Research ArticleCancer

Personalized cancer vaccine effectively mobilizes antitumor T cell immunity in ovarian cancer

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Science Translational Medicine  11 Apr 2018:
Vol. 10, Issue 436, eaao5931
DOI: 10.1126/scitranslmed.aao5931
  • Fig. 1 Production, feasibility, and safety of vaccine.

    (A) Scheme of dendritic cell (DC) vaccine production and administration. (B) Top: DC yield on day 6 is presented as percentage of seeded monocytes. Each dot represents a different patient. Bottom: DC yield correlates with the monocyte counts in the apheresis product. Correlation analysis was performed using the Spearman’s nonparametric correlation test. (C) Percentage of output DCs expressing CD86 or HLA-DR in the vaccine product. Each dot represents a different patient. The line shows the median. DCs from all patients met the release criteria of >60% positivity for both markers. (D) Number of vaccine doses produced per patient. The line shows the median. Each dot represents a different patient. (E) Example of vaccine injection under ultrasound guidance. (F) Number of adverse events in the different treatment cohorts.

  • Fig. 2 Response to immunotherapy.

    (A) Increased recognition of tumor antigens on-vaccination (Vx) treatment in all responder patients (n = 11). The frequency of interferon-γ (IFN-γ)–secreting T cells in response to autologous DCs pulsed with autologous oxidized tumor lysate measured by enzyme-linked immunospot (ELISpot) is shown. These analyses were performed with cryopreserved cells without any prior in vitro expansion. (B) Blood CD8+ and CD4+ T cell response to autologous DCs pulsed with autologous tumor lysate analyzed pre- and on-treatment shown in five patients with available cryopreserved peripheral blood mononuclear cell (PBMCs). The gating strategy is shown in fig. S7. (C) Intracellular cytokine staining showing the functional profiles of CD8+ and CD4+ T cells producing intracellular IL-2, TNFα, and/or IFN-γ in response to autologous DCs pulsed with autologous oxidized tumor lysate. These analyses (B and C) were performed with cryopreserved cells without any prior in vitro expansion. (D) Top: Representative examples of T cell responses against vaccine and autologous tumor cells detected pre- and on-vaccination. The number of spots per 105 PBMCs, as measured by IFN-γ ELISpot assay, is shown. Bottom: Fold increase in frequency of peripheral blood tumor-specific T cells over 24 months. (E) 51Cr release assay of posttherapy CD3/CD28-expanded PBMCs with autologous tumor cells. [R, responder patient (CTE-001) whose T cells produced IFN-γ in response to autologous tumor cells; NR, nonresponder (CTE-0012; *P < 0.003)]. (F) On-treatment CD3/CD28-expanded T cells suppress the growth of autologous tumor in NSG mice more potently than pretreatment T cells (*P = 0.03). (G) Example of two implants; right pelvic and presacral (arrows) regressing in on-treatment CT (bottom) relative to pretreatment CT (top). (H and I) PFS is significantly longer in patients whose on-treatment PBMCs recognized autologous tumor cells (H) or ex vivo autologous tumor lysate–pulsed DCs (I) relative to patients showing no such responses. Differences between averages of variables were compared using two-tailed t test. Percentages between groups were compared using χ2 test.

  • Fig. 3 Increased immune response and survival in patients treated with cyclophosphamide.

    (A) Frequency of T cells responding to autologous tumor lysate–pulsed DCs by IFN-γ ELISpot on-vaccination relative to prevaccination analyzed in patients treated with (n = 12) or with no (n = 10) cyclophosphamide. These analyses were performed with cryopreserved cells without any prior in vitro expansion. (B) Serum IFN-γ concentration is significantly increased relative to baseline 24 hours after the first vaccination in cyclophosphamide-treated patients (Cy; n = 6) but not in patients who did not receive cyclophosphamide (No Cy; n = 6). (C) Serum transforming growth factor–β (TGF-β) concentration is significantly decreased 6 hours after the first vaccination (and about 30 hours after the first dose of intravenous cyclophosphamide) relative to pretreatment baseline in cyclophosphamide-treated patients but not in patients who did not receive cyclophosphamide (n = 8 in each group). (D) OS of patients treated with OCDC vaccine plus bevacizumab (Bev) without (cohort 2) or with cyclophosphamide (cohort 3). (E) OS of patients treated with OCDC vaccine plus bevacizumab and cyclophosphamide (cohort 3) relative to matched historic patients (n = 56) from the same institution treated with bevacizumab and cyclophosphamide. Median survival times were computed using Kaplan-Meier methods. Differences in OS or PFS were assessed using the log-rank test.

