Peptide-drug Conjugates Research Articles

Development of bombesin-tubulysin conjugates using multicomponent chemistry to functionalize both the payload and the homing peptide

Peptide-drug conjugates (PDCs) have recently gained significant attention for the targeted delivery of anticancer therapeutics, mainly due to their cost-effective and chemically defined production and lower antigenicity compared to ADCs, among other benefits. In this study, we designed and synthesized novel PDCs by conjugating new thiol-functionalized tubulysin analogs (tubugis) to bombesin, a peptide ligand with a relevant role in cancer research. Two tubulysin analogs bearing ready-for-conjugation thiol groups were prepared by an on-resin multicomponent peptide synthesis strategy and subsequently tested for their stand-alone in vitro anti-proliferative activity against human cancer cells, which resulted in IC50 values in the nanomolar range. In addition, various fluorescently labeled [K5]-bombesin(6–14) peptides, non-lipidated and lipidated with fatty acid chains of variable length, were also synthesized using the versatile multicomponent chemistry. These bombesin derivatives were tested for their gastrin-related peptide receptor (GRPR)-mediated internalization into cancer cells using flow cytometry, proving that the lipid tail (especially C14) enhances the cell internalization. Using the tubugi toxins and bombesin peptides, three different bombesin-tubugi conjugates were synthesized with different cleavage propensity and lipophilicity. Preliminary in vitro experiments revealed that, depending on the linker and the presence of a lipid tail, these novel PDCs possess good to potent anticancer activity and moderate selectivity for GRPR-overexpressing cancer cells.

Antitumor Effect of Peptide-Camptothecin Conjugate Targeting CD133 Protein.

The peptide-drug conjugate (PDC) has emerged as one of the new approaches for cancer therapy, which has the advantages of improved drug target ability and reduced adverse effects compared with the traditional chemotherapy. CD133 is a surface antigen specific to cancer stem cells, which are thought to be responsible for the self-renewal, proliferation, metastasis, and chemoresistance of cancer cells. A PDC for CD133 was designed by us, and it consists of CD133 targeting peptide LS-7 (amino acid sequence LQNAPRS), a pH-sensitive linker (succinyl), and a cytotoxic payload, the cytotoxic molecule camptothecin (CPT) with potent toxicity in vivo and in vitro. An antitumor study exhibited that the conjugate LS-7-CPT has not only improved its cytotoxicity in tumor cells but also retained its anticancer effect in vivo. In addition, the acute toxicity in mice of LS-7-CPT has been improved and the maximum tolerated dose has been increased by at least 56.2-fold. Pull-down and in vivo fluorescent imaging results indicated that LS-7-CPT was enriched in mice tumors by targeting CD133 protein. As far as we know, this is the first report for a PDC molecule designed for CD133, which is important for the study of CPT drug development.

Structural Insights into Helix–Loop–Helix Peptides for “Ligand-Targeting” Intracellular Drug Delivery via VEGF Receptor-Mediated Endocytosis

As a new alternative to antibody-drug conjugates, we developed “ligand-targeting” peptide-drug conjugates (PDCs), in which conformationally constrained helix-loop-helix (HLH) peptide M49 targeting human vascular endothelial growth factor-A (VEGF) was used as a drug carrier. The biochemical study showed that HLH peptide M49 made a complex with VEGF in the extracellular environment, and then the M49/VEGF complex interacts with the receptor on the cell surface to induce cellular internalization via the endocytic pathway. Here, we present an X-ray crystal structure of the M49/VEGF complex at 1.5 Å resolution using a protein crystal grown in microgravity. The structure illustrated the “ligand-targeting” cellular uptake mechanism for intracellular drug delivery and the molecular basis on the peptide-VEGF binding mode with tight binding and high target specificity. In addition, mutational studies and thermodynamic analysis provided information on the driving forces of the complex formation. This work would contribute to the design of mid-size molecular-targeting peptides as well as HLH peptides, advancing the research in drug discovery and chemical biology.

Advancing Cancer Immunotherapy through Engineering New PD-L1 Degraders: A Comprehensive Study from Small Molecules to PD-L1-Specific Peptide-Drug Conjugates.

