Small Molecule Drug Conjugates Research Articles

Abstract CT166: First-in-human phase 1 study of a phosphatidylserine-targeting small molecule drug conjugate (T-1201) in patients with advanced solid tumors

Abstract Background Phosphatidylserine (PS), a phospholipid, is an essential component of bilayer cell membranes and is normally present in the inner cell membrane layer of the cells. In rapid proliferating cancer cells, PS externalizes to the outer cell membrane and has the potential to act as a tumor biomarker and therapeutic target. T-1201 is a small molecule drug conjugate comprised of a phosphatidylserine-targeting head, Zinc (II) bis-dipicolylamine (Zn-DPA), linked to bioactive topoisomerase I inhibitor SN-38. Once T-1201 via Zn-DPA targets tumor cells, then enzymatic cleavage releases the cytotoxic payload of SN38, the active metabolite of irinotecan. This leads to high accumulation of antineoplastic drug directly in the tumor microenvironment. Here, we describe a trial in progress of a first-in-human trial of T-1201 in patients with advanced solid tumors. Methods This first-in-human, open-label, multicenter, phase I trial (NCT04866641) is investigating the safety, tolerability, pharmacokinetics and antitumor activity of T-1201 administrated as monotherapy to subjects with advanced solid tumors. Eligible patients have ≥ 1 measurable lesion per RECIST v1.1. and no effective standard therapy exists. T-1201 is administered intravenously once every 4 weeks (Q4W) in 28-day cycles with dose escalation guided by an accelerated titration design. The study started with 1 subject enrolled per cohort. With 100% dose increment for the next cohort. If 1 subject experiences any ≥ Grade 2 AE or DLT at any dose level during first cycle, the dose escalation will transit to 3+3 design where dose increment will follow a modified Fibonacci sequence. Starting with Cycle 2, the dosing interval can be adjusted to once every two weeks (Q2W) and the dose level after switching to Q2W schedule will be half of that at Q4W. Intra-subject dose escalation is permitted to maximize the likelihood of responses while keeping toxicity acceptable for individual subjects. The dose can be increased after subjects complete at least 2 treatment cycles at a particular dose level without experiencing any > grade 2 drug-related toxicity. The primary endpoints are the maximum tolerated dose (MTD), recommended Phase 2 dose (RP2D) and adverse event rate as measured by CTCAE v5.0. Secondary endpoints include objective response rate (ORR), clinical benefit rate (CBR), duration of response (DOR) and time to tumor progression. This study has begun enrolling patients and is continuing as planned in Taiwan. 77% of planned patients have been enrolled as of November 2023. Citation Format: Hui-Ching Wang, Yu-Min Yeh, Jeng-Shiun Du, Tsung-Jang Yeh, Jui-Hung Tsai, Yu-Chin Gau, Shang Yin Wu, Li-Tzong Chen, Hsiao-Fang Li, Wu-Chou Su. First-in-human phase 1 study of a phosphatidylserine-targeting small molecule drug conjugate (T-1201) in patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT166.

Design and Preclinical Evaluation of a Novel Prostate-Specific Membrane Antigen Radioligand Modified with a Transthyretin Binder.

Transthyretin binders have previously been used to improve the pharmacokinetic properties of small-molecule drug conjugates and could, thus, be utilized for radiopharmaceuticals as an alternative to the widely explored "albumin binder concept". In this study, a novel PSMA ligand modified with a transthyretin-binding entity (TB-01) was synthesized and labeled with lutetium-177 to obtain [177Lu]Lu-PSMA-TB-01. A high and specific uptake of [177Lu]Lu-PSMA-TB-01 was found in PSMA-positive PC-3 PIP cells (69 ± 3% after 4 h incubation), while uptake in PSMA-negative PC-3 flu cells was negligible (<1%). In vitro binding studies showed a 174-fold stronger affinity of [177Lu]Lu-PSMA-TB-01 to transthyretin than to human serum albumin. Biodistribution studies in PC-3 PIP/flu tumor-bearing mice confirmed the enhanced blood retention of [177Lu]Lu-PSMA-TB-01 (16 ± 1% IA/g at 1 h p.i.), which translated to a high tumor uptake (69 ± 13% IA/g at 4 h p.i.) with only slow wash-out over time (31 ± 8% IA/g at 96 h p.i.), while accumulation in the PC-3 flu tumor and non-targeted normal tissue was reasonably low. Further optimization of the radioligand design would be necessary to fine-tune the biodistribution and enable its use for therapeutic purposes. This study was the first of this kind and could motivate the use of the "transthyretin binder concept" for the development of future radiopharmaceuticals.

