Nanoparticles use magnetoelectricity to target and eradicate cancer cells.

This study presents the first in vivo and in vitro evidence of an externally controlled, predictive, MRI-based nanotheranostic agent capable of cancer cell specific targeting and killing via irreversible electroporation (IRE) in solid tumors. The rectangular-prism-shaped magnetoelectric nanoparticle is a smart nanoparticle that produces a local electric field in response to an externally applied magnetic field. When externally activated, MENPs are preferentially attracted to the highly conductive cancer cell membranes, which occurs in cancer cells because of dysregulated ion flux across their membranes. In a pancreatic adenocarcinoma murine model, MENPs activated by external magnetic fields during magnetic resonance imaging (MRI) resulted in a mean three-fold tumor volume reduction (62.3% vs 188.7%; P < .001) from a single treatment. In a longitudinal confirmatory study, 35% of mice treated with activated MENPs achieved a durable complete response for 14 weeks after one treatment. The degree of tumor volume reduction correlated with a decrease in MRI T 2 * relaxation time ( r = .351; P = .039) which suggests that MENPs have a potential to serve as a predictive nanotheranostic agent at time of treatment. There were no discernable toxicities associated with MENPs at any timepoint or on histopathological analysis of major organs. MENPs are a noninvasive alternative modality for the treatment of cancer. Summary: We investigated the theranostic capabilities of magnetoelectric nanoparticles (MENPs) combined with MRI via a murine model of pancreatic adenocarcinoma. MENPs leverage the magnetoelectric effect to convert an applied magnetic field into local electric fields, which can induce irreversible electroporation of tumor cell membranes when activated by MRI. Additionally, MENPs modulate MRI relaxivity, which can be used to predict the degree of tumor ablation. Through a pilot study (n=21) and a confirmatory study (n=27), we demonstrated that, ≥300 µg of MRI-activated MENPs significantly reduced tumor volumes, averaging a three-fold decrease as compared to controls. Furthermore, there was a direct correlation between the reduction in tumor T 2 relaxation times and tumor volume reduction, highlighting the predictive prognostic value of MENPs. Six of 17 mice in the confirmatory study's experimental arms achieved a durable complete response, showcasing the potential for durable treatment outcomes. Importantly, the administration of MENPs was not associated with any evident toxicities. This study presents the first in vivo evidence of an externally controlled, MRI-based, theranostic agent that effectively targets and treats solid tumors via irreversible electroporation while sparing normal tissues, offering a new and promising approach to cancer therapy.

Targeted degradation of the HPV oncoprotein E6 reduces tumor burden in cervical cancer.

Human Papilloma Virus (HPV)-related cancers are a global health burden, yet there are no targeted therapies available for chronically infected patients. The HPV protein E6 is essential for HPV-mediated tumorigenesis and immune evasion, making it an attractive target for antiviral drug development. In this study, we developed an E6-targeting Proteolysis Targeting Chimera (PROTAC) that inhibits the growth of HPV(+) tumors. To develop E6 antagonists, we generated a panel of nanobodies targeting E6 proteins derived from the oncogenic HPV16 subtype. The highest affinity E6 nanobody, A5, was fused to Von Hippel Lindau protein (VHL) to generate a PROTAC that degrades E6 (PROTACE6). Mutational rescue experiments validated specific degradation via the CRL2VHL E3 ligase. Intralesional administration of the PROTACE6 using a clinically viable DNA vaccine reduced tumor burden in an immunocompetent mouse model of HPV(+) cancer. The inhibitory effect of the PROTACE6 was abrogated by CD4+ and CD8+ T-cell depletion, indicating that the antitumor function of the PROTACE6 relies in part on a host immune response. Overall, these results suggest that the targeted degradation of E6 inhibits its oncogenic function and stimulates a robust immune response against HPV(+) tumors, opening new opportunities for virus-specific therapies in the treatment of HPV-related cancers.

Spatially fractionated GRID radiation potentiates immune-mediated tumor control.

