Intratumoral Research Articles

Combination therapy with the oncolytic virus CF33-CD19 and blinatumomab for the treatment of advanced solid tumors.

TPS2687 Background: CF33-CD19 is a novel chimeric vaccinia virus that selectively replicates in tumor cells and can provoke anti-tumor immunity. CF33-CD19 expresses a truncated and non-signaling CD19 protein on the surface of infected tumor cells before virus-mediated tumor lysis, labeling them for CD19-targeted therapies. Preclinical studies with CF33-CD19 have shown that combination therapy with CD19 targeting chimeric antigen receptor T cells is more effective than either monotherapy in murine xenograft and syngeneic models [1]. Likewise, combination therapy with bispecific T-cell engagers (BiTE)s that bind CD3-positive T cells and infected solid tumor cells that express de novo CD19, showed pronounced recruitment of T cells to the tumor microenvironment, resulting in tumor growth inhibition in mouse models. CD19xCD3 targeting BiTEs such as blinatumomab may offer an off-the-shelf alternative to adoptive cell therapies. This phase I study, called OASIS, dose escalates CF33-CD19 administered intravenously (IV) or intratumorally (IT) combined with blinatumomab in adults with advanced or metastatic solid tumors. Methods: The OASIS study (NCT06063317) is enrolling patients with advanced or metastatic solid tumors with ≥ 2 prior lines of therapy. Patients who have received prior treatment with a poxvirus based oncolytic virus or a bispecific CD19-directed CD3 T-cell engager are excluded. A safety run-in will evaluate the safety of CF33-CD19 monotherapy administered IT or IV before initiating the combination therapy regimen. Combination therapy will be administered in 28-day cycles with CF33-CD19 on days 1 and 15. Following viral transduction to promote de novo CD19 expression, blinatumomab is given on days 2-9 and 16-23 via a 7-day continuous infusion. Patients > 45 kg will receive 9 mcg of blinatumomab during the first week of cycle 1, then 28 mcg during the third week of cycle 1 and subsequent cycles. The study consists of two parts. Part 1 follows a 3+3 dose escalation scheme independently of each route of CF33-CD19 administration (IT and IV) with dose levels of CF33-CD19 ranging from 1.0x107 to 3.0x109 PFU. Part 2 is a cohort expansion in select indications at the optimal dose. The co-primary endpoints are safety and identification of the recommended phase 2 dose. Secondary endpoints include objective response rate according to RECIST v1.1 and iRECIST. Enrollment began in October 2023. 1. Park et al. Sci Transl Med. 2020 Sep 2;12(559):eaaz1863. doi: 10.1126/scitranslmed.aaz1863. PMID: 32878978. Clinical trial information: NCT06063317 .

A randomized, controlled, multicenter, phase 3 study of vusolimogene oderparepvec (VO) combined with nivolumab vs treatment of physician’s choice in patients with advanced melanoma that has progressed on anti–PD-1 and anti–CTLA-4 therapy (IGNYTE-3).

TPS9604 Background: Melanoma is the 5th most common cancer type, with ~100,000 new cases and ~8,000 deaths estimated in the US for 2024. First-line systemic treatment with immune checkpoint inhibitors improved the objective response rate (ORR) and extended progression-free survival (PFS) and overall survival (OS) for patients with advanced disease. The combination of anti–PD-1 nivolumab (nivo) + anti–CTLA-4 ipilimumab (ipi) therapy is associated with the highest ORR and best PFS and OS in this setting. However, only about half of patients respond, and there is no standard of care for patients who progress after anti–PD-1–based therapy. VO (also referred to as RP1) is a selectively replication-competent HSV-1 oncolytic virus that expresses human GM-CSF and a fusogenic glycoprotein (GALV-GP-R–). Preliminary data from the phase 1/2 study (IGNYTE) showed that intratumoral (IT) VO plus intravenous (IV) nivolumab (nivo) was well tolerated and had meaningful antitumor activity (ORR, 31.4%) with durable responses in patients with advanced melanoma who progressed on prior anti–PD-1 therapy. This phase 3 study will evaluate the overall survival and clinical benefit as well as the safety of VO plus nivo for patients with advanced melanoma whose disease has progressed after receiving anti–PD-1 and anti–CTLA-4 therapy (or who are not eligible for anti–CTLA-4 therapy in addition to anti–PD-1) versus physician’s choice. Methods: This is a randomized, controlled, multicenter, phase 3 clinical trial comparing VO in combination with nivo vs physician’s choice of treatment in patients with unresectable or metastatic melanoma. Key eligibility criteria include age ≥12 years, stage IIIb–IV/M1a–M1d cutaneous melanoma, confirmed disease progression on an anti–PD–1 and anti–CTLA-4 treatment (administered in combination or in sequence with anti–PD-1 last), at least 1 measurable tumor (≥1 cm), and adequate hematologic, hepatic, and renal function. Patients who are not candidates for anti–CTLA-4 therapy may enroll if they have confirmed progression on anti–PD-1 therapy. Patients with BRAF V600 mutant melanoma must have received an anti-BRAF + anti-MEK targeted therapy prior to enrollment. Patients (N = ~400) will be randomized 1:1 to receive VO plus nivo or physician’s choice (nivo + relatlimab, anti–PD–1 monotherapy rechallenge [nivo or pembrolizumab], or single-agent chemotherapy [dacarbazine, temozolomide, or paclitaxel/albumin-bound paclitaxel]). VO is given IT Q2W; nivo is administered IV at 240 mg Q2W or 480 mg Q4W starting with the 2nd dose of VO. The primary endpoint of the study is OS; the key secondary endpoints are PFS and ORR. Additional endpoints include complete response rate, duration of response, disease control rate, and safety. Clinical trial information: NCT06264180 .

Lactococcus lactis subsp. cremoris C60 Upregulates Macrophage Function by Modifying Metabolic Preference in Enhanced Anti-Tumor Immunity

Lactococcus lactis subsp. cremoris C60 is a probiotic strain of lactic acid bacteria (LAB) which induces various immune modifications in myeloid lineage cells. These modifications subsequently regulate T cell function, resulting in enhanced immunity both locally and systemically. Here, we report that C60 suppresses tumor growth by enhancing macrophage function via metabolic alterations, thereby increasing adenosine triphosphate (ATP) production in a murine melanoma model. Intragastric (i.g.) administration of C60 significantly reduced tumor volume compared to saline administration in mice. The anti-tumor function of intratumor (IT) macrophage was upregulated in mice administered with C60, as evidenced by an increased inflammatory phenotype (M1) rather than an anti-inflammatory/reparative (M2) phenotype, along with enhanced antigen-presenting ability, resulting in increased tumor antigen-specific CD8+ T cells. Through this functional modification, we identified that C60 establishes a glycolysis-dominant metabolism, rather than fatty acid oxidation (FAO), in IT macrophages, leading to increased intracellular ATP levels. To address the question of why orally supplemented C60 exhibits functions in distal places, we found a possibility that bacterial cell wall components, which could be distributed throughout the body from the gut, may induce stimulatory signals in peripheral macrophages via Toll-like receptors (TLRs) signaling activation. Thus, C60 strengthens macrophage anti-tumor immunity by promoting a predominant metabolic shift towards glycolysis upon TLR-mediated stimulation, thereby increasing substantial energy production.