Despite the considerable achievements of antibodies targeting PD-1/PD-L1 in cancer immunotherapy, limitations in antitumor immune response and pharmacokinetics hinder their clinical adoption. Small molecules toward PD-L1 degradation signifies an innovative avenue to modulate PD-1/PD-L1 axis. Herein, we unveil a comprehensive engineering involving the development of new PD-L1 degraders based on the berberine (BBR) and palmatine (PMT) bioactive frameworks and explore their translational potential for cancer immunotherapy using a peptide-drug conjugate strategy. Chemical modifications at the O-9 position of PMT dramatically enhance the PD-L1 degradation capacity. Further conjugation of PMT degraders with an anti-PD-L1 peptide featuring disulfide linkers enables efficient GSH-specific prodrug activation, yielding synergistic immunotherapeutic benefits through both external PD-L1 blockade and internal PD-L1 degradation mechanisms. This work elucidates the compelling charm of the discovery and application of PD-L1 degraders, offering solutions to the challenges in advancing cancer immunotherapy in widespread clinics.

Targeted delivery of oxaliplatin to colorectal cancer using the cancer-specific cell-penetrating peptide BR2

Oxaliplatin is one of the main drugs used in chemotherapy to treat advanced colorectal cancer (CRC), but its effectiveness is limited by its weak cellular uptake, and poor targeting to cancer cells, which can lead to drug resistance and adverse effects in patients. To address these issues, we tested a cell-penetrating, cancer-specific peptide called BR2 as a new peptide-drug conjugate. We conjugated oxaliplatin with BR2 covalently using a heterobifunctional linker. The resulting conjugate (BR2-Oxal) was examined for in vitro and in vivo effectiveness against human colon cancer cells and tumors. Compared with the parent drug, the in vitro investigations showed selective cellular uptake of BR2-Oxal in cancer cells rather than in normal cells and a significantly greater reduction in colon cancer cell viability due to a higher ratio of apoptosis. We also found that BR2-Oxal exhibited a higher accumulation in tumors and greater tumor suppression than oxaliplatin in a xenograft mouse model, suggesting that BR2-Oxal was efficiently and specifically delivered to tumors in vivo. These findings indicate that using the BR2 peptide for the intracellular delivery of oxaliplatin is a promising drug-delivery strategy. This approach also has the potential to enhance the efficiency of oxaliplatin prodrugs or derivatives.

PDCdb: the biological activity and pharmaceutical information of peptide-drug conjugate (PDC).

Peptide-drug conjugates (PDCs) have emerged as a promising class of targeted therapeutics with substantial pharmaceutical advantages and market potentials, which is a combination of a peptide (selective to the disease-relevant target), a linker (stable in circulation but cleavable at target site)and a cytotoxic/radioactive drug (efficacious/traceable for disease). Among existing PDCs, those based on radiopharmaceuticals (a.k.a. radioactive drugs) are valued due to their accurate imaging and targeted destruction of disease sites. It's demanded to accumulate the biological activity and pharmaceutical information of PDCs. Herein, a database PDCdb was thus constructed to systematically describe these valuable data. Particularly, biological activities for 2036 PDCs were retrieved from literatures, which resulted in 1684, 613and 2753 activity data generated based on clinical trial, animal modeland cell line, respectively. Furthermore, the pharmaceutical information for all 2036 PDCs was collected, which gave the diverse data of (a) ADME property, plasma half-lifeand administration approach of a PDC and (b) chemical modification, primary target, mode of action, conjugating feature of the constituent peptide/linker/drug. In sum, PDCdb systematically provided the biological activities and pharmaceutical information for the most comprehensive list of PDCs among the available databases, which was expected to attract broad interest from related communities and could be freely accessible at: https://idrblab.org/PDCdb/.

185 (PB173): The novel peptide drug conjugate AVA6103 is a FAP-enabled preCISION™ medicine which targets Topoisomerase I to the tumor microenvironment via FAP cleavage

185 (PB173): The novel peptide drug conjugate AVA6103 is a FAP-enabled preCISION™ medicine which targets Topoisomerase I to the tumor microenvironment via FAP cleavage

A Peptide-Drug Conjugate-Based Nanoplatform for Immunometabolic Activation and In Situ Nerve Regeneration in Advanced-Stage Alzheimer's Disease.

