The cell adhesion molecule Nectin-4 shows elevated expression in multiple tumor types correlated with poor prognosis. Nectin-4-directed ADCs show efficacy in multiple xenograft tumor models. The ADC enfortumab vedotin (EV) delivers the antimitotic toxin MMAE to Nectin-4-expressing cells via internalization and cleavage of a valine-citrulline dipeptide linker component. Clinical validation of Nectin-4 as a target in urothelial cancer has been demonstrated with EV. In 2021, the FDA-approved EV for patients with locally advanced or metastatic urothelial cancer who had previously received a PD-1 or PD-L1 inhibitor and platinum-containing chemotherapy or were ineligible for cisplatin containing chemotherapy and had received one or more prior lines of therapy. Bicycle toxin conjugates (BTC) are structurally constrained bicyclic peptides conjugated through cleavable linkers to a toxin. Various, well-described linker toxin combinations can be incorporated into the BTC molecule (e.g., BT1718 and BT5528; refs.). They are of low molecular weight (approximately 4-4.5 kDa) and being chemically synthesized can be optimized for appropriate affinity, stability, and solubility relatively simply. Through intravenous administration, high systemic Cmax values can be attained, which, along with BTCs relatively small size, helps drive rapid diffusion into extra-vascular compartment, as reflected in a volume of distribution similar to extracellular fluid. We believe that delivery of a high number of BTCs, each carrying a reduced toxin load (peptide toxin ratio of 1:1) should improve tumor penetration and reduce the impact of the binding site barrier. BTCs show moderate clearance from the systemic vasculature, predominantly by the renal route. This overall profile marks them out from most ADCs and provides the possibility of enhanced clinical efficacy with a wider therapeutic index. BTCs are new therapeutic modality that shows a very different pharmacokinetic and structural profile to classic ADCs, whereas possessing robust tumoricidal properties. BTCs targeting MT1-MMP and EphA2 are currently in clinical trials. BT8009 is the most recent BTC to enter clinical trial. BT8009 is highly selective for Nectin-4 over other nectin family members and an extensive range of cell membrane expressed proteins. It shares the same cleavable linker and toxin combination as EV and can be cleaved by proteases in the TME, releasing cell penetrant MMAE that diffuses into tumor cells, or bystander stromal-supportive cells, evoking cell death, and tumor regression. It shows a robust and dose-dependent antitumor activity in multiple CDX and PDX models, representing lung, breast, bladder, head and neck cancers. Optimal tumor regression is associated with membrane expression of Nectin-4, in conjunction with MMAE sensitivity. Tumor regression rates are comparable for small and large tumors, indicative of deep and rapid penetration throughout the tumor. BT8009 provides tumoricidal activity with several regimens, potentially allowing for clinical titration of dose and dose interval if required.

The cell adhesion molecule Nectin-4 shows elevated expression in multiple tumor types correlated with poor prognosis. Nectin-4-directed ADCs show efficacy in multiple xenograft tumor models. The ADC enfortumab vedotin (EV) delivers the antimitotic toxin MMAE to Nectin-4-expressing cells via internalization and cleavage of a valine-citrulline dipeptide linker component. Clinical validation of Nectin-4 as a target in urothelial cancer has been demonstrated with EV. In 2021, the FDA-approved EV for patients with locally advanced or metastatic urothelial cancer who had previously received a PD-1 or PD-L1 inhibitor and platinum-containing chemotherapy or were ineligible for cisplatin containing chemotherapy and had received one or more prior lines of therapy. Bicycle toxin conjugates (BTC) are structurally constrained bicyclic peptides conjugated through cleavable linkers to a toxin. Various, well-described linker toxin combinations can be incorporated into the BTC molecule (e.g., BT1718 and BT5528; refs.). They are of low molecular weight (approximately 4-4.5 kDa) and being chemically synthesized can be optimized for appropriate affinity, stability, and solubility relatively simply. Through intravenous administration, high systemic Cmax values can be attained, which, along with BTCs relatively small size, helps drive rapid diffusion into extra-vascular compartment, as reflected in a volume of distribution similar to extracellular fluid. We believe that delivery of a high number of BTCs, each carrying a reduced toxin load (peptide toxin ratio of 1:1) should improve tumor penetration and reduce the impact of the binding site barrier. BTCs show moderate clearance from the systemic vasculature, predominantly by the renal route. This overall profile marks them out from most ADCs and provides the possibility of enhanced clinical efficacy with a wider therapeutic index. BTCs are new therapeutic modality that shows a very different pharmacokinetic and structural profile to classic ADCs, whereas possessing robust tumoricidal properties. BTCs targeting MT1-MMP and EphA2 are currently in clinical trials. BT8009 is the most recent BTC to enter clinical trial. BT8009 is highly selective for Nectin-4 over other nectin family members and an extensive range of cell membrane expressed proteins. It shares the same cleavable linker and toxin combination as EV and can be cleaved by proteases in the TME, releasing cell penetrant MMAE that diffuses into tumor cells, or bystander stromal-supportive cells, evoking cell death, and tumor regression. It shows a robust and dose-dependent antitumor activity in multiple CDX and PDX models, representing lung, breast, bladder, head and neck cancers. Optimal tumor regression is associated with membrane expression of Nectin-4, in conjunction with MMAE sensitivity. Tumor regression rates are comparable for small and large tumors, indicative of deep and rapid penetration throughout the tumor. BT8009 provides tumoricidal activity with several regimens, potentially allowing for clinical titration of dose and dose interval if required.

The cell adhesion molecule Nectin-4 shows elevated expression in multiple tumor types correlated with poor prognosis. Nectin-4-directed ADCs show efficacy in multiple xenograft tumor models. The ADC enfortumab vedotin (EV) delivers the antimitotic toxin MMAE to Nectin-4-expressing cells via internalization and cleavage of a valine-citrulline dipeptide linker component. Clinical validation of Nectin-4 as a target in urothelial cancer has been demonstrated with EV. In 2021, the FDA-approved EV for patients with locally advanced or metastatic urothelial cancer who had previously received a PD-1 or PD-L1 inhibitor and platinum-containing chemotherapy or were ineligible for cisplatin containing chemotherapy and had received one or more prior lines of therapy. Bicycle toxin conjugates (BTC) are structurally constrained bicyclic peptides conjugated through cleavable linkers to a toxin. Various, well-described linker toxin combinations can be incorporated into the BTC molecule (e.g., BT1718 and BT5528; refs.). They are of low molecular weight (approximately 4-4.5 kDa) and being chemically synthesized can be optimized for appropriate affinity, stability, and solubility relatively simply. Through intravenous administration, high systemic Cmax values can be attained, which, along with BTCs relatively small size, helps drive rapid diffusion into extra-vascular compartment, as reflected in a volume of distribution similar to extracellular fluid. We believe that delivery of a high number of BTCs, each carrying a reduced toxin load (peptide toxin ratio of 1:1) should improve tumor penetration and reduce the impact of the binding site barrier. BTCs show moderate clearance from the systemic vasculature, predominantly by the renal route. This overall profile marks them out from most ADCs and provides the possibility of enhanced clinical efficacy with a wider therapeutic index. BTCs are new therapeutic modality that shows a very different pharmacokinetic and structural profile to classic ADCs, whereas possessing robust tumoricidal properties. BTCs targeting MT1-MMP and EphA2 are currently in clinical trials. BT8009 is the most recent BTC to enter clinical trial. BT8009 is highly selective for Nectin-4 over other nectin family members and an extensive range of cell membrane expressed proteins. It shares the same cleavable linker and toxin combination as EV and can be cleaved by proteases in the TME, releasing cell penetrant MMAE that diffuses into tumor cells, or bystander stromal-supportive cells, evoking cell death, and tumor regression. It shows a robust and dose-dependent antitumor activity in multiple CDX and PDX models, representing lung, breast, bladder, head and neck cancers. Optimal tumor regression is associated with membrane expression of Nectin-4, in conjunction with MMAE sensitivity. Tumor regression rates are comparable for small and large tumors, indicative of deep and rapid penetration throughout the tumor. BT8009 provides tumoricidal activity with several regimens, potentially allowing for clinical titration of dose and dose interval if required.

