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Discovery of high-affinity ligands for prostatic acid phosphatase via DNA-encoded library screening enables targeted cancer therapy

Nature Biomedical Engineering volume 10, pages 178–191 (2026)Cite this article

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Abstract

Improving the specificity of prostate cancer treatment requires ligands that bind selectively and with ultra-high affinity to tumour-associated targets absent from healthy tissues. Prostatic acid phosphatase has emerged as an alternative target to prostate-specific membrane antigen, as it is expressed in a broader subset of prostate cancers and is not detected in healthy organs such as the salivary glands and kidneys. Here, to discover selective binders to prostatic acid phosphatase, we constructed two DNA-encoded chemical libraries comprising over 6.7 million small molecules based on proline and phenylalanine scaffolds. Screening against the purified human prostatic acid phosphatase yielded OncoACP3, a small organic ligand with picomolar binding affinity. When radiolabelled with lutetium-177, OncoACP3 selectively accumulated in enzyme-expressing tumours with a long residence time (biological half-life greater than 72 h) and a high tumour-to-blood ratio (>148 at 2 h after administration). Lutetium-177-labelled OncoACP3 cured tumours in mice at low, well-tolerated doses. Its conjugation to the cytotoxic agent monomethyl auristatin E facilitated tumour-selective payload deposition, resulting in potent anti-tumour activity. The modular structure of OncoACP3 supports flexible payload delivery for the targeted treatment of metastatic prostate cancer.

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Fig. 1: From DELs to ACP3-targeted RLTs.
Fig. 2: Synthesis and screening of DELs (SOP-DEL and FMA-DEL).
Fig. 3: Inhibitory activity and binding kinetics of DEL-derived ACP3 hits.
Fig. 4: Cellular binding and autoradiography studies of DEL-derived ACP3 ligands.
Fig. 5: In vivo biodistribution studies with ACP3-targeted RLTs.
Fig. 6: Ex vivo biodistribution studies with DEL-derived ACP3 ligands labelled with AF488.
Fig. 7: In vivo therapy studies with ACP3-targeted RLTs.
Fig. 8: From DELs to ACP3-targeted SMDCs.

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Data availability

All data supporting the findings of this study are available within the Article and its Supplementary Information. Unprocessed images for ex vivo biodistribution studies connected to Fig. 6 and Extended Data Fig. 10, as well as raw data for PET–CT in vivo biodistribution work connected to Extended Data Fig. 9, are available via figshare at https://doi.org/10.6084/m9.figshare.28668596 (ref. 55). Source data are provided with this paper.

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Acknowledgements

We thank C. Pellegrino (University Hospital Zurich) and R. De Luca (Philochem AG) for their support with the generation of hACP3+ tumour cell lines. C. Comacchio (Philochem AG) is acknowledged for the production of human recombinant TNAP. G. Neri (Philogen S.p.A.) is acknowledged for the thorough revision of the manuscript. R. Konyarska is acknowledged for preparing cover art designs and M. Satama for proofreading the manuscript to ensure clarity and accessibility for a broader audience. G. Rotta is acknowledged for providing the rendered mice images for Figs. 1 and 8. We received no specific funding for this work.

Author information

Author notes

  1. These authors contributed equally: Tony Georgiev, Francesca Migliorini.

Authors and Affiliations

  1. R&D Department, Philochem AG, Otelfingen, Switzerland

    Tony Georgiev, Francesca Migliorini, Andrea Ciamarone, Marco Mueller, Ilaria Biancofiore, Young Seo Park Kim, Lucrezia Principi, Ettore Gilardoni, Gabriele Bassi, Nicholas Favalli, Emanuele Puca, Dario Neri, Sebastian Oehler & Samuele Cazzamalli

  2. Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy

    Andrea Ciamarone

  3. Department of Surgical, Medical, Molecular Pathology and Critical Area, University of Pisa and Azienda Ospedaliero Universitaria Pisana, Pisa, Italy

