Triple-negative breast cancer (TNBC) is an aggressive disease with limited therapeutic options. Antibodies targeting programmed cell death protein 1 (PD-1)/PD-1 ligand 1 (PD-L1) have entered the therapeutic landscape in TNBC, but only a minority of patients benefit. A way to reliably enhance immunogenicity, T-cell infiltration, and predict responsiveness is critically needed.
Using mouse models of TNBC, we evaluate immune activation and tumor targeting of intratumoral IL12 plasmid followed by electroporation (tavokinogene telseplasmid; Tavo). We further present a single-arm, prospective clinical trial of Tavo monotherapy in patients with treatment refractory, advanced TNBC (OMS-I140). Finally, we expand these findings using publicly available breast cancer and melanoma datasets.
Single-cell RNA sequencing of murine tumors identified a CXCR3 gene signature (CXCR3-GS) following Tavo treatment associated with enhanced antigen presentation, T-cell infiltration and expansion, and PD-1/PD-L1 expression. Assessment of pretreatment and posttreatment tissue from patients confirms enrichment of this CXCR3-GS in tumors from patients that exhibited an enhancement of CD8+ T-cell infiltration following treatment. One patient, previously unresponsive to anti–PD-L1 therapy, but who exhibited an increased CXCR3-GS after Tavo treatment, went on to receive additional anti–PD-1 therapy as their immediate next treatment after OMS-I140, and demonstrated a significant clinical response.
These data show a safe, effective intratumoral therapy that can enhance antigen presentation and recruit CD8 T cells, which are required for the antitumor efficacy. We identify a Tavo treatment-related gene signature associated with improved outcomes and conversion of nonresponsive tumors, potentially even beyond TNBC.
This article is featured in Highlights of This Issue, p. 2367
We show that plasmid IL12 injected intratumorally followed by co-localized electroporation induces a CXCR3 gene signature (CXCR3-GS) with enhanced antigen presentation, expansion, and licensing of T cells systemically. We highlight a patient that was previously nonresponsive to atezolizumab and nab-paclitaxel, who demonstrated significant clinical response to nivolumab following tavokinogene telseplasmid therapy. We further show that expression of this CXCR3-GS is associated with improved disease-free survival and overall survival in patients with triple-negative breast cancer (TNBC) and highly expressed in patients with melanoma that had failed previous lines of immune checkpoint therapy and go on to respond to nivolumab. These data show a safe, effective intratumoral therapy that can induce expression of a gene signature that may be prognostic of improved outcomes and associated with conversion of nonresponsive tumors even beyond TNBC.
Individuals with advanced or recurrent triple-negative breast cancer (TNBC) have limited treatment options and poor overall survival (OS; ref. 1). Recent data suggest that some patients with TNBC benefit from therapies targeting the anti-programmed cell death protein 1 (PD-1)/PD-1 ligand 1 (PD-L1) axis (2–4). However, anti–PD-1/PD-L1 monotherapy strategies have limited clinical efficacy, particularly in the pretreated TNBC setting where observed response rates are <10% (3, 5). Although the anti–PD-L1 mAb atezolizumab in combination with nab-paclitaxel received U.S. regulatory approval for PD-L1–expressing metastatic TNBC in 2019, and pembrolizumab in combination with chemotherapy received U.S. regulatory approval for the treatment of PD-L1–expressing advanced TNBC in 2020, the median progression-free survival (PFS) advantage was modest (5.0 vs. 7.5 months, 5.6 vs. 9.7 months, respectively) and OS remains poor in this population (4, 6). Although anti–PD-1/PD-L1 monotherapy strategies have proven clinical benefit in a variety of cancers, sustained disease control and prolonged survival in patients with TNBC is rarely observed, highlighting the need for improved immune-based strategies particularly in poorly immunogenic tumors. Systemic combinations of immunotherapies can improve response rates and outcomes in some situations, however, these combinations are often associated with additional toxicities.
A promising alternative is the application of intratumoral immunotherapy to “license” treated lesions to yield productive ratios of infiltrating cytotoxic T cells to suppressive immune subsets while critically amplifying the PD-1/PD-L1 checkpoint axis and ultimately increasing sensitivity to anti–PD-1/PD-L1 therapeutics or other immune checkpoint inhibitors (ICI). As one of the most potent proinflammatory cytokines, IL12 is involved in the generation of adaptive immune responses, an inflammatory tumor microenvironment (TME) and is critical in eliciting a productive antitumor immune response (7, 8). IL12 has been investigated as an anticancer therapeutic using various delivery routes, but systemic delivery of IL12 has limited clinical efficacy and substantial toxicity (9). Intratumoral (IT) delivery of recombinant IL12 protein has been investigated, but rapid clearance resulted in insufficient residence time at the site of injection and poor antitumor responses (10, 11). Intratumoral injection of plasmid IL12 (tavokinogene telseplasmid; tavo) followed by electroporation (EP; collectively designated Tavo) is a gene therapy approach that drives local and immunologically relevant exposure of IL12 with minimal systemic immune-related toxicity (12–14). Studies of Tavo treatment for melanoma have shown significant preclinical and clinical efficacy, with reports of systemic, abscopal effects and local enhancement of antigen presentation, T-cell activation, and inflammatory gene expression (12, 15, 16). Other studies of a similar intratumoral plasmid IL12 treatment have shown an IFNγ-dependent eradication of established melanoma through induction of CD8 T cells (17).
