Anti-tissue factor antibody conjugated with monomethyl auristatin E or deruxtecan in pancreatic cancer models.

Antibody-drug conjugates (ADCs) have been recognized as a promising class of cancer therapeutics. Tissue factor (TF), an initiator of the blood coagulation pathway, has been investigated regarding its relationship with cancer, and several preclinical and clinical studies have presented data on anti-TF ADCs, including tisotumab vedotin, which was approved in 2021. However, the feasibility of other payloads in the design of anti-TF ADCs is still unclear because no reports have compared payloads with different cytotoxic mechanisms. For ADCs targeting other antigens, such as Her2, optimizing the payload is also an important issue in order to improve in vivo efficacy. In this study, we prepared humanized anti-TF Ab (clone.1084) conjugated with monomethyl auristatin E (MMAE) or deruxtecan (DXd), and evaluated the efficacy in several cell line- and patient-derived xenograft models of pancreatic cancer. As a result, optimizing the drug / Ab ratio was necessary for each payload in order to prevent pharmacokinetic deterioration and maximize delivery efficiency. In addition, MMAE-conjugated anti-TF ADC showed higher antitumor effects in tumors with strong and homogeneous TF expression, while DXd-conjugated anti-TF ADC was more effective in tumors with weak and heterogeneous TF expression. Analysis of a pancreatic cancer tissue array showed weak and heterogeneous TF expression in most TF-positive specimens, indicating that the response rate to pancreatic cancer might be higher for DXd- than MMAE-conjugated anti-TF ADC. Nevertheless, our findings indicated that optimizing the ADC payloads individually in each patient could maximize the potential of ADC therapeutics.

Discovery of a novel highly specific, fully human PSCA antibody and its application as an antibody-drug conjugate in prostate cancer.

Prostate stem cell antigen (PSCA) is expressed in all stages of prostate cancer, including in advanced androgen-independent tumors and bone metastasis. PSCA may associate with prostate carcinogenesis and lineage plasticity in prostate cancer. PSCA is also a promising theranostic marker for a variety of other solid tumors, including pancreatic adenocarcinoma and renal cell carcinoma. Here, we identified a novel fully human PSCA antibody using phage display methodology. The structure-based affinity maturation yielded a high-affinity binder, F12, which is highly specific and does not bind to 6,000 human membrane proteins based on a membrane proteome array assay. F12 targets PSCA amino acids 63-69 as tested by the peptide scanning microarray, and it cross-reacts with the murine PSCA. IgG1 F12 efficiently internalizes into PSCA-expressing tumor cells. The antimitotic reagent monomethyl auristatin E (MMAE)-conjugated IgG1 F12 (ADC, F12-MMAE) exhibits dose-dependent efficacy and specificity in a human prostate cancer PC-3-PSCA xenograft NSG mouse model. This is a first reported ADC based on a fully human PSCA antibody and MMAE that is characterized in a xenograft murine model, which warrants further optimizations and investigations in additional preclinical tumor models, including prostate and other solid tumors.

Natural-source payloads used in the conjugated drugs architecture for cancer therapy: Recent advances and future directions.

Drug conjugates are obtained from tumor-located vectors connected to cytotoxic agents via linkers, which are designed to deliver hyper-toxic payloads directly to targeted cancer cells. These drug conjugates include antibody-drug conjugates (ADCs), peptide-drug conjugates (PDCs), small molecule-drug conjugates (SMDCs), nucleic acid aptamer-drug conjugates (ApDCs), and virus-like drug conjugate (VDCs), which show great therapeutic value in the clinic. Drug conjugates consist of a targeting carrier, a linker, and a payload. Payloads are key therapy components. Cytotoxic molecules and their derivatives derived from natural products are commonly used in the payload portion of conjugates. The ideal payload should have sufficient toxicity, stability, coupling sites, and the ability to be released under specific conditions to kill tumor cells. Microtubule protein inhibitors, DNA damage agents, and RNA inhibitors are common cytotoxic molecules. Among these conjugates, cytotoxic molecules of natural origin are summarized based on their mechanism of action, conformational relationships, and the discovery of new derivatives. This paper also mentions some cytotoxic molecules that have the potential to be payloads. It also summarizes the latest technologies and novel conjugates developed in recent years to overcome the shortcomings of ADCs, PDCs, SMDCs, ApDCs, and VDCs. In addition, this paper summarizes the clinical trials conducted on conjugates of these cytotoxic molecules over the last five years. It provides a reference for designing and developing safer and more efficient conjugates.