Assessment of tumor hypoxia in spontaneous canine tumors after treatment with OMX, a novel H-NOX oxygen carrier, with [18F]FMISO PET/CT

BackgroundHypoxia is a detrimental factor in solid tumors, leading to aggressiveness and therapy resistance. OMX, a tunable oxygen carrier from the heme nitric oxide/oxygen-binding (H-NOX) protein family, has the potential to reduce tumor hypoxia. [18F]Fluoromisonidazole ([18F]FMISO) positron emission tomography (PET) is the most widely used and investigated method for non-invasive imaging of tumor hypoxia. In this study, we used [18F]FMISO PET/CT (computed tomography) to assess the effect of OMX on tumor hypoxia in spontaneous canine tumors.ResultsThirteen canine patients with various tumors (n = 14) were randomly divided into blocks of two, with the treatment groups alternating between receiving intratumoral (IT) OMX injection (OMX IT group) and intravenous (IV) OMX injection (OMX IV group). Tumors were regarded as hypoxic if maximum tumor-to-muscle ratio (TMRmax) was greater than 1.4. In addition, hypoxic volume (HV) was defined as the region with tumor-to-muscle ratio greater than 1.4 on [18F]FMISO PET images. Hypoxia was detected in 6/7 tumors in the OMX IT group and 5/7 tumors in the OMX IV injection group. Although there was no significant difference in baseline hypoxia between the OMX IT and IV groups, the two groups showed different responses to OMX. In the OMX IV group, hypoxic tumors (n = 5) exhibited significant reductions in tumor hypoxia, as indicated by decreased TMRmax and HV in [18F]FMISO PET imaging after treatment. In contrast, hypoxic tumors in the OMX IT group (n = 6) displayed a significant increase in [18F]FMISO uptake and variable changes in TMRmax and HV.Conclusions[18F]FMISO PET/CT imaging presents a promising non-invasive procedure for monitoring tumor hypoxia and assessing the efficacy of hypoxia-modulating therapies in canine patients. OMX has shown promising outcomes in reducing tumor hypoxia, especially when administered intravenously, as evident from reductions in both TMRmax and HV in [18F]FMISO PET imaging.

Abstract PS16-03: Intratumoral dosing of INT230-6 in Early-Stage Breast Cancer Patients Induces Tumor Cell Necrosis and Immunomodulatory Effects: A Phase II Randomized Window-Of-Opportunity Study – the INVINCIBLE Trial

Abstract Background: Larger tumors pose an increased risk for breast cancer (BC) recurrence post-surgery. In addition, most BC outside of the triple negative subtype are considered immunological quiescent and minimally responsive to immunotherapies. One potential method to combat disease recurrence risk and induce immune activation pre-surgery is through a local therapy that could cause cell death to expose tumor antigens, provide adjuvants for anti-tumor immune priming, and thus potentially increase responsiveness to immunotherapies or rates of pathological complete response in combination with chemotherapy. We have conducted a randomized, Phase 2 presurgical Window-Of-Opportunity trial for intratumoral (IT) INT230-6 comprising vinblastine, cisplatin and a diffusion enhancer (SHAO) in patients with early-stage operable BC, the INVINCIBLE trial (NCT04781725). Previous in vivo and clinical studies demonstrated that INT230-6 induces cancer cell death and halts replication while maturing dendritic cells and recruiting T-cells into the tumor. In this trial, IT injections of INT230-6 were conducted to 1) evaluate the safety of regional cytotoxic use on BC, 2) assess the drug’s ability to cause necrosis, 3) assess immune response within the tumor, microenvironment and systemically prior to surgical resection, and 4) understand the genetic pathways involved in immune activity. Methods: Women awaiting surgery for newly diagnosed early-stage intermediate or high-grade T1-T2 invasive BC were recruited to the trial. The study has two parts. Part I was a randomized (2:1) open label trial comparing 1-3 doses of INT230-6 injected weekly versus no treatment prior to surgery to evaluate safety, feasibility, and optimal drug dosing. Part II was a double-blinded randomized (2:1) trial where patients received one IT dose of INT230-6 vs saline injection IT. Results: We successfully recruited 91 patients with age ranges of 40-77 yrs (mean of 60 yrs) with tumor size ranging from 1.1 - 4.8 cm (mean 2.5 cm; SD 0.9 cm). The most common (>10%) AEs were injection site pain, injection site reaction and nausea/vomiting. Approximately 90% of AEs were grade 1. In the study INT230-6 induced necrosis in 64% of subjects (37 out of 58, range 0 to 100%); whereas saline induced partial necrosis in 25% of patients (5 out of 20, range 0 to 10%). The table below shows the results of necrosis for INT230-6 use in the entire study compared to saline injection for all subjects and those with tumors of size T2. A single injection of INT230-6 caused necrosis in various histologies, including invasive lobular carcinoma. Gene expression analysis showed significant differential gene expression between the baseline biopsy and surgical specimens using INT230-6. Pathway analysis identified genes associated with TCR signaling, B cells and T cell activation that were significantly upregulated in the post INT230-6 treatment samples. There was a relative increase in CD4 and CD8 T cells and B and mast cells. Conclusion: Preliminary evidence shows that a single dose of INT230-6 can cause significant intratumoral necrosis compared to saline especially in tumors >2. cm. INT230-6 stimulates an immune response in breast cancers prior to surgery with minimal adverse effects and good tolerability. Given its immune activation properties, INT230-6 shows promising potential in future breast cancer neoadjuvant studies. Table 1: INVINCIBLE Study Tumor Necrosis Results Intratumoral INT230-6 injection compared to IT saline injection Citation Format: Angel Arnaout, Lewis Bender, Megan Hopkins, Vanessa Lopez Ozuna, Linda Liao, Susan Robertson, Vida Talebian, Kianoosh Keyhanian, Arif Awan, Franco Abbate, Ian Walters, Gregory Pond, John Bartlett, Lazlo Radvanyi, Melanie Spears. Intratumoral dosing of INT230-6 in Early-Stage Breast Cancer Patients Induces Tumor Cell Necrosis and Immunomodulatory Effects: A Phase II Randomized Window-Of-Opportunity Study – the INVINCIBLE Trial [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS16-03.

