No abstract

The benefits of anti-cancer agents extend beyond direct tumor killing. One aspect of cell death is the potential to release antigens that initiate adaptive immune responses. Here, a diffusion enhanced formulation, INT230-6, containing potent anti-cancer cytotoxic agents, was administered intratumorally into large (approx. 300mm 3) subcutaneous murine Colon26 tumors. Treatment resulted in regression from baseline in 100% of the tumors and complete response in up to 90%. CD8 + or CD8 + /CD4 + T cell double-depletion at treatment onset prevented complete responses, indicating a critical role of T cells in promoting complete tumor regression. Mice with complete response were protected from subcutaneous and intravenous re-challenge of Colon26 cells in a CD4 + /CD8 + dependent manner. Thus, immunological T cell memory was induced by INT230-6. Colon26 tumors express the endogenous retroviral protein gp70 containing the CD8 + T-cell AH-1 epitope. AH-1-specific CD8 + T cells were detected in peripheral blood of tumor-bearing mice and their frequency increased 14 days after treatment onset. AH-1-specific CD8 + T cells were also significantly enriched in tumors of untreated mice. These cells had an activated phenotype and highly expressed Programmed cell-death protein-1 (PD-1) but did not lead to tumor regression. CD8 + T cell tumor infiltrate also increased 11 days after treatment. INT230-6 synergized with checkpoint blockade, inducing a complete remission of the primary tumors and shrinking of untreated contralateral tumors, which demonstrates not only a local but also systemic immunological effect of the combined therapy. Similar T-cell dependent inhibition of tumor growth was also found in an orthotopic 4T1 breast cancer model.

Heparin is not orally absorbed, presumably because of its size and polyanionic charge and hence is administered parenterally, either by continuous or intermittent infusion or by subcutaneous (SC) injection. However, a formulation that would result in absorption of heparin after oral administration would provide an attractive alternative to parenteral heparin. In that regard several attempts to develop effective non-parenteral heparin formulations have been reported, but they have met with limited success. The present investigation determined the molecular structure, pharmacokinetic (PK), and pharmacodynamic (PD) profiles of oral unfractionated heparin (UFH) containing oral absorption enhancer Sodium N-[8-(2-hydroxybenzoyl) amino] caprylate, salcaprozate sodium (SNAC) and assessed the safety and tolerability of orally dosed heparin solid dosage form versus other routes. Sixteen healthy men were included in this single-dose, three-way crossover, randomized, open-label study. The different arms of the study were as follow: Treatment A- Subjects randomized to Treatment A received two capsules of UFH/SNAC soft gelatin capsules for a total dose of 75,000 U heparin sodium with 150 mL of water; Treatment B: Subjects randomized to Treatment B received 1 mL of heparin sodium USP parenteral 10,000 U/mL via SC injection; Treatment C: Subjects randomized to receive 75,000 U heparin sodium milled powder for oral solution with 150 mL of water; and Treatment D: Subjects randomized to receive 0.5 mL of heparin sodium USP parenteral 5,000 U/mL via IV bolus. Each subject was randomly assigned to receive three of the four treatments so that a total of 12 subjects received each treatment. To characterize the PK of SNAC after oral heparin/SNAC administration and the PD of heparin following the administration of heparin IV, SC, PO alone and PO as heparin/SNAC, 21 blood samples were drawn from all of the subjects receiving treatment A, B, C, and D at the following time points: within 30 minutes pre-dose, at 2, 5, 10, 15, 20, 25, 30, 35, 40, 45 minutes, and at 1,1.5, 2, 2.5, 3, 3.5, 4, 5, 6, and 8 hours post-dose. Blood was drawn into sodium citrate tubes for assessment of anti-factor Xa, anti-factor IIa, aPTT, and SNAC. Disaccharide compositional analysis was performed using capillary high-performance liquid chromatography with electrospray ionization mass spectrometry detection. The PD of heparin was obtained from analysis of plasma anti-factor Xa, anti-factor IIa, aPTT, and total tissue factor pathway inhibitor (TFPI) data. The molecular weight properties and the disaccharide composition of orally administered UFH/SNAC and parenterally administered UFH are identical and consistent with the starting standard UFH administered and achieved anti-factor Xa: anti-factor IIa of 1:1. This is the first true pharmacokinetic study to measure the chemical compositions of heparin administered by different routes.

