The treatment strategy, known as neoadjuvant immunotherapy, is built on a compelling biological idea: rather than removing a tumor first and treating the patient afterward, clinicians can use the intact tumor itself as a training ground for the immune system. A tumor that has not yet been surgically disturbed still contains its full complement of cancer antigens, immune cells, and suppressive machinery. By activating immune responses inside that living tumor, physicians hope to generate a systemic anti-cancer army that can hunt down micrometastases already circulating in the body, cells that are invisible on scans but are often the seeds of later relapse.

The centerpiece of the Erlangen regimen was the TransCon TLR7/8 Agonist, an investigational drug built on a so-called transient conjugation technology platform. Toll-like receptors 7 and 8 are pattern-recognition sensors expressed by innate immune cells such as dendritic cells and monocytes. When these receptors are engaged, they trigger a cascade of signaling, classically through the NF-kB and interferon pathways, that transforms the tumor microenvironment from an immunologically cold, suppressive zone into an inflamed, antigen-presenting battlefield. Conventional TLR agonists, however, tend to flood the body quickly, causing systemic inflammatory toxicity that limits their usable dose. The TransCon approach wraps the active drug in a temporary linker that releases it slowly over days or weeks after injection, sustaining local immune activation inside the tumor while keeping blood concentrations low.

Because innate immune activation alone is rarely sufficient to eliminate established cancer, the trial combined the intratumoral agonist with a systemic partner. Patients were randomized 1:1 to receive either pembrolizumab, the widely used antibody that blocks the PD-1 checkpoint and releases the brakes on exhausted T cells, or TransCon IL-2 β/γ, an engineered long-acting version of interleukin-2 designed to preferentially expand and activate the cytotoxic T cells and natural killer cells that actually kill tumors, while avoiding the vasodilating and regulatory T-cell stimulating effects of unmodified IL-2. Each patient received two treatment cycles before undergoing standard surgical resection of the tumor.

The six patients, treated between April and December 2024 within the multicenter BelieveIT-201 phase 2 trial, had a median age of 63 years and a median follow-up of 81 weeks. The parent trial was terminated prematurely by its sponsor, Ascendis Pharma, for reasons unrelated to safety or efficacy, which is why the Erlangen cohort remained small. Three of the six patients achieved a major clinical response, meaning their tumors shrank substantially on clinical and radiographic assessment, two showed partial response, and one experienced progressive disease. On final pathological examination of the resected specimens, one patient had a complete response with no viable tumor remaining, one had a major pathological response, and four were non-responders.

Perhaps the most striking observation was a physical one. All three patients with major clinical responses developed sterile tumor-associated pseudoabscesses, fluid collections near the injection site that were inflamed but not infected. These lesions, the authors note, appeared in spatial proximity to where the TLR7/8 agonist had been delivered, suggesting that the drug was provoking a potent local immune reaction capable of liquefying tumor tissue without bacterial involvement. Such sterile inflammation is a visible signature of innate immune activation and may serve as a biomarker of drug activity in future studies.

Beneath the clinical surface, the tumors themselves were changing. The researchers performed immunohistochemical staining on paired samples taken before and after treatment, tracking markers including CD3 and CD8 for T cells and CD68 and CD163 for macrophage populations. The post-treatment tumors showed clear remodeling of the immune microenvironment, with increased T-cell infiltration that was most pronounced in the tumor center, the region typically most hostile to immune attack in untreated cancers. This shift from an immune-excluded or immune-desert phenotype toward a T-cell-inflamed state is precisely what combination immunotherapy is designed to achieve, and it provides a mechanistic explanation for the tumor shrinkage observed in half of the cohort.

Safety findings, however, temper the enthusiasm. All six patients underwent surgery, and postoperative morbidity was substantial: every patient experienced at least one grade III adverse event, and there was one postoperative death. Disentangling the contribution of the immunotherapy from the inherent risks of major oral and maxillofacial surgery in this small, descriptive cohort is impossible, and the authors are careful not to attempt it. Within the first year, two of the six patients experienced a progression-free survival event, one with progressive disease and one death. Encouragingly, after surgery none of the patients showed disease recurrence during follow-up.

