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Abstract

Objectives

CpG ODN is a Toll-like receptor 9 agonist with immunotherapeutic potential for many cancer types, including aggressive breast cancers. There is strong interest in utilizing CpG ODN as an adjuvant to improve clinical efficacy of current treatments and immunogenicity of breast cancers not traditionally responsive to active immunotherapy, such as those that are human epidermal growth factor receptor 2 (HER2)-positive. This study aimed to study the efficacy and safety of combination CpG ODN plus anti-HER2 antibody trastuzumab treatment in patients with advanced/metastatic breast cancer.

Methods

This single-arm, open-label phase II clinical trial treated patients (n = 6) with advanced/metastatic HER2-positive breast cancer with weekly subcutaneous CpG ODN and trastuzumab. Patients may have received any number of prior therapies to be enrolled (most enrolled at median 1 prior line of chemotherapy). Peripheral blood was collected at baseline and weeks 2, 6, 12, and 18 for immune analyses. Six patients were enrolled and 50% achieved stable disease (SD) response.

Results

Median PFS was 8.3 months. Three of the six patients enrolled opted to stop treatment due to tolerability issues. Multiplex assay for cytokine measurements revealed significantly higher VEGF-D levels at week 2 compared to baseline. Peripheral blood mononuclear cells analyzed by flow cytometry showed a significant increase in monocytic MDSC between weeks 6 and 12. Patients with progressive disease tended to have higher levels of week 6 monocytic MDSC and PD-1+ T cells than patients with SD. NK cell populations did not significantly change throughout treatment.

Conclusions

CpG ODN and trastuzumab treatment of metastatic HER2 + breast cancer was safe but was not tolerable for all patients. This combination did induce potentially predictive immune profile changes in treated patients with metastatic HER2 + breast cancer, the significance of which needs to be further explored.

Plain language summary

Why was the study done? Breast cancer that has metastasized (moved outside of the breast and local lymph nodes) is currently considered incurable and can be difficult to treat. Treatments that can stimulate the immune system to recognize cancer cells have been found to be useful for many types of cancers, including some types of breast cancers. This study tested a new immune stimulator (CpG ODN) in combination with a currently on-the-market antibody treatment for breast cancer (trastuzumab). What did the researchers do? The research team enrolled patients who had metastatic breast cancer and treated them all with a combination of trastuzumab and CpG ODN for 12 weeks. These patients were monitored for any side effects/toxicity, monitored for how long their breast cancer responded to this treatment, and monitored for how long they lived after beginning this treatment. Patients also had their blood drawn at different time points to observe how their immune cells and immune proteins (e.g. cytokines) changed on treatment. What did the researchers find? The research team enrolled six patients and found that the treatment was safe and that 50% of the patients treated did not have any breast cancer growth when given CpG ODN plus trastuzumab. Looking at the immune cells in the patient blood samples, some cells that are known to decrease the immune response to cancers (myeloid-derived suppressor cells) did increase towards the end of treatment. What do the findings mean? Overall, CpG ODN and trastuzumab treatment was found to be safe and potentially effective in preventing breast cancer growth.

Keywords

CpG ODN toll-like receptor 9 TLR9 trastuzumab human epidermal growth factor receptor 2 breast cancer clinical trials immunotherapy

Introduction

Approximately 15%–20% of all breast cancer cases are human epidermal growth factor receptor 2 (HER2)-positive and historically represent a group of aggressive tumors with poor outcomes.¹ The advent of HER2-targeting agents has revolutionized the treatment of HER2-positive breast cancer and has conferred significant improvements in survival rates among affected patients.² The first of these drugs was trastuzumab, a humanized monoclonal IgG1 antibody that binds to the extracellular domain of HER2. Trastuzumab has been successfully combined with chemotherapy, other HER-directed monoclonal antibodies, and tyrosine kinase inhibitors to further amplify its benefit.^3-7 Moreover, new HER2-targeting antibody-drug conjugates have shown impressive response rates in pre-treated patients with metastatic disease.^8,9 Despite the immense benefits of these treatment options, HER2-positive metastatic breast cancer remains problematic with a median overall survival rate of less than 5 years.^1,10 Therefore, there is a need for novel and more effective therapeutic options for such patients.

Several studies have shown that the antitumor effects of trastuzumab are dependent on the presence of immune effector cells that bear FcγR, such as natural killer (NK) cells.^11,12 One method to further promote the anti-HER2 immunity of trastuzumab is to add an agent that can act as an immunostimulant. A possible candidate would be a Toll-like receptor (TLR) agonist. Human TLRs are a family of ten membrane-bound receptors that recognize unique pathogen-associated molecular patterns.¹³ TLRs can be found on the cell membrane or intracellularly where they are localized to endosomes, lysosomes, or the endoplasmic reticulum. TLR activation can promote a robust innate immune response and produce anti-tumor responses. For example, TLR agonists can increase tumor antigen-specific cytotoxic T cell killing,^14-16 lead to anti-tumor Th1 polarization,^17,18 and boost anti-PD-1 adaptive immunity.^19,20 There has been considerable interest in TLR9, a receptor that recognizes unmethylated CpG oligodeoxynucleotides (ODN) commonly found in bacterial and viral genomes.²¹ TLR9 is expressed by a variety of antigen-presenting cells, T cells, and NK cells.^12,22 TLR9 is a receptor of particular interest in the breast cancer field as high levels of TLR9 expression in breast carcinomas is independently associated with improved patient outcomes.²³ TLR9 stimulation leads to a MyD88-dependent signaling cascade that induces IRF7, MAPK, and NF-kB transcription factors. TLR9 signaling in the setting of cancer can increase the amount of tumor-infiltrating lymphocytes,²⁴ inhibit angiogenic signaling,²⁵ prevent immunosuppressive T regulatory cell activities,²⁶ and improve the efficacy of immune checkpoint inhibitors.^20,24

Importantly, NK cell activity can be augmented by TLR9 activation. Our group has shown that co-stimulation through the NK cell Fc receptor and addition of a secondary activating agent (e.g. interleukin-12 (IL-12), IL-21) can lead to a robust NK cell killing response against antibody-coated tumor cells.^27-30 Specifically, we have demonstrated in murine models that activation of TLR9 on NK cells with CpG ODN can improve responses to anti-HER2 antibody-coated tumor cells leading to enhanced cytotoxicity and the secretion of interferon γ (IFN-γ), IL-8, and MIP-1α.¹² Other studies have shown that repeated administration of a TLR9 agonist and anti-CD20 monoclonal antibodies yields improved anti-CD20 activity compared with a single administration,³¹ suggesting that dual treatment may be beneficial in the clinical setting.

PF03512676 (agatolimod; Pfizer) is a class B, synthetic CpG ODN that contains 4 unmethylated (CpG) motifs, including 3 GTCGTT’ hexanucleotide motifs that are capable of being recognized by TLR9. Several cancer clinical trials have been conducted using TLR9 agonists, including PF03512676 which is the most extensively studied (results summarized in Table 1). These studies have shown PF03512676 (referred to here as CpG ODN) to be well-tolerated and a strong immune activator through such mechanisms as increasing effector memory T cells and stimulating pro-inflammatory cytokine production (Table 1). Most clinical trials have enrolled patients with cancers that tend to respond well to immune-based therapeutic agents (e.g., melanoma), however, no published breast cancer trials utilizing TLR9 agonists were found. While a few patients with breast cancer have been included in some TLR9 agonist phase I trials that enrolled a broad range of solid tumors, the current study is the first phase II study to specifically treat patients with breast cancer.