The recent development of immune-based therapies has improved the outcome for cancer patients; however, adjuvant therapies remain an important line of treatment for several cancer types. To maximize efficacy, checkpoint inhibitors are often combined with cytotoxic agents. While this approach often leads to increased tumor regression, higher off target toxicity often results in certain patients. This report describes a novel formulation comprising a unique amphiphilic molecule, 8-((2-hydroxybenzoyl)amino)octanoate (SHAO), that non-covalently interacts with payloads to increase drug dispersion and diffusion when dosed intratumorally (IT) into solid tumors. SHAO is co-formulated with cisplatin and vinblastine (referred to as INT230-6). IT dosing of the novel formulation achieved greater tumor growth inhibition and improved survival in in vivo tumor models compared to the same drugs without enhancer given intravenously or IT. INT230-6 treatment increased immune infiltrating cells in injected tumors with 10% to 20% of the animals having complete responses and developing systemic immunity to the cancer. INT230-6 was also shown to be synergistic with programmed cell death protein 1 (PD-1) antibodies at improving survival and increasing complete responses. INT230-6 induced significant tumor necrosis potentially releasing antigens to induce the systemic immune-based anti-cancer attack. This research demonstrates a novel, local treatment approach for cancer that minimizes systemic toxicity while stimulating adaptive immunity.

Background: INT230-6 is a novel formulation of cisplatin and vinblastine with an amphiphilic cell penetration enhancer that has been shown to enhance dispersion of the drug throughout tumors and allow diffusion into cells when given intratumorally. INT230-6 is being evaluated in monotherapy and in combination with immune checkpoint inhibitors (ICIs) in subjects with various advanced solid tumors, including advanced breast cancer. Methods: This phase 1/2 study evaluated INT230-6 in superficial and deep tumors with INT230-6 Q2W intratumoral injections for 5 doses alone or in combination with 200mg pembrolizumab IV Q3W for 2 years. Total INT230-6 injected in a subject ranged from 0.89 to 649 mL over 5 INT230-6 dosing sessions in each subject, except one subject who had only 2 dosing sessions. Subjects who had completed treatment in dose escalation cohorts were eligible for retreatment; and one subject was retreated with INT230-6 in multiple arms of the study. Advanced breast cancer subjects with an Eastern Cooperative Oncology Group (ECOG) performance status of 2 or below who have failed one or more approved therapies, or have no alternate approved therapy, were enrolled. Subjects must have adequate organ function and measurable disease by RECIST 1.1 criteria including one target tumor for injection. Tumor response using RECIST 1.1 was evaluated at 12 weeks from the first INT230-6 dose and then every 8 weeks. Tumor biopsies were taken prior to INT230-6 dosing on day 0 and on day 28 post dose. Results: 7 advanced triple negative breast cancer subjects (4 monotherapy, 3 pembrolizumab combination) were evaluable as of June 1, 2021. The median age was 56 (range 46-82) years old, with a median of 8 (2, 17) prior systemic therapies for metastatic disease. The intratumoral INT230-6 dose was up to 164 mL (82 mg of CIS, 16.4mg VIN) to tumors in a single dosing session. With INT230-6, 133-200% more volume is injected into the tumor and pharmacokinetics (PK) analysis shows that 95% of INT230-6 active agents remain in the tumor. Accordingly, assessment of tumor response using RECIST principles may be challenging, and even stable disease may represent a large decrease in viable tumor cells as indicated by biopsy evaluations. The most common (>20%) related treatment related adverse events (AE) were localized tumor related pain (71%), nausea (57%), anemia (29%), fatigue (29%), neck pain (29%), and vomiting (29%). AEs were mostly low grade and only one subject experienced grade 3 anemia (13%). There were no related grade 4 or 5 AEs or serious AEs. Disease control rate (DCR), defined as the percent of patients with a complete response, partial response, or stable disease at the first radiologic assessment, was 57%. Median overall survival was 12 months. Pre- and post- biopsy at 28 days after two INT230-6 doses (n= 3 evaluable, monotherapy, and combination with pembrolizumab) showed a 55% decrease in Ki67 and 69% reduction in viable cancer cells. In addition, multiplex immunofluorescence (n= 3 evaluable, combination with pembrolizumab) showed an influx of activated CD4 and CD8 T cells and in some cases a reduction in FoxP3 T-reg cells.. Conclusion: INT230-6 is a potential first-in-class intratumoral therapy for advanced breast cancer being developed in monotherapy and in combination with ICIs. There is a favorable safety profile in this population, similar to the broader metastatic solid tumor population presented elsewhere. There are early signs of cancer cell death in injected tumors and immune activation in heavily pre-treated patients. A Phase 2 expansion cohort of INT230-6 in combination with ICIs is ongoing. In addition, INT230-6 in being studied in a separate randomized Phase 2 neoadjuvant breast cancer study. Citation Format: Philippe Bedard, Lillian L Siu, Jacob Thomas, Diana Hanna, Anthony J Olszanski, Nilofer Azad, Giles Whalen, Matthew Ingham, Syed Mahmood, Lewis H Bender, Ian B Walters, Anthony El-Khoueiry. Safety and efficacy of INT230-6, a potential first-in-class intratumoral therapy, in monotherapy and in combination with pembrolizumab: Results from the IT-01 study [KEYNOTE-A10] in subjects with locally advanced, unresectable and metastatic breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-16-13.

