Intratumoral delivery of dendritic cells plus anti-HER2 therapy triggers both robust systemic antitumor immunity and complete regression in HER2 mammary carcinoma

Background Human epidermal growth factor receptor 2 (HER2) targeted antibodies in combination with chemotherapy has improved outcomes of HER2 positive (pos) breast cancer (BC) but toxicity of therapy remains a problem. High levels of tumor-infiltrating lymphocytes are associated with increased pathologic complete responses for patients treated with neoadjuvant therapy. Here we sought to investigate whether delivery of intratumoral (i.t.) multiepitope major histocompatibility complex (MHC) class II HER2 peptides-pulsed type I polarized dendritic cells (HER2-DC1) in combination with anti-HER2 antibodies without chemotherapy could enhance tumor regression by increasing anti-HER2 lymphocyte infiltration into the tumor. Methods BALB/c mice bearing orthotopic TUBO tumors, BALB/c mice bearing subcutaneous (s.c.) CT26 hHER2 tumors, or BALB-HER2/neu transgenic mice were all treated with i.t. or s.c. HER2-DC1, anti-HER2 antibodies, paclitaxel, T-DM1 or in combination. Immune response, host immune cells and effector function were analyzed using flow cytometry, interferon-γ ELISA and cytokine/chemokine arrays. The contributions of CD4+ and CD8+ T cells and antibody dependent cellular cytotoxicity (ADCC) were assessed using depleting antibodies and FcγR KO mice. Molecular changes were evaluated by immunohistochemistry and western blot. Results HER2-DC1 combined with anti-HER2 antibodies delivered i.t. compared to s.c. induced complete tumor regression in 75–80% of treated mice, with increased tumor infiltrating CD4+ and CD8+ T, B, natural killer T cells (NKT) and natural killer cells, and strong anti-HER2 responses in all HER2pos BC models tested. The therapy caused regression of untreated distant tumors. Labeled HER2-DC1 migrated prominently into the distant tumor and induced infiltration of various DC subsets into tumors. HER2-DC1 i.t. combined with anti-HER2 antibodies displayed superior antitumor response compared to standard chemotherapy with anti-HER2 antibodies. Lasting immunity was attained which prevented secondary tumor formation. The presence of CD4+ and CD8+ T cells and ADCC were required for complete tumor regression. In the HER2pos BC models, HER2-DC1 i.t. combined with anti-HER2 antibodies effectively diminished activation of HER2-mediated oncogenic signaling pathways. Conclusions HER2-DC1 i.t. with anti-HER2 antibodies mediates tumor regression through combined activation of T and B cell compartments and provides evidence that HER2-DC1 i.t. in combination with anti-HER2 antibodies can be tested as an effective alternative therapeutic strategy to current chemotherapy and anti-HER2 antibodies in HER2pos BC.

twice weekly). The multi-epitope MHC class II HER2 peptides pulsed DC1 vaccine (HER2-DC1) was prepared as previously described 29 . For combination therapy, tumor bearing mice received one injection of anti-HER2/neu antibodies on day 12. One week after, mice received HER2-DC1 s.c., concurrently with anti-HER2/neu antibodies. Mice were monitored for tumor growth and tumors were measured twice weekly using digital calipers. Tumor volume was calculated using the formula: volume (mm 3 ) = length x width 2 ÷2.
Treatment started on day 12 following tumor injection. For intratumoral (i.t.) delivery, HER2-DC1 (1X10 6 cells/50ul of PBS) were drawn into a 1ml syringe using 18G needle and gradually injected into the measurable (4x4mm) palpable tumor directly using a 27G needle. Untreated mice received i.t. injection of sterile PBS. Combination treatment groups 6 and 7 received anti-HER2 antibodies, either both clones 7.16.4+7.9.5 or clone 7.16.4 alone, respectively, on day 12. One week after, mice received HER2-DC1 i.t. BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) concurrently with anti-HER2 antibodies, either both clones 7.16.4+7.9.5 or clone 7.16.4 alone. Mice were monitored and tumor volumes were measured as described above.
Mice were monitored and tumor volumes were measured.