Table 1.

Summary of Prior Oncology Clinical Trials Using TLR9 Agonists.

Cancer Type	Phase and Enrollment	CpG ODN Administration	Additional Treatment	Patient Outcomes	Correlative Immune Changes with Study Regimen	Reference
Esophageal cancer (squamous cell carcinoma)	Phase I; 9 patients	CpG 7909/PF03512676 (Coley Pharmaceutical group) S.C. weekly	Epitope peptides derived from cancer-testis antigens (LY6K-177 & TTK-567)	Median PFS of 3.7 months. 5/9 patients showed stable disease as best response (no complete or partial responses).	IFN-γ-producing LY6K-specific T cells higher in patients who received higher dose CpG and peptide vaccine compared to those that got vaccine alone	³²
Melanoma	Phase I; 8 patients	CpG 7909/PF03512676 S.C. monthly for 4 injections	Melan-A/MART-1 analog peptide and Montanide	Not reported	Enhanced effector memory T cell populations which consisted of effector memory cells. Effector T cell populations killed in a MART-1 specific manner in vitro.	^33,34
	Phase I; 8 patients (3 patients received CpG)	CpG 7909/PF03512676 S.C. biweekly for at least 6 doses	NY-ESO-1157-165V peptide and Montanide	Median PFS of 4 months	Significant growth of effector-memory NY-ESO-1–specific CD8 + T cell population in patients who also received CpG	³⁵
	Phase II; 184 patients (half received CpG low or high dose)	CpG 7909/PF03512676 S.C. weekly	Dacarbazine	Objective response rate in CpG + dacarbazine arm was 16%, compared with 8% in the dacarbazine alone group	Serum MCP-1 tended to increase from baseline following treatment. Serum IP-10 also increased from baseline and remained detectable between 8-24 hours post-treatment.	³⁶
	Phase I; 20 patients	CpG 7909/PF03512676 S.C. on days 1 and 15 per 28 day cycle, for up to 13 cycles	MART-1, gp100, and tyrosinase peptides plus GM-CSF	Median PFS of 1.9 months. Median overall survival of 13.4 months.	9/20 patients had detectable antigen-specific CD8+ T cells at days +50 and/or + 90 of study	³⁷
	Phase I; 21 patients (17 with melanoma)	CpG 7909/PF03512676 S.C. weekly	Tremelimumab	Two patients achieved partial response that lasted at least 170 days.	No human antihuman antibody responses were detected against tremelimumab.	³⁸
	Phase I; 19 patients	CpG 7909/PF03512676 S.C. monthly	NY-ESO-1_79-108 peptide and Montanide	Patients with high IFNg-secreting NY-ESO-1 specific CD4 + and CD8 + T cells tended to have longer PFS and OS lengths	Vaccination induced strong NY-ESO-1 antigen specific CD8 + and CD4 + T-cell and antibody responses	³⁹
	Phase III; 1345 patients	CpG 7909/PF03512676 I.M. five doses every 3 weeks, followed by 8 doses every 12 weeks	Recombinant MAGE-A3 protein and AS01 B	Median DFS was 11.0 months in treatment group and 11.2 months in placebo group.	None reported	⁴⁰
	Phase Ib; 22 patients	SD-101 intratumorally (TriSalus Life Sciences) days 1, 8, 15, 22, and then every 3 weeks for 7 more doses	Pembrolizumab	12-month PFS was 88% and OS was 89%. ORR in patients naïve to anti-PD-1 therapy was 78%, but 15% in those that had prior anti-PD-1 therapy.	Increased tumor-infiltrating lymphocytes in following treatment.	⁴¹
	Phase Ib; 167 patients (144 patients receiving combo; 23 receiving CMP-001 alone)	CMP-001 (Checkmate Pharmaceuticals) intratumorally	Pembrolizumab	ORR was 24% in cominbation group and 22% with CMP-001 alone. Median duration of response not yet reached.	Possible association between serum CXCL10 and treatment response; only interim results at this time.	⁴²
Non-small cell lung cancer	Phase II; 112 patients	CpG 7909/PF03512676 S.C. days 8 and 15 of 21-day cycles	Taxane and platinum chemotherapy	38% ORR in the CpG + chemotherapy arm and 19% in the chemotherapy alone arm. Median overall survival was 12.3 months in the CpG + chemotherapy and 6.8 months in the chemotherapy-alone arm.	None reported	⁴³
	Phase I; 12 patients	CpG 7909/PF03512676 S.C. days 8 and 15 of 21-day cycles	Paclitaxel and carboplatin	33% ORR (1 complete response) and 58% patients achieved stable disease.	Significant increases in serum IP-10, IL-6, IL-12, MCP-1, CRP, and IFNa were measured following treatment.	⁴⁴
	Phase III; 828 patients	CpG 7909/PF03512676 S.C. days 8 and 15 of 21-day cycles	Paclitaxel and carboplatin	No improvement in PFS or OS in patients who received CpG + chemotherapy vs chemotherapy alone	None reported	⁴⁵
	Phase III; 839 patients	CpG 7909/PF03512676 S.C. days 8 and 15 of 21-day cycles	Gemcitabine and cisplatin	No improvement in PFS or OS in patients who received CpG + chemotherapy vs chemotherapy alone	None reported	⁴⁶
	Phase II; 43 patients (half received CpG)	CpG 7909/PF03512676 S.C. weekly	Erlotinib	Median PFS in CpG + erlotinib arm was 1.6 months; median PFS in erlotinib alone arm was 1.7 months	Increase in IP-10 from baseline	⁴⁷
	Phase Ib; 35 patients	IMO-2055 (Idera Pharmaceuticals) S.C. weekly	Erlotinib and bevacizumab	15 % of patients achieved partial response and another 61 % had stable disease.	None reported	⁴⁸
Prostate cancer	Phase I; 12 patients	CpG 7909/PF03512676 every 3 weeks I.D.	Recombinant NY-ESO-1 protein	8 patients with stable disease, 3 with progressive disease, and 2 patients remained free of detectable disease. Median PFS of 4.75 months.	NY-ESO-1-specific CD4 + and/or CD8 + T-cell responses were produced in 9 patients, 5 of which did not express NY-ESO-1 in their tumor. Anti-CpG ODN antibody formation was detectable in several patients.	^49,50
Renal cell carcinoma	Phase II; 15 patients	CpG 7909/PF03512676 weekly I.D.., then bi-weekly S.Q.	Autologous tumor cell vaccine + GM-CSF, IFNa	Response rate of 20%	Up-regulation of PD-1 and CTLA4 on effector T cells. Activation of plasmacytoid dendritic cells and non-classical monocytes.	⁵¹
Non-Hodgkin’s lymphoma	Phase I; 50 patients	CpG 7909/PF03512676 weekly I.V. for 4-20 weeks	Rituximab	Objective responses occurred of 24% overall; 50% of patients that received extended treatment (6/12)	None reported	⁵²
NY-ESO-1 expressing cancers	Phase I; 18 patients	CpG 7909/PF03512676 every 3 weeks for a total of 4 S.Q. injections	NY-ESO-1_157-165V peptide and Montanide	Didn’t report patient outcomes	All patients developed detectable NY-ESO-1 specific antibodies. NY-ESO-1 specific CD4 + (17/19 patients) and CD8 + T cells (9/18 patients) were also noted.	⁵³
NY-ESO-1 expressing cancers	Phase I; 14 patients	CpG 7909/PF03512676 every 3 weeks for a total of 4 S.Q. injections; could be extended for a second cycle	NY-ESO-1_157-165V peptide and Montanide	Most patients with NY-ESO-1 specific immune responses had long overall survival lengths (27 months or greater)	In 6 patients, antigen-specific CD8 + T-cells became detectable ex vivo following vaccination. Anti-CpG ODN antibody formation was detectable in several patients.	^50,54