An Investigation into the In Vitro Targeted Killing of CD44-Expressing Triple-Negative Breast Cancer Cells Using Recombinant Photoimmunotherapeutics Compared to Auristatin-F-Based Antibody-Drug Conjugates.

Triple-negative breast cancer (TNBC) is the deadliest form of breast cancer with limited treatment options. The persistence of highly tumorigenic CD44-expressing subpopulation referred to as cancer stem cells (CSCs), endowed with the self-renewal capacity, has been associated with therapeutic resistance, hence clinical relapses. To mitigate these undesired events, targeted immunotherapies using antibody-photoconjugate (APC) or antibody-drug conjugate (ADC), were developed to specifically release cytotoxic payloads within targeted cells overexpressing cognate antigen receptors. Therefore, an αCD44(scFv)-SNAP-tag antibody fusion protein was engineered through genetic fusion of a single-chain antibody fragment (scFv) to a SNAPf-tag fusion protein, capable of self-conjugating with benzylguanine-modified light-sensitive near-infrared (NIR) phthalocyanine dye IRDye700DX (BG-IR700) or the small molecule toxin auristatin-F (BG-AURIF). Binding of the αCD44(scFv)-SNAPf-IR700 photoimmunoconjugate to antigen-positive cells was demonstrated by confocal microscopy and flow cytometry. By switching to NIR irradiation, CD44-expressing TNBC was selectively killed through induced phototoxic activities. Likewise, the αCD44(scFv)-SNAPf-AURIF immunoconjugate was able to selectively accumulate within targeted cells and significantly reduced cell viability through antimitotic activities at nano- to micromolar drug concentrations. This study provides an in vitro proof-of-concept for a future strategy to selectively destroy light-accessible superficial CD44-expressing TNBC tumors and their metastatic lesions which are inaccessible to therapeutic light.

Development and validation of bioanalytical assays for the quantification of 9MW2821, a nectin-4-targeting antibody-drug conjugate.

We designed and developed 9MW2821, an anti-Nectin-4 antibody-drug conjugate (ADC) with an enzymatically cleavable valine-citrulline linker and monomethyl auristatin E (MMAE) as the payload. Four bioanalytical assays for total antibodies, conjugated antibodies, conjugated payload, and free payload were then developed and validated for the comprehensive evaluation of the multiple drug forms of 9MW2821. Specific sandwich enzyme-linked immunosorbent assays were used to quantify total antibodies and conjugated antibody, showing good drug-to-antibody ratio (DAR) tolerance. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine free MMAE, and conjugated MMAE was quantified using a combination of ligand-binding assay (LBA) and LC-MS/MS. Based on these four assays, we studied the serum stability and monkey pharmacokinetic profiles of 9MW2821, and the in vivo DAR of 9MW2821 was calculated and dynamically monitored. In conclusion, we developed and validated series of bioanalytical assays to quantify multiple forms of 9MW2821, a new ADC, and used the assays to evaluate the serum stability and monkey pharmacokinetic characteristics. The results indicate good linker stability and suggest that the developed assays can be further used in clinical settings.

Monomethyl auristatin E (MMAE), a payload for multiple antibody drug conjugates (ADCs), demonstrates differential red blood cell partitioning across human and animal species.