Abstract 3197: Addressing drug metabolism and pharmacokinetics (DMPK) challenges of small molecule-drug conjugates (SMDCs)

Abstract Background: SMDCs are gaining momentum with potential advantages over antibody-drug conjugates (ADCs), eg, better tumor penetration and lack of immunogenicity. We have developed a novel platform technology with tailored solutions for ADCs and SMDCs with intracellular and extracellular cleavage options. Key features of the SMDC technology comprise of a small molecule ligand such as an αvβ3 integrin binder showing efficient tumor homing and a linker extracellularly cleaved by neutrophil elastase (NE) in the tumor microenvironment allowing for seamless release of different payload classes.1 Our lead SMDC VIP2362 is currently in a Phase 1 clinical trial for the treatment of advanced solid tumors (NCT05712889). SMDCs often have a shorter half-life compared with ADCs, which may result in more frequent dosing schedules. Here we show significant reduction of clearance and drastic prolongation of half-life of SMDCs achieved by adding a second binding moiety. Methods: Payloads such as kinesin spindle protein inhibitors (KSPi), cyclin-dependent kinase 9 inhibitors (CDK9i), and monomethyl auristatin E (MMAE) have been linked via elastase-cleavable linkers to one or, via a branched linker, to two αvβ3 integrin binding moieties. Cytotoxicity of SMDCs was measured in cell lines with or without NE. Monovalent and bivalent SMDCs were investigated in DMPK studies in rats while monitoring the conjugate and the released payload. Results: Payloads were coupled to the NE-cleavable peptides via different linkages. MMAE was conjugated via sterically hindered ester bonds, CDK9i was conjugated via sulfoximide bonds, and KSPi payloads were coupled via amide bonds. Each of these SMDCs was highly stable in plasma and buffer. Without NE, in vitro cytotoxicity of SMDCs in each cell line was poor with IC50 in the micromolar range. In contrast, in the presence of NE, potency increased, and cytotoxicity (IC50) was in the low nanomolar to picomolar range depending on the cell line and payload. In each case, the SMDCs reached similar potency as the free payload indicating efficient cleavage of the SMDC by NE. In DMPK studies of SMDCs in rat, none or trace levels of free payload (&amp;lt;0.1% as compared to the respective conjugates) was measured. With each of the SMDCs and independent of the conjugated payloads, a remarkable impact on exposure was observed when attaching a second binding ligand. The SMDCs with two ligands showed a 37- to 81-fold reduced plasma clearance as compared with the respective monovalent SMDCs, which parallels a 12- to 57-fold increase in half-life (eg, 2.1 h and 28 h for mono and bivalent MMAE SMDCs respectively). Initial in vivo studies in a cell line-derived xenograft model showed efficacy of bivalent SMDCs with once weekly dosing. Conclusion: Our technology platform comprises NE-cleavable SMDCs with promising options to tune the DMPK profile for a longer half-life. 1. Lerchen et al, AACR (2023). 2. Lerchen et al, Cancers (2023). Citation Format: Anne-Sophie Rebstock, Hans-Georg Lerchen, Harvey Wong, Beatrix Stelte-Ludwig, Mareike Wiedmann, Amy J. Johnson, Raquel Izumi, Ahmed Hamdy. Addressing drug metabolism and pharmacokinetics (DMPK) challenges of small molecule-drug conjugates (SMDCs) [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 3197.