PF03512676 safety has been studied in combination with trastuzumab within phase I trials of patients with metastatic breast cancer and has been shown to be safe in doses up to 0.16 mg/kg.⁵⁵ Initial efficacy measurements from these trials suggest that the best initial responses were seen with the higher doses (0.12 and 0.16 mg/kg) of PF03512676, however, final results from these studies are not currently available from the sponsor. In the present clinical trial, the combination of subcutaneous CpG ODN and intravenous trastuzumab was studied in patients with advanced/metastatic HER2 + breast cancer who had trastuzumab-responsive disease. The primary objective was to assess the safety and tolerability of this regimen, with secondary objectives being to evaluate the effect of the drug combination on progression-free survival (PFS) and levels of immune markers including circulating cytokines and immune cells.

Materials and Methods

Study Design

A single arm, open-label phase II clinical trial was conducted at the Ohio State University Comprehensive Cancer Center under an IRB-approved protocol (IRB protocol no: 2008C0116). This was registered within the publicly accessible database ClinicalTrials.gov. The ClinicalTrials.gov ID is NCT00824733 and the registered title is “Agatolimod and Trastuzumab in Treating Patients with Locally Advanced or Metastatic Breast Cancer” (https://beta.clinicaltrials.gov/study/NCT00824733?id=NCT00824733). The sample size/accrual goal estimated was 15 patients due to limited availability of the CpG ODN agent (PF-03512676) and time constraints (formalized sample size calculation not completed). Patient accrual occurred between March 2009 and April 2014 at which point the study was terminated due to slow patient accrual. The primary objective was to determine the safety and tolerability of trastuzumab and CpG ODN treatment. Secondary objectives included the assessment of progression free survival (PFS) and measurement of cytokines and immune cell populations. Written informed consent was obtained from all study participants. Peripheral blood for correlative studies was collected at baseline and at the beginning of weeks 2, 6, 12, and 18. These timepoints were correlated with treatment schedule to include collections prior to receiving study drug, middle of treatment, end of treatment, and 6 weeks after completion.

Eligibility

Eligible patients were ≥18 years old and diagnosed with biopsy-proven metastatic, HER2 + breast cancer. HER2 overexpression was defined as 3+ by immunohistochemistry (IHC) or +2 IHC with HER2/CEP17 ratio of ≥2.2 or ≥6 copies of HER2 per nucleus. At time of enrollment, patients were required to have stable disease on trastuzumab therapy for up to 6 months prior to receiving their first dose of CpG ODN. Patients who had been receiving trastuzumab for greater than 6 months or those with disease that progressed immediately prior to enrollment were not eligible. There were no restrictions in enrollment regarding the number of prior systemic therapies given for metastatic and/or non-metastatic disease. Other eligibility criteria included Eastern Cooperative Oncology Group (ECOG) performance status of 0-2, normal organ and bone marrow function, left ventricular ejection fraction of 50% or greater, no prior history of autoimmune or antibody-mediated disorders, and absence of unstable brain metastases. Patients with treated and stable brain metastases were eligible but were required to be off any systemic corticosteroids and anti-convulsants for at least 4 weeks prior to enrollment in the study. Patients with uncontrolled human immunodeficiency virus (HIV), hepatitis B, or hepatitis C were not eligible.

Treatment Schema and Study Procedures

Patients received intravenous (IV) trastuzumab at a dose of 2 mg/kg and subcutaneous (SQ) CpG ODN at 0.16 mg/kg once weekly for 12 weeks. Patients were permitted to continue IV trastuzumab alone following completion of 12 weeks of CpG ODN. Patients who had not received trastuzumab within 4 weeks of the treatment start date or more received a loading dose of trastuzumab at 4 mg/kg in week 1. Three patients opted to continue oral anti-HER2 therapy (lapatinib) while on trial. Patients were also permitted to continue lapatinib and endocrine therapy while on study; they were not required to have progressive disease to enroll. The response to therapy was measured by performing computed tomography of the chest, abdomen, and pelvis at 12-week intervals. Assessment of left ventricular ejection fraction by echocardiogram or multi-gated acquisition scan was performed at baseline and every 12 weeks. Complete blood counts, chemistries, and hepatic function tests were obtained weekly for 12 weeks and at week 18. History and physical examination were performed at baseline (week 1) and prior to weeks 2, 6, 12, and 18. Safety signals were evaluated using the Common Terminology Criteria for Adverse Events (CTCAE) version 3.0 in use at the time.

Correlative Blood Studies

Peripheral blood mononuclear cells (PBMC) were isolated from peripheral venous blood via density gradient centrifugation with Ficoll-Paque (Cytiva) as previously described.⁵⁶ PBMC and plasma were cryopreserved and stored at −80°C until batch analysis could be performed. PBMCs were analyzed by multi-color flow cytometry to quantify the presence of immune cell subsets as previously described.^57,58 The specific fluorescent-tagged antibodies employed are listed in Supplemental File 1. NK cells were defined as CD56⁺ cells and were further categorized by expression of CD69, CD16, and/or PD-1. All T cell subsets were CD3⁺, with naïve T cells being CD44-/CD62 L+, memory T cells being CD44+/CD62 L+, and effector T cells being CD44+/CD62L-. PD-1 expression was also measured on CD3⁺ T cells. Myeloid derived suppressor cells (MDSC) were defined as the CD33+/CD11b+/HLA-DR low PBMC population. Monocytic MDSC (M-MDSC) were further phenotyped as CD14+/CD15-, polymorphonuclear MDSC (PMN-MDSC) were defined as being CD14-/CD15+, and double-positive MDSC were defined as being CD14+/CD15+. Single color staining was performed for compensation. All samples were run on a BD LSR II flow cytometer and were subsequently analyzed with FlowJo software (TreeStar, Inc). Plasma was analyzed for the presence of several cytokines thought to be associated with CpG administration/NK cell activation (GM-CSF, IFNγ, IL-1α, IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, IL-15, IL-17, IL-18, IP-10, MCP-1, MCP-2, MDC, MIG, MIP-1α, MIP-1β, RANTES, TNFα, VEGF-A, VEGF-C, VEGF-D) via custom-designed multiplex U-Plex and V-Plex assays (Meso Scale Discovery) which were performed per manufacturer’s instructions. Multiplex plates were read on a MESO QuickPlex SQ 120 instrument.