The authors are explicit about the limits of their data. With only six patients, all analyses are purely descriptive, no inferential statistical testing was performed, and no conclusions can be drawn about efficacy or comparative tolerability between the pembrolizumab and TransCon IL-2 β/γ arms. The individual patient was the unit of analysis, and the premature termination of the parent trial means the full randomized comparison will never be completed as designed. What the report offers instead is a complete, consecutive single-center experience, an unfiltered look at how this treatment actually performed in real clinical practice, including the surgical complications that are often underreported in industry-sponsored trials.

Still, the study adds an important piece to one of the most active frontiers in oncology: intratumoral immunotherapy for head and neck cancer. The concept of turning a solid tumor into its own vaccine has gained momentum as long-acting formulations solve the pharmacokinetic problems that plagued earlier innate immune agonists. The consistent immune remodeling seen here, even in a handful of patients, supports the biological plausibility of the approach and justifies continued investigation in larger, properly powered trials. For patients with locally advanced oral cancer, whose treatment options have changed little in decades, the prospect of shrinking tumors before surgery, and potentially de-escalating the devastating operations that this disease demands, remains a goal worth pursuing with rigor and caution in equal measure.

Subject of Research: Neoadjuvant intratumoral TLR7/8 agonist combined with systemic immunotherapy for locally advanced oral squamous cell carcinoma

Article Title: Neoadjuvant combined immunotherapy with intratumoral TransCon TLR7/8 Agonist and systemic TransCon IL-2 β/y or pembrolizumab in patients with locally advanced oral cancer—a monocentric experience

Article References: Neoadjuvant combined immunotherapy with intratumoral TransCon TLR7/8 Agonist and systemic TransCon IL-2 β/y or pembrolizumab in patients with locally advanced oral cancer—a monocentric experience. (n.d.). https://doi.org/10.1007/s00262-026-04515-8

Image Credits: AI Generated

DOI: 10.1007/s00262-026-04515-8

Keywords: oral squamous cell carcinoma, neoadjuvant immunotherapy, TLR7/8 agonist, TransCon IL-2 beta/gamma, pembrolizumab, tumor microenvironment, intratumoral injection, BelieveIT-201 trial, head and neck cancer, immunohistochemistry, sterile pseudoabscess, Ascendis Pharma

The scale of the problem is considerable. Canine oral tumors account for up to 12 percent of all cancers seen in dogs, with melanoma, squamous cell carcinoma, acanthomatous ameloblastoma, and fibrosarcoma representing the most common diagnoses of the oral cavity and pharynx. When these tumors are caught early, wide surgical excision remains the most effective local treatment. But many oral cancers are diagnosed at a locally advanced stage, when complete surgical resection is no longer possible. Radiation and chemotherapy become the fallback options, and while radiation can produce high response rates in tumors such as oral melanoma and squamous cell carcinoma, one-year survival rates for some of the most common oral cancers fall below 50 percent. Novel adjuvant therapies are urgently needed to make those responses last.

The drug at the heart of the study, CB101, is a proprietary hydrogel-based injectable formulation of resiquimod, a molecule known as a toll-like receptor 7/8 agonist. Toll-like receptors sit on the surface or within certain immune cells and act as sentinels for danger signals. In humans, TLR8 is uniquely expressed on myeloid dendritic cells, monocytes, and natural killer cells, making it an attractive lever for pulling the immune system into a fight against cancer. Activating these receptors may disrupt the immunosuppressive microenvironment that tumors build around themselves. Resiquimod itself has never been approved by the FDA, but topical formulations have been tested in human clinical trials for skin diseases and as a vaccine adjuvant, generating an extensive body of toxicology and pharmacology data supporting its safety profile.

The rationale for pairing this immune agonist with radiation rests on a phenomenon called immunogenic cell death. High-dose radiation, such as the 8 Gy per fraction used in some hypofractionated protocols, can kill tumor cells in a way that makes them more visible to the immune system, releasing tumor antigens that dendritic cells can engulf and cross-present to CD8-positive cytotoxic T lymphocytes. Research suggests that doses around 8 Gy per fraction are particularly well suited to synergizing with immunotherapeutics. In principle, this cascade can generate an adaptive, tumor-specific immune response capable of affecting not just the irradiated tumor but also distant, non-irradiated lesions, the so-called abscopal effect. Preliminary mouse data showed that the combination of radiation and CB101 improved local tumor control compared with either treatment alone and elicited such abscopal responses. Injecting the drug directly into the tumor is designed to limit systemic exposure and minimize off-target immune side effects.