The formidable protection of physiological barriers and unclear pathogenic mechanisms impede drug development for Alzheimer's disease (AD). As defenders of the central nervous system, immune-metabolism function, and stemness of glial cells remain dormant during degeneration, representing a significant challenge for simultaneously targeting and modulating. Here, a modular nanoplatform is presented composed of peptide-drug conjugates and an inflammation-responsive core. The nanoplatform is transported through the blood-brain barrier via transcytosis and disassembles in the oxidative stress microenvironment upon intravenous administration. The released drug-conjugated modules can specifically target and deliver hydroxychloroquine (HCQ) and all-trans retinoic acid (ATRA) to microglia and astrocytes, respectively. The immune function of chronic tolerant microglia is activated by metabolic modulation, and reactive astrocytes trans-differentiate into functional neurons. In a transgenic mouse model, nanoplatform reduces levels of toxic proteins and inflammation while increasing neuronal density. This results in the amelioration of learning and memory decline. The modular nanoplatform provides design principles for multi-cellular targeting and combination nano-therapy for inflammation-related diseases.

A CYCLIC PEPTIDE TARGETS GLIOBLASTOMA BY BINDING TO ABERRANTLY EXPOSED SNAP25.

Disease-specific changes in tumors and other diseased tissues are an important target of research because they provide clues on the pathophysiology of the disease as well as uncovering potentially useful markers for diagnosis and treatment. Here, we report a new cyclic peptide, CESPLLSEC (CES), that specifically accumulated (homed) in intracranial U87MG and the WT-GBM model of glioblastoma from intravenous (IV) injection, associating with the vasculature. Affinity chromatography of U87MG tumor extracts on insolubilized CES peptide identified Synaptosomal Associated Protein 25 (SNAP25) as a candidate target molecule (receptor) for CES. Several results supported the identification of SNAP25 as the CES receptor. IV-injected FAM-CES colocalized with SNAP25 in the tumors, and direct binding studies showed specific CES peptide binding to recombinant human SNAP25. A CES peptide-drug conjugate designed for photodynamic therapy showed selective cytotoxicity to SNAP25+ glioblastoma cell lines. Specific accumulation of systemically injected anti-SNAP25 antibody in U87MG glioblastoma, and labeling of intact U87MG cells with anti-SNAP in flow cytometry showed that SNAP25 is available from the circulation but not in normal tissues and that it is present at the cell surface. Using an array of ECM proteins and surface plasmon resonance revealed that SNAP25 binds moderately to collagen V and strongly to collagen VI. Modeling studies suggested that CES and collagen VI compete for the same binding site on SNAP25. Our results introduce CES as a valuable targeting peptide for drug delivery, and its receptor SNAP25 as a possible molecular marker of interest for glioblastoma.

Blood-brain barrier and blood-brain tumor barrier penetrating peptide-drug conjugate as targeted therapy for the treatment of lung cancer brain metastasis

Lung cancer is the leading cause of cancer deaths worldwide. Brain metastasis of lung cancer, which counts for nearly 50% of late-stage lung cancer patients, is a sign of a really poor prognosis. However, the presence of blood–brain barrier (BBB) and blood–brain tumor barrier (BBTB) limits the penetration of drugs from the blood into the brain and thus restricts their accumulation in brain tumors. Systematic delivery of drugs into brain and brain tumor lesion using BBB and BBTB penetrating vehicles represents a promising strategy to overcome the BBB and BBTB limitations. Hence, we validated one of our previously identified BBB/BBTB penetrating peptide and its drug conjugate form for the treatment of lung cancer brain metastasis. With in vitro experiment, we first validated that the receptor LRP1, which mediated the peptide penetration of the BBB, was expressed on lung cancer cells and thus can be targeted by the peptide to overcome BBTB. With this delivery peptide, we constructed peptide-paclitaxel conjugate (the PDC) and in vitro validation showed that the PDC can across the BBB and efficiently kill lung cancer cells. We therefore constructed mouse lung cancer brain metastasis xenograft. In vivo anti-tumor validations showed that the PDC efficiently inhibited the proliferation of the brain resident lung cancer cells and significantly expanded the survival of the mouse xenograft, with no visible damages to the organs. Overall, our study provided potential therapeutic drugs for the treatment of lung cancer brain metastasis that may be clinically effective in the near future.

