Partially unfolded alpha-lactalbumin forms the oleic acid complex HAMLET, with potent tumoricidal activity. Here we define a peptide-based molecular approach for targeting and killing tumor cells, and evidence of its clinical potential (ClinicalTrials.gov NCT03560479). A 39-residue alpha-helical peptide from alpha-lactalbumin is shown to gain lethality for tumor cells by forming oleic acid complexes (alpha1-oleate). Nuclear magnetic resonance measurements and computational simulations reveal a lipid core surrounded by conformationally fluid, alpha-helical peptide motifs. In a single center, placebo controlled, double blinded Phase I/II interventional clinical trial of non-muscle invasive bladder cancer, all primary end points of safety and efficacy of alpha1-oleate treatment are reached, as evaluated in an interim analysis. Intra-vesical instillations of alpha1-oleate triggers massive shedding of tumor cells and the tumor size is reduced but no drug-related side effects are detected (primary endpoints). Shed cells contain alpha1-oleate, treated tumors show evidence of apoptosis and the expression of cancer-related genes is inhibited (secondary endpoints). The results are especially encouraging for bladder cancer, where therapeutic failures and high recurrence rates create a great, unmet medical need.

Centre for Biomimetic Sensor Science Nanyang Technological University Singapore Singapore

Department of Pathology and Molecular Medicine Motol University Hospital 2nd Faculty of Medicine Charles University Praha Prague Czech Republic

Department of Urology Motol University Hospital 2nd Faculty of Medicine Charles University Praha Prague Czech Republic

Division of Microbiology Immunology and Glycobiology Department of Laboratory Medicine Faculty of Medicine Lund University Lund Sweden

School of Biological Sciences Nanyang Technological University Singapore Singapore

Trinity Biomedical Sciences Institute School of Biochemistry and Immunology Trinity College Dublin The University of Dublin Dublin Ireland

Komentář v

J Urol. 2022 Mar;207(3):727-729. doi: 10.1097/JU.0000000000002363. PubMed

Zobrazit více v PubMed

Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144:646–674. PubMed

Scott AM, Wolchok JD, Old LJ. Antibody therapy of cancer. Nat. Rev. Cancer. 2012;12:278–287. doi: 10.1038/nrc3236. PubMed DOI

Håkansson A, Zhivotovsky B, Orrenius S, Sabharwal H, Svanborg C. Apoptosis induced by a human milk protein. Proc. Natl Acad. Sci. USA. 1995;92:8064–8068. doi: 10.1073/pnas.92.17.8064. PubMed DOI PMC

Svensson M, Håkansson A, Mossberg A-K, Linse S, Svanborg C. Conversion of α-lactalbumin to a protein inducing apoptosis. Proc. Natl Acad. Sci. USA. 2000;97:4221–4226. doi: 10.1073/pnas.97.8.4221. PubMed DOI PMC

Gustafsson L, Leijonhufvud I, Aronsson A, Mossberg AK, Svanborg C. Treatment of skin papillomas with topical alpha-lactalbumin–oleic acid. N. Engl. J. Med. 2004;350:2663–2672. doi: 10.1056/NEJMoa032454. PubMed DOI

Chiti F, Dobson CM. Protein misfolding, amyloid formation, and human disease: a summary of progress over the last decade. Annu. Rev. Biochem. 2017;86:27–68. doi: 10.1146/annurev-biochem-061516-045115. PubMed DOI

Knowles TP, Vendruscolo M, Dobson CM. The amyloid state and its association with protein misfolding diseases. Nat. Rev. Mol. Cell Biol. 2014;15:384–396. doi: 10.1038/nrm3810. PubMed DOI

Ho CSJ, Rydstrom A, Manimekalai MSS, Svanborg C, Grüber G. Low resolution solution structure of HAMLET and the importance of its alpha-domains in tumoricidal activity. PLoS ONE. 2012;7:e53051. doi: 10.1371/annotation/25743e50-5a58-4fb6-b466-9a345311d4a8. PubMed DOI PMC

Pettersson-Kastberg J, et al. Alpha-lactalbumin, engineered to be nonnative and inactive, kills tumor cells when in complex with oleic acid: a new biological function resulting from partial unfolding. J. Mol. Biol. 2009;394:994–1010. doi: 10.1016/j.jmb.2009.09.026. PubMed DOI