The cell adhesion molecule Nectin-4 shows elevated expression in multiple tumor types correlated with poor prognosis. Nectin-4-directed ADCs show efficacy in multiple xenograft tumor models. The ADC enfortumab vedotin (EV) delivers the antimitotic toxin MMAE to Nectin-4-expressing cells via internalization and cleavage of a valine-citrulline dipeptide linker component. Clinical validation of Nectin-4 as a target in urothelial cancer has been demonstrated with EV. In 2021, the FDA-approved EV for patients with locally advanced or metastatic urothelial cancer who had previously received a PD-1 or PD-L1 inhibitor and platinum-containing chemotherapy or were ineligible for cisplatin containing chemotherapy and had received one or more prior lines of therapy. Bicycle toxin conjugates (BTC) are structurally constrained bicyclic peptides conjugated through cleavable linkers to a toxin. Various, well-described linker toxin combinations can be incorporated into the BTC molecule (e.g., BT1718 and BT5528; refs.). They are of low molecular weight (approximately 4-4.5 kDa) and being chemically synthesized can be optimized for appropriate affinity, stability, and solubility relatively simply. Through intravenous administration, high systemic Cmax values can be attained, which, along with BTCs relatively small size, helps drive rapid diffusion into extra-vascular compartment, as reflected in a volume of distribution similar to extracellular fluid. We believe that delivery of a high number of BTCs, each carrying a reduced toxin load (peptide toxin ratio of 1:1) should improve tumor penetration and reduce the impact of the binding site barrier. BTCs show moderate clearance from the systemic vasculature, predominantly by the renal route. This overall profile marks them out from most ADCs and provides the possibility of enhanced clinical efficacy with a wider therapeutic index. BTCs are new therapeutic modality that shows a very different pharmacokinetic and structural profile to classic ADCs, whereas possessing robust tumoricidal properties. BTCs targeting MT1-MMP and EphA2 are currently in clinical trials. BT8009 is the most recent BTC to enter clinical trial. BT8009 is highly selective for Nectin-4 over other nectin family members and an extensive range of cell membrane expressed proteins. It shares the same cleavable linker and toxin combination as EV and can be cleaved by proteases in the TME, releasing cell penetrant MMAE that diffuses into tumor cells, or bystander stromal-supportive cells, evoking cell death, and tumor regression. It shows a robust and dose-dependent antitumor activity in multiple CDX and PDX models, representing lung, breast, bladder, head and neck cancers. Optimal tumor regression is associated with membrane expression of Nectin-4, in conjunction with MMAE sensitivity. Tumor regression rates are comparable for small and large tumors, indicative of deep and rapid penetration throughout the tumor. BT8009 provides tumoricidal activity with several regimens, potentially allowing for clinical titration of dose and dose interval if required.

The cell adhesion molecule Nectin-4 shows elevated expression in multiple tumor types correlated with poor prognosis. Nectin-4-directed ADCs show efficacy in multiple xenograft tumor models. The ADC enfortumab vedotin (EV) delivers the antimitotic toxin MMAE to Nectin-4-expressing cells via internalization and cleavage of a valine-citrulline dipeptide linker component. Clinical validation of Nectin-4 as a target in urothelial cancer has been demonstrated with EV. In 2021, the FDA-approved EV for patients with locally advanced or metastatic urothelial cancer who had previously received a PD-1 or PD-L1 inhibitor and platinum-containing chemotherapy or were ineligible for cisplatin containing chemotherapy and had received one or more prior lines of therapy. Bicycle toxin conjugates (BTC) are structurally constrained bicyclic peptides conjugated through cleavable linkers to a toxin. Various, well-described linker toxin combinations can be incorporated into the BTC molecule (e.g., BT1718 and BT5528; refs.). They are of low molecular weight (approximately 4-4.5 kDa) and being chemically synthesized can be optimized for appropriate affinity, stability, and solubility relatively simply. Through intravenous administration, high systemic Cmax values can be attained, which, along with BTCs relatively small size, helps drive rapid diffusion into extra-vascular compartment, as reflected in a volume of distribution similar to extracellular fluid. We believe that delivery of a high number of BTCs, each carrying a reduced toxin load (peptide toxin ratio of 1:1) should improve tumor penetration and reduce the impact of the binding site barrier. BTCs show moderate clearance from the systemic vasculature, predominantly by the renal route. This overall profile marks them out from most ADCs and provides the possibility of enhanced clinical efficacy with a wider therapeutic index. BTCs are new therapeutic modality that shows a very different pharmacokinetic and structural profile to classic ADCs, whereas possessing robust tumoricidal properties. BTCs targeting MT1-MMP and EphA2 are currently in clinical trials. BT8009 is the most recent BTC to enter clinical trial. BT8009 is highly selective for Nectin-4 over other nectin family members and an extensive range of cell membrane expressed proteins. It shares the same cleavable linker and toxin combination as EV and can be cleaved by proteases in the TME, releasing cell penetrant MMAE that diffuses into tumor cells, or bystander stromal-supportive cells, evoking cell death, and tumor regression. It shows a robust and dose-dependent antitumor activity in multiple CDX and PDX models, representing lung, breast, bladder, head and neck cancers. Optimal tumor regression is associated with membrane expression of Nectin-4, in conjunction with MMAE sensitivity. Tumor regression rates are comparable for small and large tumors, indicative of deep and rapid penetration throughout the tumor. BT8009 provides tumoricidal activity with several regimens, potentially allowing for clinical titration of dose and dose interval if required.

The cell adhesion molecule Nectin-4 shows elevated expression in multiple tumor types correlated with poor prognosis. Nectin-4-directed ADCs show efficacy in multiple xenograft tumor models. The ADC enfortumab vedotin (EV) delivers the antimitotic toxin MMAE to Nectin-4-expressing cells via internalization and cleavage of a valine-citrulline dipeptide linker component. Clinical validation of Nectin-4 as a target in urothelial cancer has been demonstrated with EV. In 2021, the FDA-approved EV for patients with locally advanced or metastatic urothelial cancer who had previously received a PD-1 or PD-L1 inhibitor and platinum-containing chemotherapy or were ineligible for cisplatin containing chemotherapy and had received one or more prior lines of therapy. Bicycle toxin conjugates (BTC) are structurally constrained bicyclic peptides conjugated through cleavable linkers to a toxin. Various, well-described linker toxin combinations can be incorporated into the BTC molecule (e.g., BT1718 and BT5528; refs.). They are of low molecular weight (approximately 4-4.5 kDa) and being chemically synthesized can be optimized for appropriate affinity, stability, and solubility relatively simply. Through intravenous administration, high systemic Cmax values can be attained, which, along with BTCs relatively small size, helps drive rapid diffusion into extra-vascular compartment, as reflected in a volume of distribution similar to extracellular fluid. We believe that delivery of a high number of BTCs, each carrying a reduced toxin load (peptide toxin ratio of 1:1) should improve tumor penetration and reduce the impact of the binding site barrier. BTCs show moderate clearance from the systemic vasculature, predominantly by the renal route. This overall profile marks them out from most ADCs and provides the possibility of enhanced clinical efficacy with a wider therapeutic index. BTCs are new therapeutic modality that shows a very different pharmacokinetic and structural profile to classic ADCs, whereas possessing robust tumoricidal properties. BTCs targeting MT1-MMP and EphA2 are currently in clinical trials. BT8009 is the most recent BTC to enter clinical trial. BT8009 is highly selective for Nectin-4 over other nectin family members and an extensive range of cell membrane expressed proteins. It shares the same cleavable linker and toxin combination as EV and can be cleaved by proteases in the TME, releasing cell penetrant MMAE that diffuses into tumor cells, or bystander stromal-supportive cells, evoking cell death, and tumor regression. It shows a robust and dose-dependent antitumor activity in multiple CDX and PDX models, representing lung, breast, bladder, head and neck cancers. Optimal tumor regression is associated with membrane expression of Nectin-4, in conjunction with MMAE sensitivity. Tumor regression rates are comparable for small and large tumors, indicative of deep and rapid penetration throughout the tumor. BT8009 provides tumoricidal activity with several regimens, potentially allowing for clinical titration of dose and dose interval if required.

Nectin-4 is a cell adhesion molecule from the Nectin and Nectin-like family. It is a clinically validated tumor target and has been reported to be highly expressed in a wide range of solid tumors, including bladder, esophageal, pancreatic, and lung, but with limited distribution in healthy tissues.An antibody drug conjugate (PADCEV, enfortumab vedotin) that targets Nectin-4 was recently approved for the treatment of bladder cancer following the generation of positive data in a number of clinical studies. Herein we describe the discovery via phage display and subsequent chemical optimization of a Nectin-4 binding Bicycle and its incorporation into BT8009, a BTC that is currently under clinical evaluation. A detailed report of the pharmacologic properties of BT8009 has recently been described.In this, BT8009 shows potent efficacy in multiple tumor models, including patient-derived xenografts, across a variety of tumor indications and is well-tolerated in preclinical safety studies. In several models it demonstrated superior or equivalent efficacy to an analogue of the ADC PADCEV.