Enhanced efficacy of combined VEGFR peptide–drug conjugate and anti-PD-1 antibody in treating hepatocellular carcinoma

This study aimed to design a VEGFR-targeting peptide–drug conjugate with the ability to decrease tumor burden and suppress tumor angiogenesis, and to further evaluate the therapeutic effect of anti-PD-1 antibody in HCC therapy. A VEGFR-targeting peptide VEGF125 − 136 (QR) was conjugated with a lytic peptide (KLU) to form a peptide–drug conjugate QR-KLU. And the efficacy of QR-KLU in combination with anti-PD-1 antibody for HCC therapy in vivo and in vitro were evaluated. QR-KLU inhibited the proliferation and migration of mouse HCC cell line (Hepa1–6) cells under normoxic and hypoxic conditions in a dose-dependent manner. In the subcutaneous Hepa1–6 tumor model, QR-KLU combined with the anti-PD-1 antibody substantially inhibited tumor growth, promoted tumor necrosis, and prolonged the survival time of tumor-bearing mice. QR-KLU substantially inhibited hypoxia-induced expression of VEGF, promoted tumor vascular normalization, and increased cluster of differentiation 8+ (CD8+) T cell infiltration in the tumor. In addition, QR-KLU and anti-PD-1 antibody demonstrated a strong synergistic effect in promoting the activation of intratumoral CD8+ T cells, reducing the expression of immune-inhibitory factors, and increasing the expression of immune-stimulatory factors. This study proposed a novel approach for enhancing the efficacy of anti-PD-1 antibody using a VEGFR-targeting peptide–drug conjugate in HCC therapy.

In situ enzymatic peptide-based nanomedicine with combined effects for enhanced tumor radio-immunotherapy

Despite the combination of radiotherapy and immunotherapy in clinical practice have shown promising synergistic anti-tumor effect, the immunosuppressive tumor microenvironment (TME) remains to be one of stumbling block impeding the therapeutic efficacy of radio-immunotherapy. In this study, in situ self-assembled peptide-based drug delivery systems with multiple effects were constructed for enhancing tumor radio-immunotherapy. The two peptide-drug conjugates could co-assembled into peptide-based nanomedicine on the cancer cell membrane under enzyme catalysis and continuously release small molecular active drugs in the cytoplasm, which combined action lead YAP phosphorylation and nuclear transfer failure. By simutaneously suppressing glycolysis and reactivating the dysfunctional Hippo pathway, the peptide-based nanomedicine also remitted the immunosuppressive TME by reducing lactate levels and the expression of immunosuppressive factors, respectively. As a result, the pro-tumorigenic M2 phenotype of tumor-associated macrophages (TAMs) was decreased and the anti-tumorigenic M1 phenotype of TAMs was increased. Such a dual intervention strategy from cellular metabolic level and gene transcription level not only inhibited tumor cell proliferation but also reversed the immunosuppressive TME, thereby augmenting the combined efficacy of radiotherapy and immune checkpoint blockade (ICB) therapy. The work is anticipated to contribute novel insights into anti-cancer drug targeted delivery and tumor multi-modal synergistic therapy.

669P CBX-12-101: Final results of a phase I study of CBX-12, a peptide drug conjugate (PDC) in patients (pts) with metastatic solid tumors

669P CBX-12-101: Final results of a phase I study of CBX-12, a peptide drug conjugate (PDC) in patients (pts) with metastatic solid tumors

646P A phase I trial of AVA6000, a fibroblast activation protein (FAP)-released, tumor microenvironment (TME)-targeted doxorubicin peptide drug conjugate in patients with FAP-positive solid tumors

646P A phase I trial of AVA6000, a fibroblast activation protein (FAP)-released, tumor microenvironment (TME)-targeted doxorubicin peptide drug conjugate in patients with FAP-positive solid tumors

Use of a Cyclic α-Alkylidene-β-Diketone as a Cleavable Linker Strategy for Antibody-Drug Conjugates.

