit dose form, such as a tablet, capsule, vial or prefilled syringe. Suitably the unit dose form is for parenteral administration.

[0336] The pharmaceutical composition may for example contain from 0.1% to 99.99% by weight, for example from 10 to 60% by weight, of the active material, depending on the method of administration. The pharmaceutical composition may contain from 0.01% to 99% by weight, for example 40% to 90% by weight, of the carrier, depending on the method of administration. The pharmaceutical composition may contain from 0.05 mg to 2000 mg of the active material, for example from 1.0 mg to 500 mg, depending on the method of administration. The pharmaceutical composition may contain from 50 mg to 1000 mg of the carrier, for example from 100 mg to 400 mg, depending on the method of administration.

[0337] The dose of the pharmaceutical composition used will vary in the usual way with the seriousness of the cancer, the weight of the sufferer, and other similar factors. However, as a general guide, suitable unit doses may be 0.05 mg to 1000 mg, more suitably 1.0 mg to 500 mg, and such unit doses may be administered more than once a day, for example two or three a day. Such therapy may extend for a number of weeks, months or longer.

[0338] The dose provided to a subject will typically be a safe and effective dose, i.e. an amount providing an acceptable balance of desired benefits and undesired side effects. A “safe and effective amount" is intended to include an amount of a compound that is effective to achieve a desirable effect in treatment of a disease-state. A desirable effect is typically clinically significant and / or measurable, for instance in the context of (a) inhibiting the diseasestate, i.e., slowing or arresting its development; and / or (b) relieving the disease-state, i.e., causing regression of the disease state or a reduction in associated symptoms. The safe and effective amount is one that is sufficient to achieve the desirable effect.

[0339] For avoidance of doubt, a “safe and effective amount” as recited herein can be achieved by any suitable dosage regimen. Hence, for example, references herein to administering a safe and effective amount of a composition, such as by a particularadministration route, include achieving the safe and effective amount via a single dose or by plural doses, such as administered by the specified administration route. For instance, intravenously administering a safe and effective amount includes both intravenously administering a single dose and intravenously administering any plural number of doses, provided that a safe and effective amount is thereby achieved by intravenous administration.

[0340] Definitions

[0341] Binding agents

[0342] Binding agents are capable of binding to a target with an affinity (suitably expressed as a KD value, a Ka value, a kon-rate and / or a kOff-rate, as further described herein). Suitably the binding agent agonises, inversely agonises, antagonises or neutralises a specified target. In one embodiment the binding agent is a small molecule. Suitably, the binding agent comprises or consists of a binding polypeptide (also referred to as a “binding domain”). Polypeptides are said to be binding polypeptides when they comprise a domain wherein the domain comprises one or more stretches of amino acid residues which form an antigen-binding site, capable of binding to an epitope on a target with an affinity. ‘Binding polypeptide’ and ‘antigen-binding polypeptide’ are used synonymously herein. A polypeptide which binds to LTBR is synonymous with a polypeptide which is anti-LTBR, a polypeptide which binds to FAP is synonymous with a polypeptide which is anti-FAP and a polypeptide which binds to HER2 is synonymous with a polypeptide which is anti-HER2. Binding polypeptides may include antibodies and fragments thereof, such as variable domains (all of which are further described below), antibodies modified to comprise additional binding regions and antibody mimetics. Further binding polypeptides may include, for example, DARPins (Binz et al. 2003), Affimers™, Fynomers™, Centyrins, Nanofitins® and cyclic peptides.

[0343] A number of binding agents (including monospecific binding domains) described in the examples below are referred to as ‘clones’ and are assigned specific clone numbers or IDs.

[0344] Antibodies and fragments thereof

[0345] The binding agents are preferably antibodies or binding fragments thereof. Such fragments may be comprised in a non-related antibody (or antigen-binding fragment thereof) such that the construct comprising the binding agents is a chimeric antibody (or chimeric fragment thereof). A conventional antibody or immunoglobulin (Ig) is a protein comprising four polypeptide chains: two heavy (H, or HC) chains and two light (L, or LC) chains. Each chain is divided into a constant region and a variable region. The variable region comprises a heavy chain variable region paired with a light chain variable region. The heavy (H) chain variable region is abbreviated herein as VH region, and the light (L) chain variable region is abbreviatedherein as VL region. These domains, domains related thereto and domains derived therefrom, are referred to herein as variable domains. The VH and VL regions (or ‘domains’) can be further subdivided into regions of hypervariability, termed "complementarity determining regions" ("CDRs"), interspersed with regions that are more conserved, termed "framework regions" (“FRs”). The framework and complementarity determining regions have been precisely defined (Kabat et al 1991). As the invention relates to binding agents or polypeptides comprising complementarity determining regions (CDRs) and, optionally framework regions (FRs), some explanation is first provided on how such CDRs / FRs are determined. The determination of the CDR regions in a variable region sequence generally depends on the algorithm / methodology applied, such as Kabat-, Chothia-, Martin (enhanced Chothia)-, IMGT (ImMunoGeneTics information system)-, or AbM-numbering schemes. See, e.g. Kabat et al. 1991; Chothia and Lesk 1987; the AbM definition is a compromise between Kabat and Chothia as used by Oxford Molecular’s AbM antibody modelling 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.

[0346] Applying different methods to the same variable region sequence may give rise to different CDR amino acid sequences wherein the differences may reside in CDR sequence length and / or -delineation within the variable region sequence, see Figure 14 of WO2022136649A1 and Figure 8 of WO2022136647A1 which illustrate the IMGT and Kabat systems applied in parallel on the same sequence and Figure 64 of WO2022175532 A 1 which illustrates the Kabat, MacCallum, IMGT, AbM and Chothia systems applied in parallel on the same sequence. The CDRs comprised in the binding agents or polypeptides of the invention can therefore be described as the CDR sequences as present in a variable region sequence as characterized herein (more particularly an anti-LTBR or anti-FAP antibody / immunoglobulin / VHH sequence as characterized herein), or alternatively as determined or delineated according to a well-known methodology such as according to the Kabat-, Chothia-, Martin (enhanced Chothia), IMGT-, or AbM-numbering scheme or -method. The delineation of the CDRs determines the delineation of the framework regions (FRs). As used in the claims and clauses herein, in the absence of an explicitly stated definition system, the Kabat CDR definition system is intended to be applied.

[0347] In a conventional antibody, each VH and VL region is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The VH region CDRs and FRs are denoted HCDR1, HCDR2, HCDR3, HFR1, HFR2, HFR3 and HFR4. The VL region CDRs and FRs are denoted LCDR1, LCDR2, LCDR3, LFR1, LFR2, LFR3 and LFR4. The conventional antibody tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains is formed with theheavy and the light immunoglobulin chains inter-connected by e.g. disulfide bonds, and the heavy chains similarly connected. The heavy chain constant region includes three domains, CH1, CH2 and CH3. The light chain constant region is comprised of one domain, CL. The variable domain of the heavy chains and the variable domain of the light chains are binding domains that interact with an antigen. The constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g. effector cells) and the first component (C1q) of the classical complement system.

[0348] A conventional antibody comprises a homodimer of two identical heavy chains (HC) and two identical light chains (LC) (e.g. as shown in Figures 7c and 7d). Antibodies can also be produced which comprise a heterodimer of two different heavy chains (HC1 and HC2) and two identical light chains (LC) (e.g. as shown in Figures 7a and 7b).

[0349] A further exception to conventional antibody structure is found in sera of Camelidae. In addition to conventional antibodies, these sera possess special IgG antibodies. These IgG antibodies, known as heavy-chain antibodies (HCAbs), are devoid of the L chain polypeptide and lack the first constant domain (CH1). At its N-terminal region, the H chain of the homodimeric protein contains a dedicated immunoglobulin chain variable domain, referred to as the VHH, which serves to associate with its cognate antigen (Muyldermans 2013; Hamers-Casterman et al 1993).

[0350] The term “antibody” includes any substantially complete antibody protein comprising at least one antibody variable domain comprising at least one antigen binding site (ABS).

[0351] Antibodies include, but are not limited to, immunoglobulins of types IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof, e.g. lgG1). The overall structure of Immunoglobulin G (IgG) antibodies assembled from two identical heavy (H)-chain and two identical light (L)-chain polypeptides is well established and highly conserved in mammals (Padlan 1994).

[0352] A fragment of an antibody (which may also be referred to as “antibody fragment”, “antibody binding fragment”, “immunoglobulin fragment”, “antigen-binding fragment” or “antigen-binding polypeptide”) as used herein refers to a portion of an antibody that binds to the target (e.g. a molecule in which one or more immunoglobulin chains is not full length, but which binds to the target). Examples of binding fragments encompassed within the term antibody fragment include:

[0353] (i) a Fab fragment (a monovalent fragment consisting of the VL, VH, CL and CH1 domains); (ii) a F(ab')2 fragment (a bivalent fragment consisting of two Fab fragments linked by a disulphide bridge at the hinge region);

[0354] (iii) a Fd fragment (consisting of the VH and CH1 domains);(iv) a Fv fragment (a variable domain or variable region, consisting of the VL and VH domains of a single arm of an antibody wherein the VL and VH domains together form an antigen binding site);

[0355] (v) a single chain variable fragment, scFv (consisting of VL and VH domains joined, possibly using recombinant methods, by a synthetic linker (e.g. a peptide) that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules);

[0356] (vi) a VH (a heavy chain variable domain);

[0357] (vii) a VL (a light chain variable domain);

[0358] (viii) a domain antibody (dAb, consisting of either the VH or VL domain);

[0359] (ix) a minibody (consisting of a pair of scFv fragments which are linked via CH3 domains); and (x) a diabody (consisting of a noncovalent dimer of scFv fragments that consist of a VH domain from one antibody connected by a peptide linker a VL domain from another antibody);

[0360] (xi) a VHH (a heavy chain variable domain as described in detail above).

[0361] The antibody or fragment thereof may be a human antibody or derived from a human antibody. “Human antibody" refers to antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human subjects administered with said human antibodies do not generate cross-species antibody responses (for example termed HAMA responses - human-anti-mouse antibody) to the primary amino acids contained within said antibodies. Said human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g. mutations introduced by random or sitespecific mutagenesis or by somatic mutation), for example in the CDRs and in particular CDR3. However, the term is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library, antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences, may also be referred to as “recombinant human antibodies”.

[0362] Substituting at least one amino acid residue in the framework region of a non human immunoglobulin variable domain with the corresponding residue from a human variable domain is referred to as “humanisation”. Humanisation of a variable domain may reduce immunogenicity in humans.Antibodies comprise a hinge region, as do certain constructs comprising antibody domains and antibody fragments. A naturally occurring hinge region is a flexible amino acid stretch in the central part of the heavy chains of the IgG and IgA immunoglobulin classes, which links these 2 chains by disulfide bonds. As used herein, a hinge region encompasses naturally occurring hinge regions and also variants thereof wherein suitably the variants maintain adequate flexibility and rigidity to maintain integrity of the antibody, fragment or construct, while maintaining binding ability. A native hinge region may for example comprise or consist of the polypeptide sequence SEQ ID NO: 204. The variant may be a truncated version (a ‘truncated hinge region’) of a naturally occurring hinge region, such as a hinge region truncated by 5 amino acids relative to a naturally occurring hinge region, or most suitably a truncated hinge region comprising or consisting of SEQ ID NO: 203. In one embodiment a hinge region consists of 10-80 amino acids, such as 20-70 amino acids, such as 30-60 amino acids, such as 40-50 amino acids.

[0363] Specificity, affinity, avidity and potency

[0364] “Specificity” refers to the number of different types of antigens or antigenic determinants to which a particular antibody or fragment thereof can bind. The specificity of an antibody is the ability of the antibody to recognise a particular antigen as a unique molecular entity and distinguish it from another. An antibody that “specifically binds” to an antigen or an epitope is a term well understood in the art. A molecule is said to exhibit “specific binding” if it reacts more frequently, more rapidly, with greater duration and / or with greater affinity with a particular target antigen or epitope, than it does with alternative targets. An antibody “specifically binds” to a target antigen or epitope if it binds with greater affinity, avidity, more readily, and / or with greater duration than it binds to other substances. An antibody (or fragment thereof) may be considered to specifically bind to a target if the binding is statistically significant compared to a non-relevant binder. Specific binding of an antibody, or fragment thereof, to an antigen or antigenic determinant can be determined in any suitable known manner, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, equilibrium dialysis, equilibrium binding, gel filtration, ELISA, or spectroscopy (e.g. using a fluorescence assay) and the different variants thereof known in the art.

[0365] In one embodiment by ‘binding specifically’ it is meant that the target is bound to with a KD of 10'8M or less, such as 10'9M or less whereas any other entity target is bound to with a KD of 10'7M or more such as 10'6M or more. More suitably, binding to any other entity cannot be measured.“Affinity”, represented by the equilibrium constant for the dissociation of an antigen with an antigen-binding polypeptide (KD), is a measure of the binding strength between an antigenic determinant and an antigen-binding site on the antibody (or fragment thereof): the lesser the value of the KD, the stronger the binding strength between an antigenic determinant and the antigen-binding polypeptide. Alternatively, the affinity can also be expressed as the affinity constant (KA), which is 1 / KD. Affinity can be determined by known methods, depending on the specific antigen of interest. Any KD value less than 10'6M is considered to indicate binding. Suitably, constructs of the invention will bind targets with a dissociation constant of 10'6M or less, more suitably 10'7M or less, more suitably 10'8M or less and more suitably 10'9M or less.

[0366] Suitably, the KD of a binding polypeptide used in the invention is determined using surface plasmon resonance (SPR) or biolayer interferometry (“BLI”, such as by using an Octet instrument or such as by the method described in Example 4).

[0367] “Avidity” is the measure of the strength of binding between a polypeptide, an antibody or fragment thereof, and the pertinent antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen binding site on the antibody and the number of pertinent binding sites present on the antibody.

[0368] “Potency” is a measure of the activity of a therapeutic agent expressed in terms of the amount required to produce an effect of given intensity. A highly potent agent evokes a greater response at low concentrations compared to an agent of lower potency that evokes a smaller response at low concentrations. Potency is a function of affinity and efficacy. Efficacy refers to the ability of therapeutic agent to produce a biological response upon binding to a target and the quantitative magnitude of this response. The term half maximal effective concentration (EC50) refers to the concentration of a therapeutic agent which causes a response halfway between the baseline and maximum after a specified exposure time. The therapeutic agent may cause inhibition or stimulation. It is commonly used, and is used herein, as a measure of potency.

[0369]

[0370] For the purposes of comparing two closely-related polypeptide sequences, the “% sequence identity” between a first polypeptide sequence and a second polypeptide sequence may be calculated using NCBI BLAST v2.0, using standard settings for polypeptide sequences (BLASTP). For the purposes of comparing two closely-related polynucleotide sequences, the “% sequence identity” between a first nucleotide sequence and a second nucleotide sequence may be calculated using NCBI BLAST v2.0, using standard settings for nucleotide sequences (BLASTN).Polypeptide or polynucleotide sequences are said to be the same as or “identical” to other polypeptide or polynucleotide sequences if they share 100% sequence identity over their entire length. Residues in sequences are numbered from left to right, i.e. from N- to C-terminus for polypeptides; from 5’ to 3’ terminus for polynucleotides.

[0371] A “difference” between polypeptide sequences refers to an insertion, deletion or substitution of a single amino acid residue in a position of the second sequence, compared to the first sequence. Two polypeptide sequences can contain one, two or more such amino acid differences. Insertions, deletions or substitutions in a second sequence which is otherwise identical (100% sequence identity) to a first sequence result in reduced % sequence identity. For example, if the identical sequences are 9 amino acid residues long, one substitution in the second sequence results in a sequence identity of 88.9%. If first and second polypeptide sequences are 9 amino acid residues long and share 6 identical residues, the first and second polypeptide sequences share greater than 66% identity (the first and second polypeptide sequences share 66.7% identity).

[0372] As used in the clauses and claims herein, numbering of polypeptide sequences and definitions of CDRs and FRs (i.e. HCDR1 , HCDR2, HCDR3, HFR1 , HFR2, HFR3, HFR4, LCDR1, LCDR2, LCDR3, LFR1, LFR2, LFR3 and LFR4) are as defined according to the Kabat system (Kabat et al., 1991 , herein incorporated by reference for the purpose of CDR and FR definitions), unless mentioned otherwise. Alternative to the Kabat system, any of the Chothia-, Martin (enhanced Chothia)-, IMGT (ImMunoGeneTics information system)-, or AbM-numbering schemes can be applied. A “corresponding” amino acid residue between a first and second polypeptide sequence is an amino acid residue in a first sequence which shares the same position according to the Kabat system with an amino acid residue in a second sequence, whilst the amino acid residue in the second sequence may differ in identity from the first.

