Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

ROR1-targeting switchable CAR-T cells for cancer therapy

Abstract

The success of chimeric antigen receptor T cell (CAR-T) therapy in the treatment of hematologic malignancies has prompted the development of numerous CAR-T technologies, including switchable CAR-T (sCAR-T) systems that combine a universal CAR-T with bispecific adapter proteins. Owing to their controllability and versatility, sCAR-Ts have received considerable attention. To explore the therapeutic utility of sCAR-Ts targeting the receptor tyrosine kinase ROR1, which is expressed in hematologic and solid malignancies, and to identify bispecific adaptor proteins that efficiently mediate universal CAR-T engagement, a panel of switches based on ROR1-targeting Fabs with different epitopes and affinities was compared in in vitro and in vivo models of ROR1-expressing cancers. For switches targeting overlapping or identical epitopes, potency correlated with affinity. Surprisingly, however, we identified a switch targeting a unique epitope with low affinity but mediating potent and selective antitumor activity in vitro and in vivo. Converted to a conventional CAR-T, the same anti-ROR1 mAb (324) outperformed a clinically investigated conventional CAR-T that is based on an anti-ROR1 mAb (R12) with ~200-fold higher affinity. Thus, demonstrating therapeutic utility on their own, sCAR-Ts also facilitate higher throughput screening for the identification of conventional CAR-T candidates for preclinical and clinical studies.

This is a preview of subscription content, access via your institution

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: A ROR1-targeting sCAR-T system.
Fig. 2: Orthogonal binding of anti-ROR1 Fab switches to effector cells and target cells.
Fig. 3: Switch-mediated in vitro cytotoxicity of sCAR-Ts.
Fig. 4: Switch-mediated in vitro activation of sCAR-Ts.
Fig. 5: Switch-mediated in vitro cytotoxicity and activation of sCAR-Ts against TNBC cells.
Fig. 6: Switch-mediated in vivo cytotoxicity of sCAR-Ts in an NSG/JeKo-1_ffluc xenograft mouse model.
Fig. 7: In vitro and in vivo activity of sCAR-Ts targeting colon cancer cells.

References

  1. June CH, O’Connor RS, Kawalekar OU, Ghassemi S, Milone MC. CAR T cell immunotherapy for human cancer. Science. 2018;359:1361–5.

    CAS  PubMed  Article  Google Scholar 

  2. Feins S, Kong W, Williams EF, Milone MC, Fraietta JA. An introduction to chimeric antigen receptor (CAR) T-cell immunotherapy for human cancer. Am J Hematol. 2019;94:S3–S9.

    CAS  PubMed  Article  Google Scholar 

  3. Dana H, Chalbatani GM, Jalali SA, Mirzaei HR, Grupp SA, Suarez ER, et al. CAR-T cells: early successes in blood cancer and challenges in solid tumors. Acta Pharm Sin B. 2021;11:1129–47.

    CAS  PubMed  Article  Google Scholar 

  4. Hong M, Clubb JD, Chen YY. Engineering CAR-T cells for next-generation cancer therapy. Cancer Cell. 2020;38:473–88.

    CAS  PubMed  Article  Google Scholar 

  5. Depil S, Duchateau P, Grupp SA, Mufti G, Poirot L. ‘Off-the-shelf’ allogeneic CAR T cells: development and challenges. Nat Rev Drug Discov. 2020;19:185–99.

    CAS  PubMed  Article  Google Scholar 

  6. Townsend MH, Shrestha G, Robison RA, O’Neill KL. The expansion of targetable biomarkers for CAR T cell therapy. J Exp Clin Cancer Res. 2018;37:163.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  7. Moreno-Cortes E, Forero-Forero JV, Lengerke-Diaz PA, Castro JE. Chimeric antigen receptor T cell therapy in oncology —Pipeline at a glance: analysis of the ClinicalTrials.gov database. Crit Rev Oncol Hematol. 2021;159:103239.

    CAS  PubMed  Article  Google Scholar 

  8. Balakrishnan A, Goodpaster T, Randolph-Habecker J, Hoffstrom BG, Jalikis FG, Koch LK, et al. Analysis of ROR1 protein expression in human cancer and normal tissues. Clin Cancer Res. 2017;23:3061–71.