BACKGROUND: Tumor-immune interactions shape a developing tumor and its tumor immune microenvironment (TIME) resulting in either well-infiltrated, immunologically inflamed tumor beds, or immune deserts with low levels of infiltration. The pre-treatment immune make-up of the TIME is associated with treatment outcome; immunologically inflamed tumors generally exhibit better responses to radio- and immunotherapy than non-inflamed tumors. However, radiotherapy is known to induce opposing immunological consequences, resulting in both immunostimulatory and inhibitory responses. In fact, it is thought that the radiation-induced tumoricidal immune response is curtailed by subsequent applications of radiation. It is thus conceivable that spatially fractionated radiotherapy (SFRT), administered through GRID blocks (SFRT-GRID) or lattice radiotherapy to create areas of low or high dose exposure, may create protective reservoirs of the tumor immune microenvironment, thereby preserving anti-tumor immune responses that are pivotal for radiation success. METHODS: We have developed an agent-based model (ABM) of tumor-immune interactions to investigate the immunological consequences and clinical outcomes after 2 Gy × 35 whole tumor radiation therapy (WTRT) and SFRT-GRID. The ABM is conceptually calibrated such that untreated tumors escape immune surveillance and grow to clinical detection. Individual ABM simulations are initialized from four distinct multiplex immunohistochemistry (mIHC) slides, and immune related parameter rates are generated using Latin Hypercube Sampling. RESULTS: In silico simulations suggest that radiation-induced cancer cell death alone is insufficient to clear a tumor with WTRT. However, explicit consideration of radiation-induced anti-tumor immunity synergizes with radiation cytotoxicity to eradicate tumors. Similarly, SFRT-GRID is successful with radiation-induced anti-tumor immunity, and, for some pre-treatment TIME compositions and modeling parameters, SFRT-GRID might be superior to WTRT in providing tumor control. CONCLUSION: This study demonstrates the pivotal role of the radiation-induced anti-tumor immunity. Prolonged fractionated treatment schedules may counteract early immune recruitment, which may be protected by SFRT-facilitated immune reservoirs. Different biological responses and treatment outcomes are observed based on pre-treatment TIME composition and model parameters. A rigorous analysis and model calibration for different tumor types and immune infiltration states is required before any conclusions can be drawn for clinical translation.

Expansion of tumor-infiltrating and marrow-infiltrating lymphocytes from pediatric malignant solid tumors.

INTRODUCTION: The expansion of tumor-infiltrating lymphocytes (TIL) for adoptive cellular therapy is under investigation in many solid tumors of adulthood. Marrow-infiltrating lymphocytes (MIL) have demonstrated antitumor reactivity preclinically. Successful expansion of TIL/MIL has not been reported across pediatric solid tumor histologies. The objective of this study was to demonstrate successful expansion of TIL from pediatric solid tumors for translation in an adoptive cell therapy (ACT) treatment strategy. METHODS: A prospective study of TIL/MIL expansion was performed on solid tumors of pediatric patients undergoing standard-of-care procedures. TIL/MIL expansions were performed in the presence of high-dose interleukin 2. To demonstrate a full-scale expansion to clinically-relevant cell doses for TIL therapy, initial TIL culture was followed by a rapid expansion protocol for select patients. Expanded specimens were analyzed for phenotype by flow cytometry and for anti-tumor reactivity by the interferon-gamma release assay. RESULTS: Eighteen tumor samples were obtained. Initial TIL cultures were successfully generated from 14/18 samples (77.7%). A median of 5.52 × 107 (range: 2.5 × 106-3.23 × 108) cells were produced from initial cultures, with 46.9% expressing a CD3 phenotype (46.9%). Eight samples underwent rapid expansion, demonstrating a median 458-fold expansion and a CD3 phenotype of 98%. Initial MIL cultures were successfully generated from five samples, with a predominantly CD3 phenotype (45.2%). Sufficient tumor tissue was only available for seven TIL samples to be tested for reactivity; none demonstrated responsiveness to autologous tumor. CONCLUSIONS: TIL and MIL expansion from pediatric solid tumors was successful, including the full-scale expansion process. This data supports translation to an ACT-TIL treatment strategy in the pediatric population and thus a Phase I trial of ACT-TIL in pediatric high-risk solid tumors is planned.

Tumor-infiltrating lymphocytes: A new hope.