[0373] Suitably corresponding residues will share the same number (and letter) if the framework and CDRs are the same length according to Kabat definition (or according to the Chothia-, Martin (enhanced Chothia)-, IMGT (ImMunoGeneTics information system)-, or AbM-definition or delineation). Alignment can be achieved manually or by using, for example, a known computer algorithm for sequence alignment such as NCBI BLAST v2.0 (BLASTP or BLASTN) using standard settings.

[0374] Residues recited in the polypeptide sequences listed herein may be unmodified or modified. If modified, they may be for example isomerized, deaminated or oxidated.

[0375] The invention is further exemplified by the following non-limiting examples.EXAMPLES

[0376] 1. Example 1. Generation of anti-LTBR nanobodies (VHHs)

[0377] 1.1. Immunisation and library constructions

[0378] VHHs were generated by immunising llamas and alpacas with recombinant protein, essentially as described elsewhere (Pardon et al., 2014) (Henry & MacKenzie, 2018). Briefly, animals were immunised with human LTBR human IgG 1 Fc chimera (Aero Biosystems, cat. # LTR-H5251).

[0379] Phage display libraries derived from peripheral blood mononuclear cells (PBMCs) were prepared and used as previously described (Pardon et al., 2014) (Henry & MacKenzie, 2018). The VHH fragments were cloned into a M13 phagemid vector containing Myc and His6 tags. The library was rescued by infecting exponentially growing Escherichia coli TG1 cells and superinfection with VCSM13 helper phage.

[0380] The human LTBR immunised phage libraries were subjected to two consecutive selection rounds on human LTBR human lgG1 Fc chimera protein (Sino Biological, cat. #10581-H02H).

[0381] 1.2. Screening of anti-LTBR VHHs

[0382] In a binding ELISA, 830 VHH clones from the immunisation and selection campaigns were screened as crude periplasmic extracts. Plates were coated with 5pg / ml of NeutrAvidin in PBS (overnight at 4°C), followed by blocking with 1% casein in PBS (2h at RT). Next, biotinylated hLTBR (OriGene, cat. # AR51717PU-S) was captured at 10 nM in 0.1% casein in PBS (1h at RT). Detection was done with 5 pg / ml anti-c-myc antibody 9E10 (Merck, cat. # 11667203001) and 0.16 pg / ml anti-mouse IgG-HRP (Jackson Immuno Research, cat. # 715-035-150), both in 0.1% casein in PBS (1h at RT). Sequence analysis of 808 hits revealed that these belonged to 149 different clusters of related sequences. 270 unique sequences were then confirmed for binding utilising off-rate analysis on human and cynomolgus LTBR (as described below).

[0383] To confirm species cross-reactivity, either human or cynomolgus recombinant LTBR-fused on human Fc (SinoBiological, cat. # 10581-H02H and 90101-C02H) were immobilised on CM5 sensor chips (cat. # CFJB 334). 1 :5 dilutions of crude periplasmic extracts of clones expressing VHHs were injected at 30 pl / min for 2 min in HBS-EP pH 7.4 buffer to allow for binding to chip-bound antigen. Next, an HBS-EP pH7.4 buffer without VHHs was injected for 5 minutes at the same flow rate to enable spontaneous dissociation of bound VHHs. Surface regeneration was done with 10 ul of 1 mM Glycine pH 1.5 / 1 M NaCI injected twice between samples. From the sensorgrams obtained on a BiacoreT200 machine, koff values were calculated by fitting a 1:1 interaction model (Langmuir model) using Insight Evaluation Software (Biacore). Based on this analysis, four clones were selected for further evaluation,and the results are included in Table 1. The result confirmed that the selected clone recognised human and cynomolgus LTBR with comparable binding strength.

[0384] Table 1. Off-rate analysis on human and cynomolgus LTBR on selected leads by Biacore.

[0385]

[0386] Synthetic DNA fragments encoding the VHHs were subcloned into an E. coli expression vector under the control of an IPTG-inducible lac promoter in a frame with N-terminal PelB signal peptide (which directs the recombinant proteins to the periplasmic compartment) and C-terminal FLAG3 and His6 tags. VHH proteins were purified from these clones employing IMAC chromatography followed by desalting according to well-established procedures (Pardon et al., 2014).

[0387] The purified proteins were tested for binding to endogenously expressed human LTBR in HepG2 cells. The expression level was confirmed with anti-LTBR clone CBE-11 (“huCBEH” as disclosed in W02004002431). The HepG2 cells were resuspended to a final concentration of 1.0 x 106cells / ml in FACS buffer. A series dilution of purified VHHs was incubated with mouse anti-FLAG biotinylated antibody (Sigma Aldrich, cat. # F9291-1MG) at 5 pg / ml in FACS buffer for 30 min at RT with shaking. Cell suspensions were distributed into a 96-well v-bottom plate and incubated with the above-described VHH / antibody mixture for 1 hour on ice while shaking. VHH binding to cells was detected with streptavidin R-PE (Invitrogen, cat. # .SA10044) at 1:400 dilution (0.18 pg / ml) in FACS buffer, incubated for 30 minutes on ice with shaking and protection from light. The cells were acquired by flow cytometry. The data was analysed using FloJo V10 and GraphPad Prism. The EC50 values can be found in Table 2.

[0388] Table 2 EC50 values for binding curves to HepG2 cell line

[0389]

[0390] Activation of LTBR by its ligands LTaip2 or LIGHT occurs via clustering of multiple LTBR receptors leading to activation of an intracellular signal transduction cascade. Therefore, activation of the receptor with a monovalent molecule is not expected. The activation of human LTBR was evaluated in NFKB Luciferase Reporter HepG2 cells (endogenous LTBR expression Signosis, cat. # SL-0017) in the presence and absence of cross-linking anti-His antibody.

[0391] For the reporter assay, cells were plated in 96-well plates (Greiner) at a density of 3X104cells / well and cultured overnight at 37°C and 5% CO2 in Eagle's Minimum Essential Medium (EMEM, Gibco) supplemented with 10% heat-inactivated FBS and 100 U / mL penicillin and streptomycin (Gibco). Cells were then incubated with monovalent His Tag containing VHHs cross-linked with anti-His tag mAb (Jackson, cat. # 300-005-240) for 6 hours. After 6 h, a ONE-Glo Ex Solution (Promega, cat. no E8120) was added to the well and incubated for 10 min before being read on a BMG Pherastar FSX plate reader. Background-corrected RLU values were plotted against stimulant (antibody or antibody fragment) concentration for all conditions tested in GraphPad Prism, and the EC50 values can be found in Table 3. Figures 1a and 1b demonstrate that, as expected, the monovalent VHHs do not lead to activation of the LTBR unless cross-linked with anti-His Tag antibodies.

[0392] Table 3 EC50 values from the HepG2 reporter assay

[0393]

[0394] 1.3. Sequence optimisation of monovalent VHHs

[0395] The protein sequences of four selected VHHs were modified to improve their humanisation towards human IGHV3 and JH germline consensus sequences as well as their chemical and biophysical stability while minimising the impact on target binding. To this end, different mutant variants were generated for each lead, which were then compared for their capacity to compete for binding to human LTBR endogenously expressed on HepG2 cells versus their respective FLAG3-tagged parental sequences using competition flow cytometry. Melting (Tm)temperatures of the different variants were determined. The variants were also subjected to temperature stress (1 week @ 40 °C) followed by analytical size exclusion in combination with multiangle laser light scattering analysis (aSEC-MALLS) to assess the oligomerisation propensity. In addition, the VHHs were subjected to long-term temperature stress (4 weeks @ 40 °C) and forced oxidative stress (10 mM H2O2 for 3 hours @ 37 °C), followed by detailed peptide mapping mass spectrometry to assess amino acid stability. Amino acid residues with poor chemical stability (>5% modification after relevant stress) were replaced by suitable alternatives. The sequence-optimised mutants with desired properties were selected for further assessment.

[0396] 1.4. Formatting into VHH-Fc fusions and functional testing

[0397] VHH-Fc fusions were generated by combining sequence-optimised anti-LTBR VHHs with human IgG 1 Fc domain, separated by flexible Glycine Serine linkers. The constructs were cloned into an expression vector and expressed by transient CHO suspension cell transfection. VHH-Fc fusion proteins were purified from cell culture supernatants by affinity chromatography using protein A resin and followed by one polishing chromatography step: preparative size exclusion (SEC).

[0398] SEC-HPLC and mass spectrometry assessed the purity and heterogeneity of the samples. All samples were confirmed to have a monomer content of >95% and contain <10 % impurities before functional read-out. The list of constructs can be found in Table 4.

[0399] Table 4 List of VHH-Fc fusion constructs

[0400]

[0401] 1.4.1. NFKB activation of HepG2 reporter cells expressing human LTBR The ability of anti-LTBR VHH-Fc fusion proteins to activate LTBR was tested in NFKB luciferase reporter HepG2 (Signosis, cat.no SL0017-FP). Clustering endogenous LTBR on HepG2 cells leads to activation of endogenous NFKB and translocation from the cytoplasm and nucleus, which binds to the promoter region and induces luciferase expression. After that, a luciferase substrate / lysis reagent mix (ONE-GLO EX Promega) is added to cells, which allows quantification in the bioluminescence reaction, where enzymatic activity is proportional to luciferase expression.

[0402] A monospecific anti-LTBR agonist (BHA10, as disclosed in W02004002431) was used as a positive control. In short, HepG2 NFKB luciferase (Lc) (30000 cells / well) were seeded, and the 1:10 series dilution of VHH-Fc fusion proteins and controls was added to the cells and incubated for 6h at 37°C, 5% CO2, prior to the addition of detection reagents. After 6 h, a ONE-Glo Ex Solution (Promega, cat. no E8120) was added to the well and incubated for 10 min before being read on a BMG Pherastar FSX plate reader. Background-corrected RLU values were plotted against stimulant concentration for all conditions tested in GraphPad Prism, and the EC50 value can be found in Table 5. The results confirmed that formatting monomeric VHHs into bivalent molecules leads to LTBR activation with comparable or higher potency than the control monoclonal antibody (BHA10).

[0403] Table 5 EC50 values of HepG2 NFKB activation-induced luciferase activity

[0404]

[0405] 1.4.2. In vitro VHH Fc fusion protein activity in a chemokine induction assay using tumor cell line HCC1187

[0406] An activation assay measuring chemokine secretion was used to investigate the ability of anti-LTBR VHH-Fc fusion proteins to activate LTBR endogenously expressed on tumor cells (HCC1187). In short, HCC1187 (20000 / well) were seeded in their growth medium. A 1:10, 7-point serial dilution series in duplicates of antibodies and controls was prepared in assay medium as 2-fold concentration stocks, added to the cells, and incubated for24h at 37°C, 5% CO2. After incubation, the supernatants were collected and cleared by centrifugation, and thechemokine level was determined by CCL19 DuoSet ELISA (R&D system, cat. no DY361) following the manufacturer's instructions. Absorbance was read on the BMG Pherastar FSX plate reader. OD corrected values (450-540nm) for the standard curve were analysed in Prism, according to the manufacturer's recommendation, to interpolate cytokine concentrations for the test wells. Dose-response curves were plotted using GraphPad Prism Version 9, applying nonlinear fits (log(agonist) vs response (variable slope- four parameters). EC50 values can be found in Table 6.

[0407] Table 6 EC50 values of upregulation of CCL19 in HCC1187 chemokine induction assay

[0408]

[0409] Figure 2 shows that anti-LTBR VHH-Fc fusion proteins induced dose-dependent CCL19 production in HCC1887 cells.

[0410] 1.4.3. Ligand receptor inhibition assay

[0411] The ability of VHH-Fc fusion proteins to bind to endogenously expressed LTBR on the A549 tumor cell and to inhibit human LIGHT (Aero, cat.no LIT-H5242) binding to A549 cells was assessed. BHA10 was employed in the assay as a positive control. Recombinant human LIGHT was labelled with PE fluorochrome, using PE I R- Phycoerythrin Conjugation Kit -Lightning-Link®(Abcam, cat no ab102918), following the manufacturer’s instructions. 1x106cells were resuspended in the FACS buffer (1% BSA, 0.1% Sodium Azide in PBS), and 50 ul was added to each well (5x105cells / well) of a 96-U bottom plate. The cells were washed and resuspended in 25ul of antibody dilution (2x final concentration) followed by 0.5h incubation at 4°C, 25ul of PE labelled LIGHT at48nM (2x final concentration) in FACS buffer was subsequently added to the cells and additionally incubated for 1h at 4°C. The cells were washed, and cells were further stained with a secondary detection antibody, Alexa Fluor® 647-conjugated AffiniPure Fab Fragment Goat Anti-Human IgG (H+L) (Stratech, cat.no 109-607-003), for 1h incubation at 4°C. Next, cells were extensively washed and fixed with Paraformaldehyde Solution, 4% in PBS (Thermo Scientific, cat no J19943.K2) for 20 mins,following the manufacturer’s instructions. The cells were then washed, resuspended in PBS, and read on Beckman Coulter CytoFLEX. The data were analysed using FloJo V10 and GraphPad Prism v9. The IC50 and EC50 values can be found in Table 7.

[0412] Table 7 IC50 values for ligand-receptor inhibition and EC50 values of binding curves to LTBR positive cell line (A549)

[0413]

[0414] Figure 3a confirms that all tested antibodies bind strongly to endogenously express LTBR on A549 tumor cell line.

[0415] Figure 3b confirms that all tested clones demonstrated an 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 inhibition.

[0416] 1.4.4. Epitope competition assay by Octet

[0417] An epitope binning assay on Octet Red96 was employed to confirm whether the selected anti-LTBR VHHs compete for the same epitope. In short, 5 ug / ml of recombinant human LTBR was amine coupled into AR2G biosensors at pH 5.0, following manufacturer instructions. The first VHH was loaded onto pre-coated LTBR biosensors until saturation was reached, followed by the sensor’s exposure to the competitor / second VHH. Detection of any additional binding on the biosensor by the second antibody indicates an unoccupied epitope (non-com petitor). In contrast, no second antibody binding on the biosensor indicates a blocked epitope (competitor). The results suggest that only Clone 1.10 binds to a distinct epitope, whereas all the other clones bind to an overlapping epitope.

[0418] 2. Example 2 Generation of anti-LTBR monoclonal antibodies

[0419] 2.1. Immunisation

[0420] Two cohorts, 5 mice each, of transgenic humanised mice, ATX-GK Cross, were immunised with human LTBR recombinant protein or plasmid DNA encoded to express human LTBR cDNA using standard 5-week RIMMS protocol for proteins (10 pg subcutaneous dosing ofantigen emulsified in complete Freund’s adjuvant followed by 5 weekly subcutaneous dosing of antigen emulsified in incomplete Freund’s adjuvant) or an extended protocol for DNA (25 pg co-injection with pBoost4 adjuvant vector injected ID every week for six weeks with a final prefusion boost with 10 pg of recombinant protein injected IP).

[0421] Sample bleeds were taken at week four and tested for antigen-positive serum titer and purification tag-negative serum titer by ELISA. ELISA plates were coated with either 1pg / ml of human LTBR with human Fc (Aero H5259), cyno LTBR with human Fc (R&D 10759-LR), human LTBR with mouse Fc (M-300-15, produced in-house), mouse LTBR with mouse Fc (R&D 1008-LR), or internal mouse Fc control (P-110). Antigen-coated plates were incubated with 8-point 10-fold serial dilutions of sera starting at 1:100. Antibodies bound to antigen were detected by anti-mouse IgG HRP secondary antibody and one-step TMB solution. The absorbance signal at 450 nm was measured with an ELISA microplate reader.

[0422] 2.2. Hybridomas

[0423] Immune tissues from high-titer mice were harvested and preserved for antibody discovery. Hybridoma cell lines producing LTBR antibodies were produced by the fusion of single B Cells from the spleen and lymph nodes of titer-positive mice with myeloma cells. Twenty 96 well plates of hybridoma fusions were generated and expanded. Hybridomas expressing LTBR-specific antibodies were detected by antigen binding by ELISA. The affinity of antibodies in the hybridoma supernatants was measured by Bio-Layer Interferometry (BLI) using the Octet instrument. LTBR antibodies in hybridoma supernatant were loaded on a biosensor. The response was measured as a nm shift in the interference pattern and was proportional to the number of antibodies bound to the biosensor’s surface. The binding interaction of LTBR to the immobilised antibodies was measured as an association (kon). Following analyte association, the biosensor was dipped into PBS without LTBR, and the bound antigen was allowed to dissociate from the antibody (kdis), KD (M), or affinity of the antibodies for LTBR was measured as kdis / kon.

[0424] Heavy and light chains from validated hybridomas were sequenced. RNA was isolated from LTBR antibody-secreting hybridomas, and heavy and light chain variable regions were cloned by reverse transcription using gene-specific primers followed by PCR amplification with variable chain gene-specific primers. PCR products were sequenced by standard Sanger sequencing methods.

