The development of non-opioid analgesics remains a major therapeutic priority. Although peptide-based drugs offer high target specificity, their clinical utility for brain disorders is limited by poor blood-brain barrier (BBB) permeability. The neurotensin (NT) peptide elicits potent antinociception via activation of the seven-transmembrane receptors NTS1 and NTS2; however, its therapeutic potential is constrained by rapid metabolic degradation and inadequate brain penetration. Here, we report the functional characterization of An2-NT(8-13), a conjugate linking the minimal active NT fragment to the BBB-permeant shuttle peptide angiopep-2, which targets the LRP1 receptor highly expressed at the brain endothelium. An2-NT(8-13) exhibited improved plasma stability while retaining high affinity for both NTS1 and NTS2. PET-CT imaging demonstrated efficient BBB translocation of radiolabeled An2-NT(8-13) following systemic administration via an LRP1-dependent mechanism. Both intravenous and subcutaneous administration of An2-NT(8-13) produced robust antinociceptive effects in vivo, significantly reducing nocifensive behaviors in both phases of the formalin tonic pain model. In the chronic constriction injury model of neuropathic pain, intravenous treatment reversed mechanical allodynia, demonstrating efficacy in an opioid-resistant pain condition. An2-NT(8-13) did not induce marked hypotension or hypothermia following subcutaneous administration and did not cause constipation or respiratory depression. Finally, no biochemical or histopathological subacute toxicity or motor impairment was observed after repeated treatment over five consecutive days (0.25 mg/kg, i.v., twice daily). Together, these findings identify An2-NT(8-13) as a BBB-penetrant NT analog capable of producing effective central analgesia while minimizing NT- and opioid-associated adverse effects, supporting An2 conjugation as a promising strategy for developing NT-based non-opioid analgesics.

3081 Background: TH1902 is a novel SORT1 targeting PDC. It exploits the natural function of SORT1, a scavenger receptor, which rapidly internalizes its natural ligands via endocytosis (internalization half-life ≤4 min). SORT1 is highly expressed in multiple solid tumors compared to normal healthy tissues making it an attractive target for rapid delivery of anti-cancer therapeutics. In this updated analysis, further data on long-term efficacy, safety and PK is presented from Parts 1&2 of Ph1 with focus on the 300 mg/m2 q3w. Due to safety observations in 6 patients (pts) enrolled at 420 mg/m2 dose level (Gr 3 neuropathy [n=2], Gr 4 neutropenia [n=2], Gr 3 ocular [n=1] and Gr 2 skin [n=3] toxicities, the dose was reduced to 300 mg/m2 in Part 2. Methods: Primary objective of the study was to characterize the safety/tolerability of TH1902. Part 1 (modified intrapatient dose escalation) included pts with recurrent/refractory advanced solid tumors (all comers) with no limit on number of previous therapies, including taxanes. Part 2 (dose expansion) included pts with known high SORT1 expression (e.g. OVC, endometrial, TNBC, melanoma). Results: Twenty-five heavily pretreated pts were enrolled in the 300 mg/m2 group. At least one TRAE was observed in 80% of pts. Grade 3 (Gr 3) events of interest were neuropathy (12%), keratitis (8%), anemia (8%), and neutropenia (4%), for an overall incidence of 32%. All grade neuropathy was 28%. Most TRAEs were mild to moderate severity and manageable with standard supportive care or dose reductions. Safety profile of TH1902 was different from that of docetaxel. PK measures of Cmax and AUC showed that exposure to free docetaxel was much lower than that of TH1902; Cmax = 0.58 μM for free docetaxel vs 30.4 μM for TH1902 and AUC24 = 3.1 h.nmol/mL for free docetaxel vs 74.8 h.nmol/mL for TH1902. Three pts exhibited RECIST 1.1 confirmed long-term stabilizations of disease, even after drug discontinuation, which ranged from 8 to 19 mos from treatment initiation, of which one OvC pt had overall PR (with RECIST 1.1 confirmed CR in target lesions) and remained on treatment for a total of 5 months. In addition, one endometrial cancer (part 1) was dose escalated from 60-360 mg/m2 and completed 11 cycles in total. Pt remained in SD during the 8 mos of treatment, up to time of consent withdrawal. All 4 pts had prior taxane exposure. Conclusions: TH1902 induces durable disease stabilization that lasts beyond treatment completion, suggesting a unique, multimodal mechanism of action (MOA) that differs from other cancer therapeutics. TH1902 has a manageable safety profile at 300mg/m2 with few Gr3 AEs. Low levels of free docetaxel in human plasma may in part explain the low rate of taxane related AEs (i.e. neutropenia, no alopecia). Next phase of the study involves dose optimization to further limit toxicity and improve efficacy. Clinical trial information: NCT04706962 .

