Sparing Healthy Tissue Research Articles

Generating synthetic computed tomography for radiotherapy: SynthRAD2023 challenge report

Radiation therapy plays a crucial role in cancer treatment, necessitating precise delivery of radiation to tumors while sparing healthy tissues over multiple days. Computed tomography (CT) is integral for treatment planning, offering electron density data crucial for accurate dose calculations. However, accurately representing patient anatomy is challenging, especially in adaptive radiotherapy, where CT is not acquired daily. Magnetic resonance imaging (MRI) provides superior soft-tissue contrast. Still, it lacks electron density information, while cone beam CT (CBCT) lacks direct electron density calibration and is mainly used for patient positioning.Adopting MRI-only or CBCT-based adaptive radiotherapy eliminates the need for CT planning but presents challenges. Synthetic CT (sCT) generation techniques aim to address these challenges by using image synthesis to bridge the gap between MRI, CBCT, and CT. The SynthRAD2023 challenge was organized to compare synthetic CT generation methods using multi-center ground truth data from 1080 patients, divided into two tasks: (1) MRI-to-CT and (2) CBCT-to-CT. The evaluation included image similarity and dose-based metrics from proton and photon plans.The challenge attracted significant participation, with 617 registrations and 22/17 valid submissions for tasks 1/2. Top-performing teams achieved high structural similarity indices (≥0.87/0.90) and gamma pass rates for photon (≥98.1%/99.0%) and proton (≥97.3%/97.0%) plans. However, no significant correlation was found between image similarity metrics and dose accuracy, emphasizing the need for dose evaluation when assessing the clinical applicability of sCT.SynthRAD2023 facilitated the investigation and benchmarking of sCT generation techniques, providing insights for developing MRI-only and CBCT-based adaptive radiotherapy. It showcased the growing capacity of deep learning to produce high-quality sCT, reducing reliance on conventional CT for treatment planning.

A Comprehensive Review on Niosomes in Drug Delivery and Recent Advancements

The recent emphasis on nanocarrier development for drug delivery stems from the need to target specific diseased areas while sparing healthy tissues. Effective and safe drug administration has long posed challenges in medicine. Over the past decade, the emergence of vesicles as a means to enhance drug delivery has captivated researchers in the field of drug delivery systems. Among vesicular systems, niosomes have gained attention due to their nonionic features. Unlike liposomes, niosomes offer superior stability, making them a preferred choice. Non-ionic in nature niosomes offer unique advantages in drug delivery providing a versatile platform for encapsulating various drugs to enhance bioavailability and ensure controlled release. Understanding preparation techniques enables tailored applications from oral to transdermal delivery. Characterization methods such as morphology and particles size are pivotal in ensuring the stability and effectiveness of niosomes. Applications span cancer therapy, diagnostic imaging, and vaccination adjuvants, showcasing niosomes versatility. Ongoing research reflects dynamic efforts to enhance capabilities, emphasizing their pivotal role in evolving drug delivery systems. In this comprehensive review, we aim to encapsulate fundamental aspects of niosomes, encompassing diverse preparation methods, various niosomal types, methods for characterization and the advancements witnessed in niosomal research over the past decade, drawing insights from a literature review. Keywords: Niosomes, Drug delivery, Nanocarrier, Vesicles, Nanomedicines

Photothermal and radiotherapy with alginate-coated gold nanoparticles for breast cancer treatment

Radiation therapy and phototherapy are commonly used cancer treatments that offer advantages such as a low risk of adverse effects and the ability to target cancer cells while sparing healthy tissue. A promising strategy for cancer treatment involves using nanoparticles (NPs) in combination with radiation and photothermal therapy to target cancer cells and improve treatment efficacy. The synthesis of gold NPs (AuNPs) for use in biomedical applications has traditionally involved toxic reducing agents. Here we harnessed dopamine (DA)-conjugated alginate (Alg) for the facile and green synthesis of Au NPs (Au@Alg-DA NPs). Alg-DA conjugate reduced Au ions, simultaneously stabilized the resulting AuNPs, and prevented aggregation, resulting in particles with a narrow size distribution and improved stability. Injectable Au@Alg-DA NPs significantly promoted ROS generation in 4T1 breast cancer cells when exposed to X-rays. In addition, their administration raised the temperature under a light excitation of 808 nm, thus helping to destroy cancer cells more effectively. Importantly, no substantial cytotoxicity was detected in our Au@Alg-DA NPs. Taken together, our work provides a promising route to obtain an injectable combined radio enhancer and photothermally active nanosystem for further potential clinic translation.

Photodynamic priming modulates cellular ATP levels to overcome P-glycoprotein-mediated drug efflux in chemoresistant triple-negative breast cancer.