Abstract 2051: Innovations in ADC technology platform with legumain-cleavable KSP-inhibitor payloads adaptable to various aspects of cancer biology

Abstract Despite recent advances in the antibody-drug conjugate (ADC) field it remains challenging to achieve a sufficient therapeutic window. To address key issues of current ADCs such as aggregate formation and payload-related toxicities, we have developed a platform technology with tailored solutions for ADCs and small molecule-drug conjugates (SMDCs). Key features of the ADC technology comprise of kinesin spindle protein inhibitors (KSPi) as a novel payload class, a unique linker selectively cleaved by the tumor-associated protease legumain, and a CellTrapper® modification of the KSPi payload to reduce membrane permeability and provide accumulation in tumor cells beneficial for efficacy and safety1,2. Our advanced ADC VIP943 is targeting CD123 for the treatment of AML and MDS and is currently in Phase 1 clinical trial (NCT06034275). Here we show the extension and applicability of the platform in combination with various antibodies. Two case studies demonstrate how the payload-linker optimization increases the selectivity for tumor over healthy cells and potentially overcomes transporter-mediated drug resistance. Tuning the cleavable linker of cetuximab-ADCs for a higher challenge of legumain-mediated payload release resulted in a 52-fold improved selectivity of cytotoxicity against tumor cells versus HaCaT skin cells. A trastuzumab-ADC using a KSPi payload modified with the CellTrapper, shows low nanomolar potency against JIMT-1 cells resistant to trastuzumab-deruxtecan and thus may overcome resistance. An increased potency was not observed with an ADC releasing a permeable KSPi without CellTrapper. To address heterogenous target expression in solid tumors, novel antibody-2-drug conjugates (A2DCs) were synthesized to combine the advantages of intracellular accumulation of non-permeable payloads with the release of a second, membrane permeable KSPi payload. In vitro studies comparing the A2DCs with their respective mono-drug ADCs on 4 different targets shows the highest cytotoxicity achieved with A2DCs independent of the efficacy driver in mono ADCs. Lastly, synthesis of homogenous KSPi ADCs with a drug to antibody ratio of ~8 is compatible with different antibodies without significant aggregate formation. Furthermore, an easy and efficient process for ring-opening of the thiosuccinimide ring after conjugation was developed and shown to successfully avoid ADC de-conjugation via retro-Michael reaction. Selected examples of each of these subclasses were coupled to trastuzumab; all conjugates were highly efficacious and resulted in tumor regressions in the SKOV-3 model similar to trastuzumab-deruxtecan. Conclusion: Our technology platform offers options for efficient and safe cancer treatments and allows for versatile adaptations to the various aspects of cancer biology. 1. Kirchhoff et al, Cancers (2020). 2. Lerchen et al, Bioconj Chem. (2020). Citation Format: Hans-Georg Lerchen, Anne-Sophie Rebstock, Mareike Wiedmann, Beatrix Stelte-Ludwig, Amy J. Johnson, Raquel Izumi, Ahmed Hamdy. Innovations in ADC technology platform with legumain-cleavable KSP-inhibitor payloads adaptable to various aspects of cancer biology [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 2051.

A PSMA-targeted doxorubicin small-molecule drug conjugate

We developed a model small-molecule drug conjugate (SMDC) that employed doxorubicin as a representative chemotherapeutic targeted to the cell membrane biomarker PSMA (prostate-specific membrane antigen) expressed on prostate cancer cells. The strategy capitalized on the clatherin-mediated internalization of PSMA to facilitate the selective uptake and release of doxorubicin in the target cells. The SMDC was prepared and assessed for binding kinetics, plasma stability, cell toxicity, and specificity towards PSMA expressing prostate cancer cell lines. We observed high affinity of the SMDC for PSMA (IC50 5 nM) with irreversible binding, as well as specific effectiveness against PSMA(+) cells. These findings validated the strategy for a small molecule-based approach in targeted cancer therapy.

Small Molecule-Drug Conjugates: Opportunities for the Development of Targeted Anticancer Drugs.

