Table Info

Table 3.

Summary of important intrabodies in the preclinical phases of development

Compound name	Protein target	Disease	Trial stage
β42-Specific scFv	Amyloid-β	Alzheimer’s disease	Pre-clinical
Anti-α-syn—PEST nanobodies	α-Syn	Parkinson’s disease	Pre-clinical
Anti-HTT	Huntington	HD	Pre-clinical
EGFR-specific scFvs	EGFR	Epidermoid carcinoma	Pre-clinical
Anti-folate receptor	Folate receptor	Ovarian cancer	Pre-clinical
LL3 (anti-β-catenin intrabodies)	β-catenin	Familial adenomatous polyposis	Pre-clinical

α-Syn: α-synuclein; PEST: proteosome-targeting proline, aspartate or glutamate, serine, and threonine motif

Declarations

Acknowledgments

The authors acknowledge institutional infrastructure support useful for this work.

Author contributions

AD: Conceptualization, Supervision, Writing—original draft, Writing—review & editing. MJ: Formal analysis, Writing—original draft, Writing—review & editing. PD: Writing—original draft, Writing—review & editing.

Conflicts of interest

The authors declare that they have no conflicts of interest.

Ethical approval

Not applicable.

Consent to participate

Not applicable.

Consent to publication

Not applicable.

Availability of data and materials

Not applicable.

Funding

Not applicable.

Copyright

References

Zhan T, Rindtorff N, Betge J, Ebert MP, Boutros M. CRISPR/Cas9 for cancer research and therapy. Semin Cancer Biol. 2019;55:106–19. [DOI] [PubMed]

Khan SH. Genome-editing technologies: concept, pros, and cons of various genome-editing techniques and bioethical concerns for clinical application. Mol Ther Nucleic Acids. 2019;16:326–34. [DOI] [PubMed] [PMC]

Lai AC, Crews CM. Induced protein degradation: an emerging drug discovery paradigm. Nat Rev Drug Discov. 2017;16:101–14. [DOI] [PubMed] [PMC]

Mou H, Smith JL, Peng L, Yin H, Moore J, Zhang XO, et al. CRISPR/Cas9-mediated genome editing induces exon skipping by alternative splicing or exon deletion. Genome Biol. 2017;18:108. [DOI] [PubMed] [PMC]

Kapahnke M, Banning A, Tikkanen R. Random splicing of several exons caused by a single base change in the target exon of CRISPR/Cas9 mediated gene knockout. Cells. 2016;5:45. [DOI] [PubMed] [PMC]

Deribe YL, Pawson T, Dikic I. Post-translational modifications in signal integration. Nat Struct Mol Biol. 2010;17:666–72. [DOI] [PubMed]

Wu Z, Huang R, Yuan L. Crosstalk of intracellular post-translational modifications in cancer. Arch Biochem Biophys. 2019;676:108138. [DOI] [PubMed]

Xu H, Wang Y, Lin S, Deng W, Peng D, Cui Q, et al. PTMD: a database of human disease-associated post-translational modifications. Genomics Proteomics Bioinf. 2018;16:244–51. [DOI] [PubMed] [PMC]

Ciechanover A. The ubiquitin-proteasome proteolytic pathway. Cell. 1994;79:13–21. [DOI] [PubMed]

10.

Zhao L, Zhao J, Zhong K, Tong A, Jia D. Targeted protein degradation: mechanisms, strategies and application. Signal Transduct Target Ther. 2022;7:113. [DOI] [PubMed] [PMC]

11.

Filesi I, Cardinale A, Mattei S, Biocca S. Selective re-routing of prion protein to proteasomes and alteration of its vesicular secretion prevent PrP^Sc formation. J Neurochem. 2007;101:1516–26. [DOI] [PubMed]

12.

Fang Y, Wang S, Han S, Zhao Y, Yu C, Liu H, et al. Targeted protein degrader development for cancer: advances, challenges, and opportunities. Trends Pharmacol Sci. 2023;44:303–17. [DOI] [PubMed]

13.

Han X, Sun Y. PROTACs: a novel strategy for cancer drug discovery and development. MedComm (2020). 2023;4:e290. [DOI] [PubMed] [PMC]

14.

