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Intratumoral delivery of tavokinogene telseplasmid yields systemic immune responses in metastatic melanoma patients

Open AccessPublished:January 31, 2020DOI:https://doi.org/10.1016/j.annonc.2019.12.008

      Highlights

      • Intratumoral pIL-12 electroporation (Tavo) results in an ORR of 35.7% with CR in 17.9%.
      • 46% of patients have regression in at least 1 uninjected lesion.
      • 25% of patients have regression in all uninjected lesions.
      • Upregulation of immune activation and co-stimulation but also adaptive resistance.

      Background

      Interleukin 12 (IL-12) is a pivotal regulator of innate and adaptive immunity. We conducted a prospective open-label, phase II clinical trial of electroporated plasmid IL-12 in advanced melanoma patients (NCT 01502293).

      Patients and methods

      Patients with stage III/IV melanoma were treated intratumorally with plasmid encoding IL-12 (tavokinogene telseplasmid; tavo), 0.5 mg/ml followed by electroporation (six pulses, 1500 V/cm) on days 1, 5, and 8 every 90 days in the main study and additional patients were treated in two alternative schedule exploration cohorts. Correlative analyses for programmed death-ligand 1 (PD-L1), flow cytometry to assess changes in immune cell subsets, and analysis of immune-related gene expression were carried out on pre- and post-treatment samples from study patients, as well as from additional patients treated during exploration of additional dosing schedules beyond the pre-specified protocol dosing schedule. Response was measured by study-specific criteria to maximize detection of latent and potentially transient immune responses in patients with multiple skin lesions and toxicities were graded by the Common Terminology Criteria for Adverse Events version 4.0 (CTCAE v4.0).

      Results

      The objective overall response rate was 35.7% in the main study (29.8% in all cohorts), with a complete response rate of 17.9% (10.6% in all cohorts). The median progression-free survival in the main study was 3.7 months while the median overall survival was not reached at a median follow up of 29.7 months. A total of 46% of patients in all cohorts with uninjected lesions experienced regression of at least one of these lesions and 25% had a net regression of all untreated lesions. Transcriptomic and immunohistochemistry analysis showed that immune activation and co-stimulatory transcripts were up-regulated but there was also increased adaptive immune resistance.

      Conclusions

      Intratumoral Tavo was well tolerated and led to systemic immune responses in advanced melanoma patients. While tumor regression and increased immune infiltration were observed in treated as well as untreated/distal lesions, adaptive immune resistance limited the response.

      Key words

      Introduction

      The cytokine interleukin 12 (IL-12) occupies a unique niche in the cytokine repertoire bridging the innate and adaptive immune systems.
      • Vignali D.A.A.
      • Kuchroo V.K.
      IL-12 family cytokines: immunological playmakers.
      IL-12 is typically triggered upon pathogen-associated molecular pattern or danger-associated molecular pattern recognition and causes secretion of interferon-γ (IFN-γ) by T cells, natural killer (NK) cells, and dendritic cells (DCs), which in turn causes additional IL-12 production by immune cells. IL-12 causes TH1 polarization, reduces regulatory T cells, and converts myeloid-derived suppressor cells to functional DCs. In addition, IL-12 (and IFN-γ) are crucial third signals sent by cross-presenting DC (cDC1) to naive CD8+ T cells, aiding their transformation into effector T cells.
      • Curtsinger J.M.
      • Mescher M.F.
      Inflammatory cytokines as a third signal for T cell activation.
      Intravenous recombinant IL-12 (rIL-12) has shown clinical efficacy in solid tumor malignancies including renal cell cancer
      • Gollob J.A.
      • Mier J.W.
      • Veenstra K.
      • et al.
      Phase I trial of twice-weekly intravenous interleukin 12 in patients with metastatic renal cell cancer or malignant melanoma: ability to maintain IFN-gamma induction is associated with clinical response.
      and melanoma,
      • Atkins M.B.
      • Lotze M.T.
      • Dutcher J.P.
      • et al.
      High-dose recombinant interleukin 2 therapy for patients with metastatic melanoma: analysis of 270 patients treated between 1985 and 1993.
      albeit with a high level of serious adverse events (AEs). Subcutaneous and intralesional recombinant cytokines have a lower toxicity, but also a much lower efficacy.
      • Motzer R.J.
      • Rakhit A.
      • Schwartz L.H.
      • et al.
      Phase I trial of subcutaneous recombinant human interleukin-12 in patients with advanced renal cell carcinoma.
      In contrast to intralesional and systemic rIL-12, intratumoral injection of plasmid encoding IL-12 (Tavo) leads to sustained cytokine elaboration in the tumor microenvironment in vivo, with minimal systemic exposure. In the syngeneic B16 melanoma model, local IL-12 plasmid electroporation causes regression of both established local and distant (non-treated) lesions, while yielding immune memory to tumor rechallenge.
      • Heller L.
      • Merkler K.
      • Westover J.
      • et al.
      Evaluation of toxicity following electrically mediated interleukin-12 gene delivery in a B16 mouse melanoma model.
      • Heller L.C.
      • Jaroszeski M.J.
      • Coppola D.
      • et al.
      Comparison of electrically mediated and liposome-complexed plasmid DNA delivery to the skin.
      • Lucas M.L.
      • Heller R.
      IL-12 gene therapy using an electrically mediated nonviral approach reduces metastatic growth of melanoma.
      A phase I clinical trial of IL-12 plasmid electroporation established a biologically effective dose and demonstrated the safety of this approach, as well as its preliminary efficacy in increasing intratumoral IL-12 and IFN-γ, yielding sustained, global remissions in several patients after one cycle of therapy. We evaluated Tavo for efficacy and safety in an open-label, phase II trial.