Conventional chemotherapy is insufficient for precise cancer treatment due to its lack of selectivity and inevitable side effects. Targeted drugs have emerged as a promising solution for precise cancer treatment. A common strategy is to conjugate therapeutic agents with ligands that can specifically bind to tumor cells, providing targeted therapy. Similar to the more successful antibody drug conjugates (ADCs), small molecule drug conjugates (SMDCs) are another promising class of targeted drugs, consisting of three parts: targeting ligand, cleavable linker and payload. Compared to ADCs, SMDCs have the advantages of smaller size, better permeability, simpler preparation process and non-immunogenicity, making them a promising alternative to ADCs. This review describes the characteristics of the targeting ligand, linker and payload of SMDCs and the criteria for selecting a suitable one. We also discuss recently reported SMDCs and list some successful SMDCs that have entered clinical trials.

Neutrophil elastase as a versatile cleavage enzyme for activation of αvβ3 integrin-targeted small molecule drug conjugates with different payload classes in the tumor microenvironment.

Introduction: The development of bioconjugates for the targeted delivery of anticancer agents is gaining momentum after recent success of antibody drug conjugates (ADCs) in the clinic. Smaller format conjugates may have several advantages including better tumor penetration; however, cellular uptake and trafficking may be substantially different from ADCs. To fully leverage the potential of small molecule drug conjugates (SMDCs) with potent binding molecules mediating tumor homing, novel linker chemistries susceptible for efficient extracellular activation and payload release in the tumor microenvironment (TME) need to be explored. Methods: We designed a novel class of SMDCs, which target αvβ3 integrins for tumor homing and are cleaved by neutrophil elastase (NE), a serine protease active in the TME. A peptidomimetic αvβ3 ligand was attached via optimized linkers composed of substrate peptide sequences of NE connected to different functional groups of various payload classes, such as camptothecins, monomethyl auristatin E, kinesin spindle protein inhibitors (KSPi) and cyclin-dependent kinase 9 inhibitors (CDK-9i). Results: NE-mediated cleavage was found compatible with the diverse linker attachments via hindered ester bonds, amide bonds and sulfoximide bonds. Efficient and traceless release of the respective payloads was demonstrated in biochemical assays. The newly designed SMDCs were highly stable in buffer as well as in rat and human plasma. Cytotoxicity of the SMDCs in cancer cell lines was clearly dependent on NE. IC50 values were in the nanomolar or sub-nanomolar range across several cancer cell lines reaching similar potencies as compared to the respective payloads only in the presence of NE. In vivo pharmacokinetics evaluating SMDC and free payload exposures in rat and particularly the robust efficacy with good tolerability in triple negative breast and small cell lung cancer murine models demonstrate the utility of this approach for selective delivery of payloads to the tumor. Discussion: These results highlight the broad scope of potential payloads and suitable conjugation chemistries paving the way for future SMDCs harnessing the safety features of targeted delivery approaches in combination with NE cleavage in the TME.

In vivo activation of FAP-cleavable small molecule-drug conjugates for the targeted delivery of camptothecins and tubulin poisons to the tumor microenvironment

Small molecule-drug conjugates (SMDCs) are increasingly considered as a therapeutic alternative to antibody-drug conjugates (ADCs) for cancer therapy. OncoFAP is an ultra-high affinity ligand of Fibroblast Activation Protein (FAP), a stromal tumor-associated antigen overexpressed in a wide variety of solid human malignancies. We have recently reported the development of non-internalizing OncoFAP-based SMDCs, which are activated by FAP thanks to selective proteolytic cleavage of the -GlyPro- linker with consequent release of monomethyl auristatin E (MMAE) in the tumor microenvironment. In this article, we describe the generation and the in vivo characterization of FAP-cleavable OncoFAP-drug conjugates based on potent topoisomerase I inhibitors (DXd, SN-38, and exatecan) and an anti-tubulin payload (MMAE), which are already exploited in clinical-stage and approved ADCs. The Glycine-Proline FAP-cleavable technology was directly benchmarked against linkers found in Adcetris™, Enhertu™, and Trodelvy™ structures by means of in vivo therapeutic experiments in mice bearing tumors with cellular or stromal FAP expression. OncoFAP-GlyPro-Exatecan and OncoFAP-GlyPro-MMAE emerged as the most efficacious anti-cancer therapeutics against FAP-positive cellular models. OncoFAP-GlyPro-MMAE exhibited a potent antitumor activity also against stromal models, and was therefore selected for clinical development.