[0425] 2.3. Phage Display

[0426] Variable heavy and light chains were amplified from the spleen of high titer immunised mice by reverse transcription using gene-specific primers followed by PCR amplification withvariable chain gene-specific primers. Both variable regions were cloned into a phage display vector designed to express Fabs on phage g3p protein for the diverse library. For the common light chain library, only the heavy chain variable regions were cloned into the phage display vector as before. Libraries of phage expressing unique Fabs were amplified and purified. Phage were allowed to bind to biotinylated LTBR antigens (human, mouse or cyno) captured on streptavidin magnetic beads. Phage remaining bound to antigen beads after several stringent washes was eluted using a basic triethylamine solution and neutralised with Tris buffer pH 8.0. Eluted phages were reinfected into TG1 bacterial cells, amplified by co-infection with M13 helper phage, and purified by PEG precipitation. Purified phages expressing Fabs were selected for antigen binding as described. Phage from the second round was diluted and infected into TG1 cells. Polyclonal pools of phage output from two rounds of panning were tested by ELISA to confirm that the pools contained LTBR-specific phage. Variable heavy and light chain regions were sequenced from single infected bacterial colonies using a rolling circle amplification and standard Sanger sequencing.

[0427] 2.4. Antibody expression

[0428] Unique variable heavy and light chain pairs from all discovery techniques were cloned into vectors designed to express full-length antibodies as IgGs containing LALA silencing mutations in HEK293 cells under the control of a CM promoter. Antibody expression vectors were complexed with polyethylenimine and transfected into HEK293 cultures. After 5 days of shaking at 37°C in HEK293 cell culture media, antibodies were captured on agarose-based protein A resin. After several stringent washes, antibodies were eluted in glycine solution, pH 3, neutralised with HEPES, pH 9, and buffer exchanged into PBS. The purity of purified proteins was confirmed by SEC-HPLC and CE-SDS analysis. Each antibody was assigned a unique clone number.

[0429] 2.5. Binding of anti-LTBR antibodies to A375 tumor cell line

[0430] For binding to LTBR-positive cells, the A375 tumor cell line was used. At the same time, binding to LTBR-negative HEK293 cells was tested to confirm specificity. In short, the cells were resuspended in the FACS binding buffer, and live / dead efluro450 viability dye was added at a 1:1000 dilution to the cell suspension, followed by incubation at room temperature in the dark for 30 minutes. The cells were then diluted to 2 e5 / ml, and 100 ul was added to wells of a 96-well U-bottom plate as required. For generating an EC50, a 100 nM stock was prepared and then diluted 3 fold serially across 12 points. The cells were pelleted by centrifugation, and the supernatant was discarded, followed by the addition of a series of dilutions of anti-LTBR antibodies and incubation for 45 minutes at 4C in the dark. Cells were washed two times withPBS with 3% (w / v) BSA before adding 60 ul of fluorescently labelled secondary antibody (goat anti-human IgG) diluted to 1 :200 in PBS. Incubation continued at 40 for at least 30 minutes, followed by two washes in PBS pH 7.4. Cells were either fixed with 2% PFA (30 minutes at 4C) followed by washout and storage in 100 ul of PBS or immediately analysed on a Flow Cytometer. The data were analysed using FloJo V10 and GraphPad Prism. No binding to the HEK cell line was detected for any tested clones, confirming the specific interaction between anti-LTBR antibodies and LTBR receptor. The results are summarised in Table 8. Figure 4 demonstrates dose-dependent binding to the native form of LTBR, expressed on the A375 tumor cell line.

[0431] Table 8 EC50 value for binding of anti-LTBR antibodies to A375 cell line

[0432]

[0433] NP Not reaching a plateau, EC50 cannot be evaluated

[0434] 2.6. Binding kinetics of anti-LTBR antibodies to human and cynomolgus LTBR

[0435] The binding of anti-LTBR antibodies to human and cynomolgus LTBR was investigated by Surface Plasmon Resonance (SPR) on Carterra LSA. An EC30M chip was used to capture the test antibodies. Activation with EDC and sNHS occurred in MES pH 5.5 to prepare the chip for amine coupling. Goat anti-human IgG Fc antibody was then captured to the chip via incubation for 10 minutes. Excess conjugation sites were quenched with 1M Ethanolamine pH 8. To measure monovalent binding kinetics, antibodies were printed / spotted onto the chip and allowed to bind to the goat antibody, followed by subsequent washing. After the preparation, the printed chip was exposed to repeated rounds of association and dissociation with increasing analyte concentrations. In this work, recombinant proteins (Human LTBR (Aero) or cyno LTBR His (R&D)) were serially diluted across a range starting at 1500 nM with 3-fold dilutions and 10 points. The curves were fitted to global fit in a 1:1 binding model to obtain KD values. The binding affinity to both species is reported Table 9. Tested antibodies demonstrated comparable affinities to human and cynomolgus LTBR.Table 9 KD values for binding kinetics to human and cynomolgus LTBR

[0436]

[0437] 2.7. Epitope competition assay by Carterra LSA

[0438] A high-throughput epitope binning experiment was done on real-time label-free biosensors (Carterra LSA) to sort a panel of mAbs into bins based on their ability to block one another for binding to the antigen. In a pairwise epitope binning analysis, antigen and antibody 2 (analyte antibody) are sequentially applied to the sensor chip (HC200M) covalently pre-loaded with antibody 1 (ligand antibody). An increase in response upon exposure to the analyte antibody indicates non-competition between the two antibodies, whereas a lack of change in the signal indicates competition. Antibodies in the test set that have the same blocking profiles towards others are grouped into one bin. Community network plots are used to explore the clustering of mAbs that share similar but not necessarily identical competition profiles. Rather than relying strictly on the sandwiching / blocking assignments in the heat map, as the Bin network plots do, hierarchical clustering is applied to the sorted heat map to generate dendrograms, which progressively group mAbs. Community bins for tested anti-LTBR antibodies are shown in Table 10.

[0439] Table 10 Community bins of anti-LTBR antibodies defined by epitope competition assay on Carterra

[0440]

[0441]

[0442] 2.8. NFKB activation of HepG2 reporter cells expressing human LTBR

[0443] To investigate the agonistic function of anti-LTBR antibodies, the NFKB luciferase reporter HepG2 cell line (Signosis, cat.no SL0017-FP) was employed. Clustering endogenous LTBR on HepG2 cells leads to activation of endogenous NfKb and translocation from cytoplasm to the nucleus, where it binds to the promoter region and induces luciferase expression. After that, a luciferase substrate / lysis reagent mix (ONE-GLO EX Promega) is added to cells, which allows quantification in the bioluminescence reaction, where enzymatic activity is proportional to luciferase expression. Antibodies were tested in the presence and absence of cross-linking antibody (anti-human Fc, Jackson, cat no 109-005-098) in a 2:1 ratio . BHA10 was employed as a positive control in this assay.

[0444] HepG2 NF-Kb Lc (30000 cells / well) were seeded alone in their growth media. The 1:10 series dilution of monospecific antibodies, in the presence or absence of cross-linking agent was added to the cells and incubated for 6h at 37°C, 5% CO2, prior to the addition of detection reagents. After 6 h, a ONE-Glo Ex Solution (Promega, cat. no E8120) was added to the well and incubated for 10 min before being read on a BMG Pherastar FSX plate reader.

[0445] Background corrected RLU values were plotted against stimulant concentration for all conditions tested in GraphPad Prism and shown in Figures 5a (in the absence of cross-linking antibody) and 5b (in the presence of cross-linking antibody) respectively. The EC50 value can be found in Table 11.

[0446] Table 11 EC50 values for anti-LTBR antibodies agonistic activity in HepG2 reporter assay in the presence or absence of cross-linking antibody

[0447]

[0448]

[0449] A broad panel of anti-LTBR antibodies with varied agonistic activity was selected. In most cases, the presence of cross-linking antibody led to a higher potency.

[0450] 3. Example 3 Generation of anti-FAP antibodies

[0451] 3.1. Immunisation

[0452] Two cohorts, 5 mice each, of transgenic humanised mice, ATX-GK Cross, were immunised with human FAP (Aero Bioscience, cat no FAP-H5244) recombinant protein or plasmid DNA encoded to express human FAP cDNA using standard 5-week RIMMS protocol for proteins (10 pg subcutaneous dosing of antigen emulsified in complete Freund's adjuvant followed by 5 weekly subcutaneous dosing of antigen emulsified in incomplete Freund's adjuvant) or an extended protocol for DNA (25 pg co-injection with pBoost4 adjuvant vector injected ID every week for six weeks with a final pre-fusion boost with 10 pg of recombinant protein injected IP). Sample bleeds were taken at week four and tested for antigen-positive serum titer and purification tag-negative serum titer by ELISA. ELISA plates were coated with 1pg / ml of human FAP-His (Aero) or internal His tag control (CD22-His). Antigen-coated plates were incubated with 8-point 10-fold serial dilutions of sera starting at 1:100. Antibodies bound to antigen were detected by anti-mouse IgG HRP secondary antibody and one-step TMB solution. The absorbance signal at 450 nm was measured with an ELISA microplate reader. Strong titers from mice dosed with either recombinant protein or cDNA human FAP detected on Day 21 were observed with no reaction to the His Tag.

[0453] 3.2. Hybridoma

[0454] Immune tissues from high-titer mice were harvested and preserved for antibody discovery. Hybridoma cell lines producing FAP antibodies were produced by the fusion of single B Cells from the spleen and lymph nodes of titer-positive mice with myeloma cells. Twenty 96 well plates of hybridoma fusions were generated and expanded. Hybridomas expressing FAP-specific antibodies were detected by antigen binding by ELISA. The affinity of antibodies in the hybridoma supernatants was measured by Bio-Layer Interferometry (BLI) using the Octet instrument. FAP antibodies in the hybridoma supernatant were loaded on a biosensor. The response was measured as a nm shift in the interference pattern and was proportional to the number of antibodies bound to the biosensor's surface. The binding interaction of FAP to the immobilised antibodies was measured as an association (kon). Following analyte association,the biosensor was dipped into PBS without FAP, and the bound antigen was allowed to dissociate from the antibody (kdis), KD (M), or affinity of the antibodies for FAP was measured as kdis / kon.

[0455] Heavy and light chains from validated hybridomas were sequenced. RNA was isolated from FAP antibody-secreting hybridomas, and heavy and light chain variable regions were cloned by reverse transcription using gene-specific primers followed by PCR amplification with variable chain gene-specific primers. PCR products were sequenced by standard Sanger sequencing methods.

[0456] 3.3. Phage display

[0457] Variable heavy and light chains were amplified from the spleen of high titer immunised mice by reverse transcription using gene-specific primers followed by PCR amplification with variable chain gene-specific primers. Both variable regions were cloned into a phage display vector designed to express Fabs on phage g3p protein for the diverse library. For the common light chain library, only the heavy chain variable region were cloned into the phage display vector as before. Libraries of phage expressing unique Fabs were amplified and purified. Phage were allowed to bind to biotinylated FAP antigens (human, mouse or cyno) captured on streptavidin magnetic beads. After several stringent washes, the phage remaining bound to antigen beads was eluted using a basic triethylamine solution and neutralised with Tris buffer pH 8.0. Eluted phages were reinfected into TG1 bacterial cells, amplified by co-infection with M13 helper phage, and purified by PEG precipitation. Purified phages expressing Fabs were selected for antigen binding as described. Phage from the second round was diluted and infected into TG1 cells. Polyclonal pools of phage output from two rounds of panning were tested by ELISA to confirm that the pools contained FAP-specific phage. Variable heavy and light chain regions were sequenced from single infected bacterial colonies using a rolling circle amplification and standard Sanger sequencing.

[0458] 3.4. Antibody expression

[0459] Unique variable heavy and light chain pairs from all discovery techniques were cloned into vectors designed to express full-length antibodies as IgGs containing LALA silencing mutations in HEK293 cells under the control of a CMV promoter. Antibody expression vectors were complexed with polyethylenimine and transfected into HEK293 cultures. After 5 days of shaking at 37°C in 293 cell culture media, antibodies were captured on agarose-based protein A resin. After several stringent washes, antibodies were eluted in glycine solution, pH 3, neutralised with HEPES, pH 9, and buffer exchanged into PBS. The purity of purified proteinswas confirmed by SEC-HPLC and CE-SDS analysis. Each antibody was assigned a unique clone number.

[0460] 3.5. Binding of anti-FAP antibodies to FAP-Overexpressinq HEK293 cell line

[0461] The binding of antibodies to human FAP expressed on transfected HEK293 cells was measured by FACS. In addition, binding to parental HEK293 was tested to confirm antibody specificity. In short, the cells were resuspended in the FACS binding buffer and live / dead efluro450 viability dye was added at a 1:1000 dilution to the cell suspension, followed by incubation at room temperature in the dark for 30 minutes. The cells were then diluted to 2 e5 / ml, and 100 ul was added to wells of a 96-well Il-bottom plate as required. For generating an EC50, a 100 nM stock was prepared and then diluted 3 fold serially across 12 points. The cells were pelleted by centrifugation, and the supernatant was discarded, followed by the addition of a series of dilutions of anti-FAP antibodies and incubation for 45 minutes at 4°C in the dark. Cells were washed two times with PBS with 3% (w / v) BSA before adding 60 ul of fluorescently labelled secondary antibody (goat anti-human IgG) diluted to 1:200 in PBS. Incubation continued at 4C for at least 30 minutes, followed by two washes in PBS pH 7.4. Cells were either fixed with 2% PFA (30 minutes at 4°C) followed by washout and storage in 100 ul of PBS or immediately analysed on a Flow Cytometer. The data was analysed using FloJo V10 and GraphPad Prism. No binding to the HEK parental cell line was detected for any tested clones, confirming the specific interaction between anti-FAP antibodies and the FAP receptor. The results are summarised in Table 12. Figure 6 demonstrates dose-dependent binding to FAP overexpressed on the HEK293 cell line.

[0462] Table 12 EC50 values for binding of anti-FAP antibodies to FAP-HEK293 cell line

[0463]

[0464] 3.6. Binding kinetics of anti-FAP antibodies to human and cynomolqus FAP

[0465] Binding of anti-FAP antibodies to human and cynomolgus FAP was investigated by Surface Plasmon Resonance (SPR) on Carterra LSA. An EC30M chip was used to capture the test antibodies. The chip was activated for bioconjugation using EDC and sNHS in MES pH 5.5 to prepare the chip for amine coupling. Goat anti-human IgG Fc antibody was then captured tothe chip via incubation for 10 minutes. Excess conjugation sites were quenched with 1M Ethanolamine pH 8. To measure monovalent binding kinetics, antibodies were printed / spotted onto the chip and allowed to bind to the goat antibody, followed by subsequent washing. After the preparation, the printed chip was exposed to repeated rounds of association and dissociation with increasing analyte concentrations. In this work, recombinant proteins (Human FAP (Aero) or cyno FAP His (Aero)) were serially diluted across a range starting at 1500 nM with 3-fold dilutions and 10 points. The curves were fitted to global fit in a 1:1 binding model to obtain KD values. The binding affinity to both human and cynomolgus FAP is reported in Table 13. Tested antibodies demonstrated comparable affinities to human and cynomolgus FAP.

[0466] Table 13 Binding affinity of anti-FAP antibodies to human and cynomolgus FAP

[0467]

[0468] 3.7. Epitope competition assay

[0469] A high-throughput epitope binning experiment was done on real-time label-free biosensors (Carterra LSA) to sort a panel of mAbs into bins based on their ability to block one another for binding to the antigen. In a pairwise epitope binning analysis, antigen and antibody 2 (analyte antibody) were sequentially applied to the sensor chip (HC200M) covalently pre-loaded with antibody 1 (ligand antibody). An increase in response upon exposure to the analyte antibody indicates non-competition between the two antibodies, whereas a lack of change in the signal indicates competition. Antibodies in the test set that have the same blocking profiles towards others are grouped into one bin. Community network plots were used to explore the clustering of mAbs that share similar but not necessarily identical competition profiles. Rather than relying strictly on the sandwiching / blocking assignments in the heat map, as the Bin network plots do, hierarchical clustering was applied to the sorted heat map to generate dendrograms, which progressively group mAbs. Community bins for tested anti-FAP antibodies are shown in Table 14.

[0470] Table 14The results confirmed a diverse panel of anti-FAP antibodies, which were taken forward for further evaluation.Table 14 Community bin defined by epitope competition assay on Carterra

[0471]

[0472] 3.8. Sequence optimisation of anti-FAP antibodies

[0473] The variable region of an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Therefore, to decrease the immunogenicity of the variable regions of antibody (VH and VL), the framework residues were reverted to corresponding human germline sequences. In short, different mutant variants were generated for each clone, which were then compared for their capacity to bind recombinant FAP on Octet. In each case the clone which was closest to human germline sequences was selected for further evaluation.