      Methods

       Study design

      This was a prospective, multicenter, open-label, phase II trial (NCT01502293) evaluating the clinical efficacy and safety of Tavo in melanoma patients.

       Patients

      Eligible patients were required to be ≥18 years old with pathologically documented melanoma, with Eastern Cooperative Oncology Group (ECOG) performance status of 0–2, and an unresectable American Joint Committee on Cancer (AJCC) stage IIIB, IIIC, or IV A, B, or C, and two or more melanoma lesions accessible to electroporation. Any prior therapy was permitted. Any treatment-related toxicities resolved to grade 1 or better before study treatment.

       Treatment

      Tavo (IL-12 plasmid, 0.5 mg/ml) was administered on days 1, 5, and 8 of each 90-day cycle (Figure 1) by intratumoral injection at a dose volume of one-quarter of the calculated lesion volume (minimum = 0.1 ml).
      • Daud A.I.
      • DeConti R.C.
      • Andrews S.
      • et al.
      Phase I trial of interleukin-12 plasmid electroporation in patients with metastatic melanoma.
      Electroporation was carried out using six pulses of 1500 V/cm and a pulse width of 100 μs at 1-second intervals (ImmunoPulse, OncoSec Medical, Inc. San Diego, CA). Additional patients treated with the same plasmid dose but on different schedules (dose schedule exploration, supplementary Figure S1, available at Annals of Oncology online) were included in the translational and untreated lesion response analyses.
      Figure thumbnail gr1
      Figure 1(A) CONSORT diagram with the screening and treatment assignments of patients consented to study. (B) Tavokinogene telseplasmid (0.5 mg/ml) was injected at a dose-volume of one-quarter of the calculated lesion volume. Patients were treated on days 1, 5, and 8 of every 90-day treatment cycle. Tumor response assessments were made every 90 days.
      EP, electroporation; i.t., intratumoral.

       Efficacy assessment

      Tumor lesions and tumor response were assessed by the investigator according to a modified version of RECIST version 1.0 that allowed inclusion of any number of skin lesions >0.3 cm at the largest diameter to be followed as target lesions, inclusion of latent responses, and assessment of the net tumor burden in the setting of new lesions. Progression-free survival was assessed as the time from the first day of study treatment until the time that the sum of the diameters of all measurable lesions increased by at least 30% from baseline. Additional information regarding response measures for treated and untreated lesions is provided in the supplementary Materials, available at Annals of Oncology online.

       Safety evaluation

      Safety was assessed by monitoring AEs, pain assessments, clinical laboratory tests, and vital signs. AEs were graded according to the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE), Version 4.0.

       Translational medicine and statistical plan

      See the supplementary Materials, available at Annals of Oncology online.