Small molecule- and peptide-drug conjugates addressing integrins: A story of targeted cancer treatment.

Targeted cancer treatment should avoid side effects and damage to healthy cells commonly encountered during traditional chemotherapy. By combining small molecule or peptidic ligands as homing devices with cytotoxic drugs connected by a cleavable or non-cleavable linker in peptide-drug conjugates (PDCs) or small molecule-drug conjugates (SMDCs), cancer cells and tumours can be selectively targeted. The development of highly affine, selective peptides and small molecules in recent years has allowed PDCs and SMDCs to increasingly compete with antibody-drug conjugates (ADCs). Integrins represent an excellent target for conjugates because they are overexpressed by most cancer cells and because of the broad knowledge about native binding partners as well as the multitude of small-molecule and peptidic ligands that have been developed over the last 30 years. In particular, integrin αV β3 has been addressed using a variety of different PDCs and SMDCs over the last two decades, following various strategies. This review summarises and describes integrin-addressing PDCs and SMDCs while highlighting points of great interest.

Mono-amino acid linkers enable highly potent small molecule-drug conjugates by conditional release

The endosome cleavable linkers have been widely employed by antibody-drug conjugates and small molecule-drug conjugates (SMDCs) to control the accurate release of payloads. An effective linker should provide stability in systemic circulation but efficient payload release at its targeted tumor sites. This conflicting requirement always leads to linker design with increasing structural complexity. Balance of the effectiveness and structural complexity presents a linker design challenge. Here, we explored the possibility of mono-amino acid as so far the simplest cleavable linker (X-linker) for SMDC-based auristatin delivery. Within a diverse set of X-linkers, the SMDCs differed widely in bioactivity, with one (Asn-linker) having significantly improved potency (IC50= 0.1nM) and fast response to endosomal cathepsin B cleavage. Notably, this SMDC, once grafted with effector protein fragment crystallizable (Fc), demonstrated a profound invivo therapeutic effect in aspects of targetability, circulation half-life (t1/2= 73 h), stability, and anti-tumor efficacy. On the basis of these results, we believe that this mono-amino acid linker, together with the new SMDC-Fc scaffold, has significant potential in targeted delivery application.

Small Molecule-Drug Conjugates Emerge as a New Promising Approach for Cancer Treatment.

Antibody drug conjugates (ADCs) have emerged as a new promising class of anti- cancer agents. However, limitations such as higher costs and unavoidable immunogenicity due to their relatively large structures cannot be ignored. Therefore, the development of lightweight drugs such as small molecule-drug conjugates (SMDCs) based on the ADC design idea has become a new option for targeted therapy. SMDCs are derived from the coupling of small-molecule targeting ligands with cytotoxic drugs. They are composed of three parts: small-molecule targeting ligands, cytotoxic molecules, and linkers. Compared with ADCs, SMDCs can be more rapidly and evenly dispersed into tumor tissues, with low cost and no immunogenicity. In this article, we will give a comprehensive review of different types of SMDCs currently under clinical trials to provide ideas and inspirations for the development of clinically applicable SMDCs.

P-330 - Chemically Identical Pair of Theranostic Small-Molecule Drug Conjugates for Prostate Cancer Imaging and Chemotherapy

P-330 - Chemically Identical Pair of Theranostic Small-Molecule Drug Conjugates for Prostate Cancer Imaging and Chemotherapy

PSMA-targeted small-molecule drug-conjugates with valine-citrulline and phosphoramidate cleavable linkers

In this study, we present a modular synthesis and evaluation of two prostate-specific membrane antigen (PSMA) targeted small molecule drug conjugates (SMDCs) incorporating the potent chemotherapeutic agent monomethyl auristatin E (MMAE).These SMDCs are distinguished by their cleavable linker modules: one utilizing the widely known valine-citrulline linker, susceptible to cleavage by cathepsin B, and the other featuring a novel acid-labile phosphoramidate-based (PhosAm) linker. Both SMDCs maintained nanomolar affinity to PSMA. Furthermore, we confirmed the selective release of the payload and observed chemotherapeutic efficacy specifically within PSMA-positive prostate cancer cells, while maintaining cell viability in PSMA-negative cells. These findings not only validate the efficacy of our approach but also highlight the potential of the innovative pH-responsive PhosAm linker. This study contributes significantly to the field and also paves the way for future advancements in targeted cancer therapy.