    Pinuccia Faviana

  4. Department of Translational Research and Advanced Technologies in Medicine and Surgery, University of Pisa and Azienda Ospedaliero Universitaria Pisana, Pisa, Italy

    Francesco Bartoli

  5. Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology, Zurich, Switzerland

    Dario Neri

  6. Philogen S.p.A, Siena, Italy

    Dario Neri

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Contributions

All authors have contributed to the preparation of this manuscript. T.G., F.M., E.P., S.O., S.C. and D.N. designed the experiments. F.M., M.M. and S.O. synthesized the chemical libraries with support from G.B. and N.F. F.M., S.O. and G.B. performed the selections. F.M., T.G., S.O., G.B. and N.F. analysed the high-throughput screening data. Hits were synthesized and validated by T.G., F.M., A.C. and S.O. I.B. and F.M. expressed and biotinylated the proteins. In vitro and in vivo experiments were performed by T.G., F.M., M.M., S.O., Y.P. and S.C. L.P. and E.G. performed mass spectrometry experiments, including ex vivo biodistribution studies with SMDCs. P.F. and F.B. performed, analysed and interpreted ACP3 and PSMA immunohistochemistry staining on human healthy tissues and prostate cancer lesions.

Corresponding authors

Correspondence to Sebastian Oehler or Samuele Cazzamalli.

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Competing interests

D.N. is the co-founder, CEO, CSO and president of the Scientific Advisory Board of Philogen. T.G., F.M., I.B., M.M., Y.S.P.K., L.P., E.G., N.F., E.P., S.O. and S.C. are employed by Philochem AG, the research and development unit of the Philogen group. Some of the findings included in this manuscript have been patented. T.G., F.M., S.O., S.C., D.N. and Y.S.P.K. are listed in the patent application PCT/EP2024/080350 (filed on 25 October 2024). The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Additional ACP3 and PSMA IHC staining results on Prostate Cancer tissue sections (primary and metastatic lesions).

Gleason and H-scores are presented along with the specific patient ID from which the samples were obtained (University of Pisa’s biobank, Department of Surgical, Medical, Molecular Pathology, and Critical Area; approval from the Ethical Committee of the University of Pisa, PROTOCOL N. 9989 - 20th of February 2019).

Extended Data Fig. 2 Additional selection fingerprints after screening of SOP-DEL.

Selection fingerprints after screening of SOP-DEL against ACP3 (a), tissue non-specific alkaline phosphatase (TNAP, negative control, b), streptavidin-coated beads (DynabeadsTM MyOneTM Streptavidin C1) in PBS buffer (no protein, negative control, c) or TNAP buffer (no protein, negative control, d). DEL selections were performed in duplicates. Dot colour and size correspond to the normalized sequence counts. (a, left) Total Counts (TCs) = 1,461,935, Average Count (AC) = 1.02, Cut-off = 8; (a, right) TCs = 1,502,438, AC = 1.05, Cut-off = 8; (b, left) TCs = 1,556,461, AC = 1.09, Cut-off = 25; (b, right) TCs = 1,640,950, AC = 1.14, Cut-off = 25; (c) TCs = 1,050,742, AC = 0.73, Cut-off = 15; (d) TCs = 1,580,160, AC = 1.10, Cut-off = 35.

Source data

Extended Data Fig. 3 Additional selection fingerprints after screening of FMA-DEL.

Selection fingerprints after screening of FMA-DEL against prostatic acid phosphatase (ACP3, a), tissue non-specific alkaline phosphatase (TNAP, negative control, b), streptavidin-coated beads (DynabeadsTM MyOneTM Streptavidin C1) in PBS buffer (no protein, negative control, c) or TNAP buffer (no protein, negative control, d). DEL selections were performed in duplicates. Dot colour and size correspond to the normalized sequence counts. (a, left) TCs = 1,299,576, AC = 2.63, Cut-off = 10; a right) TCs = 1,258,947, AC = 2.55, Cut-off = 10; (b, left) TCs = 274,701, AC = 0.56, Cut-off = 10; (b, right) TCs = 253,457, AC = 0.51, Cut-off = 10; (c) TCs = 198,057, AC = 0.40, Cut-off = 10; (d) TCs = 286,279, AC = 0.58, Cut-off = 15.

