Background Fibroblast activation protein alpha (FAP) is a pan-tumor target highly expressed on cancer-associated fibroblasts. We developed 4AH29, a single-domain antibody binding FAP, and investigated the biodistribution of [131I]I-GMIB-4AH29 and [111In]In-DOTA-4AH29 in Göttingen minipigs. Methods Following radiopharmaceutical administration, blood activity profile was determined by gamma-counter and biodistribution kinetics were determined using SPECT/CT imaging, respectively. The data obtained with [111In]In-DOTA-4AH29 were used as a surrogate for its 177Lu- and 225Ac-labeled analogues and extrapolation to human absorbed dose was calculated for all analogues. Results Radiolabeled 4AH29 showed good tolerability within the studied time frame. It displayed fast blood clearance driven by renal excretion. Kidney clearance dynamics of [131I]I-GMIB-4AH29 and [111In]In-DOTA-4AH29 were distinct, likely driven by the different radiolabeling chemistry for halogen or metal conjugation. However, the contrasting patterns did not translate into relevant differences in mean residence time (MRT), nor was there a significant difference in bone marrow or liver MRTs when comparing halogen and metal-radiolabeled 4AH29. Extrapolated human absorbed doses for [131I]I-GMIB-4AH29 and [177Lu]Lu-DOTA-4AH29 were compared given their similar particle decay and comparable physical half-lives. In kidneys, [131I]I-GMIB-4AH29 led to an extrapolated human absorbed dose of 8.23E-01 mGy/MBq, whereas [177Lu]Lu-DOTA-4AH29 reached 5.86E-01 mGy/MBq. Consequently, the maximum tolerable administered activities were 28 and 39 GBq, respectively, to reach the renal absorbed dose limit of 23 Gy as determined by external beam radiation therapy (EBRT). In red marrow, the equivalent dose for [131I]I-GMIB-4AH29 was 3.8E-02 mGy/MBq and 1.36E-02 mGy/MBq with [177Lu]Lu-DOTA-4AH29. Thus, 53 GBq and 148 GBq can be administered, respectively, before reaching the EBRT set absorbed dose threshold of 2 Gy. Conclusion 4AH29 radiolabeled with 131I or 111In is well tolerated in Göttingen minipigs within the studied time frame. Extrapolated dosimetry of radiolabeled 4AH29 using OLINDA software indicates that its administration within a clinically relevant range is possible without exceeding toxicity limits in critical organs.

Fibroblast activation protein is a well-established target for tumour imaging, and single-domain antibodies (sdAb) offer favourable pharmacokinetic properties for PET applications. However, the translation of sdAb-based radiotracers into robust and clinically applicable manufacturing processes remains limited. Previously, preclinical protocol for the production of [68Ga]Ga-DOTA-4AH29 has been reported. In this study, this process was adapted for clinical use. Both a manual, kit-based approach and automated synthesis on two different platforms were developed. Process robustness was assessed through a stress study evaluating key parameters, and compatibility with two 68Ga generator systems was investigated. Efficient radiolabelling was achieved at 50 °C for 10 min, with radiochemical conversion exceeding 90

The HER2 targeted therapy paradigm has increasingly shifted in recent years from focussed treatment of only cancers with high HER2 expression towards those with also low HER2 expression, unlocking a larger subset of patients, in either single or combination treatment strategies. Radiotherapeutic HER2-targeting single-domain antibody (sdAb) 2Rs15d has been previously developed and characterised (pre-)clinically for HER2 high-expressing tumours. The current study explores the use of [131I]I-GMIB- and [225Ac]Ac-DOTA-labelled sdAb 2Rs15d as single treatments or as combinations with the PARP-inhibitor Olaparib in HER2 low-expressing xenografts. [131I]I-GMIB-2Rs15d and [225Ac]Ac-DOTA-2Rs15d specifically bound to HER2-low-expressing DLD-1 BRCA2−/− cells in vitro, inflicted DNA damage (elevated γH2AX) and decreased cell viability as a single agent, an effect that was invigorated upon combination with Olaparib. Radiotracers showed specific tumour uptake in DLD-1 BRCA2−/− tumour xenografts, correlating with the low HER2 expression in this model. Clear antitumour effects were observed in mice treated with fractionated [131I]I-GMIB-2Rs15d (8 × 37 MBq, p’=0.00232) and [225Ac]Ac-DOTA-2Rs15d (8 × 20 kBq, p’=0.0149) compared to vehicle control. In combination with Olaparib (28 × 75 mg/kg), synergistic effects were observed for both [131I]I-GMIB-2Rs15d and [225Ac]Ac-DOTA-2Rs15d. [131I]I-GMIB-2Rs15d and [225Ac]Ac-DOTA-2Rs15d showed therapeutic efficacy in tumour xenografts expressing low levels of HER2. Furthermore, when treatment was combined with Olaparib both radiopharmaceuticals prolonged survival further, underlining a synergistic effect.