Abstract A139: Preclinical characterization and prediction of human pharmacokinetics and efficacious dose for VIP236, a novel alpha V beta 3 binding small molecule-drug conjugate (SMDC)

Abstract VIP236 is a small molecule drug conjugate (SMDC) consisting of an alpha v beta 3 (αvβ3) integrin binder linked to an optimized camptothecin (optCPT) payload via a protease sensitive peptide sequence. The overall drug design strategy for VIP236 is targeted delivery to the tumor microenvironment (TME) with extracellular release of the opCPT by neutrophil elastase (NE) present in the TME. VIP236 is anticipated to kill tumor cells while reducing effects to normal tissue. VIP236 was characterized in preclinical pharmacokinetic (PK) and efficacy studies to predict its human PK and efficacious dose.  Briefly, VIP236 was administered intravenously (IV) to mice, rats, and dogs to characterize both its in vivo PK (1-2 mg/kg) and its routes of excretion (bile-duct cannulated [BDC] rat at 10 mg/kg).  VIP236 was evaluated in vitro for its protein binding characteristics and as a substrate of human drug transporters.   In addition, VIP236 was dosed to MX-1 (breast cancer) xenografted mice at doses ranging from 26-40 mg/kg IV on a 3 days on/4 days off regimen to characterize its antitumor activity. VIP236 had low plasma clearance in mice, rats, and dogs (CL=2.7-4.4 mL/min/kg) and low volume of distribution (Vss = 0.11 to 0.25 L/kg).  Terminal half-life ranged from 0.93 hour in mice to 3.07 hours in dogs. In BDC rats, VIP236 was primarily excreted in bile with biliary clearance accounting for essentially all the total body CL. There was minimal to no metabolites in rat bile and urine.  In vitro VIP236 exhibited low permeability in MDCKII cell monolayers and was identified as a substrate of BSEP and MRP2. VIP236 was highly protein bound to plasma protein with fraction unbound being &amp;lt;5% in all species tested. VIP236 showed robust tumor regression in MX-1 xenografted mice at all doses tested.  Allometric methods were used to predict a low human VIP236 CL (0.87-2.68 mL/min/kg) and Vss (0.10 L/kg). Translational pharmacokinetic/pharmacodynamic modeling was used to predict human efficacious doses and exposures by benchmarking VIP236 against irinotecan antitumor activity in MX-1 xenograft mice after 5 cycles of dosing.  Based on this analysis, predicted human doses ranged from 1-5 mg/kg IV on a 2 days on/5 days off regimen.  VIP236 is currently being evaluated in a first-in-human study in patients with advanced or metastatic solid tumors (NCT05712889). Citation Format: Harvey Wong, Beatrix Stelte-Ludwig, Hans-Georg Lerchen, Amy J Johnson, Melanie M Frigault, Raquel Izumi, Ahmed Hamdy. Preclinical characterization and prediction of human pharmacokinetics and efficacious dose for VIP236, a novel alpha V beta 3 binding small molecule-drug conjugate (SMDC) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A139.

Discovery of VIP236, an αvβ3-Targeted Small-Molecule-Drug Conjugate with Neutrophil Elastase-Mediated Activation of 7-Ethyl Camptothecin Payload for Treatment of Solid Tumors.