      Results

       Baseline patient characteristics

      For the main study, 38 patients were consented and 30 were eligible for the study and received at least one dose of treatment (Table 1). Of these, 28 patients were assessable for response (one withdrew before post-treatment assessment, one was deemed ineligible after initiation of treatment). Prior exposure to immunotherapy included 13 patients treated previously with systemic cytokines (high dose IL-2 or IFNα-2a), nine patients treated with anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) antibodies and four patients (13.3%) treated with anti-programmed cell death protein 1 (PD-1) antibodies. In addition, 24 patients were screened and 21 additional patients were treated in the schedule exploration cohorts (demographics are described in supplementary Table S1, available at Annals of Oncology online).
      Table 1Patient demographics and patient history
      AgeMean (SD)66.8 (10.19)
      SexMale

      Female
      16 (53.3%)

      14 (46.7%)
      ECOG PS0

      1
      21 (70.0%)

      9 (30.0%)
      StageIIIb

      IIIc

      IV M1a

      IV M1b

      IV M1c
      6 (20.0%)

      13 (43.3%)

      8 (26.7%)

      3 (10.0%)

      0
      BRAF statusMutant

      Wild-type

      Unknown
      10 (33.3%)

      13 (43.3%)

      7 (23.4%)
      Prior therapyCytokine

      CTLA-4

      PD-1/PD-L1

      Cytokine + CTLA-4

      BRAF/MEK

      Other
      13 (43.3%)

      9 (26.7%)

      4 (13.3%)

      1 (3.3%)

      3 (10%)

      5 (16.7%)
      Prior lines0

      1

      2+
      10 (33.3%)

      10 (33.3%)

      10 (33.3%)
      N = 30
      CTLA-4, cytotoxic T-lymphocyte-associated protein 4; ECOG, Eastern Cooperative Oncology Group; PD-1, programmed cell death protein 1; PD-L1, programmed death-ligand 1; PS, performance status; SD, standard deviation.

       AEs

      All treatment-emergent AEs (TEAEs) regardless of attribution observed in at least two patients and all grade 3 or higher TEAEs are described in Table 2. Transient procedural pain (n = 24, 80%) and injection site reactions were common. Constitutional symptoms were observed in a minority of patients including fatigue (n = 5, 16.7%), pyrexia (n = 2, 6.7%), and chills (n = 2, 6.7%). Grade 3 TEAEs were limited to transient procedural pain (n = 1, 3.3%) and a cerebrovascular accident that was determined to be unrelated to treatment on study. A patient in one of the schedule exploration cohorts also had grade 3, treatment-related cellulitis (supplementary Table S2, available at Annals of Oncology online).
      Table 2All treatment-emergent adverse events observed in at least two patients and all grade 3 or higher adverse events
      CategoryToxicityGrade 1Grade 2≥ Grade 3All grades
      All10 (33.3%)15 (50.0%)4 (13.3%)29 (96.7%)
      GastrointestinalNausea

      Vomiting

      Diarrhea
      4 (13.3%)

      2 (6.7%)

      1 (3.3%)
      1 (3.3%)

      2 (6.7%)

      2 (6.7%)




      5 (16.7%)

      4 (13.3%)

      3 (10.0%)
      ConstitutionalFatigue

      Injection site discoloration

      Injection site inflammation

      Chills

      Injection site discharge

      Injection site erythema

      Edema peripheral

      Pain

      Pyrexia
      4 (13.3%)

      4 (13.3%)

      3 (10.0%)

      2 (6.7%)

      2 (6.7%)

      2 (6.7%)

      2 (6.7%)



      2 (6.7%)
      1 (3.3%)



      1 (3.3%)









      2 (6.7%)

















      5 (16.7%)

      4 (13.3%)

      4 (13.3%)

      2 (6.7%)

      2 (6.7%)

      2 (6.7%)

      2 (6.7%)

      2 (6.7%)

      2 (6.7%)
      InfectiousCellulitis2 (6.7%)2 (6.7%)
      ProceduralProcedural pain23 (76.7%)1 (3.3%)24 (80.0%)
      MusculoskeletalPain in extremity

      Arthralgia

      Muscle spasms

      Musculoskeletal stiffness
      4 (13.3%)

      2 (6.7%)

      2 (6.7%)

      2 (6.7%)
      1 (3.3%)

      1 (3.3%)









      5 (16.7%)

      3 (10.0%)

      2 (6.7%)

      2 (6.7%)
      NeoplasmsNeoplasms NOS2 (6.7%)2 (6.7%)
      Nervous systemHeadache

      Dizziness

      Cerebrovascular accident
      4 (13.3%)

      2 (6.7%)

      1 (3.3%)