    CAS  PubMed  Article  Google Scholar 

  9. Baskar S, Kwong KY, Hofer T, Levy JM, Kennedy MG, Lee E, et al. Unique cell surface expression of receptor tyrosine kinase ROR1 in human B-cell chronic lymphocytic leukemia. Clin Cancer Res. 2008;14:396–404.

    CAS  PubMed  Article  Google Scholar 

  10. Yang J, Baskar S, Kwong KY, Kennedy MG, Wiestner A, Rader C. Therapeutic potential and challenges of targeting receptor tyrosine kinase ROR1 with monoclonal antibodies in B-cell malignancies. PLoS One. 2011;6:e21018.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  11. Hudecek M, Lupo-Stanghellini MT, Kosasih PL, Sommermeyer D, Jensen MC, Rader C, et al. Receptor affinity and extracellular domain modifications affect tumor recognition by ROR1-specific chimeric antigen receptor T cells. Clin Cancer Res. 2013;19:3153–64.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  12. Berger C, Sommermeyer D, Hudecek M, Berger M, Balakrishnan A, Paszkiewicz PJ, et al. Safety of targeting ROR1 in primates with chimeric antigen receptor-modified T cells. Cancer Immunol Res. 2015;3:206–16.

    CAS  PubMed  Article  Google Scholar 

  13. Srivastava S, Furlan SN, Jaeger-Ruckstuhl CA, Sarvothama M, Berger C, Smythe KS, et al. Immunogenic chemotherapy enhances recruitment of CAR-T cells to lung tumors and improves antitumor efficacy when combined with checkpoint blockade. Cancer Cell. 2021;39:193–208 e110.

    CAS  PubMed  Article  Google Scholar 

  14. Zhao Y, Zhang D, Guo Y, Lu B, Zhao ZJ, Xu X, et al. Tyrosine kinase ROR1 as a target for anti-cancer therapies. Front Oncol. 2021;11:680834.

    PubMed  PubMed Central  Article  Google Scholar 

  15. Menck K, Heinrichs S, Baden C, Bleckmann A. The WNT/ROR pathway in cancer: from signaling to therapeutic intervention. Cells. 2021;10:142.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  16. Hojjat-Farsangi M, Moshfegh A, Daneshmanesh AH, Khan AS, Mikaelsson E, Osterborg A, et al. The receptor tyrosine kinase ROR1-an oncofetal antigen for targeted cancer therapy. Semin Cancer Biol. 2014;29:21–31.

    CAS  PubMed  Article  Google Scholar 

  17. Hudecek M, Schmitt TM, Baskar S, Lupo-Stanghellini MT, Nishida T, Yamamoto TN, et al. The B-cell tumor-associated antigen ROR1 can be targeted with T cells modified to express a ROR1-specific chimeric antigen receptor. Blood. 2010;116:4532–41.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  18. Dave H, Anver MR, Butcher DO, Brown P, Khan J, Wayne AS, et al. Restricted cell surface expression of receptor tyrosine kinase ROR1 in pediatric B-lineage acute lymphoblastic leukemia suggests targetability with therapeutic monoclonal antibodies. PLoS One. 2012;7:e52655.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  19. Srivastava S, Salter AI, Liggitt D, Yechan-Gunja S, Sarvothama M, Cooper K, et al. Logic-gated ROR1 chimeric antigen receptor expression rescues T cell-mediated toxicity to normal tissues and enables selective tumor targeting. Cancer Cell. 2019;35:489–503 e488.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  20. Jensen MC, Riddell SR. Designing chimeric antigen receptors to effectively and safely target tumors. Curr Opin Immunol. 2015;33:9–15.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  21. Sahillioglu AC, Schumacher TN. Safety switches for adoptive cell therapy. Curr Opin Immunol. 2021;74:190–8.