Nectin-4 is a cell adhesion molecule from the Nectin and Nectin-like family. It is a clinically validated tumor target and has been reported to be highly expressed in a wide range of solid tumors, including bladder, esophageal, pancreatic, and lung, but with limited distribution in healthy tissues.An antibody drug conjugate (PADCEV, enfortumab vedotin) that targets Nectin-4 was recently approved for the treatment of bladder cancer following the generation of positive data in a number of clinical studies. Herein we describe the discovery via phage display and subsequent chemical optimization of a Nectin-4 binding Bicycle and its incorporation into BT8009, a BTC that is currently under clinical evaluation. A detailed report of the pharmacologic properties of BT8009 has recently been described.In this, BT8009 shows potent efficacy in multiple tumor models, including patient-derived xenografts, across a variety of tumor indications and is well-tolerated in preclinical safety studies. In several models it demonstrated superior or equivalent efficacy to an analogue of the ADC PADCEV.

Nectin-4 is a cell adhesion molecule from the Nectin and Nectin-like family. It is a clinically validated tumor target and has been reported to be highly expressed in a wide range of solid tumors, including bladder, esophageal, pancreatic, and lung, but with limited distribution in healthy tissues.An antibody drug conjugate (PADCEV, enfortumab vedotin) that targets Nectin-4 was recently approved for the treatment of bladder cancer following the generation of positive data in a number of clinical studies. Herein we describe the discovery via phage display and subsequent chemical optimization of a Nectin-4 binding Bicycle and its incorporation into BT8009, a BTC that is currently under clinical evaluation. A detailed report of the pharmacologic properties of BT8009 has recently been described.In this, BT8009 shows potent efficacy in multiple tumor models, including patient-derived xenografts, across a variety of tumor indications and is well-tolerated in preclinical safety studies. In several models it demonstrated superior or equivalent efficacy to an analogue of the ADC PADCEV.

Here we describe the development of BT5528, a bicyclic peptide (Bicycle) conjugated to the auristatin derivative maleimidocaproyl-monomethyl auristatin E to generate the Bicycle toxin conjugate BT5528. There are two potential mechanism of BT5528 bystander activity: extracellular linker cleavage and toxin penetration into neighboring cells, or receptor internalization and intracellular linker cleavage followed by release of cell penetrant toxin from lysed cells. The available data do not allow us to distinguish between these two mechanisms and it seems likely that BT5528 activity is mediated by toxin release following a combination of intracellular and extracellular linker cleavage.

Here we describe the development of BT5528, a bicyclic peptide (Bicycle) conjugated to the auristatin derivative maleimidocaproyl-monomethyl auristatin E to generate the Bicycle toxin conjugate BT5528. There are two potential mechanism of BT5528 bystander activity: extracellular linker cleavage and toxin penetration into neighboring cells, or receptor internalization and intracellular linker cleavage followed by release of cell penetrant toxin from lysed cells. The available data do not allow us to distinguish between these two mechanisms and it seems likely that BT5528 activity is mediated by toxin release following a combination of intracellular and extracellular linker cleavage.

The cancer phenotype is commonly associated with aberrant glycosylation patterns. One glycan that is directly linked to cancer is the Thomsen-Friedenreich antigen (TF or CD176). It is a disaccharide composed of a galactose β1-3 N-acetylgalactosamine, O-linked to a glycoprotein through serine or threonine residues and commonly written as Galβ1-3GalNAc--O-Ser/Thr. The TF is therapeutically attractive due to its cryptic nature in normal cells and exposure in embryonic and cancer cells. The expression of the TF has been demonstrated in 90% of primary human carcinomas, including in the lung, the breast, and the pancreas. Additionally, cancer initiating cells or cancer stem cells in the lung, liver, and colon express the TF. The peptide sequence HGRFILPWWYAFSPS (TF-peptide) is known to bind tightly to the TF (Kd = 1.2 M) and has been demonstrated to inhibit processes directly involved in TF accessibility.

As a payload for PDCs (and ADCs), analogues of the duocarmycins are attractive. Duocarmycin SA and yatakemycin rank among the most potent natural cytotoxins discovered. The cyclopropyl and prodrug seco forms are both naturally occurring and equipotent in most circumstances. Studies of the binding-driven bonding model of their interaction with DNA suggest that their utility will be enhanced when targeted to tumor cells. In fact, SYD985, an ADC that utilizes a peptide linker for a duocarmycin analogue to trastuzumab, has recently been progressed to phase III clinical trial.

The cancer phenotype is commonly associated with aberrant glycosylation patterns. One glycan that is directly linked to cancer is the Thomsen-Friedenreich antigen (TF or CD176). It is a disaccharide composed of a galactose β1-3 N-acetylgalactosamine, O-linked to a glycoprotein through serine or threonine residues and commonly written as Galβ1-3GalNAc--O-Ser/Thr. The TF is therapeutically attractive due to its cryptic nature in normal cells and exposure in embryonic and cancer cells. The expression of the TF has been demonstrated in 90% of primary human carcinomas, including in the lung, the breast, and the pancreas. Additionally, cancer initiating cells or cancer stem cells in the lung, liver, and colon express the TF. The peptide sequence HGRFILPWWYAFSPS (TFα-peptide) is known to bind tightly to the TF (Kd = 1.2 M) and has been demonstrated to inhibit processes directly involved in TF accessibility.

The cancer phenotype is commonly associated with aberrant glycosylation patterns. One glycan that is directly linked to cancer is the Thomsen-Friedenreich antigen (TF or CD176). It is a disaccharide composed of a galactose β1-3 N-acetylgalactosamine, O-linked to a glycoprotein through serine or threonine residues and commonly written as Galβ1-3GalNAc--O-Ser/Thr. The TF is therapeutically attractive due to its cryptic nature in normal cells and exposure in embryonic and cancer cells. The expression of the TF has been demonstrated in 90% of primary human carcinomas, including in the lung, the breast, and the pancreas. Additionally, cancer initiating cells or cancer stem cells in the lung, liver, and colon express the TF. The peptide sequence HGRFILPWWYAFSPS (TF-peptide) is known to bind tightly to the TF (Kd = 1.2 M) and has been demonstrated to inhibit processes directly involved in TF accessibility.

As a payload for PDCs (and ADCs), analogues of the duocarmycins are attractive. Duocarmycin SA and yatakemycin rank among the most potent natural cytotoxins discovered. The cyclopropyl and prodrug seco forms are both naturally occurring and equipotent in most circumstances. Studies of the binding-driven bonding model of their interaction with DNA suggest that their utility will be enhanced when targeted to tumor cells. In fact, SYD985, an ADC that utilizes a peptide linker for a duocarmycin analogue to trastuzumab, has recently been progressed to phase III clinical trial.

The cancer phenotype is commonly associated with aberrant glycosylation patterns. One glycan that is directly linked to cancer is the Thomsen-Friedenreich antigen (TF or CD176). It is a disaccharide composed of a galactose β1-3 N-acetylgalactosamine, O-linked to a glycoprotein through serine or threonine residues and commonly written as Galβ1-3GalNAc--O-Ser/Thr. The TF is therapeutically attractive due to its cryptic nature in normal cells and exposure in embryonic and cancer cells. The expression of the TF has been demonstrated in 90% of primary human carcinomas, including in the lung, the breast, and the pancreas. Additionally, cancer initiating cells or cancer stem cells in the lung, liver, and colon express the TF. The peptide sequence HGRFILPWWYAFSPS (TF-peptide) is known to bind tightly to the TF (Kd = 1.2 M) and has been demonstrated to inhibit processes directly involved in TF accessibility.

As a payload for PDCs (and ADCs), analogues of the duocarmycins are attractive. Duocarmycin SA and yatakemycin rank among the most potent natural cytotoxins discovered. The cyclopropyl and prodrug seco forms are both naturally occurring and equipotent in most circumstances. Studies of the binding-driven bonding model of their interaction with DNA suggest that their utility will be enhanced when targeted to tumor cells. In fact, SYD985, an ADC that utilizes a peptide linker for a duocarmycin analogue to trastuzumab, has recently been progressed to phase III clinical trial.

