Combination Immunotherapy of Glioblastoma with Dendritic Cell Cancer Vaccines, Anti-PD-1 and Poly I:C: A Case Report

doi:10.21203/rs.3.rs-2199151/v1

Glioblastoma (GBM) is a lethal cancer with limited therapeutic options. Dendritic cell (DC)-based cancer vaccines represent a promising approach for GBM treatment. Clinical studies suggest that other immunotherapeutic agents including anti-PD-1 and immune adjuvant poly I:C may be combined with DC vaccines to further enhance antitumor activity. Here we report a GBM case with combination immunotherapy consisting of DC vaccines, anti-PD-1 and poly I:C as well as a chemotherapeutic agent cyclophosphamide that were integrated with the standard chemoradiation therapy for > 5 years. The patient who received DC vaccines loaded with multiple forms of tumor antigens including mRNA-tumor associated antigens (TAAs), mRNA-neoantigens, and hypochlorous acid-oxidized tumor lysates. Furthermore, mRNA-TAAs were modified with a novel TriVac technology which fuses TAAs with a destabilization domain and inserts TAAs into a full-length LAMP-1 to enhance MHC class I and II antigen presentation, respectively. The treatment consisted of 42 DC cancer vaccine infusions, 26 anti-PD-1 antibody nivolumab administrations and 126 poly I:C injections for DC infusions. The patient also received 28 doses of cyclophosphamide for depletion of regulatory T cells. No immunotherapy related adverse events were observed during the treatment. Robust anti-tumor CD4+ and CD8+ T cell responses were detected. The patient remains free of disease progression as of July, 2022. Our results suggest that integrated combination immunotherapy is safe and feasible for long-term treatment. A large scale trial to validate these findings is warranted.

Protocol numbers: 201708152377, 20202014-F-1

Health sciences/Diseases/Cancer/Cancer therapy

Health sciences/Diseases/Cancer/CNS cancer

glioblastoma multiforme

DC vaccine

tumor associated antigens

neoantigens

anti-PD-1

poly I:C

CD4+ T cell response

CD8+ T cell response

Glioblastoma (GBM) remains one of the most lethal cancer types with limited effective treatment options ¹. Standard treatment for newly diagnosed GBM consisting of surgical resection and chemoradiotherapies yields a median overall survival of only 13.5 months ². Dendritic cell based cancer vaccines represent a promising approach for GBM treatment ³. Autologous DCs loaded with different forms of tumor antigens such as mRNAs encoding tumor associated antigens (TAAs), tumor cell lysates, or synthetic peptides derived from TAAs produced encouraging clinical outcomes ^4-7. Recently, neoantigen-based cancer vaccines have also been tested in GBM patients. Neoantigen peptides induced potent antigen-specific T cell responses and some clinical activities in GBM patients ⁸^,⁹.

Several other immunotherapeutic agents have been investigated for GBM treatment. The anti-PD-1 antibody nivolumab was combined with radiotherapy or chemoradiotherapy to treat newly diagnosed GBM with or without methylated MGMT promoter in two phase III trials ¹⁰^,¹¹. The combination did not meet the primary endpoint: improving overall survival. However, combination therapy of anti-PD-1 and personalized cancer vaccines in other cancer types including non-small cell lung cancer (NSCLC), melanoma, and bladder cancer has shown promising clinical activities ⁴^,¹²^,¹³, suggesting a need to test this combination in GBM treatment. Multiple clinical trials have tested the safety and efficacy of the adjuvant poly-ICLC, a stabilized form of poly I:C, in combination with chemoradiotherapy or cancer vaccines to treat GBM ^14-18. These trials have shown good safety profiles and some promising clinical efficacy. Nevertheless, the above combinations likely need further addition of anti-PD-1 agents as poly I:C stimulates both PD-L1 and PD-L2 expression on primary human GBM cells and PD-1 blockade invigorates the immune stimulatory effect of poly I:C ¹⁹.

Here we report a GBM case with integrated combination immunotherapy consisting of DC based-cancer vaccines, anti-PD-1 and poly I:C treatment for > 5 years. To maximize the possibility of cure, the patient received autologous DC vaccines loaded with multiple forms of tumor antigens including mRNA-TAAs, mRNA-neoantigens, and hypochlorous acid (HOCI)-oxidized tumor lysates. The TAAs were modified with a novel TriVac technology that fuses TAAs with a destabilization domain (DD) or inserts TAAs into full length LAMP-1 to enhance MHC class I and II antigen presentation ²⁰. HOCI oxidation of whole tumor cells induces necrosis and increases immunogenicity of tumor cells ²¹. Personalized cancer vaccine trials showed that autologous DC cells pulsed with HOCI-oxidized tumor lysates were safe and effective in eliciting a broad antitumor immunity in patients with advanced stage ovarian cancer ²¹^,²². To further enhance the induction of vaccine-induced antitumor specific T cell responses, the patient also received low-dose cyclophosphamide for depletion of regulatory T cells ²³^,²⁴. No immunotherapy related adverse events were observed during >5 years of treatment. The patient had robust anti-tumor CD4⁺ and CD8⁺ T cell responses and remains disease progression free in the latest MRI scan in July, 2022. Our results suggest that integrated combination immunotherapy is safe and feasible for long-term treatment. A large scale trial to validate these findings is warranted.