The anticancer efficacy of Sudocetaxel Zendusortide (TH1902), a peptide-drug conjugate internalized through a sortilin-mediated process, was assessed in a triple-negative breast cancer-derived MDA-MB-231 immunocompromised xenograft tumor model where complete tumor regression was observed for more than 40 days after the last treatment. Surprisingly, immunohistochemistry analysis revealed high staining of STING, a master regulator in the cancer-immunity cycle. A weekly administration of TH1902 as a single agent in a murine B16-F10 melanoma syngeneic tumor model demonstrated superior tumor growth inhibition than did docetaxel. A net increase in CD45 leukocyte infiltration within TH1902-treated tumors, especially for tumor-infiltrating lymphocytes and tumor-associated macrophages was observed. Increased staining of perforin, granzyme B, and caspase-3 was suggestive of elevated cytotoxic T and natural killer cell activities. Combined TH1902/anti-PD-L1 treatment led to increases in tumor growth inhibition and median animal survival. TH1902 inhibited cell proliferation and triggered apoptosis and senescence in B16-F10 cells in vitro, while inducing several downstream effectors of the cGAS/STING pathway and the expression of MHC-I and PD-L1. This is the first evidence that TH1902 exerts its antitumor activity, in part, through modulation of the immune tumor microenvironment and that the combination of TH1902 with checkpoint inhibitors (anti-PD-L1) could lead to improved clinical outcomes.

Abstract Limitations in current colorectal cancer (CRC) camptothecin (CPT)-based chemotherapy have mostly been attributed to low specificity and high systemic cytotoxic side effects. More effective therapies are therefore required to improve the clinical outcomes of patients with CRC. Here, we developed a selective and better anticancer drug delivery of CPT through conjugation to a peptide (TH19P01) that targets Sortilin (SORT1), a scavenging receptor expressed in various tumor tissues including CRC. In the current study, significant SORT1 expression was detected in various CRC cell lines as well as that of irinotecan analogs efflux pump (ABCG2) in LoVo and HT-29 cells. Considering this result, we used our proprietary peptide conjugation SORT1 technology to increase cell targeting selectivity and cell delivery efficacy of CPT analogs. Different peptide drug conjugates (PDCs) were generated linking the TH19P01 peptide to SN-38 (an irinotecan metabolite) or exatecan, which are two main CPT derivatives used in recent antibody drug conjugates (ADC) as payloads. In vitro, immunonofluorescent microscopy revealed that TH19P01 was rapidly internalized (<15 min) in a SORT1-positive human HT-29 CRC cell model. These PDCs also inhibited CRC cell proliferation at low nM concentrations (3-90 nM). In vivo, weekly administration of TH2101 (SN-38) and of TH2303 (exatecan) conjugates were well tolerated as they had little impact on mouse body weight but caused a more potent growth inhibition of the HT-29 CRC tumor xenograft model than did either unconjugated irinotecan or exatecan molecules. In fact, at their maximum tolerable dose, irinotecan and exatecan caused a tumor growth inhibition of only 48% and 45% whereas TH2101 and TH2303 inhibited the growth of HT-29 tumors by 83% and 91%, respectively. These results provide strong pre-clinical evidence for the future development of novel CPT PDCs therapeutics with targeting of SORT1-positive CRC cells. Citation Format: Sanjoy Das, Jean-Christophe Currie, Michel Demeule, Cyndia Charfi, Alain Zgheib, Amit Nayyar, Anh Minh Thao Nguyen, Bogdan Alexandru Danalache, Richard Beliveau, Christian Marsolais, Borhane Annabi. Pre-clinical evidence for new camptothecin-peptide conjugates in the treatment of sortilin-positive colorectal cancers [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 2071.

