Neoantigen-targeted CD8+ T cell responses with PD-1 blockade therapy.

Neoantigens are peptides derived from non-synonymous mutations presented by human leukocyte antigens (HLAs), which are recognized by antitumour T cells1-14. The large HLA allele diversity and limiting clinical samples have restricted the study of the landscape of neoantigen-targeted T cell responses in patients over their treatment course. Here we applied recently developed technologies15-17 to capture neoantigen-specific T cells from blood and tumours from patients with metastatic melanoma with or without response to anti-programmed death receptor 1 (PD-1) immunotherapy. We generated personalized libraries of neoantigen-HLA capture reagents to single-cell isolate the T cells and clone their T cell receptors (neoTCRs). Multiple T cells with different neoTCR sequences (T cell clonotypes) recognized a limited number of mutations in samples from seven patients with long-lasting clinical responses. These neoTCR clonotypes were recurrently detected over time in the blood and tumour. Samples from four patients with no response to anti-PD-1 also demonstrated neoantigen-specific T cell responses in the blood and tumour to a restricted number of mutations with lower TCR polyclonality and were not recurrently detected in sequential samples. Reconstitution of the neoTCRs in donor T cells using non-viral CRISPR-Cas9 gene editing demonstrated specific recognition and cytotoxicity to patient-matched melanoma cell lines. Thus, effective anti-PD-1 immunotherapy is associated with the presence of polyclonal CD8+ T cells in the tumour and blood specific for a limited number of immunodominant mutations, which are recurrently recognized over time.

Non-viral precision T cell receptor replacement for personalized cell therapy.

T cell receptors (TCRs) enable T cells to specifically recognize mutations in cancer cells1-3. Here we developed a clinical-grade approach based on CRISPR-Cas9 non-viral precision genome-editing to simultaneously knockout the two endogenous TCR genes TRAC (which encodes TCRα) and TRBC (which encodes TCRß). We also inserted into the TRAC locus two chains of a neoantigen-specific TCR (neoTCR) isolated from circulating T cells of patients. The neoTCRs were isolated using a personalized library of soluble predicted neoantigen-HLA capture reagents. Sixteen patients with different refractory solid cancers received up to three distinct neoTCR transgenic cell products. Each product expressed a patient-specific neoTCR and was administered in a cell-dose-escalation, first-in-human phase I clinical trial ( NCT03970382 ). One patient had grade 1 cytokine release syndrome and one patient had grade 3 encephalitis. All participants had the expected side effects from the lymphodepleting chemotherapy. Five patients had stable disease and the other eleven had disease progression as the best response on the therapy. neoTCR transgenic T cells were detected in tumour biopsy samples after infusion at frequencies higher than the native TCRs before infusion. This study demonstrates the feasibility of isolating and cloning multiple TCRs that recognize mutational neoantigens. Moreover, simultaneous knockout of the endogenous TCR and knock-in of neoTCRs using single-step, non-viral precision genome-editing are achieved. The manufacture of neoTCR engineered T cells at clinical grade, the safety of infusing up to three gene-edited neoTCR T cell products and the ability of the transgenic T cells to traffic to the tumours of patients are also demonstrated.

Poxvirus-based active immunotherapy synergizes with CTLA-4 blockade to increase survival in a murine tumor model by improving the magnitude and quality of cytotoxic T cells.