Abstract CT153: Phase I trial of intratumoral administration of autologous CCL21 gene-modified dendritic cells in combination with pembrolizumab for advanced NSCLC: Feasibility of repeated IT injections

Abstract Background: Effective immunotherapy options are lacking for patients with advanced non-small cell lung cancer (NSCLC) who progress on a programmed cell death-(ligand)1 [PD-(L)1] inhibitor and for those who are epidermal growth factor receptor (EGFR) mutation or anaplastic lymphoma kinase (ALK) rearrangement positive after progression on tyrosine kinase inhibitor (TKI) therapy. One potential approach to improve immune checkpoint efficacy in these patient populations is to promote cytolytic T cell infiltration into tumors. This can be accomplished via in situ vaccination with chemokine gene-engineered functional antigen presenting cells (APCs) which can take advantage of the full repertoire of tumor antigens and convert the tumor into a lymph node-like environment to promote both local and systemic antitumor responses. The chemokine CCL21 promotes co-localization of naive T cells and antigen-experienced dendritic cells (DCs) to facilitate T cell activation. Our preclinical studies and phase I trial of intratumoral (IT) administration of DC genetically modified to overexpress CCL21 (CCL21-DC) revealed augmentation of tumor antigen presentation in situ, resulting in systemic antitumor immunity. However, increased PD-L1 expression was observed in some patient tumors, suggesting that tumor-mediated impairment of T cell function may be forestalling a more robust CCL21-DC mediated antitumor response. Similarly, improved PD-(L)1 inhibitor efficacy may be possible with enhanced T cell infiltration and augmented APC function following IT CCL21-DC. Therefore, we are conducting a phase I trial, combining IT CCL21-DC with pembrolizumab in patients with advanced NSCLC. Methods: Phase I, dose-escalating, multi-cohort trial followed by dose expansion. Maximum of 24 patients (9-12 escalation + 12 expansion) with stage IV NSCLC will be evaluated who have tumors accessible for IT injection and are either (1) EGFR/ALK wild-type after progression on a PD-(L)1 inhibitor or (2) EGFR/ALK mutant after progression on TKI therapy. Three IT injections of autologous CCL21-DC (days 0, 21, 42) will be concurrently administered with pembrolizumab, followed by pembrolizumab once every three weeks for up to 1 year. Primary objective of dose escalation is safety and determination of maximum tolerated dose (MTD) of IT CCL21-DC when combined with pembrolizumab. Primary objective of dose expansion is objective response rate at MTD. Secondary objectives include adverse event profiling and determination of drug target activity by immune monitoring studies. This trial, NCT03546361, is currently open for enrollment, and this updated abstract will highlight the feasibility of repeated IT injections in treated patients. Citation Format: Bin Liu, Aaron Lisberg, Ramin Salehi-Rad, Michael Oh, Jay M. Lee, Linh M. Tran, Kostyantyn Krysan, Raymond J. Lim, Camelia Dumitras, Zhe Jing, Fereidoun Abtin, Robert D. Suh, Scott J. Genshaft, Scott S. Oh, Gregory A. Fishbein, Anita Kaul, Kanwarpal S. Kahlon, Shahryar A. Ashouri, Jonathan W. Goldman, David A. Elashoff, Edward Garon, Steven M. Dubinett. Phase I trial of intratumoral administration of autologous CCL21 gene-modified dendritic cells in combination with pembrolizumab for advanced NSCLC: Feasibility of repeated IT injections [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 CT153.

Abstract LB439: Intra-tumoral administration of releasable hydrogel microsphere drug conjugates for long acting therapeutics

Abstract Background. Intra-tumoral (IT) injections of therapeutics hold promise for the treatment of solid tumors. However, drugs administered directly in the tumor have short IT residence times potentially limiting their exposure and therefore efficacy. We are developing a platform of novel hydrogel microsphere (MS) conjugates that enables long-acting tumor residence after a single injection of the conjugate. We demonstrate this approach not only solves the problem of short acting intra-tumoral therapy, but also allows for the safe administration of two therapies with overlapping toxicities such as SN-38 and a PARP inhibitor (PARPi). Methods. Hydrogel microsphere (MS) conjugates were prepared and characterized. Using MS conjugates labeled with rhodamine and fluorescein, we established methods to directly measure the IT concentration and half-life of the released drug after IT administration into CT26 tumor bearing mice. We next measured the tissue pharmacokinetics and efficacy of MSs attached to SN-38, a potent topoisomerase 1 inhibitor and immunomodulator, using NSG mice bearing 22Rv1 ATM-/- xenografts. Finally, we measured the anti-tumor efficacy of MS~SN-38 in combination with an oral PARPi, talazoparib, in 22Rv1 ATM-/- xenografts. Results. We successfully established methods to quantify the concentration of released drug and MS bound drug from tumor biopsies using fluorescent surrogates. When applied to MS~SN-38 conjugates, locally injected tissues showed high levels of SN-38 with a long half-life of about one week. In contrast, the systemic half-life of free SN-38 is ~30 minutes. IT MS~SN-38 was ~10-fold more efficacious as an anti- tumor agent than systemic SN-38 from subcutaneous administration of MS~SN-38. Finally, we show high anti-tumor synergy from the SN-38 localized in a tumor by IT MS~SN-38 in combination with systemic administration of the PARPi talazoparib. Conclusions. This microsphere technology allows for sustained delivery of high local concentrations of drugs administered intratumorally. Here, IT MS~SN-38 resulted in an intra-tumoral half-life of ~1 week and marked anti-tumor efficacy in 22Rv1 ATM-/- xenografts. We also demonstrated that long-acting IT injections enables safe use of drug combinations with well-described overlapping toxicities such as SN-38 and PARPi. The microsphere technology may offer an alternative strategy for localizing and sustaining drugs in tumors and may mitigate toxicities from combination therapy where two agents have a similar toxicological profile. Other combinations, including small molecules and immuno-oncology agents, are being explored. Citation Format: John A. Hangasky, Jeff Henise, Deborah Charych, Christopher W. Carreras, Gary Ashley, Daniel V. Santi. Intra-tumoral administration of releasable hydrogel microsphere drug conjugates for long acting therapeutics [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 LB439.