[0474] 4. Example 4 Generation of bispecific antibodies targeting FAP and LTBR

[0475] 4.1. Production of human and mouse-specific FAP / LTBR bispecific antibodies

[0476] The anti-LTBR VHHs identified in Example 1 were shown to have agonistic activity in an NFKB reporter assay when cross-linked by an external cross-linking agent (e.g. an anti-His antibody or when formatted as a bivalent molecule on a human lgG1 Fc region). These clones presented the most favourable functional properties for bispecific antibody evaluation.

[0477] A highly diverse panel of anti-FAP antibodies was identified in the previous examples with a range of affinities that bind to different huFAP regions.

[0478] The human cynomolgus cross-reactive anti-LTBR agonist VHHs (as described in Example 1) or anti-LTBR agonist antibodies (as described in Example 2) and anti-FAP antibodies (as described in Example 3) were used to generate constructs comprising a FAP binding agent and an LTBR binding agent. These constructs were FAP / LTBR bispecific agonistic antibodies. Different formats of bispecific antibodies were prepared with either bivalent or monovalent binding for LTBR and monovalent or bivalent binding for FAP, i.e. FAP: LTBR 1:1 format, 2:1 format, or 2:2 format. Schematic illustrations of the different bispecific formats produced are represented in Figures 7a-7d. These FAP / LTBR bispecific agonistic antibodies comprised one or more FAP binding agents and one or more LTBR binding agents variously in the form of (a) one or two anti-FAP Fab region(s) and (b) one or two anti-LTBR VHH(s) or one or two anti-LTBR scFv(s). Each construct was assigned a unique clone number.The variable regions of human-specific constructs were cloned into expression vectors containing the constant region of human lgG1. In some cases, to generate either 2:1 or 2:2 formats, scFvs containing sequences of variable regions anti-LTBR antibodies (Example 2) were fused to the C-terminus of an Fc by a G4S linker (SEQ ID NO: 3). As scFv fragments could have stability issues, an additional disulfide bond was introduced in the VH44:VL100 position (Weatherill et al., 2012). A well-established knob-into-holes strategy was employed to ensure the correct assembly of heavy chains (Merchant et al., 1998). In addition, to reduce the binding to FcyRs, LALA (Leu234Ala and Leu235Ala) or PGLALA (Pro329Gly, Leu234Ala, and Leu235Ala), Schlothauer et al. 2016, were introduced in the Fc region.

[0479] To produce murine surrogate antibodies, the variable regions of mouse-specific constructs were cloned into expression vectors containing the constant region of mouse lgG2a or mouse lgG1. To ensure the correct assembly of light and heavy chains, published mutations in CH1 / CL to promote sufficient light chain pairing and electrostatic steering mutations into the CH3 domain were introduced to ensure specific heavy chain pairing (Wang et al., 2020) were introduced. Like human constructs to prevent the binding to FcyRs, PGLALA (Pro329Gly, Leu234Ala, and Leu235Ala, Schlothauer et al. 2016) were introduced in the Fc region. In some cases, to generate either 2:1 or 2:2 formats, scFv containing sequences of variable regions 5G11 were fused to the C-terminus of an Fc by a G4S linker (SEQ ID NO: 3). As scFv fragments could have stability issues, an additional disulfide bond was introduced in the VH44:VL100 position (Weatherill et al., 2012).

[0480] In parallel, (i) a monospecific control antibody, (ii) anti-human LTBR control clones BHA10, and CBE-11 (as disclosed in WQ2004002431), (iii) anti-FAP clone 28H1 (as disclosed in US10577429B2) and (iv) anti-mouse LTBR control (clone 5G11) were cloned into expression vectors containing either the constant region of human IgG 1 , rat lgG2a or mouse lgG1. PGLALA mutations (Pro329Gly, Leu234Ala, and Leu235Ala, (Schlothauer et al., 2016)) were introduced in the constant region of the human lgG1 / mouse lgG2a heavy chains to abrogate binding to Fc gamma receptors.

[0481] In addition, FAP / LTBR bispecific comparative example antibodies were produced, clones: P1AH5886, P1AG5459 and P1AG5461 (as disclosed in WO2023 / 117834A1).

[0482] The bispecific and monospecific controls were expressed by transient transfection of CHO suspension cells. Antibodies were purified from cell culture supernatants by affinity chromatography using protein A resin and followed by one or two chromatography steps: preparative size exclusion (SEC) and / or ion exchange to enrich the heterodimer.

[0483] The purity and heterogeneity of the samples were assessed by SEC-HPLC and mass spectrometry. All samples were confirmed to have a monomer content of >95% and contain <10 % impurities prior to functional read-out.The resulting bispecific and monospecific antibodies are listed in Table 15, and the sequences of listed clone variable regions can be found in Table 16.

[0484] Table 15 List of bispecific antibodies and monospecific controls

[0485]

[0486]

[0487]

[0488] All 1:1 format constructs detailed in the table above included a truncated hinge region in HC1 (DKTHTCPPCP, SEQ ID NO: 203) and a native hinge region in HC2 (EPKSCDKTHTCP, SEQ ID NO: 204). The constructs of other formats included native hinge regions in the HC (EPKSCDKTHTCP, SEQ ID NO: 204).

[0489] Table 16 Amino acid sequences of variable regions of FAP and LTBR clones

[0490]

[0491]

[0492]

[0493]

[0494]

[0495]

[0496] Table 17A. Amino acid sequences of CDRs of FAP and LTBR clones. For VHHs, HCDR sequences are provided. For paired VH / VLs clones HCDR sequences are provided in the first row for each clone, followed by LCDR sequences in the second row for the same clone.

[0497]

[0498]

[0499]

[0500] The heavy chain and light chain polypeptide sequences for Clones 4.1 to 4.10 are as follows, wherein for homodimers, HC = heavy chain, LC = light chain. In the case of heterodimers, polypeptide sequences for the first (HC1) and second (HC2) heavy chains are provided in addition to the LC polypeptide sequence.

[0501] Clone 4.1 HC1: SEQ ID NO: 175, HC2: SEQ ID NO: 176, LC: SEQ ID NO: 177Clone 4.2 HC1: SEQ ID NO: 178, HC2: SEQ ID NO: 179, LC: SEQ ID NO: 180 Clone 4.3 HC1: SEQ ID NO: 181, HC2: SEQ ID NO: 182, LC: SEQ ID NO: 183

[0502] Clone 4.4 HC1: SEQ ID NO: 184, HC2: SEQ ID NO: 185, LC: SEQ ID NO: 186

[0503] Clone 4.5 HC1: SEQ ID NO: 187, HC2: SEQ ID NO: 188, LC: SEQ ID NO: 189

[0504] Clone 4.6 HC1: SEQ ID NO: 190, HC2: SEQ ID NO: 191, LC: SEQ ID NO: 192

[0505] Clone 4.7 HC1: SEQ ID NO: 193, HC2: SEQ ID NO: 194, LC: SEQ ID NO: 195

[0506] Clone 4.8 HC: SEQ ID NO: 196, LC: SEQ ID NO: 197

[0507] Clone 4.9 HC: SEQ ID NO: 198, LC: SEQ ID NO: 199

[0508] Clone 4.10 HC1: SEQ ID NO: 200, HC2: SEQ ID NO: 201, LC: SEQ ID NO: 202

[0509] Table 17B. Alternative amino acid sequences of CDRs or LTBR VHHs. Alternative CDRs resulting from combination of nearly identical individual CDRs are provided with their amino acid sequence and SEQ ID NO:.

[0510]

[0511] 4.2. Binding of FAP / LTBR bispecific antibodies to human FAP and LTBR-expressinq cells To test the binding of FAP / LTBR bispecific antibodies to human FAP on cells, a binding assay using a FAP-over expressing CHO-K1 recombinant cell line (BPS, cat no. 79947) or a FAP overexpressing transient HEK293 cell line was used. To test the binding of FAP / LTBR bispecific antibodies to human LTBR on cells, a binding assay using the endogenously LTBR-expressing A549 (ATCC, cat. no CCL-185) or A375 tumor cell line was used. In this experiment, an LTBR monospecific antibody (BHA10) was used as a control.

[0512] In some cases, the secondary antibody interfered with detection; therefore, the bispecific antibodies were labelled directly with Alexa Fluor® 647 Conjugation Kit (Fast) - Lightning-Link® (Abeam, cat no ab269823), following manufacturer instructions. In that case, the staining protocol was as described below, apart from adding a secondary antibody.In short, 1x106cells were resuspended in the FACS buffer (1% BSA, 0.1% Sodium Azide in PBS), and 50 pl was added to each well (5x104cells / well) of a 96-U bottom plate. The cells were washed once and resuspended in 50pl / well of FACS buffer containing a 1 :3, 11 -point series dilutions of constructs 4.1-4.6 and clones 4.8-4.9 followed by 1h incubation at 4°C. After extensive washing, cells were further stained with a secondary detection antibody, R-Phycoerythrin-conjugated AffiniPure Goat Anti-Human IgG, Fey Fragment Specific (Stratech, cat.no 109-115-098-JIR), for 1h incubation at4°C. Next, cells were washed and resuspended in FACS buffer and read on Beckman Coulter CytoFLEX. The data were analysed using FloJo V10 and GraphPad Prism v9. Clone 4.7 was tested for binding to A375 (LTBR positive cell line) and the HEK293-FAP overexpressing cell line, as described in examples 2.4 and 3.5. The EC50 values can be found in Table 18.

[0513] Table 18 EC50 values for binding of FAP / LTBR bispecific antibodies to LTBR and FAP positive cell lines

[0514]

[0515] NP = Not reaching a plateau, EC50 cannot be evaluated.

[0516] As demonstrated in the table above, all the FAP / LTBR bispecific antibodies bind to LTBR-positive cells with a lower potency than control BHA10, which has an EC50 value in the pM range. As discussed above, a lower binding affinity may be beneficial in the context of an agonistic antibody.4.3. NFKB activation of HepG2 reporter cells expressing human LTBR co-cultured with a FAP-overexpressing CHO cell line

[0517] For FAP-mediated cross-linking, a FAP-CHO-K1 recombinant cell line (BPS, cat no.

[0518] 79947) was used.

[0519] To assess the ability of the FAP / LTBR bispecific antibodies to activate LTBR conditionally, the molecules were tested in a NFKB luciferase reporter HEPG2 assay (Signosis, cat.no SL0017-FP) in the presence or absence of a CHO- overexpressing FAP cell line (FAP-CHO-K1). Clustering endogenous LTBR on HepG2 cells leads to activation of endogenous NFKB and translocation from the cytoplasm and nucleus, where it binds to the promoter region and induces luciferase expression. After that, a luciferase substrate / lysis reagent mix (ONE-GLO EX Promega) is added to cells, which allows guantification in the bioluminescence reaction, where enzymatic activity is proportional to luciferase expression.

[0520] The monospecific anti-LTBR agonist CBE-11 was used as a positive control.

[0521] HepG2 NFKB Lc (30000 cells / well) were either co-seeded with FAP overexpressing CHO cells (15000 cells / well) or seeded alone in their growth media. The 1:10 series dilution of bispecific antibodies and controls was added to the cells and incubated for 6h at 37°C, 5% CO2, prior to the addition of detection reagents. After 6 h ONE-Glo Ex Solution (Promega, cat. no E8120) was added to the well and incubated for 10 min before being read on a BMG Pherastar FSX plate reader. Background-corrected RLU values were plotted against stimulant concentration for all conditions tested in GraphPad Prism, and the EC50 value can be found in Table 19.

[0522] Table 19 EC50 values of HepG2 NFKB activation-induced luciferase activity

[0523]

[0524]

[0525] The results confirmed that the FAP / LTBR bispecific antibodies containing a monovalent LTBR arm (1:1 and 2:1 format) activate LTBR in a FAP-dependent way, with minimal or no activity in the absence of FAP, as demonstrated by representative data in Figures 8a, be (monoculture), 8b and 8d (co-culture) performed in the HEPG2 Reporter assay. This is in contrast to the control monospecific antibody CBE-11, which activates HepG2 cells to a similar extent in the presence or absence of FAP-positive cells.

[0526] The FAP / LTBR bispecific antibodies (clones 4.8 and 4.9) and comparative example antibody clone P1AH5886 (WO2023117834) containing two LTBR binding sites retained more LTBR activity in the absence of FAP. In contrast to monospecific control, the activity is further increased in the presence of FAP-positive cells. As discussed above, this conditional activation phenomenon should allow for reduced toxicity due to activation of LTBR-positive cells in the presence of FAP in the tumour (and not in tissues where FAP is not expressed).

[0527] Due to the complexity of the bispecific, the minimal activity in the monoculture could be due to the presence of some residual impurities in the sample.

[0528] 4.4. In vitro FAP / LTBR bispecific antibody activity in a chemokine induction assay using tumor cell line HCC1187 cultured with FAP-positive primary human breast CAFs An activation assay measuring chemokine secretion was used to evaluate the ability of the LTBR / FAP bispecific antibodies to conditionally cluster and activate LTBR endogenously expressed on cells (tumor cell line HCC1187).

[0529] The primary human breast CAFs were used as a source of FAP to determine the biological activities of the LTBR / FAP bispecific antibodies in a physiologically relevant setting.

[0530] The expression of LTBR on HCC1187 and primary human breast CAFs was confirmed by flow cytometry.

[0531] HCC1187 (20000 / well) were co-seeded either with primary human breast CAFs, isolated from cancer patients (20000 / well) or alone in their growth medium. A 1:10, 7-point serial dilution series in duplicates of bispecific antibodies and controls were prepared in assaymedium as 2-fold concentration stocks, added to the cells, and incubated for 24h at 37°C, 5% CO2.

[0532] After incubation, the supernatants were collected and cleared by centrifugation, and the chemokine level was determined by CCL19 DuoSet ELISA (R&D system, cat. no DY361) following the manufacturer's instructions. Absorbance was read on the BMG Pherastar FSX plate reader. OD corrected values (450-540nm) for the standard curves were analysed in Prism, according to the manufacturer's recommendation, in order to interpolate cytokine concentrations for the test wells. Dose-response curves were plotted using GraphPad Prism Version 9, applying non-linear fits (log(agonist) vs response (variable slope- four parameters).

[0533] The FAP / LTBR bispecific antibodies induce secretion of CCL19 on HCC1187 cells in a dose-dependent manner (Figures 9a-9f). These data show that LTBR activation occurs only upon clustering LTBR in the presence of FAP-positive cells (primary human breast CAFs, in that case) for molecules with a single LTBR binding site. Similarly, as shown in example 4.3, bispecific constructs with two binding sites for LTBR retained some function in the monoculture. EC50 values can be found in Table 20.

[0534] Surprisingly, the FAP / LTBR bispecific antibodies (clones 4.1-4.6) showed much greater potency in the co-culture compared to the control antibody CBE-11 which could be explained by lower binding affinity to LTBR-positive cells. As reported in the literature, low-affinity anti-4-1BB, CD40 monoclonal antibodies mediate higher agonistic activity, suggesting that low-affinity antibody-induced agonism is a conserved feature among TNFRs (Yu et al., Nature 2023).

[0535] Table 20 EC50 values of upregulation of CCL19 on HCC1187 in the presence and absence of FAP positive primary human breast CAFs

[0536]

[0537]

[0538] 4.5. In vitro FAP / LTBR bispecific antibody activity in an ICAM-1 upregulation assay on human breast CAFs

[0539] It has been reported that stimulation of LTBR leads to the activation of canonical NF-KB1-RelA and the alternative NF-KB2-RelB pathways (Benezech et al. 2012), which result in the induction of chemokines CXCL13, CCI21, and CCL19 and adhesion molecules ICAM1 and VCAM1.

[0540] A monoculture assay with primary human breast CAFs was performed to verify if activation of LTBR with the FAP / LTBR bispecific antibodies leads to upregulation of adhesion molecules. ICAM-1 expression was evaluated by immunofluorescence staining.

[0541] The expression of both targets on primary human breast CAFs was confirmed by flow cytometry using control antibodies.

[0542] Human breast CAFs co-expressing both receptors (FAP and LTBR) were seeded at 20k / well in a 96-well plate and left to adhere for 24h. Following this, a 7-point, 10-fold dilution series of bsAb and control LTBR agonist were prepared as x10 stocks in culture medium, added to cells and incubated at 37°C, 5% CO2 for 24h. After stimulation, the cells were fixed by the addition of 100 ul 8% formaldehyde (4% final) for 20 minutes and washed with PBS. Cells were incubated with blocking buffer (3% BSA / 0.3% Triton X-100 in PBS (W / V / V)) for 1h at RT to prevent non-specific binding. For immunostaining, cells were incubated with rabbit monoclonal affinity purified antibody to ICAM-1 (Abeam, cat. no ab282575 diln 1 / 500 in 3% BSA in PBS (W / V)) O / N at 4°C. Cells were then washed three times with 100 ul wash buffer (10 mM sodium phosphate, 0.15 M NaCI, 0.05% Tween-20 buffer) followed by incubation with secondary antibody AF546 conjugated anti-Rabbit IgG (H+L) polyclonal antibody donkey IgG for 1h at RT (Invitrogen, cat. no A10040, 1 / 500 diln in 3% BSA in PBS (W / V)). In parallel, nuclei were counterstained with the nuclear stain Hoechst 33342 (Invitrogen, H3570, 1 / 10000 diln). Cells were washed a further three times with wash buffer before being transferred into PBS for image acquisition. Fluorescence images were captured on Cell Insight CX7 HCS platform. Image acquisition and analysis were performed with Thermo Scientific HCS studio: cellomics scan V6.6.2 software using the Spot Detector Bioapplication V4. This bioapplication measures fluorescence intensities on a single cell level, allowing rapid protein expressionanalysis. Data are presented as average fluorescence intensity per cell (ICAM1 expression) minus intensity from untreated conditions (baseline subtracted) and plotted with GraphPad Prism Version 9.