    PubMed  Article  CAS  Google Scholar 

  22. Hoyos V, Savoldo B, Quintarelli C, Mahendravada A, Zhang M, Vera J, et al. Engineering CD19-specific T lymphocytes with interleukin-15 and a suicide gene to enhance their anti-lymphoma/leukemia effects and safety. Leukemia. 2010;24:1160–70.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. Wu CY, Roybal KT, Puchner EM, Onuffer J, Lim WA. Remote control of therapeutic T cells through a small molecule-gated chimeric receptor. Science. 2015;350:aab4077.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  24. Cho JH, Collins JJ, Wong WW. Universal chimeric antigen receptors for multiplexed and logical control of T cell responses. Cell. 2018;173:1426–1438 e1411.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  25. Rodgers DT, Mazagova M, Hampton EN, Cao Y, Ramadoss NS, Hardy IR, et al. Switch-mediated activation and retargeting of CAR-T cells for B-cell malignancies. Proc Natl Acad Sci USA. 2016;113:E459–468.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  26. Cao Y, Rodgers DT, Du J, Ahmad I, Hampton EN, Ma JS, et al. Design of switchable chimeric antigen receptor T cells targeting breast cancer. Angew Chem Int Ed Engl. 2016;55:7520–4.

    CAS  PubMed  Article  Google Scholar 

  27. Viaud S, Ma JSY, Hardy IR, Hampton EN, Benish B, Sherwood L, et al. Switchable control over in vivo CAR T expansion, B cell depletion, and induction of memory. Proc Natl Acad Sci USA. 2018;115:E10898–E10906.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  28. Darowski D, Kobold S, Jost C, Klein C. Combining the best of two worlds: highly flexible chimeric antigen receptor adaptor molecules (CAR-adaptors) for the recruitment of chimeric antigen receptor T cells. mAbs. 2019;11:621–31.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  29. Raj D, Yang MH, Rodgers D, Hampton EN, Begum J, Mustafa A, et al. Switchable CAR-T cells mediate remission in metastatic pancreatic ductal adenocarcinoma. Gut. 2019;68:1052–64.

    CAS  PubMed  Article  Google Scholar 

  30. Qi J, Tsuji K, Hymel D, Burke TR Jr., Hudecek M, Rader C, et al. Chemically programmable and switchable CAR-T therapy. Angew Chem Int Ed Engl. 2020;59:12178–85.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  31. Zahnd C, Spinelli S, Luginbuhl B, Amstutz P, Cambillau C, Pluckthun A. Directed in vitro evolution and crystallographic analysis of a peptide-binding single chain antibody fragment (scFv) with low picomolar affinity. J Biol Chem. 2004;279:18870–7.

    CAS  PubMed  Article  Google Scholar 

  32. Liu X, Jiang S, Fang C, Yang S, Olalere D, Pequignot EC, et al. Affinity-tuned ErbB2 or EGFR chimeric antigen receptor T cells exhibit an increased therapeutic index against tumors in mice. Cancer Res. 2015;75:3596–607.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  33. Benmebarek MR, Karches CH, Cadilha BL, Lesch S, Endres S, Kobold S. Killing mechanisms of chimeric antigen receptor (CAR) T cells. Int J Mol Sci. 2019;20:1283.

    CAS  PubMed Central  Article  Google Scholar 

  34. Larson RC, Maus MV. Recent advances and discoveries in the mechanisms and functions of CAR T cells. Nat Rev Cancer. 2021;21:145–61.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  35. Ghorashian S, Kramer AM, Onuoha S, Wright G, Bartram J, Richardson R, et al. Enhanced CAR T cell expansion and prolonged persistence in pediatric patients with ALL treated with a low-affinity CD19 CAR. Nat Med. 2019;25:1408–14.

    CAS  PubMed  Article  Google Scholar 

  36. Greenman R, Pizem Y, Haus-Cohen M, Goor A, Horev G, Denkberg G, et al. Shaping functional avidity of CAR T cells: affinity, avidity, and antigen density that regulate response. Mol Cancer Ther. 2021;20:872–84.

    CAS  PubMed  Article  Google Scholar 

  37. Peng H, Nerreter T, Chang J, Qi J, Li X, Karunadharma P, et al. Mining naive rabbit antibody repertoires by phage display for monoclonal antibodies of therapeutic utility. J Mol Biol. 2017;429:2954–73.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. Yam PY, Li S, Wu J, Hu J, Zaia JA, Yee JK. Design of HIV vectors for efficient gene delivery into human hematopoietic cells. Mol Ther. 2002;5:479–84.