As a payload for PDCs (and ADCs), analogues of the duocarmycins are attractive. Duocarmycin SA and yatakemycin rank among the most potent natural cytotoxins discovered. The cyclopropyl and prodrug seco forms are both naturally occurring and equipotent in most circumstances. Studies of the binding-driven bonding model of their interaction with DNA suggest that their utility will be enhanced when targeted to tumor cells. In fact, SYD985, an ADC that utilizes a peptide linker for a duocarmycin analogue to trastuzumab, has recently been progressed to phase III clinical trial.

Drug delivery systems (DDS) are promising tools for targeted tumor therapy providing the selective delivery of cytotoxic drugs to malignant cells, while side-effects and systemic toxicity are reduced. In addition to monoclonal antibodies (mAb), peptide ligands with a high affinity for tumor-specific cell surface compartments (e.g., receptors) can be used as carriers for cytotoxic payloads, as they provide beneficial features such as good tissue penetration, low immunogenicity and structural simplicity which enables their cost-efficient production by chemical synthesi. Receptors for the human gonadotropin releasing hormone (GnRH-I, <EHWSYGLRPG-NH2, <E is pyroglutamic acid) were not only identified in the pituitary, but also in various reproductive system-related cancers, such as breast, prostate and ovarian cancers, as well as non-reproductive cancers, such as colon and lung cancer. Thus, GnRH-related peptides are promising homing devices to deliver cytotoxic drugs selectively to cancer cells. A natural isoform of GnRH-I is the sea lamprey analog GnRH-III. This weak GnRH agonist binds to GnRH receptors (GnRH-R) on cancer cells and induces, like GnRH-I, a direct antitumor activity on several cancer cell lines, but its gonadotropin releasing activity is 500-1000 times lower in vitro and in vivo. Due to the direct anticancer activity and the low endocrine effect, GnRH-III and its derivatives have been successfully used as homing devices in in vitro and in vivo experiments. Encouraged by these promising findings, we report on the synthesis and biochemical characterization of eight cleavable self-immolative linker containing GnRH-III-drug conjugates. Of particular interest was the comparison of (1) two GnRH-III targeting moieties (GnRH-III-[4Lys(Bu)] (I) and GnRH-III-[2His,3D-Tic,4Lys(Bu)] (II)), (2) two cathepsin B-cleavable dipeptidyl-PABC linkers (Val-Ala and Val-Cit) and (3) two traditional anticancer drugs with different modes of action (Dau and PTX). For a better comparison and to demonstrate the proof of concept, four corresponding non-cleavable GnRH-III-Dau and -PTX conjugates have been developed and analyzed. The 8Lys of the targeting peptide was used as the ligation site. In the case of the Dau conjugates, the amino group of the daunosamine sugar has been used for attachment to the linker, while in the case of PTX, the C2-OH group was exploited for this purpose. All synthesized GnRH-III-Dau and -PTX conjugates were studied for their anticancer activity on A2780 ovarian and Panc-1 pancreatic cancer cells. Furthermore, the release of the drug by lysosomal enzymes and the GnRH-R binding affinities of the SMDC were examined.

Drug delivery systems (DDS) are promising tools for targeted tumor therapy providing the selective delivery of cytotoxic drugs to malignant cells, while side-effects and systemic toxicity are reduced. In addition to monoclonal antibodies (mAb), peptide ligands with a high affinity for tumor-specific cell surface compartments (e.g., receptors) can be used as carriers for cytotoxic payloads, as they provide beneficial features such as good tissue penetration, low immunogenicity and structural simplicity which enables their cost-efficient production by chemical synthesi. Receptors for the human gonadotropin releasing hormone (GnRH-I, <EHWSYGLRPG-NH2, <E is pyroglutamic acid) were not only identified in the pituitary, but also in various reproductive system-related cancers, such as breast, prostate and ovarian cancers, as well as non-reproductive cancers, such as colon and lung cancer. Thus, GnRH-related peptides are promising homing devices to deliver cytotoxic drugs selectively to cancer cells. A natural isoform of GnRH-I is the sea lamprey analog GnRH-III. This weak GnRH agonist binds to GnRH receptors (GnRH-R) on cancer cells and induces, like GnRH-I, a direct antitumor activity on several cancer cell lines, but its gonadotropin releasing activity is 500-1000 times lower in vitro and in vivo. Due to the direct anticancer activity and the low endocrine effect, GnRH-III and its derivatives have been successfully used as homing devices in in vitro and in vivo experiments. Encouraged by these promising findings, we report on the synthesis and biochemical characterization of eight cleavable self-immolative linker containing GnRH-III-drug conjugates. Of particular interest was the comparison of (1) two GnRH-III targeting moieties (GnRH-III-[4Lys(Bu)] (I) and GnRH-III-[2His,3D-Tic,4Lys(Bu)] (II)), (2) two cathepsin B-cleavable dipeptidyl-PABC linkers (Val-Ala and Val-Cit) and (3) two traditional anticancer drugs with different modes of action (Dau and PTX). For a better comparison and to demonstrate the proof of concept, four corresponding non-cleavable GnRH-III-Dau and -PTX conjugates have been developed and analyzed. The 8Lys of the targeting peptide was used as the ligation site. In the case of the Dau conjugates, the amino group of the daunosamine sugar has been used for attachment to the linker, while in the case of PTX, the C2-OH group was exploited for this purpose. All synthesized GnRH-III-Dau and -PTX conjugates were studied for their anticancer activity on A2780 ovarian and Panc-1 pancreatic cancer cells. Furthermore, the release of the drug by lysosomal enzymes and the GnRH-R binding affinities of the SMDC were examined.

Drug delivery systems (DDS) are promising tools for targeted tumor therapy providing the selective delivery of cytotoxic drugs to malignant cells, while side-effects and systemic toxicity are reduced. In addition to monoclonal antibodies (mAb), peptide ligands with a high affinity for tumor-specific cell surface compartments (e.g., receptors) can be used as carriers for cytotoxic payloads, as they provide beneficial features such as good tissue penetration, low immunogenicity and structural simplicity which enables their cost-efficient production by chemical synthesi. Receptors for the human gonadotropin releasing hormone (GnRH-I, <EHWSYGLRPG-NH2, <E is pyroglutamic acid) were not only identified in the pituitary, but also in various reproductive system-related cancers, such as breast, prostate and ovarian cancers, as well as non-reproductive cancers, such as colon and lung cancer. Thus, GnRH-related peptides are promising homing devices to deliver cytotoxic drugs selectively to cancer cells. A natural isoform of GnRH-I is the sea lamprey analog GnRH-III. This weak GnRH agonist binds to GnRH receptors (GnRH-R) on cancer cells and induces, like GnRH-I, a direct antitumor activity on several cancer cell lines, but its gonadotropin releasing activity is 500-1000 times lower in vitro and in vivo. Due to the direct anticancer activity and the low endocrine effect, GnRH-III and its derivatives have been successfully used as homing devices in in vitro and in vivo experiments. Encouraged by these promising findings, we report on the synthesis and biochemical characterization of eight cleavable self-immolative linker containing GnRH-III-drug conjugates. Of particular interest was the comparison of (1) two GnRH-III targeting moieties (GnRH-III-[4Lys(Bu)] (I) and GnRH-III-[2His,3D-Tic,4Lys(Bu)] (II)), (2) two cathepsin B-cleavable dipeptidyl-PABC linkers (Val-Ala and Val-Cit) and (3) two traditional anticancer drugs with different modes of action (Dau and PTX). For a better comparison and to demonstrate the proof of concept, four corresponding non-cleavable GnRH-III-Dau and -PTX conjugates have been developed and analyzed. The 8Lys of the targeting peptide was used as the ligation site. In the case of the Dau conjugates, the amino group of the daunosamine sugar has been used for attachment to the linker, while in the case of PTX, the C2-OH group was exploited for this purpose. All synthesized GnRH-III-Dau and -PTX conjugates were studied for their anticancer activity on A2780 ovarian and Panc-1 pancreatic cancer cells. Furthermore, the release of the drug by lysosomal enzymes and the GnRH-R binding affinities of the SMDC were examined.