BALB-HER2/neu transgenic mice treatments and MRI imaging
We utilized BALB-HER2 transgenic mouse model, a classic model of human HER2 pos BC, which express rat HER2. The spontaneous focal mammary carcinoma develops in the mammary glands of these mice that slowly progresses from microscopic lesions to invasive tumors.
BALB-HER2/neuT mice at 8-9 weeks of age were given HER2-DC1 i.t. (once weekly for six weeks), anti-HER2/neu antibodies (both clones 7.16.4+7.9.5, i.p., once weekly) or combination of both. HER2-DC1 i.t. delivery was assisted by ultrasound guidance. Mice were examined for spontaneous tumor growth in mammary glands at different timepoints by magnetic resonance imaging (MRI). Mice were anesthetized with 2% isoflurane delivered in 1.5-liter/min oxygen in an induction chamber. Next, mice were transferred and imaged on a 7-Tesla horizontal MRI scanner (Bruker Biospin, Inc. BioSpec AV3HD) using a 35mm Litzcage coil (Doty Scientific). While imaging, ventilation was provided through a nose cone and respiration range was maintained at a range of 40-60 breaths per minute. The core body temperature was monitored and maintained at 37°C by MRI-compatible Small Rodent Heater System (SAII ® , SA Instruments, Stony Brook, NY). Anatomical T2-weighted coronal images were acquired using a TurboRARE sequence with field of view (FOV) = 75x35mm 2 , echo time/repetition time (TR/TE) = 4513/38ms and slice thickness of 1.2mm and 19 slices.

CT26 hHER2 tumor model and treatments
The CT26 cell line expressing human HER2 was used to induce clinically relevant 90μg, i.p., every five days) and paclitaxel (7.5mg/kg, i.p., every three days). For paclitaxel combined with anti-HER2 antibodies treatment, mice received two injections of paclitaxel followed by concurrent treatment with anti-HER2 antibodies (trastuzumab and pertuzumab). HER2-DC1 i.t. and anti-HER2 antibodies combination treatment was carried out as described above.

Immunohistochemistry
Immunohistochemistry was performed as previous described 25 . Briefly, tumor tissues were collected from the experimental mice, fixed in formalin, and embedded in paraffin.
Five micrometer thick paraffin tumor tissue sections were deparaffinized with xylene and rehydrated in graded ethanol. Antigen retrieval was performed using Tris-EDTA buffer and slides were incubated with 3% hydrogen peroxidase for 30 minutes to block endogenous peroxidase activity. Slides were then washed and incubated with 10% normal goat serum in Tris-buffered saline (TBS). Thereafter, slides were incubated with anti-rabbit HER2 antibody (Cat. No. 4290S, Cell Signaling Technologies, Danvers, MA) for overnight. Slides were washed and incubated with secondary antibody labeled with HRP for 1 hour at 37°C. The color development was detected using 3,3'diaminobenzidine (DAB) and the slides were counter-stained with hematoxylin. Slides were scanned using a Leica Aperio™ AT2 scanner (Vista, CA) at the Microscopy Core Facility at H. Lee Moffitt Cancer Center and Research Institute.

CD4 + and CD8 + T cell depletion experiments
Anti-CD4 and anti-CD8 antibodies were used to deplete host CD4 + and CD8 + T cells.
Mice were injected with or without 300 ug of depleting antibody (i.p.) twice a week beginning three days prior to TUBO cells (3x10 4 cells/mouse) inoculation in the MFP BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) J Immunother Cancer doi: 10.1136/jitc-2022-004841 :e004841. and was continued until the endpoint. Non-depleted mice were injected i.p. with rat IgG2b isotype antibody. After establishment of TUBO tumors, mice were treated as described above. Tumor volume was measured twice a week.

Western blot analysis
Whole protein extracts from tumor samples were prepared by homogenization in RIPA buffer Millipore,Billerica,MA)