[0543] Figure 10a and 10b show that the FAP / LTBR bispecific antibodies, apart from comparative example P1AH5886 (WO2023117834) and clone 4.10, achieved potent LTBR activation measured by upregulation of ICAM-1 on primary fibroblasts. Comparative example P1AH5886 did not achieve potent LTBR activation in this assay.

[0544] This led to the conclusion that the anti-FAP / LTBR bispecific antibodies can function in cis by co-engaging FAP and LTBR on the same cells, as well as in trans by targeting LTBR expressed on one cell (e.g. stroma) and FAP on adjacent cells, as demonstrated in Figure 9a-9d. The data is summarised in Table 21.

[0545] The FAP / LTBR bispecific antibodies in 2:2 format (clones 4.8 and 4.9) have the lowest EC50 suggesting a higher potency than the FAP / LTBR bispecific antibodies in 2:1 or 1:1 format. However, the activity of 2:2 formats, as shown in the previous examples, could be FAP-dependent and FAP-independent, as the bivalent LTBR format retained some agonistic function (Examples 4.3 and 4.4).

[0546] Surprisingly, the FAP / LTBR bispecific antibody clone 4.10 is unable to upregulate ICAM-1 in circumstances where FAP and LTBR are expressed on the same cell, even though it contains the same functional LTBR binding agent as clones 4.1 , 4.3, 4.4 and 4.5. However, clone 4.10 contains a different FAP binding agent (binding to a distinct epitope). This distinct epitope appears to be instrumental in achieving ICAM-1 upregulation in a FAP / LTBR bispecific antibody.

[0547] Wishout wishing to be bound by theory, this observation may indicate that ICAM-1 upregulation can be achieved by binding to both FAP and LTBR simultaneously on the same cell (‘cis’) (while binding to FAP on a first cell and binding to LTBR on a second cell simultaneously (‘trans’) may not give rise to ICAM-1 upregulation). This FAP epitope may facilitate binding to both FAP and LTBR simultaneously on the same cell.

[0548] Table 21 EC50 values for ICAM1 upregulation in CAFs monoculture assay

[0549]

[0550]

[0551] 4.6. In vitro FAP / LTBR bispecific antibody activity in a chemokine induction assay using tumor cell line RPMI-7951

[0552] To investigate if the FAP / LTBR bispecific antibodies can activate LTBR efficiently on double-positive cells in cis (such as Fibroblast Reticular cells which are essential for forming and maintaining TLS), a monoculture assay was performed with tumor cell line RPMI-7951. The expression of FAP and LTBR was confirmed by flow cytometry. Briefly, RPMI-7951 were seeded at 50 K / well into 96- V bottom plates in culture media. A 7-point, 10-fold dilution series for the FAP / LTBR bispecific antibodies and control LTBR agonist antibody CBE11 were prepared as x10 stocks in culture medium, added to cells and incubated at 37°C, 5% CO2 for 24h. After 24h, plates were centrifuged, and the supernatant was collected. Following the manufacturer's instructions, the chemokine CCL5 level in the supernatant was determined by human CCL5 DuoSet ELISA (R&D system, cat.no DY278). Absorbance was read on the BMG Pherastar FSX plate reader. OD corrected values (450-540nm) for the standard curve were analysed in GraphPad Prism, according to the manufacturer's recommendation, in order to interpolate cytokine concentrations for the test wells. Dose-response curves were plotted using GraphPad Prism Version 9, applying non-linear fits (log(agonist) vs response (variable slope-four parameters). The data is summarised in Table 22.

[0553] Figure 11a and 11b show potent LTBR activation with the FAP / LTBR bispecific antibodies and the LTBR agonist control antibody, resulting in pro-inflammatory CCL5 chemokine upregulation. This is in contrast to the comparative example bispecific antibody P1AH5886, which does not lead to the secretion of CCL5 on RPMI-7951. From the results obtained, it can be concluded that the FAP / LTBR bispecific antibodies can sufficiently cluster LTBR even if both receptors are co-expressed. Similarly, as was described in Example 4.5, clones 4.8 and 4.9 induced significantly higher levels of CCL5 compared to the FAP / LTBR bispecifics in 1:1 or 2:1 format.

[0554] Taken together, these data confirm that the FAP / LTBR bispecific antibodies can activate LTBR in cis sufficiently (when both receptors co-expressed) and in trans byneighbouring cells in a FAP-dependent manner, leading to the induction of chemokines, such as CCL19 and CCL5 and upregulation of adhesion molecules, such as ICAM-1.

[0555] Table 22 EC50 values for chemokine induction in RPMI-7951

[0556]

[0557] 4.7. In vitro FAP / LTBR bispecific antibody trans activity in a chemokine induction assay using low-FAP expressing cells (RPMI-7951) when in co-culture with HCC-1187 The potency of the FAP / LTBR bispecific antibodies was determined in the presence of a low-FAP expressing cell line (RPMI-7951). The expression level was confirmed by flow cytometry, and FAP expression was around 4-fold lower than on previously used primary human breast CAFs (Example 4.4). Since RPMI-7951 and HCC1187 secrete unigue chemokines upon LTBR stimulation, as described in examples 4.4 and 4.6, it was possible to evaluate cis and trans activation simultaneously.

[0558] Since HCC1187 are single positive cells (LTBR is present on the cell surface, but not FAP), for FAP / LTBR bispecific antibodies to cluster LTBR and subseguently activate downstream signalling pathways, these bispecific antibodies must co-engage FAP expressed on RPMI-7951 cells and LTBR expressed on HCC1187 cells. If CCL19 secretion is observed in this assay upon adding FAP / LTBR bispecific antibodies, this suggests trans activation of LTBRoccurs. Cis activation was characterised in parallel as RPMI-7951 cells express both FAP and LTBR and secrete CCL5 upon LTBR stimulation.

[0559] In short, RPMI-7951 cells (20,000 cells / well) were seeded in 96-well plates for RPMI-7951 monoculture and co-culture plates alongside media-only plates for HCC1187 monoculture. Plates were then incubated for 24 hours at 37°C and 5% CO2 before adding HCC1187 cells and compounds. The next day, HCC1187 cells (20,000 cells / well) were seeded into media-only plates, and RPMI-7951 co-culture plates were prepared on day 1. A 7-point, 10-fold dilution series for the FAP / LTBR bispecific antibodies and control LTBR agonist antibody (CBE-11) were prepared as x10 stocks in culture medium and added to the cell plates. After 24h, plates were centrifuged, and the supernatant was collected. Following the manufacturer's instructions, the chemokine levels of CCL5 and CCL19 were determined in the supernatant by human CCL5 DuoSet ELISA (R&D system, cat.no DY278) and human CCL19 DuoSet ELISA (R&D system, cat.no DY361). Absorbance was read on the BMG Pherastar FSX plate reader. OD corrected values (450-540nm) for the standard curve were analysed in GraphPad Prism, according to the manufacturer's recommendation, in order to interpolate cytokine concentrations for the test wells. Dose-response curves were plotted using GraphPad Prism Version 9, applying non-linear fits (log(agonist) vs response (variable slope- four parameters). The data are provided in Table 23.

[0560] Table 23 EC50 values for cis and trans activation in the HCC1187:RPMI-7951 co-culture assay

[0561]

[0562]

[0563] Figure 12a suggests that the FAP / LTBR bispecific antibodies in 1:1 format (see bispecific antibodies 4.1 , 4.2 and 4.3) can induce the secretion of CCL19 as a result of FAP-mediated activation of LTBR, even in the presence of low levels of FAP.

[0564] Figure 12b confirms the previous result described in Example 4.6 that the FAP / LTBR bispecific antibodies can cluster LTBR sufficiently on double-positive cells (FAP and LTBR positive), which leads to dose-dependent CCL5 secretion.

[0565] Taking together these data and Example 4.4 suggests that the potency of the FAP / LTBR bispecific antibodies correlates with FAP expression density on cells.

[0566] 4.8. The binding affinity of LTBR / FAP bispecific antibodies to human and cynomolgus recombinant LTBR and FAP by Octet

[0567] To confirm species cross-reactivity, binding affinity to human and cynomolgus recombinant proteins for both targets was measured by Octet. Such cross-reactivity is advantageous, as it allows dosing and safety testing of the antibody molecules to be performed in cynomolgus monkeys during preclinical development.

[0568] Briefly, Biotinylated hLTBR-His-Avitag (Aero, cat no LTR-H82E9-25pg) was loaded on SA Streptavidin biosensors (Fortebio) in kinetics buffer (ForteBio) followed by a 2-fold dilution series starting at 50 nM of FAP / LTBR bispecific antibodies. Binding kinetics were studied in a 1x Kinetics buffer where the association was allowed for 100 seconds, followed by dissociation for 300 seconds. Data generated were referenced by subtracting a parallel buffer blank, the baseline was aligned with the y-axis, inter-step correlation by alignment against dissociation was performed, and the data were smoothed by a Savitzky-Golay filter in the data analysis software. The processed data were fitted using a 1 :1 Langmuir binding model with RA2 as a measurement of fitting accuracy. The KD and RA2 values can be found in Table 24.

[0569] A 2-fold series dilution starting at 50 nM of cynomologus recombinant LTBR was immobilised on the surface of ARG biosensors (Fortebio), followed by FAP / LTBR bispecific at 25nM. Binding kinetics were studied in a 1x Kinetics buffer where the association was allowed for 180 seconds, followed by dissociation for 100 seconds. Sensor tips were regenerated using ethylene glycol. Data generated were referenced by subtracting a parallel buffer blank, the baseline was aligned with the y-axis, inter-step correlation by alignment against dissociation was performed, and the data were smoothed by a Savitzky-Golay filter in the data analysis software. The processed data were fitted using a 1 :1 Langmuir binding model with RA2 as a measurement of fitting accuracy. The KD values can be found in Table 24.Binding kinetics to both targets for clone 4.7 was carried out as described in Example 2.6 and 3.6, and the KD value can be found in Table 24.

[0570] Binding to recombinant FAP proteins was investigated by Bio-Layer Interferometry (BLI) using the Octet instrument. Using standard amine coupling chemistry, an anti-Fc antibody was immobilised on the surface of ARG biosensors (Fortebio). The FAP / LTBR bispecific antibodies were loaded on the previously coated surface with anti-Fc capture antibody, followed by a 2-fold series dilution starting at 50 nM of either cyno FAP (Aero, cat no FAP-C82Q5) or human FAP (Aero, cat no FAP-H82Q6). Binding kinetics were studied in a 1x Kinetics buffer where the association was allowed for 200 seconds, followed by dissociation for 400 seconds. Sensor tips were regenerated using 10 mM Glycine pH 2.0. Data generated were referenced by subtracting a parallel buffer blank, the baseline was aligned with the y-axis, inter-step correlation by alignment against dissociation was performed, and the data were smoothed by a Savitzky-Golay filter in the data analysis software. The processed data were fitted using a 1 :1 Langmuir binding model with RA2 as a measurement of fitting accuracy. The Results are summarised in Table 24. Avidity was observed as FAP is naturally a dimeric antigen, and the limited detection by Octet with these molecules has been reached. Species cross-reactivity has been confirmed for all test samples.

[0571] Table 24 Kinetics parameters for FAP / LTBR bispecific antibody binding to human and cyno LTBR and FAP measured by Octet

[0572]

[0573] *values outside instrument specifications4.9. In vitro surrogate FAP / LTBR bispecific antibody activity in a chemokine induction assay using tumor cell line 4T1 cultured with FAP positive CHO-overexpressing line Since the anti-FAP antibody (clone 28H1) selectively binds to both human and murine antigens, it is possible to use the CHO-human FAP-K1 line as a source of FAP in the following assay.

[0574] To assess the ability of surrogate mouse FAP / LTBR bispecific antibodies to activate LTBR conditionally, FAP / LTBR bispecific antibodies and monoclonal LTBR agonist antibodies were tested in 4T 1 (ATCC, cat no. CRL-2539) in the presence or absence of FAP-CHO-K1 cell line. In addition, the monospecific monoclonal anti-LTBR antibodies were tested in the presence or absence of a cross-linking agent to induce sufficient LTBR clustering. Anti-Fc antibody was used as a cross-linking agent.

[0575] Briefly, 4T 1 (20000 / well) were co-seeded either with FAP-CHO-K1 (20000 / well) or alone in their growth medium. A 1:10, 7-point serial dilution series in duplicates of bispecific antibodies and controls were prepared in assay medium as 10-fold concentration stocks, added to the cells, and incubated for 24h at 37°C, 5% CO2.

[0576] After incubation, the supernatants were collected and cleared by centrifugation, and the chemokine level was determined by CCL5 DuoSet ELISA (DY478-05) following the manufacturer's instructions. Absorbance was read on the BMG Pherastar FSX plate reader. OD corrected values (450-540nm) for the standard curves were analysed in Prism, according to the manufacturer's recommendation, in order to interpolate cytokine concentrations for the test wells. Dose-response curves were plotted using GraphPad Prism Version 9, applying nonlinear fits (log(agonist) vs response (variable slope- four parameters). EC50 values can be found in Table 25.

[0577] Table 25 EC50 values for chemokine induction in 4T1

[0578]

[0579]

[0580] Figures 13a and 13b show that the surrogate FAP / LTBR bispecific antibodies, in contrast to the non-targeted LTBR bispecific (clone 4.13), conditionally activate LTBR, with limited activity in the absence of FAP. The results show that the FAP-dependent activity of clone 4.11 is more potent, demonstrated by lower EC50 values and increased max activity compared to cross-linked monospecific LTBR agonist antibody (clone 4.14).

[0581] Figure 13c shows that monospecific LTBR agonist antibody 5G11, regardless of the Fc backbone, demonstrates the same potency in the 4T 1 monoculture assay, resulting in dosedependent secretion of CCL5 (results with clone 4.14 and 4.15).

[0582] Activation of LTBR using the monospecific LTBR agonist antibody was achieved in the absence of FAP, and it could potentially lead to toxicity due to the broad expression of LTBR in normal tissue. A higher activation level of LTBR was achieved when the monospecific antibody was cross-linked by anti-Fc antibody.

[0583] In contrast to surrogate FAP / LTBR bispecific antibody (clone 4.12) Figure 13d shows that comparative surrogate bispecific molecules (FAP targeted P1AG5459 and non-targeted P1AG55461) activate LTBR to the same extent as the monospecific LTBR agonist control antibody (clone 4.14) in 4T1 monoculture assay without crosslinking. The potency of FAP targeted comparative molecules is being enhanced upon co-culture with FAP positive cell line (Figure 13e) in contrast to non-targeted control, which demonstrated comparable activity in the mono and co-culture.

[0584] 4.10. In vitro activity of mouse LTBR / FAP bispecific agonist antibody in primary mouse lung fibroblasts

[0585] A monoculture assay with primary mouse lung fibroblasts (PMLF) was employed to understand if stimulation with mouse FAP / LTBR bispecific antibody agonists of fibroblasts results in the upregulation of adhesion molecules, such as ICAM-1.Primary mouse lung fibroblasts (PMLF) were derived from naive (untreated) mice. PMLF were seeded (20k / well) in 96 well plates and left to adhere for 24h. Following this, a 7-point, 10-fold dilution series of FAP / LTBR bsAb and control LTBR agonist antibody were prepared as x10 stocks in culture medium, added to cells and incubated at 37°C, 5% CO2 for 48h. After stimulation, the cells were fixed by the addition of 100 ul 8% formaldehyde (4% final) for 20 minutes and washed with PBS. Cells were incubated with blocking buffer (3% BSA / 0.3% Triton X-100 in PBS (WA / / V)) for 1h at RT to prevent non-specific binding. For immunostaining, cells were incubated with rat monoclonal affinity purified antibody to ICAM-1 (Biolegend, cat no 116102) diln 1 / 100 in 3% BSA in PBS (W / V)) O / N at 4°C. Cells were then washed three times with 100 ul wash buffer (10 mM sodium phosphate, 0.15 M NaCI, 0.05% Tween-20 buffer) followed by incubation with secondary antibody anti-Rat IgG (H+L) AF488 pAb, Donkey IgG for 1h at RT (Invitrogen, cat. no A-21208, 1 / 500 diln in 3% BSA in PBS (W / V)). In parallel, nuclei were counterstained with the nuclear stain Hoechst 33342 (Invitrogen, H3570, 1 / 10000 diln). Cells were washed a further three times with wash buffer before being transferred into PBS for image acquisition. Fluorescence images were captured on Cell Insight CX7 HCS platform. Image acquisition and analysis were performed with Thermo Scientific HCS studio: cellomics scan V6.6.2 software using the Spot Detector Bioapplication V4. This bioapplication measures fluorescence intensities on a single cell level, allowing rapid protein expression analysis. Data are presented as average fluorescence intensity per cell (ICAM1 expression) minus intensity from untreated condition (baseline subtracted) and plotted with GraphPad Prism Version 9. Stimulation with LTBR / FAP bispecific agonist antibodies results in the concentrationdependent upregulation of adhesion molecules, as represented in Figures 14 and 15.