Fjell CD, Hiss JA, Hancock RE, Schneider G. Designing antimicrobial peptides: form follows function. Nat. Rev. Drug Discov. 2011;11:37–51. doi: 10.1038/nrd3591. PubMed DOI

Mok KH, Nagashima T, Day IJ, Hore PJ, Dobson CM. Multiple subsets of side-chain packing in partially folded states of α-lactalbumins. Proc. Natl Acad. Sci. USA. 2005;102:8899–8904. doi: 10.1073/pnas.0500661102. PubMed DOI PMC

Fischer W, et al. Human alpha-lactalbumin made lethal to tumor cells (HAMLET) kills human glioblastoma cells in brain xenografts by an apoptosis-like mechanism and prolongs survival. Cancer Res. 2004;64:2105–2112. doi: 10.1158/0008-5472.CAN-03-2661. PubMed DOI

Hien TT, et al. Bladder cancer therapy without toxicity—a dose-escalation study of alpha1–oleate. Int. J. Can. 2020;147:2479–2492. doi: 10.1002/ijc.33019. PubMed DOI

Mossberg AK, Hou Y, Svensson M, Holmqvist B, Svanborg C. HAMLET treatment delays bladder cancer development. J. Urol. 2010;183:1590–1597. doi: 10.1016/j.juro.2009.12.008. PubMed DOI

Puthia M, Storm P, Nadeem A, Hsiung S, Svanborg C. Prevention and treatment of colon cancer by peroral administration of HAMLET (human α-lactalbumin made lethal to tumour cells) Gut. 2014;63:131–142. doi: 10.1136/gutjnl-2012-303715. PubMed DOI PMC

Mossberg AK, et al. Bladder cancers respond to intravesical instillation of (HAMLET human α-lactalbumin made lethal to tumor cells) Int. J. Can. 2007;121:1352–1359. doi: 10.1002/ijc.22810. PubMed DOI

Barlowe C, et al. COPII: a membrane coat formed by Sec proteins that drive vesicle budding from the endoplasmic reticulum. Cell. 1994;77:895–907. doi: 10.1016/0092-8674(94)90138-4. PubMed DOI

Jensen D, Schekman R. COPII-mediated vesicle formation at a glance. J. Cell. Sci. 2010;124:1–4. doi: 10.1242/jcs.069773. PubMed DOI

Sato K, Nakano A. Dissection of COPII subunit-cargo assembly and disassembly kinetics during Sar1p-GTP hydrolysis. Nat. Struct. Mol. Biol. 2005;12:167–174. doi: 10.1038/nsmb893. PubMed DOI

Nadeem A, et al. Protein receptor-independent plasma membrane remodeling by HAMLET: a tumoricidal protein–lipid complex. Sci. Rep. 2015;5:16432. doi: 10.1038/srep16432. PubMed DOI PMC

Kang M, Jeong CW, Kwak C, Kim HH, Ku JH. Single, immediate postoperative instillation of chemotherapy in non-muscle invasive bladder cancer: a systematic review and network meta-analysis of randomized clinical trials using different drugs. Oncotarget. 2016;7:45479–45488. doi: 10.18632/oncotarget.9991. PubMed DOI PMC

Sylvester RJ, et al. Systematic review and individual patient data meta-analysis of randomized trials comparing a single immediate instillation of chemotherapy after transurethral resection with transurethral resection alone in patients with stage pTa–pT1 urothelial carcinoma of the bladder: which patients benefit from the instillation? Eur. Urol. 2016;69:231–244. doi: 10.1016/j.eururo.2015.05.050. PubMed DOI

Khare SR, et al. Quality indicators in the management of bladder cancer: a modified Delphi study. Urol. Oncol. 2017;35:328–334. doi: 10.1016/j.urolonc.2016.12.003. PubMed DOI

Ho J, Nadeem A, Rydström A, Puthia M, Svanborg C. Targeting of nucleotide-binding proteins by HAMLET—a conserved tumor cell death mechanism. Oncogene. 2016;35:897–907. doi: 10.1038/onc.2015.144. PubMed DOI

Lawler PR, Lawler J. Molecular basis for the regulation of angiogenesis by thrombospondin-1 and -2. Cold Spring Harb. Perspect. Med. 2012;2:a006627. doi: 10.1101/cshperspect.a006627. PubMed DOI PMC

Antoni S, et al. Bladder cancer incidence and mortality: a global overview and recent trends. Eur. Urol. 2017;71:96–108. doi: 10.1016/j.eururo.2016.06.010. PubMed DOI

