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.

  • Brief Communication
  • Published:

Simultaneous multiple interaction T-cell engaging (SMITE) bispecific antibodies overcome bispecific T-cell engager (BiTE) resistance via CD28 co-stimulation

Leukemia volume 32, pages 1239–1243 (2018)Cite this article

Subjects

The efficacy of early bispecific antibodies redirecting T-cells to eradicate cancer cells was partly limited because of suboptimal effector cell engagement [1]. More efficient T-cell activation has been obtained with single-chain variable fragment (scFv) antibodies, notably bispecific T-cell engagers (BiTEs) [2]. Activity of the CD19/CD3 BiTE blinatumomab in adults and children with chemotherapy-resistant CD19+ B-cell acute lymphoblastic leukemia (B-ALL) led to regulatory drug approval in Europe and the United States. Many other BiTEs, all relying on CD3 signaling without providing co-stimulation, are in clinical testing in several solid tumors and hematologic malignancies [2, 3].

To develop our platform, we generated a series of CD3-directed and CD28-directed bispecific antibodies targeting the cancer cell-associated antigens CD19 and receptor tyrosine kinase-like orphan receptor 1 (ROR1) [9] as well as a CD28-directed bispecific antibody targeting PD-L1. Briefly (see Online Supplement for detailed methods), we used variable domain sequences available from the literature (Supplementary Table 1) for CD19 (blinatumomab), ROR1 (clone R12), CD3 (blinatumomab), CD28 (TGN1412), and PD-L1 (atezolizumab) to build molecules in the canonical BiTE format (Supplementary Fig. 1a). Protein sequences were reverse-translated using human codons and cloned into a modified pCVL lentiviral vector which was then used to transduce FreestyleTM 293-F cells. Secreted protein was extracted from the conditioned media via immobilized metal-affinity chromatography and subsequently polished via size exclusion chromatography (Supplementary Fig. 1b–d). Fractions corresponding to the monomeric proteins were pooled, quantitated, and further analyzed by SDS-PAGE (Supplementary Fig. 1b–d, insets). As source for T-cells, unstimulated peripheral blood mononuclear cells were collected from healthy adult volunteers via leukapheresis under research protocols approved by the Fred Hutch Institutional Review Board after written informed consent was obtained. T-cells were enriched via negative selection through magnetic cell sorting (Pan T-Cell Isolation Kit; Miltenyi Biotec, Auburn, CA, USA), and stored in aliquots in liquid nitrogen [10]. Thawed cell aliquots were used, unlabeled or labeled with CellVue dye (eBioscience, San Diego, CA, USA), in assays without prior pre-stimulation [6, 10,11,12]. To obtain well-controlled experimental models, sublines of human myeloid K562 and human lymphoid CD19+ RCH-ACV and REH cells overexpressing ROR1 were generated through transduction with a pMP71 retrovirus (kindly provided by Dr. Stanley R. Riddell, Fred Hutchinson Cancer Research Center, Seattle, WA). Sublines of cells overexpressing PD-L1 were generated through transduction with a pRRLsin.cPPT.MSCV lentivirus [11, 13, 14] containing a human PD-L1-IRES-Enhanced Green Fluorescent Protein cassette [6]. To quantify T-cell activation, target antigen-negative and target antigen-positive cancer cells were labeled with CellVue dye and incubated in 96-well plates together with unlabeled healthy-donor T-cells at an effector:target (E:T) ratio of 1:1. Parallel cultures were treated with a CD3 antibody (clone OKT3, low endotoxin/azide-free; BioLegend, San Diego, CA, USA) or a CD3 BiTE in combination with either a CD28 antibody (clone CD28.2, low endotoxin/azide-free; BioLegend) or a CD28 BiTE. After 12–24 h, T-cell activation was assessed by flow cytometry after staining of cells with fluorescently labeled antibodies recognizing CD3 (clone UCHT1, FITC-labeled; BD Biosciences, San Jose, CA, USA), CD4 (clone SK3, BV786-labeled; BD Biosciences), CD8 (clone RPA-T8, PE-Cy7-labeled; BD Biosciences), CD25 (clone M-A2451, APC-labeled; BD Biosciences), and CD69 (clone FN50, PE-labeled; BD Biosciences). Using 4’,6-diamidino-2-phenylindole (DAPI) to separate non-viable cells, induction of CD25 and CD69 was analyzed on CellVue dye-negative cells with FlowJo Software (Tree Star, Ashland, OR). To quantify antibody-induced cytotoxicity, cancer cells were incubated in 96-well plates with various concentrations of monoclonal or bispecific antibodies as well as CellVue dye-labeled T-cells at different E:T cell ratios [6, 10,11,12]. After 48 h, cell numbers and drug-induced cytotoxicity, using DAPI to detect non-viable cells, were determined by flow cytometry. AML cells were identified by forward/side scatter properties and negativity for the CellVue dye. Repeated measures one-way or two-way ANOVA method with Tukey’s multiple comparison testing was used for statistical analysis with provision of two-sided P-values (Prism 7.0c; GraphPad [La Jolla, CA, USA]).