Three dogs with histologically confirmed head and neck cancers were prospectively enrolled in the pilot study. Each animal received a baseline CT scan for radiation planning, followed by palliative radiation therapy delivered as four weekly 8 Gy fractions over weeks one through four. CB101 was administered intratumorally at a fixed dose of 10 micrograms in 500 to 1,000 microliters of volume at one-week intervals during weeks two through five, with the first dose given immediately before the second radiation fraction. Treatment planning relied on CT-based software, and dogs were anesthetized and immobilized in vacuum cushions with bite blocks to ensure reproducible setup. A follow-up CT scan at week 12 assessed tumor response, and serial thoracic radiographs at week 24 monitored for distant metastasis. All owners provided informed consent under institutional animal care protocols.

Feasibility proved to be the study’s clearest success. Little to no difficulty occurred during the intratumoral injections, and leakage of the formulation was negligible. Tumors arising from or containing bone were more challenging to infuse; one dog with mandibular fibrosarcoma required both intratumoral and peritumoral administration because of the density of the tumor and its proximity to the normal mandible. Injections were generally performed under general anesthesia for patient comfort, and no pain was noted after the procedure. The volume of each injection was chosen based on the expected ability of the hydrogel to diffuse into the tumor and surrounding tissue, guided by physical examination, CT evidence of bone infiltration, and three-dimensional tumor measurements.

Safety outcomes were equally encouraging. Toxicity was graded using the Veterinary Cooperative Oncology Group criteria and a veterinary radiation morbidity scoring scheme. Only one adverse event was attributable to CB101 itself: a case of grade 1, mild pain during a fourth injection performed under light sedation rather than general anesthesia. Every other adverse event documented was an expected side effect of palliative radiation, and no dog experienced toxicity higher than grade II. One dog developed grade I skin and mucosal toxicity along with grade II ocular toxicity requiring eye drops and antibiotics, an outcome anticipated from the radiation dose distribution, which included a large portion of the left eye. Another dog experienced no toxicity at all.

Tumor responses varied, as expected in such a small and histologically diverse cohort. The dog with oronasal squamous cell carcinoma showed a partial response at week 12 but developed distant progressive disease, first in lymph nodes and then in the lungs, between weeks 12 and 24, and was euthanized 198 days after enrollment. The dog with oral melanoma experienced local progression at week 12 and pulmonary metastasis by week 24, surviving 254 days. The dog with oral fibrosarcoma had stable disease at the week 12 scan, with eventual progression outside the radiation field at week 26 and a survival time of 371 days. All three dogs were free of distant metastases when treatment began.

Intriguingly, serum cytokine analysis revealed negligible changes in the immune signaling molecules the team measured, including TNF-alpha, IL-6, IL-15, interferon-alpha, IL-1 beta, and IL-12p40. The researchers suspect the sampling schedule missed any transient cytokine peaks, which could be expected within hours to three days after dosing rather than a full week later. The absence of clinical signs of cytokine release suggests the weekly 10 microgram dose was well tolerated but may have fallen below a therapeutic threshold. The starting dose was chosen conservatively because a human trial using 12.5 micrograms intralesionally had reportedly triggered cytokine storm and hypotension in some patients. Other veterinary researchers have used far higher resiquimod doses, with a University of Kansas group treating dogs with cutaneous mast cell tumors using doses ranging from 0.07 to 1.36 milligrams and observing one complete response and three partial responses among six dogs.

The Penn team acknowledges several confounders, including the use of NSAIDs in two of the three dogs, which could have either blunted the innate immune response or improved outcomes through COX-2 inhibition, and antimicrobial treatment in one dog, which in human melanoma patients has been associated with worse survival on immunotherapy. Future studies, the authors say, should standardize these medications, sample blood at multiple early time points after injection, and consider analyzing tumor tissue to capture local immune effects within the microenvironment. A dose escalation study appears warranted, and the researchers envision eventually combining TLR agonists with checkpoint inhibitors, an approach that could shift immunologically cold tumors toward hot ones. As academic and industry groups work toward commercializing canine checkpoint inhibitors, this modest pilot study in three dogs may mark the first step of a translational path that runs from the veterinary clinic to human oncology and back again.