Abstract The potential impact of the immune tumor microenvironment (iTME) was assessed with respect to the anticancer efficacy of sudocetaxel zendusortide (TH1902), a peptide-drug conjugate internalized through a sortilin (SORT1)-mediated endocytic pathway. In the triple-negative breast cancer (TNBC)-derived MDA-MB-231 immunocompromised xenograft tumor model, TH1902 induced complete tumor regression for more than 40 days after the last treatment. Surprisingly, immunohistochemistry analysis revealed higher staining of CD45 leukocytes and of STING, a master regulator in the cancer-immunity cycle, in comparison to docetaxel. Additionally, in a murine B16-F10 melanoma syngeneic tumor model, weekly administration of TH1902 as a single agent also demonstrated superior tumor growth inhibition than did docetaxel. Although B16-F10 is considered a non-immunogenic “cold” tumor model, we observed a net increase in CD45 leukocyte infiltration within TH1902-treated tumors, especially for tumor-infiltrating lymphocytes and tumor-associated macrophages. Moreover, in TH1902-treated tumors, increased staining of perforin, granzyme B, and caspase-3 was suggestive of elevated cytotoxic T and natural killer cell activities. Combined TH1902/anti-PD-L1 treatment led to increased tumor growth inhibition and median animal survival. Accordingly, TH1902 inhibited cell proliferation and triggered apoptosis and senescence in B16-F10 cells in vitro, while inducing several downstream effectors of the cGAS/STING pathway and the cell surface expression of MHC-I and PD-L1. This is the first evidence that TH1902 exerts its antitumor activity, in part, through modulation of the iTME and that the combination of TH1902 with checkpoint inhibitors (anti-PD-L1) could lead to improved clinical outcomes. Citation Format: Michel Demeule, Jean-Christophe Currie, Cyndia Charfi, Alain Zgheib, Isabelle Cousineau, Veronique Lullier, Richard Beliveau, Christian Marsolais, Borhane Annabi. Sudocetaxel Zendusortide (TH1902) triggers the cGAS/STING pathway and potentiates anti-PD-L1 immune-mediated tumor cell killing [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 3717.

A. In vitro binding of ANG4043 and Anti-HER2 mAb to the HER2/Fc chimera protein. B, C. Lack of effect of ANG4043 and Anti-HER2 on the proliferation of HER2-negative MCF-7 (B) and U-87 (C) cells using the [3H] thymidine incorporation assay.

Sortilin (SORT1), or neurotensin receptor-3, is a scavenging receptor in the Vacuolar Protein Sorting 10 protein (Vps10p) family. SORT1 is involved in the internalization and trafficking of its ligands through an endocytic process and is associated with cancer cell survival and progression, making SORT1 a candidate for novel drug delivery. We recently reported on the pattern and prevalence of SORT1 expression in endometrial, breast, ovarian, colorectal, pancreas cancers, and skin melanoma. To better understand SORT1 expression, we screened tissues from different cancer types using the same immunohistochemistry (IHC) method. A total of 19 cancer tissue microarrays (TMAs) with 1394 evaluable cancer cores were screened. Each cancer core was scored using an H-score ranging from 0 to 300, where 0 corresponds to no cell stained for SORT1, while 300 corresponds to strong SORT1 staining in all cells. The table below summarizes the % of cores with moderate to high SORT1 expression (defined as H-score ≥100) as well as the average H-Score for each cancer type evaluated. Sub-analyses of SORT1 expression by tumor histological sub-type, stage and grade are also being performed. A total of 234 healthy or normal adjacent tissues cores were also assessed. Weak or null staining was observed in these tissues. Moderate staining was observed in specific cell types in kidney tubules and glomeruli, colonic mucosa, splenic sinusoidal spaces in red pulp, blood vessels in smooth muscle of spleen and colon, dendritic and axonal extensions of pyramidal-type neurons in brain, and testicular seminiferous tubules. SORT1 is currently being studied as a cancer target in a first-in-human (FIH) study of a peptide-drug conjugate (clinicaltrial.gov: NCT04706962). These results suggest that SORT1 is highly expressed in multiple tumors and is a promising target for the delivery and internalization of cancer therapeutic agents. Table 1. Cancer Type No. evaluable cores % of indication with H-score ≥ 100 Average H-Score Endometrial 94 90 197 Thyroid 108 92 188 Melanoma 155 83 184 Lung 152 58 112 SCLC 44 95 183 NSCLC 108 43 82 Bladder 118 81 156 Testis 40 100 116 Small intestine 54 63 102 Eye 26 46 83 Cervix 376 38 75 Prostate 150 39 71 Liver 121 23 52 Citation Format: Guylaine Roy, Pratik Kadekar, Lynn Marie Douglas, Maude Frappier, Jean-Christophe Currie, Jess Dhillon, Gregory Cesarone, Richard Siderits, Karen Kirchner, Michel Demeule, Christian Marsolais. Differential expression of a novel transport receptor, SORT1 (sortilin), in cancer versus healthy tissues that can be utilized for targeted delivery of anti-cancer drugs. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3942.