In the fast-evolving landscape of targeted cancer therapies, the revolutionary class of biotherapeutics known as antibody-drug conjugates (ADCs) are taking center stage. Most clinically approved ADCs utilize cleavable linkers to temporarily attach potent cytotoxic payloads to antibodies, allowing selective payload release under tumor-specific conditions. In this study, we explored the utilization of 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde), a cyclic β-diketone featuring an active alkylidene group, to develop a novel chemically labile linker. This linker was designed to exploit the difference in reduction potential between the intracellular compartment and plasma. Upon reduction of an azido trigger strategically installed neighboring the cyclic β-diketone, the resulting nucleophilic primary amine reacts with the alkylidene group facilitated by a favorable ring closure reaction in accordance with Baldwin's rules. Consequently, this reaction enables the simultaneous release of the attached cytotoxic payload. The therapeutic utility of this novel linker strategy was demonstrated by separate conjugation of the linker to two epidermal growth factor receptor (EGFR)-targeting ligands to afford a peptide-drug conjugate and an ADC. This work comprises a significant contribution to the bioconjugation field by introducing the alkylidene cyclic β-diketone as a tunable scaffold used for the temporary conjugation of therapeutic agents to peptides and proteins.

Developing New Peptides and Peptide-Drug Conjugates for Targeting the FGFR2 Receptor-Expressing Tumor Cells and 3D Spheroids.

In this work, we utilized a biomimetic approach for targeting KATO (III) tumor cells and 3D tumoroids. Specifically, the binding interactions of the bioactive short peptide sequences ACSAG (A-pep) and LPHVLTPEAGAT (L-pep) with the fibroblast growth factor receptor (FGFR2) kinase domain was investigated for the first time. Both peptides have been shown to be derived from natural resources previously. We then created a new fusion trimer peptide ACSAG-LPHVLTPEAGAT-GASCA (Trimer-pep) and investigated its binding interactions with the FGFR2 kinase domain in order to target the fibroblast growth factor receptor 2 (FGFR2), which is many overexpressed in tumor cells. Molecular docking and molecular dynamics simulation studies revealed critical interactions with the activation loop, hinge and glycine-rich loop regions of the FGFR2 kinase domain. To develop these peptides for drug delivery, DOX (Doxorubicin) conjugates of the peptides were created. Furthermore, the binding of the peptides with the kinase domain was further confirmed through surface plasmon resonance studies. Cell studies with gastric cancer cells (KATO III) revealed that the conjugates and the peptides induced higher cytotoxicity in the tumor cells compared to normal cells. Following confirmation of cytotoxicity against tumor cells, the ability of the conjugates and the peptides to penetrate 3D spheroids was investigated by evaluating their permeation in co-cultured spheroids grown with KATO (III) and colon tumor-associated fibroblasts (CAFs). Results demonstrated that Trimer-pep conjugated with DOX showed the highest permeation, while the ACSAG conjugate also demonstrated reasonable permeation of the drug. These results indicate that these peptides may be further explored and potentially utilized to create drug conjugates for targeting tumor cells expressing FGFR2 for developing therapeutics.

The emerging role of melflufen and peptide-conjugates in multiple myeloma.

The past two decades have witnessed an impressive expansion in the treatment landscape of multiple myeloma, leading to significant improvements in progression-free; as well as overall survival. However, almost all patients still experience multiple relapses during their disease course, with biological and cytogenetic heterogeneity affecting response to subsequent treatments. The purpose of this review is to provide a historical background regarding the role of alkylating agents and an updated data regarding the use of peptide-drug conjugates such as melflufen for patients with multiple myeloma. The combination of daratumumab-melflufen-dexamethasone evaluated in the LIGHTHOUSE study showed a statistically significant improvement in progression-free survival compared to single-agent daratumumab (not reached vs. 4.9 months respectively; P = 0.0032), with improvement in overall response rate to 59% vs. 30% respectively; P = 0.03. There have been an interest in developing and utilizing peptide-drug conjugates such as melflufen for treatment of patients with multiple myeloma, especially in the relapsed setting given historical results with alkylating agents, the use of which has been limited by dose-related toxicities in a disease that remains largely incurable. Single agent melflufen initially showed promising results especially in specific subgroups of heavily pretreated patients before the decision to suspend all clinical trials evaluating this agent after results from the OCEAN phase 3 trial. Subsequent reported analyses especially for melflufen-based combinations appear promising and suggest a potential use of peptide-drug conjugates provided optimal patient selection, as well as identification of the best companion agent.