2520 Background: INT230-6 is a new product with a unique dual anti-cancer mechanism. The drug is comprised of cisplatin (CIS) and vinblastine (VIN) co-formulated with an amphiphilic molecule that enables drug dispersion throughout a tumor and passive diffusion into cancer cells following intratumoral (IT) delivery. A neoadjuvant study in breast cancer confirms that a single injection can kill 95% of an injected tumor and recruit TILs. Methods: INT230-6 treatments are Q2W for up to 5 treatments followed by maintenance dosing every 9 weeks. Dose is set by the tumor’s longest diameter or volume. One arm received INT230-6 plus PEM 200mg IV Q3W. Biopsies from injected tumor are taken pretreatment and Day 28 for immunohistochemistry (IHC) analysis. Results: Sixty-two subjects received INT230-6 alone (median age 61, with 4 prior treatments), and 21 INT230-6 + PEM (median age 70, with 3 prior treatments). To these subjects over 575 image guided INT230-6 IT injections were given (320 to visceral tumors such as lung, liver, pancreas). Doses ranged from 0.14 to 175mL (87.5 mg of CIS, 17.5 mg VIN - higher than typical IV doses). Pharmacokinetic data shows > 95% of the INT230-6 active agents remain in the tumor. The most common ( > 25%) adverse events (AEs) related to INT230-6 were localized pain (58%), nausea (40%), and fatigue (29%). The most common AEs attributed to the PEM combination were nausea (62%), localized pain (57%), vomiting (57%), decreased appetite (43%), fatigue (43%) and constipation (29%). The incidence of grade 3 AEs for the INT230-6 arm was 11% and for the PEM combination was 14%. There were no related grade 4 or 5 AEs in the INT230-6 arm; and one grade 4 neutrophil count decrease was seen on the PEM combination. There were no dose limiting adverse events. No patient discontinued therapy due to toxicities related to either drug or injection procedure. The monotherapy arm enrolled patients from 17 tumor types; while the PEM combo recruited primarily pancreatic, CRC, triple negative breast, or bile duct cancer. IHC results confirm a marked reduction in proliferating tumor cells with influx of CD4 and CD8 T-cells. Seven of the INT230-6 monotherapy patients had non injected tumor shrinkage in 9 visceral/deep lesions. Estimated median overall survival (mOS) was over 1 year for both arms. Conclusions: In this clinical trial, deep and superficial tumor injections into patients with widely metastatic disease was feasible and well tolerated. Biopsies confirm the dual anti-cancer mechanism, and study patients live longer than would be expected for these refractory populations. INT230-6’s rapid tumor killing and immune activation properties may offer an alternative to control refractory patients (even those that are chemotherapy refractory) and the product is moving into randomized controlled registration trials. Clinical trial information: NCT03058289.