Folate receptor alpha (FRα) overexpression is seen in many cancers. Radioligand therapy (RLT) has emerged as a promising tool to target FRα and has been investigated previously, but further progression was limited due to high kidney retention and, subsequently, toxicity. To circumvent this, we present here the development of a [131I]I-GMIB-conjugated anti-human FRα (hFRα) single-domain antibody (sdAb), with intrinsically fast renal clearance and concomitant low kidney retention. We report the hit-to-lead development of an anti-hFRα sdAb. We evaluated its potential in vitro and assessed its targeting ability using SPECT imaging in hFRα-knockin and tumour-bearing mice. The toxicity and therapeutic efficacy of the [131I]I-GMIB-sdAb were investigated in mouse models. The lead anti-hFRα sdAb 2BD42 was developed with picomolar affinities, low koff, and radiolabelled using [131I]I with yields of > 41

A detailed description of the materials and methods used for the HER2-2Rs15d structure determination, the radioiodination of sdAbs, and all flow cytometry experiments.

Supplementary Table S2 shows the interactions between 2Rs15d and HER2 domain I.

Supplementary Table S3 shows the in vivo biodistribution of 131I-2Rs15d after 1, 4 and 24h in mice with BT474/M1 and SKOV-3 xenografts.

Supplementary figure S1 details the HER2-epitope recognized by 2Rs15d compared to those reported for other described HER2-binders and a view of HER2-ÂÃ,â­2Rs15d interactions.

Fibroblast activation protein alpha (FAP) is highly expressed on cancer-associated fibroblasts of epithelial-derived cancers. Breast, colon, and pancreatic tumors often show strong desmoplastic reactions, which result in a dominant presence of stromal cells. FAP has gained interest as a target for molecular imaging and targeted therapies. Single-domain antibodies (sdAbs) are the smallest antibody-derived fragments with beneficial pharmacokinetic properties for molecular imaging and targeted therapy. Methods: We describe the generation, selection, and characterization of a sdAb against FAP. In mice, we assessed its imaging and therapeutic potential after radiolabeling with tracer-dose I-131 and Ga-68 for SPECT and PET imaging, respectively, and with I-131 and Ac-225 for targeted radionuclide therapy. Results: The lead sdAb, 4AH29, exhibiting picomolar affinity for a distinct FAP epitope, recognized both purified and membrane-bound FAP protein. Radiolabeled versions, including [Ga-68]Ga-DOTA-4AH29, [Ac-225]Ac-DOTA-4AH29, and [I-131]I-guanidinomethyl iodobenzoate (GMIB)-4AH29, displayed radiochemical purities exceeding 95% and effectively bound to recombinant human FAP protein and FAP-positive GM05389 human fibroblasts. These radiolabeled compounds exhibited rapid and specific accumulation in human FAP-positive U87-MG glioblastoma tumors, with low but specific uptake in lymph nodes, uterus, bone, and skin (similar to 2-3 percentage injected activity per gram of tissue [%IA/g]). Kidney clearance of unbound [I-131]I-GMIB-4AH29 was fast (<1 %IA/g after 24 h), whereas [Ac-225]Ac-DOTA-4AH29 exhibited slower clearance (8.07 +/- 1.39 %IA/g after 24 h and 2.47 +/- 0.18 %IA/g after 96 h). Mice treated with [Ac-225]Ac-DOTA-4AH29 and [I-131]I-GMIB-4AH29 demonstrated prolonged survival compared with those receiving vehicle solution. Conclusion: [Ga-68]Ga-DOTA-4AH29 and [I-131]I-GMIB-4AH29 enable precise FAP-positive tumor detection in mice. Therapeutic [Ac-225]Ac-DOTA-4AH29 and [I-131]I-GMIB-4AH29 exhibit strong and sustained tumor targeting, resulting in dose-dependent therapeutic effects in FAP-positive tumor-bearing mice, albeit with kidney toxicity observed later for [Ac-225]Ac-DOTA-4AH29. This study confirms the potential of radiolabeled sdAb 4AH29 as a radiotheranostic agent for FAP-positive cancers, warranting clinical evaluation.

Supplementary Table S1 shows the X-ray crystallographic data collection and refinement statistics.

Current cancer therapeutics suffer from a lack of specificity in targeting tumor cells and cause severe side effects. Therefore, the design of highly specialized drugs comprising antibody derivatives inducing apoptosis in targeted cancer cells is considered to be a promising strategy. Drugs acting on death receptor 5 (DR5) such as DR5 agonist antibodies replacing "TNF-related apoptosis-inducing ligand" (TRAIL) offer feasible opportunities in this direction. Although such agonists provided good antitumor activity in preclinical studies, they were less effective in clinical studies, possibly due to a disturbed Fc interaction with Fc-γ receptors. Thus, multimerized antigen binding fragments without Fc have been proposed to increase their efficacy. We generated nanobodies (Nbs), recombinant variable domains of heavy chain-only antibodies of camelids, against the DR5 ectodomain. Nb24 and Nb28 had an affinity in the nM and sub-nM range, but only Nb28 competes with TRAIL for binding to DR5. Bivalent, trivalent, and tetravalent constructs were generated, as well as an innovative pentameric Nb complex, to provoke avidity effects. In our cellular assays, these trimeric, tetrameric, and pentameric Nbs have a higher apoptotic capacity than monomeric Nbs and seem to mimic the activity of the natural TRAIL ligand on various cancer cells.