Integrated combination immunotherapy with DC vaccines plus anti-PD-1 and poly I:C

The overall combination immunotherapy strategy was based on our phase I trial of personalized cancer vaccine therapy ⁴ aiming to maximize possibility of cure. All immunotherapeutic interventions were integrated into the standard treatment procedure for GBM patients (Figure 1A). DC vaccine therapy started one week after completion of the concurrent chemoradiation therapy and was administered every 2-4 weeks in the first two years with some long intervals in the last three years (Figure 1A). The patient received a total of 42 DC cancer vaccines loaded with mRNA-TAAs, mRNA-neoantigens or HOCI-oxidized lysates from October 17, 2017 to May 4, 2022 (Figure 1A and Table 1). The doses for the DC vaccines ranged from 2x10⁶-3.5x10⁷ cells per infusion with most of the vaccinations at >1.5 x10⁷ cells (Table 1). Low-dose cyclophosphamide (200 mg/m²) was administered intravenously at 24 hours before each vaccination in the first 30 months (28 injections for a total of 8.4 g) to deplete Tregs ²³^,²⁴ (Figure 1A). Cyclophosphamide was discontinued at the discretion of treating physicians. To further enhance the clinical efficacy of the DC cancer vaccines, the anti-PD-1 antibody nivolumab was administered from April 2018 to April 2022 at 100 mg (26 injections) during vaccinations. Poly I:C, an approved immune adjuvant in China ⁴, was injected at 3 mg intramuscularly 3 times every two days after each DC vaccination (Figure 1A).

The patient was regularly monitored. MRI was performed every three months to monitor disease and peripheral blood was drawn at indicated times to monitor immune responses (Figure 1A). The patient’s Karnofsky Performance Status (KPS) score was 70 immediately after the surgery and the symptoms of speech and word-finding difficulty improved gradually. The patient continues to live a normal life with a KPS of 90-100 at one year after the surgery. MRI results showed no signs of recurrence in the latest scan on July 21, 2022 (Figure 1B).

Safety of the combination immunotherapy

The combination of DC cancer vaccines, anti-PD-1 and poly I:C treatment was well tolerated. No reportable adverse event associated with DC vaccinations, anti-PD-1 and poly I:C treatment was observed. One temporary syncope and four periods of short-time lack of verbal fluency were recorded and attributed to postoperative seizures. The patient’s peripheral lymphocyte counts gradually decreased during the first 20 months of treatment, attributed to chemoradiation and 12 cycles of TMZ.

Tumor antigen identification and modification

To maximize the possibility of disease cure, three types of tumor antigens, mRNA-TAAs, HOCI-oxidized lysates, and mRNA-neoantigens, were used to load autologous DCs. A high throughput qPCR assay identified over-expressed TAAs in the patient’s tumor tissue (Figure 2A). The following criteria were used to select TAAs for DC vaccine: a >2-fold expression compared to that in para-tumor tissue, published studies reporting detection of TAA-specific T cells in cancer patients, and low relative expression in normal human tissues as reported in the Gencode site (www.gencodegenes.org). A total of 7 mRNA-TAAs including ADAMTSL1, PIM1, MTFR2, SP17, SOX3, CSPG4, and SOX11 were used to load autologous DCs (Figure 2A and Table 1). In addition, mRNA-CMVpp65 was included as a target antigen for GBM treatment in the first 3 immunizations based on previous clinical trials ²⁵^,²⁶. In the first phase of immunizations, 9 DC vaccines with mRNA-TAAs fused with the sorting signal (transmembrane and cytoplasmic domains) of LAMP-1 were infused (#1-3, 7-12, Table 1). In the second phase of immunizations, 22 DC vaccines with mRNA-TAAs modified with a new TriVac platform were infused ²⁰ (#20-41, Table 1). Full-length TAAs were inserted between the luminal domain and sorting signal (transmembrane and cytoplasmic domains) of a full-length LAMP-1 as this modification elicited stronger cellular and humoral immune responses than fusing antigen only to the LAMP-1 sorting signal ²⁷. Furthermore, we devised a new strategy to enhance MHC class I antigen presentation by targeting antigen to proteasomal degradation with fusion of TAAs to DD. The 7 TAAs were either inserted into a full-length LAMP-1 or fused with DD to generate individual mRNA-TAAs. DC cells were divided into equal portions for mRNA-TAA electroporation and pooled for infusion.