Background: Monomethyl auristatin E (MMAE) has been used as a payload for several Food and Drug Administration (FDA) approved antibody-drug conjugates (ADCs). It is known that MMAE is released from the ADC following binding, internalisation and proteolytic degradation in target tissues. A striking discrepancy in systemic MMAE levels has been observed across species with 50-fold higher MMAE levels in human than that in rodents when normalised by ADC dose with unknown mechanism.Hypothesis and purpose: Multiple factors could affect systemic MMAE levels such as production and elimination of unconjugated MMAE following ADC dosing. In this study, we have explored whether MMAE displays differential red blood cell (RBC) partitioning across species that may contribute to the different MMAE levels seen between human and animals.Experiments: To determine MMAE RBC partitioning, tritium labelled MMAE ([3H]-MMAE) was incubated in whole blood from mice, rats, monkeys and humans in vitro, then RBC partitioning was determined and compared across species. To test whether MMAE released from the ADC would show any difference in RBC partitioning, pinatuzumab vedotin or polatuzumab vedotin was administered to mice, rats, and monkeys. MMAE levels were measured in both blood and plasma, and the ratios of MMAE levels were calculated as blood-to-plasma ratio (in vivo RBC partitioning).Results: Our in vitro data showed that unconjugated MMAE has a species-dependent RBC partitioning with strong RBC partitioning in mouse, rat, followed by monkey blood, whereas minimal RBC partitioning was seen in human blood. Incubation of 2 nM of MMAE in mouse blood resulted in a blood-to-plasma ratio of 11.8 ± 0.291, followed by rat, monkey, and human at 2.36 ± 0.0825, 1.57 ± 0.0250, and 0.976 ± 0.0620, respectively. MMAE RBC partitioning is also concentration-dependent, with an inverse relationship between RBC partitioning and MMAE concentration (higher RBC partitioning at lower concentration). In vivo dosing of pinatuzumab vedotin in mouse displayed systemic MMAE at about a 5-fold higher blood concentration compared to plasma concentration once MMAE reached a pseudo-equilibrium, while systemic MMAE from blood and plasma concentration showed a 1.65-fold difference in rat.Implication and conclusion: These data demonstrated that MMAE has a distinct RBC partitioning across different species, which may contribute to, at least in part, to the differential in the systemic MMAE levels observed in vivo between preclinical and clinical studies. These findings highlight the importance of fully characterising the ADME properties of both the ADC and its payload, to enable better translation from animals to human for ADC development.

Development of a generalized pharmacokinetic model to characterize clinical pharmacokinetics of monomethyl auristatin E-based antibody-drug conjugates.

AIMS: This study aims to develop a generalized pharmacokinetic (PK) model for monomethyl auristatin E (MMAE)-based antibody-drug conjugates (ADCs) that can simultaneously capture the PK of multiple ADC analytes commonly measured in the clinic. METHODS: A comprehensive literature review was conducted to collect PK data on MMAE-based ADCs from clinical trials. From each study, PK profiles of total antibody, the ADC, conjugated MMAE, and unconjugated MMAE, were extracted. These data were pooled and dose-normalized to evaluate the generalizability of PK across various ADCs and dose levels. Upon confirming PK generalizability, a generalized PK model for MMAE-based ADCs was developed using the entire dataset. Furthermore, exposure metrics ( C max and AUC) reported across the range of doses were combined to establish linear relationships between dose and exposure metrics for MMAE-based ADCs. RESULTS: A total of 109 PK profiles from 18 distinct MMAE-based ADCs were gathered. The dose-normalized PK profiles supported the generalizability of PK for MMAE-based ADCs. A generalized PK model was developed, which enabled capturing the PK data for 4 ADC analytes across all collected MMAE-based ADCs. A linear relationship between dose and PK exposure metrics was established, enabling the prediction of typical exposure values across different doses for MMAE-based ADCs. CONCLUSIONS: This study comprehensively analysed clinical PK data from different valine-citrulline (vc)-MMAE-based ADCs. The generalized PK model developed here serves as an important tool for a priori prediction of the PK for multiple ADC analytes in clinical settings and lays the foundation for establishing generalized exposure-response and exposure-toxicity correlations for MMAE-based ADCs.

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 = 0.1 nM) and fast response to endosomal cathepsin B cleavage. Notably, this SMDC, once grafted with effector protein fragment crystallizable (Fc), demonstrated a profound in vivo 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.