Sievert KD, et al. Economic aspects of bladder cancer: what are the benefits and costs? World J. Urol. 2009;27:295–300. doi: 10.1007/s00345-009-0395-z. PubMed DOI PMC

van Rhijn BW, et al. Recurrence and progression of disease in non–muscle-invasive bladder cancer: from epidemiology to treatment strategy. Eur. Urol. 2009;56:430–442. doi: 10.1016/j.eururo.2009.06.028. PubMed DOI

Hu Q, Sun W, Wang C, Gu Z. Recent advances of cocktail chemotherapy by combination drug delivery systems. Adv. Drug Deliv. Rev. 2016;98:19–34. doi: 10.1016/j.addr.2015.10.022. PubMed DOI PMC

Sharma P, Allison JP. Immune checkpoint targeting in cancer therapy: toward combination strategies with curative potential. Cell. 2015;161:205–214. doi: 10.1016/j.cell.2015.03.030. PubMed DOI PMC

Ourfali S, et al. Recurrence rate and cost consequence of the shortage of Bacillus Calmette-Guérin connaught strain for bladder cancer patients. Eur. Urol. Focus. 2021;7:111–116. doi: 10.1016/j.euf.2019.04.002. PubMed DOI

Dang CV. MYC on the path to cancer. Cell. 2012;149:22–35. doi: 10.1016/j.cell.2012.03.003. PubMed DOI PMC

Chevallier D, Carette D, Segretain D, Gilleron J, Pointis G. Connexin 43 a check-point component of cell proliferation implicated in a wide range of human testis diseases. Cell. Mol. Life Sci. 2013;70:1207–1220. PubMed PMC

Ghosh S, Kumar A, Chandna S. Connexin-43 downregulation in G2/M phase enriched tumour cells causes extensive low-dose hyper-radiosensitivity (HRS) associated with mitochondrial apoptotic events. Cancer Lett. 2015;363:46–59. doi: 10.1016/j.canlet.2015.03.046. PubMed DOI

Kamat AM, et al. KEYNOTE-676: Phase III study of BCG and pembrolizumab for persistent/recurrent high-risk NMIBC. Future Oncol. 2020;16:507–516. doi: 10.2217/fon-2019-0817. PubMed DOI

Lamm DL. Intravesical therapy for superficial bladder cancer: slow but steady progress. J. Clin. Oncol. 2003;21:4259–4260. doi: 10.1200/JCO.2003.08.099. PubMed DOI

Kamat AM, et al. Evidence-based assessment of current and emerging bladder-sparing therapies for non–muscle-invasive bladder cancer after Bacillus Calmette-Guerin therapy: a systematic review and meta-analysis. Eur. Urol. Oncol. 2020;3:318–340. doi: 10.1016/j.euo.2020.02.006. PubMed DOI

de Jong JJ, Hendricksen K, Rosier M, Mostafid H, Boormans JL. Hyperthermic intravesical chemotherapy for BCG unresponsive non-muscle invasive bladder cancer patients. Bladder Cancer. 2018;4:395–401. doi: 10.3233/BLC-180191. PubMed DOI PMC

Colombo R, et al. Multicentric study comparing intravesical chemotherapy alone and with local microwave hyperthermia for prophylaxis of recurrence of superficial transitional cell carcinoma. J. Clin. Oncol. 2003;21:4270–4276. doi: 10.1200/JCO.2003.01.089. PubMed DOI

Arends TJH, et al. Results of a randomised controlled trial comparing intravesical chemohyperthermia with mitomycin C versus Bacillus Calmette-Guérin for adjuvant treatment of patients with intermediate- and high-risk non-muscle-invasive bladder cancer. Eur. Urol. 2016;69:1046–1052. doi: 10.1016/j.eururo.2016.01.006. PubMed DOI

Boorjian SA, et al. Intravesical nadofaragene firadenovec gene therapy for BCG-unresponsive non-muscle-invasive bladder cancer: a single-arm, open-label, repeat-dose clinical trial. Lancet Oncol. 2021;22:107–117. doi: 10.1016/S1470-2045(20)30540-4. PubMed DOI PMC

Babjuk M, et al. EAU guidelines on non–muscle-invasive urothelial carcinoma of the bladder: update 2016. Eur. Urol. 2017;71:447–461. doi: 10.1016/j.eururo.2016.05.041. PubMed DOI

Rosenthal, D. L., Wojcik, E. M. & Kurtycz, D. F. In The Paris System for Reporting Urinary Cytology (eds Rosenthal, D. L., Wojcik, E. M. & Kurtycz, D. F.) (Springer, 2016).