To identify potential neoantigens as vaccine targets, we performed WES and RNAseq of the patient’s tumor tissue. WES and RNAseq revealed 21 missense mutations with 10 of the mutated genes transcribed (Figure 2B). To further select these tentative neoantigens, we stimulated the patient’s PBMCs with synthesized wild type and neoantigen peptides. Four of the 10 predicted neoantigens including PRKRIR, FLGN, HUWE1, and RUNX1 stimulated specific T cell responses equal or greater than wild type antigenic peptides (Figure 2C). mRNA-neoantigens for these 4 neoantigens were generated and electroporated into autologous DCs for immunization (#13-19, 24, 42) (Table 1).

Although encouraging results were reported on using HOCI-oxidized tumor lysates in DC-based cancer vaccine clinical trials ²¹^,²², the antigens are limited by the amount of tumor tissue obtained from resection. The patient’s HOCI-oxidized tumor lysates were only sufficient for use in three immunizations (#4-6)(Table 1).

Specific T cell responses to TAAs and neoantigens

To examine tumor antigen-specific T cell responses to the immunized TAAs and neoantigens, peripheral T cells taken at different time points during the treatment (Figure 1A) were stimulated with mRNA-TAAs or mRNA-neoantigen transfected autologous DCs in vitro and assayed for intracellular production of IFN-γ. Among the 7 TAAs, 5 TAAs (SOX11, PIM1, SP17, SOX3, and CSPG4) modified with the sorting signal of LAMP-1 or full-length LAMP-1 and -DD were used in the first and second phase immunizations respectively (Figure 3A and Table 1). Two TAAs (MTFR2 and ADAMTSL1) modified only with the new TriVac technology (full-length LAMP-1 and DD) were used in the second phase immunization (Figure 3A and Table 1). DC vaccines induced strong antigen-specific CD4⁺ and CD8⁺ T cell responses to all 7 TAAs (Figure 3A). The antigen specific T cell responses to the five TAAs were relatively weak after the first phase immunization but substantially enhanced during the second phase immunization (Figure 3A). Interestingly, anti-MTFR2 and -ADAMTSL1 specific T cell responses were strongly induced upon immunization in the second phase (Figure. 3A). Specific T cell responses to these TAAs were maintained until the cut-off time of this study (Figure 3A).

Antigen-specific CD4⁺ and CD8⁺ T cells to the immunized neoantigens were readily detectable upon DC vaccinations (Figure 3B). The frequency of antigen-specific T cells were much increased at late phase (Figure 3B), suggesting antigen-specific T cell expansion during the interval.

Immune cell and cytokine dynamics during the 5-year treatment

Circulating immune cell subsets and cytokine levels were monitored during the course of the treatment. Total counts of peripheral lymphocytes including CD4⁺ and CD8⁺ T cells, B cells and NK cells displayed a gradual decrease during the first 20 months of treatment (Figure 4A). The immune cell subsets began to increase at 20 months and stabilized at 26 months (Figure 4A). Interestingly, these cells had a transient increase coincidental with 6 DC vaccine immunizations during this time period (Figure 4A and Table 1).

A total of 12 cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-17, IFNα, IFN-γ, TNF-α) in circulation were monitored. Four cytokines (TNF-α, IL-6, IL-8, and IL-10) displayed changes above baseline levels (Figure 4B) while the other 8 cytokines did not (data not shown). The increased expression of these four cytokines was observed during therapy (Figure 4B), suggesting that DC vaccination activated cytokine responses.

In this report, we present a patient with GBM treated with long-term combination immunotherapy consisting of DC vaccines, anti-PD-1 and poly I:C as well as a chemotherapeutic agent cyclophosphamide. Several novel aspects were observed in this case study. First, this case represents the first study combining three immunotherapeutic agents in GBM treatment. Although all three immunotherapeutic agents have been tested in GBM individually, there was no report of combining them. Second, the combination immunotherapy can be performed long-term and appears to be safe and effective. The objectives of the study were to test safety and tolerability of combination immunotherapy with the aim to maximize the possibility of cure. The patient’s >5 years of progression free survival is encouraging. Third, the different forms of tumor antigens including mRNA-TAAs, mRNA-neoantigens and HOCI-oxidized tumor lysates for priming antitumor T cell responses may complement each other to promote vaccine efficacy. Fourth, TriVac technology-based modification of tumor antigens appears to be safe and induce robust antitumor specific CD4⁺ and CD8⁺ T cell responses in the patient.