      1 (3.3%)
      5 (16.7%)

      2 (6.7%)

      1 (3.3%)
      PsychiatricAnxiety1 (3.3%)2 (6.7%)3 (10.0%)
      RespiratoryCough1 (3.3%)1 (3.3%)2 (6.7%)
      CutaneousPruritus

      Rash

      Ecchymosis

      Skin disorder
      2 (6.7%)

      3 (10.0%)

      2 (6.7%)

      1 (3.3%)
      1 (3.3%)





      1 (3.3%)






      3 (10.0%)

      3 (10.0%)

      2 (6.7%)

      2 (6.7%)
      VascularLymphedema2 (6.7%)2 (6.7%)
      NOS, not otherwise specified.
      N = 30.

       Clinical response

      The best overall response rate at any time point for Tavo treatment in the main study population (cohort A) was 35.7 % (waterfall plot, Figure 2A). Seven patients had disease progression before the first response assessment at 90 days and are represented as having a 100% change in tumor burden for graphic purposes. Responses included five complete responses and responses in patients with extensive in-transit/satellite metastases (Figure 2E and F). The clinical response rate was 26.7% in cohort B (4/15) and none of the four patients treated in cohort C responded. The best overall response rate for patients in all cohorts was 29.8% (supplementary Figure S2, available at Annals of Oncology online). Per-patient lesion and response data are presented in supplementary Table S3, available at Annals of Oncology online.
      Figure thumbnail gr2
      Figure 2(A) Best overall response in all assessable patients (n = 28) assessed as the sum of diameters of target lesions by a modified version of RECIST version 1.0 (* = disease progression due to progression of non-targets). (B) A spider plot demonstrating durables and transient responses in responding patients (n = 8). (C) Kaplan–Meier plots for progression-free survival (PFS) and (D) overall survival (OS). The median PFS was 3.7 months (95% confidence interval 0.6–6.9 months) and the median OS was not reached at a median follow-up of 29.7 months. (E) A patient with extensive satellite lesions before treatment demonstrates (F) complete regression of treated and untreated lesions by day 270.

       Adaptive resistance and response to checkpoint therapy

      The median progression-free survival was 3.72 months [95% confidence interval (CI) 0.55–6.89], 3.2 months (95% CI 2.41–3.97), and 2.5 months (95% CI not defined) in cohorts A (main study), B, and C, respectively. The median overall survival was not reached in any cohort (Figure 2C and D, supplementary Figure S2B, available at Annals of Oncology online). Treatment with Tavo induced adaptive resistance as demonstrated by increases in programmed death-ligand 1 (PD-L1) expression by immunohistochemistry (Figure 3A). As a possible consequence, although durable responses were seen in four patients, transient responses were observed in six others (Figure 2B). In some patients, however, Tavo increased the total number of tumor infiltrating lymphocytes and the CD8:FoxP3 ratio suggesting an increase in the relative abundance of effector T cells versus regulatory cells (e.g. Figure 3C–E), and, in a retrospective analysis, six of eight patients progressing on Tavo responded to pembrolizumab immediately thereafter (Figure 3B, F–J).
      Figure thumbnail gr3
      Figure 3(A) Treatment with tavokinogene telseplasmid was associated with an increase in PD-L1 IHC scores suggesting induction of adaptive resistance. (B) Responses to Tavo and subsequent anti-PD-1 antibody therapy in 8 patients treated with Tavo followed immediately by an anti-PD1 antibody (ϕ denotes patient response assessed by FDG-PET). (C–E) Treatment with Tavo was associated with an increase in CD8+ cells and an increase in the CD8+:Foxp3+ cells in a responding patient. (F–J) A patient with a transient response to Tavo followed by a durable response to pembrolizumab by FDG-PET.
      FDG, [18F]2-fluoro-2-deoxy-D-glucose; IHC, immunohistochemistry; PD-1, programmed cell death protein 1; PD-L1, programmed death-ligand 1; PET, positron emission tomography.