Abstract LB353: Repurposing varicella zoster virus and human papillomavirus vaccines for local immunotherapy against solid tumors

Abstract Local immunotherapy against solid tumors is considered a viable approach to stimulate the tumor microenvironment (TME) and anti-tumor immunity. We assessed in situ vaccination with licensed subunit vaccines to leverage preexisting anti-vaccine immunity in the TC-1 tumor model expressing HPV16 viral oncogenes E6 and E7. We chose Shingrix, a VZV vaccine containing the glycoprotein E (gE), and Gardasil-9, an HPV vaccine containing L1 virus-like particles (VLP), to preferentially induce CD4 and CD8 T cell responses, respectively. Intratumoral (IT) injection of Shingrix in Shingrix prevaccinated mice led to complete regression in 20% to 50% of treated mice and elicited CD8 T cell responses against the viral oncoprotein E7. IT injection of an MHC-II-restricted gE peptide with polyI:C also led to durable remission, suggesting that gE-specific CD4 T cells played a significant role in tumor control. In contrast, IT injection of Gardasil-9 in Gardasi-9 prevaccinated animals did not delay tumor growth. However, IT injection of an MHC-I-restricted L1 peptide with Shingrix consistently led to complete remissions perhaps by overcoming preexisting antibodies against native HPV VLP which appeared to inhibit the cross-presentation of VLP derived antigens. TME analysis showed that IT injection of Shingrix with Gardasil-derived peptide induced IFN-gamma, TNF-alpha and CXCL9. This treatment also dramatically reshaped the immune cell infiltrate causing the recruitment of T cells and neutrophils and the decrease of tumor-associated macrophage and eosinophils. We are investigating now how these changes may contribute to tumor clearance and epitope spreading against tumor-derived antigens. Finally, IT injection of Shingrix and a E7 viral neoantigen peptide led to the eradication of all primary injected and abscopal tumors. Our results indicate that the ability of Shingrix to induce exceptionally strong Th1 responses makes it a versatile component for in-situ vaccination which can be combined with peptides derived from licensed vaccines or tumor antigens. Because this approach relies on relatively low cost existing vaccines and inexpensively manufactured minimal CD8 restricted peptides, it has the potential for a broadly applicable cancer immunotherapy strategy for use in resource-constrained settings. The approach is currently being evaluated in a phase I clinical trial in companion dogs with spontaneously arising cancers. Citation Format: Nicolas Cuburu, Shiv K. Sethi, Claire E. Bradley, Lukas Bialkowski, Cynthia D. Thompson, Douglas R. Lowy, John T. Schiller. Repurposing varicella zoster virus and human papillomavirus vaccines for local immunotherapy against solid tumors [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 LB353.

Abstract CT186: A first-in-human phase 1 dose-escalation and expansion study of intratumoral oncolytic reovirus (RC402) as a monotherapy or in combination with pembrolizumab in advanced solid tumors

Abstract Background: RC402 is an oncolytic reovirus that selectively infects and destroys cancer cells. Preclinical studies have shown that RC402 remodels the tumor immune microenvironments and elicits potent anti-tumor immunity, thereby enhancing immune checkpoint inhibitors. Methods: This Phase I clinical trial (ACTRN12620000777998) was designed to evaluate the safety profiles and antitumor efficacy of intratumoral (IT) RC402 in patients with advanced solid tumors refractory to standard therapy and with accessible tumor lesions for IT injection of RC402. For the dose-escalation part (Phase 1a), the RC402 monotherapy dose was evaluated at 3 dose levels in Part A and the RC402 combination therapy dose with pembrolizumab was evaluated at 2 dose levels in part B using an accelerated titration method followed by a standard 3+3 dose escalation. In the dose-expansion part (Phase 1b), patients were treated with IT RC402 in combination with pembrolizumab at the recommended dose selected from the dose escalation part. RC402 was IT injected on day 1 of each treatment cycle. Fixed-dose pembrolizumab (200mg Q3W) was intravenously administered on day 8 of each treatment cycle for Phase 1a Part B and Phase 1b. Viral shedding was assessed serially in serum, urine, sputum, and stool samples, and neutralizing antibodies to Reovirus Type 3 Dearing were analyzed in serum samples. Results: Nine patients (2 gastric cancer and 1 cutaneous squamous cell carcinoma [cSCC] in Phase 1a Part A, 1 cSCC and 1 colorectal cancer [CRC] in Phase 1a Part B, and 1 cSCC and 3 CRC in Phase 1b) were enrolled and treated with at least two doses of RC402. IT administration of RC402 was generally well tolerated with no dose-limiting toxicities at all dose levels (1.6 × 108, 1.4 × 109, and 1.4 ×1010 PFU) as a monotherapy or in combination with pembrolizumab. The maximum tolerated dose was not reached. There were no treatment-emergent adverse events (TEAEs) leading to treatment discontinuation and no RC402-related serious TEAEs. The most common AEs were chills, fatigue, injection site reactions (erythema and pain) and nausea. Response evaluation showed two complete responses (one patients with cSCC in Phase 1a Part A and another patient with cSCC in the dose expansion part) lasting longer than 90 weeks and 36 weeks, respectively. No viral shedding was detected in any serum, urine, sputum, and stool samples on days 1, 2, and 15 of cycle 1, on day 2 of subsequent cycles and at the end of treatment. All patients showed an initial increase in antireoviral antibody titers on day 1 of cycle 2, followed by a decrease in subsequent cycles. Conclusion: This first-in-human study demonstrated IT RC402 is well tolerated and has a manageable safety profile with preliminary clinical activity as a monotherapy and in combination with pembrolizumab. These results warrant further clinical development of RC402. Citation Format: Mark Voskoboynik, Jia Liu, Ki-Hoon Song, Dong-Guk Park, Min-Hee Ryu, Hong Jae Chon, Chan Kim. A first-in-human phase 1 dose-escalation and expansion study of intratumoral oncolytic reovirus (RC402) as a monotherapy or in combination with pembrolizumab in advanced solid tumors [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 CT186.