Tumor-infiltrating lymphocytes (TILs) can be massively expanded from resected tumors and used as a cellular treatment for advanced malignancies. TILs require a preparative non-myeloablative chemotherapy followed by an abbreviated course of interleukin-2. Here, we review the historical development of TIL therapy and discuss potential solutions to ongoing roadblocks that may result in broader and improved efficacy for patients afflicted with treatment-refractory, advanced cancer.

Accelerating the development of genetically engineered cellular therapies: a framework for extrapolating data across related products.

BACKGROUND: Significant advancements have been made in the field of cellular therapy as anti-cancer treatments, with the approval of chimeric antigen receptor (CAR)-T cell therapies and the development of other genetically engineered cellular therapies. CAR-T cell therapies have demonstrated remarkable clinical outcomes in various hematological malignancies, establishing their potential to change the current cancer treatment paradigm. Due to the increasing importance of genetically engineered cellular therapies in the oncology treatment landscape, implementing strategies to expedite development and evidence generation for the next generation of cellular therapy products can have a positive impact on patients. METHODS: We outline a risk-based methodology and assessment aid for the data extrapolation approach across related genetically engineered cellular therapy products. This systematic data extrapolation approach has applicability beyond CAR-T cells and can influence clinical development strategies for a variety of immune therapies such as T cell receptor (TCR) or genetically engineered and other cell-based therapies (e.g., tumor infiltrating lymphocytes, natural killer cells and macrophages). RESULTS: By analyzing commonalities in manufacturing processes, clinical trial designs, and regulatory considerations, key learnings were identified. These insights support optimization of the development and regulatory approval of novel cellular therapies. CONCLUSIONS: The field of cellular therapy holds immense promise in safely and effectively treating cancer. The ability to extrapolate data across related products presents opportunities to streamline the development process and accelerate the delivery of novel therapies to patients.

First-in-Human Stage III/IV Melanoma Clinical Trial of Immune Priming Agent IFx-Hu2.0.

IFx-Hu2.0 was designed to encode part of the Emm55 protein contained within a plasmid in a formulation intended for transfection into mammalian cells. IFx-Hu2.0 promotes both adaptive and innate immune responses in animal studies. Furthermore, previous studies have demonstrated safety/efficacy in equine, canine, and murine species. We present the first-in-human study of IFx-Hu2.0, administered by intralesional injection into melanoma tumors of seven patients with stage III/IV unresectable melanoma. No dose-limiting toxicities attributable to IFx-Hu2.0 were observed. Grade 1/2 injection site reactions were observed in five of seven patients. IgG and IgM responses to Emm55 peptides and known melanoma antigens were seen in the peripheral blood, suggesting that IFx-Hu2.0 acts as an individualized "in situ vaccine." Three of four patients previously refractory to anti-PD1 experienced clinical benefit upon subsequent anti-PD1-based treatment. Therefore, this approach is feasible, and clinical/correlative outcomes warrant further investigation for treating patients with metastatic melanoma with an immune priming agent.

Histology-guided mathematical model of tumor oxygenation: sensitivity analysis of physical and computational parameters.

A hybrid off-lattice agent-based model has been developed to reconstruct the tumor tissue oxygenation landscape based on histology images and simulated interactions between vasculature and cells with microenvironment metabolites. Here, we performed a robustness sensitivity analysis of that model's physical and computational parameters. We found that changes in the domain boundary conditions, the initial conditions, and the Michaelis constant are negligible and, thus, do not affect the model outputs. The model is also not sensitive to small perturbations of the vascular influx or the maximum consumption rate of oxygen. However, the model is sensitive to large perturbations of these parameters and changes in the tissue boundary condition, emphasizing an imperative aim to measure these parameters experimentally.

L-DOS47 Elevates Pancreatic Cancer Tumor pH and Enhances Response to Immunotherapy.

Acidosis is an important immunosuppressive mechanism that leads to tumor growth. Therefore, we investigated the neutralization of tumor acidity to improve immunotherapy response. L-DOS47, a new targeted urease immunoconjugate designed to neutralize tumor acidity, has been well tolerated in phase I/IIa trials. L-DOS47 binds to CEACAM6, a cell-surface protein that is highly expressed in gastrointestinal cancers, allowing urease to cleave endogenous urea into two NH4+ and one CO2, thereby raising local pH. To test the synergetic effect of neutralizing tumor acidity with immunotherapy, we developed a pancreatic orthotopic murine tumor model (KPC961) expressing human CEACAM6. Using chemical exchange saturation transfer-magnetic resonance imaging (CEST-MRI) to measure the tumor extracellular pH (pHe), we confirmed that L-DOS47 raises the tumor pHe from 4 h to 96 h post injection in acidic tumors (average increase of 0.13 units). Additional studies showed that combining L-DOS47 with anti-PD1 significantly increases the efficacy of the anti-PD1 monotherapy, reducing tumor growth for up to 4 weeks.