P-glycoprotein (P-gp, ABCB1) is a well-researched ATP-binding cassette (ABC) drug efflux transporter linked to the development of cancer multidrug resistance (MDR). Despite extensive studies, approved therapies to safely inhibit P-gp in clinical settings are lacking, necessitating innovative strategies beyond conventional inhibitors or antibodies to reverse MDR. Photodynamic therapy is a globally approved cancer treatment that uses targeted, harmless red light to activate non-toxic photosensitizers, confining its cytotoxic photochemical effects to disease sites while sparing healthy tissues. This study demonstrates that photodynamic priming (PDP), a sub-cytotoxic photodynamic therapy process, can inhibit P-gp function by modulating cellular respiration and ATP levels in light accessible regions. Using chemoresistant (VBL-MDA-MB-231) and chemosensitive (MDA-MB-231) triple-negative breast cancer cell lines, we showed that PDP decreases mitochondrial membrane potential by 54.4% ± 30.4 and reduces mitochondrial ATP production rates by 94.9% ± 3.46. Flow cytometry studies showed PDP can effectively improve the retention of P-gp substrates (calcein) by up to 228.4% ± 156.3 in chemoresistant VBL-MDA-MB-231 cells, but not in chemosensitive MDA-MB-231 cells. Further analysis revealed that PDP did not alter the cell surface expression level of P-gp in VBL-MDA-MB-231 cells. These findings indicate that PDP can reduce cellular ATP below the levels that is required for the function of P-gp and improve intracellular substrate retention. We propose that PDP in combination with chemotherapy drugs, might improve the efficacy of chemotherapy and overcome cancer MDR.

A phase I/IIa of [212Pb]VMT01 targeted a-particle therapy for unresectable or metastatic melanoma.

TPS9610 Background: Melanoma is an aggressive skin cancer with potential to metastasize early in disease development. Unresectable and metastatic melanoma accounts for 12% of cases and, despite recent advances in treatment, 5-year overall survival for this disease remains low at 35%. The melanocortin 1 receptor (MC1R) is highly expressed in melanoma with low expression in normal tissues and is therefore a target for radiopharmaceutical therapy. We developed [212Pb]VMT01, a MC1R-targeting peptide conjugated to a lead-specific chelator. Targeted therapy with 212Pb has the potential to be efficacious due to the high relative biological effectiveness of α-particles emitted upon its decay. The short physical half-life of 212Pb (10.6 h) and fast tumor targeting and clearance properties of [212Pb]VMT01 can achieve highly efficient delivery of α-particles to MC1R-expressing cancers while sparing healthy tissues. We hypothesize that [212Pb]VMT01 can be safely administered to patients with metastatic or unresectable melanoma and can enable disease control. Methods: This is an open-label, multi-center, dose-escalation/dose expansion, phase I/IIa clinical trial evaluating the safety, tolerability, pharmacokinetics, and efficacy of [212Pb]VMT01 (NCT05655312). Phase I of the trial follows a mTPI-2 design. The first two cohorts incorporate dosimetry, where participants receive up to 925 MBq or 278 MBq of the therapeutic surrogate [203Pb]VMT01 or [68Ga]VMT02, respectively, prior to receiving treatment cycles. In these participants, SPECT/CT or PET/CT imaging is obtained up to 24 hours post-injection. Up to 3 treatment cycles of [212Pb]VMT01, co-administered with renal protective amino acids, are given 8 weeks apart, with a dose-limiting toxicity observation period of 6 weeks. The cohort 1 dose was 111 MBq, while the ongoing cohort 2 utilizes 185 MBq. Eligibility criteria include MC1R+ disease as determined by SPECT or PET, melanoma stage III or IV and progressive disease by RECIST 1.1. Study participants must have progressed on at least one prior SOC therapy. As part of the study, CT and/or MRI, and FDG PET are acquired at baseline, during, and at the end of treatment. Safety is assessed weekly during cycle 1 and bi-weekly for subsequent cycles. The total in-trial follow-up period is 18 months following final administration of drug. Efficacy is assessed by RECIST 1.1 criteria. The primary objectives are evaluation of the safety of [212Pb]VMT01, determination of the maximum tolerated or maximum feasible dose, and determination of the anti-tumor efficacy of [212Pb]. All primary, secondary, and exploratory objectives’ data will be collected for all participants. An interim futility evaluation may be performed after ~10 participants in the dose escalation phase for participants who have completed their first post-treatment response assessment. The trial is open for recruitment. Clinical trial information: NCT05655312 .

Indocyanine green (ICG)-guided robotic resection for liver adenoma: combined technologies for precision surgery.