Ishida T, Ciulli A. E3 ligase ligands for PROTACs: how they were found and how to discover new ones. SLAS Discov. 2021;26:484–502. [DOI] [PubMed] [PMC]

15.

Troup RI, Fallan C, Baud MGJ. Current strategies for the design of PROTAC linkers: a critical review. Explor Target Antitumor Ther. 2020;1:273–312. [DOI] [PubMed] [PMC]

16.

Bemis TA, La Clair JJ, Burkart MD. Unraveling the role of linker design in proteolysis targeting chimeras. J Med Chem. 2021;64:8042–52. [DOI] [PubMed]

17.

Sakamoto KM, Kim KB, Kumagai A, Mercurio F, Crews CM, Deshaies RJ. Protacs: chimeric molecules that target proteins to the Skp1–Cullin–F box complex for ubiquitination and degradation. Proc Natl Acad Sci U S A. 2001;98:8554–9. [DOI] [PubMed] [PMC]

18.

Sun X, Wang J, Yao X, Zheng W, Mao Y, Lan T, et al. A chemical approach for global protein knockdown from mice to non-human primates. Cell Discov. 2019;5:10. [DOI] [PubMed] [PMC]

19.

Dai Y, Yue N, Gong J, Liu C, Li Q, Zhou J, et al. Development of cell-permeable peptide-based PROTACs targeting estrogen receptor α. Eur J Med Chem. 2020;187:111967. [DOI] [PubMed]

20.

Hines J, Gough JD, Corson TW, Crews CM. Posttranslational protein knockdown coupled to receptor tyrosine kinase activation with phosphoPROTACs. Proc Natl Acad Sci U S A. 2013;110:8942–7. [DOI] [PubMed] [PMC]

21.

Naito M, Ohoka N, Shibata N. SNIPERs—hijacking IAP activity to induce protein degradation. Drug Discov Today Technol. 2019;31:35–42. [DOI] [PubMed]

22.

Itoh Y, Ishikawa M, Naito M, Hashimoto Y. Protein knockdown using methyl bestatin−ligand hybrid molecules: design and synthesis of inducers of ubiquitination-mediated degradation of cellular retinoic acid-binding proteins. J Am Chem Soc. 2010;132:5820–6. [DOI] [PubMed]

23.

Ohoka N, Okuhira K, Ito M, Nagai K, Shibata N, Hattori T, et al. In vivo knockdown of pathogenic proteins via specific and nongenetic inhibitor of apoptosis protein (IAP)-dependent protein erasers (SNIPERs)^*. J Biol Chem. 2017;292:4556–70. [DOI] [PubMed] [PMC]

24.

Okuhira K, Shoda T, Omura R, Ohoka N, Hattori T, Shibata N, et al. Targeted degradation of proteins localized in subcellular compartments by hybrid small molecules. Mol Pharmacol. 2017;91:159–66. [DOI] [PubMed]

25.

Buckley DL, Raina K, Darricarrere N, Hines J, Gustafson JL, Smith IE, et al. HaloPROTACS: use of small molecule PROTACs to induce degradation of HaloTag fusion proteins. ACS Chem Biol. 2015;10:1831–7. [DOI] [PubMed] [PMC]

26.

Tovell H, Testa A, Maniaci C, Zhou H, Prescott AR, Macartney T, et al. Rapid and reversible knockdown of endogenously tagged endosomal proteins via an optimized HaloPROTAC degrader. ACS Chem Biol. 2019;14:882–92. [DOI] [PubMed] [PMC]

27.

Simpson LM, Macartney TJ, Nardin A, Fulcher LJ, Röth S, Testa A, et al. Inducible degradation of target proteins through a tractable affinity-directed protein missile system. Cell Chem Biol. 2020;27:1164–80.E5. [DOI] [PubMed] [PMC]

28.

Li X, Pu W, Chen S, Peng Y. Therapeutic targeting of RNA-binding protein by RNA-PROTAC. Mol Ther. 2021;29:1940–2. [DOI] [PubMed] [PMC]

29.