One hypothesis for how Tavo could convert poorly immunogenic/low tumor-infiltrating lymphocyte (TIL) tumors into highly inflamed immunologically active lesions is through the coordinated production of chemokines that regulate the migration, differentiation, and activation of both innate and adaptive immune cells. One such axis that is upregulated in response to IFNγ production is CXCL9/10/11/CXCR3 (18). Recently published results from two clinical trials treating patients with melanoma with Tavo or Tavo plus pembrolizumab both highlighted significantly increased on-treatment intratumoral expression of CXCR3 (12, 13). This axis has been extensively studied as a potential prognostic biomarker for improved clinical outcomes and a potential predictive biomarker for responses to ICI in patients with melanoma (19, 20). As preclinical studies have linked responsiveness to ICI therapy to an induction of the CXCL9/10/11/CXCR3 axis, most clinical studies focus on it as a predictive biomarker (21) in patients with melanoma or non–small cell lung cancer (NSCLC; ref. 19), with few studies in TNBC.
To better understand the immunomodulatory effects of Tavo and associated mechanism of action in TNBC, we conducted both preclinical studies and a single-arm, prospective clinical trial of Tavo monotherapy in participants with pretreated advanced TNBC (OMS-I140; NCT02531425). In preclinical studies, we demonstrate the Tavo therapy induces changes in the TME resulting in dramatic expansion and activation of CD8+ T cells which is characterized by a CXCR3 gene signature (CXCR3-GS). PD-1/PD-L1 expression after Tavo treatment is also profoundly increased, sensitizing treated mice to anti–PD-1 and leading to complete tumor regression and long-term survival. A single-arm, prospective clinical trial of Tavo in patients with treatment refractory, advanced TNBC demonstrates safety and feasibility of this treatment, with pretreatment and posttreatment tissue assessment demonstrating the CXCR3-GS in tumors that respond to treatment. We highlight one patient, who previously received atezolizumab therapy in combination with nab-paclitaxel without any objective tumor regression from baseline. After being treated with Tavo, this patient exhibited an increase in our novel CXCR3-GS and went on to clinically respond to the anti–PD-1 agent nivolumab, which was their immediate next therapy. Analyzing genomic databases, we show that expression of our CXCR3-GS is associated with improved disease-free survival (DFS) and OS in patients with TNBC and highly expressed in patients with melanoma nonresponsive to ipilimumab who respond to nivolumab. These data show a safe, effective intratumoral therapy that can induce expression of a gene signature that may be prognostic of improved outcomes and associated with conversion of nonresponsive tumors even beyond TNBC.
Patients and Methods
Clinical trial details
Eligible patients had a histologically confirmed diagnosis of inoperable locally advanced or metastatic TNBC previously treated with at least one line of systemic therapy for advanced disease with accessible tumor for intratumoral injection involving the breast or chest wall. TNBC was defined as estrogen receptor (ER) and progesterone receptor (PR) < 10% and HER2- negative (IHC score of 0 to 1+, or negative by ISH). The trial was conducted in accordance with International Conference on Harmonization Guidelines for Good Clinical Practice and the Code of Federal Regulations. The protocol was approved by the Stanford University (Stanford, CA) Institutional Review Board (IRB34299) and Stanford Administrative Panel on Biosafety (APB-3266-NT0220). All patients provided written, informed consent at the time of screening.
Tavo (IL12 plasmid, 0.5 mg/mL) was administered by intratumoral injection at a dose volume of one-fourth of the calculated lesion volume with a minimum dose volume per lesion of 0.1 mL for lesions of volume <0.4 cm3. Tumor volume was calculated using the formula: Vtumor = ab (2)/2, where a is the longest diameter of the lesion and b is the length of the axis perpendicular to a. An applicator containing either a 1.0-cm or 0.5-cm diameter array of six stainless steel needles was colocalized at the site(s) and depth of plasmid injection and electroporation was performed using six pulses at field strengths of 1,500 V/cm and pulse width of 100 μs at 300-ms intervals.
Preclinical plasmids and reagents
The murine IL12 plasmid (pIL12-P2A), where picornavirus 2A-linked with IL12p35 and p40 genes inserted to pUMVC3 plasmid (16), and the control pUMVC3 plasmid were provided by OncoSec Medical, Inc. For depletions, 250-μg anti-CD8ß antibody (clone 53-5.8; BE0223) or IgG1 isotype control (clone HRPN; BE0088) was given on days −1, 1, 3, and 9. For PD-1 blockade, anti-mouse PD-1 antibody (Clone RMP1-14, BE0146) or the rat IgG2a isotype control (Clone 2A3, BE0089) was given on days 2, 6, 9, and 13. These antibodies were purchased from Bio X Cell.
Female BALB/c mice were purchased from Jackson Lab and maintained in the Duke University Cancer Center Isolation Facility (DU-CCIF; Durham, NC), and used for 4T1 experiments. Given the propensity of breast cancer to occur in females, no males were used in these studies. HER3-transgenic mice with BALB/c background were established from MMTV-neu/MMTV-hHER3 mice, a kind gift from Stan Gerson (Case Western Reserve University, Cleveland, OH). They were bred in the DU-CCIF and used between 8 and 16 weeks of age. All animal studies described were approved by the Duke University Medical Center (Durham, NC) Institutional Animal Care and Use Committee (A198-18-08 and A164-20-08) and performed in accordance with established guidelines.