Statistics

Linear mixed effect modeling was used to model repeated measures on percentage of various immune cells/cytokines over time and between multiple time points. Data distribution comparing immune cell and cytokine levels over time were determined by linear mixed modeling to allow for multiple testing corrections. To decrease the chance of false-positive results with this testing, Tukey’s method was also used to adjust P-values for multiple comparisons between time points. All P-values were double sided and statistical significance was set at alpha of 0.05. Adverse reactions were tabulated based on grade using CTCAE version 3.0 and attribution to study therapy. Response was assessed using Response Evaluation Criteria in Solid Tumors (RECIST) criteria version 1.1. Overall survival times were measured from date of first study treatment to date of death. Overall survival of stable and progressive disease patient groups were compared by log-rank (Mantel-Cox) analysis.

Results

Patient Demographics

This study enrolled six patients with metastatic HER2 + breast cancer. Table 2 lists the demographic characteristics of the patients enrolled in this study. The median age of study patients was 46 years (range 32-56), with two patients being pre-menopausal. Five patients were Caucasian, and one was African American. All six patients had hormone receptor (HR) positive breast cancer. Metastatic sites of disease included visceral sites (n = 5), bone (n = 3), and central nervous system (n = 1). Most patients had received only one prior line of chemotherapy (range 1-6) and all had previously received trastuzumab in combination with other agents. Four patients had prior endocrine therapy exposure. As allowed per protocol, two patients opted to continue lapatinib that they were already receiving, one patient opted to continue endocrine therapy, and one patient opted to continue both lapatinib and endocrine therapy while on study.

Table 2.

Patient Demographics and Tumor Characteristics.

	Patient 1	Patient 2	Patient 3	Patient 4	Patient 5	Patient 6
Age at study entry	56	42	50	56	40	32
Menopausal status	Post-menopausal	Post-menopausal	Post-menopausal	Post-menopausal	Pre-menopausal	Pre-menopausal
ECOG performance status	0	1	1	1	0	1
Race/ethnicity	Caucasian	Caucasian	Caucasian	Caucasian	African American	Caucasian
Hormone receptor expression	Positive	Positive	Positive	Positive	Positive	Positive
Sites of metastatic disease	Viscera	Viscera, bone	Viscera, bone	Viscera, bone	CNS	Viscera
Prior lines of chemotherapy	1	2	1	6	1	1
Prior HER2 targeted agents	Trastuzumab	Trastuzumab, lapatinib	Trastuzumab	Trastuzumab, lapatinib	Trastuzumab, lapatinib	Trastuzumab
Prior endocrine agents	Anastrozole	None	Anastrozole	Letrozole	Tamoxifen	None
Number of CpG ODN injections received (max 12)	4	12	5	12	12	3
Best disease response	Stable disease	Progressive disease	Stable disease	Progressive disease	Stable disease	Progressive disease

Safety

The most common toxicity of weekly trastuzumab and CpG ODN was injection site reaction, which occurred in all patients on study (Table 3). One patient had a grade 3 reaction and was taken off study. One patient with a grade 2 injection site reaction was offered a lower dose but refused and opted to discontinue study therapy after four CpG ODN treatments. Another patient had grade 3 fatigue and a grade 2 injection site reaction; she received dose reduction but did not feel like this adequately resolved symptoms and chose to stop CpG ODN treatments after five injections. The only other grade 3 adverse reaction was lymphopenia in one patient. Multiple mild grade 2 adverse events were observed and most often resolved quickly, the most common of which being fever (n = 2), pain (n = 2), and alanine aminotransferase elevation (n = 2).

Table 3.

Summary of Adverse Events. All Grade 2 or Higher Adverse Events Deemed to be at Least Possibly Related to Study Treatment are Included.

	Grade 2 [N(%)]	Grade 3 [N(%)]
Constitutional
Fatigue		1 (16.7%)
Fever	2 (33.3%)
Pain	2 (33.3%)
Dermatologic
Injection site reaction	2 (33.3%)	1 (16.7%)
Hematologic
Leukopenia	1 (16.7%)
Lymphopenia		1 (16.7%)
Neutropenia	1 (16.7%)
Metabolic
Alanine aminotransferase elevation	2 (33.3%)
Hyperglycemia	1 (16.7%)
Hypoalbuminemia	1 (16.7%)
Neurologic
Encephalopathy	1 (16.7%)
Sensory neuropathy	1 (16.7%)

Clinical Responses

Of the six patients enrolled on this study, there were no objective responses, but three patients (50%) experienced stable disease (SD) at 12 weeks as their best response. Median progression free survival of study patients was 8.3 months (Figure 1A). Of note, all three stable disease patients remained free of disease progression for at least 12 months from the start of study treatment. One patient with stable disease was also on endocrine therapy, one patient with stable disease was on lapatinib (no endocrine therapy), and the last patient with stable disease was on neither additional medication. Median overall survival (OS) of all patients was 84.6 months (Figure 1B). Median OS for patients with SD has not been reached, while median OS for patients with progressive disease (PD) was 40.5 months (P = 0.0588).

Figure 1.

Survival analysis. Kaplan-Meier graphs are shown demonstrating the (A) progression-free survival and (B) overall survival rates of study patients. The overall survival graph also contains lines denoting patients who had either stable or progressive disease as their best treatment response.

Circulating Plasma Cytokine Levels

Peripheral blood for correlative studies was available from five patients. Plasma cytokine multiplex analysis revealed that VEGF-D levels significantly increased among patients from off-treatment baseline to week 2 on treatment (P = 0.0392) (Figure 2). CpG-TLR9 signaling and NK cell activation-related cytokines including MCP-1, MCP-2, MIP-1α, MIP-1β, and RANTES levels were also measured, however, no significant trends between time points were elucidated. No statistically significant changes were observed in plasma levels of the other cytokines included within the multiplex panel, including GM-CSF, IFNγ, IL-1α, IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, IL-15, IL-17, IL-18, IP-10, MDC, MIG, TNFα, VEGF-A, VEGF-C, and VEGF-D (Supplemental File 2).

Figure 2.

Plasma cytokine levels of patients receiving CpG ODN and trastuzumab. Blood specimens were collected from patients at the beginning of weeks 1 (baseline), 2, 6, 12, and 18 while they were on study. Plasma was isolated and batch analyzed by multiplex cytokine analysis. Individual lines/shapes denote different patients’ cytokine trajectories. Data distribution comparing cytokine levels over time were determined by linear mixed modeling to allow for multiple testing corrections. Tukey’s method was used to adjust P-values for multiple comparisons between time points. Statistical significance was set at an alpha of .05. P-value of <.05 is denoted by *.

Immune Cell Subset Measurements

PBMC were isolated, stained, and analyzed by flow cytometry to determine immune cell populations over the course of treatment. Several T cell subsets were measured, including naïve CD8⁺ T cells, effector CD8⁺ T cells, memory CD8⁺ T cells, and PD-1+ T cells (Figure 3A). No significant trends were noted in the level of these subsets while patients were on treatment. NK cell subsets were also analyzed by flow cytometry within patient PBMC samples. Activated CD16⁺ and CD69⁺ NK cells were quantified as were exhausted PD-1+ NK cells (Figure 3B). There were no significant differences in levels of NK cell populations across the different trial time points. Total MDSC levels were measured as were the PMN-MDSC and M-MDSC subtypes (Figure 3C). Total MDSC tended to increase towards the end of overall CpG ODN treatment, but this finding did not reach statistical significance. However, levels of M-MDSC were significantly higher at week 12 compared to week 6 of treatment (P = 0.0099) and appeared to further increase at week 18 (6 weeks off CpG ODN treatment). Double-positive (CD14+/CD15+) MDSC were also identified, however, no appreciable differences in these cells were found between time points (data not shown).