HCA resection is crucial to prevent bleeding and malignant transformation. The aim of this study was to enhance the precision of tumor resection in hepatocellular adenoma (HCA) through the combination of intraoperative ultrasound (IOUS) and indocyanine green (ICG) fluorescence imaging. ICG was intravenously injected 24h before surgery, enabling positive staining of HCA nodules. IOUS guided the parenchymal transection performed using the RoboLap approach. IOUS combined with ICG effectively demarcated lesions, allowing precision surgery while sparing healthy liver tissue. Intraoperative frozen examination further validated the potential of ICG to identify previously undetected lesions. The study showed promising advantages of ICG in HCA resections, potentially reducing the risk of recurrence and malignant transformation. The combined robotic and laparoscopic approach improved the feasibility of parenchymal-sparing surgery, offering a cautious assessment of HCA lesions.

Helium Ion Therapy for Advanced Juvenile Nasopharyngeal Angiofibroma.

Helium ion therapy (HRT) is a promising modality for the treatment of pediatric tumors and those located close to critical structures due to the favorable biophysical properties of helium ions. This in silico study aimed to explore the potential benefits of HRT in advanced juvenile nasopharyngeal angiofibroma (JNA) compared to proton therapy (PRT). We assessed 11 consecutive patients previously treated with PRT for JNA in a definitive or postoperative setting with a relative biological effectiveness (RBE) weighted dose of 45 Gy (RBE) in 25 fractions at the Heidelberg Ion-Beam Therapy Center. HRT plans were designed retrospectively for dosimetric comparisons and risk assessments of radiation-induced complications. HRT led to enhanced target coverage in all patients, along with sparing of critical organs at risk, including a reduction in the brain integral dose by approximately 27%. In terms of estimated risks of radiation-induced complications, HRT led to a reduction in ocular toxicity, cataract development, xerostomia, tinnitus, alopecia and delayed recall. Similarly, HRT led to reduced estimated risks of radiation-induced secondary neoplasms, with a mean excess absolute risk reduction of approximately 30% for secondary CNS malignancies. HRT is a promising modality for advanced JNA, with the potential for enhanced sparing of healthy tissue and thus reduced radiation-induced acute and long-term complications.

CARTAR: a comprehensive web tool for identifying potential targets in chimeric antigen receptor therapies using TCGA and GTEx data.

Chimeric antigen receptor (CAR) therapy has emerged as a ground-breaking advancement in cancer treatment, harnessing the power of engineered human immune cells to target and eliminate cancer cells. The escalating interest and investment in CAR therapy in recent years emphasize its profound significance in clinical research, positioning it as a rapidly expanding frontier in the field of personalized cancer therapies. A crucial step in CAR therapy design is choosing the right target as it determines the therapy's effectiveness, safety and specificity against cancer cells, while sparing healthy tissues. Herein, we propose a suite of tools for the identification and analysis of potential CAR targets leveraging expression data from The Cancer Genome Atlas and Genotype-Tissue Expression Project, which are implemented in CARTAR website. These tools focus on pinpointing tumor-associated antigens, ensuring target selectivity and assessing specificity to avoid off-tumor toxicities and can be used to rationally designing dual CARs. In addition, candidate target expression can be explored in cancer cell lines using the expression data for the Cancer Cell Line Encyclopedia. To our best knowledge, CARTAR is the first website dedicated to the systematic search of suitable candidate targets for CAR therapy. CARTAR is publicly accessible at https://gmxenomica.github.io/CARTAR/.

Gamma-Delta (γδ) CAR-T Cells targeting CD33 and lacking the CD3ζ signaling domain enhance killing of AML cells without toxicity to normal hematopoietic cells

Abstract Extending CAR-T therapy to myeloid malignancies has proven challenging due to co-expression of targetable antigens on hematopoietic progenitors. We leveraged the enhanced targeting conferred by CAR-T technology with the broad stress-associated antigen recognition and killing capabilities of γδ T cells by employing a non-signaling CAR (nsCAR) that excludes the CD3ζ domain, thereby facilitating targeted tumor recognition while sparing healthy tissues. Jurkat cells expressing a CD3ζ+ CD33+ CAR showed significant upregulation of CD69 in co-culture with KG1a AML cells but nsCAR-expressing Jurkat cells did not upregulate CD69. A time-dependent reduction (43%) in the CD3ζ+CD33CAR+ Jurkat population was also noted over a 7-day coculture while the nsCAR+ Jurkat population remained stable, suggesting improved durability of the nsCAR modified Jurkat cells. nsCAR-expressing expanded and activated γδ T cells from healthy donors exhibited enhanced killing against AML lines HL-60 (1.3x) and K-562 (1.6x) compared to unmodified γδ T cells (UTD) in a 24-hour cytotoxicity assay. Incorporation of sIL-15 also increased killing (1.8x for HL-60, 2.6x for KG-1a, 2.0x for MOLM-13 2.0x for K-562). Minimal cytotoxicity (<10%) was observed for nsCARs or UTD γδ T cells against CD34+ HPCs. Our findings suggest that combining nsCAR constructs with γδ T cells may widen the therapeutic window to enable of CAR-T therapy for cancers with target antigen expression on healthy tissues.