In a single tumor model, cancer cells were injected subcutaneously into the right flank of mice (1 × 106 cells). In a bilateral tumor model, cancer cells were injected subcutaneously into bilateral flanks of mice (5 × 105) cells/site. The measurement of tumor size was performed every 2 days using calipers and volumes were calculated as follows: volume = width*width*(length/2). For the intratumoral delivery of IL12 gene, mice were anesthetized using 97% oxygen and 3% isoflurane, and tumors were injected with 50-μL (1 μg/μL) plasmid DNA in sterile saline using a a 27-gauge needle. Electroporation was carried out using eight unidirectional (low voltage: 400 V/cm, 100 μs pulse width, 300 ms delay) pulses (APOLLO generator, OncoSec Medical, Inc.).
Data are presented as mean ± SEM. Tumor volumes from experiments with three or more treatment groups were analyzed by one-way ANOVA with Bonferroni multiple comparisons test. Comparisons were made to untreated or control group unless otherwise indicated. A two-tailed, unpaired Student t test was used for experiments with only two groups. Group sizes for animal tumor growth experiments were determined on the basis of preliminary datasets. All subjects were randomized into a treatment or control group. Proportion tests were used to test the null hypothesis that the proportions of cells in various groups are the same. The resulting P values were computed from Pearson χ2 test. Kaplan–Meier methods were used to generate time-to-event plots, and groups were compared using the log-rank test. Graphs were generated and statistical analysis was performed using GraphPad Prism (RRID:SCR_002798). Human patient NanoString data were log transformed and quantile normalized before applying our gene expression signatures. Unpaired two-sided Wilcoxon rank-sum tests were used for pairwise comparisons. For the univariate forest plots, HRs along with 95% confidence intervals were derived from Cox proportional hazard survival models with endpoints of DFS and OS.
Intratumoral administration of Tavo significantly suppressed tumor growth and improved survival
Intratumoral delivery of IL12 plasmid has demonstrated antitumor potential in preclinical and clinical studies of melanoma (12–14, 22). To explore the potential efficacy of this therapy in TNBC, murine 4T1 TNBC cells were implanted into BALB/c mice. When tumor diameter reached 6–7 mm, mice were randomized into control plasmid or Tavo treatment group (day 0). On days 0, 4, and 7, the mice underwent intratumoral administration of plasmid, followed by in vivo electroporation (Fig. 1A). This regimen led to substantial production of IL12 protein in treated tumors, with no detectable IL12 in control treated tumors or systemically in either group (Supplementary Fig. S1A and S1B). Significant suppression of tumor growth was observed in the Tavo-treated group that corresponded to increased survival compared with control treatment (Fig. 1B and C). The same effect was observed in other murine TNBC models using E0771 and JC-HER3 tumor cells (Supplementary Fig. S2A–S2D). Evaluation of TILs in the treated tumors revealed a significant increase in CD8+ T-cell infiltration that corresponded to a concomitant increase in the activation/exhaustion markers PD-1, PD-L1, LAG-3, and ICOS (Supplementary Fig. S2B, S2E–S2G). Interestingly, there was also a systemic increase in the frequency of effector CD8+ T cells observed in the spleen of treated mice (Supplementary Fig. S2H).
Given that this local therapy was having a systemic impact on immunity, we next determined whether any abscopal effect could be seen in treated mice. Tumor cells were implanted bilaterally into BALB/c mice prior to randomization into treatment groups. The treatment schedule remained the same as above, with only one of the tumors receiving treatment at the indicated times. As before, we observed significant tumor growth suppression not only in the treated tumors, but also in the contralateral tumors that did not receive any treatment (Fig. 1D). This abscopal effect points to not just a change in the treated TME, but also a robust priming and systemic engagement of the immune system.
TME induced by intratumoral administration of Tavo
To better understand treatment-related changes in tumor infiltration and associated gene expression within the TME, we sorted CD45+ cells from tumors receiving either control plasmid or Tavo. We performed single-cell RNA sequencing (scRNA-seq) on these cells and evaluated changes in the immune cell types present in the tumor. Cell types were clustered using graph-based clustering and classified using expression of canonical cell type gene markers (Fig. 1E; Supplementary Fig. S3A and S3B). Quantification of the proportions of each cell type in control and treated tumors show a significant increase in CD8+ T cells, CD4+ T cells, and dendritic cells (DC), with a decrease in neutrophils (Fig. 1E inset). Interestingly, although natural killer (NK) cells are highly responsive to IL12 and expanded but Tavo in other tumor models, there was no distinct NK-cell cluster or change in this model (17, 23). In addition to gene expression (GEX) libraries, T-cell receptor (TCR) libraries were also sequenced (Fig. 1F). The induction of a significant IFN-stimulated gene signature occurred in Tavo-treated tumors in both innate and adaptive immune cells (Fig. 1G). Cells containing both a GEX and TCR were reclustered and further analyzed (Supplementary Fig. S4).
The increase in CD8+ T cells is characterized by an expansion in both the breadth and depth of the repertoire. There were significantly more clones in Tavo-treated tumors that had expanded (>10 cells sharing the same TCR, shown in red) as well as significantly more unique clones present (Fig. 1H). Control treated mice had 234 unique clonotypes while 842 were isolated from Tavo-treated tumors. We examined the gene expression patterns following treatment in T cells and see a notable exhaustion signature is present in both control and Tavo treated (Fig. 1I; Supplementary Table S1). Importantly, a distinct cluster of highly activated CD8+ T cells were only present in Tavo-treated tumors (Fig. 1J; Supplementary Table S1).