The dramatic clinical benefit of immune checkpoint blockade for a fraction of cancer patients suggests the potential for further clinical benefit in a broader cancer patient population by combining immune checkpoint inhibitors with active immunotherapies. The anti-tumor efficacy of MVA-BN-HER2 poxvirus-based active immunotherapy alone or in combination with CTLA-4 checkpoint blockade was investigated in a therapeutic CT26-HER-2 lung metastasis mouse model. MVA-BN-HER2 immunotherapy significantly improved the median overall survival compared to untreated controls or CTLA-4 blockade alone (p < 0.001). Robust synergistic efficacy was achieved with the combination therapy (p < 0.01). Improved survival following MVA-BN-HER2 administration was accompanied by increased tumor infiltration by HER-2-specific cytotoxic T lymphocytes (CTL). These tumor-specific CTL had characteristics similar to antiviral CTL, including strong expression of activation markers and co-expression of IFNγ and TNFα. Combination with CTLA-4 blockade significantly increased the magnitude of HER-2-specific T cell responses, with a higher proportion co-expressing TNFα and/or IL-2 with IFNγ. Furthermore, in mice treated with MVA-BN-HER2 (alone or in combination with CTLA-4 blockade), the inducible T cell co-stimulator (ICOS) protein was expressed predominantly on CD4 and CD8 effector T cells but not on regulatory T cells (T(reg)). In contrast, mice left untreated or treated solely with CTLA-4 blockade harbored elevated ICOS(+) Treg, a phenotype associated with highly suppressive activity. In conclusion, poxvirus-based active immunotherapy induced robust tumor infiltration by highly efficient effector T cells. Combination with CTLA-4 immune checkpoint blockade amplified this response resulting in synergistically improved efficacy. These hypothesis-generating data may help elucidate evidence of enhanced clinical benefit from combining CTLA-4 blockade with poxvirus-based active immunotherapy.

Poxvirus-Based Active Immunotherapy with PD-1 and LAG-3 Dual Immune Checkpoint Inhibition Overcomes Compensatory Immune Regulation, Yielding Complete Tumor Regression in Mice.

Poxvirus-based active immunotherapies mediate anti-tumor efficacy by triggering broad and durable Th1 dominated T cell responses against the tumor. While monotherapy significantly delays tumor growth, it often does not lead to complete tumor regression. It was hypothesized that the induced robust infiltration of IFNγ-producing T cells into the tumor could provoke an adaptive immune evasive response by the tumor through the upregulation of PD-L1 expression. In therapeutic CT26-HER-2 tumor models, MVA-BN-HER2 poxvirus immunotherapy resulted in significant tumor growth delay accompanied by a robust, tumor-infiltrating T cell response that was characterized by low to mid-levels of PD-1 expression on T cells. As hypothesized, this response was countered by significantly increased PD-L1 expression on the tumor and, unexpectedly, also on infiltrating T cells. Synergistic benefit of anti-tumor therapy was observed when MVA-BN-HER2 immunotherapy was combined with PD-1 immune checkpoint blockade. Interestingly, PD-1 blockade stimulated a second immune checkpoint molecule, LAG-3, to be expressed on T cells. Combining MVA-BN-HER2 immunotherapy with dual PD-1 plus LAG-3 blockade resulted in comprehensive tumor regression in all mice treated with the triple combination therapy. Subsequent rejection of tumors lacking the HER-2 antigen by treatment-responsive mice without further therapy six months after the original challenge demonstrated long lasting memory and suggested that effective T cell immunity to novel, non-targeted tumor antigens (antigen spread) had occurred. These data support the clinical investigation of this triple therapy regimen, especially in cancer patients harboring PD-L1neg/low tumors unlikely to benefit from immune checkpoint blockade alone.

Elucidating immunologic mechanisms of PROSTVAC cancer immunotherapy.