Dosimetric study for breathing-induced motion effects in an abdominal pancreas phantom for carbon ion mini-beam radiotherapy.

Particle mini-beam therapy exhibits promise in sparing healthy tissue through spatial fractionation, particularly notable for heavy ions, further enhancing the already favorable differential biological effectiveness at both target and entrance regions. However, breathing-induced organ motion affects particle mini-beam irradiation schemes since the organ displacements exceed the mini-beam structure dimensions, decreasing the advantages of spatialfractionation. In this study, the impact of breathing-induced organ motion on the dose distribution was examined at the target and organs at risk(OARs) during carbon ion mini-beam irradiation for pancreaticcancer. As a first step, the carbon ion mini-beam pattern was characterized with Monte Carlo simulations. To analyze the impact of breathing-induced organ motion on the dose distribution of a virtual pancreas tumor as target and related OARs, the anthropomorphic Pancreas Phantom for Ion beam Therapy (PPIeT) was irradiated with carbon ions. A mini-beam collimator was used to deliver a spatially fractionated dose distribution. During irradiation, varying breathing motion amplitudes were induced, ranging from 5to 15mm. Post-irradiation, the 2D dose pattern was analyzed, focusing on the full width at half maximum (FWHM), center-to-center distance (ctc), and the peak-to-valley dose ratio (PVDR). The mini-beam pattern was visible within OARs, while in the virtual pancreas tumor a more homogeneous dose distribution was achieved. Applied motion affected the mini-beam pattern within the kidney, one of the OARs, reducing the PVDR from 3.78 0.12 to 1.478 0.070 for the 15mm motion amplitude. In the immobile OARs including the spine and the skin at the back, the PVDR did not change within 3.4% comparing reference and motionconditions. This study provides an initial understanding of how breathing-induced organ motion affects spatial fractionation during carbon ion irradiation, using an anthropomorphic phantom. A decrease in the PVDR was observed in the right kidney when breathing-induced motion was applied, potentially increasing the risk of damage to OARs. Therefore, further studies are needed to explore the clinical viability of mini-beam radiotherapy with carbon ions when irradiating abdominalregions.

Abstract LB042: A potent FGFR4-targeted antibody-drug conjugate therapy for patients with rhabdomyosarcoma and other cancers expressing FGFR4

Abstract Background Rhabdomyosarcoma (RMS) is the most common pediatric sarcoma, representing 3-4% of childhood and adolescent cancers. Although multimodal therapies have significantly improved the overall survival of patients with localized disease, 5-year overall survival for patients with relapsed and metastatic disease has remained dismal. Therefore, new therapies are desperately needed for this deadly disease. Antibody-drug conjugates (ADCs) combine the specificity of monoclonal antibodies with the cytotoxic potency of chemotherapy drugs to selectively deliver potent anticancer agents to tumor cells while sparing healthy tissues. FGFR4 is a cell-surface receptor tyrosine kinase highly expressed in RMS and other human cancers such as hepatocellular carcinoma (HCC) and some breast cancers. It is expressed at much lower levels in normal tissues, making it a promising immune target. We hypothesize that an FGFR4-targeted ADC would have potent activity against RMS and other cancers expressing FGFR4 with minimal toxicity toward normal tissues. Methods We developed high-affinity binders to human FGFR4 and determined their internalization ability in RMS cancer cell lines. Our top candidate 3A11, a murine monoclonal antibody, was conjugated to monomethyl auristatin E (MMAE), a potent cytotoxic agent, using a valine-citrulline-p-aminocarbamate linker (VC-PABC), to generate an FGFR4-targeted ADC (3A11-vc-MMAE). We tested the in-vitro efficacy of 3A11-vc-MMAE against multiple cell lines of RMS, triple-negative breast cancer, HCC, and FGFR4 negative controls using a live-cell analysis system Incucyte. Western blots were performed to validate 3A11-vc-MMAE’s mechanism of action. We finally tested the in vivo efficacy of 3A11-vc-MMAE in subcutaneous xenograft models of a fusion-positive RMS (RH4) and a fusion-negative RMS with an FGFR4 V550L activating mutation (RMS559). Results We showed that 3A11 was internalized by FGFR4-expressing cell lines and trafficked to lysosomes. Cell viability assays demonstrated that 3A11-vc-MMAE selectively killed FGFR4-expressing cells with minimal effect on low or non-expression cells. Western blot confirmed that 3A11-vc-MMAE induced apoptosis specifically in FGFR4-expressing RMS cell lines. Mice treated with one cycle of 3A11-vc-MMAE (3mg/kg twice a week for 2 weeks) demonstrated a significantly increased survival rate of 30% for the RH4 model with a median survival of 43 days for 3A11-vc-MMAE and 23 days for control, vehicle-treated mice (p-value = 0.0038), and 70% for the RMS559 model, with a median survival not reached for of 3A11-vc-MMAE treated compared with 23 days for control mice (p-value < 0.0001). Of note, tumors were eradicated in 4 out of 10 mice bearing RMS559 xenografts. Conclusions and Future Directions Our results demonstrate that the ADC 3A11-vc-MMAE provides a potent and specific precision immunotherapy for rhabdomyosarcoma and other cancers with a high expression of FGFR4, which warrants further development for clinical translation. Citation Format: Meijie Tian, Katrina Jia, Jerry T. Wu, Jun S. Wei, Adam T. Cheuk, Zhongmei Zhang, Eleanor G. Pope, Boris Shor, L. Nathan Tumey, Javed Khan. A potent FGFR4-targeted antibody-drug conjugate therapy for patients with rhabdomyosarcoma and other cancers expressing FGFR4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB042.