VandenBussche C. A review of the paris system for reporting urinary cytology. Cytopathology. 2016;27:153–156. doi: 10.1111/cyt.12345. PubMed DOI

Storm P, et al. A unifying mechanism for cancer cell death through Ion channel activation by HAMLET. PLoS ONE. 2013;8:e58578. doi: 10.1371/journal.pone.0058578. PubMed DOI PMC

States DJ, Haberkorn RA, Ruben DJ. A two-dimensional nuclear overhauser experiment with pure absorption phase in four quadrants. J. Magn. Reson. 1982;48:286–292.

Pelta MD, Barjat H, Morris GA, Davis AL, Hammond SJ. Pulse sequences for high-resolution diffusion-ordered spectroscopy (HR-DOSY) Magn. Reson. Chem. 1998;36:706–714. doi: 10.1002/(SICI)1097-458X(199810)36:10<706::AID-OMR363>3.0.CO;2-W. DOI

Jones JA, Wilkins DK, Smith LJ, Dobson CM. Characterisation of protein unfolding by NMR diffusion measurements. J. Biomol. NMR. 1997;10:199–203. doi: 10.1023/A:1018304117895. DOI

Shimizu A, Ikeguchi M, Sugai S. Appropriateness of DSS and TSP as internal references for (1)H NMR studies of molten globule proteins in aqueous media. J. Biomol. NMR. 1994;4:859–862. doi: 10.1007/BF00398414. PubMed DOI

SEC: Size Exclusion Chromatography (Tosoh Bioscience GmbH, 2017), https://www.separations.eu.tosohbioscience.com/solutions/hplc-products/size-exclusion.

Yang J, et al. The I-TASSER Suite: protein structure and function prediction. Nat. Methods. 2015;12:7–8. doi: 10.1038/nmeth.3213. PubMed DOI PMC

Lindorff-Larsen K, et al. Improved side-chain torsion potentials for the Amber ff99SB protein force field. Proteins. 2010;78:1950–1958. doi: 10.1002/prot.22711. PubMed DOI PMC

Jorgensen WL, Chandrasekhar J, Madura JD, Impey RW, Klein ML. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 1983;79:926–935. doi: 10.1063/1.445869. DOI

Gaussian 09 (Gaussian, Inc., 2016).

Bayly CI, Cieplak P, Cornell W, Kollman PA. A well-behaved electrostatic potential based method using charge restraints for deriving atomic charges: the RESP model. J. Phys. Chem. A. 1993;97:10269–10280. doi: 10.1021/j100142a004. DOI

Van Der Spoel D, et al. GROMACS: fast, flexible, and free. J. Comput. Chem. 2005;26:1701–1718. doi: 10.1002/jcc.20291. PubMed DOI

Darden T, York D, Pedersen L. Particle mesh Ewald: an N log(N) method for Ewald sums in large systems. J. Chem. Phys. 1993;98:10089–10092. doi: 10.1063/1.464397. DOI

Bussi G, Donadio D, Parrinello M. Canonical sampling through velocity rescaling. J. Chem. Phys. 2007;126:014101. doi: 10.1063/1.2408420. PubMed DOI

Wang L, Friesner RA, Berne BJ. Replica exchange with solute scaling: a more efficient version of replica exchange with solute tempering (REST2) J. Phys. Chem. B. 2011;115:9431–9438. doi: 10.1021/jp204407d. PubMed DOI PMC

Mu Y, Nguyen PH, Stock G. Energy landscape of a small peptide revealed by dihedral angle principal component analysis. Proteins. 2005;58:45–52. doi: 10.1002/prot.20310. PubMed DOI

Kabsch W, Sander C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers. 1983;22:2577–2637. doi: 10.1002/bip.360221211. PubMed DOI

Clinical and molecular response to alpha1-oleate treatment in patients with bladder cancer

Similar immune responses to alpha1-oleate and Bacillus Calmette-Guérin treatment in patients with bladder cancer

BAMLET administration via drinking water inhibits intestinal tumor development and promotes long-term health

A scientific journey from discovery to validation of efficacy in cancer patients: HAMLET and alpha1-oleate

Zobrazit více v PubMed

ClinicalTrials.gov
NCT03560479