Personal neoantigen vaccines have been envisioned as an effective approach to induce, amplify and diversify antitumor T cell responses. To define the long-term effects of such a vaccine, we evaluated ...the clinical outcome and circulating immune responses of eight patients with surgically resected stage IIIB/C or IVM1a/b melanoma, at a median of almost 4 years after treatment with NeoVax, a long-peptide vaccine targeting up to 20 personal neoantigens per patient ( NCT01970358 ). All patients were alive and six were without evidence of active disease. We observed long-term persistence of neoantigen-specific T cell responses following vaccination, with ex vivo detection of neoantigen-specific T cells exhibiting a memory phenotype. We also found diversification of neoantigen-specific T cell clones over time, with emergence of multiple T cell receptor clonotypes exhibiting distinct functional avidities. Furthermore, we detected evidence of tumor infiltration by neoantigen-specific T cell clones after vaccination and epitope spreading, suggesting on-target vaccine-induced tumor cell killing. Personal neoantigen peptide vaccines thus induce T cell responses that persist over years and broaden the spectrum of tumor-specific cytotoxicity in patients with melanoma.
High-throughput genome sequencing and computation have enabled rapid identification of targets for personalized medicine, including cancer vaccines. Synthetic peptides are an established mode of ...cancer vaccine delivery, but generating the peptides for each patient in a rapid and affordable fashion remains difficult. High-throughput peptide synthesis technology is therefore urgently needed for patient-specific cancer vaccines to succeed in the clinic. Previously, we developed automated flow peptide synthesis technology that greatly accelerates the production of synthetic peptides. Herein, we show that this technology permits the synthesis of high-quality peptides for personalized medicine. Automated flow synthesis produces 30-mer peptides in less than 35 minutes and 15- to 16-mer peptides in less than 20 minutes. The purity of these peptides is comparable with or higher than the purity of peptides produced by other methods. This work illustrates how automated flow synthesis technology can enable customized peptide therapies by accelerating synthesis and increasing purity. We envision that implementing this technology in clinical settings will greatly increase capacity to generate clinical-grade peptides on demand, which is a key step in reaching the full potential of personalized vaccines for the treatment of cancer and other diseases.
Effective anti-tumour immunity in humans has been associated with the presence of T cells directed at cancer neoantigens, a class of HLA-bound peptides that arise from tumour-specific mutations. They ...are highly immunogenic because they are not present in normal tissues and hence bypass central thymic tolerance. Although neoantigens were long-envisioned as optimal targets for an anti-tumour immune response, their systematic discovery and evaluation only became feasible with the recent availability of massively parallel sequencing for detection of all coding mutations within tumours, and of machine learning approaches to reliably predict those mutated peptides with high-affinity binding of autologous human leukocyte antigen (HLA) molecules. We hypothesized that vaccination with neoantigens can both expand pre-existing neoantigen-specific T-cell populations and induce a broader repertoire of new T-cell specificities in cancer patients, tipping the intra-tumoural balance in favour of enhanced tumour control. Here we demonstrate the feasibility, safety, and immunogenicity of a vaccine that targets up to 20 predicted personal tumour neoantigens. Vaccine-induced polyfunctional CD4
and CD8
T cells targeted 58 (60%) and 15 (16%) of the 97 unique neoantigens used across patients, respectively. These T cells discriminated mutated from wild-type antigens, and in some cases directly recognized autologous tumour. Of six vaccinated patients, four had no recurrence at 25 months after vaccination, while two with recurrent disease were subsequently treated with anti-PD-1 (anti-programmed cell death-1) therapy and experienced complete tumour regression, with expansion of the repertoire of neoantigen-specific T cells. These data provide a strong rationale for further development of this approach, alone and in combination with checkpoint blockade or other immunotherapies.
Neoantigens, which are derived from tumour-specific protein-coding mutations, are exempt from central tolerance, can generate robust immune responses
and can function as bona fide antigens that ...facilitate tumour rejection
. Here we demonstrate that a strategy that uses multi-epitope, personalized neoantigen vaccination, which has previously been tested in patients with high-risk melanoma
, is feasible for tumours such as glioblastoma, which typically have a relatively low mutation load
and an immunologically 'cold' tumour microenvironment
. We used personalized neoantigen-targeting vaccines to immunize patients newly diagnosed with glioblastoma following surgical resection and conventional radiotherapy in a phase I/Ib study. Patients who did not receive dexamethasone-a highly potent corticosteroid that is frequently prescribed to treat cerebral oedema in patients with glioblastoma-generated circulating polyfunctional neoantigen-specific CD4
and CD8
T cell responses that were enriched in a memory phenotype and showed an increase in the number of tumour-infiltrating T cells. Using single-cell T cell receptor analysis, we provide evidence that neoantigen-specific T cells from the peripheral blood can migrate into an intracranial glioblastoma tumour. Neoantigen-targeting vaccines thus have the potential to favourably alter the immune milieu of glioblastoma.