The emerging field of small-molecule-drug conjugates (SMDCs) using small-molecule biomarker-targeted compounds for tumor homing may provide new perspectives for targeted delivery. Here, for the first time, we disclose the structure and the synthesis of VIP236, an SMDC designed for the treatment of metastatic solid tumors by targeting αvβ3 integrins and extracellular cleavage of the 7-ethyl camptothecin payload by neutrophil elastase in the tumor microenvironment. Imaging studies in the Lewis lung mouse model using an elastase cleavable quenched substrate showed pronounced elastase activity in the tumor. Pharmacokinetics studies of VIP236 in tumor-bearing mice demonstrated high stability of the SMDC in plasma and high tumor accumulation of the cleaved payload. Studies in bile-duct-cannulated rats showed that biliary excretion of the unmodified conjugate is the primary route of elimination. Treatment- and time-dependent phosphorylation of H2AX, a marker of DNA damage downstream of topoisomerase 1 inhibition, verified the on-target activity of the payload cleaved from VIP236 in vivo. Treatment with VIP236 resulted in long-lasting tumor regression in subcutaneous patient-derived xenograft (PDX) models from patients with non-small-cell lung, colon, and renal cancer as well as in two orthotopic metastatic triple-negative breast cancer PDX models. In these models, a significant reduction of brain and lung metastases also was observed.

Design and synthesis of multivalent drug delivery system with CA IX inhibitors as ligands

A multivalent ligand delivery system holds tremendous potential in the field of tumor-targeted drug delivery. It addresses the challenges posed by the low affinity between small molecule ligand receptors and the rapid metabolism of small molecule drug conjugates (SMDCs) in vivo. Notably, existing multivalent ligand systems have demonstrated significant anti-tumor activity in various tumor models. In this study, we have developed a novel multivalent ligand delivery system for SN38, utilizing acetazolamide, a carbonic anhydrase IX (CA IX) inhibitor, as the target ligand. Our multivalent ligand delivery systems exhibited superior metabolic stability and enhanced targeting specificity compared to SMDC molecules. Furthermore, they demonstrated improved anti-proliferation activity, addressing the existing challenges associated with the low receptor affinity and rapid metabolism of SMDCs.

Synthesis and Evaluation of ePSMA-DM1: A New Theranostic Small-Molecule Drug Conjugate (T-SMDC) for Prostate Cancer.

Small-molecule drug conjugates (SMDCs) are compounds in which a therapeutic payload is conjugated to a targeting vector, for specific delivery to the tumor site. This promising approach can be translated to the treatment of prostate cancer by selecting a targeting vector which binds to the prostate-specific membrane antigen (PSMA). Moreover, the addition of a bifunctional chelator to the molecule allows for the use of both diagnostic and therapeutic radionuclides. In this way, the distribution of the SMDC in the body can be monitored, and combination therapy regimes can be implemented. We combined a glutamate-urea-lysine vector to the cytotoxic agent DM1 and a DOTA chelator via an optimized linker to obtain the theranostic SMDC (T-SMDC) ePSMA-DM1. ePSMA-DM1 retained a high binding affinity to PSMA and demonstrated PSMA-specific uptake in cells. Glutathione stability assays showed that the half-life of the T-SMDC in a reducing environment was 2 h, and full drug release was obtained after 6 h. Moreover, 100 nM of ePSMA-DM1 reduced the cell viability of the human PSMA-positive LS174T cells by >85% after 72 h of incubation, which was comparable to a 10-fold higher dose of free DM1. [111In]In-ePSMA-DM1 and [177Lu]Lu-ePSMA-DM1 were both obtained in high radiochemical yields and purities (>95%), with >90% stability in PBS and >80% stability in mouse serum for up to 24 h post incubation at 37 °C. SPECT/CT imaging studies allowed for a faint tumor visualization of [111In]In-ePSMA-DM1 at 1 h p.i., and the ex vivo biodistribution showed tumor uptake (2.39 ± 0.29% ID/g) at 1 h p.i., with the compound retained in the tumor for up to 24 h. Therefore, ePSMA-DM1 is a promising T-SMDC candidate for prostate cancer, and the data obtained so far warrant further investigations, such as therapeutic experiments, after further optimization.

Synthesis, and biological evaluation of EGFR/HER2-NAMPT conjugates for tumor treatment.