A. Confirmation of HER2-positive expression status of BT-474 and SKBR3 cells and HER2-negative status of MCF-7, U-87 and HBMEC cells by WB analysis using an anti-HER2 mAb. B. Immunodetection of LRP1 by WB using an antibody directed against the 85 kDa subunit of LRP1 in U-87 and HBMEC. LRP1 expression is not detected in BT-474 cells.

Legends to supplemental figures 1-4.

SDS-PAGE of anti-HER2 and ANG4043 under reducing conditions with Coomassie Blue staining and immunodetection of An2 by WB. The An2 peptide is associated with two distinct bands corresponding to heavy and light chains of ANG4043.

Abstract Tumor-infiltrating immune cells are involved in the control of cancer and are closely related to clinical outcomes. Sudocetaxel Zendusortide (TH1902), a peptide-drug conjugate (PDC) of the sortilin (SORT1)-binding peptide TH19P01 ester-linked to two docetaxel moieties, has been shown to exert superior anti-cancer activities in multiple cancer models including melanoma syngeneic and xenograft murine models. Melanomas express elevated levels of SORT1 and are considered as one of the most immunogenic tumors where immune checkpoint inhibitors are among the standard of care treatment used. Here, we first questioned whether TH1902 anti-cancer effects involved infiltration of immune cells in a murine SORT1-positive syngeneic, non-immunogenic and highly aggressive B16-F10 melanoma model in C57BL/6 mice. Weekly administration of docetaxel reduced by half the growth of B16-F10 tumor allografts, while administration of TH1902 as a single agent and at equivalent docetaxel doses, was well tolerated and resulted in tumor regression after only 2 treatments. Surprisingly, in this immunologically cold tumor, immunohistochemistry analysis of tumors for immune cell infiltration showed a net increase in total leukocytes (CD45+) infiltration within TH1902-treated tumors compared with docetaxel-treated tumors, especially with for tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs). Furthermore, the increased staining intensities for Perforin and Granzyme B were indicative of the elevated cytotoxic T and natural killer (NK) cells activity in TH1902-treated tumors. This corroborated with increased caspase-3 apoptotic activity. TH1902 and docetaxel doses were next halved and combined with the checkpoint inhibitor anti-PD-L1. In a first study, the TH1902/anti-PD-L1 combination resulted in increased tumor growth inhibition when compared with both agents alone. Interestingly, TH1902 as a single agent showed better tumor growth inhibition than docetaxel or docetaxel/anti-PD-L1 combination. In a second study, the TH1902/anti-PD-L1 combination significantly increased animal survival over either anti-PD-L1 or TH1902 as single agents (21 days increased median survival compared to 2.5 and 12.5 days respectively). We conclude that the superiority of TH1902 anticancer activity over docetaxel involves, in part, the modulation of infiltrating immune cells within the tumor microenvironment. This is the first demonstration that immune cell infiltration patterns play a pivotal role in the TH1902-associated anti-tumoral response. Combination of TH1902 with checkpoint inhibitors (anti-PD-L1) further reveals that this may lead to improved clinical outcomes in future immunotherapy translational approaches. Citation Format: Michel Demeule, Jean-Christophe Currie, Cyndia Charfi, Alain Zgheib, Isabelle Cousineau, Richard Béliveau, Christian Marsolais, Borhane Annabi. The peptide-drug conjugate sudocetaxel zendusortide (TH1902) potentiates anti-tumoral activity of the anti-PD-L1 checkpoint inhibitor and induces immune cell infiltration in a B16-F10 syngeneic melanoma model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4499.