Neoantigen-specific CD4+ tumor-infiltrating lymphocytes are potent effectors identified within adoptive cell therapy products for metastatic melanoma patients.

BACKGROUND: Adoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TILs) is a promising immunotherapeutic approach for patients with advanced solid tumors. While numerous advances have been made, the contribution of neoantigen-specific CD4+T cells within TIL infusion products remains underexplored and therefore offers a significant opportunity for progress. METHODS: We analyzed infused TIL products from metastatic melanoma patients previously treated with ACT for the presence of neoantigen-specific T cells. TILs were enriched on reactivity to neoantigen peptides derived and prioritized from patient sample-directed mutanome analysis. Enriched TILs were further investigated to establish the clonal neoantigen response with respect to function, transcriptomics, and persistence following ACT. RESULTS: We discovered that neoantigen-specific TIL clones were predominantly CD4+ T cells and were present in both therapeutic responders and non-responders. CD4+ TIL demonstrated an effector T cell response with cytotoxicity toward autologous tumor in a major histocompatibility complex class II-dependent manner. These results were validated by paired TCR and single cell RNA sequencing, which elucidated transcriptomic profiles distinct to neoantigen-specific CD4+ TIL. CONCLUSIONS: Despite methods which often focus on CD8+T cells, our study supports the importance of prospective identification of neoantigen-specific CD4+ T cells within TIL products as they are a potent source of tumor-specific effectors. We further advocate for the inclusion of neoantigen-specific CD4+ TIL in future ACT protocols as a strategy to improve antitumor immunity.

Targeting myeloid-derived suppressor cells with gemcitabine to enhance efficacy of adoptive cell therapy in bladder cancer.

Background: New therapeutics in development for bladder cancer need to address the recalcitrant nature of the disease. Intravesical adoptive cell therapy (ACT) with tumor infiltrating lymphocytes (TIL) can potentially induce durable responses in bladder cancer while maximizing T cells at the tumor site. T cells infused into the bladder directly encounter immunosuppressive populations, such as myeloid derived suppressor cells (MDSCs), that can attenuate T cell responses. Intravesical instillation of gemcitabine can be used as a lymphodepleting agent to precondition the bladder microenvironment for infused T cell products. Methods: Urine samples from bladder cancer patients and healthy donors were analyzed by flow cytometry and cytometric bead array for immune profiling and cytokine quantification. MDSCs were isolated from the urine and cocultured with stimulated T cells to assess effects on proliferation. An orthotopic murine model of bladder cancer was established using the MB49-OVA cell line and immune profiling was performed. MDSCs from tumor-bearing mice were cocultured with OT-I splenocytes to assess T cell proliferation. Mice received intravesical instillation of gemcitabine and depletion of immune cells was measured via flow cytometry. Bladder tumor growth of mice treated with intravesical gemcitabine, OT-I transgenic T cells, or combination was monitored via ultrasound measurement. Results: In comparison to healthy donors, urine specimen from bladder cancer patients show high levels of MDSCs and cytokines associated with myeloid chemotaxis, T cell chemotaxis, and inflammation. T cells isolated from healthy donors were less proliferative when cocultured with MDSCs from the urine. Orthotopic murine bladder tumors also presented with high levels of MDSCs along with enrichment of cytokines found in the patient urine samples. MDSCs isolated from spleens of tumor-bearing mice exerted suppressive effects on the proliferation of OT-I T cells. Intravesical instillation of gemcitabine reduced overall immune cells, MDSCs, and T cells in orthotopic bladder tumors. Combination treatment with gemcitabine and OT-I T cells resulted in sustained anti-tumor responses in comparison to monotherapy treatments. Conclusion: MDSCs are enriched within the microenvironment of bladder tumors and are suppressive to T cells. Gemcitabine can be used to lymphodeplete bladder tumors and precondition the microenvironment for intravesical ACT.