Robustness analysis of dynamic trajectory radiotherapy and volumetric modulated arc therapy plans for head and neck cancer

Background and purposeDynamic trajectory radiotherapy (DTRT) has been shown to improve healthy tissue sparing compared to volumetric arc therapy (VMAT). This study aimed to assess and compare the robustness of DTRT and VMAT treatment-plans for head and neck (H&N) cancer to patient-setup (PS) and machine-positioning uncertainties. Materials and methodsThe robustness of DTRT and VMAT plans previously created for 46 H&N cases, prescribed 50–70 Gy to 95 % of the planning-target-volume, was assessed. For this purpose, dose distributions were recalculated using Monte Carlo, including uncertainties in PS (translation and rotation) and machine-positioning (gantry-, table-, collimator-rotation and multi-leaf collimator (MLC)). Plan robustness was evaluated by the uncertainties’ impact on normal tissue complication probabilities (NTCP) for xerostomia and dysphagia and on dose-volume endpoints. Differences between DTRT and VMAT plan robustness were compared using Wilcoxon matched-pair signed-rank test (α = 5 %). ResultsAverage NTCP for moderate-to-severe xerostomia and grade ≥ II dysphagia was lower for DTRT than VMAT in the nominal scenario (0.5 %, p = 0.01; 2.1 %, p < 0.01) and for all investigated uncertainties, except MLC positioning, where the difference was not significant. Average differences compared to the nominal scenario were ≤ 3.5 Gy for rotational PS (≤ 3°) and machine-positioning (≤ 2°) uncertainties, <7 Gy for translational PS uncertainties (≤ 5 mm) and < 20 Gy for MLC-positioning uncertainties (≤ 5 mm). ConclusionsDTRT and VMAT plan robustness to the investigated uncertainties depended on uncertainty direction and location of the structure-of-interest to the target. NTCP remained on average lower for DTRT than VMAT even when considering uncertainties.

Abstract 5934: Ferroptosis induction therapy for castration resistant prostate cancer