Abstract CT182: Oncolytic virus CF33-hNIS for the treatment of advanced cancer

Abstract Background: CF33-hNIS is a novel vaccinia virus engineered with the human sodium-iodide symporter (hNIS) gene. CF33-hNIS selectively replicates in tumor cells and promotes anti-tumor immunity. Here, we report results on a first-in-human phase 1 dose escalation study of CF33-hNIS, administered intratumorally (IT) or intravenously (IV) in adult patients with metastatic or advanced solid tumors (MAST). Methods: The MAST study is evaluating the safety of CF33-hNIS administered IT or IV, alone or in combination with pembrolizumab in patients with advanced or metastatic solid tumors with ≥ 2 prior lines of therapy (NCT05346484). CF33-hNIS is administered in 21-day cycles on C1D1 and C1D8, then D1 of each cycle thereafter. Pembrolizumab begins C2D1 for the combination groups and is administered Q3W. The study consists of two parts. Part 1 follows a 3+3 dose escalation scheme independent of each route of CF33-hNIS administration (IT and IV) and for each therapy regimen (monotherapy and combination therapy) with up to 7 dose levels of CF33-hNIS ranging from 8.6x105 to 3.0x109 PFU. Part 2 is a cohort expansion in select indications at the optimal dose. The co-primary endpoints are safety and identification of the recommended phase 2 dose. Secondary endpoints include objective response rate according to RECIST v1.1 and iRECIST, and assessment of viral replication in tumor lesions via Single-Photon Emission Computerized Tomography (SPECT). Results: As of Dec 2023, 36 patients have been treated in Part 1 of the study with a median age of 59 (range 23-81). Twenty-two (61%) patients were heavily pretreated with ≥3 prior lines of systemic therapy, and 14 (39%) received prior treatment with checkpoint blockade. Treatment-related adverse events consisted predominantly of ‘flu-like’ symptoms, such as grade 1/2: fatigue (31%), pyrexia (22%), and chills (14%). No dose-limiting toxicities have occurred. The highest dose treated to date is 1.1x108 PFU by IT and IV delivery; dose escalation continues. In the IT cohorts (14 patients), 7 of 15 (47%) injected lesions had a reduction in tumor burden, three lesions were completely eradicated. Three patients (21%) had an objective response: one complete response by iRECIST in a patient with cholangiocarcinoma; and two partial responses in patients with melanoma by RECIST. In IV cohorts (17 patients), 53% of patients achieved stable disease as their best response. Patients who received prior checkpoint blockade therapy derived clinical benefit with and without pembrolizumab. Viral replication, assessed by SPECT was higher in patients that saw a reduction in tumor burden. Immunological changes in peripheral blood and tumor biopsies will be presented. Conclusions: CF33-hNIS alone or in combination with pembrolizumab is a safe treatment option for advanced cancer patients. Encouraging efficacy has warranted advancement to part 2 of this study with a cohort expansion of patients with cholangiocarcinoma and other tumor types. Citation Format: Daneng Li, Anthony F. Shields, Hirva Mamdani, Patrick Travis, Gavin Wright, Wallace Akerley, Alex Spira, Gregory Daniels, Jaime Merchan, Abhijeet Kumar, Emerson Lim, Colt Egelston, Oscar Flores, Stanley Hamilton, Janet Austria, Amanda Seiz, Sharon Yavrom, Seymour Fein, John Byon, Paul Woodard, Grey A. Wilkinson, Jennifer Leddon. Oncolytic virus CF33-hNIS for the treatment of advanced cancer [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 CT182.

Tigilanol tiglate is an oncolytic small molecule that induces immunogenic cell death and enhances the response of both target and non-injected tumors to immune checkpoint blockade

BackgroundTigilanol tiglate (TT) is a protein kinase C (PKC)/C1 domain activator currently being developed as an intralesional agent for the treatment of various (sub)cutaneous malignancies. Previous work has shown that...

Immunochemistry-based quantification of tumor-infiltrating lymphocytes and immunoscore as prognostic biomarkers in bladder cancer

BackgroundTumor-infiltrating lymphocytes (TILs) and the derived immunoscore (IS) have gained considerable attention over the last decade as prognostic markers in many solid cancers. However, in bladder cancer (BC), their prognostic value is not clearly established.MethodsThe present study aimed to quantify the TILs rates in BC, assess the derived immunoscore, and investigate their prognostic value. An immunochemistry-based quantification of the different subtypes of TILS was performed on paraffin-embedded blocks from patients with invasive urothelial carcinoma of the bladder. We have assessed the rates of TILs, respectively, on peri-tumoral (PT) and intra-tumoral (IT) areas and calculated for each case the corresponding IS which is the index: CD8+/CD3+ TILs. The IS was then classified as low (I0, I1) or high (I2, I3, I4). We included 30 cases in the analysis.ResultsThe median age of patients was 65 years with a sex ratio of 9. TILs densities and distribution were significantly variable between IT and PT areas CD3+ (p = 0.03) and CD8+ (p = 0.004) with the highest rates on the PT areas. In univariate analysis, a low density of CD8+ TILs was significantly associated with an advanced age (p = 0.05), with the presence of lympho-vascular invasion (p = 0.02) and with the absence of specific histological subtype (p = 0.05). A low immunoscore was significantly associated with the presence of lympho-vascular invasion (p = 0.004). No significant association was found between TILs subpopulations, the IS, and the other clinicopathological and survival data. The overall survival (OS) and disease-free survival (DFS) medians were slightly superior in highly T (CD3+/CD8+)-cell infiltrated tumors as well as tumors with a high IS densities. However, the univariate analysis showed that TILs and immunoscore did not impact overall survival (OS) and disease-free survival (DFS).ConclusionTILs and immunoscore might be effective prognostic tools in BC. However, standardized quantification methods and further investigation on larger samples are highly recommended to definitively attest the prognostic value of TILs and IS in BC.

Abstract 3734: Tumoral and systemic immune modulation with neoadjuvant (NeoAd) intratumoral (IT) TAVO-EP (plasmid IL-12 electro gene transfer) and nivolumab (NIVO) in patients (pts) with operable locoregionally advanced melanoma