A Dual Bispecific Hydrolysis Peptide-Drug Conjugate Responsive to Micro-Acidic and Reduction Circumstance Promotes Antitumor Efficacy in Triple-Negative Breast Cancer.

Paclitaxel and its derivates are the first-line chemotherapeutic agents of breast cancer, which also showed tremendous clinical value in many other diseases including ovarian cancer, lung cancer etc. However, there are many drawbacks for almost all paclitaxel or its derivates, including extremely short half-life, poor solubility and adverse events, which significantly limits their clinical applications. In this work, we designed and constructed a bispecific hydrolysis PAP-SS-PTX (term as PDC), consisting with pro-apoptosis peptide (PAP) and paclitaxel (PTX) that were conjugated together via disulfide and ester bonds. On the one hand, PAP could improve the solubility of PTX and promote cellular uptake for drugs. On the other hand, it was able to prolong the PTX half-life. We performed series of chemo-dynamical assays and showed that PDC would release active drug molecules under micro-acidic and reduction circumstance. The further assays elucidated that PDC could interrupt DNA synthesis and arrest cell division through downregulating CDK4/6 and Histone methylation that inhibit tumor growth in vitro. What's more, it could not only inhibit 4T1 breast tumor growth, but also prolong the survival time of mice and exert antitumor efficacy in vivo. It may provide a new research idea for cancer therapies via controlled release strategy in tumor microenvironment.

In vitro and in vivo evaluation of Bombesin-MMAE conjugates for targeted tumour therapy

Targeted tumour therapy has proved to be an efficient alternative to overcome the limitations of conventional chemotherapy. The upregulation of the bombesin receptor 2 (BB2) in several malignancies and the advantages offered by peptide drug conjugates over antibody drug conjugates in terms of production and tumour targeting motivated us to synthesise and test bombesin conjugates armed with the tubulin binder monomethyl auristatin E. The widely used Val-Cit-PABC was initially included as cathepsin cleavable self-immolative linker for the release of the free drug. However, the poor stability of the Val-Cit-conjugates in mouse plasma encouraged us to consider the optimised alternatives Glu-Val-Cit-PABC and Glu-Gly-Cit-PABC. Conjugate BN-EVcM1, featuring Glu-Val-Cit-PABC, combined suitable stability (t(½) in mouse and human plasma: 8.4 h and 4.6 h, respectively), antiproliferative activity in vitro (IC50 = 29.6 nM on the human prostate cancer cell line PC-3) and the full release of the free payload within 24 h. Three conjugates, namely BN-EGcM1, BN-EVcM1 and BN-EVcM2, improved the accumulation of MMAE in PC-3 human prostate cancer xenograft mice models, compared to the administration of the free drug. Among them, BN-EVcM1 also stood out for the significantly extended survival of mice in in vivo acute efficacy studies and for the significant inhibition of the growth of a PC-3 tumour in mice in both acute and chronic efficacy studies.

Recent Progress of pH-Responsive Peptides, Polypeptides, and Their Supramolecular Assemblies for Biomedical Applications.

Peptides and polypeptides feature a variety of active functional groups on their side chains (including carboxylic acid, hydroxyl, amino, and thiol groups), enabling diverse chemical modifications. This versatility makes them highly valuable in stimuli-responsive systems. Notably, pH-responsive peptides and polypeptides, due to their ability to respond to pH changes, hold significant promise for applications in cellular pathology and tumor targeting. Extensive researches have highlighted the potentials of low pH insertion peptides (pHLIPs), peptide-drug conjugates (PDCs), and antibody-drug conjugates (ADCs) in biomedicine. Peptide self-assemblies, with their structural stability, ease of regulation, excellent biocompatibility, and biodegradability, offer immense potentials in the development of novel materials and biomedical applications. We also explore specific examples of their applications in drug delivery, tumor targeting, and tissue engineering, while discussing future challenges and potential advancements in the field of pH-responsive self-assembling peptide-based biomaterials.