    CAS  PubMed  Article  Google Scholar 

  39. Wang X, Chang WC, Wong CW, Colcher D, Sherman M, Ostberg JR, et al. A transgene-encoded cell surface polypeptide for selection, in vivo tracking, and ablation of engineered cells. Blood. 2011;118:1255–63.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  40. Qi J, Li X, Peng H, Cook EM, Dadashian EL, Wiestner A, et al. Potent and selective antitumor activity of a T cell-engaging bispecific antibody targeting a membrane-proximal epitope of ROR1. Proc Natl Acad Sci USA. 2018;115:E5467–E5476.

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Nerreter T, Letschert S, Gotz R, Doose S, Danhof S, Einsele H, et al. Super-resolution microscopy reveals ultra-low CD19 expression on myeloma cells that triggers elimination by CD19 CAR-T. Nat Commun. 2019;10:3137.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  42. Lee YG, Chu H, Lu Y, Leamon CP, Srinivasarao M, Putt KS, et al. Regulation of CAR T cell-mediated cytokine release syndrome-like toxicity using low molecular weight adapters. Nat Commun. 2019;10:2681.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  43. Minutolo NG, Sharma P, Poussin M, Shaw LC, Brown DP, Hollander EE, et al. Quantitative control of gene-engineered T-cell activity through the covalent attachment of targeting ligands to a universal immune receptor. J Am Chem Soc. 2020;142:6554–68.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  44. Rennert P, Su L, Dufort F, Birt A, Sanford T, Wu L, et al. A novel CD19-anti-CD20 bridging protein prevents and reverses CD19-negative relapse from CAR19 T cell treatment in vivo. Blood. 2019;134:252.

    Article  Google Scholar 

  45. Arndt C, Fasslrinner F, Loureiro LR, Koristka S, Feldmann A, Bachmann M. Adaptor CAR platforms—next generation of T cell-based cancer immunotherapy. Cancers. 2020;12:1302.

    CAS  PubMed Central  Article  Google Scholar 

  46. Minutolo NG, Hollander EE, Powell DJ Jr. The emergence of universal immune receptor T cell therapy for cancer. Front Oncol. 2019;9:176.

    PubMed  PubMed Central  Article  Google Scholar 

  47. Hombach AA, Schildgen V, Heuser C, Finnern R, Gilham DE, Abken H. T cell activation by antibody-like immunoreceptors: the position of the binding epitope within the target molecule determines the efficiency of activation of redirected T cells. J Immunol. 2007;178:4650–7.

    CAS  PubMed  Article  Google Scholar 

  48. Chmielewski M, Hombach A, Heuser C, Adams GP, Abken H. T cell activation by antibody-like immunoreceptors: increase in affinity of the single-chain fragment domain above threshold does not increase T cell activation against antigen-positive target cells but decreases selectivity. J Immunol. 2004;173:7647–53.

    CAS  PubMed  Article  Google Scholar 

  49. Schmid DA, Irving MB, Posevitz V, Hebeisen M, Posevitz-Fejfar A, Sarria JC, et al. Evidence for a TCR affinity threshold delimiting maximal CD8 T cell function. J Immunol. 2010;184:4936–46.

    CAS  PubMed  Article  Google Scholar 

  50. Lindner SE, Johnson SM, Brown CE, Wang LD. Chimeric antigen receptor signaling: functional consequences and design implications. Sci Adv. 2020;6:eaaz3223.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  51. Picanco-Castro V, Swiech K, Malmegrim KCR, Covas DT. CAR-T cells for cancer treatment: current design and next frontiers. Methods Mol Biol. 2020;2086:1–10.

    CAS  PubMed  Article  Google Scholar 

  52. Jayaraman J, Mellody MP, Hou AJ, Desai RP, Fung AW, Pham AHT, et al. CAR-T design: elements and their synergistic function. EBioMedicine. 2020;58:102931.

    PubMed  PubMed Central  Article  Google Scholar 

  53. Hudecek M, Sommermeyer D, Kosasih PL, Silva-Benedict A, Liu L, Rader C, et al. The nonsignaling extracellular spacer domain of chimeric antigen receptors is decisive for in vivo antitumor activity. Cancer Immunol Res. 2015;3:125–35.