Throughout the reported applications of EGFR inhibitors, it is usually employed with HDAC or other targets to design multi-target inhibitors for cancer treatment. In this paper, we designed a drug conjugate that targeted EGFR&HER2 and had inhibitory activity of NAMPT simultaneously. Compound 20c significantly inhibited the EGFR&HER2 and NAMPT enzyme activities, and had comparable or even higher anti-proliferative activity than lapatinib in various cancer cells with over-expressed EGFR and HER2. Importantly, 20c was expected to increase sensitivity to EGFR inhibitor-resistant cells. In Osimertinib-resistant cells (NCI-1975 cells with the L858R/T790M/C797S triple mutation and Ba/F3 cells with the Del19/T790M/C797S triple mutation), the anti-proliferative activity of compound 20c was increased by more than twofold compared with Osimertinib, so as to obtain better curative effect. This strategy is a promising method of embedding multiple pharmacophores into a single molecule, which lays a good foundation for the design and synthesis of small molecule drug conjugates with strong targeting ability and high cytotoxicity.

Optimizing the enzymatic release of MMAE from isoDGR-based small molecule drug conjugate by incorporation of a GPLG-PABC enzymatically cleavable linker.

Antibody-Drug Conjugates (ADCs) and Small Molecule-Drug Conjugates (SMDCs) represent successful examples of targeted drug-delivery technologies for overcoming unwanted side effects of conventional chemotherapy in cancer treatment. In both strategies, a cytotoxic payload is connected to the tumor homing moiety through a linker that releases the drug inside or in proximity of the tumor cell, and that represents a key component for the final therapeutic effect of the conjugate. Here, we show that the replacement of the Val-Ala-p-aminobenzyloxycarbamate linker with the Gly-Pro-Leu-Gly-p-aminobenzyloxycarbamate (GPLG-PABC) sequence as enzymatically cleavable linker in the SMDC bearing the cyclo[DKP-isoDGR] αVβ3 integrin ligand as tumor homing moiety and the monomethyl auristatin E (MMAE) as cytotoxic payload led to a 4-fold more potent anti-tumoral effect of the final conjugate on different cancer cell lines. In addition, the synthesized conjugate resulted to be significantly more potent than the free MMAE when tested following the "kiss-and-run" protocol, and the relative potency were clearly consistent with the expression of the αVβ3 integrin receptor in the considered cancer cell lines. In vitro enzymatic cleavage tests showed that the GPLG-PABC linker is cleaved by lysosomal enzymes, and that the released drug is observable already after 15min of incubation. Although additional data are needed to fully characterize the releasing capacity of GPLG-PABC linker, our findings are of therapeutic significance since we are introducing an alternative to other well-established enzymatically sensitive peptide sequences that might be used in the future for generating more efficient and less toxic drug delivery systems.

Old Drug, New Delivery Strategy: MMAE Repackaged.

Targeting therapy is a concept that has gained significant importance in recent years, especially in oncology. The severe dose-limiting side effects of chemotherapy necessitate the development of novel, efficient and tolerable therapy approaches. In this regard, the prostate specific membrane antigene (PSMA) has been well established as a molecular target for diagnosis of, as well as therapy for, prostate cancer. Although most PSMA-targeting ligands are radiopharmaceuticals used in imaging or radioligand therapy, this article evaluates a PSMA-targeting small molecule-drug conjugate, and, thus, addresses a hitherto little-explored field. PSMA binding affinity and cytotoxicity were determined in vitro using cell-based assays. Enzyme-specific cleavage of the active drug was quantified via an enzyme-based assay. Efficacy and tolerability in vivo were assessed using an LNCaP xenograft model. Histopathological characterization of the tumor in terms of apoptotic status and proliferation rate was carried out using caspase-3 and Ki67 staining. The binding affinity of the Monomethyl auristatin E (MMAE) conjugate was moderate, compared to the drug-free PSMA ligand. Cytotoxicity in vitro was in the nanomolar range. Both binding and cytotoxicity were found to be PSMA-specific. Additionally, complete MMAE release could be reached after incubation with cathepsin B. In vivo, the MMAE conjugate displayed good tolerability and dose-dependent inhibition of tumor growth. Immunohistochemical and histological studies revealed the antitumor effect of MMAE.VC.SA.617, resulting in the inhibition of proliferation and the enhancement of apoptosis. The developed MMAE conjugate showed good properties in vitro, as well as in vivo, and should, therefore, be considered a promising candidate for a translational approach.