Abstract Introduction: IT TAVO-EP (tavokinogene telseplasmid delivered by electroporation) results in localized expression of IL12 in the tumor microenvironment (TME). This study (NCT04526730) evaluated NeoAd TAVO-EP and NIVO. All patients provided a written informed consent (Advarra IRB Pro00041794). Biomarkers of tumoral and systemic immune responses were conducted and correlated with clinical outcomes. Methods: NeoAd phase comprised up to 3 × 4-week cycles of IT TAVO-EP and iv NIVO followed by surgery. Endpoints included pathologic complete response (pCR), near pCR, major response (pMR; pCR + near pCR) and nonresponse (pNR). Biospecimens were collected at screening, C1D8, C2D1 (~30 days from treatment start), pre-surgery (~90 days). Slides cut from FFPE tissue were analyzed by chromogenic immunohistochemistry (IHC) for CD8+ tumor infiltrating lymphocytes (TIL) and PD-L1 (22C3 CDx assay). RNA extracted from FFPE tissue was subjected to NanoString’s IO360 transcriptomic analysis, including Tumor Inflammation Signature (TIS). PBMCs were stained and analyzed via flow cytometry on an LSR Fortessa X-20. Serum samples were analyzed for cytokine levels using Luminex® MAGPIX® platform (15-plex). Results: 16 pts were treated; 1 with PR declined surgery, 1 with early distant PD did not have surgery, 14 had surgery: 2 pNR, 3 near pCR, 9 pCR; pMR rate 12/15 (80%). In tumor, at baseline, 9/11 pts tested had <20% CD8+ TIL, 7/11 <10% PD-L1 tumor proportion score (TPS) and 6/10 TIS of ≤0, predicting non-response to anti-PD-1. In 5 pts with evaluable matched tissue at baseline and C2D1: 5/5 had increased peritumoral CD8+ T cells, 4/5 increased CD8+ TIL, 2/5 increased PD-L1, 4/5 increased TIS at C2D1 compared to baseline. Tumoral transcriptome profile revealed significant upregulation of genes involved in innate and adaptive immune responses (IFN-gamma, APM, granzymes, CD8, PD-L1, JAK1, chemokines, others) at C2D1. In blood, proliferating Ki-67+/PD-1+/CD8+ T cells expanded at C2D1 while total PD1+/CD8+ cells decreased. Decrease of PD1+CD8+ cells and other subtypes in blood at C2D1 coincided with TME infiltration by CD8+ cells. Serum effector cytokines including IL12, IFN-gamma and IL2, showed no biologically meaningful changes following treatment or differences between responders and non-responders. Conclusions: At baseline, most patients exhibited low CD8+ TIL, PD-L1 and TIS, with enhanced immune activation following treatment in the TME and blood including increased immune-related gene expression, CD8+ TIL, peritumoral CD8+ T cells and TIS. Four of 5 pts with negative predictive baseline biomarkers [CD8+TIL/PD-L1/TIS]low experienced pCRs supporting activity of IL12/anti-PD1 based regimens in this setting. Citation Format: Ahmad A. Tarhini, Zeynep Eroglu, Jonathan S. Zager, Ricardo J. Gonzalez, Amod A. Sarnaik, C Wayne Cruse, Deanryan B. De Aquino, Edith Abraham, Diana M. Acevedo, Allison Richards, Michael J. Schell, Denise Kalos, Pei-Ling Chen, Jane L. Messina, David A. Canton, Vernon K. Sondak. Tumoral and systemic immune modulation with neoadjuvant (NeoAd) intratumoral (IT) TAVO-EP (plasmid IL-12 electro gene transfer) and nivolumab (NIVO) in patients (pts) with operable locoregionally advanced melanoma [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 3734.

Abstract 6552: Intratumoral (ITu) delivery of mRNA-2752 encoding human OX40L/IL-23/IL-36γ in combination with durvalumab induces an immunostimulatory effect within the tumor microenvironment (TME) of patients with advanced solid tumors

Abstract Background mRNA-2752 is a first-in-class ITu therapy composed of mRNA encoding human OX40L/IL-23/IL-36γ in a lipid nanoparticle. The combination of T cell costimulation and proinflammatory cytokines were developed to modulate the TME and allow a more robust immune response. Here we show evidence of the pharmacodynamic effect of ITu administration of mRNA-2752 in combination with PD-L1 antibody. Methods In this ongoing phase 1 study (NCT03739931), patients (pts) with advanced solid tumors received 0.25-8 mg mRNA-2752 alone (Arm A) or in combination with 1500 mg IV durvalumab (Arm B)1,2. Pre and on-treatment peripheral and tumor samples were collected to monitor changes in cytokines by ligand binding assays, gene signatures by RNA sequencing, and T cell infiltration by immunohistochemistry. Tumor biopsies were taken at baseline, 24h, 15 and 28 days post treatment. Results mRNA-2752 administration in combination with durvalumab revealed ≥2-fold plasma level increases of IL-23 (34/40, 85%) and IFN-γ (50/65, 77%) at 24h after first injection; exposure was induced in a dose-dependent manner. We observed induction of CD8 T cell abundance (20/49, 41%) and proliferation (23/49, 47%), and OX40L protein expression (28/49, 57%) in pts with evaluable post-treatment injected lesion biopsies. CD8 T cell abundance increased in 2 pts with evaluable post-treatment un-injected lesion biopsies. A positive correlation was observed between T cell abundance and T cell effector genes. All three mRNA (IL-23, IL-36γ, and OX40L) encoded genes were detected post treatment in tumor samples from 43 evaluable patients. In 18 pts with injected lesion biopsy samples at baseline and day 28 samples, the genes related to cytotoxic function (IFNG, GZMB, PRF1), T cell migration (CCL21, CCR7, KLF2), T cell activation/memory phenotype (TCF7, CD69, ITGAE), and conventional type 1 dendritic cells (CLEC9A, XCR1) were induced 28 days post-treatment in pts with complete/partial response or stable disease (CR/PR/SD, n=7) compared to progressive disease (PD, n=11) pts. RNA-Seq transcriptome analysis showed increases in the immune inflamed gene signatures related to antitumor activity in 24/43 (56%) pts post-treatment, and the pre-existing immune inflamed phenotype within the TME was associated with clinical benefit. Conclusions mRNA-2752 ITu injection induced a systemic immunomodulatory effect by elevating effector cytokines. This was associated with pharmacodynamic changes within the TME, including increases in T cell abundance and proliferation, and upregulation of immune gene signatures that correlate with antitumor response.

Abstract 4151: Development of a cancer-activated biologic imaging platform for early lung cancer diagnosis

