Patient accrual details and demographics

From December 2019 to March 2022, 12 patients were consented. Three patients did not meet eligibility criteria and nine patients were accrued to the study (figure 1A: study schema; figure 1B: Consolidated Standards of Reporting Trials diagram). The study was designed as an accelerated titration study. One patient was accrued at dose level 1 (#1 DL1), one patient at dose level 2 (#2 DL2), four patients at dose level 3 (#3 DL3, #4 DL3, #5 DL3, #6 DL3) and three patients at dose level 4 (#7 DL4, #8 DL4, #9 DL4). One patient (#4 DL3) was non-compliant with the required number of CKM doses (minimum of six doses) and thus, only eight patients were considered evaluable for the primary endpoint of DLTs. Patient demographics are shown in table 1.

Figure 1

Study design. (A) Study schema. Systemic chemokine modulation (CKM; 30 min-long IFN-α2b infusion followed by 2.5-hour-long infusion of rintatolimod) was given as nine daily infusions (3 days per week) over 3 weeks (days 0, 1, 2, days 7, 8, 9, and days 14, 15, 16) along with weekly paclitaxel 80 mg/m2. CKM consisted of intravenous rintatolimod (200 mg) and oral celecoxib (two doses of 200 mg)±IFN-⍺2b at dose-escalation from 0 (no IFN-⍺2b; dose level 1) through 5 MU/m2 (dose level 2) and 10 MU/m2 (dose level 3) to 20 MU/m2 (dose level 4). Stars represent the timing of tumor biopsies performed before and after the CKM at the higher dose levels. (B) Consolidated Standards of Reporting Trials flow diagram. This depicts screening, enrollment and follow-up of participants in the trial. The trial enrolled nine patients, eight of whom were evaluable for the primary endpoint of safety. IFN, interferon.

Table 1

Baseline characteristics of the study population

Safety of CKM combination with weekly paclitaxel

The data cut-off for the safety and efficacy analyses was February 16, 2024. There were no AEs that qualified as DLT or irAE at any of the dose levels tested. Apart from patient #4 DL3 (who forgot to take celecoxib), all other patients completed at least 6 doses of IFN-α2b and rintatolimod and 12 doses of celecoxib taken twice a day (overall 6 doses of CKM) and were considered evaluable for the primary endpoint (online supplemental figure S1). A summary of AEs is presented in online supplemental table 2. The most common worst-grade AEs, which were possibly, probably, or definitely attributed to study therapy (CKM, paclitaxel, doxorubicin and cyclophosphamide) are listed in table 2 (CKM), (online supplemental figure 2A) (paclitaxel), and online supplemental figure 2B) (doxorubicin or cyclophosphamide), respectively.

Table 2

Treatment-related adverse events (TRAEs) attributed to components of chemokine modulaton regimen (CKM)

The most frequent AEs attributed to CKM were nausea, chills, fatigue, fever, alanine transaminase/aspartate transaminase increase, myalgia, dizziness, headache, constipation, and hypotension, which were grade 1 or 2. Grade 3 treatment-related adverse events included neutropenia (4/9 patients), attributed to CKM (1/9 patients) or paclitaxel (3/9 patients) or doxorubicin/cyclophosphamide (1/9 patients), pneumonia (1/9 patients) and anemia (1/9 patients) attributed to doxorubicin/cyclophosphamide. The only serious AE in the study was pneumonia (2/9 patients), one attributed to doxorubicin/cyclophosphamide (1/9 patients), and the other one was a grade 3 event, unrelated to study treatment. Both the events of pneumonia were managed with antibiotics.

Clinical outcomes in patients receiving CKM/NAC regimen: Rates of pCR, microinvasive disease (ypTmic) and interim breast MRI responses

The pCR rate (ypT0/is N0) was 55% (5/9 patients), which included wo of the two node-positive patients. Among the remaining four patients who did not achieve a pCR, two (patients #3 DL3, #8 DL4) were classified as residual cancer burden-I (RCB-I), 1 of which was ypTmic. The other two patients (#1 DL1, #4 DL3) were classified as RCB-II. Three patients received adjuvant capecitabine and one patient with germline Breast Cancer gene (BRCA) mutation received adjuvant olaparib. Figure 2 shows the waterfall plot (left) with the per cent change in tumor size by breast MRI from baseline to pre-surgery and the swimmer’s plot (right) with dose levels, clinical stage of the tumors, RCB status, RFS and OS.