The tumor microenvironment of numerous prevalent cancer types is abundantly infiltrated with tumor-associated macrophages (TAMs). Macrophage mannose receptor (MMR or CD206) expressing TAMs have been shown to be key promoters of tumor progression and major opponents of successful cancer therapy. Therefore, depleting MMR+ TAMs is an interesting approach to synergize with current antitumor therapies. We studied the potential of single-domain antibodies (sdAbs) specific for MMR to target proteins to MMR+ TAMs. Anti-MMR sdAbs were genetically coupled to a reporter protein, mWasabi (wasabi green, WG), generating sdAb “drug” fusion proteins (SFPs), referred to as WG-SFPs. The resulting WG-SFPs were highly efficient in targeting MMR+ macrophages both in vitro and in vivo. As we showed that second mitochondria-derived activator of caspase (SMAC) mimetics modulate MMR+ macrophages, we further coupled the anti-MMR sdAb to an active form of SMAC, referred to as tSMAC. The resulting tSMAC-SFPs were able to bind and upregulate caspase3/7 activity in MMR+ macrophages in vitro. In conclusion, we report the proof-of-concept of an elegant approach to conjugate anti-MMR sdAbs to proteins, which opens new avenues for targeted manipulation of MMR+ tumor-promoting TAMs.

Immune checkpoint inhibition (ICI) is a promising cancer therapy, which has progressed rapidly from a preclinical concept to clinical implementation. Commonly considered targets in ICI are CTLA-4, PD-1/PD-L1, and LAG-3, and the list grows. As ICI is generally only beneficial for a subset of patients, there is a need to select patients that are eligible for therapy as well as to monitor therapy response. There is growing interest to do this noninvasively, by molecular imaging with target-specific tracers. To this day, noninvasive imaging has focused on CTLA-4 and PD-1/PD-L1, while there is no noninvasive tool available to accurately assess LAG-3 expression in vivo. In this proof-of-concept study, we developed nanobodies, the smallest functional fragments from camelid heavy chain-only antibodies, to noninvasively evaluate mouse LAG-3 expression using single photon emission computed tomography (SPECT)/CT imaging. The in vitro characterization of 114 nanobodies led to the selection of nine nanobodies binding to mouse LAG-3. The injection of 99mTechnetium-labeled nanobodies in healthy mice showed specific uptake in immune peripheral organs like the spleen and lymph nodes, which was not observed in LAG-3 gene knock-out mice. Moreover, nanobody uptake could be visualized using SPECT/CT and correlated to the presence of LAG-3 as assessed in flow cytometry and immunohistochemistry. SPECT/CT scans of tumor bearing mice further confirmed the diagnostic potential of the nanobodies. These findings substantiate the approach to use nanobodies as a tool to image inhibitory immune checkpoints in the tumor environment.

Tumor-associated macrophages (TAMs) with high expression levels of the Macrophage Mannose Receptor (MMR, CD206) exhibit a strong angiogenic and immune suppressive activity. Thus, they are a highly attractive target in cancer immunotherapy, with the aim to modulate their protumoral behavior. Here, we introduce polymer nanogels as potential drug nanocarriers which were site-specifically decorated with a Nanobody (Nb) specific for the MMR. Using azide-functionalized RAFT chain transfer agents, they provide access to amphiphilic reactive ester block copolymers that self-assemble into micelles and are afterwards core-cross-linked toward fully hydrophilic nanogels with terminal azide groups on their surface. MMR-targeting Nb can site-selectively be functionalized with one single cyclooctyne moiety by maleimide-cysteine chemistry under mildly reducing conditions which enables successful chemoorthogonal conjugation to the nanogels. The resulting Nb-functionalized nanogels were highly efficient in targeting MMR-expressing cells and TAMs both in vitro and in vivo. We believe that these findings pave the road for targeted eradication or modulation of pro-tumoral MMRhigh TAMs.

Single-domain antibody fragments, also called nanobodies (Nbs), are increasingly being used as targeting molecular tools for imaging and/or targeted radionuclide therapy. To translate these tools to the clinic, it is preferred to obtain a homogeneous, well-defined, and well-characterized product. It has been shown that Sortase A, a transpeptidase found in Staphylococcus aureus, catalyzes the site-specific conjugation between a recognition oligopeptide (LPXTG, known as sortag) and an oligoglycine functionalized probe. This versatile technique manages to couple various molecular reagents, such as biotin, fluorophores, bifunctional chelators, etc., to the target protein containing the sortag. This chapter focuses on the site-specific coupling of a bifunctional chelator (e.g., CHX-A"-DTPA) to a Nb equipped with a C-terminal sortag. The chelator conjugated to the Nb can be radiolabeled with 111In or 177Lu for SPECT imaging or targeted radionuclide therapy, respectively.