CD8+ T cells were not the only cell population to be altered following treatment. We further analyzed differentially expressed genes from all cell types. Differential gene expression analysis identified more than 1,600 genes that were significantly upregulated following Tavo electroporation (Fig. 2A; Supplementary Table S2). Notably, these included genes associated with the increased CD8+ T-cell infiltration and activation (Cd3e/g/d, Cd8a, Gzmb, Prf1, Ifng, Tnf, Nkg7), trafficking (Cxcr3, Ccr5, Cxcl9/10/11), antigen presentation (B2m, H2-K1, H2-k2, Cd74, Klrc1, Klrd1), and exhaustion (Pdcd1, Lag3, Cd274, Icos, Tigit, Ctla4). These data are consistent with transcription analysis of biopsies from patients with melanoma treated with Tavo clinically (12, 13). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of these differentially expressed genes further highlight the enrichment of antigen presentation, cytokine, chemokine, and PD-L1 pathways in Tavo-treated tumors confirming the induction of IL12, IFNγ, and IFN-stimulated genes demonstrated above (Fig. 2B). Pathway and gene set enrichment analysis of isolated dendritic cells highlights the enhancement of antigen presentation, cytokine production, and cell adhesion molecules stimulated by IL12 and IFNγ production (Supplementary Fig. S5A and S5B). Similarly, CD8 T cells show IFNγ-specific pathways and cytokine gene sets significantly enriched following Tavo treatment (Supplementary Fig. S5C and S5D).
An advantage of single-cell transcriptional information is that we can precisely map receptor-ligand interactions between specific cell compartments. Interaction scores were computed, as described previously (24), for each ligand-receptor pair and compared across cell types. Relative interaction scores were computed as a ratio between Tavo and control groups. From these interactomes, we identified an enrichment of interactions between antigen presenting, myeloid populations and CD8 T cells (Fig. 2C). Within the top interactions, there is a striking enrichment of chemokines from the CCL5/CXCL9/10/11/CXCR3 family, which is consistent with the gene expression data of both expanded T-cell clones and the overall TME. A closer look at the top interactions between receptors on macrophages and ligands on CD8+ T cells again highlights the CXCL9/10/11/CXCR3 axis, as well as other immune checkpoints like Cd274/Pdcd1 and CD47/Sirpa (Fig. 2D).
Focusing on this CXCR3 axis, we identified a gene signature of the top 50 genes with the highest correlation with the reference gene, CXCR3 (Supplementary Table S1). We included genes that had increased expression associated with CXCR3 across all cell types in each sample. Tavo-treated tumors had a significantly higher CXCR3-GS score than cells from control treated tumors (Fig. 2E). To determine whether the recruitment of activated CD8 T cells was critical to responses to Tavo as seen in other tumor models (17), we depleted CD8 T cells at the time of IT-Tavo treatment and see a complete loss of protection (Fig. 2F and G), supporting the critical role of CD8 T cells in this therapeutic response.
Combination of Tavo with PD-1/PD-L1 blockade
Although CD8 T cells are clearly enhanced by Tavo treatment, the mixed expression of activation and exhaustion markers and enrichment of PD-1/PD-L1 indicate this therapy may be rationally combined with ICIs to maximize therapeutic efficacy (25, 26). To test this, we combined anti–PD-1 antibodies with our Tavo treatment (Fig. 2H). This combination not only significantly slowed tumor growth in multiple tumor models of TNBC (Supplementary Fig. S6A and S6B), it also led to complete tumor regression and long-term, tumor-free survival (Fig. 2I). This transition from a delay in tumor growth to complete tumor regression and long-term survival is the ultimate goal of an immunotherapeutic for cancer and highlights to potentially significant impact this combination could have clinically.
Prospective Evaluation of Tavo in Patients with Pretreated Advanced TNBC
We conducted a phase I, nonrandomized, open-label trial evaluating the pharmacodynamic effects and safety of Tavo in patients with pretreated, inoperable locally advanced or metastatic TNBC (NCT02531425). Patients received Tavo by intratumoral injection on days 1, 5, and 8 of a single 28-day cycle into accessible lesions (Fig. 3A). Patients were required to have at least two anatomically distinct lesions accessible for biopsy with at least one cutaneous or subcutaneous lesion accessible for injection and electroporation. At least one lesion remained untreated for the duration of the study. One tumor biopsy was obtained at screening (S) and two biopsies were obtained posttreatment on day 28 [end of study (EOS)] of both treated and untreated lesions.
The primary objective was to evaluate the potential of one cycle of Tavo to promote a proinflammatory molecular and histologic signature. Safety and tolerability of Tavo in subjects with TNBC was a secondary objective. Toxicity was graded according to the NCI Common Terminology Criteria for Adverse Events v4.0. Pain was assessed on numeric pain rating scale of 0–10 and captured prior to, immediately following and 5 minutes after the procedure. All patients who received at least one Tavo treatment were included in the safety population. Patients who discontinued prior to receiving all three planned IT-pIL-12 EP treatments or without providing at least one set of posttreatment tumor biopsy samples were not be considered evaluable for the primary objective.
Patient characteristics and safety
Ten eligible patients were enrolled and completed study therapy; one patient did not complete follow-up. Median age was 61 years (range, 35–84), all were women and seven patients had distant metastases at enrollment (Supplementary Table S3). Median number of prior therapies for advanced TNBC was two (range, 1–4 prior lines of treatment). Treatment emergent adverse events are shown in Supplementary Table S4. Treatment-related adverse events included pain associated with electroporation (grade 1) in nine patients and fatigue (grade 1), pruritis (grade 1), and tremors/involuntary shaking (grade 1) in one patient each. Median pain score (range, 0–10) immediately after treatment was 2 (range, 0–10) and 5 minutes posttreatment was 1 (range, 0–7).