Drug delivery systems (DDS) are promising tools for targeted tumor therapy providing the selective delivery of cytotoxic drugs to malignant cells, while side-effects and systemic toxicity are reduced. In addition to monoclonal antibodies (mAb), peptide ligands with a high affinity for tumor-specific cell surface compartments (e.g., receptors) can be used as carriers for cytotoxic payloads, as they provide beneficial features such as good tissue penetration, low immunogenicity and structural simplicity which enables their cost-efficient production by chemical synthesi. Receptors for the human gonadotropin releasing hormone (GnRH-I, <EHWSYGLRPG-NH2, <E is pyroglutamic acid) were not only identified in the pituitary, but also in various reproductive system-related cancers, such as breast, prostate and ovarian cancers, as well as non-reproductive cancers, such as colon and lung cancer. Thus, GnRH-related peptides are promising homing devices to deliver cytotoxic drugs selectively to cancer cells. A natural isoform of GnRH-I is the sea lamprey analog GnRH-III. This weak GnRH agonist binds to GnRH receptors (GnRH-R) on cancer cells and induces, like GnRH-I, a direct antitumor activity on several cancer cell lines, but its gonadotropin releasing activity is 500-1000 times lower in vitro and in vivo. Due to the direct anticancer activity and the low endocrine effect, GnRH-III and its derivatives have been successfully used as homing devices in in vitro and in vivo experiments. Encouraged by these promising findings, we report on the synthesis and biochemical characterization of eight cleavable self-immolative linker containing GnRH-III-drug conjugates. Of particular interest was the comparison of (1) two GnRH-III targeting moieties (GnRH-III-[4Lys(Bu)] (I) and GnRH-III-[2His,3D-Tic,4Lys(Bu)] (II)), (2) two cathepsin B-cleavable dipeptidyl-PABC linkers (Val-Ala and Val-Cit) and (3) two traditional anticancer drugs with different modes of action (Dau and PTX). For a better comparison and to demonstrate the proof of concept, four corresponding non-cleavable GnRH-III-Dau and -PTX conjugates have been developed and analyzed. The 8Lys of the targeting peptide was used as the ligation site. In the case of the Dau conjugates, the amino group of the daunosamine sugar has been used for attachment to the linker, while in the case of PTX, the C2-OH group was exploited for this purpose. All synthesized GnRH-III-Dau and -PTX conjugates were studied for their anticancer activity on A2780 ovarian and Panc-1 pancreatic cancer cells. Furthermore, the release of the drug by lysosomal enzymes and the GnRH-R binding affinities of the SMDC were examined.

Drug delivery systems (DDS) are promising tools for targeted tumor therapy providing the selective delivery of cytotoxic drugs to malignant cells, while side-effects and systemic toxicity are reduced. In addition to monoclonal antibodies (mAb), peptide ligands with a high affinity for tumor-specific cell surface compartments (e.g., receptors) can be used as carriers for cytotoxic payloads, as they provide beneficial features such as good tissue penetration, low immunogenicity and structural simplicity which enables their cost-efficient production by chemical synthesi. Receptors for the human gonadotropin releasing hormone (GnRH-I, <EHWSYGLRPG-NH2, <E is pyroglutamic acid) were not only identified in the pituitary, but also in various reproductive system-related cancers, such as breast, prostate and ovarian cancers, as well as non-reproductive cancers, such as colon and lung cancer. Thus, GnRH-related peptides are promising homing devices to deliver cytotoxic drugs selectively to cancer cells. A natural isoform of GnRH-I is the sea lamprey analog GnRH-III. This weak GnRH agonist binds to GnRH receptors (GnRH-R) on cancer cells and induces, like GnRH-I, a direct antitumor activity on several cancer cell lines, but its gonadotropin releasing activity is 500-1000 times lower in vitro and in vivo. Due to the direct anticancer activity and the low endocrine effect, GnRH-III and its derivatives have been successfully used as homing devices in in vitro and in vivo experiments. Encouraged by these promising findings, we report on the synthesis and biochemical characterization of eight cleavable self-immolative linker containing GnRH-III-drug conjugates. Of particular interest was the comparison of (1) two GnRH-III targeting moieties (GnRH-III-[4Lys(Bu)] (I) and GnRH-III-[2His,3D-Tic,4Lys(Bu)] (II)), (2) two cathepsin B-cleavable dipeptidyl-PABC linkers (Val-Ala and Val-Cit) and (3) two traditional anticancer drugs with different modes of action (Dau and PTX). For a better comparison and to demonstrate the proof of concept, four corresponding non-cleavable GnRH-III-Dau and -PTX conjugates have been developed and analyzed. The 8Lys of the targeting peptide was used as the ligation site. In the case of the Dau conjugates, the amino group of the daunosamine sugar has been used for attachment to the linker, while in the case of PTX, the C2-OH group was exploited for this purpose. All synthesized GnRH-III-Dau and -PTX conjugates were studied for their anticancer activity on A2780 ovarian and Panc-1 pancreatic cancer cells. Furthermore, the release of the drug by lysosomal enzymes and the GnRH-R binding affinities of the SMDC were examined.

Drug delivery systems (DDS) are promising tools for targeted tumor therapy providing the selective delivery of cytotoxic drugs to malignant cells, while side-effects and systemic toxicity are reduced. In addition to monoclonal antibodies (mAb), peptide ligands with a high affinity for tumor-specific cell surface compartments (e.g., receptors) can be used as carriers for cytotoxic payloads, as they provide beneficial features such as good tissue penetration, low immunogenicity and structural simplicity which enables their cost-efficient production by chemical synthesi. Receptors for the human gonadotropin releasing hormone (GnRH-I, <EHWSYGLRPG-NH2, <E is pyroglutamic acid) were not only identified in the pituitary, but also in various reproductive system-related cancers, such as breast, prostate and ovarian cancers, as well as non-reproductive cancers, such as colon and lung cancer. Thus, GnRH-related peptides are promising homing devices to deliver cytotoxic drugs selectively to cancer cells. A natural isoform of GnRH-I is the sea lamprey analog GnRH-III. This weak GnRH agonist binds to GnRH receptors (GnRH-R) on cancer cells and induces, like GnRH-I, a direct antitumor activity on several cancer cell lines, but its gonadotropin releasing activity is 500-1000 times lower in vitro and in vivo. Due to the direct anticancer activity and the low endocrine effect, GnRH-III and its derivatives have been successfully used as homing devices in in vitro and in vivo experiments. Encouraged by these promising findings, we report on the synthesis and biochemical characterization of eight cleavable self-immolative linker containing GnRH-III-drug conjugates. Of particular interest was the comparison of (1) two GnRH-III targeting moieties (GnRH-III-[4Lys(Bu)] (I) and GnRH-III-[2His,3D-Tic,4Lys(Bu)] (II)), (2) two cathepsin B-cleavable dipeptidyl-PABC linkers (Val-Ala and Val-Cit) and (3) two traditional anticancer drugs with different modes of action (Dau and PTX). For a better comparison and to demonstrate the proof of concept, four corresponding non-cleavable GnRH-III-Dau and -PTX conjugates have been developed and analyzed. The 8Lys of the targeting peptide was used as the ligation site. In the case of the Dau conjugates, the amino group of the daunosamine sugar has been used for attachment to the linker, while in the case of PTX, the C2-OH group was exploited for this purpose. All synthesized GnRH-III-Dau and -PTX conjugates were studied for their anticancer activity on A2780 ovarian and Panc-1 pancreatic cancer cells. Furthermore, the release of the drug by lysosomal enzymes and the GnRH-R binding affinities of the SMDC were examined.

Drug delivery systems (DDS) are promising tools for targeted tumor therapy providing the selective delivery of cytotoxic drugs to malignant cells, while side-effects and systemic toxicity are reduced. In addition to monoclonal antibodies (mAb), peptide ligands with a high affinity for tumor-specific cell surface compartments (e.g., receptors) can be used as carriers for cytotoxic payloads, as they provide beneficial features such as good tissue penetration, low immunogenicity and structural simplicity which enables their cost-efficient production by chemical synthesi. Receptors for the human gonadotropin releasing hormone (GnRH-I, <EHWSYGLRPG-NH2, <E is pyroglutamic acid) were not only identified in the pituitary, but also in various reproductive system-related cancers, such as breast, prostate and ovarian cancers, as well as non-reproductive cancers, such as colon and lung cancer. Thus, GnRH-related peptides are promising homing devices to deliver cytotoxic drugs selectively to cancer cells. A natural isoform of GnRH-I is the sea lamprey analog GnRH-III. This weak GnRH agonist binds to GnRH receptors (GnRH-R) on cancer cells and induces, like GnRH-I, a direct antitumor activity on several cancer cell lines, but its gonadotropin releasing activity is 500-1000 times lower in vitro and in vivo. Due to the direct anticancer activity and the low endocrine effect, GnRH-III and its derivatives have been successfully used as homing devices in in vitro and in vivo experiments. Encouraged by these promising findings, we report on the synthesis and biochemical characterization of eight cleavable self-immolative linker containing GnRH-III-drug conjugates. Of particular interest was the comparison of (1) two GnRH-III targeting moieties (GnRH-III-[4Lys(Bu)] (I) and GnRH-III-[2His,3D-Tic,4Lys(Bu)] (II)), (2) two cathepsin B-cleavable dipeptidyl-PABC linkers (Val-Ala and Val-Cit) and (3) two traditional anticancer drugs with different modes of action (Dau and PTX). For a better comparison and to demonstrate the proof of concept, four corresponding non-cleavable GnRH-III-Dau and -PTX conjugates have been developed and analyzed. The 8Lys of the targeting peptide was used as the ligation site. In the case of the Dau conjugates, the amino group of the daunosamine sugar has been used for attachment to the linker, while in the case of PTX, the C2-OH group was exploited for this purpose. All synthesized GnRH-III-Dau and -PTX conjugates were studied for their anticancer activity on A2780 ovarian and Panc-1 pancreatic cancer cells. Furthermore, the release of the drug by lysosomal enzymes and the GnRH-R binding affinities of the SMDC were examined.