BACKGROUND: PROSTVAC®, an active immunotherapy currently studied for the treatment of metastatic castration-resistant prostate cancer (mCRPC), consists of a heterologous prime-boost regimen with two different poxvirus-based vectors to provoke productive immune responses against prostate specific antigen (PSA) as the target tumor antigen. A Phase 2 study of PROSTVAC immunotherapy showed significantly improved median overall survival by 8.5 months and is currently being validated in a global Phase 3 study (PROSPECT; NCT01322490). Here, preclinical models were explored to investigate the mechanism of action and immune signatures of anti-tumor efficacy with PROSTVAC immunotherapy with the goal to identify potential immune correlates of clinical benefit. METHODS: PROSTVAC-induced immune responses and anti-tumor efficacy were studied in male BALB/c mice. Functionality of the induced T cell response was characterized by interferon-gamma (IFNγ) ELISPOT, cytotoxic degranulation, multi-cytokine intracellular staining, and in vivo T cell depletion. Tumor infiltrating lymphocytes (TILs) were evaluated phenotypically by flow cytometry. RESULTS: The heterologous prime-boost regimen of the two PROSTVAC vectors significantly enhanced the magnitude and quality of activated PSA-specific CD4 and CD8 T cell responses compared to homologous, single vector regimens. PROSTVAC-activated CD4 and CD8 T cells were highly functional as evidenced by expression of activation markers, production of multiple cytokines, and amplified cytotoxic T cell activity. Importantly, PROSTVAC immunotherapy resulted in significant anti-tumor efficacy in a transplantable prostate cancer mouse model. Antigen-spreading occurred in PROSTVAC-treated animals that rejected PSA-expressing tumors, as shown by subsequent rejection of PSA-negative tumors. In vivo CD4 and CD8 depletion revealed that both T cell subsets contributed to anti-tumor efficacy. Characterization of TILs demonstrated that PROSTVAC immunotherapy greatly increased the intra-tumoral ratio of activated effector to regulatory T cells. CONCLUSIONS: PROSTVAC immunotherapy activates broad, highly functional T cell immunity to PSA and to endogenous tumor antigens via immune-mediated antigen spreading. These preclinical results further elucidate the mode of action of PROSTVAC immunotherapy and its potential causal relationship to extended overall survival as observed in the PROSTVAC Phase 2 study. The clinical validation is ongoing in the PROSPECT Phase 3 clinical study.

Oh the irony: Iron as a cancer cause or cure?

Iron-oxide nanoparticles facilitate cancer diagnosis through enhanced contrast, selectively enhance tumor cell death with magnetic hyperthermia, and improve drug delivery with magnetic drug targeting. One application that remains largely unexplored is using the iron-oxide nanoparticles themselves to selectively inhibit tumor growth. In this leading opinion paper, we propose that high doses of iron-oxide nanoparticles can be used as a treatment for cancer by generating an oxidative assault against cancer. This proposal may be met with resistance considering the controversy surrounding iron in the field of cancer. Iron generates reactive oxygen species through the Fenton reaction, which may both cause - or cure cancer. Additionally, high demand for iron by cancer cells leads to contradictory therapeutic approaches: iron deprivation or overdose are both potential cancer therapies.

Tumor ablation and nanotechnology.

Next to surgical resection, tumor ablation is a commonly used intervention in the treatment of solid tumors. Tumor ablation methods include thermal therapies, photodynamic therapy, and reactive oxygen species (ROS) producing agents. Thermal therapies induce tumor cell death via thermal energy and include radiofrequency, microwave, high intensity focused ultrasound, and cryoablation. Photodynamic therapy and ROS producing agents cause increased oxidative stress in tumor cells leading to apoptosis. While these therapies are safe and viable alternatives when resection of malignancies is not feasible, they do have associated limitations that prevent their widespread use in clinical applications. To improve the efficacy of these treatments, nanoparticles are being studied in combination with nonsurgical ablation regimens. In addition to better thermal effect on tumor ablation, nanoparticles can deliver anticancer therapeutics that show a synergistic antitumor effect in the presence of heat and can also be imaged to achieve precision in therapy. Understanding the molecular mechanism of nanoparticle-mediated tumor ablation could further help engineer nanoparticles of appropriate composition and properties to synergize the ablation effect. This review aims to explore the various types of nonsurgical tumor ablation methods currently used in cancer treatment and potential improvements by nanotechnology applications.

PEG-functionalized magnetic nanoparticles for drug delivery and magnetic resonance imaging applications.