Intratumoral immune response
To assess immunologic changes within the tumors following Tavo treatment, tumor tissue was collected before and after treatment. Lesion-matched pretreatment and posttreatment biopsies were assessed with quantitative multiparametric IHC (mIHC). Given the highly variable nature of chest wall/breast/cutaneous disease involvement in this cohort, collection of matched biopsies of treated lesions was not always possible. For this analysis, matched biopsies were used regardless of whether the specific lesion was treated with Tavo or not (Table 1). Adequate tissue was only available for mIHC analysis of nine of the treated patients. Analysis of lesion-matched samples demonstrated treatment-related increased density of CD8+ TILs at the EOS in four patients (highlighted green; Fig. 3B; Table 1). Although an on-treatment increase in TILs was defined in the protocol as a >10% increase in the mean response of matched paired biopsies, we analyzed the data using a more sensitive measure of > 2-fold on treatment increase to define an increase in CD8+ TILs. Furthermore, mIHC analysis demonstrated increased ratios of CD8+ T cells to suppressive immune subsets, including CD8+:CD163+ macrophages and CD8+:FoxP3+ regulatory T cells. Each of these populations were increased in six of nine patients, although they were not increased in the same six patients (Table 1). Increased levels of PD-L1 were shown in four of nine patients at EOS compared with baseline, consistent with data from our preclinical model (Table 1).
|Patient .||Treated .||CD8+ at S (cells/mm2) .||CD8+ at EOS (cells/mm2) .||Fold change to CD8s .||CD163+ at S (cells/mm2) .||CD163+ at EOS (cells/mm2) .||Ratio at S CD8/CD163 .||Ratio at EOS CD8/CD163 .||Fold change CD8:CD163 .||FoxP3+ at S (cells/mm2) .||FoxP3+ at EOS (cells/mm2) .||Ratio at S CD8/FoxP3 .||Ratio at EOS CD8/FoxP3 .||Fold change CD8:FoxP3 .||Total PD-L1 at S (cells/mm2) .||Total PD-L1 at EOS (cells/mm2) .||Fold change in PD-L1 .|
|Patient .||Treated .||CD8+ at S (cells/mm2) .||CD8+ at EOS (cells/mm2) .||Fold change to CD8s .||CD163+ at S (cells/mm2) .||CD163+ at EOS (cells/mm2) .||Ratio at S CD8/CD163 .||Ratio at EOS CD8/CD163 .||Fold change CD8:CD163 .||FoxP3+ at S (cells/mm2) .||FoxP3+ at EOS (cells/mm2) .||Ratio at S CD8/FoxP3 .||Ratio at EOS CD8/FoxP3 .||Fold change CD8:FoxP3 .||Total PD-L1 at S (cells/mm2) .||Total PD-L1 at EOS (cells/mm2) .||Fold change in PD-L1 .|
Note: Values shown in bold signify patients with a ≥2-fold change in CD8 T cells at EOS.
Abbreviation: EOS, end of study.
aSlides from patient 1 did not contain enough tissue to complete the analysis.
Expression of an immune-based gene set was measured for all 10 patients with lesion-matched biopsies pre- and post-Tavo treatment using a NanoString assay. Here we divided patients into two groups: those with no change or a decrease in CD8+ T cells after treatment and those with an increase in CD8+ T cells (Table 1). The data shown are from the matched EOS biopsy regardless of whether that lesion was treated or not (Table 1). Immune-related genes involved in chemotaxis (VCAM1, CCL2/5/13/18/19, CXCL9/12, ICAM1/2/3), T-cell activation (CD4, TCF7, ZAP70, GZMA, GZMK, ITGAL), antigen presentation (HLA-A/B/C, HLA-DRB1, HLA-DMA/B, B2M, TAP1/2, CD74, PSMB8/9/10), and cytokines/cytokine signaling pathways (IL1R1, IL2RB/RG, IL7R, TRAF3/6, TNFSF10/12, IFNAR2, STAT1/2/3, IRF1/4/7, JAK1/2/3) are significantly upregulated in biopsies from patients with an increase in CD8+ T cells as measured by mIHC (Fig. 3C).
NanoString gene expression data were also used to evaluate expression of the CXCR3-GS that was identified in our preclinical studies (Fig. 2E). For this signature analysis, all EOS values are from the treated lesions. Given the limited scope of genes evaluated by this assay, only 19 of the 50 genes from our original gene signature were included (Supplementary Table S1). Strikingly, the CXCR3-GS score increased from screening to EOS in eight of 10 of the patients (Fig. 3D). One of the patients for whom the signature decreased slightly over the course of treatment already had a high signature score at screening. When the CXCR3-GS scores from the EOS biopsies are stratified on the basis of TIL infiltration as defined by mIHC (Table 1), there is a significantly higher expression in patients that had an increase in TILs (red) than in those patients that did not (blue; Fig. 3E).
Evaluation of prognostic value of CXCR3-GS in METABRIC dataset
To assess the prognostic value of our newly identified gene signature, we utilized gene expression and survival data from the publicly available Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) dataset (27). Here we see that patients with TNBC have the highest expression of the CXCR3-GS (Fig. 3F). When patients with TNBC were divided on the basis of CXCR3-GS expression (top 25% shown in blue, bottom 75% shown in red), high CXCR3-GS expression was significantly associated with improved DFS and OS (Fig. 3G and H). HRs calculated for DFS show that the CXCR3-GS is as prognostic as several other published immune-based signatures (Fig. 3I; refs. 28–30).