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1
Fig. 2

References

  1. Riethmüller G. Symmetry breaking: bispecific antibodies, the beginnings, and 50 years on. Cancer Immun. 2012;12:12.

    PubMed  PubMed Central  Google Scholar 

  2. Yuraszeck T, Kasichayanula S, Benjamin JE. Translation and clinical development of bispecific T-cell engaging antibodies for cancer treatment. Clin Pharmacol Ther. 2017;101:634–45.

    Article  CAS  Google Scholar 

  3. Brinkmann U, Kontermann RE. The making of bispecific antibodies. MAbs. 2017;9:182–12.

    Article  CAS  Google Scholar 

  4. Topp MS, Gökbuget N, Stein AS, Zugmaier G, O’Brien S, Bargou RC, et al. Safety and activity of blinatumomab for adult patients with relapsed or refractory B-precursor acute lymphoblastic leukaemia: a multicentre, single-arm, phase 2 study. Lancet Oncol. 2015;16:57–66.

    Article  CAS  Google Scholar 

  5. von Stackelberg A, Locatelli F, Zugmaier G, Handgretinger R, Trippett TM, Rizzari C, et al. Phase I/phase II study of blinatumomab in pediatric patients with relapsed/refractory acute lymphoblastic leukemia. J Clin Oncol. 2016;34:4381–89.

    Article  Google Scholar 

  6. Laszlo GS, Gudgeon CJ, Harrington KH, Walter RB. T-cell ligands modulate the cytolytic activity of the CD33/CD3 BiTE antibody construct, AMG 330. Blood Cancer J. 2015;5:e340.

    Article  CAS  Google Scholar 

  7. Feucht J, Kayser S, Gorodezki D, Hamieh M, Döring M, Blaeschke F, et al. T-cell responses against CD19+ pediatric acute lymphoblastic leukemia mediated by bispecific T-cell engager (BiTE) are regulated contrarily by PD-L1 and CD80/CD86 on leukemic blasts. Oncotarget. 2016;7:76902–19.

    Article  Google Scholar 

  8. Suntharalingam G, Perry MR, Ward S, Brett SJ, Castello-Cortes A, Brunner MD, et al. Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412. N Engl J Med. 2006;355:1018–28.

    Article  CAS  Google Scholar 

  9. 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.

    Article  CAS  Google Scholar 

  10. Reusch U, Harrington KH, Gudgeon CJ, Fucek I, Ellwanger K, Weichel M, et al. Characterization of CD33/CD3 tetravalent bispecific tandem diabodies (TandAbs) for the treatment of acute myeloid leukemia. Clin Cancer Res. 2016;22:5829–38.

    Article  CAS  Google Scholar 

  11. Laszlo GS, Gudgeon CJ, Harrington KH, Dell’Aringa J, Newhall KJ, Means GD, et al. Cellular determinants for preclinical activity of a novel CD33/CD3 bispecific T-cell engager (BiTE) antibody, AMG 330, against human AML. Blood. 2014;123:554–61.

    Article  CAS  Google Scholar 

  12. Harrington KH, Gudgeon CJ, Laszlo GS, Newhall KJ, Sinclair AM, Frankel SR, et al. The broad anti-AML activity of the CD33/CD3 BiTE antibody construct, AMG 330, is impacted by disease stage and risk. PLoS One. 2015;10:e0135945.