  • Fig. 4 Effect of vaccination on neoepitope-specific T cell response.

    (A) Schematic representation of neoantigen prediction and validation algorithm. (B and C) Representative examples of neoepitope-specific T cell responses detected pre- and on-vaccination. (B) CD8+ T cell response detected in patient CTE-0011 pre- and on-vaccination (left) against the mutant (mut) neoepitope from the SEPT9 gene, but not the native [wild type (wt)] peptide (right). These analyses were performed with cryopreserved cells after one round of in vitro stimulation. Validation of SEPT9-specific CD8+ T cells is shown by flow cytometry using multimer (No Ag, no peptide; PHA, phytohemagglutinin). The analysis of multimer staining was performed with cryopreserved cells after multiple rounds of in vitro stimulation, as described in Materials and Methods. (C) CD8+ T cell responses in patient CTE-0019 against two neoepitopes. PLK3-specific T cells are detected only on-vaccination; ODZ3-specific T cells are detected also prevaccination, but their frequency markedly increased after immunization (left). These analyses were performed with cryopreserved cells after one round of in vitro stimulation. The right panels show the lack of cross-reactivity against native (wt) peptides for both PLK3 and ODZ3. These analyses required a second round of in vitro stimulation. For (B) and (C), significant differences in magnitude compared to either prevaccination or to wt peptides are identified with “*” and refer to criteria described in Materials and Methods. Out-of-scale T cell responses (for positive controls) are identified with “>”. (D) Representative intracellular detection of IL-2, TNFα, and/or IFN-γ in viable CD8+ T cells on-vaccination after exposure to neoepitopes. (E) SPICE analysis shows polyfunctional response by neoepitope-specific CD8+ T cells harvested on-vaccination. These analyses (D and E) were performed with cryopreserved cells after one round of in vitro stimulation, as described in Materials and Methods. The gating strategy is shown in fig. S7. (F) Increased recognition of tumor neoepitopes on-vaccination in all analyzed patients for whom material was available. Change in preexisting responses (left) and newly detected responses to neoepitopes (right) are shown separately. These data show analyses performed with cryopreserved cells after one round of in vitro stimulation, as described in Materials and Methods. Differences between averages of variables were compared using two-tailed t test. Percentages between groups were compared using χ2 test.

  • Fig. 5 Neoantigen analysis on patient CTE-0013 who achieved clinical benefit.

    (A) Expansion of preexisting CD8+ T cell response to neoepitope (from ODZ3; patient CTE-0019) on treatment (left) and increase in the functional avidity of neoepitope ODZ3-specific T cell clones pretreatment and on-treatment (right). (B) Expansion of preexisting CD8+ T cell response to neoepitope (from HHAT; patient CTE-0013) on treatment (left) and increase in the functional avidity of neoepitope HHAT-specific T cell clones pretreatment and on-reatment (right). Data from (A) and (B) show analyses performed with cryopreserved cells after one round of in vitro stimulation. (C) Cumulative data showing the marked increase in the functional avidity of neoepitope-specific (HHAT) T cell clones pretreatment (pre) and on-treatment (on). (D) T cell receptor–β (TCRβ) V-J segments recombination landscape of sorted HHAT neoepitope-specific CD8+ T cells from patient CTE-0013 [neoepitope-specific T cell responses pre- and on-treatment were fluorescence-activated cell sorting–sorted using peptide–major histocompatibility complex (pMHC) multimer complexes]. V and J segments are represented according to their chromosomal location on the x and y axes; the frequency of each recombination is shown on the z axis and highlighted by colors. Analysis of TCR repertoires pre- and on-treatment showed a remarkable diversity of the two populations, with few distinct dominant clones, supporting the hypothesis of new priming of neoepitope-specific T cells by the treatment. TCRα V-J segments recombination are shown in fig. S8. (E) Calculated binding mode of peptide KQWLVWLFL on HLA-A*0206. The peptide is shown in ball and stick, colored according to the atom types; with carbon in dark and light gray for residues buried or exposed to the TCR, respectively. The TCR (D83-102501-311993) was removed for clarity. Trp3 and Trp6 are predicted to be buried in the same pocket [see (F)]. Anchor residues Gln2 and Leu9 are deeply buried in the MHC surface. (F) Calculated binding mode of peptide KQWLVWLFL on HLA-A*0206, centered on the peptide Trp3 and Trp6 residues. MHC residues are shown in thick lines, colored according to the atom types, with carbon colored in brown. The side chains of peptide residues Leu4 and Val5 were removed for clarity. (G) Calculated TCR/pMHCs for D1 (pre-Vx)–TCRα (111105)–TCRβ (139954), on the left, and D83 (on-Vx)–TCRα (79822)–TCRβ (139954) on the right. TCRα is colored in light blue. Hydrogen bonds and ionic interactions are shown as thin blue lines. Residues are labeled in brown, black, and blue for MHC, peptide, and TCRα, respectively. More numerous favorable interactions can be found between pMHC and TCRα (79822) (right) than with TCRα (111105) (left) (see table S5). EC50, median effective concentration.