Figure 2

Radiologic and pathologic responses post neoadjuvant treatment. Breast MRI responses (left) after completion of neoadjuvant treatment were measured by RECIST V.1.1 and irRECIST for the nine patients on dose levels (DL) 1–4. Waterfall plot shows the % changes in tumor size from baseline to pre-surgery. The clinical stage of the tumors is depicted using AJCC staging. The pathological stage (right) is reported as RCB-0, RCB-I and RCB-II. As of the data cut-off of February 16, 2024, two patients on follow-up progressed and developed metastatic disease and died. The table lists the doses of celecoxib, IFN-α2b, rintatolimod at each DL and occurrences of grade 3 or higher treatment-related adverse events (TRAEs), attributed to paclitaxel, CKM or doxorubicin and cyclophosphamide (AC). Abbreviations: AC, doxorubicin (Adriamycin) and cyclophosphamide; AJCC, American Joint Committee on Cancer; CKM, chemokine modulatory regimen (rintatolimod, IFN-α2b, celecoxib); IFN, interferon; irRECIST, immune related Response Evaluation Criteria in Solid Tumours; RCB, residual cancer burden; RECIST, Response Evaluation Criteria in Solid Tumors.

With a median follow-up time of 29.9 months (range: 21.6–43.9 months), there were two distant BC recurrences, which both resulted in BC-related mortality. There were no recurrence events in RCB 0–1 patients. Median RFS was not reached (NR; 95% CI, 15.0 months to NR) with a 2-year rate of 75%. Median OS was also NR (95% CI, 21.7 months to NR) with a 2-year rate of 75%.

The breast MRI response was evaluated after completion of neoadjuvant CKM and paclitaxel and pre-surgery using RECIST V.1.1 and irRECIST. There was concordance in measurements by RECIST and irRECIST (although only one MRI scan is available for irRECIST measurement, and a follow-up repeat MRI was not performed). Among the 7/9 patients who underwent breast MRI after CKM and paclitaxel, one attained radiographic complete response (CR), one had partial response, four had stable disease, while one experienced progressive disease. Thus, the interim breast MRI response was 28.6% (online supplemental figure 3).

Systemic CKM and paclitaxel induced transient decreases in circulating CTLs. Blood samples were obtained pretreatment (day 0) and on the same day post-treatment after completion of CKM and paclitaxel. Similar to the observations from our prior TNBC clinical trial (NCT03599453), which included no chemotherapy component,30 longitudinal flow cytometry analysis of the circulating immune cells demonstrated rapid decreases in the numbers of circulating effector-type CD3+CD4−CD8+ CTLs and of CD3+CD4−CD8+GrB+ CTLs. These decreases were statistically significant both in patients with pCR (p<0.001 and p=0.002 for each cell type) and in patients with residual disease (respectively, p=0.004 and p=0.011) (figure 3A). These results were mirrored by real-time quantitative polymerase chain reaction (RT-qPCR), which showed consistent decreases in CD8α (to an average of 0.12 and median of 0.16 of baseline; p=0.004) and CD8β (average of 0.25 and median of 0.19 of baseline; p=0.005). Interestingly, there was no difference in GrB expression (p=0.108) (figure 3B), which may suggest a compensatory (GrB-activating) effect of CKM on the remaining CTLs.

