Abstract
T cells directed against mutant neo-epitopes drive cancer immunity. However, spontaneous immune recognition of mutations is inefficient. We recently introduced the concept of individualized mutanome vaccines and implemented an RNA-based poly-neo-epitope approach to mobilize immunity against a spectrum of cancer mutations1,2. Here we report the first-in-human application of this concept in melanoma. We set up a process comprising comprehensive identification of individual mutations, computational prediction of neo-epitopes, and design and manufacturing of a vaccine unique for each patient. All patients developed T cell responses against multiple vaccine neo-epitopes at up to high single-digit percentages. Vaccine-induced T cell infiltration and neo-epitope-specific killing of autologous tumour cells were shown in post-vaccination resected metastases from two patients. The cumulative rate of metastatic events was highly significantly reduced after the start of vaccination, resulting in a sustained progression-free survival. Two of the five patients with metastatic disease experienced vaccine-related objective responses. One of these patients had a late relapse owing to outgrowth of β2-microglobulin-deficient melanoma cells as an acquired resistance mechanism. A third patient developed a complete response to vaccination in combination with PD-1 blockade therapy. Our study demonstrates that individual mutations can be exploited, thereby opening a path to personalized immunotherapy for patients with cancer.
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Acknowledgements
We thank J. de Graaf, I. Eichelbrönner, L. Leppin, L. Giese and S. Vogler, D. Becker, M. Dorner, J. Grützner, M. Hossainzadeh, A. Selmi, S. Wessel, C. Ecker, M. Lochschmitt, B. Schmitz, C. Anft, N. Bidmon, H. Schröder, D. Barea Roldán, C. Walter, S. Wöll, C. Rohde, O. Renz, F. Bayer, C. Kröner, B. Otte, T. Stricker, M. Drude S. Petri, M. Mechler, L. Hebich, B. Steege, A. Oelbermann, J. Schwarz, C. Britten, J. C. Castle and B. Pless for technical support, project management and advice. We thank A. Tüttenberg for support with a figure. We thank I. Mellman, L. Delamarre and G. Fine for critical reading of the manuscript. We thank K. Sahin for her advice. The study was supported by the CI3 cluster program of the Federal Ministry of Education and Research (BMBF).
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Contributions
U.S. conceptualized the work and strategy. E.D., Pe.Si., T.O., I.O., I.V., S.A., A.R. and B.K. planned and analysed experiments. E.G., R.R., A.B., C.T. and A.H. did experiments. M.L., V.B., A.D.T., B.S., C.A., A.P. and Pa.So. performed and analysed NGS runs. A.N.K., J.B. and J.C. manufactured the RNA vaccines, O.W., M.W. and M.Z. performed quality assurance. M.M., B.K., S.H., K.H.S., F.M., A.K.-B., D.L. and S.B. managed sample logistics. R.N., C.G., S.G., C.H., J.U. are clinical investigators. C.L. is the principal clinical investigator. U.L., J.D., M.D. and S.K. supported clinical grade assays. U.S., ÖT supported by E.D., M.V., Pe.Si., M.L, M.M., B.S. interpreted data and wrote the manuscript. All authors supported the revision of the manuscript.
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Some of the authors are employees at BioNTech AG (Mainz, Germany) as mentioned in the affiliations. U.S. is stock owner of BioNTech AG (Mainz, Germany). U.S., M.L., B.S., M.V., A.N.K., M.D., A.T., Ö.T. and S.K. are inventors on patents and patent applications, which cover parts of this article.
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Extended data figures and tables
Extended Data Figure 2 Characterization of and examples for CD4+CD8+ and wild-type cross-reactive T cell responses induced by neo-epitope vaccination.
a, Pre- and post-vaccination T cell frequencies against neo-epitope-encoding RNAs (nine patients). In vitro stimulation of T cells as in Fig. 1f, except autologous DCs for read-out were loaded with RNA encoding single neo-epitopes instead of OLPs. 88% (110 out of 125) concordance rate of both measurements, with slightly higher sensitivity of the OLP read-out. b, c, Pre- and post-vaccination CD4+ and CD8+ T-cell-enriched cultures of patient P19 stimulated with the patient’s pentatope RNAs read-out against autologous DCs loaded with OLPs representing mutated neo-epitopes in the ST5 and UTP6 proteins. b, Example for an exclusive de novo CD4+ T cell response. c, Example for a de novo response recognized by both CD4+ and CD8+ T cells at different portions of the mutated sequence. d, CD4+ and CD8+ T cell cultures were quality controlled for purity after stimulation by flow cytometry to exclude cross contamination of the separated in vitro stimulation cultures. e, Wild-type epitope cross-reactivity of post-vaccine CD4+ (top left) and CD8+ (top right) T cells (tested for 55 neo-epitopes in total). Autologous DCs loaded with RNA (not marked), with different OLPs (P1, P2) of the OLP pool encoding the mutated or the wild-type sequence used as read out (marked with ‘P’). Examples of reactivity with mutated and wild-type epitopes analysed by ELISpot is shown for P04 CDC37L1(P186L). f–h, Three examples of wild-type epitope cross-reactive responses. For all three responses immune recognition of autologous DCs that naturally express the wild-type genes was not detected. g, Sanger sequencing of P05 FAM135B in control DCs used in ELISpot (f). Control RNA, luciferase. h, Left, example for wild-type reactivity observed with encoding RNA only but not with OLPs. Right, differential cross-reactivity depended on the assessed OLP in a single T cell response.