PURPOSE: Polyethylene glycol (PEG) functionalized magnetic nanoparticles (MNPs) were tested as a drug carrier system, as a magnetic resonance imaging (MRI) agent, and for their ability to conjugate to an antibody. METHODS: An iron oxide core coated with oleic acid (OA) and then with OA-PEG forms a water-dispersible MNP formulation. Hydrophobic doxorubicin partitions into the OA layer for sustained drug delivery. The T(1) and T(2) MRI contrast properties were determined in vitro and the circulation of the MNPs was measured in mouse carotid arteries. An N-hydroxysuccinimide group (NHS) on the OA-PEG-80 was used to conjugate the amine functional group on antibodies for active targeting in the human MCF-7 breast cancer cell line. RESULTS: The optimized formulation had a mean hydrodynamic diameter of 184 nm with an ~8 nm iron-oxide core. The MNPs enhance the T(2) MRI contrast and have a long circulation time in vivo with 30% relative concentration 50 min post-injection. Doxorubicin-loaded MNPs showed sustained drug release and dose-dependent antiproliferative effects in vitro; the drug effect was enhanced with transferrin antibody-conjugated MNPs. CONCLUSION: PEG-functionalized MNPs could be developed as a targeted drug delivery system and MRI contrast agent.

Optical imaging and magnetic field targeting of magnetic nanoparticles in tumors.

To address efficacy issues of cancer diagnosis and chemotherapy, we have developed a magnetic nanoparticle (MNP) formulation with combined drug delivery and imaging properties that can potentially be used in image-guided drug therapy. Our MNP consists of an iron-oxide magnetic core coated with oleic acid (OA) and stabilized with an amphiphilic block copolymer. Previously, we reported that our MNP formulation can provide prolonged contrast for tumor magnetic resonance imaging and can be loaded with hydrophobic anticancer agents for sustained drug delivery. In this study, we developed MNPs with optical imaging properties using new near-infrared dyes to quantitatively determine their long-term biodistribution and tumor localization with and without an external magnetic field in mice with xenograft breast tumors. MNPs localized slowly in the tumor, reaching a peak 48 h post-injection before slowly declining over the next 11 days. One hour exposure of the tumor to a magnetic field further enhanced MNP localization to tumors. Our MNPs can be developed with combined drug delivery and multimodal imaging properties to improve cancer diagnosis, provide sustained treatment, and monitor therapeutic effects in tumors over time.

Magnetic resonance imaging of multifunctional pluronic stabilized iron-oxide nanoparticles in tumor-bearing mice.

We are investigating the magnetic resonance imaging characteristics of magnetic nanoparticles (MNPs) that consist of an iron-oxide magnetic core coated with oleic acid (OA), then stabilized with a pluronic or tetronic block copolymer. Since pluronics and tetronics vary structurally, and also in the ratio of hydrophobic (poly[propylene oxide]) and hydrophilic (poly[ethylene oxide]) segments in the polymer chain and in molecular weight, it was hypothesized that their anchoring to the OA coating around the magnetic core could significantly influence the physical properties of MNPs, their interactions with biological environment following intravenous administration, and ability to localize to tumors. The amount of block copolymer associated with MNPs was seen to depend upon their molecular structures and influence the characteristics of MNPs. Pluronic F127-modified MNPs demonstrated sustained and enhanced contrast in the whole tumor, whereas that of Feridex IV was transient and confined to the tumor periphery. In conclusion, our pluronic F127-coated MNPs, which can also be loaded with anticancer agents for drug delivery, can be developed as an effective cancer theranostic agent, i.e. an agent with combined drug delivery and imaging properties.

The impact of intravenous corticosteroids with third molar surgery in patients at high risk for delayed health-related quality of life and clinical recovery.

PURPOSE: To compare recovery for clinical and health-related quality of life (HRQOL) outcomes after third molar surgery in patients predicted to be at risk for delayed recovery, treated with or without intravenous (IV) corticosteroids at surgery. PATIENTS AND METHODS: Patients at least 18 years of age and with all 4 third molars below the occlusal plane were given IV corticosteroids just before third molar surgery. Clinical and HRQOL outcomes of these patients were compared with those of a nonconcurrent control group who did not receive corticosteroids. No antibiotics were administered. The control group was selected using the same criteria and treated under the same surgical protocol as the corticosteroid group. Differences between the groups were assessed with Cochran-Mantel-Haenszel row mean score statistics. RESULTS: Sixty patients were in each cohort. The incidence of delayed clinical recovery, a postsurgery visit with treatment, was higher in the control group compared with the corticosteroid group. In the corticosteroid group, 6 patients (10%) had 1 postsurgery visit with treatment. In the control group without corticosteroids, 17 patients (28%) had at least 1 postsurgery visit with treatment (P = .01). Compared with the control group, nausea tended to bother patients less on postsurgery day 1 (P = .07); sleep was improved on postsurgery days 1 through 4 (P < .05). Though not statistically significant, corticosteroids reduced the patients reported recovery by at least 1 day for pain, lifestyle, and oral function. CONCLUSION: Administration of IV corticosteroids before third molar surgery without antibiotics does not hamper clinical recovery even when healthy adult patients are predicted to have delayed recovery. Overall, IV corticosteroid administration had a limited, but beneficial effect on HRQOL outcomes.