    CAS  PubMed  Article  Google Scholar 

  54. Shabani M, Naseri J, Shokri F. Receptor tyrosine kinase-like orphan receptor 1: a novel target for cancer immunotherapy. Expert Opin Ther Targets. 2015;19:941–55.

    CAS  PubMed  Article  Google Scholar 

  55. Kamrani A, Mehdizadeh A, Ahmadi M, Aghebati-Maleki L, Yousefi M. Therapeutic approaches for targeting receptor tyrosine kinase like orphan receptor-1 in cancer cells. Expert Opin Ther Targets. 2019;23:447–56.

    CAS  PubMed  Article  Google Scholar 

  56. Peng H. Perspectives on the development of antibody-drug conjugates targeting ROR1 for hematological and solid cancers. Antib Ther. 2021;4:222–7.

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Choi MY, Widhopf GF 2nd, Ghia EM, Kidwell RL, Hasan MK, Yu J, et al. Phase I trial: cirmtuzumab inhibits ROR1 signaling and stemness signatures in patients with chronic lymphocytic leukemia. Cell Stem Cell. 2018;22:951–959 e953.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  58. Wang ML, Barrientos JC, Furman RR, Mei M, Barr PM, et al. Zilovertamab vedotin targeting of ROR1 as therapy for lymphoid cancers. N. Engl J Med Evid. 2022;1:1–11.

    Google Scholar 

  59. Labanieh L, Majzner RG, Klysz D, Sotillo E, Fisher CJ, et al. Enhanced safety and efficacy of protease-regulated CAR-T cell receptors. Cell. 2022;185:1745–63.

    CAS  PubMed  Article  Google Scholar 

  60. Acevedo-Rocha CG, Reetz MT, Nov Y. Economical analysis of saturation mutagenesis experiments. Sci Rep. 2015;5:10654.

    PubMed  PubMed Central  Article  Google Scholar 

  61. Rydzek J, Nerreter T, Peng H, Jutz S, Leitner J, Steinberger P, et al. Chimeric antigen receptor library screening using a novel NF-kappaB/NFAT reporter cell platform. Mol Ther. 2019;27:287–99.

    CAS  PubMed  Article  Google Scholar 

  62. Goydel RS, Weber J, Peng H, Qi J, Soden J, Freeth J, et al. Affinity maturation, humanization, and co-crystallization of a rabbit anti-human ROR2 monoclonal antibody for therapeutic applications. J Biol Chem. 2020;295:5995–6006.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  63. Charan J, Kantharia ND. How to calculate sample size in animal studies? J Pharm Pharmacother. 2013;4:303–6.

    Article  Google Scholar 

Download references

Acknowledgements

CR acknowledges support by National Institutes of Health (NIH) grants R01 CA174844, R01 CA181258, R01 CA204484, R21 CA229961, and R21 CA263240, and by the Klorfine Foundation.

Author information

Authors and Affiliations

Authors

Contributions

HP and CR conceived and designed the research. HP conducted the in vitro and in vivo experiments with switches and sCAR-Ts. Supervised by MH, KM and JW selected affinity variants. JC assisted with protein production. TN and MH provided lentiviral vectors and advice for generating CAR-Ts. HP, TN, and CR wrote the manuscript. All authors read, edited, and approved the manuscript.

Corresponding authors

Correspondence to Haiyong Peng or Christoph Rader.

Ethics declarations

Competing interests

CR and HP are named inventors on a licensed patent family (assignee, University of Florida and Boehringer Ingelheim) that claims a set of anti-ROR1 mAbs used in this study including 324 (United States Patent 10,618,959). CR is named inventor on a licensed patent family (assignee, United States of America) that claims another set of anti-ROR1 mAbs used in this study including R12 (United States Patent 9,758,586).

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Peng, H., Nerreter, T., Mestermann, K. et al. ROR1-targeting switchable CAR-T cells for cancer therapy. Oncogene 41, 4104–4114 (2022). https://doi.org/10.1038/s41388-022-02416-5

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41388-022-02416-5

Search

Quick links