Over the last decade, the targeting of specific cell surface receptors such as human epidermal growth factor receptor 2 (Her2) in breast cancer cells has allowed the emergence of innovative strategies for the delivery of anticancer agents. High expression levels of the sortilin receptor (SORT1) have been reported in various tumors of breast cancer patients, including triple-negative breast cancer (TNBC), HR+ and Her2+ breast cancers. Given SORT1 function in ligand internalization, we addressed the sorting and trafficking of the newly designed TH19P01 peptide recognizing SORT1, and of TH1902, an anticancer TH19P01-docetaxel conjugate (PDC), and whether such PDC could target SORT1+ breast cancers. In vitro, high expression of SORT1 was found in several TNBC and Her2+ breast cancer cell lines as well as in in more than 88% of cases (H-score >100) from commercial breast cancer tissue microarrays. SORT1-mediated cell surface binding and internalization of TH19P01 was first investigated in TNBC-derived MDA-MB-231 cells. The binding and uptake of Alexa488-labeled TH19P01, at 4 and 37°C respectively, were found significantly reduced upon siRNA-mediated SORT1 silencing. This demonstrates that functional SORT1 processes are required for both cell surface recognition and internalization of TH19P01. Moreover, the internalization of TH19P01 and TH1902 in MDA-MB-231 cells were monitored by fluorescence microscopy using a polyclonal antibody generated against TH19P01. Both TH19P01 and TH1902 showed a rapid uptake and co-localized in the perinuclear region with the late endosomal marker Rab-7 and with the lysosomal marker Lamp-1 within 30 minutes indicating that both compounds are internalized through a receptor-mediated endocytosis pathway. In addition, TH19P01 internalization after 1 hour appeared significantly associated within intracellular compartments of MDA-MB-231 cells whereas Herceptin remained mostly localized at the cell surface of Her2+ BT-474 cells. In vivo, weekly administration of intravenous bolus of TH1902 (35 mg/kg) at an equivalent docetaxel MTD dose (15 mg/kg) led to complete tumor regression, while docetaxel only inhibited tumor growth by half in a murine MDA-MB-231 xenograft tumor model. Furthermore, in mice bearing Her2+ HCC-1954 breast tumor xenografts, TH1902 induced complete tumor regression in contrast to docetaxel and Herceptin. Taken together, these preclinical data demonstrate the high anticancer properties of TH1902 against SORT1+ TNBC and Herceptin resistant Her2+ breast cancers. These results demonstrate that TH1902 can be a promising avenue for personalized therapy in the treatment of all SORT1+ breast cancers. Citation Format: Cyndia Charfi, Michel Demeule, Jean-Christophe Currie, Alain Zgheib, Bogdan Alexandru Danalache, Richard Béliveau, Christian Marsolais, Borhane Annabi. Sudocetaxel Zendusortide (TH1902), a peptide-drug conjugate for the treatment of sortilin-positive (SORT1+) TNBC and Her2-positive breast cancers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4493.

Tumor-infiltrating immune cells are involved in the control of cancer and are closely related to clinical outcomes. Sudocetaxel Zendusortide (TH1902), a peptide-drug conjugate (PDC) of the sortilin (SORT1)-binding peptide TH19P01 ester-linked to two docetaxel moieties, has been shown to exert superior anti-cancer activities in multiple cancer models including melanoma syngeneic and xenograft murine models. Melanomas express elevated levels of SORT1 and are considered as one of the most immunogenic tumors where immune checkpoint inhibitors are among the standard of care treatment used. Here, we first questioned whether TH1902 anti-cancer effects involved infiltration of immune cells in a murine SORT1-positive syngeneic, non-immunogenic and highly aggressive B16-F10 melanoma model in C57BL/6 mice. Weekly administration of docetaxel reduced by half the growth of B16-F10 tumor allografts, while administration of TH1902 as a single agent and at equivalent docetaxel doses, was well tolerated and resulted in tumor regression after only 2 treatments. Surprisingly, in this immunologically cold tumor, immunohistochemistry analysis of tumors for immune cell infiltration showed a net increase in total leukocytes (CD45+) infiltration within TH1902-treated tumors compared with docetaxel-treated tumors, especially with for tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs). Furthermore, the increased staining intensities for Perforin and Granzyme B were indicative of the elevated cytotoxic T and natural killer (NK) cells activity in TH1902-treated tumors. This corroborated with increased caspase-3 apoptotic activity. TH1902 and docetaxel doses were next halved and combined with the checkpoint inhibitor anti-PD-L1. In a first study, the TH1902/anti-PD-L1 combination resulted in increased tumor growth inhibition when compared with both agents alone. Interestingly, TH1902 as a single agent showed better tumor growth inhibition than docetaxel or docetaxel/anti-PD-L1 combination. In a second study, the TH1902/anti-PD-L1 combination significantly increased animal survival over either anti-PD-L1 or TH1902 as single agents (21 days increased median survival compared to 2.5 and 12.5 days respectively). We conclude that the superiority of TH1902 anticancer activity over docetaxel involves, in part, the modulation of infiltrating immune cells within the tumor microenvironment. This is the first demonstration that immune cell infiltration patterns play a pivotal role in the TH1902-associated anti-tumoral response. Combination of TH1902 with checkpoint inhibitors (anti-PD-L1) further reveals that this may lead to improved clinical outcomes in future immunotherapy translational approaches. Citation Format: Michel Demeule, Jean-Christophe Currie, Cyndia Charfi, Alain Zgheib, Isabelle Cousineau, Richard Béliveau, Christian Marsolais, Borhane Annabi. The peptide-drug conjugate sudocetaxel zendusortide (TH1902) potentiates anti-tumoral activity of the anti-PD-L1 checkpoint inhibitor and induces immune cell infiltration in a B16-F10 syngeneic melanoma model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4499.