Figure 3

Decreased CD8+T cell counts in the blood of patients directly after CKM with paclitaxel treatment. Acute changes in the immune cell subset composition of peripheral blood were measured at the end (same day) of treatment with CKM and paclitaxel. (A) Multiparameter flow cytometry analysis of circulating lymphocytes (N=8) for patients (PTS) 2–9. The blue lines represent changes in blood in patients who attained a pCR and the red lines represent blood changes in patients with non-pCR. A statistically significant decrease in CD8+ T cells and GrB+ CD8+ T cells was observed. (B) Expression of cytotoxic T lymphocyte markers (CD8α and CD8β) and their effector status (GrB) was measured using RT-PCR for PTS 3–9 (N=7). Data is expressed as ratios of the individual markers to the housekeeping gene, HPRT1. Decreases in both CD8α and CDβ transcripts are observed with no change in GrB transcript measured by RT-PCR (N=7). (C) Short-term changes in the immune signature in peripheral blood mononuclear cells induced by CKM and paclitaxel. Note the changes in chemokines, chemokine receptors, IFN-inducible and IFN-regulatory genes which are upregulated post-treatment (N=7) for PTS 3–9 (>1.2-fold change, FDR<0.1). (D) Combination of CKM and paclitaxel downregulates expression of the Wnt family member 7A, granzyme M, granzyme K, CXCL8, CXCL2, CXCR3, CD8A and CD8B (N=7) for PTS 3–9 (>1.2-fold change, FDR<0.1). (E) xCell immune cell average enrichment scores for the various immune cell subtypes across the pretreatment and same day post-treatment points for PTS 3–9 in the pCR and non-pCR groups. The left panel legend shows the cell subtypes in the order they appear in each stacked bar from left to right. The right panel shows post-treatment decreases in the signatures of the CD4+ and CD8+ T cells, and a concomitant increase in B cells and macrophage signatures, consistent with their preferential retention within the circulation. CKM, chemokine modulatory regimen; FDR; false discovery rate; IFN, interferon; pCR, pathological complete response RT-qPCR, real-time quantitative polymerase chain reaction.

We also observed statistically significant decreases in the number of circulating total CD4+ T cells. Unexpectedly, this same trend was also observed in total FoxP3+ cells and CXCR4+ FoxP3+ cells in the circulation, among both patients with pCR and those without (online supplemental figure 4A), which may suggest a direct Treg-reprogramming effect of CKM. These results were mirrored by RT-PCR (online supplemental figure 4B) which revealed decreases in total circulating CD4+ T cells (to an average of 0.54/median of 0.32 of baseline; p=0.026), which was statistically significant only among the patients with pCR (average 0.36/median 0.36 of baseline, p=0.038). These changes resulted in a significant decrease in the ratios of CD8α/FoxP3 (average 0.11/median 0.05 of baseline; p=0.014), and CD8β/FoxP3 (average 0.18/median 0.16; p=0.002). We also observed an average 15.9-fold/median=13.8-fold increase in programmed death-ligand 1 (PD-L1) (p<0.001) and an average 7.04-fold/median=7.25-fold increase in PD-L2 (p<0.001) in the circulation among all patients, suggestive of upregulation of PD-L1 on myeloid cells. Unexpectedly, there was also a decrease in programmed cell death protein-1 (PD-1) in the PBMCs of patients with non-pCR (average 0.35/median 0.39 of baseline, p=0.012).

CKM and paclitaxel induce differential changes in circulating immune markers. The post-treatment blood samples showed higher expression of immune genes, such as, IFN-stimulated genes; chemokines CXCL10, CXCL11, CXCL9, and CCL5; chemokine receptor XCR1; IFN inducible genes, IFN regulatory factors, signal transducer and activator of transcription (STAT), and the transmembrane genes, as determined by bulk RNA-seq (figure 3C).

Consistent with the results of the RT-PCR and flow cytometry, we observed downregulation of Wnt family member 7A, granzyme M, granzyme K, CXCL8, CXCL2, CXCR3, CD8A and CD8B (figure 3D), consistent with the efflux of CXCR3+CTLs from the circulation to the tumor. Similarly, GSEA showed upregulation of IFN-α, IFN-γ and inflammatory response pathways (online supplemental figure 5A). Immune deconvolution analysis using xCell showed a decrease in CD4+ T cells, CD8+ T cells, but a concomitant expansion of B cells and M1 and M2 macrophages in post-treatment versus pretreatment samples (figure 3E).