Glypican-1-targeted antibody-drug conjugate inhibits the growth of glypican-1-positive glioblastoma.

Glioblastoma is the deadliest form of brain tumor. The presence of the blood-brain barrier (BBB) significantly hinders chemotherapy, necessitating the development of innovative treatment options for this tumor. This report presents the in vitro and in vivo efficacy of an antibody-drug conjugate (ADC) that targets glypican-1 (GPC1) in glioblastoma. The GPC1-ADC was created by conjugating a humanized anti-GPC1 antibody (clone T2) with monomethyl auristatin E (MMAE) via maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl linkers. Immunohistochemical staining analysis of a glioblastoma tissue microarray revealed that GPC1 expression was elevated in more than half of the cases. GPC1-ADC, when bound to GPC1, was efficiently and rapidly internalized in glioblastoma cell lines. It inhibited the growth of GPC1-positive glioma cell lines by inducing cell cycle arrest in the G2/M phase and triggering apoptosis in vitro. We established a heterotopic xenograft model by subcutaneously implanting KALS-1 and administered GPC1-ADC intravenously. GPC1-ADC significantly inhibited tumor growth and increased the number of mitotic cells. We also established an orthotopic xenograft model by intracranially implanting luciferase-transfected KS-1-Luc#19. After injecting Evans blue and resecting brain tissues, dye leakage was observed in the implantation area, confirming BBB disruption. We administered GPC1-ADC intravenously and measured the luciferase activity using an in vivo imaging system. GPC1-ADC significantly inhibited tumor growth and extended survival. In conclusion, GPC1-ADC demonstrated potent intracranial activity against GPC1-positive glioblastoma in an orthotopic xenograft model. These results indicate that GPC1-ADC could represent a groundbreaking new therapy for treating glioblastoma beyond the BBB.

Clinical pharmacology strategies to accelerate the development of polatuzumab vedotin and summary of key findings.

The favorable benefit-risk profile of polatuzumab vedotin, as demonstrated in a pivotal Phase Ib/II randomized study (GO29365; NCT02257567), coupled with the need for effective therapies in relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL), prompted the need to accelerate polatuzumab vedotin development. An integrated, fit-for-purpose clinical pharmacology package was designed to support regulatory approval. To address key clinical pharmacology questions without dedicated clinical pharmacology studies, we leveraged non-clinical and clinical data for polatuzumab vedotin, published clinical data for brentuximab vedotin, a similar antibody-drug conjugate, and physiologically based pharmacokinetic and population pharmacokinetic modeling approaches. We review strategies and model-informed outcomes that contributed to regulatory approval of polatuzumab vedotin plus bendamustine and rituximab in R/R DLBCL. These strategies made polatuzumab vedotin available to patients earlier than previously possible; depending on the strength of available data and the regulatory/competitive environment, they may also prove useful in accelerating the development of other agents.

Polatuzumab vedotin pharmacokinetics in a hemodialysis patient with diffuse large B-cell lymphoma.

PURPOSE: Chemotherapy for the hemodialysis (HD) patient is a challenging situation because it requires special considerations including dose modifications and timing of drug administration in relation with HD sessions. Polaltuzumab vedotin (PV), an antibody-drug conjugate in which monomethyl auristatin E (MMAE) is linked to an anti-CD79b monoclonal antibody, is an extremely promising therapeutic for treating diffuse large B cell lymphoma (DLBCL), but the pharmacokinetics are unknown in HD patients. METHODS: We carried out pharmacokinetic studies of PV when administered at 1.2 mg/kg to a DLBCL patient on HD, and compared the results with that of non-HD patients. PV was administered in conjunction with bendamustine and rituximab. RESULTS: Serum concentration-time curves of both antibodyconjugated and unconjugated MMAE in the presented HD patient were similar compared to that of non-HD patients. We also demonstrate that elimination of both antibody-conjugated and unconjugated MMAE through HD is limited. PV administration at 1.2 mg/kg to an HD patient was also clinically feasible, and no signs of peripheral neuropathy were observed. CONCLUSIONS: PV therapy may be a relatively safe treatment method for DLBCL patients on HD.