Liu J, Chen H, Kaniskan HÜ, Xie L, Chen X, Jin J, et al. TF-PROTACs enable targeted degradation of transcription factors. J Am Chem Soc. 2021;143:8902–10. [DOI] [PubMed] [PMC]

30.

Samarasinghe KTG, Jaime-Figueroa S, Burgess M, Nalawansha DA, Dai K, Hu Z, et al. Targeted degradation of transcription factors by TRAFTACs: TRAnscription factor targeting chimeras. Cell Chem Biol. 2021;28:648–61.E5. [DOI] [PubMed] [PMC]

31.

Estimated number of new cancer cases by world area, 2018^* [Internet]. Source: GLOBOCAN 2018; [cited 2023 May 8]. Available from: https://www.cancer.org/content/dam/cancer-org/research/cancer-facts-and-statistics/global-cancer-facts-and-figures/global-cancer-facts-and-figures-4th-edition.pdf

32.

Dey P, Rathod M, De A. Targeting stem cells in the realm of drug-resistant breast cancer. Breast Cancer (Dove Med Press). 2019;11:115–35. [DOI] [PubMed] [PMC]

33.

Kelm JM, Pandey DS, Malin E, Kansou H, Arora S, Kumar R, et al. PROTAC’ing oncoproteins: targeted protein degradation for cancer therapy. Mol Cancer. 2023;22:62. [DOI] [PubMed] [PMC]

34.

Giardina SF, Valdambrini E, Peel M, Bacolod MD, Rothenberg ML, Lanman RB, et al. Cure-PROs: next-generation targeted protein degraders. J Clin Oncol. 2023;41:e15101. [DOI]

35.

Zhao Q, Lan T, Su S, Rao Y. Induction of apoptosis in MDA-MB-231 breast cancer cells by a PARP1-targeting PROTAC small molecule†. Chem Commun (Camb). 2019;55:369–72. [DOI] [PubMed]

36.

Cromm PM, Samarasinghe KTG, Hines J, Crews CM. Addressing kinase-independent functions of Fak via PROTAC-mediated degradation. J Am Chem Soc. 2018;140:17019–26. [DOI] [PubMed]

37.

Tanjoni I, Walsh C, Uryu S, Tomar A, Nam JO, Mielgo A, et al. PND-1186 FAK inhibitor selectively promotes tumor cell apoptosis in three-dimensional environments. Cancer Biol Ther. 2010;9:764–77. [DOI] [PubMed] [PMC]

38.

Palmer WS, Poncet-Montange G, Liu G, Petrocchi A, Reyna N, Subramanian G, et al. Structure-guided design of IACS-9571, a selective high-affinity dual TRIM24-BRPF1 bromodomain inhibitor. J Med Chem. 2016;59:1440–54. [DOI] [PubMed] [PMC]

39.

Brand M, Jiang B, Bauer S, Donovan KA, Liang Y, Wang ES, et al. Homolog-selective degradation as a strategy to probe the function of CDK6 in AML. Cell Chem Biol. 2019;26:300–6.E9. [DOI] [PubMed] [PMC]

40.

Zhang Q, Khan S, Zhang X, Kuruvilla V, Ghotbaldini S, Wells J, et al. Targeting BCL-XL by protac DT2216 effectively eliminates leukemia cells in T-ALL pre-clinical models. Blood. 2019;134:3870. [DOI]

41.

Degorce SL, Tavana O, Banks E, Crafter C, Gingipalli L, Kouvchinov D, et al. Discovery of proteolysis-targeting chimera molecules that selectively degrade the IRAK3 pseudokinase. J Med Chem. 2020;63:10460–73. [DOI] [PubMed]

42.

Hantschel O, Warsch W, Eckelhart E, Kaupe I, Grebien F, Wagner KU, et al. BCR-ABL uncouples canonical JAK2-STAT5 signaling in chronic myeloid leukemia. Nat Chem Biol. 2012;8:285–93. [DOI] [PubMed]

43.

Lai AC, Toure M, Hellerschmied D, Salami J, Jaime-Figueroa S, Ko E, et al. Modular PROTAC design for the degradation of oncogenic BCR-ABL. Angew Chem Int Ed Engl. 2016;55:807–10. [DOI] [PubMed] [PMC]

44.