Gene expression only elevated in post-treatment blood samples of pCR patients was analyzed. This included elevated expression of XCR1, CCL5, CCR7, CCR2, TNFRSF17, IL-6, IL-6-AS1, IL-2RA, LTF, and immunoglobulin heavy and light chains (online supplemental figure 5B), raising the possibility that CKM-driven immune patterns can act as early predictors of pCR. In contrast, we could not identify any pretreatment predictors of pCR.

Online supplemental figure 5C shows the differential post-treatment expression of genes in patients who subsequently attained pCR versus non-pCR. We observed higher expression of immunoglobulin genes, IGLV8-61, IGHG3 and IGLV6-57, IL-12RB2 and XCR1 (expressed on cDC1 subset of dendritic cells involved in antigen presentation46) in patients who subsequently attained pCR, providing additional indication that CKM/paclitaxel-induced immune signature may be used as a pCR predictor. Similarly, GSEA showed upregulation in cell proliferation (E2F targets, G2M checkpoint, mitotic spindle, MYC targets) and immune signaling (IFN-γ response, interleukin (IL-6)-Janus kinase-STAT 3) pathways, combined with the downregulation of angiogenesis-signaling, hypoxia-signaling and tumor necrosis factor (TNF)-signaling pathways, in the post-treatment blood samples of pCR patients, compared with non-pCR patients.

Improved chemokine production patterns and CTL markers in the TME of patients receiving systemic CKM and paclitaxel

Since CD8+ T-cell infiltration is a prognostic marker that predicts pCR and improved long-term outcomes in patients with TNBC, our exploratory analyses involved treatment-associated changes in intratumoral CTL markers in the total biopsy volume. Paired pre-CKM and post-CKM/paclitaxel biopsies (weeks 0 vs 3) were successfully obtained in four patients with pCR: patients (#5 DL3, #6 DL3, #7 DL4, #9 DL4), but only in one patient with non-pCR (#8 DL4), limiting our ability to interpret the non-pCR data (figure 4A). Comparison of the pre/post CKM/paclitaxel mRNAs showed a trend towards an overall increase in CD8α (average 6.88-fold/median 2.86-fold; p=0.312) and an average 5.9-fold/median 5.39-fold increase in CD8β (p=0.094). The increase in CD8β was statistically significant in the patients with pCR (average 7.07-fold/median 5.67-fold increase; p=0.016); with a trend towards CD8α increase in the same subset of patients (8.3-fold average/3.03-fold median increase, p=0.096). We further noted significant increases in the ratios of CD8α/FoxP3 (average 2.11-fold/median 2.15-fold; p=0.018), which was particularly significant in patients with pCR (average 2.18-fold increase and median 2.21-fold increase, p=0.009).

Figure 4

Intratumoral increases in cytotoxic T lymphocyte markers after systemic CKM and paclitaxel. (A) Intratumoral levels of CD8⍺, CD8β and chemokine expression (normalized for HPRT1) were measured using RT-PCR in tumor biopsies obtained at baseline and at 3 weeks of CKM/paclitaxel regimen (see figure 1 for study schema) for patients on the higher CKM dose levels (PTS 5–9). The blue lines represent patients who attained a pCR (N=4) and the red line represents the non-pCR patient (N=1; dose level 4). Statistically significant increases in CD8β, CD8α/FoxP3, CCL5 and CXCL12 mRNA were observed. (B) Gene Set Enrichment Analysis (GSEA) expression level changes (normalized enrichment scores (NES)) for PTS 5–9 identified multiple immune signaling groups which increased significantly post CKM/paclitaxel, while proliferation gene families were significantly decreased (FDR<0.05). CKM, chemokine modulatory regimen; FDR; false discovery rate; pCR, pathological complete response; PTS, patients; RECIST, Response Evaluation Criteria in Solid Tumors.

To gain insight into the mechanism of the CKM-driven enhancement of the CTL signature, we analyzed changes in the expression of CD8+T cell-attracting and Treg-attracting chemokines (figure 4A). The average intratumoral expression of CCL5 mRNA (chemokine binding to CTL-expressed CCR5) showed a trend to post-treatment increase overall (average 4.73-fold/median=2.88-fold; p=0.099) but was statistically significant in the patients with pCR (average 5.49-fold/median 4.06-fold; p=0.030).