Abstract Current modalities, such as LDCT and fluorodeoxyglucose positron emission tomography (FDG PET), for early lung cancer imaging, rely upon proxies resulting in poor specificity and/or sensitivity. They are also unreliable for discriminating benign versus malignant lesions resulting in the requirement of longitudinal analyses euphemistically called “watchful waiting”. Lung biopsies are marred by significant false negative rates and pose complications of collapsed lungs or bleeding in up to 22% of procedures and death rates up to 1%. To overcome these obstacles, Earli is developing a non-invasive, highly specific cancer-activated imaging platform that usurps dysregulated gene expression within malignancies to force only cancer cells to produce a synthetic biomarker that can be localized using PET imaging. EARLI-204 is a lipid nanoparticle that contains a DNA nanoplasmid comprised of a cancer-activated promoter driving selective expression of a functionally inactivated somatostatin receptor 2 (SSTR2fi) gene. Abrogation of internalization and signal transduction allows the safe use of SSTR2fi as an ectopically expressed PET reporter gene (PRG). Following administration, EARLI-204 transfects tumor and normal cells but transcriptional activation only within cancer cells results in selective cell-surface expression of SSTR2fi that can be imaged with FDA-approved SSTR2 PET tracers. H1299 human lung cancer cells engineered to express SSTR2 (H1299-SSTR2) revealed a discernable PET signal with as little as 31,000 engineered cells. Mixed cell titration studies using subcutaneous (SQ) tumors with H1299-SSTR2 and non-expressing H1299-wt cells suggest tumors with between 0.1% and 1% H1299-SSTR2 cells had a significant PET signal, compared to tumors with 100% H1299-wt. This result sets a benchmark transfection of ~1% to achieve a PET signal. Intratumoral (IT) dose titration of EARLI-204 revealed that 5 ug of EARLI-204 generates a significant PET signal. Consistent with the H1299-SSTR2 studies, IHC shows ~1% of tumor cells express the PRG at a 5 ug IT dose level. Furthermore, we measured 70 copies of EARLI-204 DNA per cell resulting in 22,629 SSTR2 mRNA copies/25 ng at a 5 ug IT dose level. IV dosing of EARLI-204 in SQ and intralung tumor models results in cancer-activated expression of SSTR2fi in both SQ and small (<4 mm) lung tumors with significantly increased (p < 0.05) PET signal. For lung tumors, analyses showed 1 copy of EARLI-204 DNA/cell resulting in 780 SSTR2 mRNA copies/25 ng. The results demonstrate an effective molecular imaging platform for drug discovery ranging from in vivo screening to building PK-PD-efficacy relationships and enabling human dose projection for the Earli PET localization drug. The benchmarks and findings established with this preclinical imaging pipeline are currently being evaluated in large animals such as dogs and pigs, and will be translated to humans in planned clinical trials. Citation Format: Mohammed Goryawala, Hung-Yu Henry Lee, Ling Tong, Trupti Patil, Alex Harwig, Chloe Xia, Suthara Ramachandran, Dariusz Wodziak, Dean Felsher, Tim Sproul, David Suhy. Development of a cancer-activated biologic imaging platform for early lung cancer diagnosis [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 4151.

Abstract 725: Listeria-inspired phagosome escape drives STING responses to CD47 blockade

Abstract Purpose: Through phagocytosis, antigen-presenting cells (APCs) engulf and neutralize malignant cells. ​However, engulfed tumor cells are destroyed in phagolysosomes leaving few peptides to enter cytosol, where cross-presentation of antigens and immune activation occurs. Listeria (L.) monocytogenes escape phagolysosomes by secreting a pore-forming protein Listeriolysin O (LLO). Our breakthrough centers on harnessing LLO to create LLO-CD47, a novel protein-antibody conjugate that fuses the myeloid checkpoint anti-CD47 to LLO. By promoting tumor cell-phagocytosis and the release of tumor peptides and DNA from phagolysosomes, we hypothesize that LLO-CD47 enhances the immune functionalities of APCs. Procedures: Anti-CD47 was conjugated to LLO using a water-soluble SPDP crosslinker and purified by affinity chromatography. Transmission electron microscopy (TEM) was used to visualize the integrity of macrophage phagolysosomes following treatment. C57BL/6 mouse bone marrow-derived macrophages (BMDMs) were used to study M2-to-Ml polarization, tumor cell phagocytosis, STING activation, and antigen presentation. Mice bearing orthotopically implanted spontaneously metastatic murine triple-negative 4T1 tumors and E0771 tumors received intratumoral (IT) or intraperitoneal (IP) injection. CD8+ T cells or tumor-associated macrophages (TAMs) were depleted from tumor-bearing mice using an anti-CD8 antibody or anti-CSF-1R antibody prior to treatment. Results: 1. LLO-CD47 skews BMDMs from M2-to-M1 inflammatory phenotypes and enhances the phagocytosis of E0771 tumor cells. 2. BMDMs visualized by TEM show breaches in phagosome membranes following LLO-CD47, but not anti-CD47, treatment. 3. LLO-CD47 increases levels of phosphorylated STING, IFN, and TNFa in BMDMs in vitro and TAMs in vivo, relative to anti-CD47. 4. IT delivery of LLO-CD47 inhibits the growth and metastasis of orthotopically implanted murine breast tumors relative to anti-CD47. Harvested tumors show increased M1 polarized macrophages and CD8+ T cells, which express high levels of PD-1. 5. Co-administration of IP anti-PD-1 and LLO-CD47 delays the progression of breast tumor metastasis and prolongs animal survival relative to animals treated with anti-PD-1 and anti-CD47. 6. The elimination of CD8+ T cells or TAMs abrogates the antitumor effect of LLO-CD47. 7. IP administration of LLO-CD47 showed acceptable toxicity with stable body weight gain and no long-term hematological, renal, or hepatic toxicities. Conclusion: Conjugation of LLO to anti-CD47 enhances macrophage STING activation, antigen presentation, and tumor cell phagocytosis. Our work represents the first antibody-drug conjugate specifically designed to enhance immune activation against cancer.​ Citation Format: Benjamin R. Schrank, Yifan Wang, Abin Antony, Wen Jiang. Listeria-inspired phagosome escape drives STING responses to CD47 blockade [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 725.

Abstract 5004: Oncolytic Jurona-driven systemic and intratumoral immunotherapy combined with immune checkpoint blockade boost immune response and survival in hepatocellular carcinoma models

Abstract The members of the vesiculovirus genus have recently become attractive immunotherapeutic agents due to their natural selectivity for tumors and lack of toxicity in humans. Here, the research shows Intratumoral (IT) or Intraperitoneal (IP) injection of JURV was found to induce dynamic tumor regression in human hepatocellular carcinoma (HCC) xenografts and syngeneic models. Furthermore, IT injections of JURV were also shown to recruit and activate cytotoxic T lymphocytes and decrease tumor-associated macrophage infiltration, resulting in the delay of tumor growth in both local and distant murine HCC tumors established in a syngeneic model. Additionally, the concurrent use of JURV and anti-PD-1 antibodies was found to synergistically modulate the tumor microenvironment (TME) via increased tumor-infiltrating CD4+ T cells and depleted CD8+ PD-1+ and NK cells. Furthermore, using RIL-175, an aggressive metastatic HCC tumor mouse model, we show that intraperitoneal (IP) (systemic) administration of JURV and anti-PD1 significantly improved long-term survival (90%) compared to the (20%) JURV or anti-PD1 alone. Through proteogenomic analysis, the activation of different effectors of the immune system was observed to cooperate to alter the TME to a favorable anti-tumor immune state. These findings further support the development of JURV as an immunovirotherapy platform for HCC, with the potential to induce abscopal effects via the activation of several tumor suppressor genes and the mechanism regulating the T helper cell responses. Citation Format: Mulu Z. Tesfay, Khandoker U. Ferdous, Aleksandra Cios, Randal S. Shelton, Camila C. Simoes, Steven R. Post, Thomas J. Kelly, Martin J. Cannon, Alexei Basnakian, Bolni M. Nagalo. Oncolytic Jurona-driven systemic and intratumoral immunotherapy combined with immune checkpoint blockade boost immune response and survival in hepatocellular carcinoma models [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 5004.