Shibata N, Miyamoto N, Nagai K, Shimokawa K, Sameshima T, Ohoka N, et al. Development of protein degradation inducers of oncogenic BCR-ABL protein by conjugation of ABL kinase inhibitors and IAP ligands. Cancer Sci. 2017;108:1657–66. [DOI] [PubMed] [PMC]

45.

King HM, Rana S, Kubica SP, Mallareddy JR, Kizhake S, Ezell EL, et al. Aminopyrazole based CDK9 PROTAC sensitizes pancreatic cancer cells to venetoclax. Bioorg Med Chem Lett. 2021;43:128061. [DOI] [PubMed] [PMC]

46.

Choi HS, Rucker PV, Wang Z, Fan Y, Albaugh P, Chopiuk G, et al. (R)-2-phenylpyrrolidine substituted imidazopyridazines: a new class of potent and selective pan-TRK inhibitors. ACS Med Chem Lett. 2015;6:562–7. [DOI] [PubMed] [PMC]

47.

Chen L, Chen Y, Zhang C, Jiao B, Liang S, Tan Q, et al. Discovery of first-in-class potent and selective tropomyosin receptor kinase degraders. J Med Chem. 2020;63:14562–75. [DOI] [PubMed]

48.

He M, Lv W, Rao Y. Opportunities and challenges of small molecule induced targeted protein degradation. Front Cell Dev Biol. 2021;9:685106. [DOI] [PubMed] [PMC]

49.

Chen Y, Ning Y, Bai G, Tong L, Zhang T, Zhou J, et al. Design, synthesis, and biological evaluation of IRAK4-targeting PROTACs. ACS Med Chem Lett. 2021;12:82–7. [DOI] [PubMed] [PMC]

50.

Cao F, de Weerd S, Chen D, Zwinderman MRH, van der Wouden PE, Dekker FJ. Induced protein degradation of histone deacetylases 3 (HDAC3) by proteolysis targeting chimera (PROTAC). Eur J Med Chem. 2020;208:112800. [DOI] [PubMed]

51.

Wen L, Wei Q. ASK1 inhibitor in chronic kidney disease therapy: from bench to bedside. Kidney360. 2022;3:1128–31. [DOI] [PubMed] [PMC]

52.

Nelson CH, Etchevers K, Yi S, Breckenridge D, Hepner M, Patel U, et al. Pharmacokinetics, safety, and tolerability of selonsertib, an apoptosis signal-regulating kinase 1 (ASK1) inhibitor, following first-in-human single and multiple ascending doses in healthy subjects. Clin Pharmacokinet. 2020;59:1109–17. [DOI] [PubMed]

53.

Barbier P, Zejneli O, Martinho M, Lasorsa A, Belle V, Smet-Nocca C, et al. Role of Tau as a microtubule-associated protein: structural and functional aspects. Front Aging Neurosci. 2019;11:204. [DOI] [PubMed] [PMC]

54.

Kargbo RB. Treatment of Alzheimer’s by PROTAC-Tau protein degradation. ACS Med Chem Lett. 2019;10:699–700. [DOI] [PubMed] [PMC]

55.

Silva MC, Ferguson FM, Cai Q, Donovan KA, Nandi G, Patnaik D, et al. Targeted degradation of aberrant tau in frontotemporal dementia patient-derived neuronal cell models. Elife. 2019;8:e45457. [DOI] [PubMed] [PMC]

56.

Luo G, Li Z, Lin X, Li X, Chen Y, Xi K, et al. Discovery of an orally active VHL-recruiting PROTAC that achieves robust HMGCR degradation and potent hypolipidemic activity in vivo. Acta Pharm Sin B. 2021;11:1300–14. [DOI] [PubMed] [PMC]

57.

Kashima D, Kageoka M, Kimura Y, Horikawa M, Miura M, Nakakido M, et al. A novel cell-based intracellular protein–protein interaction detection platform (SOLIS) for multimodality screening. ACS Synth Biol. 2021;10:990–9. [DOI] [PubMed]

58.