[0586] These data show that the surrogate FAP / LTBR bispecific antibodies are capable of inducing clustering and signalling of LTBR in a FAP-dependent manner (due to FAP binding) resulting in chemokine induction and adhesion molecule upregulation on cells, similar to human constructs, as opposed to irrelevant-target control (clone 4.13, which does not bind to FAP) which demonstrated reduced adhesion molecule upregulation (Figure 14 and 15) and no chemokine induction (Figure 13a and 13b).

[0587] Table 26 EC50 ICAM1 induction in PMLF

[0588]

[0589]

[0590] 5. Example 5. In vivo efficacy of an LTBR binding agent in combination with a topoisomerase inhibitor in orthotopic breast cancer models

[0591] To investigate the effect of administering an LTBR binding agent in combination with a topoisomerase I inhibitor a combination study was performed using the EMT6-human HER2 syngeneic mouse model wherein the EMT6 cell line used was modified to express human HER2. This mouse model was selected for its expression of the human HER2 antigen, making it suitable for evaluating the therapeutic efficacy of trastuzumab deruxtecan (“DS-8201a”, which consists of the humanized monoclonal anti-HER2 antibody trastuzumab covalently linked to the topoisomerase I inhibitor deruxtecan; DS-8201a binds human HER2) in combination with a FAP / LTBR bispecific antibody (clone 4.11).

[0592] Briefly, BALB / c mice were inoculated orthotopically in the mammary fat pad with EMT6-human HER2 (Crown Bioscience, cat no ECL-00086) tumor cells (5 x 105cells) for tumor development. Mice were randomised into treatment groups when the mean tumor size reached approximately 100mm3. Mice were treated with (a) isotype control mouse lgG2a (BioXcell, cal no BP0085 / 910824J1), (b) vehicle control (PBS), (c) trastuzumab deruxtecan (DS-8201a) (MCE, cat no 386767) and PBS, (d) FAP / LTBR bispecific antibody clone 4.11 and PBS, or (e) a combination of DS-8201a together with FAP / LTBR bispecific antibody clone 4.11 and PBS. FAP / LTBR bispecific antibody was dosed at evenly-spaced intervals three times per week at 10mg / kg by i.p. administration, whereas DS-8210a was administrated as a single dose at 3mg / kg. The study was terminated when the mice in the control group reached humane endpoints. On the day of termination, tumors from all groups (if possible) were processed for FFPE blocks. Details of the treatment schedule are provided in Table 27.

[0593] Table 27 Treatment schedule in orthotopic EMT6-huHER2 mouse model

[0594]

[0595]

[0596] T umour volumes were established at various timepoints and tumour weight was established at endpoint. The combination of FAP / LTBR bispecific antibody, clone 4.11 and DS-8201a (group 5), resulted in highly significant tumour growth inhibition in terms of both volume and weight compared to monotherapy arms (groups 3 and 4) (see Figures 16 and 17). The tumor growth curves were plotted using GraphPad Prism Version 9. The statistical analysis was done with one-way ANOVA (* p<0.05, ** p<0.01, *** p<0.001 **** p<0.0001).

[0597] This surprising combined effect represents a potential novel approach to cancer therapy.

[0598] Throughout the specification and the claims which follow, unless the context requires otherwise, the word ‘comprise’, and variations such as ‘comprises’ and ‘comprising’, will be understood to imply the inclusion of a stated integer, step, group of integers or group of steps but not to the exclusion of any other integer, step, group of integers or group of steps.

[0599] Concentration percentages stated herein are w / v unless specified otherwise. The unit prefixes p and u are used interchangeably herein for ‘micro’.

[0600] The application of which this description and claims forms part may be used as a basis for priority in respect of any subsequent application. The claims of such subsequent application may be directed to any feature or combination of features described herein. They may take the form of product, composition, process, or use claims and may include, by way of example and without limitation, the claims which follow.

[0601] All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth.Clauses of the invention:

[0602] A series of clauses setting out embodiments of the invention is as follows.

[0603] Clause 1. A composition comprising a first construct comprising an LTBR binding agent and a second construct comprising a topoisomerase inhibitor.

[0604] Clause 2. The composition of clause 1 wherein the composition is for use in the treatment of cancer.

[0605] Clause 3. A first construct comprising an LTBR binding agent for use in the treatment of cancer in combination with a second construct comprising a topoisomerase inhibitor.

[0606] Clause 4. A second construct comprising a topoisomerase inhibitor for use in the treatment of cancer in combination with a first construct comprising an LTBR binding agent.

[0607] Clause 5. A method of treating cancer in a subject comprising administering to the subject a first construct comprising an LTBR binding agent and a second construct comprising a topoisomerase inhibitor.

[0608] Clause 6. Use of a first construct comprising an LTBR binding agent in the manufacture of a medicament for the treatment of cancer in combination with a second construct comprising a topoisomerase inhibitor.

[0609] Clause 7. Use of a second construct comprising a topoisomerase inhibitor in the manufacture of a medicament for the treatment of cancer in combination with a first construct comprising an LTBR binding agent.

[0610] Clause 8. The first construct for use, second construct for use, method, use or composition of any one of clauses 1 to 7 wherein the first construct comprises only one LTBR binding agent.

[0611] Clause 9. The first construct for use, second construct for use, method, use or composition of any one of clauses 1 to 7 wherein the first construct comprises more than one LTBR binding agent.

[0612] Clause 10. The first construct for use, second construct for use, method, use or composition of clause 9 wherein the first construct comprises two LTBR binding agents.

[0613] Clause 11. The first construct for use, second construct for use, method, use or composition of clause 10 wherein the first construct comprises only two LTBR binding agents.

[0614] Clause 12. The first construct for use, second construct for use, method, use or composition of any one of clauses 1 to 11 wherein one or all of the LTBR binding agents comprise or consist of a binding polypeptide which binds to LTBR.

[0615] Clause 13. The first construct for use, second construct for use, method, use or composition of any one of clauses 1 to 12, wherein the first construct comprises an anti-LTBR antibody.Clause 14. The first construct for use, second construct for use, method, use or composition of clause 13 wherein the anti-LTBR antibody is a bispecific antibody.

[0616] Clause 15. The first construct for use, second construct for use, method, use or composition of either clause 13 or 14 wherein the anti-LTBR antibody is a bivalent antibody.

[0617] Clause 16. The first construct for use, second construct for use, method, use or composition of clause 15 wherein the anti-LTBR antibody consists essentially of a bispecific, bivalent antibody.

[0618] Clause 17. The first construct for use, second construct for use, method, use or composition of clause 16 wherein the anti-LTBR antibody consists of a bispecific, bivalent antibody.

[0619] Clause 18. The first construct for use, second construct for use, method, use or composition of any one of clauses 13 to 17, wherein the anti-LTBR antibody is an lgG1 antibody.

[0620] Clause 19. The first construct for use, second construct for use, method, use or composition of any one of clauses 1 to 19, wherein the first construct comprises one or more anti-LTBR antibody fragments.

[0621] Clause 20. The first construct for use, second construct for use, method, use or composition of clause 19, wherein the one or more anti-LTBR antibody fragments are independently selected from an scFv, Fv, Fab, Fab’, F(ab')2, variable domain (e.g. VH, VL, VNAR or VHH), diabody or minibody.

[0622] Clause 21. The first construct for use, second construct for use, method, use or composition of clause 20 wherein the first construct comprises or consists of two scFvs. Clause 22. The first construct for use, second construct for use, method, use or composition of either clause 20 or 21 wherein the scFvs comprise a disulfide bond in the VH44:VL100 position.

[0623] Clause 23. The first construct for use, second construct for use, method, use or composition of any one of clauses 1 to 22 wherein one or all of the LTBR binding agents comprise a variable domain which binds to LTBR.

[0624] Clause 24. The first construct for use, second construct for use, method, use or composition of clause 23, wherein the variable domain which binds to LTBR is selected from a VH, VL, VNAR or VHH.

[0625] Clause 25. The first construct for use, second construct for use, method, use or composition of clause 24 wherein the variable domain which binds to LTBR is a heavy chain variable domain.Clause 26. The first construct for use, second construct for use, method, use or composition of clause 25 wherein the heavy chain variable domain which binds to LTBR is a VHH.

[0626] Clause 27. The first construct for use, second construct for use, method, use or composition of any one of clauses 23 to 26 wherein the variable domain which binds 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 sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater 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 with SEQ ID NO: 57 or 166, HCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 205, 58 or 167 or wherein HCDR2 comprises or consists of a polypeptide sequence having 3, 2 or 1 amino acid sequence difference with SEQ ID NO: 205, 58 or 167, and HCDR3 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 59 or 168 or wherein HCDR3 comprises or consists of a polypeptide sequence having 3, 2 or 1 amino acid sequence difference with SEQ ID NO: 59 or 168.

[0627] Clause 28. The first construct for use, second construct for use, method, use or composition of clause 27 wherein HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 57 or 166, HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 205, 58 or 167 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 59 or 168.

[0628] Clause 29. The first construct for use, second construct for use, method, use or composition of any one of clauses 23 to 26 wherein the variable domain which binds 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 sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater 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 difference with SEQ ID NO: 60 or 172, HCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 61 or 173 or wherein HCDR2 comprises or consists of a polypeptide sequence having 3, 2 or 1 amino acid sequence difference with SEQ ID NO: 61 or 173, and HCDR3 comprises or consists of apolypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 62 or 173 or wherein HCDR3 comprises or consists of a polypeptide sequence having 3, 2 or 1 amino acid sequence difference with SEQ ID NO: 62 or 173.

[0629] Clause 30. The first construct for use, second construct for use, method, use or composition of clause 29 wherein HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 60 or 172, HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 61 or 173 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 62 or 174.

[0630] Clause 31. The first construct for use, second construct for use, method, use or composition of any one of clauses 23 to 26 wherein the variable domain which binds 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 sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater 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 difference with SEQ ID NO: 63 or 163, HCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 64 or 164 or wherein HCDR2 comprises or consists of a polypeptide sequence having 3, 2 or 1 amino acid sequence difference with SEQ ID NO: 64 or 164, and HCDR3 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 207, 65 or 165 or wherein HCDR3 comprises or consists of a polypeptide sequence having 3, 2 or 1 amino acid sequence difference with SEQ ID NO: 207, 65 or 165.

[0631] Clause 32. The first construct for use, second construct for use, method, use or composition of clause 31 wherein HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 63 or 163, HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 64 or 164 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 207, 65 or 165.

[0632] Clause 33. The first construct for use, second construct for use, method, use or composition of any one of clauses 23 to 26 wherein the variable domain which binds 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 sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 66 or 169 or whereinHCDR1 comprises or consists of a polypeptide sequence having 3, 2 or 1 amino acid sequence difference with SEQ ID NO: 66 or 169, HCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 206, 67 or 170 or wherein HCDR2 comprises or consists of a polypeptide sequence having 3, 2 or 1 amino acid sequence difference with SEQ ID NO: 206, 67 or 170, and HCDR3 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 208, 68 or 171 or wherein HCDR3 comprises or consists of a polypeptide sequence having 3, 2 or 1 amino acid sequence difference with SEQ ID NO: 208, 68 or 171.

[0633] Clause 34. The first construct for use, second construct for use, method, use or composition of clause 33 wherein HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 66 or 169, HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 206, 67 or 170 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 208, 68 or 171.

[0634] Clause 35. The first construct for use, second construct for use, method, use or composition of any one of clauses 23 to 26 wherein the variable domain which binds to LTBR comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 23 or 160.

[0635] Clause 36. The first construct for use, second construct for use, method, use or composition of clause 35 wherein the variable domain which binds to LTBR comprises or consists of a polypeptide sequence of SEQ ID NO: 23 or 160.

[0636] Clause 37. The first construct for use, second construct for use, method, use or composition of any one of clauses 23 to 26 wherein the variable domain which binds to LTBR comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 24 or 162.

[0637] Clause 38. The first construct for use, second construct for use, method, use or composition of clause 37 wherein the variable domain which binds to LTBR comprises or consists of a polypeptide sequence of SEQ ID NO: 24 or 162.

[0638] Clause 39. The first construct for use, second construct for use, method, use or composition of any one of clauses 23 to 26 wherein the variable domain which binds to LTBR comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 25 or 159.Clause 40. The first construct for use, second construct for use, method, use or composition of clause 39 wherein the variable domain which binds to LTBR comprises or consists of a polypeptide sequence of SEQ ID NO: 25 or 159.

[0639] Clause 41. The first construct for use, second construct for use, method, use or composition of any one of clauses 23 to 26 wherein the variable domain which binds to LTBR comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 26 or 161.

[0640] Clause 42. The first construct for use, second construct for use, method, use or composition of clause 41 wherein the variable domain which binds to LTBR comprises or consists of a polypeptide sequence of SEQ ID NO: 26 or 161.

[0641] Clause 43. The first construct for use, second construct for use, method, use or composition of any one of clauses 1 to 22 wherein one or all of the LTBR binding agents comprise or consist of a paired heavy chain variable domain and light chain variable domain which bind to LTBR.

[0642] Clause 44. The first construct for use, second construct for use, method, use or composition of clause 43, wherein one or all of the LTBR binding agents is independently selected from an scFv, Fv, Fab, Fab’, F(ab')2, diabody or minibody.

[0643] Clause 45. The first construct for use, second construct for use, method, use or composition of either clause 43 or 44 wherein the heavy chain variable domain which binds 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 sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 69, HCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 70 and HCDR3 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 71 and the light chain variable domain which binds to LTBR comprises or consists of three complementarity determining regions (LCDR1-LCDR3) and four framework regions (LFR1-LFR4), wherein LCDR1 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 72, LCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 73 and LCDR3 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80%or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 74.

[0644] Clause 46. The first construct for use, second construct for use, method, use or composition of clause 45 wherein HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 69, HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 70 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 71 and the light chain variable domain comprises or consists of three complementarity determining regions (LCDR1-LCDR3) and four framework regions (LFR1-LFR4), wherein LCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 72, LCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 73 and LCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 74.

[0645] Clause 47. The first construct for use, second construct for use, method, use or composition of either clause 43 or 44 wherein the heavy chain variable domain which binds 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 sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 75, HCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 76 and HCDR3 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 77 and the light chain variable domain which binds to LTBR comprises or consists of three complementarity determining regions (LCDR1-LCDR3) and four framework regions (LFR1-LFR4), wherein LCDR1 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 78, LCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 79 and LCDR3 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 80.

[0646] Clause 48. The first construct for use, second construct for use, method, use or composition of clause 47 wherein HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 75, HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 76 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 77 and the light chain variable domain comprises or consists of three complementarity determining regions(LCDR1-LCDR3) and four framework regions (LFR1-LFR4), wherein LCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 78, LCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 79 and LCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 80.

[0647] Clause 49. The first construct for use, second construct for use, method, use or composition of either clause 43 or 44 wherein the heavy chain variable domain which binds 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 sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 81 , HCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 82 and HCDR3 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 83 and the light chain variable domain which binds to LTBR comprises or consists of three complementarity determining regions (LCDR1-LCDR3) and four framework regions (LFR1-LFR4), wherein LCDR1 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 84, LCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 85 and LCDR3 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 86.

[0648] Clause 50. The first construct for use, second construct for use, method, use or composition of clause 49 wherein HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 81 , HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 82 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 83 and the light chain variable domain comprises or consists of three complementarity determining regions (LCDR1-LCDR3) and four framework regions (LFR1-LFR4), wherein LCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 84, LCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 85 and LCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 86.

[0649] Clause 51. The first construct for use, second construct for use, method, use or composition of either clause 43 or 44 wherein the heavy chain variable domain which binds 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 sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 87, HCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 88 and HCDR3 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 89 and the light chain variable domain which binds to LTBR comprises or consists of three complementarity determining regions (LCDR1-LCDR3) and four framework regions (LFR1-LFR4), wherein LCDR1 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 90, LCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 91 and LCDR3 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 92.