Drug delivery systems (DDS) are promising tools for targeted tumor therapy providing the selective delivery of cytotoxic drugs to malignant cells, while side-effects and systemic toxicity are reduced. In addition to monoclonal antibodies (mAb), peptide ligands with a high affinity for tumor-specific cell surface compartments (e.g., receptors) can be used as carriers for cytotoxic payloads, as they provide beneficial features such as good tissue penetration, low immunogenicity and structural simplicity which enables their cost-efficient production by chemical synthesi. Receptors for the human gonadotropin releasing hormone (GnRH-I, <EHWSYGLRPG-NH2, <E is pyroglutamic acid) were not only identified in the pituitary, but also in various reproductive system-related cancers, such as breast, prostate and ovarian cancers, as well as non-reproductive cancers, such as colon and lung cancer. Thus, GnRH-related peptides are promising homing devices to deliver cytotoxic drugs selectively to cancer cells. A natural isoform of GnRH-I is the sea lamprey analog GnRH-III. This weak GnRH agonist binds to GnRH receptors (GnRH-R) on cancer cells and induces, like GnRH-I, a direct antitumor activity on several cancer cell lines, but its gonadotropin releasing activity is 500-1000 times lower in vitro and in vivo. Due to the direct anticancer activity and the low endocrine effect, GnRH-III and its derivatives have been successfully used as homing devices in in vitro and in vivo experiments. Encouraged by these promising findings, we report on the synthesis and biochemical characterization of eight cleavable self-immolative linker containing GnRH-III-drug conjugates. Of particular interest was the comparison of (1) two GnRH-III targeting moieties (GnRH-III-[4Lys(Bu)] (I) and GnRH-III-[2His,3D-Tic,4Lys(Bu)] (II)), (2) two cathepsin B-cleavable dipeptidyl-PABC linkers (Val-Ala and Val-Cit) and (3) two traditional anticancer drugs with different modes of action (Dau and PTX). For a better comparison and to demonstrate the proof of concept, four corresponding non-cleavable GnRH-III-Dau and -PTX conjugates have been developed and analyzed. The 8Lys of the targeting peptide was used as the ligation site. In the case of the Dau conjugates, the amino group of the daunosamine sugar has been used for attachment to the linker, while in the case of PTX, the C2-OH group was exploited for this purpose. All synthesized GnRH-III-Dau and -PTX conjugates were studied for their anticancer activity on A2780 ovarian and Panc-1 pancreatic cancer cells. Furthermore, the release of the drug by lysosomal enzymes and the GnRH-R binding affinities of the SMDC were examined.

Drug delivery systems (DDS) are promising tools for targeted tumor therapy providing the selective delivery of cytotoxic drugs to malignant cells, while side-effects and systemic toxicity are reduced. In addition to monoclonal antibodies (mAb), peptide ligands with a high affinity for tumor-specific cell surface compartments (e.g., receptors) can be used as carriers for cytotoxic payloads, as they provide beneficial features such as good tissue penetration, low immunogenicity and structural simplicity which enables their cost-efficient production by chemical synthesi. Receptors for the human gonadotropin releasing hormone (GnRH-I, <EHWSYGLRPG-NH2, <E is pyroglutamic acid) were not only identified in the pituitary, but also in various reproductive system-related cancers, such as breast, prostate and ovarian cancers, as well as non-reproductive cancers, such as colon and lung cancer. Thus, GnRH-related peptides are promising homing devices to deliver cytotoxic drugs selectively to cancer cells. A natural isoform of GnRH-I is the sea lamprey analog GnRH-III. This weak GnRH agonist binds to GnRH receptors (GnRH-R) on cancer cells and induces, like GnRH-I, a direct antitumor activity on several cancer cell lines, but its gonadotropin releasing activity is 500-1000 times lower in vitro and in vivo. Due to the direct anticancer activity and the low endocrine effect, GnRH-III and its derivatives have been successfully used as homing devices in in vitro and in vivo experiments. Encouraged by these promising findings, we report on the synthesis and biochemical characterization of eight cleavable self-immolative linker containing GnRH-III-drug conjugates. Of particular interest was the comparison of (1) two GnRH-III targeting moieties (GnRH-III-[4Lys(Bu)] (I) and GnRH-III-[2His,3D-Tic,4Lys(Bu)] (II)), (2) two cathepsin B-cleavable dipeptidyl-PABC linkers (Val-Ala and Val-Cit) and (3) two traditional anticancer drugs with different modes of action (Dau and PTX). For a better comparison and to demonstrate the proof of concept, four corresponding non-cleavable GnRH-III-Dau and -PTX conjugates have been developed and analyzed. The 8Lys of the targeting peptide was used as the ligation site. In the case of the Dau conjugates, the amino group of the daunosamine sugar has been used for attachment to the linker, while in the case of PTX, the C2-OH group was exploited for this purpose. All synthesized GnRH-III-Dau and -PTX conjugates were studied for their anticancer activity on A2780 ovarian and Panc-1 pancreatic cancer cells. Furthermore, the release of the drug by lysosomal enzymes and the GnRH-R binding affinities of the SMDC were examined.

Drug delivery systems (DDS) are promising tools for targeted tumor therapy providing the selective delivery of cytotoxic drugs to malignant cells, while side-effects and systemic toxicity are reduced. In addition to monoclonal antibodies (mAb), peptide ligands with a high affinity for tumor-specific cell surface compartments (e.g., receptors) can be used as carriers for cytotoxic payloads, as they provide beneficial features such as good tissue penetration, low immunogenicity and structural simplicity which enables their cost-efficient production by chemical synthesi. Receptors for the human gonadotropin releasing hormone (GnRH-I, <EHWSYGLRPG-NH2, <E is pyroglutamic acid) were not only identified in the pituitary, but also in various reproductive system-related cancers, such as breast, prostate and ovarian cancers, as well as non-reproductive cancers, such as colon and lung cancer. Thus, GnRH-related peptides are promising homing devices to deliver cytotoxic drugs selectively to cancer cells. A natural isoform of GnRH-I is the sea lamprey analog GnRH-III. This weak GnRH agonist binds to GnRH receptors (GnRH-R) on cancer cells and induces, like GnRH-I, a direct antitumor activity on several cancer cell lines, but its gonadotropin releasing activity is 500-1000 times lower in vitro and in vivo. Due to the direct anticancer activity and the low endocrine effect, GnRH-III and its derivatives have been successfully used as homing devices in in vitro and in vivo experiments. Encouraged by these promising findings, we report on the synthesis and biochemical characterization of eight cleavable self-immolative linker containing GnRH-III-drug conjugates. Of particular interest was the comparison of (1) two GnRH-III targeting moieties (GnRH-III-[4Lys(Bu)] (I) and GnRH-III-[2His,3D-Tic,4Lys(Bu)] (II)), (2) two cathepsin B-cleavable dipeptidyl-PABC linkers (Val-Ala and Val-Cit) and (3) two traditional anticancer drugs with different modes of action (Dau and PTX). For a better comparison and to demonstrate the proof of concept, four corresponding non-cleavable GnRH-III-Dau and -PTX conjugates have been developed and analyzed. The 8Lys of the targeting peptide was used as the ligation site. In the case of the Dau conjugates, the amino group of the daunosamine sugar has been used for attachment to the linker, while in the case of PTX, the C2-OH group was exploited for this purpose. All synthesized GnRH-III-Dau and -PTX conjugates were studied for their anticancer activity on A2780 ovarian and Panc-1 pancreatic cancer cells. Furthermore, the release of the drug by lysosomal enzymes and the GnRH-R binding affinities of the SMDC were examined.