Figure 3.

Measurement of peripheral blood immune cell subsets from patients receiving CpG ODN and trastuzumab. PBMC were isolated from patient peripheral blood specimens then stained and measured for immune cell subsets by flow cytometry. (A) T cell, (B) NK cell, and (C) MDSC. (D) PD-1+ T cell and total MDSC levels were evaluated in the context of clinical outcome where patients were grouped as having either stable disease or progressive disease as their best clinical response. Individual lines/shapes denote different patients’ cytokine trajectories. Statistical significance was set at an alpha of .05. P-value of <.01 is denoted by **.

Trends comparing immune cell levels of patients who had stable disease versus progressive disease were also conducted. While not powered for statistical comparison, patients with progressive disease on treatment tended to have higher levels of PD-1+ T cells and total MDSC (Figure 3D).

Discussion

Combinations of CpG ODN with a variety of chemotherapeutics and biologic therapies have shown it to be a safe and tolerable treatment option for most patients; however, there is no combination that has been proven effective in a late-stage disease trial. In the present study, we report the results of a phase II clinical trial which enrolled patients with metastatic HER2 + breast cancer and treated them with the HER2 monoclonal antibody trastuzumab and CpG ODN. Unfortunately, this trial closed after recruiting only 6 of the planned 10-15 patients due to slow accrual, in part due to patient concern over the frequency of drug administration. There were also limitations on the correlative studies performed due to only two of the patients completing all the pre-specified blood draw time points. Nevertheless, several important findings can be gleaned from this study.

Patients experienced adverse events that were similar in type and frequency to those previously described in other CpG ODN clinical studies.⁴⁵ The most common symptoms were injection site reaction, fatigue, and vomiting. These symptoms were all short-lived, but for a small number of patients they were significant enough for the patient to stop treatment. Of the patients on this study, half experienced stable disease as their best clinical disease response, while the other half had progressive disease. All three patients with stable disease were able to continue with trastuzumab treatment and they all experienced stability for at least 12 months prior to disease progression. This outcome is particularly encouraging when it is noted that all study patients had received trastuzumab treatment in the past. Compared to those with progressive disease following CpG ODN/trastuzumab treatment, patients with stable disease also tended to have longer durations of overall survival.

It was observed that patient serum VEGF-D levels significantly increased within one week of receiving CpG ODN and trastuzumab compared to baseline. VEGF expression has been found to be upregulated by activation of several TLR, including TLR9 within an in vitro model of pancreatic carcinoma and an in vivo model of lung carcinoma.^59,60 While a TLR9-stimulated increase in VEGF expression was generally found to promote angiogenesis and metastasis, it should be noted that TLR9 expression has been identified as a poor prognostic marker in several cancers. However, increased TLR9 expression indicates an improved prognosis in patients with breast cancer.²³ It is not known if this brief elevation of VEGF-D expression is enough to confer additional risk for tumor progression, particularly as the spike returned to baseline levels after the initial week. Many other cytokines were found to fluctuate over the course of CpG ODN/trastuzumab treatment, but it was difficult to identify specific cytokine expression trends within any group of patients on this study.

Patient peripheral blood M-MDSC increased significantly between weeks 6 and 12 of CpG ODN/trastuzumab treatment. While not statistically significant, this trend continued into the post-treatment period at week 18. Also, patients with stable disease tended to have overall lower amounts of MDSC compared to those with progressive disease throughout the course of treatment. M-MDSC are typically considered to be an immunosuppressive subset of cells whose presence are negatively associated with cancer survival outcomes, including breast cancer.⁶¹ Notably, M-MDSC are known to express TLR9 and it has been found that CpG treatment of M-MDSC leads to their differentiation into tumoricidal macrophages and promotion of a Th1 response.⁶² While M-MDSC levels are often thought to be associated with poorer cancer-associated prognoses, it is possible that the increase in M-MDSC populations towards the end of CpG ODN treatment is indicative of disease progression more than CpG ODN effects or whether this portends that the therapy is having unintended immune consequences. In future studies, it would also be interesting to study the tumor microenvironment changes that occur after CpG ODN treatment (e.g. tumor-infiltrating lymphocyte characterization) as these may be unique compared to trends seen in peripheral blood PBMCs.

Patients with stable disease also tended to have lower amounts of circulating PD-1+ T cells than patients with progressive disease. Our group has previously shown increased levels of intratumoral PD-1+ T cells to be associated with improved HER2 + breast cancer outcomes.⁶³ Alternatively, it has been suggested that circulating PD-1+ T cells are in fact a negative prognostic marker in breast cancer.⁶⁴

All patients who were enrolled in this trial had metastatic hormone (estrogen and progesterone) receptor-positive (HR), HER2-positive breast cancer. Patients with metastatic HR-positive, HER2-positive breast cancers tend to have longer rates of overall survival than those with HR-negative, HER2-positive breast cancer.⁶⁵ With the exception of adding endocrine therapy to HR-positive, HER2-positive breast cancer, there are no current differences in treatment management of these patients compared to those with HR-negative, HER2-positive metastatic disease.⁶⁶ It is important to highlight that recent research suggests that the tumor microenvironment of HER2-positive breast cancers may differ depending on HR receptor expression. For example, in the CALGB 40601 and PAMELA trials was noted that HER2-positive breast cancers that were also HR-negative had higher proportions of tumor infiltrating lymphocytes than HR-positive tumors.⁶⁷ This may suggest that HR-negative HER2-positive tumors have a more immunogenic environment than their HR-positive counterparts and thus may be more responsive to immunotherapeutic responses.

Ultimately the results of this study show that the regimen of trastuzumab and CpG ODN was found to be safe and associated with fluctuations in circulating cytokine and immune cells levels. Discontinuation rates were higher than other CpG ODN-based clinical studies, but the true proportion of patients who would tolerate this treatment regimen may be higher if expanded to a larger study size. An encouraging overall survival rate was observed among treated patients; however, due to this study’s small sample size this combination requires further investigation for verification of outcomes and correlative results. Of note, it is unclear what the effect of concomitant treatments (e.g. lapatinib, endocrine therapy) could have on the efficacy of these agents. With the current HER2 + breast cancer landscape in mind, CpG ODN would be a relevant adjuvant to study in combination with current immune checkpoint inhibitors (ICI) or other novel immunotherapies to expand options for breast cancer treatment. It is known that HER2 + breast cancers are not typically responsive to chemotherapy plus ICI alone,⁶⁸ therefore the addition of an agent that can further prime/boost the immune response may be beneficial. Additionally, CpG ODN may be provide a stronger response to earlier lines of metastatic HER2-positive breast cancer treatment, such as adding it to standard-of-care docetaxel plus trastuzumab and pertuzumab. Within the larger treatment context for breast cancer, many opportunities to improve upon anti-tumor immunity remain.