Our preliminary data here support the notion that combination therapy with different immunotherapeutic agents may be needed to treat GBM. Although two recent phase III trials failed to demonstrate improved OS with nivolumab combining radiotherapy or chemoradiotherapy to treat newly diagnosed GBM ¹⁰^,¹¹, addition of anti-PD-1 to personalized cancer vaccines has shown promising clinical activities in NSCLC, melanoma, and bladder cancer ⁴^,¹²^,¹³. In our study, addition of nivolumab serves two purposes. First, mature DCs, the final products for infusion, upregulate both PD-L1 and PD-L2 (unpublished data). Second, poly I:C also stimulates both PD-L1 and PD-L2 expression on primary human GBM cells ¹⁹. Thus, nivolumab blocks the inhibitory signals of PD-L1/2 on mDCs and GBM tumor cells from being delivered to T cells.

This case study shows a promising disease progression free status and an excellent safety profile in the patient upon long-term combination immunotherapy. The DC vaccination was initiated at one week after the completion of concurrent chemoradiation and continued for 5 years with the intention to achieve a cure status. Consistent with our previous phase I trial safety profile ⁴, no immunotherapy related adverse events were observed. Cyclophosphamide was discontinued out of precaution by the physician team based on an 8.4 g cumulative dose. Although the results are encouraging, other favorable factors including young age, IDH1 mutation, and MGMT promoter methylation may be contributing to the improved survival. Therefore, a large trial to validate the efficacy of this combination immunotherapy in newly diagnosed GBM is needed.

Three approaches for the preparation of tumor antigens were used in this case study to induce a wide array of antitumor T cells required to cover heterogenous expression of tumor antigens in GBM ⁴^,²⁸. These approaches, each with advantages and disadvantages, may complement each other to serve as targets for long-term cancer vaccinations. Although HOCI-oxidized tumor lysates with enhanced immunogenicity have a major advantage as a source of tumor antigens containing both TAAs and neoantigens, procurement of sufficient amount of tumor tissue for long-term vaccination is not feasible. In contrast, in vitro synthesized mRNA-encoded TAAs and neoantigens provide unlimited supplies. However, the use of TAAs is hampered by their poor immunogenicity when compared to neoantigens due to host tolerance mechanisms ²⁰. Modification of TAAs with the TriVac technology enhances both MHC class I and II antigen presentations and T cell stimulation ²⁰. Neoantigen-based cancer vaccines induce a potent antigen-specific T cell response and may have some clinical activities in GBM patients ⁸^,⁹. Nevertheless, two major issues exist for the use of neoantigens in GBM treatment. First, GBM has a low tumor mutation burden with few neoantigens to select and second, manufacturing GMP grade mRNA-neoantigens takes time. Therefore, combined use of mRNA-TAAs and neoantigens in this case is necessary to resolve the above issues.

Our data support further investigation in clinical application of the TriVac technology ²⁰. We previously tested the capacity of mRNA-TAAs modified with the sorting signal of LAMP-1 in inducing antigen specific T cells in cancer patients ⁴. The new TriVac technology platform uses a full-length LAMP-1 and a DD to modify tumor antigens to enhance antigen presentation to both MHC class I and II complexes. Consistent with this notion, two TAAs (MTFR2 and ADAMTSL1) modified only with this new technology induced robust antigen specific T cell responses. In contrast, five TAAs (SOX11, PIM1, SP17, SOX3, and CSPG4) modified with the sorting signal of LAMP-1 in the first phase of immunization induced relatively weak antigen-specific T cell responses while these 5 TAAs induced much stronger T cell responses in the second phase of immunization after modification with the TriVac-technology. Alternatively, the enhanced antigen-specific T cell responses could result from a general elevated systemic immune activation status derived from nivolumab and poly I:C treatment. These possibilities need to be examined in more detailed clinical studies. The mRNA-neoantigen vaccines also induced significant levels of antigen-specific T cell responses. Whether these antigen-specific T cells play any roles in the patient’s disease-free status remain to be determined. In summary, we report here a GBM case with long-term integrated combination immunotherapy characterized by good safety and >5 years of disease progression free survival. Future large-scale studies are warranted to validate our findings.

Clinical study and case description

A combination immunotherapy study for GBM was approved by the Scientific Review Committee and the Ethics Committee of Xijing Hospital as a compassionate use protocol based on our previous phase I trial (NCT02709616)⁴. The protocol numbers of the study are 201708152377 and 20202014-F-1. The primary objective was to test the safety and tolerability of personalized DC cancer vaccines pulsed with different types of tumor antigens combining anti-PD-1 and poly I:C in patients with GBM. The secondary objectives were to investigate clinical efficacy of an integrated combination immunotherapy and the degree of DC cancer vaccine induced-CD4⁺ and CD8⁺ T cell responses with the goal to maximize possibility of cure.