       Inflammatory gene expression

      Tavo induced significant increases in multiple immune transcripts (Figure 4A), including modules associated with immune activation (Figure 4B), NK cell activity, antigen presentation and adaptive resistance (Figure 4C), as well as T cell trafficking (Figure 4D); all characteristic of an antitumor immune response. Specific findings included increased expression of CD3E, CD8, STAT4, IL-2RB and IL-12RB1 as well as effector molecules such as GZMA, consistent with the known effect of IL-12 on T cell and NK cell activation.
      • Vignali D.A.A.
      • Kuchroo V.K.
      IL-12 family cytokines: immunological playmakers.
      A significant increase in transcripts associated with cross-presenting DCs
      • Zitvogel L.
      • Kroemer G.
      CD103+ dendritic cells producing interleukin-12 in anticancer immunosurveillance.
      such as CIITA, BATF3, PSMB7 and TAPBP1
      • Spranger S.
      • Dai D.
      • Horton B.
      • et al.
      Tumor-residing Batf3 dendritic cells are required for effector T cell trafficking and adoptive T cell therapy.
      were also noted. Additionally, the chemokine receptor CXCR3, expressed on TH1-polarized T cells, as well as chemokines and adhesion molecules were significantly increased. However, this global increase in genes associated with productive antitumor immunity was accompanied by increases in genes associated with adaptive resistance including CD274 (PD-L1), TRFB1 and TRAIL. IFN-γ gene expression increased overall in patients benefitting from treatment, but not in patients with progressive disease as the best treatment response (Figure 4E). Overall, these results suggest that Tavo induced NK cell and DC activation, recruitment, and activation of CD4+ T cell and CD8+ T cells, as well as the compensatory development of adaptive resistance.
      Figure thumbnail gr4
      Figure 4Tavokinogene telseplasmid-induced productive antitumor immune responses. (A) Volcano plot of both non-responding and responding patients based on transcriptional analysis of biopsies collected at screening and post-treatment. In particular, intratumoral expression of genes associated with (B) immune activation (C) natural killer (NK) cell activity, antigen presentation, adaptive resistance, and (D) T cell trafficking was increased after treatment. (E) Interferon-γ gene expression increased overall and in patients benefitting from treatment, but not in patients with progressive disease as the best treatment response (n = 28 including 14 patients with pre-/post-biopsy specimens).
      IFN, interferon.

       Systemic immune response

      We assessed systemic immune activity after treatment with Tavo. Analysis of serum inflammatory markers showed an increase in IL-1β and MIP-1α in responders but not in non-responders (Figure 5A and B). Systemic increases were seen in proliferating effector memory CD8+ T cells (Ki-67+CCR7−CD45RA−, Figure 5C) and in circulating cytolytic NK cells (CD56dimCD16+, Figure 5D) in responding but not in non-responding patients (P < 0.05).
      Figure thumbnail gr5
      Figure 5Signs of systemic immune activity after treatment with tavokinogene telseplasmid (Tavo). Tavo increased circulating levels of (A) IL-1β and (B) MIP-1α as well as (C) the proportion of proliferating effector T cells in the periphery in responding patients. (D) Tavo increased the frequency of NK cells in the periphery in the responding population. (E) Best overall response in treated and (F) untreated lesions as assessed as the sum of diameters of all lesions in each categories for patients treated in the main study and in the additional cohorts (B and C). Regression of at least one untreated lesions was observed in 46% of patients.
      IL-1β, interleukin 1-beta; MIP-1α, macrophage inflammatory protein-1α.
      For patients in the main study and expansion cohorts, responses in untreated lesions were common. In 40 patients with uninjected pre- and post-treatment tumor measurements available, the best overall response for untreated lesions was 25% (n = 40, Figure 5F) compared with a 43.8% response rate in treated lesions (n = 45 patients, Figure 5E). The per-lesion response rate for treated lesions was 62.7% (64/102) and for untreated lesions it was 17.4% (20/115).