Supplemental Material

Supplemental Material - An Open-Label Study of Subcutaneous CpG Oligodeoxynucleotide (PF03512676) in Combination with Trastuzumab in Patients with Metastatic HER2+ Breast Cancer

Supplemental Material for An Open-Label Study of Subcutaneous CpG Oligodeoxynucleotide (PF03512676) in Combination with Trastuzumab in Patients with Metastatic HER2+ Breast Cancer by Dionisia Quiroga, Robert Wesolowski, Sara Zelinskas, Ashley Pinette, Brooke Benner, Emily Schwarz, Himanshu Savardekar, Courtney Johnson, Andrew Stiff, Lianbo Yu, Erin Macrae, Maryam Lustberg, Ewa Mrozek, Bhuvaneswari Ramaswamy, and William E. Carson in Cancer Control

Supplemental Material

Supplemental Material - An Open-Label Study of Subcutaneous CpG Oligodeoxynucleotide (PF03512676) in Combination with Trastuzumab in Patients with Metastatic HER2+ Breast Cancer

Footnotes

Acknowledgments

The authors would like to thank the patients and their families, the investigators, research nurses, study coordinators, and operations staff who contributed to this study. The authors would like to thank the Shared Resources Core at The Ohio State University James Comprehensive Cancer Center.

Authors Note

The original research in this manuscript was accepted and presented as a poster presentation at the National Comprehensive Cancer Network 2022 Annual Meeting. This original research and manuscript have not otherwise been submitted or accepted for publication in another journal.

Author’s Contribution

Conceptualization/supervision (BR, WEC); formal analysis (DQ, LY); funding acquisition (BR, WEC); investigation (DQ, RW, SZ, AP, BB, ES, HS, AS); methodology (DQ, BR, WEC); resources/patient enrollment (EO, ML, EM, BR); visualization (DQ, CJ); writing – original draft (DQ); writing – review and editing (DQ, CJ, BR, WEC).

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by NIH Grants P01 CA095426 (M. Caligiuri, WEC III), P30 CA016058 (M. Caligiuri, R. Pollock), R21 CA084402 (WEC III), K24 CA093670 (WEC III), T32 CA009338 (AP), and T32 CA247815 (DQ). Pfizer provided drug support for PF03512676.

Ethical Statement

Clinical Trials Registration

ClinicalTrials.gov Registration: Agatolimod and Trastuzumab in Treating Patients with Locally Advanced or Metastatic Breast Cancer (NCT00824733) URL: https://beta.clinicaltrials.gov/study/NCT00824733?id=NCT00824733&rank=1

Off-Label Use/Unapproved Drugs or Products

This manuscript discusses a clinical trial in which the investigational drug PF03512676 (CpG ODN) is being tested in combination with trastuzumab. PF03512676 is not currently an approved treatment for breast cancer and was used in the scope of a clinical trial.

ORCID iD

Dionisia Quiroga

Data Availability Statement

The datasets generated during and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request. It is confirmed that other researchers may reproduce the methodology presented in this manuscript from the description given in the Methods section and that corresponding author is available for assistance on reasonable request.^32-44,46-54

Supplemental Material

Supplemental material for this article is available online.

Appendix

References

Howlader

Cronin

Kurian

Andridge

. Differences in breast cancer survival by molecular subtypes in the United States. Cancer Epidemiol Biomarkers Prev. 2018;27(6):619-626. doi:10.1158/1055-9965.EPI-17-0627.

Hurvitz

Gelmon

Tolaney

. Optimal management of early and advanced HER2 breast cancer. Am Soc Clin Oncol Educ Book. 2017;37:76-92. doi:10.1200/EDBK_175630.

Slamon

Leyland-Jones

Shak

, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med. 2001;344(11):783-792. doi:10.1056/NEJM200103153441101.

Geyer

Forster

Lindquist

, et al. Lapatinib plus capecitabine for HER2-positive advanced breast cancer. N Engl J Med. 2006;355(26):2733-2743. doi:10.1056/NEJMoa064320.

Baselga

Cortés

Kim

, et al. Pertuzumab plus trastuzumab plus docetaxel for metastatic breast cancer. N Engl J Med. 2012;366(2):109-119. doi:10.1056/NEJMoa1113216.

Swain

Baselga

Kim

, et al. Pertuzumab, trastuzumab, and docetaxel in HER2-positive metastatic breast cancer. N Engl J Med. 2015;372(8):724-734. doi:10.1056/NEJMoa1413513.

Martin

Holmes

Ejlertsen

, et al. Neratinib after trastuzumab-based adjuvant therapy in HER2-positive breast cancer (ExteNET): 5-year analysis of a randomised, double-blind, placebo-controlled, phase 3 trial. Lancet Oncol. 2017;18(12):1688-1700. doi:10.1016/S1470-2045(17)30717-9.

Modi

Saura

Yamashita

, et al. Trastuzumab deruxtecan in previously treated HER2-positive breast cancer. N Engl J Med. 2020;382(7):610-621. doi:10.1056/NEJMoa1914510.

Verma

Miles

Gianni

, et al. Trastuzumab emtansine for HER2-positive advanced breast cancer. N Engl J Med. 2012;367(19):1783-1791. doi:10.1056/NEJMoa1209124.

10.

Swain

Miles

Kim

, et al. Pertuzumab, trastuzumab, and docetaxel for HER2-positive metastatic breast cancer (CLEOPATRA): end-of-study results from a double-blind, randomised, placebo-controlled, phase 3 study. Lancet Oncol. 2020;21(4):519-530. doi:10.1016/S1470-2045(19)30863-0.

11.

Jaime-Ramirez

Mundy-Bosse

Kondadasula

, et al. IL-12 enhances the antitumor actions of trastuzumab via NK cell IFN-γ production. J Immunol. 2011;186(6):3401-3409. doi:10.4049/jimmunol.1000328.

12.

Roda

Parihar

Carson

. CpG-containing oligodeoxynucleotides act through TLR9 to enhance the NK cell cytokine response to antibody-coated tumor cells. J Immunol. 2005;175(3):1619-1627. doi:10.4049/jimmunol.175.3.1619.

13.

Akira

Takeda

. Toll-like receptor signalling. Nat Rev Immunol. 2004;4(7):499-511. doi:10.1038/nri1391.

14.

Quiroga

Aldhamen

Appledorn

Godbehere

Amalfitano

. Strengthened tumor antigen immune recognition by inclusion of a recombinant Eimeria antigen in therapeutic cancer vaccination. Cancer Immunol Immunother. 2015;64(4):479-491. doi:10.1007/s00262-015-1659-7.

15.

Takeda

Kataoka

Yamagishi

Ogawa

Seya

Matsumoto

. A TLR3-specific adjuvant relieves innate resistance to PD-L1 blockade without cytokine toxicity in tumor vaccine immunotherapy. Cell Rep. 2017;19(9):1874-1887. doi:10.1016/j.celrep.2017.05.015.

16.

Urban-Wojciuk

Khan

Oyler

, et al. The role of TLRs in anti-cancer immunity and tumor rejection. Front Immunol. 2019;10:2388. doi:10.3389/fimmu.2019.02388.

17.

Mullins

Vasilakos

Deschler

, et al. Intratumoral immunotherapy with TLR7/8 agonist MEDI9197 modulates the tumor microenvironment leading to enhanced activity when combined with other immunotherapies. J Immunother Cancer. 2019;7(1):244. doi:10.1186/s40425-019-0724-8.

18.

Lee

Fitzpatrick

Sharma

Graves

Czerniecki

. Inhibition of CD4+CD25+ regulatory T cell function and conversion into Th1-like effectors by a Toll-like receptor-activated dendritic cell vaccine. PLoS One. 2013;8(11):e74698. doi:10.1371/journal.pone.0074698.

19.