Drug delivery systems (DDS) are promising tools for targeted tumor therapy providing the selective delivery of cytotoxic drugs to malignant cells, while side-effects and systemic toxicity are reduced. In addition to monoclonal antibodies (mAb), peptide ligands with a high affinity for tumor-specific cell surface compartments (e.g., receptors) can be used as carriers for cytotoxic payloads, as they provide beneficial features such as good tissue penetration, low immunogenicity and structural simplicity which enables their cost-efficient production by chemical synthesi. Receptors for the human gonadotropin releasing hormone (GnRH-I, <EHWSYGLRPG-NH2, <E is pyroglutamic acid) were not only identified in the pituitary, but also in various reproductive system-related cancers, such as breast, prostate and ovarian cancers, as well as non-reproductive cancers, such as colon and lung cancer. Thus, GnRH-related peptides are promising homing devices to deliver cytotoxic drugs selectively to cancer cells. A natural isoform of GnRH-I is the sea lamprey analog GnRH-III. This weak GnRH agonist binds to GnRH receptors (GnRH-R) on cancer cells and induces, like GnRH-I, a direct antitumor activity on several cancer cell lines, but its gonadotropin releasing activity is 500-1000 times lower in vitro and in vivo. Due to the direct anticancer activity and the low endocrine effect, GnRH-III and its derivatives have been successfully used as homing devices in in vitro and in vivo experiments. Encouraged by these promising findings, we report on the synthesis and biochemical characterization of eight cleavable self-immolative linker containing GnRH-III-drug conjugates. Of particular interest was the comparison of (1) two GnRH-III targeting moieties (GnRH-III-[4Lys(Bu)] (I) and GnRH-III-[2His,3D-Tic,4Lys(Bu)] (II)), (2) two cathepsin B-cleavable dipeptidyl-PABC linkers (Val-Ala and Val-Cit) and (3) two traditional anticancer drugs with different modes of action (Dau and PTX). For a better comparison and to demonstrate the proof of concept, four corresponding non-cleavable GnRH-III-Dau and -PTX conjugates have been developed and analyzed. The 8Lys of the targeting peptide was used as the ligation site. In the case of the Dau conjugates, the amino group of the daunosamine sugar has been used for attachment to the linker, while in the case of PTX, the C2-OH group was exploited for this purpose. All synthesized GnRH-III-Dau and -PTX conjugates were studied for their anticancer activity on A2780 ovarian and Panc-1 pancreatic cancer cells. Furthermore, the release of the drug by lysosomal enzymes and the GnRH-R binding affinities of the SMDC were examined.

Drug delivery systems (DDS) are promising tools for targeted tumor therapy providing the selective delivery of cytotoxic drugs to malignant cells, while side-effects and systemic toxicity are reduced. In addition to monoclonal antibodies (mAb), peptide ligands with a high affinity for tumor-specific cell surface compartments (e.g., receptors) can be used as carriers for cytotoxic payloads, as they provide beneficial features such as good tissue penetration, low immunogenicity and structural simplicity which enables their cost-efficient production by chemical synthesi. Receptors for the human gonadotropin releasing hormone (GnRH-I, <EHWSYGLRPG-NH2, <E is pyroglutamic acid) were not only identified in the pituitary, but also in various reproductive system-related cancers, such as breast, prostate and ovarian cancers, as well as non-reproductive cancers, such as colon and lung cancer. Thus, GnRH-related peptides are promising homing devices to deliver cytotoxic drugs selectively to cancer cells. A natural isoform of GnRH-I is the sea lamprey analog GnRH-III. This weak GnRH agonist binds to GnRH receptors (GnRH-R) on cancer cells and induces, like GnRH-I, a direct antitumor activity on several cancer cell lines, but its gonadotropin releasing activity is 500-1000 times lower in vitro and in vivo. Due to the direct anticancer activity and the low endocrine effect, GnRH-III and its derivatives have been successfully used as homing devices in in vitro and in vivo experiments. Encouraged by these promising findings, we report on the synthesis and biochemical characterization of eight cleavable self-immolative linker containing GnRH-III-drug conjugates. Of particular interest was the comparison of (1) two GnRH-III targeting moieties (GnRH-III-[4Lys(Bu)] (I) and GnRH-III-[2His,3D-Tic,4Lys(Bu)] (II)), (2) two cathepsin B-cleavable dipeptidyl-PABC linkers (Val-Ala and Val-Cit) and (3) two traditional anticancer drugs with different modes of action (Dau and PTX). For a better comparison and to demonstrate the proof of concept, four corresponding non-cleavable GnRH-III-Dau and -PTX conjugates have been developed and analyzed. The 8Lys of the targeting peptide was used as the ligation site. In the case of the Dau conjugates, the amino group of the daunosamine sugar has been used for attachment to the linker, while in the case of PTX, the C2-OH group was exploited for this purpose. All synthesized GnRH-III-Dau and -PTX conjugates were studied for their anticancer activity on A2780 ovarian and Panc-1 pancreatic cancer cells. Furthermore, the release of the drug by lysosomal enzymes and the GnRH-R binding affinities of the SMDC were examined.

Towards these ends, we report on the design of a modular and user-friendly approach to enable bio-orthogonal conjugation of synthetic amanitins to three different linkers that are elaborated to three potent peptide-amanitin conjugates that differ by their mode of toxin release, as defined by the linker. Octreotate (TATE-N3, 2) was chosen as it is a circulation-stable octapeptide somatostatin analog that agonizes somatostatin receptors (Kd &tide; 0.4 nM for sstr2). Targeting sstr2 has been successfully exploited for drug delivery, cancer imaging, and radiotherapy owing to the high levels of sstr2 expression on neuroendocrine tumors (e.g. carcinoids, pancreatic islet cell tumors, thyroid carcinomas, and small lung cancer to name a few). While this is the first report of an octreotate-amanitin conjugate, herein, octreotate serves as an exemplar to highlight the potential for developing peptide-amanitin conjugates in targeted applications.

Towards these ends, we report on the design of a modular and user-friendly approach to enable bio-orthogonal conjugation of synthetic amanitins to three different linkers that are elaborated to three potent peptide-amanitin conjugates that differ by their mode of toxin release, as defined by the linker. Octreotate (TATE-N3, 2) was chosen as it is a circulation-stable octapeptide somatostatin analog that agonizes somatostatin receptors (Kd &tide; 0.4 nM for sstr2). Targeting sstr2 has been successfully exploited for drug delivery, cancer imaging, and radiotherapy owing to the high levels of sstr2 expression on neuroendocrine tumors (e.g. carcinoids, pancreatic islet cell tumors, thyroid carcinomas, and small lung cancer to name a few). While this is the first report of an octreotate-amanitin conjugate, herein, octreotate serves as an exemplar to highlight the potential for developing peptide-amanitin conjugates in targeted applications.

Drug delivery systems (DDS) are promising tools for targeted tumor therapy providing the selective delivery of cytotoxic drugs to malignant cells, while side-effects and systemic toxicity are reduced. In addition to monoclonal antibodies (mAb), peptide ligands with a high affinity for tumor-specific cell surface compartments (e.g., receptors) can be used as carriers for cytotoxic payloads, as they provide beneficial features such as good tissue penetration, low immunogenicity and structural simplicity which enables their cost-efficient production by chemical synthesi. Receptors for the human gonadotropin releasing hormone (GnRH-I, <EHWSYGLRPG-NH2, <E is pyroglutamic acid) were not only identified in the pituitary, but also in various reproductive system-related cancers, such as breast, prostate and ovarian cancers, as well as non-reproductive cancers, such as colon and lung cancer. Thus, GnRH-related peptides are promising homing devices to deliver cytotoxic drugs selectively to cancer cells. A natural isoform of GnRH-I is the sea lamprey analog GnRH-III. This weak GnRH agonist binds to GnRH receptors (GnRH-R) on cancer cells and induces, like GnRH-I, a direct antitumor activity on several cancer cell lines, but its gonadotropin releasing activity is 500-1000 times lower in vitro and in vivo. Due to the direct anticancer activity and the low endocrine effect, GnRH-III and its derivatives have been successfully used as homing devices in in vitro and in vivo experiments. Encouraged by these promising findings, we report on the synthesis and biochemical characterization of eight cleavable self-immolative linker containing GnRH-III-drug conjugates. Of particular interest was the comparison of (1) two GnRH-III targeting moieties (GnRH-III-[4Lys(Bu)] (I) and GnRH-III-[2His,3D-Tic,4Lys(Bu)] (II)), (2) two cathepsin B-cleavable dipeptidyl-PABC linkers (Val-Ala and Val-Cit) and (3) two traditional anticancer drugs with different modes of action (Dau and PTX). For a better comparison and to demonstrate the proof of concept, four corresponding non-cleavable GnRH-III-Dau and -PTX conjugates have been developed and analyzed. The 8Lys of the targeting peptide was used as the ligation site. In the case of the Dau conjugates, the amino group of the daunosamine sugar has been used for attachment to the linker, while in the case of PTX, the C2-OH group was exploited for this purpose. All synthesized GnRH-III-Dau and -PTX conjugates were studied for their anticancer activity on A2780 ovarian and Panc-1 pancreatic cancer cells. Furthermore, the release of the drug by lysosomal enzymes and the GnRH-R binding affinities of the SMDC were examined.