Abstract Prostate cancer (PCa) is the most common non-skin cancer in men, and second leading cause of death. The fundamental treatment for treating PCa is androgen deprivation therapy which is an effective form of treatment initially. Nevertheless, the disease reoccurs in most men; this is referred to as castration-resistant PCa (CRPC), for which the standard treatment is enzalutamide. However, some develop resistance to this enzalutamide anti-androgen agent. Ferroptosis is an iron-dependent lipid peroxidation form of regulated cell death being investigated as a novel potential therapy for PCa using ferroptosis inducers such as RSL3; they have shown promise in selectively triggering cell death in cancer cells while sparing healthy tissues. We hypothesize that RSL3- induced ferroptosis will overcome enzalutamide resistance in PCa cells. To test this, we utilized C4-2B MDVR cells which represents a cell model for aggressive PCa that metastasized to the bone and is resistant to both castration therapy and the enzalutamide drug treatment. C42B-MDVR cells were treated with increasing concentrations of RSL3 followed by analysis of cell viability with MTS assay. A dose dependent reduction in cell viability was observed which was reversed by ferroptosis inhibitor, ferrostatin-1. Western blot analysis revealed a decreased expression of the antioxidant enzyme glutathione peroxidase 4 (GPX4) with ferroptosis induction indicating a mechanism of the RSL3-induced cell death. The limitations to our research are that the research was performed in vitro and have not been tested on mouse models to visualize the effects of the treatments in animals. In conclusion, ferroptosis inducer RSL3 decreases GPX4 expression and decreases proliferation of PCa cells while ferrostatin-1 reverses the effects of the RSL3 treatment. Thus, ferroptosis inducers present a potential novel therapeutic approach for CRPC patients who have developed resistance to conventional androgen therapies. NIH NIGMS RISE 5R25GM058904, NIH/NIMHD U54MD007590; U54MD013376 Citation Format: Mojisoluwa Awolowo, Precious Elechi Dike, Valerie Odero-Marah. Ferroptosis induction therapy for castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5934.

Abstract 5227: Gamma-delta (γδ) CAR-T cells lacking the CD3ζ signaling domain enhance targeted killing of AML cells and preserve healthy tissues

Abstract Chimeric antigen receptor T cell (CAR-T) therapy has shown remarkable efficacy against B cell malignancies, offering hope to patients with limited treatment options. However, extending this therapy to myeloid malignancies and solid tumors has proven challenging due to co-expression of targetable antigens on hematopoietic progenitors and healthy tissues. In this context, γδ T cells show promise, as they can directly identify and eliminate malignant cells via recognition of multiple tumor-associated stress antigens rarely expressed on normal tissues. We leveraged the tumor-sensing capabilities of γδ T cells with enhanced tumor localization by employing a non-signaling CAR (nsCAR) that excludes the CD3ζ domain, facilitating targeted tumor cell killing while sparing healthy tissues. We designed second-generation lentiviral constructs for anti-CD33-scfv for in vitro evaluation against acute myeloid leukemia (AML) lines HL-60, KG-1a, and MOLM-13 and the chronic myeloid leukemia (CML) line K-562 Additionally, we tested CD33/CD123 dual-targeting nsCAR constructs (ns-dCAR) to determine if the addition of CD123 targeting enhanced the therapeutic index. We also tested whether a secreted IL-15 (sIL-15) would enhance CAR-T cell fitness. We observed significant upregulation of CD69 in Jurkat cells transduced with signaling CARs in co-culture with KG-1a cells but not in those with nsCAR constructs. We also noticed a time-dependent reduction (43%) in the CD3ζ+ CD33CAR+ population over a 7-day extended coculture while the nsCAR+ Jurkat population remained stable, suggesting potential mitigation of activation-induced cell death (AICD). Expanded and activated γδ T cells from healthy donors were then transduced with nsCAR lentiviral vectors. The ns33CAR+ γδ T cells exhibited enhanced killing capability against HL-60 (up to 1.3x) and K-562 (up to 1.6x) compared to unmodified γδ T cells (UTD) in a 24-hour cytotoxicity assay. Incorporation of sIL-15 into the nsCD33 CAR construct also increased killing across all 4 cell lines (up to 1.8x for HL-60, up to 2.6x for KG-1a, up to 2.0x for MOLM-13 and up to 2.0x for K-562). Minimal cytotoxicity (&amp;lt;10%) was observed for nsCARs or UTDs against normal CD33+/CD123+ cells from healthy donor PBMC or CD34+ HPSCs. The ns-dCAR constructs did not improve in vitro AML killing compared to ns33CAR alone, although ns-dCARs generally exhibited lower transduction efficiency. In summary, our findings suggest that combining nsCAR constructs with γδ T cells may widen the therapeutic window to expand the reach of CAR-T therapy to cancers with limited target antigen expression on critical healthy tissues. Further optimization to improve integration and incorporation of membrane-bound IL-15 co-expression holds the potential to enhance both the efficacy and safety profiles of next-generation adoptive cell therapies against a broader spectrum of cancers. Citation Format: Lei Ding, Yanjie Li, Jiexue Li, Mariska Ter Haak, Lawrence Lamb. Gamma-delta (γδ) CAR-T cells lacking the CD3ζ signaling domain enhance targeted killing of AML cells and preserve healthy tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5227.