Subject of Research: Combining intratumoral resiquimod immunotherapy with hypofractionated radiation therapy in canine head and neck cancer

Article Title: Preliminary evaluation of the safety and feasibility of toll-like receptor ligand CB101 combined with hypofractionated radiation therapy in canine head and neck cancer: a pilot study

Article References: Ghanian, A., DiBona, J., Duda, L., Xu, X., Lukens, J. N., Pearce, T., Ehrhardt, M., Rook, A., Durham, A., Maity, A., & Flesner, B. (2025). Preliminary evaluation of the safety and feasibility of toll-like receptor ligand CB101 combined with hypofractionated radiation therapy in canine head and neck cancer: a pilot study. Veterinary Oncology, 2(1), Article 18. https://doi.org/10.1186/s44356-025-00031-6

Image Credits: AI Generated

DOI: 10.1186/s44356-025-00031-6

Keywords: canine cancer, head and neck cancer, radiation therapy, resiquimod, CB101, toll-like receptors, immunotherapy, intratumoral injection, veterinary oncology, dendritic cells, abscopal effect, pilot study

The study addressed a fundamental blind spot in interventional oncology. Most therapeutic molecules cannot be seen on CT, MRI, or ultrasound, so clinicians injecting drugs directly into tumors have no way of knowing whether the agent stayed in the lesion or escaped into surrounding tissue. This uncertainty is compounded by the physics of interstitial injection: fluid pushed into tissue at clinically practical rates generates pressures that can exceed tumor interstitial pressure by an order of magnitude, driving convective flow away from the needle tip. When that pressure becomes great enough, it can fracture the tissue architecture, creating preferential pathways along which drug escapes rather than distributing uniformly. Until now, there have been no standardized technical guidelines governing needle selection, injection volume, or infusion rate for intratumoral delivery.

The researchers’ strategy was elegant in its simplicity. They co-injected iodixanol, a widely used iodinated CT contrast agent, together with fluorescently labeled albumin, which served as a surrogate for a real drug. Fluorescent albumin was chosen specifically because its size approximates that of LMB-100, an antibody-toxin conjugate being tested against mesothelioma in a clinical trial. Because iodixanol is radiopaque, its distribution could be mapped in three dimensions by CT within two minutes of injection, while the fluorescent albumin could be visualized afterward with fluorescence microscopy of thinly sectioned frozen tissue. Comparing the two distributions side by side would reveal whether a contrast agent visible on a clinical scanner can reliably predict where a therapeutic protein actually travels.

The physical chemistry of the two agents, however, turned out to be strikingly different. Dynamic light scattering measurements showed that iodixanol has a hydrodynamic diameter of just 2.7 nanometers, compared with 17.0 nanometers for the fluorescent albumin, a statistically significant difference. Their surface charges diverged as well: iodixanol carried a nearly neutral zeta potential of 2.3 millivolts, while the albumin was strongly negative at minus 17.7 millivolts. These differences matter enormously in the confined geometry of the interstitial space, where a molecule’s size determines whether it can weave between cells and collagen fibers, and its charge determines whether it binds to or repels components of the extracellular matrix.

When the team injected mixtures of the two agents into blocks of ex vivo bovine liver at volumes of 1, 2, or 4 milliliters, delivered at 1 milliliter per minute, the consequences of those physicochemical differences became immediately apparent. Iodixanol spread through the tissue over a volume roughly seven times larger than that occupied by the fluorescent albumin. The contrast agent’s concentration was highest at the center of the injectate, matching the concentration loaded into the syringe, and then declined gradually with distance, a profile suggestive of diffusive permeation through the interstitial gel. The albumin behaved differently: it accumulated in highly concentrated pockets, sometimes exceeding the injected concentration at the distribution’s core, and then dropped off sharply at a well-defined boundary, as though the large, negatively charged protein had been physically restrained by the tissue matrix and by binding to collagen and positively charged proteins.