Systemic immune responses
Having demonstrated significant abscopal effects and changes to systemic immunity preclinically, whole blood was collected from six of 10 treated patients at screen, C1D8, C1D15 (optional), and EOS and immune cell subsets were analyzed. Of the evaluable patients, three showed treatment-related increases in short-lived effector cells (SLECs; CD3+CD8+KLRG1+CD127−; Fig. 4A). SLECs, a key antitumor immune subset, are driven by IL12-mediated upregulation of the transcription factors T-bet and Blimp-1 (31, 32). For two of these patients, that increase was sustained through the EOS (Fig. 4A). The levels of SLECs in the remaining patients remained approximately constant throughout the treatment window. We also examined changes in the frequency of peripheral granulocytic myeloid-derived suppressor cells (PMN-MDSC; CD45+Lin−HLA−CD15+CD11b+), which can limit productive antitumor responses and were decreased in both the preclinical mouse models of breast cancer and melanoma following intratumoral delivery of Tavo (Fig. 4B; refs. 33, 34). From our longitudinal studies of peripheral blood mononuclear cell (PBMC), we found that four patients had decreases in the levels of these suppressive cells while the other two had little to no change in PMN-MDSC levels (Fig. 4B).
Another measure of the systemic impact of Tavo treatment is the evaluation of circulating cytokine levels, which were assessed in all 10 patients. Serum from three of 10 patients and plasma from seven of 10 patients was examined for posttreatment changes in cytokine levels compared with baseline via Luminex MAGPIX. Consistent with Tavo safety profile, changes in systemic cytokine levels were very modest across all patients; however, significant posttreatment increases were observed in levels of IL2 and TNFα (Fig. 4C). Importantly, no increases in serum levels of bioactive IL12p70 were detected throughout the treatment window.
Post-Tavo response to nivolumab in a patient nonresponsive to nab-paclitaxel and atezolizumab
This trial did not specifically enroll or treat any patients with a combination of Tavo and a PD-1/PD-L1 inhibitor, however, a subset of patients (n = 4) went on to receive anti–PD-1 monotherapy as their next immediate treatment with clinically meaningful responses observed. Only one patient (patient 6) had received prior anti–PD-1/PD-L1 therapy. She was a 64-year-old woman who was diagnosed with a T2N0M0 stage IIA metaplastic carcinoma of the right breast, ER 2%, PR 2%, HER2 negative. She was treated with preoperative chemotherapy with dose-dense doxorubicin and cyclophosphamide, to which she had no response, followed by dose-dense paclitaxel with gemcitabine, to which she had minimal clinical response. She was treated with lumpectomy with 2 cm of residual carcinoma in the breast with negative lymph nodes. She completed whole breast radiotherapy and 12 months later was diagnosed with metastatic TNBC with multiple bilateral pulmonary nodules and bone metastases. She was treated with weekly nab-paclitaxel and atezolizumab for 18 months with stable disease as best response and no decrease from baseline in target lesions. She was taken off therapy given progression of disease with an enlarging right breast nodule and development of multiple ulcerated scalp metastases (Fig. 5A). She enrolled in OMS-I140 and received one cycle of Tavo. She started nivolumab 7 days after her end of treatment biopsy. Tumor assessment at 12 weeks revealed healing of multiple scalp metastases, interval decrease in bilateral spiculated pulmonary nodules, increased sclerosis of widespread osseous metastatic lesions reflecting posttreatment response and decrease in the size of nodular soft tissue in the right breast (Fig. 5B).
Evaluation of the CXCR3-GS in patients with melanoma
Melanoma was the first malignancy to show profound responses to immunotherapy, and as such, a large amount of genomic and survival data associated with responsiveness to checkpoint inhibitor therapy. To determine whether the induction of a CXCR3-GS is more broadly applicable to tumors beyond TNBC, we utilized a previously published data from patients with metastatic melanoma that went on to be treated with nivolumab (35). Baseline tumor samples were taken prior to treatment with nivolumab and response to ICI was reported. The CXCR3-GS was significantly higher in patients that went on to respond to nivolumab than in nonresponders (Fig. 5C). Interestingly, when the patients were stratified on the basis of previous treatment, specifically whether they had previously been treated with ipilimumab, the patients that had previously progressed on ipilimumab but went on to respond to nivolumab had the highest expression of the CXCR3-GS score (Fig. 5D). This analysis provides additional evidence that expression of a high CXCR3-GS may be indicative of an anti-PD1–responsive tumor.
The composition and quantity of tumor-infiltrating immune cells is recognized as both a prognostic and predictive biomarker in many types of cancer. Across tumor types, so-called immunologically “hot” tumors are characterized by T-cell infiltration and molecular signatures of immune activation, whereas “cold” tumors show striking features of T-cell absence or exclusion (36). In general, inflamed tumors achieve higher response rates to immunotherapy, prompting many studies to focus on converting noninflamed cold tumors into hot ones (37–39). IL12 is a pivotal antitumor cytokine, which induces a positive feedback loop to prime NK (40) and T cells (41) to produce IFNγ that in turn primes DCs to produce more IL12 (42–44). Despite its importance, very few strategies for safely increasing local IL12 exist. Here we demonstrate in preclinical models that intratumoral injection of Tavo followed by electroporation had a dramatic impact on the immune cell composition and activation within the tumor (Figs. 1 and 2). Dramatic enhancement of antigen presentation, cytokine, and chemokine pathways, characterized by IFNγ led to increased infiltration and activation of CD8 T cells. This increase in antigen presentation and T-cell activation was confirmed in clinical studies in tumor biopsies obtained from patients following Tavo monotherapy (Fig. 3) with similar pathways identified in a recent study in melanoma (12). We also demonstrate systemic antitumor immune responses on distant, untreated tumors without any evidence of cytokine storm or other toxicity.