Syngeneic mouse models provide an effective approach for studying how cancer therapies perform in the presence of a functional immune system. Here TH1902, a peptide-drug conjugate (PDC) which consists of two molecules of docetaxel attached by a cleavable succinyl linker to a peptide (TH19P01) that targets the scavenger receptor sortilin (SORT1), was investigated using subcutaneous implantation of murine B16-F10 melanoma cells within the flanks of syngeneic C57BL/6 mice. Tumor growth was at most inhibited by half upon weekly administration of docetaxel compared to vehicle-treated mice, while administration of TH1902 was well tolerated and led to tumor regression. The B16-F10 SORT1-expressing melanoma cell line is also known to produce distant lung metastases when injected intravenously into the mice. Tail vein injection of these cells (1.25x105 cells per implantation) created a model system for evaluating TH1902’s effects on metastatic nodules formation. Each implantation produced 70 to 100 visible, black surface nodules on the lungs at 13 days following cancer cell implantation, appropriate for nodule quantification. No black nodules were detected elsewhere within the mice tissues other than those detected in the lungs. This model was next used to demonstrate that the formation of lung metastases could be sharply curtailed by bi-weekly intravenous administration of either 10 mg/kg docetaxel or 23 mg/kg TH1902 (equivalent to the docetaxel injection in terms of docetaxel content) during this period. Administration of docetaxel routinely decreased the number of metastatic lung nodules by about 50%, whereas administration of TH1902 decreased the number of nodules by more than 85%. Similar results were obtained whether the test articles were administered i) one time as a bolus at one hour prior to implantation of the cells, ii) as a bolus twice weekly beginning 3 days post-implantation, or iii) both prior to and following implantation as described. Although little weight loss was recorded over the 13-day treatment duration, all TH1902-treated mice showed better tolerability profile in terms of weight compared to docetaxel for all conditions tested. In a subsequent experiment using prolonged administration at the same dosage (up to 24 days of treatment), TH1902 was able to inhibit metastatic formation over a longer period compared to docetaxel. In fact, after six cycles of treatment, TH1902-treated mice significantly sustained its inhibitory effect on melanoma lungs metastasis formation (-77%) whereas docetaxel was unable to maintain its effect (-7%). Moreover, considerable weight loss was associated with docetaxel treatment over 24 days while TH1902 treatments resulted in no net change in mouse body weights. In this syngeneic model, TH1902 is more tolerated and effective than docetaxel at inhibiting both melanoma xenograft growth and metastatic formation. Citation Format: Michel Demeule, Jean-Christophe Currie, Cyndia Charfi, Alain Larocque, Alain Zgheib, Sophie Kozelko, Richard Béliveau, Christian Marsolais, Borhane Annabi. The peptide-drug conjugate TH1902 inhibits growth of subcutaneous melanoma xenografts and formation of lung metastases in a syngeneic mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1076.