Drug delivery systems (DDS) are promising tools for targeted tumor therapy providing the selective delivery of cytotoxic drugs to malignant cells, while side-effects and systemic toxicity are reduced. In addition to monoclonal antibodies (mAb), peptide ligands with a high affinity for tumor-specific cell surface compartments (e.g., receptors) can be used as carriers for cytotoxic payloads, as they provide beneficial features such as good tissue penetration, low immunogenicity and structural simplicity which enables their cost-efficient production by chemical synthesi. Receptors for the human gonadotropin releasing hormone (GnRH-I, <EHWSYGLRPG-NH2, <E is pyroglutamic acid) were not only identified in the pituitary, but also in various reproductive system-related cancers, such as breast, prostate and ovarian cancers, as well as non-reproductive cancers, such as colon and lung cancer. Thus, GnRH-related peptides are promising homing devices to deliver cytotoxic drugs selectively to cancer cells. A natural isoform of GnRH-I is the sea lamprey analog GnRH-III. This weak GnRH agonist binds to GnRH receptors (GnRH-R) on cancer cells and induces, like GnRH-I, a direct antitumor activity on several cancer cell lines, but its gonadotropin releasing activity is 500-1000 times lower in vitro and in vivo. Due to the direct anticancer activity and the low endocrine effect, GnRH-III and its derivatives have been successfully used as homing devices in in vitro and in vivo experiments. Encouraged by these promising findings, we report on the synthesis and biochemical characterization of eight cleavable self-immolative linker containing GnRH-III-drug conjugates. Of particular interest was the comparison of (1) two GnRH-III targeting moieties (GnRH-III-[4Lys(Bu)] (I) and GnRH-III-[2His,3D-Tic,4Lys(Bu)] (II)), (2) two cathepsin B-cleavable dipeptidyl-PABC linkers (Val-Ala and Val-Cit) and (3) two traditional anticancer drugs with different modes of action (Dau and PTX). For a better comparison and to demonstrate the proof of concept, four corresponding non-cleavable GnRH-III-Dau and -PTX conjugates have been developed and analyzed. The 8Lys of the targeting peptide was used as the ligation site. In the case of the Dau conjugates, the amino group of the daunosamine sugar has been used for attachment to the linker, while in the case of PTX, the C2-OH group was exploited for this purpose. All synthesized GnRH-III-Dau and -PTX conjugates were studied for their anticancer activity on A2780 ovarian and Panc-1 pancreatic cancer cells. Furthermore, the release of the drug by lysosomal enzymes and the GnRH-R binding affinities of the SMDC were examined.

Drug delivery systems (DDS) are promising tools for targeted tumor therapy providing the selective delivery of cytotoxic drugs to malignant cells, while side-effects and systemic toxicity are reduced. In addition to monoclonal antibodies (mAb), peptide ligands with a high affinity for tumor-specific cell surface compartments (e.g., receptors) can be used as carriers for cytotoxic payloads, as they provide beneficial features such as good tissue penetration, low immunogenicity and structural simplicity which enables their cost-efficient production by chemical synthesi. Receptors for the human gonadotropin releasing hormone (GnRH-I, <EHWSYGLRPG-NH2, <E is pyroglutamic acid) were not only identified in the pituitary, but also in various reproductive system-related cancers, such as breast, prostate and ovarian cancers, as well as non-reproductive cancers, such as colon and lung cancer. Thus, GnRH-related peptides are promising homing devices to deliver cytotoxic drugs selectively to cancer cells. A natural isoform of GnRH-I is the sea lamprey analog GnRH-III. This weak GnRH agonist binds to GnRH receptors (GnRH-R) on cancer cells and induces, like GnRH-I, a direct antitumor activity on several cancer cell lines, but its gonadotropin releasing activity is 500-1000 times lower in vitro and in vivo. Due to the direct anticancer activity and the low endocrine effect, GnRH-III and its derivatives have been successfully used as homing devices in in vitro and in vivo experiments. Encouraged by these promising findings, we report on the synthesis and biochemical characterization of eight cleavable self-immolative linker containing GnRH-III-drug conjugates. Of particular interest was the comparison of (1) two GnRH-III targeting moieties (GnRH-III-[4Lys(Bu)] (I) and GnRH-III-[2His,3D-Tic,4Lys(Bu)] (II)), (2) two cathepsin B-cleavable dipeptidyl-PABC linkers (Val-Ala and Val-Cit) and (3) two traditional anticancer drugs with different modes of action (Dau and PTX). For a better comparison and to demonstrate the proof of concept, four corresponding non-cleavable GnRH-III-Dau and -PTX conjugates have been developed and analyzed. The 8Lys of the targeting peptide was used as the ligation site. In the case of the Dau conjugates, the amino group of the daunosamine sugar has been used for attachment to the linker, while in the case of PTX, the C2-OH group was exploited for this purpose. All synthesized GnRH-III-Dau and -PTX conjugates were studied for their anticancer activity on A2780 ovarian and Panc-1 pancreatic cancer cells. Furthermore, the release of the drug by lysosomal enzymes and the GnRH-R binding affinities of the SMDC were examined.

Drug delivery systems (DDS) are promising tools for targeted tumor therapy providing the selective delivery of cytotoxic drugs to malignant cells, while side-effects and systemic toxicity are reduced. In addition to monoclonal antibodies (mAb), peptide ligands with a high affinity for tumor-specific cell surface compartments (e.g., receptors) can be used as carriers for cytotoxic payloads, as they provide beneficial features such as good tissue penetration, low immunogenicity and structural simplicity which enables their cost-efficient production by chemical synthesi. Receptors for the human gonadotropin releasing hormone (GnRH-I, <EHWSYGLRPG-NH2, <E is pyroglutamic acid) were not only identified in the pituitary, but also in various reproductive system-related cancers, such as breast, prostate and ovarian cancers, as well as non-reproductive cancers, such as colon and lung cancer. Thus, GnRH-related peptides are promising homing devices to deliver cytotoxic drugs selectively to cancer cells. A natural isoform of GnRH-I is the sea lamprey analog GnRH-III. This weak GnRH agonist binds to GnRH receptors (GnRH-R) on cancer cells and induces, like GnRH-I, a direct antitumor activity on several cancer cell lines, but its gonadotropin releasing activity is 500-1000 times lower in vitro and in vivo. Due to the direct anticancer activity and the low endocrine effect, GnRH-III and its derivatives have been successfully used as homing devices in in vitro and in vivo experiments. Encouraged by these promising findings, we report on the synthesis and biochemical characterization of eight cleavable self-immolative linker containing GnRH-III-drug conjugates. Of particular interest was the comparison of (1) two GnRH-III targeting moieties (GnRH-III-[4Lys(Bu)] (I) and GnRH-III-[2His,3D-Tic,4Lys(Bu)] (II)), (2) two cathepsin B-cleavable dipeptidyl-PABC linkers (Val-Ala and Val-Cit) and (3) two traditional anticancer drugs with different modes of action (Dau and PTX). For a better comparison and to demonstrate the proof of concept, four corresponding non-cleavable GnRH-III-Dau and -PTX conjugates have been developed and analyzed. The 8Lys of the targeting peptide was used as the ligation site. In the case of the Dau conjugates, the amino group of the daunosamine sugar has been used for attachment to the linker, while in the case of PTX, the C2-OH group was exploited for this purpose. All synthesized GnRH-III-Dau and -PTX conjugates were studied for their anticancer activity on A2780 ovarian and Panc-1 pancreatic cancer cells. Furthermore, the release of the drug by lysosomal enzymes and the GnRH-R binding affinities of the SMDC were examined.