Bioanalysis of the Bicycle® toxin conjugate BT5528 and released monomethyl auristatin E via liquid chromatography-tandem mass spectrometry.

Background: The Bicycle® toxin conjugate BT5528 is a novel peptide therapeutic conjugated to the cytotoxic agent monomethyl auristatin E (MMAE). A bioanalytical assay was developed to quantify BT5528 and unconjugated MMAE in human plasma. Methodology: BT5528 quantitation used a protein precipitation procedure followed by LC-MS/MS detection. Quantitation of MMAE required a selective offline and online solid-phase extraction with detection via LC-MS/MS. Results: BT5528 was quantified over the assay range of 5-2500 ng/ml and free MMAE was quantified over the assay range of 0.05-50 ng/ml. Conclusion: Bioanalytical methods were used in the bioanalysis of intact BT5528 and released MMAE, in a phase I/IIa clinical trial; to date, over 2000 human patient samples have been analyzed.

A novel human single-domain antibody-drug conjugate targeting CEACAM5 exhibits potent in vitro and in vivo antitumor activity.

Leveraging the specificity of antibody to deliver cytotoxic agent into tumor, antibody-drug conjugates (ADCs) have become one of the hotspots in the development of anticancer therapies. Although significant progress has been achieved, there remain challenges to overcome, including limited penetration into solid tumors and potential immunogenicity. Fully human single-domain antibodies (UdAbs), with their small size and human nature, represent a promising approach for addressing these challenges. Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) is a glycosylated cell surface protein that rarely expressed in normal adult tissues but overexpressed in diverse cancers, taking part in tumorigenesis, progression, and metastasis. In this study, we investigated the therapeutic potential of UdADC targeting CEACAM5. We performed biopanning in our library and obtained an antibody candidate B9, which bound potently and specifically to CEACAM5 protein (KD = 4.84 nM) and possessed excellent biophysical properties (low aggregation tendency, high homogeneity, and thermal stability). The conjugation of B9 with a potent cytotoxic agent, monomethyl auristatin E (MMAE), exhibited superior antitumor efficacy against CEACAM5-expressing human gastric cancer cell line MKN-45, human pancreatic carcinoma cell line BxPC-3 and human colorectal cancer cell line LS174T with IC50 values of 38.14, 25.60, and 101.4 nM, respectively. In BxPC-3 and MKN-45 xenograft mice, administration of UdADC B9-MMAE (5 mg/kg, i.v.) every 2 days for 4 times markedly inhibited the tumor growth without significant change in body weight. This study may have significant implications for the design of next-generation ADCs.

Design, synthesis of auristatins-glucuronide conjugates targeting the ß-glucuronidase in tumor microenvironment.

Auristatins-glucuronide conjugates designed targeting the ß-Glucuronidase in tumor microenvironment were synthesized and evaluated on stabilities, the release of auristatins and the antitumor activities in this study. Conjugates 20 and 21 showed remarkable stabilities in phosphate buffer and bovine serum solution, and excellent selectivity between the in vitro antiproliferative activities against ß-glucuronidase pretreated and untreated cancer cells (IC50 = 5.7 nM âˆ¼ 9.7 nM, IC50 (-Enz) > 1 µM). Furthermore, conjugate 20 showed potent antitumor efficacy in HCT-116 xenograft mouse model without inducing side effects.

Mitochondrial trafficking as a protective mechanism against chemotherapy drug-induced peripheral neuropathy: Identifying the key site of action.