How the genomic features of a patient's cancer relate to individual disease kinetics remains poorly understood. Here we used the indolent growth dynamics of chronic lymphocytic leukaemia (CLL) to ...analyse the growth rates and corresponding genomic patterns of leukaemia cells from 107 patients with CLL, spanning decades-long disease courses. We found that CLL commonly demonstrates not only exponential expansion but also logistic growth, which is sigmoidal and reaches a certain steady-state level. Each growth pattern was associated with marked differences in genetic composition, the pace of disease progression and the extent of clonal evolution. In a subset of patients, whose serial samples underwent next-generation sequencing, we found that dynamic changes in the disease course of CLL were shaped by the genetic events that were already present in the early slow-growing stages. Finally, by analysing the growth rates of subclones compared with their parental clones, we quantified the growth advantage conferred by putative CLL drivers in vivo.
Recent studies have highlighted the promise of targeting tumor neoantigens to generate potent antitumor immune responses and provide strong motivation for improving our understanding of ...antigen-T-cell receptor (TCR) interactions. Advances in single-cell sequencing technologies have opened the door for detailed investigation of the TCR repertoire, providing paired information from TCRα and TCRβ, which together determine specificity. However, a need remains for efficient methods to assess the specificity of discovered TCRs. We developed a streamlined approach for matching TCR sequences with cognate antigen through on-demand cloning and expression of TCRs and screening against candidate antigens. Here, we first demonstrate the system's capacity to identify viral-antigen-specific TCRs and compare the functional avidity of TCRs specific for a given antigen target. We then apply this system to identify neoantigen-specific TCR sequences from patients with melanoma treated with personalized neoantigen vaccines and characterize functional avidity of neoantigen-specific TCRs. Furthermore, we use a neoantigen-prediction pipeline to show that an insertion-deletion mutation in a putative chronic lymphocytic leukemia (CLL) driver gives rise to an immunogenic neoantigen mut-MGA, and use this approach to identify the mut-MGA-specific TCR sequence. This approach provides a means to identify and express TCRs, and then rapidly assess antigen specificity and functional avidity of a reconstructed TCR, which can be applied for monitoring antigen-specific T-cell responses, and potentially for guiding the design of effective T-cell-based immunotherapies.
•A variable chain plasmid library enables rapid TCR cloning and expression to assess specificity and functional avidity.•Antigen-specific T-cell responses to neoantigens in CLL and after personalized neoantigen vaccine in melanoma are characterized.
Display omitted
Abstract
Recent advances in single cell T cell receptor (TCR) sequencing have allowed detailed analysis of TCRαβ pairs. However, efficient methods for probing the specificity of discovered TCRs ...remain limited. We developed a streamlined approach for cloning and expressing TCRs and screening against candidate antigens. A key innovation was the establishment of a plasmid library to encode all variable (V) regions of the TCR, from which any TCR of interest can be assembled with only custom synthesis of short CDR3 regions. A reporter cell system allows rapid detection of TCR antigen specificity and assessment of functional avidity based on cytokine production.
We applied this pipeline to study the intra-tumoral TCR repertoire of a patient with glioblastoma treated with personal neoantigen vaccine. The vaccine consisted of up to 20 synthetic long peptides containing predicted neoepitopes and poly-ICLC adjuvant. Single cell TCR sequencing of CD3+ tumor infiltrating lymphocytes (TILs) and peripheral T cells in vitro expanded against immunizing peptides showed that 4 CD4+ and 2 CD8+ T cell clonotypes in peripheral blood were identical to TILs. The cloning and expression system was used to test their specificity. We identified a TCR from a CD4+ TIL specific for ARHGAP35, a neoantigen targeted by vaccination, and capable of discriminating between mutant and wildtype peptide. This result provides the first demonstration that neoepitope-sensitized T cells can traffic from the periphery to an intracranial GBM. Overall, we demonstrated our ability to identify TCRαβ pairs, express them on demand, and probe antigen specificity. Ongoing studies are characterizing the functional avidity of neoepitope-specific TCRs and reactivity against autologous tumor.