Kovalenko OV, Olland A, Piché-Nicholas N, Godbole A, King D, Svenson K, et al. Atypical antigen recognition mode of a shark immunoglobulin new antigen receptor (IgNAR) variable domain characterized by humanization and structural analysis. J Biol Chem. 2013;288:17408–19. [DOI] [PubMed] [PMC]

59.

Liu JL, Zabetakis D, Brown JC, Anderson GP, Goldman ER. Thermal stability and refolding capability of shark derived single domain antibodies. Mol Immunol. 2014;59:194–9. [DOI] [PubMed]

60.

Messer A, Butler DC. Optimizing intracellular antibodies (intrabodies/nanobodies) to treat neurodegenerative disorders. Neurobiol Dis. 2020;134:104619. [DOI] [PubMed]

61.

Marschall AL, Dübel S, Böldicke T. Specific in vivo knockdown of protein function by intrabodies. MAbs. 2015;7:1010–35. [DOI] [PubMed] [PMC]

62.

Deng C, Xiong J, Gu X, Chen X, Wu S, Wang Z, et al. Novel recombinant immunotoxin of EGFR specific nanobody fused with cucurmosin, construction and antitumor efficiency in vitro. Oncotarget. 2017;8:38568–80. [DOI] [PubMed] [PMC]

63.

Pol J, Kroemer G, Galluzzi L. First oncolytic virus approved for melanoma immunotherapy. Oncoimmunology. 2015;5:e1115641. [DOI] [PubMed] [PMC]

64.

Zhang J, Shrivastava S, Cleveland RO, Rabbitts TH. Lipid-mRNA nanoparticle designed to enhance intracellular delivery mediated by shock waves. ACS Appl Mater Interfaces. 2019;11:10481–91. [DOI] [PubMed] [PMC]

65.

Moghimi B, Zolotukhin I, Sack BK, Herzog RW, Cao O. High efficiency ex vivo gene transfer to primary murine B cells using plasmid or viral vectors. J Genet Syndr Gene Ther. 2011;2:1000103. [DOI] [PubMed] [PMC]

66.

Bachy E, Le Gouill S, Di Blasi R, Sesques P, Manson G, Cartron G, et al. A real-world comparison of tisagenlecleucel and axicabtagene ciloleucel CAR T cells in relapsed or refractory diffuse large B cell lymphoma. Nat Med. 2022;28:2145–54. [DOI] [PubMed] [PMC]

67.

Aires da Silva F, Santa-Marta M, Freitas-Vieira A, Mascarenhas P, Barahona I, Moniz-Pereira J, et al. Camelized rabbit-derived VH single-domain intrabodies against Vif strongly neutralize HIV-1 infectivity. J Mol Biol. 2004;340:525–42. [DOI] [PubMed]

68.

Colby DW, Chu Y, Cassady JP, Duennwald M, Zazulak H, Webster JM, et al. Potent inhibition of huntingtin aggregation and cytotoxicity by a disulfide bond-free single-domain intracellular antibody. Proc Natl Acad Sci U S A. 2004;101:17616–21. Erratum in: Proc Natl Acad Sci U S A. 2005;102:955. [DOI] [PubMed] [PMC]

69.

Reimer E, Somplatzki S, Zegenhagen D, Hänel S, Fels A, Bollhorst T, et al. Molecular cloning and characterization of a novel anti-TLR9 intrabody. Cell Mol Biol Lett. 2013;18:433–46. [DOI] [PubMed] [PMC]

70.

Quevedo CE, Cruz-Migoni A, Bery N, Miller A, Tanaka T, Petch D, et al. Small molecule inhibitors of RAS-effector protein interactions derived using an intracellular antibody fragment. Nat Commun. 2018;9:3169. [DOI] [PubMed] [PMC]

71.

Sokolova EA, Shilova ON, Kiseleva DV, Schulga AA, Balalaeva IV, Deyev SM. HER2-specific targeted toxin DARPin-LoPE: immunogenicity and antitumor effect on intraperitoneal ovarian cancer xenograft model. Int J Mol Sci. 2019;20:2399. [DOI] [PubMed] [PMC]

72.