      Discussion

      Prior therapeutic approaches to rIL-12, including intralesional and systemic administration, have had limited efficacy due to transient exposures associated with intralesional therapy
      • Mahvi D.M.
      • Henry M.B.
      • Albertini M.R.
      • et al.
      Intratumoral injection of IL-12 plasmid DNA--results of a phase I/IB clinical trial.
      and severe toxicity associated with systemic administration.
      • Atkins M.B.
      • Robertson M.J.
      • Gordon M.
      • et al.
      Phase I evaluation of intravenous recombinant human interleukin 12 in patients with advanced malignancies.
      We previously described a phase I intratumoral dose-escalation pIL-12 electroporation trial demonstrating that a plasmid concentration of 0.5 mg/ml was well tolerated and showed clinical effectiveness with abscopal responses and systemic immune activation.
      • Daud A.I.
      • DeConti R.C.
      • Andrews S.
      • et al.
      Phase I trial of interleukin-12 plasmid electroporation in patients with metastatic melanoma.
      In the current report, we confirm these findings in a phase II expansion, demonstrating a 35.7% overall response rate in the main study and a 29.8% overall response rate in all cohorts.
      Recently, several intratumoral therapies have been explored, either in combination or alone, with a goal of demonstrating that an ‘in situ’ immunization strategy can yield systemic immune effects. For example, a retrospective analysis of the modified herpes virus, Talimogene laherparepvec, administered intratumorally, demonstrated an objective response rate of 26%,
      • Senzer N.N.
      • Kaufman H.L.
      • Amatruda T.
      • et al.
      Phase II clinical trial of a granulocyte-macrophage colony-stimulating factor-encoding, second-generation oncolytic herpesvirus in patients with unresectable metastatic melanoma.
      ,
      • Andtbacka R.H.I.
      • Kaufman H.L.
      • Collichio F.
      • et al.
      Talimogene laherparepvec improves durable response rate in patients with advanced melanoma.
      with regression of some baseline uninjected lesions.
      • Kaufman H.L.
      • Amatruda T.
      • Reid T.
      • et al.
      Systemic versus local responses in melanoma patients treated with talimogene laherparepvec from a multi-institutional phase II study.
      In the current phase II trial of Tavo, we used different response criteria, but regression of treated lesions was common. Overall, Tavo induced regression of at least one uninjected lesion in nearly half of patients, demonstrating clinical evidence of systemic antitumor immunity. In addition, a major benefit of the plasmid electroporation platform is that it can be modified relatively easily, based on translational data, to create next-generation therapies. Indeed, preclinical testing of a next-generation plasmid that induces expression of IL-12, CXCL9, and tumor membrane-anchored anti-CD3 is ongoing.
      • Han M.
      • Mukhopadhyaya A.
      • Twitty C.G.
      Intratumoral electroporation of plasmid IL-12 and CXCL9 with membrane-bound anti-CD3 elicits robust anti-tumor immunity.
      Intratumoral Tavo electroporation was well tolerated, and it did not induce the systemic symptoms associated with intravenous cytokine administration and even constitutional symptoms were mild and infrequent. No grade 4 adverse effects were noted, and only six patients had grade 3 adverse effects (local pain, five patients and cellulitis, one patient). While systemic cytokine administration induces fever, chills, and pyrexia suggesting a systemic inflammatory response, despite a high rate of regression of untreated lesions, these symptoms were not observed in patients treated with Tavo.
      Intratumoral IL-12, as generated by Tavo, induces cDC1 and establishes DC-T cell crosstalk that mediates tumor rejection.
      • Barry K.C.
      • Hsu J.
      • Broz M.L.
      • et al.
      A natural killer-dendritic cell axis defines checkpoint therapy-responsive tumor microenvironments.
      Since cDC1 play a crucial role in recruiting and activating CD8+ T cells into the tumor microenvironment
      • Zitvogel L.
      • Kroemer G.
      CD103+ dendritic cells producing interleukin-12 in anticancer immunosurveillance.
      ,
      • Spranger S.
      • Dai D.
      • Horton B.
      • et al.
      Tumor-residing Batf3 dendritic cells are required for effector T cell trafficking and adoptive T cell therapy.
      ,
      • Barry K.C.
      • Hsu J.
      • Broz M.L.
      • et al.
      A natural killer-dendritic cell axis defines checkpoint therapy-responsive tumor microenvironments.
      and are in turn induced by NK cells,
      • Barry K.C.
      • Hsu J.
      • Broz M.L.
      • et al.
      A natural killer-dendritic cell axis defines checkpoint therapy-responsive tumor microenvironments.
      we explored the effect of Tavo, in this publication, in a study by Garris et al.,
      • Garris C.S.
      • Arlauckas S.P.
      • Kohler R.H.
      • et al.
      Successful anti-PD-1 cancer immunotherapy requires T cell-dendritic cell crosstalk involving the cytokines IFN-γ and IL-12.
      and in combination with PD-1 (manuscript submitted for review). Tavo induces activation across multiple classes of immune transcripts (Figure 4), including immune activation (Figure 4B), NK cell (Figure 4C), and antigen presentation (Figure 4D). The immune activation produced by Tavo results in both increased inflammatory gene expression, including expression of IFN-γ associated genes, and adaptive immune resistance, with increased expression of PD-L1 and TGFβ. This induction of adaptive resistance through PD-1/PD-L1 could explain the high proportion of responses to subsequent PD-1 blockade in patients progressing on Tavo (Figure 3B). Based on these findings, patients have now been treated on two prospective phase II clinical trials of Tavo in combination with the anti-PD-1 antibody pembrolizumab. A study of patients with few partially exhausted (PD1hiCTLA-4hiCD8+) cells has been completed and results will be reported elsewhere (submitted for publication) and a larger single-arm study in patients with documented progression on PD-1 blockade (KEYNOTE-695) is currently ongoing.
      In summary, Tavo treatment drives changes in the immune microenvironment resulting in both local and global immune responses with minimal systemic toxicity. Our data demonstrate that this in situ tumor vaccination strategy can be a safe and effective approach to inducing multiple sustained, productive changes in the immune microenvironment that would be too toxic using similar systemic agents.