Abstract
Personal vaccines directed at neoantigens arising from tumor mutations can induce neoepitope-specific T cell responses in patients with highly mutated tumors such as melanoma. It remains ...unknown if this approach can be successfully applied in tumors with low mutation frequency. We conducted a phase 1/1b study to determine the safety and feasibility of patient-specific neoantigen-targeting vaccination in patients with newly diagnosed, methylguanine methyltransferase (MGMT) unmethylated glioblastoma, administered following maximum surgical resection and conventional radiotherapy. Tumor-specific mutations were identified by whole-exome sequencing. Each vaccine, composed of up to 20 synthetic long peptides containing predicted tumor neoepitopes admixed with poly-ICLC, was administered subcutaneously on a prime-boost schedule. Among 8 treated patients, adverse events were limited to mild injection site reactions. Seven patients (88%) received a vaccine with ≥10 neoepitope peptides (median 12, range, 7-20), with median time to vaccine initiation of 18.6 weeks from surgery. All patients died from progressive disease. Median progression-free and overall survival was 7.5 months (95% CI: 6.2, 9.7) and 16.8 months (90% CI: 9.6, 21.3), respectively. 3 patients dropped out of the study due to disease progression. We analyzed vaccine responses on 5 patients that got at least one booster immunization. All 3 patients who required dexamethasone during vaccine priming failed to generate interferon-γ responses in peripheral blood mononuclear cells. In contrast, 2 patients who did not receive dexamethasone during vaccine priming, generated robust de novo immune responses against multiple personal neoantigens. Circulating vaccine-induced polyfunctional neoantigen-specific CD4+ and CD8+ T cell responses were enriched for memory and activated phenotypes, and increased numbers of tumor-infiltrating CD4+ and CD8+ T cells were detected in these 2 patients. T cell receptor analysis identified identical clonotypes isolated from post-vaccination glioblastoma tissue and peripheral blood including a clonotype specific for ARHGAP35, a neoantigen targeted by vaccination. To our knowledge we provide the first evidence that tumor specific T cells can traffic from the periphery into glioblastoma tumors and that neoantigen-targeting vaccines can favorably alter the tumor immune milieu of glioblastoma. In conclusion, individualized, multi-neoepitope vaccination is feasible, safe, and generates neoantigen-specific T cell responses in the periphery and intracranial tumors of patients with glioblastoma.
Citation Format: Derin B. Keskin, Itay Tirosh, Annabelle Anandappa, Jing Sun, Nathan D. Mathewson, Sachet A. Shukla, Evisa Gjini, Shuqiang Li, Letitia Li, Anita Giobbie-Hurder, Phuong M. Le, Zhuting Hu, Wandi Zhang, Oriol Olive, Christine McCluskey, Heather Daley, Patrick Y. Wen, Antonio E. Chiocca, Maegan Harden, Niall Lennon, Stacey Gabriel, Gad Getz, Donna Neuberg, Jerome Ritz, Eric S. Lander, Aviv Regev, Kai Wucherpfennig, Mario Suva, Edward F. Fritsch, Rodig Scott, Keith L. Ligon, Kenneth J. Livak, Hacohen Nir, Patrick A. Ott, Catherine J. Wu, David A. Reardon. Personal neoantigen-targeting vaccination generates neoepitope-specific T cell responses in tumors of patients with glioblastoma abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5631.
Abstract
This abstract is being presented as a short talk in the scientific program. A full abstract is printed in the Proffered Abstracts section (PR015) of the Conference Program/Proceedings.
...Citation Format: David A. Braun, Derin B. Keskin, Sachet A. Shukla, Bradley A. McGregor, Nicholas R. Schindler, Eryn Blass, Susan Klaeger, Lucas Pomerance, Siranush Sarkizova, Shuqiang Li, Jackson Southard, Giorgia Moranzoni, Christina B. Pedersen, Yiwen Liu, Steven L. Chang, Michelle S. Hirsch, Nicole R. LeBoeuf, Matthew Mossanen, Vipheaviny Chea, Isabel Carulli, Oriol Olive, Ambica Mehndiratta, Haley Greenslade, Giacomo Oliveira, J. Bryan Iorgulescu, Sabina Signoretti, Jon C. Aster, Liudmila Elagina, Ignaty Leshchiner, Gad Getz, Maegan Harden, Stacey Gabriel, Lars R. Olsen, Donna S. Neuberg, Edward F. Fritsch, Nir Hacohen, Kenneth J. Livak, Steven Carr, Patrick A. Ott, Catherine J. Wu, Toni K. Choueiri. Tumor-specific immunity generated by a personalized neoantigen vaccination incorporating locally delivered ipilimumab in renal cell carcinoma abstract. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr A013.
PDF