Background: Breast and ovarian cancer stem cells (CSC) can contribute to the invasive and chemoresistance phenotype of tumors. TH1902, a newly developed sortilin (SORT1)-targeted peptide-docetaxel conjugate is currently in phase-1 clinical trial. Whether TH1902 impacts the chemoresistance phenotype of human triple-negative breast CSC (hTNBCSC) and ovarian CSC (hOvCSC) is unknown. Methods and Results: Immunophenotyping of hTNBCSC and hOvCSC was performed by flow cytometry and confirmed the expression of SORT1, and of CSC markers CD133, NANOG, and SOX2. Western blotting demonstrated the expression of the drug efflux pumps from the P-gp family members, ABCB1 and ABCB5. The cellular uptake of the fluorescent Alexa488-peptide from TH1902 was inhibited upon siRNA-mediated repression of SORT1 or upon competition with SORT1 ligands. In contrast to docetaxel, TH1902 inhibited in vitro migration, induced cell apoptosis and lead to G2/M cell cycle arrest of the hTNBCSC. These events were unaffected by the presence of the P-gp inhibitors cyclosporine A or PSC-833. In vivo, using immunosuppressed nude mice xenografts, TH1902 significantly inhibited the growth of hTNBCSC and hOvCSC xenografts (~80% vs. ~35% for docetaxel) when administered weekly as intravenous bolus for three cycles at 15 mg/kg, a dose equivalent to the maximal tolerated dose of docetaxel. Therapeutic efficacy was further observed when carboplatin was combined to TH1902. Conclusions: Overall, TH1902 exerts a superior anticancer activity than the unconjugated docetaxel, in part, by circumventing the CSC drug resistance phenotype that could potentially reduce cancer recurrence attributable to CSC.

Background: Sortilin (SORT1), or neurotensin receptor-3, is a scavenging receptor in the Vacuolar Protein Sorting 10 protein (Vps10p) family. SORT1 is involved in the internalization and trafficking of its ligands through an endocytic process and is reported to play a role in cancer cell survival and progression, making SORT1 a candidate for novel drug delivery. The pattern and prevalence of SORT1 expression in different healthy and cancer tissues is still not well understood.

Cancer stem cells (CSC), typically associated with CD133 expression, represent a small proportion of cells residing within most tumors and which have high metastatic potential and enhanced drug resistance capacities. In cancer indications such as breast and ovarian cancers, CSC have been shown to be involved in vasculogenic mimicry (VM) and to express the receptor protein sortilin (SORT1). The newly developed SORT1-targeted peptide-docetaxel conjugate (TH1902) has been reported to inhibit both the growth of xenograft tumors in mice and the formation of VM. This work now examines the efficacy of TH1902 against CSC and its potential to circumvent some of the known drug resistance phenotype associated with this population of cells. The effects of TH1902 on human triple-negative breast cancer or ovarian CSC (hTNBCSC and hOvCSC respectively) were explored using two commercial CD133-positive cell lines which are highly resistant to docetaxel and doxorubicin, and which express SORT1 along with the drug efflux pump P-gp and other CSC markers such as CD44, Oct4, Nanog and Sox2. The cellular uptake of the fluorescent Alexa488-peptide from TH1902 was inhibited by SORT1 ligands and in cells where SORT1 expression was repressed using siRNA. In contrast to docetaxel, the addition of TH1902 in vitro induced a marked increase in cell apoptosis and produced cell cycle arrest in the G2/M phase. Interestingly, TH1902-mediated cell cycle arrest was unaffected by the presence of the P-gp inhibitors cyclosporine A or PSC-833. However, the cell cycle which was unaffected by docetaxel was arrested in the G2/M phase in the presence of P-gp inhibitors to levels similar to those of TH1902 alone. These results suggest that TH1902 bypasses the P-gp drug efflux pump. Also, in contrast to docetaxel, exposure to TH1902 inhibited in vitro migration of the hTNBCSC in a wound-healing assay. In vivo, in both CSC models, TH1902 significantly inhibited the growth of CSC-tumor xenografts when administered weekly as intravenous bolus for three cycles at a dose equivalent to the maximal tolerated dose of docetaxel (15 mg/kg) and TH1902-treated mice showed an increased tolerability when compared to those treated with docetaxel. A decrease of approximately 80% of hTNBCSC or hOvCSC tumor growth was observed in TH1902-treated animals while tumor volumes were diminished by about 35% in docetaxel-treated mice. Overall, these results demonstrate that TH1902 exerts superior anticancer activity than the unconjugated docetaxel in two CD133-positive CSC animal models. Furthermore, these results show that TH1902 can circumvent the chemoresistance phenotype of CSC through use of a SORT1-mediated internalization process and therefore could possibly reduce the potential recurrence of cancers associated with CSC. Citation Format: Christian Marsolais, Cyndia Charfi, Michel Demeule, Jean-Christophe Currie, Alain Zgheib, Alain Larocque, Richard Béliveau, Borhane Annabi. TH1902, a SORT1 docetaxel peptide-drug conjugate, inhibits tumor growth of human cancer stem-like cells (CD133+) from both triple-negative breast cancers and ovarian cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1853.