Abstract 3193: Utilizing a pH sensitive peptide (pHLIP) for antigen-directed T cell therapy

Abstract Many targeted therapies, such as antibody drug conjugates (ADCs) and chimeric antigen receptor (CAR) T cell therapies, rely on the identification of an optimal antigen as the cell marker for targeting cancer cells. However, there is a paucity of antigen candidates that are sufficiently specific to epithelial cancers. Therefore, technologies that can mark tumor cells for immune recognition could prove beneficial in the treatment of these solid cancers. pH low insertion peptides (pHLIPs) act as a unique delivery platform that can specifically target the acidic microenvironment of tumors, sparing healthy tissue in the process. We developed a pHLIP-peptide conjugate (pHLIP-SIINFEKL) to deliver the SIINFEKL peptide, an immunogenic fragment of ovalbumin, to investigate antigen delivery and recognition to tumors in vivo. When processed intracellularly, SIINFEKL is presented for immune recognition through the major histocompatibility complex (MHC) class I pathway. We observed selective delivery of pHLIP-SIINFEKL both in vitro and in vivo using fluorescently labeled constructs. In vitro, treatment of melanoma tumor cells with pHLIP-SIINFEKL resulted in recognition by SIINFEKL-specific T cells (OT1), leading to T cell activation and effector function. Mechanistically, we show that this recognition by OT1 cells was abrogated by siRNA knockdown of the β2 microglobulin (β2m) component of MHC class I in the target tumor cells, indicating that an intact antigen processing pathway in the cancer cells is necessary to mediate the effect of pHLIP-directed SIINFEKL delivery. In vivo, pHLIP-SIINFEKL treatment of tumor-bearing mice resulted in recruitment of OT1 T cells and suppression of tumor growth in two syngeneic tumor models in immunocompetent mice, with no effect when mutating either the pHLIP or SIINFEKL components of the conjugate. These results suggest that pHLIP-mediated peptide delivery can provide a novel approach to decorate solid tumors with engineered antigens for enhanced immune recognition and anti-tumor efficacy. Citation Format: Annali M. Yurkevicz, Yanfeng Liu, Peter M. Glazer. Utilizing a pH sensitive peptide (pHLIP) for antigen-directed T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3193.

Abstract 1216: Pan-RAS inhibition by a tumor-targeted biotherapeutic

Abstract Oncogenic RAS signaling drives tumor growth in about 30% of human malignancies. In recent years the first mutant-specific inhibitors of KRAS G12C (sotorasib and adagrasib) entered the clinic. While some durable responses were noted in KRAS-mutant non-small cell lung cancer, the clinical experience of KRAS G12C inhibitors is marked by rapid drug resistance, either intrinsic or acquired, causing re-activation of the MAPK pathway. Common resistance mechanisms include compensatory signaling through the related RAS isoforms NRAS and HRAS, as well as the selection of non-G12C KRAS mutations, among others. The bacterial peptidase RRSP potently cleaves all RAS mutants and isoforms in the conserved Switch I region. We have shown that receptor-mediated intracellular delivery of RRSP using an engineered protein delivery system (RASx) can inactivate all RAS signaling in a cell, causing apoptosis in RAS-addicted tumor cells in vitro and in vivo. Since systemic pan-RAS inhibition is not likely to be tolerated therapeutically, precise tumor targeting is a necessary feature of a true pan-RAS inhibitor. Here, we describe the unique ability of the RASx system to specify the cell surface receptor used by RRSP to enter cells, allowing exquisite targeting and killing of KRAS, NRAS and HRAS-driven tumor cells while sparing healthy tissues. We screened a panel of patient-derived tumor organoids (PDOs), to demonstrate broad tumor cell killing across pancreatic and colorectal cancer-derived PDOs. 97% of KRAS mutant organoids were sensitive to RASx, independent of the specific mutation. Intriguingly, 46% of wildtype RAS PDOs were sensitive to RASx, including organoids with activating mutations in EGFR. In contrast, PDOs with mutations downstream of RAS such as BRAF V600E were resistant to RASx. Indeed, RASx is effective not only against RAS-addicted tumors, but also wildtype RAS tumors driven by upstream receptor tyrosine kinases, potentially expanding the clinical utility of this platform. RASx is a first-in-class, tumor-targeted pan-RAS inhibitor that represents a unique entry into the growing armamentarium against oncogenic RAS. Citation Format: Greg L. Beilhartz, Huazhu Liang, Molly Udaskin, Christine Ng, Nikolina Radulovich, Roman A. Melnyk. Pan-RAS inhibition by a tumor-targeted biotherapeutic [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1216.