Despite the unequivocal role that IL12 plays in the activation of NK cells, our scRNA-seq analysis did not reveal a distinct NK-cell cluster. In addition, depletion of CD8 T cells completely abrogated the antitumor effect in this model, suggesting a minor or secondary role for NK cells. This may be a model specific observation or may be related to the timing of analysis. Despite this, other models have shown a role for NK cells in IL12-driven immune responses (17, 45, 46) and their impact clinically in Tavo responses should continue to be investigated in future trials.
Although ICIs have revolutionized cancer treatment, therapeutic nonresponse or acquired resistance occurs in the majority of treated patients. As there are many other antibodies, cytokines, and small-molecule drugs targeting the immune system or cancer cells, an overabundance of competing options for combination therapies can be tested, often with limited preclinical data (47). Our preclinical studies demonstrate a strong rationale and efficacy data for combining Tavo with PD-1/PD-L1 inhibitors, as well as identifying a potential predictive biomarker in TNBC characterized by the induction of IFNγ (a classic inducer of PD-L1). Tavo treatment is associated with an increase in PD-1+CD8+ T cells in the tumor, and an enrichment for the PD-1/PD-L1 pathway (Figs. 1 and 2). We further present a patient with metastatic TNBC previously unresponsive to anti–PD-L1 therapy and chemotherapy; however, after receiving Tavo, patient was immediately treated with a different PD-1 inhibitor and exhibited a significant clinical response (Fig. 5). This case is consistent with our hypothesis and studies in melanoma that Tavo treatment can license immunologically “cold” tumors, triggering activation of T cells to fight local as well as distant lesions (12). Furthermore, analysis of published genomic data from a prospective study of melanoma tumors that had previously progressed on ipilimumab (35) revealed that all subsequent responders to nivolumab expressed a high baseline level of our CXCR3-GS. These data not only support the rationale combination of Tavo with a PD-1/PD-L1 inhibitor, they highlight the role for sophisticated preclinical biomarker analyses like scRNA-seq pathway enrichment and receptor-ligand interactomes.
One advantage of our unbiased preclinical scRNA-seq analysis is the ability to identify specific pathways that are enhanced by treatment, such as the CXCL9/10/11/CXCR3 axis that was dramatically increased in Tavo-treated tumors (Fig. 2). These chemokines have been a major focus of cancer therapeutic research due to their pivotal role in the differentiation, migration, and activation of T cells (20). Our scRNA-seq receptor-ligand interaction analysis shows that the CXCL9/10/11 is being produced by multiple populations of myeloid cells including macrophages, DCs, and neutrophils (Fig. 2), which is consistent with a recent report showing that macrophage derived CXCR3 ligands are required for responsiveness to ICI (21). This pathway has been linked to responsiveness to PD-1–targeting therapies largely in melanoma and NSCLC (48, 49). A recent study reported activation of the STAT1 pathway as being pivotal and predicting response to ICI and showed that pretreatment with IFNγ, poly(I:C), and anti-IL10 could sensitize nonresponsive mice to ICI (39). This is mechanistically consistent with our data, but in contrast to Tavo monotherapy, requires a complicated dosing of three different therapies. Another study supporting the importance of this axis demonstrated that epigenetic silencing of CXCL9/10 was enough to inhibit response to ICI and that modifying this epigenetic suppression could sensitize mice (50).
Although the clinical study of Tavo monotherapy shows a favorable safety profile, the limited number of patients requires further studies to better understand the mechanisms of response/nonresponse and to clearly define biomarkers. This study evaluated a single cycle of Tavo monotherapy and while some patients received anti–PD-1 therapy as their immediate next therapy, this treatment was not protocol specified. Furthermore, the trial was limited to participants with heavily pretreated, advanced TNBC for whom response rates to immunotherapy are historically very low. An additional caveat to the analysis of this trial was the heterogeneity seen in the presentation, location, and size of lesions that complicated or prohibited the ability to longitudinally track matched tumors. For many reasons, we would predict that patients with earlier stage disease may have a greater response to immunotherapy and derive more benefit from Tavo treatment. As such, we have an ongoing trial OMS-I141/KEYNOTE-890 (NCT03567720) evaluating repeated dosing of Tavo intratumorally with intravenous pembrolizumab in patients with pretreated metastatic TNBC in addition to a separate cohort of previously untreated patients who will receive Tavo, pembrolizumab, and nab-paclitaxel in the first-line metastatic setting. Furthermore, we were able to utilize published datasets to evaluate the CXCR3-GS in TNBC and melanoma, confirming its prognostic potential.
Overall, this work demonstrates that Tavo treatment can overcome an immunologically “cold” tumor microenvironment by increasing antigen presentation, expanding and activating T cells, and minimizing the infiltration of potentially suppressive granulocytic cells. Furthermore, we show an increase in expression of PD-1/PD-L1 and the CXCL9/10/11/CXCR3 pathways, which have both been shown to sensitize tumors to PD-1–targeting therapies. Taken together, these data show a safe, effective intratumoral therapy that can induce expression of a gene signature that may be prognostic of improved outcomes and associated with conversion of ICI nonresponsive tumors even beyond TNBC.