A 37-year-old patient with glioblastoma multiforme (WHO grade IV) was enrolled in this study at Xijing Hospital, Xi’an, China. The patient presented with a lack of verbal fluency and word-finding difficulty on July 17, 2017. MRI revealed a 3.8 x 5.8 x 3.6 cm contrast-enhancing lesion in the left frontal lobe. Histopathology showed that the patient had GBM (WHO IV) with an isocitrate dehydrogenase 1 (IDH1) R132H mutation, chromosome loss of heterozygosity (LOH) 1p /19q -/- and 52% promoter methylation of O6-methylguanine-DNA methyltransferase (MGMT). The patient underwent surgical resection on July 20, 2017, followed by a 6-week standard chemoradiotherapy and 12 cycles of adjuvant chemotherapy. The first leukapheresis to collect peripheral blood mononuclear cells (PBMCs) for autologous DC generation was performed before the chemoradiotherapy. Tumor tissues from surgical resection were stored at -80^oC for subsequent analysis of TAA/neoantigen expression and generation of HOCI-oxidized lysates. Conformal radiotherapy was performed with a 6 MV X-ray fractionated-dose of 2.0 Gy up to a total dose of 60 Gy in 30 fractions. The concurrent chemotherapy was administered with temozolomide (TMZ) at a dose of 75 mg/m² per day. The 12 cycles of chemotherapy with temozolomide at 200 mg/m² orally per day for 5 consecutive days per 28-day treatment cycles was completed in September 2018. Oxcarbazepine was prescribed in March 2018 at an initial dose of 0.9 g/d, gradually increased to 1.35 g/d, then tapered and withdrawn in December 2021 to control postoperative seizures. Oxiracetam was used to improve memory impairments from March 2018 to May 2019. No steroids were used during the entire treatment. The process of the integrated combination immunotherapy is described in the Results section. The patient was evaluated every 3 months for treatment response and disease progression using iRANO criteria. Adverse events were evaluated and reported using CTCAE v4.

Generation of autologous DC cancer vaccine

Autologous DC cancer vaccines were generated as previously described in our personalized cancer vaccine phase I trials ⁴. Briefly, PBMCs were obtained via leukapheresis, further purified by density gradient centrifugation, and cryopreserved. The cells were thawed for each DC vaccine preparation a week before the vaccination. PBMCs were cultured in 30 ml AIM-V containing 800 IU/ml GM-CSF and 500 IU/ml IL-4 to induce immature DCs (iDCs). On day 6, iDCs were harvested and transfected with the individual mRNA-TAAs and mRNA-neoantigens by electroporation, or pulsed with HOCI-oxidized tumor lysates. The antigen loaded iDCs were matured in AIM-V containing 800 IU/ml GM-CSF, 500 IU/ml IL-4, 160 ng/ml IL-6, 5 ng/ml IL-1β, 5 ng/ml TNF-α and 1 µg/ml PGE2 for 18-20 hours and pooled for quality control before injection. The DC cancer vaccines were infused intradermally and intravenously at a 1:5 route ratio. The detailed information on the tumor antigens used for each immunization and the number of infused DCs are shown in Table 1. The sources for all reagents including cytokines, antibodies and therapeutic agents used in this study are listed in Supplemental Table 1.

Preparation of autologous HOCl-oxidized tumor lysates and antigen loading of DCs

HOCl-oxidized tumor lysate was prepared as previously described ²¹. HOCl concentration was determined by spectrophotometer at an absorbance of 252 nm. Final concentration of 60 mM HOCl solution was prepared by diluting the stock NaOCl reagent (Sigma-Aldrich) with PBS (Gibco). HOCl solution was added immediately to the tumor cells to a final density of 10⁶ cells/ml. The tumor cell suspension was incubated at 37°C, 5% CO₂ for 1 hour with gentle agitation every 30 mins to induce oxidation-dependent tumor cell death. After incubation, tumor cells were harvested, washed twice with PBS and frozen/thawed for 6 cycles. The oxidized tumor lysates were tested for complete removal of the HOCl solution before loading into DCs. Aliquoted HOCI-oxidized tumor lysates were stored at -80°C. HOCl-oxidized tumor cell lysates were cocultured with normal donor DCs (at a 1:1 ratio of original tumor cells:DCs) for 20-24 hours at 37°C for antigen loading. The antigen-loaded DCs were harvested, washed, and injected.

Whole-exome DNA and RNA sequencing

Genomic DNA and total RNA were extracted from paired tumor and para-tumor samples for whole-exome sequencing and transcriptome sequencing (Anoroad Genome, Ltd). Genomic DNA was fragmented to a target size of 150~200 bp. Exons were captured using the Agilent SureSelect Human All Exon V6 kit (Agilent), subsequently sequenced on a HiSeq 2500 (Illumina) in a Paired End sequencing of 150 bp forward and 150 bp reverse. The sequencing used the HiSeq SBS Kit v4 (Illumina) with a target coverage of 100× for tumor sample and 80× for paired para-tumor sample. Sequence data were analyzed with the GATK Best Practice pipeline. Total RNA from paired tumor and para-tumor samples were extracted and sequenced by Hiseq2500 system. RAW data was analyzed with the Mapsplice-RSEM-Ebseq pipeline.