[0650] Clause 52. The first construct for use, second construct for use, method, use or composition of clause 51 wherein HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 87, HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 88 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 89 and the light chain variable domain comprises or consists of three complementarity determining regions (LCDR1-LCDR3) and four framework regions (LFR1-LFR4), wherein LCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 90, LCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 91 and LCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 92.

[0651] Clause 53. The first construct for use, second construct for use, method, use or composition of either clause 43 or 44 wherein the heavy chain variable domain which binds 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 sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 93, HCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 94 and HCDR3 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identitywith SEQ ID NO: 95 and the light chain variable domain which binds to LTBR comprises or consists of three complementarity determining regions (LCDR1-LCDR3) and four framework regions (LFR1-LFR4), wherein LCDR1 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 96, LCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 97 and LCDR3 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 98.

[0652] Clause 54. The first construct for use, second construct for use, method, use or composition of clause 53 wherein HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 93, HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 94 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 95 and the light chain variable domain comprises or consists of three complementarity determining regions (LCDR1-LCDR3) and four framework regions (LFR1-LFR4), wherein LCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 96, LCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 97 and LCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 98.

[0653] Clause 55. The first construct for use, second construct for use, method, use or composition of either clause 43 or 44 wherein the heavy chain variable domain which binds 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 sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 99, HCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 100 and HCDR3 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 101 and the light chain variable domain which binds to LTBR comprises or consists of three complementarity determining regions (LCDR1-LCDR3) and four framework regions (LFR1-LFR4), wherein LCDR1 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 102, LCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 103 and LCDR3comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 104.

[0654] Clause 56. The first construct for use, second construct for use, method, use or composition of clause 55 wherein HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 99, HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 100 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 101 and the light chain variable domain comprises or consists of three complementarity determining regions (LCDR1-LCDR3) and four framework regions (LFR1-LFR4), wherein LCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 102, LCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 103 and LCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 104.

[0655] Clause 57. The first construct for use, second construct for use, method, use or composition of any one of either clause 43 or 44 wherein the heavy chain variable domain which binds 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 sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 105, HCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 106 and HCDR3 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 107 and the light chain variable domain which binds to LTBR comprises or consists of three complementarity determining regions (LCDR1-LCDR3) and four framework regions (LFR1-LFR4), wherein LCDR1 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 108, LCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 109 and LCDR3 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 110.

[0656] Clause 58. The first construct for use, second construct for use, method, use or composition of clause 57 wherein HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 105, HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 106 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 107 and thelight chain variable domain comprises or consists of three complementarity determining regions (LCDR1-LCDR3) and four framework regions (LFR1-LFR4), wherein LCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 108, LCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 109 and LCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 110.

[0657] Clause 59. The first construct for use, second construct for use, method, use or composition of either clause 43 or 44 wherein the heavy chain variable domain which binds 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 sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 111, HCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 112 and HCDR3 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 113 and the light chain variable domain which binds to LTBR comprises or consists of three complementarity determining regions (LCDR1-LCDR3) and four framework regions (LFR1-LFR4), wherein LCDR1 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 114, LCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 115 and LCDR3 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 116.

[0658] Clause 60. The first construct for use, second construct for use, method, use or composition of clause 59 wherein HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 111, HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 112 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 113 and the light chain variable domain comprises or consists of three complementarity determining regions (LCDR1-LCDR3) and four framework regions (LFR1-LFR4), wherein LCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 114, LCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 115 and LCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 116.

[0659] Clause 61. The first construct for use, second construct for use, method, use or composition of either clause 43 or 44 wherein the heavy chain variable domain which binds toLTBR comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 27 and the light chain variable domain which binds to LTBR comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 28.

[0660] Clause 62. The first construct for use, second construct for use, method, use or composition of clause 61 wherein the heavy chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 27 and the light chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 28.

[0661] Clause 63. The first construct for use, second construct for use, method, use or composition of either clause 43 or 44 wherein the heavy chain variable domain which binds to LTBR comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 29 and the light chain variable domain which binds to LTBR comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 30.

[0662] Clause 64. The first construct for use, second construct for use, method, use or composition of clause 63 wherein the heavy chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 29 and the light chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 30.

[0663] Clause 65. The first construct for use, second construct for use, method, use or composition of either clause 43 or 44 wherein the heavy chain variable domain which binds to LTBR comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 31 and the light chain variable domain which binds to LTBR comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 32.

[0664] Clause 66. The first construct for use, second construct for use, method, use or composition of clause 65 wherein the heavy chain variable domain comprises or consists of apolypeptide sequence of SEQ ID NO: 31 and the light chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 32.

[0665] Clause 67. The first construct for use, second construct for use, method, use or composition of either clause 43 or 44 wherein the heavy chain variable domain which binds to LTBR comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 33 and the light chain variable domain which binds to LTBR comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 34.

[0666] Clause 68. The first construct for use, second construct for use, method, use or composition of clause 67 wherein the heavy chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 33 and the light chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 34.

[0667] Clause 69. The first construct for use, second construct for use, method, use or composition of either clause 43 or 44 wherein the heavy chain variable domain which binds to LTBR comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 35 and the light chain variable domain which binds to LTBR comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 36.

[0668] Clause 70. The first construct for use, second construct for use, method, use or composition of clause 69 wherein the heavy chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 35 and the light chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 36.

[0669] Clause 71. The first construct for use, second construct for use, method, use or composition of either clause 43 or 44 wherein the heavy chain variable domain which binds to LTBR comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 37 and the light chain variable domain which binds to LTBR comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% orgreater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 38.

[0670] Clause 72. The first construct for use, second construct for use, method, use or composition of clause 71 wherein the heavy chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 37 and the light chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 38.

[0671] Clause 73. The first construct for use, second construct for use, method, use or composition of either clause 43 or 44 wherein the heavy chain variable domain which binds to LTBR comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 39 and the light chain variable domain which binds to LTBR comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 40.

[0672] Clause 74. The first construct for use, second construct for use, method, use or composition of clause 73 wherein the heavy chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 39 and the light chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 40.

[0673] Clause 75. The first construct for use, second construct for use, method, use or composition of either clause 43 or 44 wherein the heavy chain variable domain which binds to LTBR comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 41 and the light chain variable domain which binds to LTBR comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 42.

[0674] Clause 76. The first construct for use, second construct for use, method, use or composition of clause 75 wherein the heavy chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 41 and the light chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 42.

[0675] Clause 77. The first construct for use, second construct for use, method, use or composition of any one of clauses 1 to 26 wherein the LTBR-binding agent comprises at least one CDR, wherein the CDR is a HCDR1, HCDR2 or HCDR3 comprised in a heavy chain variable domain of SEQ ID NOs: 23-26 or 159-162.Clause 78. The first construct for use, second construct for use, method, use or composition of clause 77 wherein the LTBR-binding agent comprises at least such HCDR1 and HCDR2, such HCDR1 and HCDR3, or such HCDR2 and HCDR3.

[0676] Clause 79. The first construct for use, second construct for use, method, use or composition of clause 77 wherein the LTBR-binding agent comprises such HCDR1, HCDR2 and HCDR3.

[0677] Clause 80. The first construct for use, second construct for use, method, use or composition of any one of clauses 77 to 79 wherein the HCDR1, HCDR2 and HCDR3 are delineated according to the Kabat-, Chothia-, Martin-, IMGT-, or AbM-method.

[0678] Clause 81. The first construct for use, second construct for use, method, use or composition of any one of clauses 77 to 79 wherein the HCDR1 is chosen from SEQ ID NOs: 57, 60, 63, 66, 163, 166, 169 or 172 as defined by the Kabat-method; the HCDR2 is chosen from SEQ ID NOs: 58, 61, 64, 67, 164, 167, 170, 173, 205 or 206 as defined by the Kabat-method; and / or the HCDR3 is chosen from SEQ ID NOs: 59, 62, 65, 68, 165, 168, 171, 174, 207 or 208 as defined by the Kabat-method.

[0679] Clause 82. The first construct for use, second construct for use, method, use or composition of any one of clauses 1 to 81 wherein if multiple LTBR binding agents are present, the LTBR binding agents are directly linked, or indirectly linked (e.g. via a linker such as a peptide linker).

[0680] Clause 83. The first construct for use, second construct for use, method, use or composition of any one of clauses 1 to 82 wherein the first construct comprises an Fc region, wherein optionally one or all of the LTBR binding agents are linked by a peptide linker to the Fc region, for example to the CH3 region.

[0681] Clause 84. The first construct for use, second construct for use, method, use or composition of clause 83 wherein the peptide linker of the first construct linking the LTBR binding agents to the Fc is no longer than 50 amino acids, such as no longer than 40 amino acids, such as no longer than 30 amino acids, such as no longer than 20 amino acids, such as no longer than 10 amino acids, such as no longer than 9 amino acids, such as no longer than 8 amino acids, such as no longer than 7 amino acids, such as no longer than 6 amino acids, such as no longer than 5 amino acids.

[0682] Clause 85. The first construct for use, second construct for use, method, use or composition of either clause 83 or 84 wherein the peptide linker of the first construct linking the LTBR binding agents to the Fc is shorter than 6 amino acids, such as shorter than 7 amino acids, such as shorter than 8 amino acids, such as shorter than 9 amino acids, such as shorter than 10 amino acids, such as shorter than 20 amino acids, such as shorter than 30 amino acids, such as shorter than 40 amino acids, such as shorter than 50 amino acids.Clause 86. The first construct for use, second construct for use, method, use or composition of any one of clauses 83 to 85 wherein the peptide linker of the first construct linking the LTBR binding agents to the Fc comprises or consists of glycine and serine residues, such as comprising or consisting of a G4S linker (SEQ ID NO: 3).

[0683] Clause 87. The first construct for use, second construct for use, method, use or composition of any one of clauses 1 to 86 wherein one or all of the LTBR binding agents specifically bind to LTBR.

[0684] Clause 88. The first construct for use, second construct for use, method, use or composition of clause 87 wherein one or all of the LTBR binding agents specifically bind to LTBR in that any other entity is bound to with a KD of 10'7M or more, such as 10'6M or more.

[0685] Clause 89. The first construct for use, second construct for use, method, use or composition of any one of clauses 1 to 88 wherein one or all of the LTBR binding agents specifically bind to the extracellular domain of LTBR.

[0686] Clause 90. The first construct for use, second construct for use, method, use or composition of any one of clauses 1 to 89, wherein the LTBR is on the surface of stromal cells.

[0687] Clause 91. The first construct for use, second construct for use, method, use or composition of any one of clauses 1 to 90, wherein the LTBR is on the surface of cancer cells, myeloid cells and / or macrophages.

[0688] Clause 92. The first construct for use, second construct for use, method, use or composition of any one of any one of clauses 1 to 91 wherein one or all of the LTBR binding agents is an LTBR agonist.

[0689] Clause 93. The first construct for use, second construct for use, method, use or composition of any one of clause 92 wherein the LTBR agonist is an agonist of NFKB pathways.

[0690] Clause 94. The first construct for use, second construct for use, method, use or composition of any one of clause 93 wherein the LTBR agonist (a) induces clustering of LTBR and / or (b) activates the classical NFKB pathway (such as leading to expression of adhesion molecules such as ICAMs, such as ICAM-1, VCAM-1 and / or MAdCAM-1) and / or activates the alternative NFKB pathway (such as leading to expression of chemokines, such as CCL5, CCL19, CCL21 and / or CXCL13).

[0691] Clause 95. The first construct for use, second construct for use, method, use or composition of any one of clauses 92 to 94 wherein the LTBR binding agent agonises LTBR in that the LTBR binding agent has an EC50 of 1.0E-08 M or less.

[0692] Clause 96. The first construct for use, second construct for use, method, use or composition of any one of clause 95 wherein the LTBR binding agent agonises LTBR in that the LTBR binding agent has an EC50 of 9.0E-09 M or less.Clause 97. The first construct for use, second construct for use, method, use or composition of any one of clause 96 wherein the LTBR binding agent agonises LTBR in that the LTBR binding agent has an EC50 of 8.0E-09 M or less, such as 7.0E-09 M or less, such as 6.0E-09 M or less, such as 5.0E-09 M or less, such as 4.0E-09 M or less, such as 3.0E-09 M or less, such as 2.0E-09 M or less, such as 1.0E-09 M or less, such as 9.0E-10 M or less, such as 8.0E-10 M or less, such as 7.0E-10 M or less, such as 6.0E-10 M or less, such as 5.0E-10 M or less, such as 4.0E-10 M or less, such as 3.0E-10 M or less, such as 2.0E-10 M or less, such as 1.0E-10 M or less, such as 9.0E-11 M or less, such as 8.0E-11 M or less, such as 7.0E-11 M or less, such as 6.0E-11 M or less, such as 5.0E-11 M or less, such as 4.0E-11 M or less, such as 3.0E-11 M or less, such as 2.0E-11 M or less, such as 1.0E-11 M or less, such as 9.0E-12 M or less, such as 8.0E-12 M or less, such as 7.0E-12 M or less, such as 6.0E-12 M or less, such as 5.0E-12 M or less, such as 4.0E-12 M or less, such as 3.0E-12 M or less, such as 2.0E-12 M or less, such as 1.OE-12 M or less.

[0693] Clause 98. The first construct for use, second construct for use, method, use or composition of any one of clauses 95 to 97 wherein the EC50 is established using a chemokine induction assay, for example assayed using an ELISA.

[0694] Clause 99. The first construct for use, second construct for use, method, use or composition of any one of clauses 95 to 98 wherein the EC50 is established using an assay set out in any one of the Examples.

[0695] Clause 100. The first construct for use, second construct for use, method, use or composition of any one of clauses 1 to 99 wherein the LTBR is human, Macaca fascicularis or mouse LTBR.

[0696] Clause 101. The first construct for use, second construct for use, method, use or composition of clause 100 wherein the LTBR is human LTBR.

[0697] Clause 102. The first construct for use, second construct for use, method, use or composition of any one of clauses 1 to 101 wherein one or all of the LTBR binding agents bind to LTBR with an affinity (KD) as recited in Table 24 or less.

[0698] Clause 103. The first construct for use, second construct for use, method, use or composition of any one of clauses 1 to 102 wherein one or all of the LTBR binding agents bind to LTBR with an affinity (KD) of 20 nM or less, such as 15 nM or less, such as 10 nM or less, such as 9 nM or less, such as 8 nM or less, such as 7 nM or less, such as 6 nM or less, such as 5 nM or less, such as 4 nM or less, such as 3 nM or less, such as 2 nM or less, such as 1 nM or less.

[0699] Clause 104. The first construct for use, second construct for use, method, use or composition of any one of clauses 1 to 103 wherein one or all of the LTBR binding agents have an EC50 of 9.0E-09 M or less, such as 8.0E-09 M or less, such as 7.0E-09 M or less, such as6.0E-09 M or less, such as 5.0E-09 M or less, such as 4.0E-09 M or less, such as 3.0E-09 M or less, such as 2.0E-09 M or less, such as 1.0E-09 M or less, such as 9.0E-10 M or less, such as 8.0E-10 M or less, such as 7.0E-10 M or less, such as 6.0E-10 M or less, such as 5.0E-10 M or less, such as 4.0E-10 M or less, such as 3.0E-10 M or less, such as 2.0E-10 M or less, such as 1.0E-10 M or less, such as 9.0E-11 M or less, such as 8.0E-11 M or less, such as 7.0E-11 M or less, such as 6.0E-11 M or less, such as 5.0E-11 M or less, such as 4.0E-11 M or less, such as 3.0E-11 M or less, such as 2.0E-11 M or less, such as 1.0E-11 M or less, such as 9.0E-12 M or less, such as 8.0E-12 M or less, such as 7.0E-12 M or less, such as 6.0E-12 M or less, such as 5.0E-12 M or less, such as 4.0E-12 M or less, such as 3.0E-12 M or less, such as 2.0E-12 M or less, such as 1.OE-12 M or less.

[0700] Clause 105. The first construct for use, second construct for use, method, use or composition of any one of clauses 1 to 104 wherein the construct is multivalent for LTBR and binds to LTBR with an avidity of 1.0E-08 M or less, such as 9.0E-09 M or less, such as 7.0E-09 M or less, such as 6.7E-09 M or less, such as 5.0E-09 M or less, such as 3.0E-09 M or less.

[0701] Clause 106. The first construct for use, second construct for use, method, use or composition of clause 105 wherein the construct binds to LTBR with an avidity of 2.0E-09 M or less, such as 1.0E-09 M or less.

[0702] Clause 107. The first construct for use, second construct for use, method, use or composition of any one of clauses 102 to 106 wherein the LTBR binding affinity or avidity is established as set out in Example 2 or Example 4.