Extended Data Figure 3 Characterization of selected TCRs obtained by single-cell cloning.
a, Specificity of NARFL(E62K)-specific TCRs cloned from CD8+ T cells of patient P01. CD8+ T cells transfected with four TCRs directed against a mutation in the NARFL gene were tested by ELISpot for recognition of HLA-A*3101-transfected K562 cells pulsed with individual 15mer OLPs representing the mutant or the wild-type sequence. Control, irrelevant OLPs. b–g, Cloning and characterization of TCRs directed against mutations in PPFIA4 and HPN proteins of P02. b, e, Activation-induced IFNγ-secretion-based single-cell sorting from in vitro stimulation cultures of neo-epitope-specific T cells after co-culture with autologous DCs transfected with RNA (b) or pulsed with OLPs (e) encoding the respective neo-epitope. Control, RNA or OLPs encoding an irrelevant neo-epitope. c, d, f, g, Determination of HLA-restriction and specificity of the cloned TCRs by ELISpot with healthy donor-derived T cells co-transfected with RNAs encoding the identified TCR-α/β chains and peptide-pulsed K562 cells expressing single HLA molecules of the patient. Controls, OLPs encoding an irrelevant neo-epitope (c), HIV-gag (f), target cells without OLPs (d, g), Staphylococcal-enterotoxin-B (SEB) (g).
Extended Data Figure 4 Kinetics and specificity of selected vaccine-induced T cell responses.
a, Kinetics of vaccine-induced responses ex vivo by ELISpot (first three) or multimer staining (last two). b, Reactivity of CD8+ T cells of patient P05 against autologous DCs loaded with individual OLPs or with the predicted HLA-B*0702-restricted minimal epitope (ME, bold sequence). c, Detection of CD8+ T cells recognizing minimal epitope in the same in vitro stimulation cultures by HLA multimer staining. d–f, CD8+ T cell responses of patient P17 against two different HLA-restricted T cell epitopes generated by the same mutation. d, Detection of CD8+ T cells recognizing HSCVMASLR, the best-predicted HLA-A*6801-restricted minimal epitope within P17 RETSAT(P546) (contained in OLP 3 and 4) in post-vaccination TILs from patient P17 by multimer staining. e, ELISpot assay of post-vaccine CD8+ T cells of P17 on autologous DCs loaded with individual neo-epitope OLPs. f, Specificity of two HLA-B*3701-restricted RETSAT(P546S) TCRs obtained from TILs of patient P17 recognizing OLP 1 and 2. Control, HIV-gag OLPs. g, Reactivity of three unvaccinated healthy blood bank donors with predicted neo-epitopes found to be strongly recognized by patients (four neo-epitopes from two patients) by multimer staining.
Extended Data Figure 5 Characterization of vaccine-associated immune responses, tumour immune infiltrates and B2M loss in P04.
a, Time-course analysis of selected T cell responses in blood PBMCs by multimers. b, c, TCRs cloned from neo-epitope-specific CD8+ T cells transfected into healthy donor T cells analysed by ELISpot assay for recognition of K562-A*0201 cells transfected with RNAs encoding the neo-epitopes (b) and loaded with minimal epitopes (c) (FLNA(P639L), HIAKSLFEV; CDC37L1(P183L), FLSDHLYLV). Triplicates (mean ± s.d.) are shown. Controls, respective other minimal peptide epitope. d–f, Comparison of pre-vaccination (lymph node metastasis inguinal right; used for vaccine neo-epitope selection) and post-vaccination (lymph node metastasis iliacal right). d, T cell infiltrates analysed by immunohistochemistry. Tumour cells visualized by anti-Melan-A co-staining. e, Differential gene expression patterns comparing pre- and post-vaccination tumour samples. f, Complete B2M locus deletion in MZ-GaBa-018 but not in the pre-vaccination tumour resectate and autologous PBMCs as shown in coverage profiles of exome data. Deletion start localized in an intron in the PATL2 locus upstream of B2M in the exome capture. g, Killing of MZ-GaBa-018 melanoma cells by neo-epitope RNA-stimulated T cells. Untreated, IFNγ pre-treated or B2M-RNA-transfected melanoma cells used as targets for autologous CD8+ or CD4+ T cells from stimulation cultures (effector:target ratio 50:1). Effects on HLA class I/II surface staining analysed by flow cytometry (lower panel). h, Neo-epitope RNA transcript levels expressed by the autologous tumour cell line MZ-GaBa-018 as analysed from RNA-seq data and plotted as RPKM values.
Supplementary information
Supplementary Table 1
This table lists the neo-epitope vaccine sequence for patient P04. (XLSX 17 kb)
Supplementary Table 2
This table summarizes all identified neo-epitopes across patients. (XLSX 36 kb)
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Sahin, U., Derhovanessian, E., Miller, M. et al. Personalized RNA mutanome vaccines mobilize poly-specific therapeutic immunity against cancer. Nature 547, 222–226 (2017). https://doi.org/10.1038/nature23003
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DOI: https://doi.org/10.1038/nature23003
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