L-DOS47 enhances response to immunotherapy in pancreatic cancer tumor.

Acidosis is an important immunosuppressive mechanism that leads to tumor growth. Therefore, we investigated the neutralization of tumor acidity to improve immunotherapy response. L-DOS47, a new targeted urease immunoconjugate designed to neutralize tumor acidity, has been well tolerated in phase I/IIa trials. L-DOS47 binds CEACAM6, a cell surface protein highly expressed in gastrointestinal cancers, allowing urease to cleave endogenous urea into two NH4+ and one CO2, thereby raising local pH. To test the synergetic effect of neutralizing tumor acidity with immunotherapy, we developed a pancreatic orthotopic murine tumor model (KPC961) expressing human CEACAM6. Our results demonstrate that combining L DOS47 with anti-PD1 significantly increases the efficacy of anti-PD1 monotherapy, reducing tumor growth for up to 4 weeks.

SARS-CoV-2 antibody response duration and neutralization following natural infection.

Background: The role of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) neutralizing antibody response from natural infection and vaccination, and the potential determinants of this response are poorly understood. Characterizing this antibody response and the factors associated with neutralization can help inform future prevention efforts and improve clinical outcomes in those infected. Objectives: The goals of this study were to prospectively evaluate SARS-CoV-2 antibody levels and the neutralizing antibody responses among naturally infected adults and to determine demographic and behavioral factors independently associated with these responses. Methods: Serum was collected from seropositive individuals at baseline, four-weeks, and three-months following their first study visit to be evaluated for antibody levels. Detection of neutralizing antibodies was performed at baseline. Participant demographic and behavioral information was collected via web questionnaire prior to their first visit. Results: At baseline, higher antibody levels were associated with better neutralization capacity, with 83% of participants having detectable neutralizing antibodies. We found an age-dependent effect on antibody level and neutralization capacity with participants over 65 years having significantly higher levels. Ethnicity, heart disease, autoimmune disease, and COVID symptoms were associated with higher antibody levels, but not with increased neutralization capacity. Work environment during the pandemic correlated with increased neutralization capacity, while kidney or liver disease and traveling out of state after February 2020 correlated with decreased neutralization capacity, however neither correlated with antibody levels. Conclusions: Our data show that natural infection by SARS-CoV-2 can induce a humoral response reflected by high antibody levels and neutralization capacity.

Modulating the polyamine/hypusine axis controls generation of CD8+ tissue-resident memory T cells.

Glutaminolysis is a hallmark of the activation and metabolic reprogramming of T cells. Isotopic tracer analyses of antigen-activated effector CD8+ T cells revealed that glutamine is the principal carbon source for the biosynthesis of polyamines putrescine, spermidine, and spermine. These metabolites play critical roles in activation-induced T cell proliferation, as well as for the production of hypusine, which is derived from spermidine and is covalently linked to the translation elongation factor eukaryotic translation initiation factor 5A (eIF5A). Here, we demonstrated that the glutamine/polyamine/hypusine axis controlled the expression of CD69, an important regulator of tissue-resident memory T cells (Trm). Inhibition of this circuit augmented the development of Trm cells ex vivo and in vivo in the BM, a well-established niche for Trm cells. Furthermore, blocking the polyamine/hypusine axis augmented CD69 expression as well as IFN-γ and TNF-α production in (a) human CD8+ T cells from peripheral blood and sarcoma tumor infiltrating lymphocytes and (b) human CD8+ CAR-T cells. Collectively, these findings support the notion that the polyamine-hypusine circuit can be exploited to modulate Trm cells for therapeutic benefit.

Safety and immunogenicity of a third dose of mRNA-1273 vaccine among cancer patients.