The impact of third molar symptoms, pain, and swelling on oral health-related quality of life.

PURPOSE: This study was designed to assess the impact of "pain and swelling" associated with third molars on patients' quality of life before surgery. PATIENTS AND METHODS: The data for these analyses were obtained from a larger ongoing study designed to examine the surgical and medical management of problems associated with third molars. Data from 480 patients with 4 third molars scheduled for removal were used in the analysis. Questionnaires administered presurgery assessed patients' medical and dental history, their reasons for seeking third molar removal, and sociodemographic characteristics. Adverse impacts on oral health-related quality of life were measured using the 14-item Oral Health Impact Profile (OHIP) questionnaire. The primary outcome variable was the percentage of people reporting 1 or more of the 12 non-pain-specific OHIP items "fairly often" or "very often" during the 3 months before enrollment. RESULTS: One third (178 of 480) of patients said they were seeking third molar surgery because of current or previous symptoms of pain/swelling, and 17% reported 1 or more of the 12 non-pain-specific OHIP items. In the multivariate logistic regression model, the odds of one or more impacts was greater for people who presented because of symptoms (odds ratio [OR], 2.9; 95% confidence interval [CI], 1.7 to 4.8), who were aged 25 years or more (OR, 1.9; 95% CI, 1.1-3.3), and who had a self-reported history of tooth loss due to pathology or trauma (OR, 2.9; 95% CI, 1.9 to 5.5). CONCLUSIONS: Adverse impacts on quality of life occurred for 1 in 8 patients seeking third molar surgery, and the odds increased 3-fold for patients who had experienced pain/swelling compared with those who were asymptomatic.

The impact of intravenous antibiotics on health-related quality of life outcomes and clinical recovery after third molar surgery.

PURPOSE: We sought to compare recovery for clinical and health-related quality of life (HRQOL) outcomes after third molar surgery in patients treated with or without intravenous antibiotics at surgery. PATIENTS AND METHODS: Fifty-six patients at least 18 years of age and with all 4 third molars below the occlusal plane, treated at 3 clinical centers, were given intravenous antibiotics just before third molar surgery. Clinical and HRQOL outcomes of these patients were compared with those of a nonconcurrent control group (n = 60 patients) who did not receive antibiotics. The control group was selected using the same criteria and treated under the same surgical protocol as the antibiotic group. Differences between the groups were assessed with Cochran-Mantel-Haenszel row mean score statistics. RESULTS: The incidence of delayed clinical recovery defined as a postsurgery visit with treatment was higher in the control group compared with the antibiotic group. In the antibiotic group, 4% had 1 postsurgery visit with treatment; no patient had 2 visits. In the control group without antibiotics, 28% had at least 1 postsurgery visit with treatment (P <.0001) and 13% had at least 2 postsurgery visits with treatment. No statistically significant differences in HRQOL outcomes were found between the 2 groups. CONCLUSIONS: Administration of intravenous antibiotics before third molar surgery may improve clinical recovery in healthy adult patients with all 4 third molars below the occlusal plane, a presenting characteristic that has been suggested as a risk factor for delayed recovery. The findings from this exploratory trial indicate that evaluation of the effectiveness of systemic antibiotic administration with third molar surgery in a randomized, multi-intervention, explanatory clinical trial is warranted.