Neoantigen prediction and selection

HLA typing was performed using the PCR sequencing-based typing method. The reads are aligned to reference HLA sequences from the IPD-IMGT/HLA Database version 3.16. Somatic mutations of the tumor tissue were identified by MuTect. Missense mutation expression was identified using transcriptome sequencing and validated with qRT-PCR. Expressed mutations were selected for immunogenicity prediction. Firstly, all mutated sequences and wild-type sequences were translated to strings of 27 amino acids, with the mutated amino acid centrally situated. Secondly, all mutant and wild-type MHC-I and MHC-II epitope binding affinities were evaluated by NetMHCpan 4.0 (http://tools.immuneepitope.org/mhci/) and Consensus method (http://tools.immuneepitope. org/mhcii/), respectively. All mutations with binding scores below 500 nM were defined as neoantigens, and subsequently selected for mRNA and peptide synthesis. A total of 49 somatic mutations were identified in the tumor sample, including 21 missense mutations and 28 nonsense mutations. The mRNA expression of all mutated genes were examined by qPCR and 10 of the mutated genes were detected in the patient’s tumor sample. The 10 putative neoantigens and their corresponding wild type peptides were synthesized (Genscript) and tested for in vitro T cell stimulation. Four neoantigens with equal or greater T cell stimulation capability compared to their wild type counterparts were selected to generate mRNA-neoantigens for immunization.

Measurement of mRNA expression of TAAs

Total RNA was extracted from tumor samples obtained by resection and reverse transcribed. The cDNAs were analyzed by quantitative polymerase chain reaction (qPCR) for the expression of TAAs. All reactions were performed in triplicate. The primers for the tested TAA genes were previously described ⁴. The mRNA expression levels of TAAs in the tumor samples were compared with those in para-tumor and shown as mean fold changes.

Tumor antigen DNA constructs and GMP mRNA production

To enhance tumor antigen presentation by MHC class I and class II pathways, full-length TAAs were modified with a novel TriVac technology ²⁰. The TAAs were either inserted between the sorting signal and the luminal domain of a full-length LAMP-1 or fused with a destabilizing domain (DD) as described ²⁰. Mutations in human FKBP12 created unstable proteins that subject the proteins fused with the mutants to rapid proteasomal degradation, therefore functioning as a destabilizing domain ²⁹. We tested antigen presentation by MHC class I complexes with TAAs fused with DD, which resulted in rapid proteasomal degradation and enhanced MHC class I antigen presentation (unpublished data). Modification of TAAs with the TriVac technology also enhanced anti-tumor activities of cancer vaccines in mouse models (unpublished data). For neoantigen constructs, a string of 27 amino acids with the mutation residue centrally situated was inserted between the sorting signal and the luminal domain of a full-length LAMP-1. Plasmids were all linearized by digestion with SpeI-HF (NEB) and used as templates in in vitro transcription reactions. The in vitro transcription was performed with T7 RNA polymerase (mMESSAGE mMACHINE T7 Transcription kit; Ambion) according to the manufacturer’s instructions. The transcribed RNA was purified after TURBO DNase (Ambion) digestion on RNeasy columns (Qiagen) according to the manufacturer’s instructions. RNA quality was verified by the Agilent 2100 Bioanalyzer to ensure that the levels of endotoxin contamination were below 0.25 EU/mg. RNA concentration was measured by Nanodrop. Manufactured mRNA-TAAs or mRNA-neoantigens were stored at −80℃ in aliquots. All mRNAs encoding full-length TAAs were pre-manufactured while mRNAs encoding neoantigens were manufactured after their identification and selection in a GMP facility.

In vitro stimulation of PBMCs to select neoantigens

For in vitro T cell stimulation to select neoantigens, day 6 iDCs were pulsed with 10 neoantigen or wildtype peptides for 24 hours at 2 µg/ml and matured for 18-20 hours. Then the non-adherent T cells and antigen loaded DCs were cocultured at an effector to target (E:T) ratio of 10:1 in the presence of 10 ng/ml IL-7. Three days after stimulation, fresh culture medium containing 100 IU/ml IL-2 and 10 ng/ml IL-7 was added every 2-3 days. After 12-14 days of stimulation, cells were restimulated with peptide-pulsed DCs and analyzed via flow cytometry.