  • Table 1 Clinical data.

    Patient identification number, age, tumor histologic type (ovarian, epithelial ovarian cancer; peritoneal, primary peritoneal serous carcinoma), FIGO stage, number of prior chemotherapy lines (chemos), number of prior recurrences, protocol cohort, combination therapy with vaccine (bev, bevacizumab; cy/bev, cyclophosphamide and bevacizumab), total number of vaccine doses received, clinical response, remission inversion status after vaccination, vaccine, tumor, and new antigen immune response are reported. Vx, vaccine; PD, progressive disease; SD, stable disease; PR, partial response; Y, yes; N, no; NR, nonresponder; R, responder.

    IDAgeTypeStageNo. of
    prior
    chemos
    No. of prior
    recurrences
    CohortTreatmentNumber
    of
    vaccines
    received
    Clinical
    response
    EOS
    Clinical
    response
    Remission
    inversion
    Last
    relapse—
    free time/
    current
    relapse—
    free time
    ratios
    Vaccine immune
    response
    Tumor
    immune
    response
    Neoantigen immune
    response
    LineagePrePost
    CTE-002048OvarianIIIC421Vx3PDPDN0.23
    CTE-000146OvarianIV521Vx28SDSDY1.64NRR
    CTE-000249OvarianIV411Vx7SDSDY7.64RR
    CTE-000363OvarianIIC941Vx5PDSDN0.96NR
    CTE-000459PeritonealIIIC311Vx6SDPDN0.45NRNR
    CTE-000560OvarianIV512Vx/Bev10SDPRY4.95NRR
    CTE-000663OvarianIV632Vx/Bev4PDPDN0.49
    CTE-002159OvarianIIIC412Vx/Bev5PDPDN0.68NR
    CTE-002254OvarianIV632Vx/Bev2SDSDN0.78
    CTE-000760OvarianIIIC212Vx/Bev18SDSDY1.18NRR
    CTE-000854OvarianIIIC312Vx/Bev5PDPDN0.69RCD4NRSEMA3GP>A
    CTE-000972PeritonealIIIC512Vx/Bev8PDPDN0.78NRR
    CTE-001061PeritonealIIIC522Vx/Bev10SDSDY1.20RRTRIM26G>W,
    CHD3L>V,
    EBF4I>S,
    DCHS1P>L
    TRIM26G>W,
    CHD3L>V,
    DCHS1P>L
    CTE-001163OvarianIIIC412Vx/Bev5PDPDN0.21NRNRSEPT9R>H
    CTE-001268OvarianIIIC512Vx/Bev16SDSDN0.52NR
    CTE-001349OvarianIIIB213Vx/Bev/Cy15SDSDY1.06RRHHATL>FHHATL>F
    CTE-001443PeritonealIIIC423Vx/Bev/Cy8PDPDN0.35NR
    CTE-001547OvarianIIIC433Vx/Bev/Cy9PDPDN0.62RNRZCCHC11P>H,
    OR2T3C>Y,
    TNRC6AP>Q
    ZCCHC11P>H,
    TNRC6AP>Q
    CTE-001667OvarianIIIC213Vx/Bev/Cy5SDSDY1.10RR
    CTE-002344OvarianIIIC423Vx/Bev/Cy10SDSDY4.37RCD4+CD8
    CTE-002453OvarianIV313Vx/Bev/Cy11SDPDN0.78RCD4
    CTE-001738OvarianIIIC323Vx/Bev/Cy10SDPRY1.49RCD4+CD8
    CTE-002555OvarianIIIC333Vx/Bev/Cy10SDSDY2.09RCD4+CD8
    CTE-001865OvarianIIIC533Vx/Bev/Cy10SDSDN0.33NR
    CTE-001961OvarianIIIC423Vx/Bev/Cy5SDSDY1.61RRODZ3A>VP2RX5A>G,
    PLK3S>L,
    MUC4H>D,
    DNAAF2S>L,
    KDM5CE>K,
    ODZ3A>V
  • Table 2 Neoepitope analysis of clinical study patients.