[0703] Clause 108. The first construct for use, second construct for use, method, use or composition of any one of clauses 1 to 107 wherein one or all of the LTBR binding agents have an EC50 as recited for any of the clones specified in the tables of the Examples or less.

[0704] Clause 109. The first construct for use, second construct for use, method, use or composition of any one of clauses 104 to 108 wherein the EC50 of one or all of the LTBR binding agents is established in an assay as set out in the Examples.

[0705] Clause 110. The first construct for use, second construct for use, method, use or composition of any one of clauses 95 to 109 wherein the EC50 of one or all of the LTBR binding agents is established in respect of binding to a HepG2 cell line.

[0706] Clause 111. The first construct for use, second construct for use, method, use or composition of clause 110 wherein the EC50 of one or all of the LTBR binding agents is established in a HepG2 reporter assay.

[0707] Clause 112. The first construct for use, second construct for use, method, use or composition of any one of clauses 109 to 111 wherein the EC50 of one or all of the LTBR binding agents is established in a HepG2 NF-KB activation-induced luciferase activity.Clause 113. The first construct for use, second construct for use, method, use or composition of any one of clauses 95 to 109 wherein the EC50 of one or all of the LTBR binding agents is established in respect of upregulation of CCL19 on HCC1187 cells.

[0708] Clause 114. The first construct for use, second construct for use, method, use or composition of any one of clauses 95 to 109 wherein the EC50 of one or all of the LTBR binding agents is established in respect of binding to LTBR positive A549 cell line.

[0709] Clause 115. The first construct for use, second construct for use, method, use or composition of any one of clauses 95 to 109 wherein the EC50 of one or all of the LTBR binding agents is established in respect of binding to the A375 tumour cell line.

[0710] Clause 116. The first construct for use, second construct for use, method, use or composition of any one of clauses 95 to 109 wherein the EC50 of one or all of the LTBR binding agents is established in respect of binding to an LTBR and FAP positive cell line (e.g. A549 or A375, or CHO-FAP or Hek-FAP).

[0711] Clause 117. The first construct for use, second construct for use, method, use or composition of any one of clauses 95 to 109 wherein the EC50 of one or all of the LTBR binding agents is established in respect of ICAM1 upregulation in a CAFs monoculture assay.

[0712] Clause 118. The first construct for use, second construct for use, method, use or composition of clause 117 wherein the EC50 of one or all of the LTBR binding agents is established in respect of ICAM1 induction in PMLF.

[0713] Clause 119. The first construct for use, second construct for use, method, use or composition of any one of clauses 95 to 109 wherein the EC50 of one or all of the LTBR binding agents is established in a HCC1187:RPMI-7951 co-culture assay.

[0714] Clause 120. The first construct for use, second construct for use, method, use or composition of any one of clauses 95 to 109 wherein the EC50 of one or all of the LTBR binding agents is established in a chemokine induction assay.

[0715] Clause 121. The first construct for use, second construct for use, method, use or composition of clause 120 wherein the chemokine induction is measured with an ELISA.

[0716] Clause 122. The first construct for use, second construct for use, method, use or composition of either clause 120 or 121 wherein the chemokine is selected from CCL2, CCL5 orCCL19.

[0717] Clause 123. The first construct for use, second construct for use, method, use or composition of clause 122 wherein the chemokine is CCL19 on HCC1187 cells in the presence or absence of CAFs.

[0718] Clause 124. The first construct for use, second construct for use, method, use or composition of any one of clauses 120 to 123 wherein the chemokine induction is measured in RPMI-7951.Clause 125. The first construct for use, second construct for use, method, use or composition of any one of clauses 120 to 123 wherein the chemokine induction is measured in 4T 1 monoculture or 4T 1 :CHO-FAP co-culture.

[0719] Clause 126. The first construct for use, second construct for use, method, use or composition of any one of clauses 1 to 125 wherein the first construct induces clustering of LTBR.

[0720] Clause 127. The first construct for use, second construct for use, method, use or composition of any one of clauses 1 to 126 wherein the first construct comprises a FAP binding agent.

[0721] Clause 128. The first construct for use, second construct for use, method, use or composition of clause 127 wherein the first construct comprises only one FAP binding agent.

[0722] Clause 129. The first construct for use, second construct for use, method, use or composition of clause 127 wherein the first construct comprises more than one FAP binding agent.

[0723] Clause 130. The first construct for use, second construct for use, method, use or composition of clause 129 wherein the first construct comprises two FAP binding agents.

[0724] Clause 131. The first construct for use, second construct for use, method, use or composition of clause 130 wherein the first construct comprises only two FAP binding agents.

[0725] Clause 132. The first construct for use, second construct for use, method, use or composition of any one of clauses 127 to 131 wherein one or all of the FAP binding agents comprise or consist of a binding polypeptide which binds to FAP.

[0726] Clause 133. The first construct for use, second construct for use, method, use or composition of any one of clauses 127 to 132 wherein one or all of the FAP binding agents comprise or consist of a paired heavy chain variable domain and light chain variable domain which binds to FAP.

[0727] Clause 134. The first construct for use, second construct for use, method, use or composition of clause 133, wherein one or all of the FAP binding agents are independently selected from an scFv, Fv, Fab, Fab’, F(ab')2, diabody or minibody.

[0728] Clause 135. The first construct for use, second construct for use, method, use or composition of either clause 133 or 134 the heavy chain variable domain which binds to FAP 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 sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 117, HCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 118 andHCDR3 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 119 and the light chain variable domain which binds to FAP comprises or consists of three complementarity determining regions (LCDR1-LCDR3) and four framework regions (LFR1-LFR4), wherein LCDR1 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 120, LCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 121 and LCDR3 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 122.

[0729] Clause 136. The first construct for use, second construct for use, method, use or composition of clause 135 wherein HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 117, HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 118 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 119 and the light chain variable domain comprises or consists of three complementarity determining regions (LCDR1-LCDR3) and four framework regions (LFR1-LFR4), wherein LCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 120, LCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 121 and LCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 122.

[0730] Clause 137. The first construct for use, second construct for use, method, use or composition of either clause 133 or 134 wherein the heavy chain variable domain which binds to FAP 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 sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 123, HCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 124 and HCDR3 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 125 and the light chain variable domain which binds to FAP comprises or consists of three complementarity determining regions (LCDR1-LCDR3) and four framework regions (LFR1-LFR4), wherein LCDR1 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 126, LCDR2 comprises or consists of apolypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 127 and LCDR3 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 128.

[0731] Clause 138. The first construct for use, second construct for use, method, use or composition of clause 137 wherein HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 123, HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 124 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 125 and the light chain variable domain comprises or consists of three complementarity determining regions (LCDR1-LCDR3) and four framework regions (LFR1-LFR4), wherein LCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 126, LCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 127 and LCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 128.

[0732] Clause 139. The first construct for use, second construct for use, method, use or composition of either clause 133 or 134 wherein the heavy chain variable domain which binds to FAP 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 sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 129, HCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 130 and HCDR3 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 131 and the light chain variable domain which binds to FAP comprises or consists of three complementarity determining regions (LCDR1-LCDR3) and four framework regions (LFR1-LFR4), wherein LCDR1 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 132, LCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 133 and LCDR3 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 134.

[0733] Clause 140. The first construct for use, second construct for use, method, use or composition of clause 139 wherein HCDR1 comprises or consists of a polypeptide sequenceof SEQ ID NO: 129, HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 130 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 131 and the light chain variable domain comprises or consists of three complementarity determining regions (LCDR1-LCDR3) and four framework regions (LFR1-LFR4), wherein LCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 132, LCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 133 and LCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 134.

[0734] Clause 141. The first construct for use, second construct for use, method, use or composition of either clause 133 or 134 wherein the heavy chain variable domain which binds to FAP 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 sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 135, HCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 136 and HCDR3 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 137 and the light chain variable domain which binds to FAP comprises or consists of three complementarity determining regions (LCDR1-LCDR3) and four framework regions (LFR1-LFR4), wherein LCDR1 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 138, LCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 139 and LCDR3 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 140.

[0735] Clause 142. The first construct for use, second construct for use, method, use or composition of clause 141 wherein HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 135, HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 136 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 137 and the light chain variable domain comprises or consists of three complementarity determining regions (LCDR1-LCDR3) and four framework regions (LFR1-LFR4), wherein LCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 138, LCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 139 and LCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 140.Clause 143. The first construct for use, second construct for use, method, use or composition of any either clause 133 or 134 wherein the heavy chain variable domain which binds to FAP 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 sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 141, HCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 142 and HCDR3 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 143 and the light chain variable domain which binds to FAP comprises or consists of three complementarity determining regions (LCDR1-LCDR3) and four framework regions (LFR1-LFR4), wherein LCDR1 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 144, LCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 145 and LCDR3 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 146.

[0736] Clause 144. The first construct for use, second construct for use, method, use or composition of clause 143 wherein HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 141 , HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 142 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 143 and the light chain variable domain comprises or consists of three complementarity determining regions (LCDR1-LCDR3) and four framework regions (LFR1-LFR4), wherein LCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 144, LCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 145 and LCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 146.

[0737] Clause 145. The first construct for use, second construct for use, method, use or composition of either clause 133 or 134 wherein the heavy chain variable domain which binds to FAP 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 sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 147, HCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater,85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 148 and HCDR3 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 149 and the light chain variable domain which binds to FAP comprises or consists of three complementarity determining regions (LCDR1-LCDR3) and four framework regions (LFR1-LFR4), wherein LCDR1 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 150, LCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 151 and LCDR3 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 152.

[0738] Clause 146. The first construct for use, second construct for use, method, use or composition of clause 145 wherein HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 147, HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 148 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 149 and the light chain variable domain comprises or consists of three complementarity determining regions (LCDR1-LCDR3) and four framework regions (LFR1-LFR4), wherein LCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 150, LCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 151 and LCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 152.

[0739] Clause 147. The first construct for use, second construct for use, method, use or composition of either clause 133 or 134 wherein the heavy chain variable domain which binds to FAP 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 sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 153, HCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 154 and HCDR3 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 155 and the light chain variable domain which binds to FAP comprises or consists of three complementarity determining regions (LCDR1-LCDR3) and four framework regions (LFR1-LFR4), wherein LCDR1 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or95% or greater sequence identity with SEQ ID NO: 156, LCDR2 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 157 and LCDR3 comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater or 95% or greater sequence identity with SEQ ID NO: 158.

[0740] Clause 148. The first construct for use, second construct for use, method, use or composition of clause 147 wherein HCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 153, HCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 154 and HCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 155 and the light chain variable domain comprises or consists of three complementarity determining regions (LCDR1-LCDR3) and four framework regions (LFR1-LFR4), wherein LCDR1 comprises or consists of a polypeptide sequence of SEQ ID NO: 156, LCDR2 comprises or consists of a polypeptide sequence of SEQ ID NO: 157 and LCDR3 comprises or consists of a polypeptide sequence of SEQ ID NO: 158.

[0741] Clause 149. The first construct for use, second construct for use, method, use or composition of either clause 133 or 134 wherein the heavy chain variable domain which binds to FAP comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 7 and the light chain variable domain which binds to FAP comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 8.

[0742] Clause 150. The first construct for use, second construct for use, method, use or composition of clause 149 wherein the heavy chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 7 and the light chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 8.

[0743] Clause 151. The first construct for use, second construct for use, method, use or composition of either clause 133 or 134 wherein the heavy chain variable domain which binds to FAP comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 43 and the light chain variable domain which binds to FAP comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% orgreater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 44.

[0744] Clause 152. The first construct for use, second construct for use, method, use or composition of clause 151 wherein the heavy chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 43 and the light chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 44.

[0745] Clause 153. The first construct for use, second construct for use, method, use or composition of either clause 133 or 134 wherein the heavy chain variable domain which binds to FAP comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 45 and the light chain variable domain which binds to FAP comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 46.

[0746] Clause 154. The first construct for use, second construct for use, method, use or composition of clause 153 wherein the heavy chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 45 and the light chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 46.

[0747] Clause 155. The first construct for use, second construct for use, method, use or composition of either clause 133 or 134 wherein the heavy chain variable domain which binds to FAP comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 47 and the light chain variable domain which binds to FAP comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 48.

[0748] Clause 156. The first construct for use, second construct for use, method, use or composition of clause 155 wherein the heavy chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 47 and the light chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 48.

[0749] Clause 157. The first construct for use, second construct for use, method, use or composition of either clause 133 or 134 wherein the heavy chain variable domain which binds to FAP comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97%or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 49 and the light chain variable domain which binds to FAP comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 50.

[0750] Clause 158. The first construct for use, second construct for use, method, use or composition of clause 157 wherein the heavy chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 49 and the light chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 50.

[0751] Clause 159. The first construct for use, second construct for use, method, use or composition of either clause 133 or 134 wherein the heavy chain variable domain which binds to FAP comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 51 and the light chain variable domain which binds to FAP comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 52.

[0752] Clause 160. The first construct for use, second construct for use, method, use or composition of clause 159 wherein the heavy chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 51 and the light chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 52.

[0753] Clause 161. The first construct for use, second construct for use, method, use or composition of either clause 133 or 134 wherein the heavy chain variable domain which binds to FAP comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 53 and the light chain variable domain which binds to FAP comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 54.

[0754] Clause 162. The first construct for use, second construct for use, method, use or composition of clause 161 wherein the heavy chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 53 and the light chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 54.Clause 163. The first construct for use, second construct for use, method, use or composition of either clause 133 or 134 wherein the heavy chain variable domain which binds to FAP comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 55 and the light chain variable domain which binds to FAP comprises or consists of a polypeptide sequence sharing 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with SEQ ID NO: 56.

[0755] Clause 164. The first construct for use, second construct for use, method, use or composition of clause 163 wherein the heavy chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 55 and the light chain variable domain comprises or consists of a polypeptide sequence of SEQ ID NO: 56.

[0756] Clause 165. The first construct for use, second construct for use, method, use or composition of any one of clauses 127 to 164 wherein one or all of the FAP binding agents specifically bind to FAP.

[0757] Clause 166. The first construct for use, second construct for use, method, use or composition of clause 165 wherein one or all of the FAP binding agents specifically bind to FAP in that any other entity is bound to with a KD of 10'7M or more, such as 10'6M or more.

[0758] Clau...

Claims

CLAIMS1. A composition comprising a first construct comprising an LTBR binding agent and a second construct comprising a topoisomerase inhibitor.

2. A composition for use in the treatment of cancer wherein the composition comprises a first construct comprising an LTBR binding agent and a second construct comprising a topoisomerase inhibitor.

3. A first construct comprising an LTBR binding agent for use in the treatment of cancer in combination with a second construct comprising a topoisomerase inhibitor.

4. The first construct for use or composition of any one of claims 1 to 3, wherein the first construct comprises an anti-LTBR antibody or one or more anti-LTBR antibody fragments.

5. The first construct for use or composition of any one of claims 1 to 4, wherein the LTBR binding agent is an LTBR agonist.

6. The first construct for use or composition of claim 5, wherein the LTBR binding agent agonises LTBR in that the LTBR binding agent has an EC50 of 1.0E-08 M or less.

7. The first construct for use or composition of claim 6, wherein the EC50 of one or all of the LTBR binding agents is established in a chemokine induction assay.

8. The first construct for use or composition of any one of claims 1 to 7, wherein the first construct comprises a FAP binding agent, such as an anti-FAP antibody or one or more anti-FAP antibody fragments.

9. The first construct for use or composition of any one of claims 1 to 8, wherein the topoisomerase inhibitor is a topoisomerase I inhibitor.

10. The first construct for use or composition of any one of claims 1 to 9, wherein the topoisomerase inhibitor is exatecan or a derivative thereof, such as deruxtecan.

11. The first construct for use or composition of any one of claims 1 to 10, wherein the second construct comprises a TAA binding agent.

12. The first construct for use or composition of claim 11, wherein the second construct comprises a HER2 binding agent, such as an anti-HER2 antibody.

13. The first construct for use or composition of claim 12, wherein the second construct is trastuzumab deruxtecan.

14. The first construct for use or composition of any one of claims 1 to 13, wherein the cancer is a tumour.

15. The first construct for use or composition of either claim 2 or 3, whereinthe first construct comprises an anti-LTBR antibody or one or more anti-LTBR antibody fragments,the anti-LTBR antibody or one or more anti-LTBR antibody fragments is an LTBR agonist,the first construct comprises an anti-FAP antibody or one or more anti-FAP antibody fragments,the topoisomerase inhibitor is a topoisomerase I inhibitor,the second construct comprises a HER2 binding agent andthe cancer is a tumour.

16. The first construct for use or composition of any one of claims 1 to 3, wherein the first construct is a bispecific antibody comprising one or more variable domains which bind to LTBR and one or more variable domains which bind to FAP and wherein the topoisomerase inhibitor is a topoisomerase I inhibitor.

17. The first construct for use or composition of claim 16 wherein the second construct is an anti-HER2 antibody.

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