Source data

Extended Data Fig. 4 Additional colorimetric ACP3 inhibition assays.

(a) Experiments performed with conjugates d9, d10, d11, and d12 from SOP-DEL. (b) Experiment performed with oligonucleotides bearing building block A2723 (d13 and d14) and (c) oligonucleotides bearing building block A2855 (d15 and d16) from FMA-DEL. Colorimetric ACP3 inhibition assay of B1326 (SOP-DEL), B125 (FMA-DEL), and their regioisomer derivatives were further performed (d, e). Possible exit-vector modifications of the benchmark scaffold were explored on the phenyl side (d) with compounds s13a-c, or benzyl side (e) with compounds s14a-c. ProXBM-DOTAGA, derived from compound s13c, was selected as the most potent conjugate, and used for further in vitro and in vivo studies. IC50 values are given as mean, with error bars indicating standard deviations of the replicates (n = 3).

Source data

Extended Data Fig. 5 Selectivity studies with DEL-derived hits by fluorescence polarization.

In comparison to (a) benchmark compound s56, DEL-derived-compounds (b) ProX2-(S)-Fluo (s27a), (c) ProX2-(R)-Fluo (s27b), (d) ProX1-(SS).Fluo (8), (e) ProX3-(S)-Fluo (9), (f) ProX3-(R)-Fluo (s33) presented >50-fold improvement of binding affinity. The molecules presented no-cross reactivity with alternative phosphatases (PLAP and TNAP) and bound to albumins (HSA and MSA) in the micromolar concentration range. Final compound concentration used corresponds to 5 nM for test items. Data is plotted as mean, with error bars indicating standard deviations of the replicates (n = 3, except for PLAP and TNAP in (d) where n = 2).

Source data

Extended Data Fig. 6 Radioligand bead- and cell-based assays.

(a) Radioligand bead-based assay with 177Lu-OncoACP3 (s44) and 177Lu-ProX3-(S)-DOTAGA (12). Magnetic Streptavidin-coated DynabeadsTM M-280 were functionalized with recombinant ACP3 and exposed to test compounds s44 and 12, without (full bars) or with (dashed bars) a 5,000-fold molar excess of blocking cold ACP3 ligands. Assay was performed in accordance with prior literature56. (b) Radioligand cell-based assay with 177Lu-OncoACP3 (s44) and 177Lu-ProX3-(S)-DOTAGA (12). Cells were exposed to test compounds s44 and 12, without (full bars) or with (dashed bars) a 1,000-fold molar excess of blocking cold ACP3 ligands. Assay was performed in accordance with prior literature57. Data is plotted as mean, with error bars indicating standard deviations of the replicates (n = 3).

Source data

Extended Data Fig. 7 Additional Autoradiography results.

Human prostate cancer tissue samples were exposed to ACP3-targeted 177Lu-labelled compounds s44 and 12. Patient IDs are presented on the left (University of Pisa’s tissue biobank).

Source data

Extended Data Fig. 8 Additional in vivo biodistribution studies with ACP3-targeted RLTs in tumour-bearing mice.