    Article  Google Scholar 

  13. Walter RB, Raden BW, Kamikura DM, Cooper JA, Bernstein ID. Influence of CD33 expression levels and ITIM-dependent internalization on gemtuzumab ozogamicin-induced cytotoxicity. Blood. 2005;105:1295–302.

    Article  CAS  Google Scholar 

  14. Laszlo GS, Harrington KH, Gudgeon CJ, Beddoe ME, Fitzgibbon MP, Ries RE, et al. Expression and functional characterization of CD33 transcript variants in human acute myeloid leukemia. Oncotarget. 2016;7:43281–94.

    Article  Google Scholar 

  15. Krupka C, Kufer P, Kischel R, Zugmaier G, Lichtenegger FS, Köhnke T, et al. Blockade of the PD-1/PD-L1 axis augments lysis of AML cells by the CD33/CD3 BiTE antibody construct AMG 330: reversing a T-cell-induced immune escape mechanism. Leukemia. 2016;30:484–91.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from the Leukemia & Lymphoma Society (Translational Research Program grant no. 6489-16), Alex’s Lemonade Stand Foundation/Cure4Cam Childhood Cancer Foundation (Innovation Grant), Hyundai Hope on Wheels (Scholar Grant), Bezos Family Immunotherapy Initiative (Pilot Award), and the National Institute of Diabetes and Digestive and Kidney Diseases/National Institutes of Health (NIDDK/NIH: P30-DK56465, Co-operative Center of Excellence in Hematology). C.D.G. is supported by a fellowship training grant from the National Heart, Lung, and Blood Institute/NIH (NHLBI/NIH: T32-HL007093). R.B.W. is a Leukemia & Lymphoma Society Scholar in Clinical Research.

Author information

Authors and Affiliations

  1. Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA

    Colin E. Correnti, George S. Laszlo, Willem J. de van der Schueren, Ashok Bandaranayake, Melanie A. Busch, Chelsea J. Gudgeon, Olivia M. Bates, James M. Olson, Christopher Mehlin & Roland B. Walter

  2. Hematology/Oncology Fellowship Program, University of Washington, Seattle, WA, USA

    Colin D. Godwin

  3. Department of Pediatrics, Division of Hematology, University of Washington, Seattle, WA, USA

    James M. Olson

  4. Department of Medicine, Division of Hematology, University of Washington, Seattle, WA, USA

    Roland B. Walter

  5. Department of Epidemiology, University of Washington, Seattle, WA, USA

    Roland B. Walter

Authors
  1. Colin E. Correnti
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. George S. Laszlo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Willem J. de van der Schueren
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Colin D. Godwin
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ashok Bandaranayake
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Melanie A. Busch
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Chelsea J. Gudgeon
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Olivia M. Bates
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. James M. Olson
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Christopher Mehlin
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Roland B. Walter
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Christopher Mehlin or Roland B. Walter.

Ethics declarations

Conflict of interest

RBW has received laboratory research grants and/or clinical trial support from Amgen Inc., Amphivena Therapeutics, Inc., Covagen AG, Aptevo Therapeutics, Inc., and Seattle Genetics, Inc.; has ownership interests with Amphivena Therapeutics, Inc.; and is (or has been) a consultant to Amphivena Therapeutics, Inc., Covagen AG, Emergent Biosolutions, Inc. (now Aptevo Therapeutics, Inc.), Pfizer, Inc., and Seattle Genetics, Inc. The remaining authors declare that they have no conflict of interest.

Additional information

Colin E. Correnti and George S. Laszlo contributed equally to this work.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Correnti, C.E., Laszlo, G.S., de van der Schueren, W.J. et al. Simultaneous multiple interaction T-cell engaging (SMITE) bispecific antibodies overcome bispecific T-cell engager (BiTE) resistance via CD28 co-stimulation. Leukemia 32, 1239–1243 (2018). https://doi.org/10.1038/s41375-018-0014-3

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41375-018-0014-3

This article is cited by

Search

Advanced search

Quick links