11557 Background: Patients have limited treatment options following initial chemotherapy failure. INT230-6, a novel formulation of cisplatin (CIS) and vinblastine (VIN) with an amphiphilic cell penetration enhancer, is designed for intratumoral (IT) administration. Study IT-01 (BMS # CA184-592, NCT 03058289) evaluates INT230-6 alone or in combination with ipilimumab (IPI), an antibody to CTLA-4. INT230-6 dosing is set by a % of the volume of the tumor to be injected. The product has been shown to disperse throughout an injected tumor and diffuse into cancer cells. Cell death leads to recruitment of dendritic and T cells, the effect of which may be augmented by CTLA-4 inhibition as evidenced by increased efficacy of the combination in preclinical models. Historically, checkpoint inhibitors have limited activity in sarcoma. Considering the large volume of drug injected and retained in the tumor, coupled with immune infiltration on biopsies, RECIST response methodology may not capture the benefits of INT230-6 treatment. Methods: IT-01 is an open-label phase 1/2 study that is enrolling adult subjects with locally advanced, unresectable or metastatic sarcoma. INT230-6 was administered IT Q2W for 5 doses alone or with IPI 3mg/kg IV Q3W for 4 doses. The study objectives are to assess the safety and efficacy of IT INT230-6 alone and in combination with IPI. Results: 16 heterogenous sarcoma subjects (13 monotherapy, 3 IPI combination) having a median of 3 prior therapies (0, 8) were enrolled to date. The INT230-6 dose was up to 145 mL (72.5 mg of CIS, 14.5 mg VIN) in a single session (an amount of each agent in excess of standard IV doses). The most common ( > 20%) related TEAEs in sarcoma subjects (n = 16) were localized pain (63%), fatigue (38%), decreased appetite (31%), nausea (31%), and vomiting (25%) most of which were low grade; with only grade 3 TEAE above 5% being anemia (13%). There were no related grade 4 or 5 TEAEs. In 11 evaluable monotherapy subjects, the disease control rate (DCR = CR+PD+SD) was 82%. Basket studies of sarcomas, including chordoma, with Royal Marsden Hospital index (RMHI) scores of 2 or higher report median overall survival (mOS) of 4 months. In this study 75% of monotherapy subjects had a RMHI score of 2 and preliminary estimates of mOS was 21.3 (4.67, NA) months. Pilot immunohistochemistry analysis of 5 paired (pre- and 28 days post-dose) biopsy samples showed substantial tumor necrosis, reduction of viable cancer, a decreased cancer proliferation as measured by Ki67, and increased TILs. Conclusions: Preliminary data shows that INT230-6 administered intratumorally alone or in combination with ipilimumab is well-tolerated in this small, heterogenous sarcoma population. The preclinical cancer cell death and immune infiltration mechanism of action appears to translate to sarcoma subjects. There are early signs of efficacy, DCR and potentially OS, that need to be confirmed in randomized studies. Clinical trial information: 03058289.

Standard care for many types of cancer involves systemic administration of cytotoxic agents. This may result in low drug concentration at tumor sites, which limits cell killing. More recently it has been shown that cytotoxic formulations designed for intratumoral delivery improve drug efficacy presumably by increasing drug concentration at the tumor site. Furthermore, it has been discovered that the mechanisms of anticancer agents extend beyond direct tumor cell lysis. One major aspect is that cell death often induces an immune response. Different types of cell death such as necrosis, apoptosis and autophagic cell death induced by cytotoxic agents trigger immune responses with varying degrees of inflammation and involving different types of immune cells. The ideal immune responses that may give maximum benefit to patients would be strong and long lasting anti-tumor T cell responses. In this study, a novel, tissue and cell diffusive cytotoxic formulation, INT230-6, was administered intratumorally over 5 sequential days into subcutaneous 300mm3 murine Colon26 tumors. Treatment resulted in regression from baseline of 100% of the tumors and up to 80% complete response (CR). We then analyzed the T cell responses in the protection induced by INT230-6. No animals achieved a CR after CD8 or CD8/CD4 double depletion at treatment onset, indicating a critical T-cell role in tumor regression. Mice with CRs were protected from re-challenge by either subcutaneous or intravenous re-inoculation of the Colon26. The protection was abrogated by CD4/CD8 double depletion prior to the re-challenge, indicating that immunological T cell memory was induced and was necessary for protection. Hence, INT230-6 given locally to treat tumors induces tumor-specific protective T cell immunity. Colon26 tumors express the endogenous retroviral protein gp70 containing the AH-1 CTL epitope. AH-1-specific CD8 T cells were detected in tumors of mice without treatment. However, these highly expressed PD-1. This was consistent with the observation that the PD-1 blockade enhanced the efficacy of INT230-6. Conversely, INT230-6 enhances the efficacy of checkpoint inhibitors. Citation Format: Anja C. Bloom, Lewis H. Bender, Ian B. Walters, Katharine T. Clark, Masaki Terabe, Jay A. Berzofsky. Tumor cell death caused by INT230-6 induces protective T cell immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5660. doi:10.1158/1538-7445.AM2017-5660