(a) Comparison of 177Lu-ProX1-(SS)-DOTAGA (11) and 177Lu-ProX1-(RR)-DOTAGA (s39) at the 24 h time point and 62.5 nmol/kg dose. (b) Comparison of 177Lu-ProX2-DOTAGA enantiomers (s40 or s41) and 177Lu-ProX3-DOTAGA enantiomers (12 or s42) at the 2 h time point and 62.5 nmol/kg dose. (c) Comparison of 177Lu-ProX1-(SS)-DOTAGA (11), 177Lu-ProX3-(S)-DOTAGA (12), and 177Lu-ProXBM-DOTAGA (10) at the 2 h time point and 62.5 nmol/kg dose. Biodistribution studies were performed with BALB/c nu/nu female (a and b) and male (c and d) mice, at a molar activity ranging from 0.05 and 0.8 MBq/nmol (~1 MBq/mouse). Data for all biodistribution studies are presented as mean of %ID g−1 ± standard error of the mean (SEM) (n = 3 mice/group). Overall, differences in tumour uptake of ~10-20% ID/g at early time points were observed when repeating in vivo biodistribution experiments using the HT1080.hACP3 cell line (see also Fig. 5). This observation could be explained by differences in vascular permeability that naturally occur when implanting tumour cells in different animal batches. (d) In vivo biodistribution studies with 68Ga-OncoACP3 (s45, 62.5 nmol/kg) at the 1 h time point. Mice in the pre-blocking group were injected with cold OncoACP3 (50 nmol/mouse, ~2.5 µmol/kg – corresponding to a 40-fold molar excess as compared to the radioactive compound) 30 min before the administration of 68Ga-OncoACP3 (s45). Individual values are represented by circles for which bars display the average group %ID g−1 values. Error bars indicate the standard error of the mean (SEM) (n = 3 mice/group for the 68Ga-OncoACP3, n = 2 mice/group for the pre-blocking and ACP3-negative SK-RC-52.wt groups).

Source data

Extended Data Fig. 9 In vivo biodistribution and PET/CT imaging studies with 68Ga-OncoACP3 in tumour-bearing mice.

(a) In vivo biodistribution study with 68Ga-OncoACP3 (s45, 62.5 nmol/g, ~1 MBq/mouse) at 1 and 2 h post-injection in BALB/c nu/nu male mice (n = 3 mice/group). (b) Maximum intensity projection (MIP) obtained 1 h after injection (15 min exposure time). The arrows indicate the urinary bladder (U; highlighting renal excretion of the compound), the gallbladder (G; highlighting hepatobiliary excretion of the compound), and the tumour (T). (c) MicroPET images were obtained 1 h post-injection of 68Ga-OncoACP3 (s45) in the HT1080.hACP3 xenograft model. The subcutaneous tumour is highlighted by the cross section of the green (coronal), red (transversal) and yellow (para sagittal) planes. Dose: 62.5 nmol/kg (600 MBq/kg). A scale bar is included, displaying %ID g−1 (equal to 1/[g body weight]*SUV*100).

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Extended Data Fig. 10 Additional ex vivo biodistribution studies with DEL-derived ACP3 ligands labelled with AF488.

Ex vivo biodistribution of ProX1-(SS)-AF488 (a, compound 14) and ProX3-(S)-AF488 (b, compound 15) in HT1080.hACP3 tumour-bearing mice, 2 h after intravenous administration. Compounds were administered at the 30 nmol/mouse dose (150 µL/animal, PBS solution). Green = compound 14 or 15, Blue = DAPI staining. Scale bar = 400 µm (full scale) or 100 µm (zoomed-in pictures).

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Detailed information on general procedures, Synthetic protocols, Compound characterization, Instrumentations and Supplemental results.

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Source Data For Figs. 2–5, 7 and 8 and Extended Data Figs. 2–9a (download ZIP )

Source data: DEL selections (.txt files), enzymatic assays, SPR measurements, biodistribution studies, therapy experiments, FP studies, radioligand cell- and bead-based binding assays, flow cytometry analysis, unprocessed confocal microscopy and autoradiography images.

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Georgiev, T., Migliorini, F., Ciamarone, A. et al. Discovery of high-affinity ligands for prostatic acid phosphatase via DNA-encoded library screening enables targeted cancer therapy. Nat. Biomed. Eng 10, 178–191 (2026). https://doi.org/10.1038/s41551-025-01432-6

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