Abstract 2458: Targeting the CXCR3 axis in HER2+ breast cancer to enhance anti-tumor immunity

Abstract Breast cancer (BC) is the most frequently diagnosed cancer in women worldwide with more than 2 million new cases in 2020. The efficacy of immunotherapies for BC is limited, with response rates of <10% for anti-PD-1/PDL1 monotherapy in pre-treated triple-negative breast cancer (TNBC) patients and even lower in subtypes that express human epidermal growth factor receptor 2 (HER2+) and/or hormone receptors. Hence, there is an urgent need to improve immune based strategies for BC. One possible approach to enhance response is the application of intratumoral (IT) immunotherapy utilizing IL-12; a potent pro-inflammatory cytokine that is critical in producing an anti-tumor response. However, systemic delivery of IL-12 has substantial toxicity and localized delivery of protein has insufficient residence time. To circumvent these issues, we have previously reported the efficacy of intratumorally injected plasmid IL-12 (pIL-12-EP) followed by electroporation in a TNBC mouse model and in clinical trials. Following pIL-12-EP, treated tumor expressed genes that were associated with increased CD8+ T cell infiltration, activation, trafficking, antigen presentation, and exhaustion. Single-cell RNA sequencing (scRNAseq) of tumor infiltrating immune cells from treated tumors revealed a striking enrichment of the CXCL9/10/11/CXCR3 family. This CXCR3 axis is upregulated in response to IFNγ production and has been studied extensively in melanoma and NSCLC patients as a potential predictive biomarker for responses to immune checkpoint inhibitors but has not been well characterized in BC. We hypothesized that this axis may be the critical component downstream of pIL-12-EP treatment that is responsible for anti-tumor responses and that targeting it will be effective in other molecular subtypes of BC beyond TNBC. We first evaluated the expression of CXCR3 by tumor infiltrating lymphocytes (TILs) in biopsies of human HER2+ BC. Using a single cell resolution spatial transcriptomic Xenium instrument we find not only expression of CXCR3 by TILs, but can also track the distance and gradients of the chemokine ligands CXCL9 and CXCL10 in this tumor microenvironment. Having confirmed the activity of this axis in human HER2+ BC, we used our well-established spontaneous HER2-driven mouse model to compare treatment of HER2+ tumors with pIL-12-EP or pCXCL9-EP intratumorally. We observed tumor growth inhibition in both treatment groups as compared to control treated tumors. Interestingly, the abscopal effect previously seen with pIL-12-EP was not evident for distant tumors in mice treated with pCXCL9-EP. These data suggest that IT CXCL9 therapies may benefit from combination with a systemic immune activator like a vaccine or immunogenic chemotherapy. Ongoing studies will continue to evaluate ways to better target this axis therapeutically and define clinically active combination therapies. Citation Format: Anchit Bhagat, Jesuchristopher Joseph, Joshua D. Ginzel, Christopher Rabiola, Amy C. Hobeika, Joshua C. Snyder, Herbert K. Lyerly, Erika J. Crosby. Targeting the CXCR3 axis in HER2+ breast cancer to enhance anti-tumor immunity [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 2458.

CXCL9/10-engineered dendritic cells promote T cell activation and enhance immune checkpoint blockade for lung cancer

Immune checkpoint blockade (ICB) with PD-1/PD-L1 inhibition has revolutionized the treatment of non-small cell lung cancer (NSCLC). Durable responses, however, are observed only in a subpopulation of patients. Defective antigen presentation and an immunosuppressive tumor microenvironment (TME) can lead to deficient Tcell recruitment and ICB resistance. We evaluate intratumoral (IT) vaccination with CXCL9- and CXCL10-engineered dendritic cells (CXCL9/10-DC) as a strategy to overcome resistance. IT CXCL9/10-DC leads to enhanced Tcell infiltration and activation in the TME and tumor inhibition in murine NSCLC models. The antitumor efficacy of IT CXCL9/10-DC is dependent on CD4+ and CD8+ Tcells, as well as CXCR3-dependent Tcell trafficking from the lymph node. IT CXCL9/10-DC, in combination with ICB, overcomes resistance and establishes systemic tumor-specific immunity in murine models. These studies provide a mechanistic understanding of CXCL9/10-DC-mediated host immune activation and support clinical translation of IT CXCL9/10-DC to augment ICB efficacy in NSCLC.

Accuracy of sentinel node mapping in Marjolin ulcer: Comparison of three different injection techniques

BackgroundMarjolin ulcer is a specific type of squamous cell cancer that can benefit from the use of lymphoscintigraphy.The purpose of this study was to evaluate 3 different injection techniques for sentinel node biopsy in patients with Marjolin ulcer. MethodsForty-eight patients with Marjolin ulcer (27 male and 21 female) ranging in age from 24 to 85 years were included in our study. Intratumoral (IT), peritumoral (PT) and periscar (PS) tissue injections of radiotracer were done in 9, 10, and 29 patients respectively. Injections were done 2–4 h before surgery. Lymphoscintigraphy was done for mapping the lymphatic drainage. During surgery, lymphatic mapping and sentinel node biopsy was performed using a handheld gamma probe. After harvesting sentinel nodes, regional lymph node dissection was done. ResultSentinel node detection rate was higher in the PS group as compared to the IT and PT groups (89.6% vs. 50% and 22.2%) respectively. False negative rate was 0%. Pathologically involved sentinel nodes were detected in 16% of the included patients of our study which led to management change. None of the patients with pathologically non-involved sentinel nodes developed regional lymph node recurrence. ConclusionLymphatic mapping and sentinel node biopsy is feasible in Marjolin ulcer with high detection rate and low false negative cases which by detection of regional lymph node involvement can potentially change the management of patients. Injection site may be an important factor influencing the success of the procedure and injection of the mapping material in the peri-scar normal skin proximal to the lesion is the best technique.