Sato-Kaneko

Yao

Ahmadi

, et al. Combination immunotherapy with TLR agonists and checkpoint inhibitors suppresses head and neck cancer. JCI Insight. 2017;2(18):93397. doi:10.1172/jci.insight.93397.

20.

Carbone

Piro

Agostini

, et al. Intratumoral injection of TLR9 agonist promotes an immunopermissive microenvironment transition and causes cooperative antitumor activity in combination with anti-PD1 in pancreatic cancer. J Immunother Cancer. 2021;9(9):e002876. doi:10.1136/jitc-2021-002876.

21.

Bauer

Kirschning

Häcker

, et al. Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition. Proc Natl Acad Sci USA. 2001;98(16):9237-9242. doi:10.1073/pnas.161293498.

22.

Karapetyan

Luke

Davar

. Toll-like receptor 9 agonists in cancer. OncoTargets Ther. 2020;13:10039-10060. doi:10.2147/OTT.S247050.

23.

González-Reyes

Marín

González

, et al. Study of TLR3, TLR4 and TLR9 in breast carcinomas and their association with metastasis. BMC Cancer. 2010;10:665. doi:10.1186/1471-2407-10-665.

24.

Wang

Campos

Gallotta

, et al. Intratumoral injection of a CpG oligonucleotide reverts resistance to PD-1 blockade by expanding multifunctional CD8+ T cells. Proc Natl Acad Sci USA. 2016;113(46):E7240-E7249. doi:10.1073/pnas.1608555113.

25.

Damiano

Caputo

Bianco

, et al. Novel toll-like receptor 9 agonist induces epidermal growth factor receptor (EGFR) inhibition and synergistic antitumor activity with EGFR inhibitors. Clin Cancer Res. 2006;12(2):577-583. doi:10.1158/1078-0432.CCR-05-1943.

26.

Pasare

Medzhitov

. Toll pathway-dependent blockade of CD4+CD25+ T cell-mediated suppression by dendritic cells. Science. 2003;299(5609):1033-1036. doi:10.1126/science.1078231.

27.

Parihar

Dierksheide

Carson

. IL-12 enhances the natural killer cell cytokine response to Ab-coated tumor cells. J Clin Invest. 2002;110(7):983-992. doi:10.1172/JCI15950.

28.

McMichael

Jaime-Ramirez

Guenterberg

, et al. IL-21 enhances natural killer cell response to cetuximab-coated pancreatic tumor cells. Clin Cancer Res. 2017;23(2):489-502. doi:10.1158/1078-0432.CCR-16-0004.

29.

Roda

Joshi

Butchar

, et al. The activation of natural killer cell effector functions by cetuximab-coated, epidermal growth factor receptor positive tumor cells is enhanced by cytokines. Clin Cancer Res. 2007;13(21):6419-6428. doi:10.1158/1078-0432.CCR-07-0865.

30.

Roda

Parihar

Lehman

Mani

Tridandapani

Carson

. Interleukin-21 enhances NK cell activation in response to antibody-coated targets. J Immunol. 2006;177(1):120-129. doi:10.4049/jimmunol.177.1.120.

31.

Buhé

Guerrier

Youinou

Berthou

Loisel

. CpG ODN enhances the efficacy of rituximab in non-Hodgkin lymphoma. Ann N Y Acad Sci. 2009;1173:858-864. doi:10.1111/j.1749-6632.2009.04615.x.

32.

Iwahashi

Katsuda

Nakamori

, et al. Vaccination with peptides derived from cancer-testis antigens in combination with CpG-7909 elicits strong specific CD8+ T cell response in patients with metastatic esophageal squamous cell carcinoma. Cancer Sci. 2010;101(12):2510-2517. doi:10.1111/j.1349-7006.2010.01732.x.

33.

Speiser

Liénard

Rufer

, et al. Rapid and strong human CD8+ T cell responses to vaccination with peptide, IFA, and CpG oligodeoxynucleotide 7909. J Clin Invest. 2005;115(3):739-746. doi:10.1172/JCI23373.

34.

Lövgren

Baumgaertner

Wieckowski

, et al. Enhanced cytotoxicity and decreased CD8 dependence of human cancer-specific cytotoxic T lymphocytes after vaccination with low peptide dose. Cancer Immunol Immunother. 2012;61(6):817-826. doi:10.1007/s00262-011-1140-1.

35.

Fourcade

Kudela

Andrade Filho

, et al. Immunization with analog peptide in combination with CpG and montanide expands tumor antigen-specific CD8+ T cells in melanoma patients. J Immunother. 2008;31(8):781-791. doi:10.1097/CJI.0b013e318183af0b.

36.

Weber

Zarour

Redman

, et al. Randomized phase 2/3 trial of CpG oligodeoxynucleotide PF-3512676 alone or with dacarbazine for patients with unresectable stage III and IV melanoma. Cancer. 2009;115(17):3944-3954. doi:10.1002/cncr.24473.

37.

Tarhini

Leng

Moschos

, et al. Safety and immunogenicity of vaccination with MART-1 (26-35, 27L), gp100 (209-217, 210M), and tyrosinase (368-376, 370D) in adjuvant with PF-3512676 and GM-CSF in metastatic melanoma. J Immunother. 2012;35(4):359-366. doi:10.1097/CJI.0b013e31825481fe.

38.

Millward

Underhill

Lobb

, et al. Phase I study of tremelimumab (CP-675 206) plus PF-3512676 (CPG 7909) in patients with melanoma or advanced solid tumours. Br J Cancer. 2013;108(10):1998-2004. doi:10.1038/bjc.2013.227.

39.

Baumgaertner

Costa Nunes

Cachot

, et al. Vaccination of stage III/IV melanoma patients with long NY-ESO-1 peptide and CpG-B elicits robust CD8+ and CD4+ T-cell responses with multiple specificities including a novel DR7-restricted epitope. OncoImmunology. 2016;5(10):e1216290. doi:10.1080/2162402X.2016.1216290.

40.

Dreno

Thompson

Smithers

, et al. MAGE-A3 immunotherapeutic as adjuvant therapy for patients with resected, MAGE-A3-positive, stage III melanoma (DERMA): a double-blind, randomised, placebo-controlled, phase 3 trial. Lancet Oncol. 2018;19(7):916-929. doi:10.1016/S1470-2045(18)30254-7.

41.

Ribas

Medina

Kummar

, et al. SD-101 in combination with pembrolizumab in advanced melanoma: results of a phase ib, multicenter study. Cancer Discov. 2018;8(10):1250-1257. doi:10.1158/2159-8290.CD-18-0280.

42.

Milhem

Zakharia

Davar

, et al. O85 Durable responses in anti-PD-1 refractory melanoma following intratumoral injection of a toll-like receptor 9 (TLR9) agonist, CMP-001, in combination with pembrolizumab. J Immunother Cancer. 2020;8(Suppl 1):A2. doi:10.1136/LBA2019.4.

43.

Manegold

Gravenor

Woytowitz

, et al. Randomized phase II trial of a toll-like receptor 9 agonist oligodeoxynucleotide, PF-3512676, in combination with first-line taxane plus platinum chemotherapy for advanced-stage non–small-cell lung cancer. J Clin Orthod. 2008;26(24):3979-3986. doi:10.1200/JCO.2007.12.5807.

44.