Sortilin (SORT1) receptor-mediated endocytosis functions were exploited for this new approach for effective and safe treatments of gynecological cancers. Here, high expression of SORT1 was found in >75% of the clinically annotated ovarian and endometrial tumors analyzed by immunohistochemistry. Therefore, the anticancer properties of the peptide-drug conjugate TH1902, a peptide that targets SORT1 and which is linked to docetaxel molecules, were investigated both in vitro using ovarian and endometrial cancer cell cultures and in vivo using xenograft models. In vitro, TH1902 inhibited cell proliferation and triggered higher SORT1-dependent cell apoptosis than unconjugated docetaxel did in ES-2 and SKOV3 ovarian cancer cell lines. The uptake of the Alexa488-TH19P01 peptide from TH1902 was reduced upon siRNA-mediated silencing of SORT1. In vivo, weekly administration of TH1902 showed better tolerability compared to equivalent docetaxel doses and inhibited tumor growth in ovarian and endometrial xenograft mice models. TH1902 as a single agent inhibited ovarian tumor growth more than either of the unconjugated taxanes or carboplatin. Furthermore, TH1902 combination with carboplatin also demonstrated better efficacy when compared to both taxanes-carboplatin combinations. Overall, TH1902 shows better in vivo efficacy, compared to that of docetaxel and even paclitaxel, against SORT1-positive ovarian and endometrial cancers and could be safely combined with carboplatin.

Effective, safe chemotherapy treatments for gynecological cancers remain a daunting challenge. One strategy to achieve greater selectivity and better anticancer drug delivery into cancer cells is to conjugate cytotoxic agents to specific peptide ligands that selectively target receptors abundantly expressed on these cells. Increased expression of sortilin (SORT1), a scavenging receptor, has been shown to be highly expressed in many cancers, including ovarian and endometrial cancers. The peptide-drug conjugate TH1902 (a peptide which targets SORT1 and is bis-linked to two docetaxel molecules) was investigated to determine whether it could be efficacious against SORT1-positive ovarian (ES-2, A-2780 and SKOV-3) and endometrial (AN3-CA) cancer models. The Alexa488-fluorescent peptide used to generate TH1902 was transported into ES-2 and SKOV-3 cells by a process requiring clathrin-coated vesicles, and which was strongly diminished by siRNA-mediated SORT1 silencing or by competition with SORT1 ligands. In vitro, TH1902 enabled an increase in apoptosis of over 2-fold compared to that of unconjugated docetaxel. The effects of TH1902 administration were also examined in multiple subcutaneous xenograft tumor models (ES-2, SKOV-3, A-2780 or AN3-CA cells). Following two weeks treatment at equivalent docetaxel doses, the ES-2 xenograft sizes in mice treated with vehicle or with docetaxel were statistically indistinguishable but there was a significant decrease in tumor size by 78% for mice treated with TH1902. In SKOV-3 xenografts, treatment with either drug both stopped tumor growth and induced similar tumor regressions. In AN3-CA xenografts, treatments administered at equivalent docetaxel maximum tolerated dose (MTD) induced similar initial tumor regressions. However, TH1902-treated mice showed prolonged tumors regression whereas tumors started to regrow upon docetaxel treatment interruption. At one quarter of the docetaxel MTD, TH1902 significantly inhibited AN3-CA tumor growth whereas docetaxel had minimal effect (-73% vs -11%, respectively). Mice bearing A-2780 xenograft tumors were treated with TH1902, paclitaxel or docetaxel alone, as well as with each in combination with carboplatin. Comparison of the tumor sizes between the various treatments showed that TH1902 alone caused stronger A-2780 tumor growth inhibition than did either of the unconjugated taxanes or carboplatin. Moreover, TH1902 combined with carboplatin also demonstrated better efficacy than did either of the taxane-carboplatin combinations. Overall, the results indicate that TH1902 possesses an in vivo efficacy superior to those of docetaxel against ovarian and endometrial cancers in the animal models tested, and that TH1902 could be safely combined with carboplatin to reach optimal inhibition of tumor growth. Citation Format: Christian Marsolais, Michel Demeule, Cyndia Charfi, Jean-Christophe Currie, Alain Larocque, Alain Zgheib, Richard Béliveau, Borhane Annabi. Anti-cancer efficacy of TH1902, a SORT1 docetaxel peptide-drug conjugate, against ovarian and endometrial cancers xenografts alone or in combination with carboplatin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1079.