      Funding

      This study was supported by OncoSec Medical, Inc . (no grant number).

      Disclosure

      AA is a paid advisor to OncoSec Medical, Inc. and he holds stock options in the company. He is also a paid advisor to Array, Regeneron, and Valitor. He also receives research funding from Acerta, Amgen, AstraZeneca, BMS, Dynavax, Genentech, Idera, Incyte, Idera, ISA, LOXO, Merck, Novartis, Regeneron, Sensei, and Tessa. He previously received research funding from Amgen, Celldex, GlaxoSmithKline, Lilly, Medimmune, Plexxicon, Roche, OncoSec Medical, Inc. SB, research funding from OncoSec Inc and Merck Inc. SA, research funding from OncoSec Inc. and Merck Inc. KL receives research funding from OncoSec Inc. and Merck Inc. MF receives research funding from OncoSec Inc; Advisor Boards of Novartis, Pulse Bioscience, Array Bioscience, Bristol Myers Squibb, Sanofi. LF receives research funding from OncoSec, Merck, AbbVie, Bavarian Nordic, BMS, Dendreon, Janssen, Roche/Genentech. CBB is advisor to PrimeVax, BMS. Stock ownership in BMS, Patent US20180322632A1: image processing systems and methods for displaying multiple images of a biological specimen. DB is an employee of OncoSec Medical, Inc. RT is an employee of OncoSec Medical, Inc. EB is an employee of OncoSec Medical, Inc. MHL is a former employee of OncoSec Medical, Inc. Consulting: Immunomic Therapeutics, Inc., Pulse Biosciences, Inc., Juno Therapeutics, Inc., Genexine, Inc., NKarta Therapeutics, Inc., Seattle Genetics, Inc., Ideaya Biosciences, Inc., Apros Therapeutics, Inc., IgM Biosciences, Inc., Immune Design Corporation, Plexxikon, Inc., Curis, Inc. She is married to RHP. with conflict listed next. RHP is a former employee of OncoSec Medical, Inc. with equity. He also has consulting income from Immunomic Therapeutics, Inc., Pulse Biosciences, Sensei Biotherapeutics, AbbVie, Calithera Biosciences, Minerva Biosciences, AstraZeneca, Curis, Inc. He has received research funding from Exicure Therapeutics, X4-Pharma, Incyte Pharmaceuticals. He is married to MHL with conflicts listed earlier. SG is a former employee of OncoSec Medical, Inc., currently employed by Alloplex Biotherapeutics, Inc. KKT receives research funding from OncoSec, Merck Inc., Takeda, and Regeneron; consulting for Compugen, Pulse Biosciences. CT is an employee of OncoSec Medical, Inc. with equity. AD receives research funding from OncoSec Inc., Merck, BMS, Pfizer, Novartis, Roche/Genentech, Xencor, Consultant for Incyte, Novartis, Amgen, Caris Inc. All other authors have declared no conflicts of interest.