BACKGROUND: Compared to the general population, cancer patients are at higher risk of morbidity and mortality following SARS-CoV-2 infection. The immune response to a two-dose regimen of mRNA vaccines in cancer patients is generally lower than in immunocompetent individuals. Booster doses may meaningfully augment immune response in this population. We conducted an observational study with the primary objective of determining the immunogenicity of vaccine dose three (100 µg) of mRNA-1273 among cancer patients and a secondary objective of evaluating safety at 14 and 28 days. METHODS: The mRNA-1273 vaccine was administered ∼7 to 9 months after administering two vaccine doses (i.e., the primary series). Immune responses (enzyme-linked immunosorbent assay [ELISA]) were assessed 28 days post-dose three. Adverse events were collected at days 14 (± 5) and 28 (+5) post-dose three. Fisher exact or X2 tests were used to compare SARS-CoV-2 antibody positivity rates, and paired t-tests were used to compare SARS-CoV-2 antibody geometric mean titers (GMTs) across different time intervals. RESULTS: Among 284 adults diagnosed with solid tumors or hematologic malignancies, dose three of mRNA-1273 increased the percentage of patients seropositive for SARS-CoV-2 antibody from 81.7% pre-dose three to 94.4% 28 days post-dose three. GMTs increased 19.0-fold (15.8-22.8). Patients with lymphoid cancers or solid tumors had the lowest and highest antibody titers post-dose three, respectively. Antibody responses after dose three were reduced among those who received anti-CD20 antibody treatment, had lower total lymphocyte counts and received anticancer therapy within 3 months. Among patients seronegative for SARS-CoV-2 antibody pre-dose three, 69.2% seroconverted after dose three. A majority (70.4%) experienced mostly mild, transient adverse reactions within 14 days of dose three, whereas severe treatment-emergent events within 28 days were very rare (<2%). CONCLUSION: Dose three of the mRNA-1273 vaccine was well-tolerated and augmented SARS-CoV-2 seropositivity in cancer patients, especially those who did not seroconvert post-dose two or whose GMTs significantly waned post-dose two. Lymphoid cancer patients experienced lower humoral responses to dose three of the mRNA-1273 vaccine, suggesting that timely access to boosters is important for this population.

Epigenetic state determines the in vivo efficacy of STING agonist therapy.

While STING-activating agents have shown limited efficacy in early-phase clinical trials, multiple lines of evidence suggest the importance of tumor cell-intrinsic STING function in mediating antitumor immune responses. Although STING signaling is impaired in human melanoma, its restoration through epigenetic reprogramming can augment its antigenicity and T cell recognition. In this study, we show that reversal of methylation silencing of STING in murine melanoma cell lines using a clinically available DNA methylation inhibitor can improve agonist-induced STING activation and type-I IFN induction, which, in tumor-bearing mice, can induce tumor regression through a CD8+ T cell-dependent immune response. These findings not only provide mechanistic insight into how STING signaling dysfunction in tumor cells can contribute to impaired responses to STING agonist therapy, but also suggest that pharmacological restoration of STING signaling through epigenetic reprogramming might improve the therapeutic efficacy of STING agonists.

Fucosylation of HLA-DRB1 regulates CD4+ T cell-mediated anti-melanoma immunity and enhances immunotherapy efficacy.

Immunotherapy efficacy is limited in melanoma, and combinations of immunotherapies with other modalities have yielded limited improvements but also adverse events requiring cessation of treatment. In addition to ineffective patient stratification, efficacy is impaired by paucity of intratumoral immune cells (itICs); thus, effective strategies to safely increase itICs are needed. We report that dietary administration of L-fucose induces fucosylation and cell surface enrichment of the major histocompatibility complex (MHC)-II protein HLA-DRB1 in melanoma cells, triggering CD4+ T cell-mediated increases in itICs and anti-tumor immunity, enhancing immune checkpoint blockade responses. Melanoma fucosylation and fucosylated HLA-DRB1 associate with intratumoral T cell abundance and anti-programmed cell death protein 1 (PD1) responder status in patient melanoma specimens, suggesting the potential use of melanoma fucosylation as a strategy for stratifying patients for immunotherapies. Our findings demonstrate that fucosylation is a key mediator of anti-tumor immunity and, importantly, suggest that L-fucose is a powerful agent for safely increasing itICs and immunotherapy efficacy in melanoma.

Intraperitoneal Delivery of Iopamidol to Assess Extracellular pH of Orthotopic Pancreatic Tumor Model by CEST-MRI.