    Patient identification number, total number of nonsynonymous somatic mutations, HLA class I haplotypes, and number of predicted neoepitopes.

    PatientNonsynonymous
    mutations
    HLA-AHLA-BHLA-CPredicted
    neoepitopes*
    CTE-000811724:0269:0115:0151:0103:0316:02101
    CTE-00109002:0131:0115:0135:0203:0304:01133
    CTE-001110811:0124:0215:1845:0106:0207:0484
    CTE-00135602:0633:0335:0158:0103:0203:0379
    CTE-001510511:0102:1407:0227:0502:0207:02119
    CTE-00196402:1102:1140:0640:0615:0215:0250

    *Based on netMHC.

    Supplementary Materials

    • www.sciencetranslationalmedicine.org/cgi/content/full/10/436/eaao5931/DC1

      Materials and Methods

      Fig. S1. Schema of the clinical study.

      Fig. S2. Analysis of DC vaccine product.

      Fig. S3. Immune response in patient CTE-0017.

      Fig. S4. Immune competency of patients.

      Fig. S5. Clinical response does not depend on prevaccination T cell gene expression.

      Fig. S6. Validation of CD8+ T cell responses against neoepitopes and wild-type peptides.

      Fig. S7. Representative example of the gating strategy applied for intracellular cytokine staining analyses.

      Fig. S8. TCRα V-J segments recombination landscape of sorted HHAT neoepitope-specific CD8+ T lymphocytes.

      Table S1. Adverse events detected in cohorts 1 to 3.

      Table S2. Comparative parameters in immune responder and nonresponder patients.

      Table S3. Gene set enrichment analysis between clinical responders and nonresponders does not find immune-related pathways differentiating both groups of patients before the vaccination.

      Table S4. Description of validated HLA class I neoepitopes identified.

      Table S5. Protein Data Bank entries used as templates to model the TCRα, TCRβ, MHC, β-microglobulin, and the peptide epitope.

      Table S6. Molecular interactions between the peptide epitope (KQWLVWLFL) and MHC HLA-A*0206, as predicted by homology modeling.

      Table S7. Molecular interactions between the TCR and pMHC, as predicted by homology modeling.

      References (6166)

    • Supplementary Material for:

      Personalized cancer vaccine effectively mobilizes antitumor T cell immunity in ovarian cancer

      Janos L. Tanyi, Sara Bobisse, Eran Ophir, Sandra Tuyaerts, Annalisa Roberti, Raphael Genolet, Petra Baumgartner, Brian J. Stevenson, Christian Iseli, Denarda Dangaj, Brian Czerniecki, Aikaterini Semilietof, Julien Racle, Alexandra Michel, Ioannis Xenarios, Cheryl Chiang, Dimitri S. Monos, Drew A. Torigian, Harvey L. Nisenbaum, Olivier Michielin, Carl H. June, Bruce L. Levine, Daniel J. Powell Jr., David Gfeller, Rosemarie Mick, Urania Dafni, Vincent Zoete, Alexandre Harari, George Coukos, Lana E. Kandalaft*

      *Corresponding author. Email: ana.kandalaft{at}chuv.ch

      Published 11 April 2018, Sci. Transl. Med. 10, eaao5931 (2018)
      DOI: 10.1126/scitranslmed.aao5931

      This PDF file includes:

      • Materials and Methods
      • Fig. S1. Schema of the clinical study.
      • Fig. S2. Analysis of DC vaccine product.
      • Fig. S3. Immune response in patient CTE-0017.
      • Fig. S4. Immune competency of patients.
      • Fig. S5. Clinical response does not depend on prevaccination T cell gene expression.
      • Fig. S6. Validation of CD8+ T cell responses against neoepitopes and wild-type peptides.
      • Fig. S7. Representative example of the gating strategy applied for intracellular cytokine staining analyses.
      • Fig. S8. TCRα V-J segments recombination landscape of sorted HHAT neoepitope-specific CD8+ T lymphocytes.
      • Table S1. Adverse events detected in cohorts 1 to 3
      • Table S2. Comparative parameters in immune responder and nonresponder patients.
      • Table S3. Gene set enrichment analysis between clinical responders and nonresponders does not find immune-related pathways differentiating both groups of patients before the vaccination.

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