      Supplementary Figure Legends

      References

        • Vignali D.A.A.
        • Kuchroo V.K.
        IL-12 family cytokines: immunological playmakers.
        Nat Immunol. 2012; 13: 722-728
        • Curtsinger J.M.
        • Mescher M.F.
        Inflammatory cytokines as a third signal for T cell activation.
        Curr Opin Immunol. 2010; 22: 333-340
        • Gollob J.A.
        • Mier J.W.
        • Veenstra K.
        • et al.
        Phase I trial of twice-weekly intravenous interleukin 12 in patients with metastatic renal cell cancer or malignant melanoma: ability to maintain IFN-gamma induction is associated with clinical response.
        Clin Cancer Res. 2000; 6: 1678-1692
        • Atkins M.B.
        • Lotze M.T.
        • Dutcher J.P.
        • et al.
        High-dose recombinant interleukin 2 therapy for patients with metastatic melanoma: analysis of 270 patients treated between 1985 and 1993.
        J Clin Oncol. 1999; 17: 2105-2116
        • Motzer R.J.
        • Rakhit A.
        • Schwartz L.H.
        • et al.
        Phase I trial of subcutaneous recombinant human interleukin-12 in patients with advanced renal cell carcinoma.
        Clin Cancer Res. 1998; 4: 1183-1191
        • Heller L.
        • Merkler K.
        • Westover J.
        • et al.
        Evaluation of toxicity following electrically mediated interleukin-12 gene delivery in a B16 mouse melanoma model.
        Clin Cancer Res. 2006; 12: 3177-3183
        • Heller L.C.
        • Jaroszeski M.J.
        • Coppola D.
        • et al.
        Comparison of electrically mediated and liposome-complexed plasmid DNA delivery to the skin.
        Genet Vaccines Ther. 2008; 6: 16
        • Lucas M.L.
        • Heller R.
        IL-12 gene therapy using an electrically mediated nonviral approach reduces metastatic growth of melanoma.
        DNA Cell Biol. 2003; 22: 755-763
        • Daud A.I.
        • DeConti R.C.
        • Andrews S.
        • et al.
        Phase I trial of interleukin-12 plasmid electroporation in patients with metastatic melanoma.
        J Clin Oncol. 2008; 26: 5896-5903
        • Zitvogel L.
        • Kroemer G.
        CD103+ dendritic cells producing interleukin-12 in anticancer immunosurveillance.
        Cancer Cell. 2014; 26: 591-593
        • Spranger S.
        • Dai D.
        • Horton B.
        • et al.
        Tumor-residing Batf3 dendritic cells are required for effector T cell trafficking and adoptive T cell therapy.
        Cancer Cell. 2017; 31: 711-723.e4
        • Mahvi D.M.
        • Henry M.B.
        • Albertini M.R.
        • et al.
        Intratumoral injection of IL-12 plasmid DNA--results of a phase I/IB clinical trial.
        Cancer Gene Ther. 2007; 14: 717-723
        • Atkins M.B.
        • Robertson M.J.
        • Gordon M.
        • et al.
        Phase I evaluation of intravenous recombinant human interleukin 12 in patients with advanced malignancies.
        Clin Cancer Res. 1997; 3: 409-417
        • Senzer N.N.
        • Kaufman H.L.
        • Amatruda T.
        • et al.
        Phase II clinical trial of a granulocyte-macrophage colony-stimulating factor-encoding, second-generation oncolytic herpesvirus in patients with unresectable metastatic melanoma.
        J Clin Oncol. 2009; 27: 5763-5771
        • Andtbacka R.H.I.
        • Kaufman H.L.
        • Collichio F.
        • et al.
        Talimogene laherparepvec improves durable response rate in patients with advanced melanoma.
        J Clin Oncol. 2015; 33: 2780-2788
        • Kaufman H.L.
        • Amatruda T.
        • Reid T.
        • et al.
        Systemic versus local responses in melanoma patients treated with talimogene laherparepvec from a multi-institutional phase II study.
        J Immunother Cancer. 2016; 4: 12
        • Han M.
        • Mukhopadhyaya A.
        • Twitty C.G.
        Intratumoral electroporation of plasmid IL-12 and CXCL9 with membrane-bound anti-CD3 elicits robust anti-tumor immunity.
        (In: AACR [Internet]. Atlanta)2019 (Available at)
        • Barry K.C.
        • Hsu J.
        • Broz M.L.
        • et al.
        A natural killer-dendritic cell axis defines checkpoint therapy-responsive tumor microenvironments.
        Nat Med. 2018; 24: 1178-1191
        • Garris C.S.
        • Arlauckas S.P.
        • Kohler R.H.
        • et al.
        Successful anti-PD-1 cancer immunotherapy requires T cell-dendritic cell crosstalk involving the cytokines IFN-γ and IL-12.
        Immunity. 2018; 49: 1148-1161.e7