High dose proton and photon-based radiation therapy for 213 liver lesions: a multi-institutional dosimetric comparison with a clinical perspective

BackgroundStereotactic radiotherapy (SRT) and Proton therapy (PT) are both options in the management of liver lesions. Limited clinical-dosimetric comparison are available. Moreover, dose-constraint routinely used in liver PT and SRT considers only the liver spared, while optimization strategies to limit the liver damaged are poorly reported.MethodsPrimary endpoint was to assess and compare liver sparing of four contemporary RT techniques. Secondary endpoints were freedom from local recurrence (FFLR), overall survival (OS), acute and late toxicity. We hypothesize that Focal Liver Reaction (FLR) is determined by a similar biologic dose. FLR was delineated on follow-up MRI. Mean C.I. was computed for all the schedules used. A so-called Fall-off Volume (FOV) was defined as the area of healthy liver (liver-PTV) receiving more than the isotoxic dose. Fall-off Volume Ratio (FOVR) was defined as ratio between FOV and PTV.Results213 lesions were identified. Mean best fitting isodose (isotoxic doses) for FLR were 18Gy, 21.5 Gy and 28.5 Gy for 3, 5 and 15 fractions. Among photons, an advantage in terms of healthy liver sparing was found for Vmat FFF with 5mm jaws (p = 0.013) and Cyberknife (p = 0.03). FOV and FOVR resulted lower for PT (p < 0.001). Three years FFLR resulted 83%. Classic Radiation induced liver disease (RILD, any grade) affected 2 patients.ConclusionsCyberknife and V-MAT FFF with 5mm jaws spare more liver than V-MAT FF with 10 mm jaws. PT spare more liver compared to photons. FOV and FOVR allows a quantitative analysis of healthy tissue sparing performance showing also the quality of plan in terms of dose fall-off.

Photodynamic Therapy for Atherosclerosis.

Atherosclerosis, which currently contributes to 31% of deaths globally, is of critical cardiovascular concern. Current diagnostic tools and biomarkers are limited, emphasizing the need for early detection. Lifestyle modifications and medications form the basis of treatment, and emerging therapies such as photodynamic therapy are being developed. Photodynamic therapy involves a photosensitizer selectively targeting components of atherosclerotic plaques. When activated by specific light wavelengths, it induces localized oxidative stress aiming to stabilize plaques and reduce inflammation. The key advantage lies in its selective targeting, sparing healthy tissues. While preclinical studies are encouraging, ongoing research and clinical trials are crucial for optimizing protocols and ensuring long-term safety and efficacy. The potential combination with other therapies makes photodynamic therapy a versatile and promising avenue for addressing atherosclerosis and associated cardiovascular disease. The investigations underscore the possibility of utilizing photodynamic therapy as a valuable treatment choice for atherosclerosis. As advancements in research continue, photodynamic therapy might become more seamlessly incorporated into clinical approaches for managing atherosclerosis, providing a blend of efficacy and limited invasiveness.

FLASH Radiation Therapy — Key physical irradiation parameters and beam characteristics

FLASH-RT represents a novel therapeutic radiation modality that holds remarkable potential for mitigating radiation therapy's adverse side effects. This cutting-edge technology allows for sparing healthy tissue while precisely targeting cancerous cells. This is possible by administering an ultra-high-dose-rate in less than a few hundred milliseconds. FLASH-RT has demonstrated impressive results in small-animal models, prompting scientists to adapt and advance existing technologies to make it a viable treatment option for humans. However, producing the ultra-high-dose-rate radiation required for the therapy remains a significant challenge. Several radiation sources, such as very high energy electrons (VHEEs), low energy electrons, x-rays, and protons, have been studied for their ability to deliver the necessary dose. Among them, FLASH-x-ray has gained the most attention owing to its capacity to penetrate deep-seated tumors. Despite the complexity of the process, the potential advantages of FLASH-RT made it an exciting area of research. To achieve the FLASH effect, high-frequency, pulsed irradiated accelerator technology can be employed. Sparing healthy tissue may allow for more aggressive and effective cancer treatments, leading to a better quality of life for patients. Ongoing research and development will be necessary to refine and optimize this approach to radiation therapy.

Radiopharmaceuticals for Cancer Diagnosis and Therapy: New Targets, New Therapies-Alpha-Emitters, Novel Targets.

Radiopharmaceutical therapy has emerged as a promising approach for the treatment of various cancers. The exploration of novel targets such as tumor-specific antigens, overexpressed receptors, and intracellular biomolecules using antibodies, peptides, or small molecules has expanded the scope of radiopharmaceutical therapy, enabling precise and effective cancer treatment for an increasing number of tumor types. Alpha emitters, characterized by their high linear energy transfer and short path length, offer unique advantages in targeted therapy due to their potent cytotoxicity against cancer cells while sparing healthy tissues. This article reviews recent advancements in identifying novel targets for radiopharmaceutical therapy and applications in utilizing α-emitters for targeted treatment.