Tanaka T, Rabbitts TH. Intrabodies based on intracellular capture frameworks that bind the RAS protein with high affinity and impair oncogenic transformation. EMBO J. 2003;22:1025–35. [DOI] [PubMed] [PMC]

73.

Paz K, Brennan LA, Iacolina M, Doody J, Hadari YR, Zhu Z. Human single-domain neutralizing intrabodies directed against Etk kinase: a novel approach to impair cellular transformation. Mol Cancer Ther. 2005;4:1801–9. [DOI] [PubMed]

74.

Van Impe K, Bethuyne J, Cool S, Impens F, Ruano-Gallego D, De Wever O, et al. A nanobody targeting the F-actin capping protein CapG restrains breast cancer metastasis. Breast Cancer Res. 2013;15:R116. [DOI] [PubMed] [PMC]

75.

Slastnikova TA, Ulasov AV, Rosenkranz AA, Sobolev AS. Targeted intracellular delivery of antibodies: the state of the art. Front Pharmacol. 2018;9:1208. [DOI] [PubMed] [PMC]

76.

Kabayama H, Takeuchi M, Tokushige N, Muramatsu SI, Kabayama M, Fukuda M, et al. An ultra-stable cytoplasmic antibody engineered for in vivo applications. Nat Commun. 2020;11:336. [DOI] [PubMed] [PMC]

77.

Xue B, Li H, Guo M, Wang J, Xu Y, Zou X, et al. TRIM21 promotes innate immune response to RNA viral infection through Lys27-linked polyubiquitination of MAVS. J Virol. 2018;92:e00321–18. [DOI] [PubMed] [PMC]

78.

Clift D, So C, McEwan WA, James LC, Schuh M. Acute and rapid degradation of endogenous proteins by Trim-Away. Nat Protoc. 2018;13:2149–75. Erratum in: Nat Protoc. 2019;14:2596. [DOI] [PubMed]

79.

Clift D, McEwan WA, Labzin LI, Konieczny V, Mogessie B, James LC, et al. A method for the acute and rapid degradation of endogenous proteins. Cell. 2017;171:1692–706.E18. [DOI] [PubMed] [PMC]

80.

Nag S, Rahaman S, Guha A, Ray PS. An AKT1-and TRIM21-mediated phosphodegron controls proteasomal degradation of HuR enabling cell survival under heat shock. iScience. 2023;26:106307. [DOI] [PubMed] [PMC]

81.

Dey P, Joshi M, Mujawar A, Malhotra R, De A. Direct knockdown of phospho-PTM targets mediated by TRIM21 can improve personalized treatment in breast cancer. Cell Oncol (Dordr). 2022;45:873–91. [DOI] [PubMed]

82.

Chen G, Kong Y, Li Y, Huang A, Wang C, Zhou S, et al. A promising intracellular protein-degradation strategy: TRIMbody-Away technique based on nanobody fragment. Biomolecules. 2021;11:1512. [DOI] [PubMed] [PMC]

83.

Du S, Liew SS, Zhang CW, Du W, Lang W, Yao CCY, et al. Cell-permeant bioadaptors for cytosolic delivery of native antibodies: a “Mix-and-Go” approach. ACS Cent Sci. 2020;6:2362–76. [DOI] [PubMed] [PMC]

84.

Mevissen TE, Prasad AV, Walter JC. Cell-free Trim-Away reveals the mechanism of antibody-mediated protein degradation by TRIM21. BioRxiv [Preprint]. 2022 [cited 2023 Jan 8]. Available from: https://www.biorxiv.org/content/10.1101/2022.07.23.501259v1

85.

Liu YX, Wan S, Yang XQ, Wang Y, Gan WJ, Ye WL, et al. TRIM21 is a druggable target for the treatment of metastatic colorectal cancer through ubiquitination and activation of MST2. Cell Chem Biol. 2023;30:709–25.E6. [DOI] [PubMed]

86.

Mao S, Cai X, Niu S, Wei J, Jiang N, Deng H, et al. TRIM21 promotes ubiquitination of SARS-CoV-2 nucleocapsid protein to regulate innate immunity. J Med Virol. 2023;95:e28719. [DOI] [PubMed]