Detection of tumor antigen specific T cells after DC vaccination

PBMCs collected at different times plated at 5×10⁵/well in 48-well plates were stimulated with autologous TAA-mRNAs or neoantigen-mRNA transfected-mature DC (mDC) at a 10:1 ratio in the presence of 10 ng/ml IL-7. On day 12, non-adherent T cells were restimulated with tumor antigen mRNA transfected DCs for 6 hours in RPMI 1640 with 10% FBS and Golgistop. Tumor antigen-specific T cells were determined using intracellular staining of IFN-γ and TNF-α. The in vitro stimulated T cells were stained with LIVE/DEAD-AmCyan, and a panel of cell surface markers including CD3-FITC, CD4-PE/Cy7, and CD8-PerCP, followed by fixation/permeabilization and staining with PE-anti-IFN-γ and APC-anti-TNF-α.

iDC Immature DC

mDCs Mature dendritic cells

DD Destabilization domain

GBM Glioblastoma multiforme

GMP Good manufacturing practice

HOCI Hypochlorous acid

NSCLC Non-small cell lung cancer

PBMCs Peripheral blood mononuclear cells

qPCR Quantitative polymerase chain reaction

TAAs Tumor-associated antigens

Acknowledgements

We thank the patient’s many contributions to this study. We thank Dr. Edward F. Patz Jr. and Dr. Elizabeth B. Gottlin for critical reading of the manuscript.

Authors’ contributions

PZ, ZNC and YWH conceived and designed the study. PZ, SYL, and HJB supervised the study. SNS, JLM, XL, BW, KZ, SP and SYL performed DC vaccine work and monitoring of immune cells and cytokines. SNS, JJ and SYL performed antigen detection and selection as well as GMP production of mRNAs. PZ, JD, ZHZ, SZL and ZF carried patient treatment and care. YWH, JOL and JTA are co-inventors of the TriVac technology. PZ, SNS, JD, SYL, ZNC and YWH analyzed and interpreted the data. SNS, JD and YWH drafted the manuscript with contributions from all authors. PZ, SNS, JD, JOL, ZNC, and YWH critically reviewed or revised the manuscript.

All authors reviewed the interim drafts and the final version of the manuscript and agreed with its content and submission. All authors had access to all the relevant study data and related analyses and vouch for the completeness and accuracy of the presented data. All authors agree to be accountable for all aspects of the work and will ensure that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors read and approved the final manuscript.

Funding statement

The study was supported by 2019ZY-CXPT-03-01 to PZ and 2020ZDLSF03-02 to ZNC and HB as well as tricision Biotherapeutics Inc. The funders did not have any roles in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the author contributions’ section.

Availability of data and materials

All data generated or analyzed during this study are included in this article and its supplementary information file.

Ethical approval and ethical standards

Written informed consent was obtained from the enrolled patient before any of the study procedures were conducted. This study was approved by Xijing Hospital as a compassionate use protocol based on a registered phase I trial (⁴, NCT02709616). The protocol numbers of the study are 201708152377 and 20202014-F-1. All treatment procedures were performed in accordance with the ethical standards of the institutional and/or national research regulations and with the Helsinki declaration ethical standards.

Conflict of interest

You-Wen He and Shi-You Li are co-founders of tricision Biotherapeutics Inc. Sheng-Nan Sun, Jun Jiang, Qian-Ting Wang, Shi-You Li are full-time employee of tricision Biotherapeutics Inc. You-Wen He and Jun O. Liu are co-inventors of the TriVac technology patented by Duke University and Johns Hopkins University and co-founders of TriVac Inc.

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Table 1. Detailed information for each DC cancer vaccination