Unexpectedly, we also observed a 5.23-fold average/median 4.45-fold increase in the undesirable chemokine CXCL12 (p=0.021), particularly significant in patients with pCR (average 6.06-fold/median 4.93-fold; p=0.005; figure 4A). We also observed trends towards increased ratios of CD8α/CD4 (average 2.67-fold/median 2.31-fold; p=0.054) and CCL5/CCL22 (average 5.09-fold/median 2.85-fold; p=0.058) selectively among tumors with pCR (online supplemental figure 6). GSEA (figure 4B) revealed elevated immune signaling pathways associated with allograft rejection, IFN-α-response, IFN-γ-response, complement, IL-6-Janus kinase-STAT 3, IL-2-STAT 5, and general inflammatory signature in the post-treatment TMEs. Reduced proliferation-related genes (E2F targets, G2M checkpoints, MYC targets, mammalian target of rapamycin complex1, glycolysis and cholesterol hemostasis) were also noted, interestingly with reduced early and late estrogen response signature (figure 4B).

We used multiplex IHC (figure 5) for spatial profiling, to compare the intratumoral distribution and frequencies of immune cells before and after the CKM/paclitaxel regimen. Figure 5A–B show representative pre/post-treatment tumor samples and trends towards the increased proximity of CD8+ T cells to PanCK+ cancer cells post-treatment, preferentially seen in the patients who subsequently attained pCR. Cell enumeration (figure 5C) demonstrated a post CKM/paclitaxel overall increase in intratumoral T cells (p=0.028) being particularly pronounced in tumors from patients who achieved pCR (p=0.009). This increase included both CD8+ T cells and CD4+ T cells, but not Tregs. The patients with pCR also demonstrated strong decreases in stromal and vascular marker, α-smooth muscle actin (αSMA) expression in whole tumor tissue and in their stromal areas (respectively, p=0.006 and p=0.02; figure 5C). αSMA is a marker of epithelial to mesenchymal transition and an increase in αSMA expression in the tumor with non-pCR while decrease post-treatment in tumors with pCR is consistent with the role of αSMA positive cells in promoting tumor growth.47 No significant changes in other markers (PD-L1, PD-1, FOXP3, COX2, CD68, CD20) were observed (data not shown).

Figure 5

CKM and paclitaxel treatment decreases stromal vascular markers while increasing cytotoxic T lymphocytes in the tumors. MICSSS was performed for high-dimensional tissue analysis on tumors at baseline and at 3 weeks at the completion of CKM and paclitaxel. The whole tumor area (excluding normal, necrosis or fibrosis), tumor nests (epithelial) and stromal elements were analyzed. (A) Pseudo-immunofluorescence images from MICSSS used to visualize each marker, are shown for a representative patient with pCR (PT 5) and non-pCR (PT 8), before and after the CKM/paclitaxel. Note that the patient with pCR, but not the non-pCR patient, showed post-treatment CD3 and CD8 infiltration, and decreases in the PanCK-positive and α-smooth muscle actin (α-SMA)-positive cells. Each ROI) is 500 μm × 400 μm. (B) The blue lines represent patients who attained a pCR (N=4) and the red line represents the non-pCR patient (N=1; dose level 4). Treatment with CKM and paclitaxel induces a trend towards closer apposition of CD8+T cells to the nearest PanCK+ cancer cells in patients with pCR (patients 5, 6, 7, 9) but not in the single patient with non-pCR (PT 8). (C) The blue lines represent patients who attained a pCR (N=4) and the red line represents the non-pCR patient (N=1). There were consistent post-treatment increases in total T cells, CD8+ T cells, and CD8− T cells (identified as CD4+ T cells) but no changes in regulatory T cells (Tregs) in the epithelial and stromal/vascular areas of the tumors and associated decreases in the prevalence of the cells expressing αSMA, selectively affecting patients who subsequently attained pCR. CKM, chemokine modulation; Multiplexed Immunohistochemical Consecutive Staining on Single Slide, MICSSS; NK, natural killer; pCR, pathological complete response; PT, patient; ROI, region of interest.