Despite these differences, the two distributions were strongly correlated. The distribution volumes measured by CT and by fluorescence microscopy tracked each other with a coefficient of determination of 0.89, and a leave-one-out analysis confirmed that this relationship was robust, with correlation values ranging from 0.65 to 0.98 across all subsets of the data. In other words, while the contrast agent cannot be treated as an exact replica of the drug, its CT footprint provides a meaningful, quantifiable estimate of where a similarly sized therapeutic will end up. That is a powerful tool for preclinical development and for planning clinical injection protocols, because it converts an invisible process into something a radiologist can measure in real time.

Perhaps the most clinically consequential finding concerned injection volume. When the researchers normalized the distribution volume by the injected volume, the average value stayed roughly constant across all three injection sizes, but the variance ballooned with larger volumes. One-milliliter injections produced tightly clustered, predictable distributions, whereas 4-milliliter injections showed wildly variable spread, with contrast visibly leaking out of the tissue blocks in some cases. The fluorescent albumin, meanwhile, stayed remarkably localized at all volumes, with normalized distribution volumes averaging just 0.12. The implication is clear: dividing a total dose into multiple small-volume injections at different sites, a strategy known as dose fractionation, minimizes local fluid pressure, reduces the risk of tissue fracture and leakage, and yields far more predictable drug localization than a single large bolus.

This benchtop insight translated directly into the clinic. The same team applied a fractionation strategy in a Phase 1 trial testing intratumoral injection of LMB-100, an anti-mesothelin immunotoxin, combined with the immunotherapy ipilimumab in patients with mesothelioma. Rather than delivering the full 4-milliliter dose at one site, clinicians used a multi-pronged needle with three curved injection tines that deploy from the sides of the main cannula, extending up to a 5-centimeter array diameter. The tines were advanced in incremental stages, partially retracted, and the base needle was rotated 60 degrees before redeployment, allowing small aliquots to be deposited across the entire tumor volume. Ultrasound provided real-time visualization of tine position and could detect extravasation the moment it occurred, prompting the operator to halt injection at that site. PET-CT fusion imaging identified metabolically active tumor regions to target.

The broader significance of this work extends well beyond one immunotoxin. The imageable surrogate methodology could be used to optimize needle selection, injection spacing, infusion rates, and drug concentrations for any locally delivered therapy, from oncolytic viruses to nanoparticles to gelling polymer depots. It could also underpin the development of the standardized technical guidelines that intratumoral injection currently lacks, replacing intuition with quantitative data about how injectate properties and injection parameters interact. The authors caution that their experiments were performed in normal ex vivo liver, which lacks the vascular clearance, lymphatic drainage, and elevated interstitial pressures of living tumors, and that differences in spatial resolution between CT and fluorescence microscopy introduce some uncertainty. Larger, more charged drugs such as doxorubicin or oncolytic viruses would likely deviate even further from the contrast agent’s distribution than albumin did.

Even with those caveats, the study marks a turning point in how direct injection into tumors can be understood and controlled. It demonstrates that contrast co-injection can serve as an imageable proxy for drug distribution, that physicochemical properties like size and charge govern how far a molecule travels through tissue, and that small, multifocal injections outperform large single boluses in predictability. For a field where treatment success has long depended on where a needle tip happens to deposit its payload, making the invisible visible may prove to be one of the most practical advances in image-guided drug delivery in years.

Subject of Research: CT-based mapping of drug distribution after direct interstitial injection in ex vivo liver tissue

Article Title: Mapping drug distribution using CT imaging following direct tissue injection in ex vivo liver: informing clinical implementation

Article References: Morhard, R., Mauda-Havakuk, M., Delgado, J. F., Kassin, M. T., Ghafoor, A., Pastan, I., Hassan, R., Karanian, J. W., Pritchard, W. F., Wood, B. J., & Mikhail, A. S. (2025). Mapping drug distribution using CT imaging following direct tissue injection in ex vivo liver: informing clinical implementation. CVIR Oncology, 1(1), Article 27. https://doi.org/10.1007/s44343-025-00027-x

Image Credits: AI Generated

DOI: 10.1007/s44343-025-00027-x

Keywords: intratumoral injection, CT contrast, iodixanol, drug distribution, image-guided delivery, dose fractionation, fluorescent albumin, immunotoxin LMB-100, mesothelioma, interventional oncology, tissue fracture, ex vivo liver