The extracellular pH (pHe) of solid tumors is often acidic, as a consequence of the Warburg effect, and an altered metabolic state is often associated with malignancy. It has been shown that acidosis can promote tumor progression; thus, many therapeutic strategies have been adopted against tumor metabolism; one of these involves alkalinization therapies to raise tumor pH to inhibit tumor progression, improve immune surveillance, and overcome resistance to chemotherapies. Chemical exchange saturation transfer-magnetic resonance imaging (CEST-MRI) is a noninvasive technique that can measure pH in vivo using pH-sensitive contrast agents. Iopamidol, an iodinated contrast agent, clinically used for computed tomography (CT), contains amide group protons with pH-dependent exchange rates that can reveal the pHe of the tumor microenvironment. In this study, we optimized intraperitoneal (IP) delivery of iopamidol to facilitate longitudinal assessments of orthotopic pancreatic tumor pHe by CEST-MRI. Following IV-infusion and IP-bolus injections, we compared the two protocols for assessing tumor pH. Time-resolved CT imaging was used to evaluate the uptake of iopamidol in the tumor, revealing that IP-bolus delivered a high amount of contrast agent 40 min postinjection, which was similar to the amounts reached with the IV-infusion protocol. As expected, both IP and IV injection protocols produced comparable measurements of tumor pHe, showing no statistically significant difference between groups (p=0.16). In addition, we showed the ability to conduct longitudinal monitoring of tumor pHe using CEST-MRI with the IP injection protocol, revealing a statistically significant increase in tumor pHe following bicarbonate administration (p < 0.001). In conclusion, this study shows the capability to measure pHe using an IP delivery of iopamidol into orthotopic pancreatic tumors, which is important to conduct longitudinal studies.

Branched-chain keto acids promote an immune-suppressive and neurodegenerative microenvironment in leptomeningeal disease.

Leptomeningeal disease (LMD) occurs when tumors seed into the leptomeningeal space and cerebrospinal fluid (CSF), leading to severe neurological deterioration and poor survival outcomes. We utilized comprehensive multi-omics analyses of CSF from patients with lymphoma LMD to demonstrate an immunosuppressive cellular microenvironment and identified dysregulations in proteins and lipids indicating neurodegenerative processes. Strikingly, we found a significant accumulation of toxic branched-chain keto acids (BCKA) in the CSF of patients with LMD. The BCKA accumulation was found to be a pan-cancer occurrence, evident in lymphoma, breast cancer, and melanoma LMD patients. Functionally, BCKA disrupted the viability and function of endogenous T lymphocytes, chimeric antigen receptor (CAR) T cells, neurons, and meningeal cells. Treatment of LMD mice with BCKA-reducing sodium phenylbutyrate significantly improved neurological function, survival outcomes, and efficacy of anti-CD19 CAR T cell therapy. This is the first report of BCKA accumulation in LMD and provides preclinical evidence that targeting these toxic metabolites improves outcomes.

Neutralizing Antibody Response following a Third Dose of the mRNA-1273 Vaccine among Cancer Patients.

Cancer patients are at an increased risk of morbidity and mortality from SARS-CoV-2 infection and have a decreased immune response to vaccination. We conducted a study measuring both the neutralizing and total antibodies in cancer patients following a third dose of the mRNA-1273 COVID-19 vaccine. Immune responses were measured with an enzyme-linked immunosorbent assay (ELISA) and neutralization assays. Kruskal-Wallis tests were used to evaluate the association between patient characteristics and neutralization geometric mean titers (GMTs), and paired t-tests were used to compare the GMTs between different timepoints. Spearman correlation coefficients were calculated to determine the correlation between total antibody and neutralization GMTs. Among 238 adults diagnosed with cancer, a third dose of mRNA-1273 resulted in a 37-fold increase in neutralization GMT 28 days post-vaccination and maintained a 14.6-fold increase at 6 months. Patients with solid tumors or lymphoid cancer had the highest and lowest neutralization GMTs, respectively, at both 28 days and 6 months post-dose 3. While total antibody GMTs in lymphoid patients continued to increase, other cancer types showed decreases in titers between 28 days and 6 months post-dose 3. A strong correlation (p < 0.001) was found between total antibody and neutralization GMTs. The third dose of mRNA-1273 was able to elicit a robust neutralizing antibody response in cancer patients, which remained for 6 months after administration. Lymphoid cancer patients can benefit most from this third dose, as it was shown to continue to increase total antibody GMTs 6 months after vaccination.