M.L. Telli reports grants from OncoSec Medical during the conduct of the study; grants and personal fees from Merck, Pfizer, Genentech, and AbbVie; personal fees from Natera, Immunomedics, Lilly, Celgene, G1 Therapeutics, Daiichi Sankyo, and Aduro; grants from Bayer, AstraZeneca, Calithera, Biothera, EMD Serono, Vertex, Tesaro, and PharmaMar outside the submitted work. I. Wapnir reports grants from Oncosec during the conduct of the study; I. Wapnir also reports grants from Oncosec Inc outside the submitted work. K. Zablotsky reports other from Oncosec Medical during the conduct of the study; K. Zablotsky also reports other from Oncosec Medical outside the submitted work. B.A. Fox reports grants from OncoSec during the conduct of the study; personal fees from AstraZeneca, Boehringer Ingelheim, and Ultivue; grants and other from Bristol Myers Squibb; other from Incyte; grants from Macrogenics and Shimadzu; nonfinancial support from PerkinElmer/Akoya and NanoString; personal fees and other from PrimeVax and UbiVac outside the submitted work. C.B. Bifulco reports stock ownership and scientific board membership with PrimeVax, scientific board membership with BioAI, and patent US20180322632A1 for image processing systems and methods for displaying multiple images of a biological specimen. S.M. Jensen reports grants from OncoSec, Bristol Myers Squibb, and MacroGenics; S.M. Jensen also reports nonfinancial support from Akoya Biosciences during the conduct of the study. C. Ballesteros-Merino reports grants from ONCOSEC, BMS, and Macrogenics and other from Akoya during the conduct of the study. M.H. Le reports other from OncoSec Medical, Inc. and OncoSec Medical, Inc during the conduct of the study. R.H. Pierce reports other from OncoSec Medical during the conduct of the study; in addition, R.H. Pierce has a patent for multiple patents pertaining to IL12 and its use in immuno-oncology indications pending and issued to OncoSec Medical, which owns all of the relevant intellectual property pertaining to this publication. E. Browning reports personal fees from OncoSec Medical, Inc. during the conduct of the study. R. Hermiz reports personal fees from OncoSec Immunotherapies during the conduct of the study; in additon, R. Hermiz reports personal fees from OncoSec Immunotherapies outside the submitted work. D. Bannavong reports personal fees from OncoSec Medical Inc. during the conduct of the study. K. Jaffe is an employee at OncoSec and receives a salary and stock options. M. Foerter reports other from OncoSec during the conduct of the study; M. Foerter also reports other from OncoSec outside the submitted work. D.A. Canton is a current employee of Oncosec Medical Inc. C.G. Twitty is an employee of Oncosec and receives a salary and stock options. H.K. Lyerly reports nonfinancial support and other from Oncosec Medical during the conduct of the study; H.K. Lyerly also reports other from AlphaVax, nonfinancial support and other from REMD Biotherapeutics and Replicate Bioscience outside the submitted work; in addition, H.K. Lyerly has a patent for Biomarkers of Response pending; and is a member of the Board of Directors, Oncosec Medical. E.J. Crosby reports a patent for “CXCR3 gene signature induced by local immunotherapy predicts response to checkpoint inhibitors” pending. No disclosures were reported by the other authors.
M.L. Telli: Conceptualization, resources, formal analysis, supervision, investigation, writing–original draft, project administration, writing–review and editing. H. Nagata: Formal analysis, investigation, visualization, methodology, writing–original draft, writing–review and editing. I. Wapnir: Investigation, writing–review and editing. C.R. Acharya: Formal analysis, validation, investigation, visualization, writing–original draft, writing–review and editing. K. Zablotsky: Investigation, writing–review and editing. B.A. Fox: Supervision, validation, investigation, writing–review and editing. C.B. Bifulco: Investigation, writing–review and editing. S.M. Jensen: Investigation, writing–review and editing. C. Ballesteros-Merino: Conceptualization, investigation, writing–review and editing. M.H. Le: Conceptualization, investigation, writing–review and editing. R.H. Pierce: Conceptualization, resources, writing–review and editing. E. Browning: Conceptualization, validation, investigation, writing–original draft, writing–review and editing. R. Hermiz: Conceptualization, validation, investigation, writing–original draft, writing–review and editing. L. Svenson: Conceptualization, validation, investigation, writing–original draft, writing–review and editing. D. Bannavong: Conceptualization, validation, investigation, writing–original draft, writing–review and editing. K. Jaffe: Conceptualization, resources, validation, investigation, writing–original draft, writing–review and editing. J. Sell: Conceptualization, validation, investigation, writing–original draft, writing–review and editing. K. Malloy Foerter: Conceptualization, supervision, investigation, writing–original draft, writing–review and editing. D.A. Canton: Conceptualization, resources, supervision, investigation, writing–original draft, writing–review and editing. C.G. Twitty: Conceptualization, resources, supervision, investigation, writing–original draft, writing–review and editing. T. Osada: Formal analysis, supervision, investigation, methodology, writing–original draft, writing–review and editing. H. Kim Lyerly: Conceptualization, resources, supervision, funding acquisition, writing–original draft, writing–review and editing. E.J. Crosby: Conceptualization, supervision, validation, investigation, visualization, methodology, writing–original draft, project administration, writing–review and editing.
OncoSec Medical provided research support for the conduct of the OMS-I140 clinical trial. Research reported in this article was supported by Susan G Komen for the Cure (grant 180062, to M.L. Telli), National Center For Advancing Translational Sciences of the NIH under Award Number UL1TR003142 (to M.L. Telli). We would like to acknowledge Tao Wang and Christopher Rabiola for their technical assistance with animal models, immune monitoring, and flow cytometry. We would also like to acknowledge the tireless efforts of the coordinators in assisting with patient management on the clinical trial and all the patients and their families who participated in this trial.
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