Yamada

Nakao

Fukuyama

, et al. Phase I study of TLR9 agonist PF-3512676 in combination with carboplatin and paclitaxel in patients with advanced non-small-cell lung cancer. Cancer Sci. 2010;101(1):188-195. doi:10.1111/j.1349-7006.2009.01361.x.

45.

Hirsh

Paz-Ares

Boyer

, et al. Randomized phase III trial of paclitaxel/carboplatin with or without PF-3512676 (Toll-like receptor 9 agonist) as first-line treatment for advanced non-small-cell lung cancer. J Clin Oncol. 2011;29(19):2667-2674. doi:10.1200/JCO.2010.32.8971.

46.

Manegold

van Zandwijk

Szczesna

, et al. A phase III randomized study of gemcitabine and cisplatin with or without PF-3512676 (TLR9 agonist) as first-line treatment of advanced non-small-cell lung cancer. Ann Oncol. 2012;23(1):72-77. doi:10.1093/annonc/mdr030.

47.

Belani

Nemunaitis

Chachoua

, et al. Phase 2 trial of erlotinib with or without PF-3512676 (CPG 7909, a Toll-like receptor 9 agonist) in patients with advanced recurrent EGFR-positive non-small cell lung cancer. Cancer Biol Ther. 2013;14(7):557-563. doi:10.4161/cbt.24598.

48.

Smith

Conkling

Richards

, et al. Antitumor activity and safety of combination therapy with the Toll-like receptor 9 agonist IMO-2055, erlotinib, and bevacizumab in advanced or metastatic non-small cell lung cancer patients who have progressed following chemotherapy. Cancer Immunol Immunother. 2014;63(8):787-796. doi:10.1007/s00262-014-1547-6.

49.

Karbach

Neumann

Atmaca

, et al. Efficient in vivo priming by vaccination with recombinant NY-ESO-1 protein and CpG in antigen naive prostate cancer patients. Clin Cancer Res. 2011;17(4):861-870. doi:10.1158/1078-0432.CCR-10-1811.

50.

Karbach

Neumann

Wahle

Brand

Gnjatic

Jäger

. Therapeutic administration of a synthetic CpG oligodeoxynucleotide triggers formation of anti-CpG antibodies. Cancer Res. 2012;72(17):4304-4310. doi:10.1158/0008-5472.CAN-12-0257.

51.

Koster

Santegoets

SJAM

Harting

, et al. Autologous tumor cell vaccination combined with systemic CpG-B and IFN-α promotes immune activation and induces clinical responses in patients with metastatic renal cell carcinoma: a phase II trial. Cancer Immunol Immunother. 2019;68(6):1025-1035. doi:10.1007/s00262-019-02320-0.

52.

Leonard

Link

Emmanouilides

, et al. Phase I trial of toll-like receptor 9 agonist PF-3512676 with and following rituximab in patients with recurrent indolent and aggressive non Hodgkin’s lymphoma. Clin Cancer Res. 2007;13(20):6168-6174. doi:10.1158/1078-0432.CCR-07-0815.

53.

Valmori

Souleimanian

Tosello

, et al. Vaccination with NY-ESO-1 protein and CpG in Montanide induces integrated antibody/Th1 responses and CD8 T cells through cross-priming. Proc Natl Acad Sci U S A. 2007;104(21):8947-8952. doi:10.1073/pnas.0703395104.

54.

Karbach

Gnjatic

Bender

, et al. Tumor-reactive CD8+ T-cell responses after vaccination with NY-ESO-1 peptide, CpG 7909 and Montanide ISA-51: association with survival. Int J Cancer. 2010;126(4):909-918. doi:10.1002/ijc.24850.

55.

Pfizer

CUD

. C015: a phase 1/2 Open-Label, multi-center, dose-escalation study of subcutaneous PF-03512676 (CPG 7909) Plus Herceptin® in patients with metastatic breast cancer; 2008.

56.

Lesinski

Kondadasula

Crespin

, et al. Multiparametric flow cytometric analysis of inter-patient variation in STAT1 phosphorylation following interferon Alfa immunotherapy. J Natl Cancer Inst. 2004;96(17):1331-1342. doi:10.1093/jnci/djh252.

57.

Sun

Benner

Savardekar

, et al. Effect of immune checkpoint blockade on myeloid-derived suppressor cell populations in patients with melanoma. Front Immunol. 2021;12:740890. doi:10.3389/fimmu.2021.740890.

58.

Wesolowski

Stiff

Quiroga

, et al. Exploratory analysis of immune checkpoint receptor expression by circulating T cells and tumor specimens in patients receiving neo-adjuvant chemotherapy for operable breast cancer. BMC Cancer. 2020;20(1):445. doi:10.1186/s12885-020-06949-4.

59.

Grimmig

Moench

Kreckel

, et al. Toll like receptor 2, 4, and 9 signaling promotes autoregulative tumor cell growth and VEGF/PDGF expression in human pancreatic cancer. Int J Mol Sci. 2016;17(12):E2060. doi:10.3390/ijms17122060.

60.

Belmont

Rabbe

Antoine

, et al. Expression of TLR9 in tumor-infiltrating mononuclear cells enhances angiogenesis and is associated with a worse survival in lung cancer. Int J Cancer. 2014;134(4):765-777. doi:10.1002/ijc.28413.

61.

Bergenfelz

Larsson

von Stedingk

, et al. Systemic monocytic-MDSCs are generated from monocytes and correlate with disease progression in breast cancer patients. PLoS One. 2015;10(5):e0127028. doi:10.1371/journal.pone.0127028.

62.

Shirota

Klinman

. Intratumoral injection of CpG oligonucleotides induces the differentiation and reduces the immunosuppressive activity of myeloid-derived suppressor cells. J Immunol. 2012;188(4):1592-1599. doi:10.4049/jimmunol.1101304.

63.

Hou

Nitta

Wei

, et al. PD-L1 expression and CD8-positive T cells are associated with favorable survival in HER2-positive invasive breast cancer. Breast J. doi:10.1111/tbj.13112. Published online 19 September 2018.

64.

Noda

Masuda

Ito

, et al. Circulating PD-1 mRNA in peripheral blood is a potential biomarker for predicting survival of breast cancer patients. Ann Surg Oncol. 2020;27(10):4035-4043. doi:10.1245/s10434-020-08375-z.

65.

Arciero

Guo

Jiang

, et al. ER+/HER2+ breast cancer has different metastatic patterns and better survival than ER-/HER2+ breast cancer. Clin Breast Cancer. 2019;19(4):236-245. doi:10.1016/j.clbc.2019.02.001.

66.

Giordano

Temin

Chandarlapaty

, et al. Systemic therapy for patients with advanced human epidermal growth factor receptor 2-positive breast cancer: ASCO clinical Practice guideline update. J Clin Oncol. 2018;36(26):2736-2740. doi:10.1200/JCO.2018.79.2697.

67.

Fernandez-Martinez

Pascual

Singh

, et al. Prognostic and predictive value of immune-related gene expression signatures vs tumor-infiltrating lymphocytes in early-stage ERBB2/HER2-positive breast cancer: a correlative analysis of the CALGB 40601 and PAMELA trials. JAMA Oncol. 2023;9(4):490-499. doi:10.1001/jamaoncol.2022.6288.

68.

Agostinetto

Montemurro

Puglisi

, et al. Immunotherapy for HER2-positive breast cancer: clinical evidence and future perspectives. Cancers. 2022;14(9):2136. doi:10.3390/cancers14092136.

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