	Date	Cell number	IV	ID（Groin）		Antigen
	Date	Cell number	IV	Left	Right	Antigen
1	2017-10-17	1.5*10⁷	√	√		TAA: PP65, SOX11-L(t/c), SP17-L(t/c)
2	2017-10-31	2.1*10⁷	√		√	TAA: PP65, SOX11-L(t/c), SP17-L(t/c)
3	2017-11-14	1.8*10⁷	√	√		TAA: PP65, SOX11-L(t/c), SP17-L(t/c)
4	2017-12-12	1.7*10⁷	√		√	HOCI-lysates
5	2017-12-26	1.1*10⁷	√	√		HOCI-lysates
6	2018-01-09	1.1*10⁷	√		√	HOCI-lysates
7	2018-01-23	2.4*10⁷	√	√		TAA: CSPG4-L(t/c), SOX3-L(t/c), PIM1-L(t/c)
8	2018-02-06	2.6*10⁷	√		√	TAA: CSPG4-L(t/c), SOX3-L(t/c), PIM1-L(t/c)
9	2018-02-13	3.3*10⁷	√	√		TAA: CSPG4-L(t/c), SOX3-L(t/c), PIM1-L(t/c)
10	2018-04-11	2.8*10⁷	√		√	TAA: CSPG4-L(t/c), SOX3-L(t/c), PIM1-L(t/c)
11	2018-05-04	3.1*10⁷	√	√		TAA: CSPG4-L(t/c), SOX3-L(t/c), PIM1-L(t/c)
12	2018-05-15	1.6*10⁷	√		√	TAA: CSPG4-L(t/c), SOX3-L(t/c), PIM1-L(t/c)
13	2018-07-12	3.4*10⁷	√	√		Neoantigen: PRKRIR
14	2018-07-24	1.7*10⁷	√		√	Neoantigen: PRKRIR
15	2018-08-07	2.3*10⁷	√	√		Neoantigen: PRKRIR
16	2018-09-26	1.2*10⁷	√		√	Neoantigen: PRKRIR, FLGN, HUWE1, RUNX1
17	2018-10-09	1.8*10⁷	√	√		Neoantigen: PRKRIR, FLGN, HUWE1, RUNX1
18	2018-10-23	2.2*10⁷	√		√	Neoantigen: PRKRIR, FLGN, HUWE1, RUNX1
19	2018-12-27	2.9*10⁷	√		√	Neoantigen: PRKRIR, FLGN, HUWE1, RUNX1
20	2019-01-30	3.1*10⁷	√	√		TAA: PIM1-DD, PIM1-L, SOX3-DD, SOX3-L
21	2019-02-27	3.5*10⁷	√		√	TAA: MTFR2-DD, MTFR2-L, ADAMTSL-DD, ADAMTSL -L
22	2019-03-19	2.2*10⁷	√	√		TAA: MTFR2-DD, MTFR2-L, ADAMTSL-DD, ADAMTSL -L
23	2019-04-03	3.1*10⁷	√		√	TAA: MTFR2-DD, MTFR2-L, ADAMTSL-DD, ADAMTSL -L
24	2019-04-17	2.5*10⁷	√	√		Neoantigen: PRKRIR, FLGN, HUWE1, RUNX1
25	2019-05-15	2.4*10⁷	√	√		TAA: PIM1-DD, PIM1-L, SOX3-DD, SOX3-L, SP17-DD, SP17-L
26	2019-05-29	3.3*10⁷	√		√	TAA: PIM1-DD, PIM1-L, SOX3-DD, SOX3-L, SP17-DD, SP17-L
27	2019-06-12	3.4*10⁷	√	√		TAA: PIM1-DD, PIM1-L, SOX3-DD, SOX3-L, SP17-DD, SP17-L
28	2019-11-20	2*10⁶	√		√	TAA: MTFR2-DD, MTFR2-L, ADAMTSL-DD, ADAMTSL -L, SOX11-DD, SOX11-L
29	2019-12-4	4.67*10⁶	√	√		TAA: MTFR2-DD, MTFR2-L, ADAMTSL-DD, ADAMTSL -L, SOX11-DD, SOX11-L
30	2020-7-22	7*10⁶	√		√	TAA: SOX3-L, SP17-L, MTFR2-DD, ADAMTSL -L
31	2020-8-5	1.25*10⁷	√		√	TAA: SOX3-L, SP17-L, MTFR2-DD, ADAMTSL -L
32	2020-8-19	2.37*10⁷	√		√	TAA: SOX3-L, SP17-L, MTFR2-DD, ADAMTSL -L
33	2020-9-3	1.1*10⁷	√	√		TAA: PIM1-L, SOX11-L
34	2020-9-16	1.5*10⁷	√		√	TAA: PIM1-L, SOX11-L
35	2020-9-30	2.4*10⁷	√	√		TAA: PIM1-L, SOX11-L
36	2021-7-21	1.01*10⁷	√	√		TAA: PIM-1-L, SP17-L, SOX3-L
37	2021-8-4	1.26*10⁷	√	√		TAA: PIM-1-L, SP17-L, SOX3-L
38	2021-8-18	1.04*10⁷	√		√	TAA: SOX11-L, CSPG4-L
39	2021-9-1	1.4*10⁷	√	√		TAA: SOX11-L, CSPG4-L
40	2022-4-6	1.9*10⁷	√		√	TAA: SP17-L, SOX3-L
41	2022-4-20	3.3*10⁷	√	√		TAA: ADAMTSL1-L, MTFR2-L
42	2022-5-4	3.5*10⁷	√		√	Neoantigen: PRKRIR, FLGN, HUWE1, RUNX1

IV: intravenously. ID: Intradermally. DCs were infused at 5:1 ratio IV:ID. For ID administration, right or left groin region was used as listed. TAAs with a -L(t/c) tag indicates modification with the sorting domain of LAMP-1 while TAAs with a -L or -DD indicates modification with full-length LAMP-1 or the destabilization domain.

Yes there is potential Competing Interest. You-Wen He and Shi-You Li are co-founders of tricision Biotherapeutics Inc. Sheng-Nan Sun, Jun Jiang, Qian-Ting Wang, Shi-You Li are full-time employee of tricision Biotherapeutics Inc. You-Wen He and Jun O. Liu are co-inventors of the TriVac technology patented by Duke University and Johns Hopkins University and co-founders of TriVac Inc.

SupplementalTable1.xls
Supplemental Tabel 1

Combination Immunotherapy of Glioblastoma with Dendritic Cell Cancer Vaccines, Anti-PD-1 and Poly I:C: A Case Report

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