AIMS: Chemotherapy induced peripheral neuropathy (CIPN) is a common side effect seen in patients who have undergone most chemotherapy treatments to which there are currently no treatment methods. CIPN has been shown to cause axonal degeneration leading to Peripheral Neuropathy (PN), which can lead to major dosage reduction and may prevent further chemotherapy treatment due to oftentimes debilitating pain. Previously, we have determined the site-specific action of Paclitaxel (PTX), a microtubule targeting agent, as well as the neuroprotective effect of Fluocinolone Acetonide (FA) against Paclitaxel Induced Peripheral Neuropathy (PIPN). MAIN METHODS: Mitochondrial trafficking analysis was determined for all sample sets, wherein FA showed enhanced anterograde (axonal) mitochondrial trafficking leading to neuroprotective effects for all samples. KEY FINDINGS: Using this system, we demonstrate that PTX, Monomethyl auristatin E (MMAE), and Vincristine (VCR), are toxic at clinically prescribed levels when treated focally to axons. However, Cisplatin (CDDP) was determined to have a higher toxicity when treated to cell bodies. Although having different targeting mechanisms, the administration of FA was determined to have a significant neuroprotective effect for against all chemotherapy drugs tested. SIGNIFICANCE: This study identifies key insights regarding site of action and neuroprotective strategies to further development as potential therapeutics against CIPN. FA was treated alongside each chemotherapy drug to identify the neuroprotective effect against CIPN, where FA was found to be neuroprotective for all drugs tested. This study found that treatment with FA led to an enhancement in the anterograde movement of mitochondria based on fluorescent imaging.

CSPG4 as a target for the specific killing of triple-negative breast cancer cells by a recombinant SNAP-tag-based antibody-auristatin F drug conjugate.

PURPOSE: Triple-negative breast cancer (TNBC) is phenotypic of breast tumors lacking expression of the estrogen receptor (ER), the progesterone receptor (PgR), and the human epidermal growth factor receptor 2 (HER2). The paucity of well-defined molecular targets in TNBC, coupled with the increasing burden of breast cancer-related mortality, emphasizes the need to develop targeted diagnostics and therapeutics. While antibody-drug conjugates (ADCs) have emerged as revolutionary tools in the selective delivery of drugs to malignant cells, their widespread clinical use has been hampered by traditional strategies which often give rise to heterogeneous mixtures of ADC products. METHODS: Utilizing SNAP-tag technology as a cutting-edge site-specific conjugation method, a chondroitin sulfate proteoglycan 4 (CSPG4)-targeting ADC was engineered, encompassing a single-chain antibody fragment (scFv) conjugated to auristatin F (AURIF) via a click chemistry strategy. RESULTS: After showcasing the self-labeling potential of the SNAP-tag component, surface binding and internalization of the fluorescently labeled product were demonstrated on CSPG4-positive TNBC cell lines through confocal microscopy and flow cytometry. The cell-killing ability of the novel AURIF-based recombinant ADC was illustrated by the induction of a 50% reduction in cell viability at nanomolar to micromolar concentrations on target cell lines. CONCLUSION: This research underscores the applicability of SNAP-tag in the unambiguous generation of homogeneous and pharmaceutically relevant immunoconjugates that could potentially be instrumental in the management of a daunting disease like TNBC.

A mouse model of sensory neuropathy induced by a long course of monomethyl-auristatin E treatment.

Antibody-drug conjugates (ADCs) are anticancer drugs consisting of a monoclonal antibody, targeting selective tumor antigens, to which has been frequently associated a highly potent cytotoxic agent, the monomethyl auristatin E (MMAE) using a chemical linker. MMAE is a tubulin polymerization inhibitor derived from dolastin-10. These MMAE-ADCs are responsible for peripheral nerve toxicities. Our objective was to develop and characterize a mouse model of MMAE-induced peripheral neuropathy induced by free MMAE injections. MMAE was injected in Swiss mice at 50 µg/kg i.p. every other day for 7 weeks. Assessments of motor and sensory nerve functions were performed once a week on MMAE and Vehicle-treated mice. Sciatic nerve and paw skin were removed at the end of experiment for subsequent immunofluorescence and morphological analysis. MMAE did not affect motor coordination, muscular strength and heat nociception, but significantly induced tactile allodynia in MMAE-treated mice compared with Vehicle-treated mice from day 35 to day 49. MMAE significantly reduced myelinated and unmyelinated axon densities in sciatic nerves and led to a loss of intraepidermal nerve fiber in paw skin. In summary, long course of low dose of MMAE induced a peripheral sensory neuropathy associated with nerve degeneration, without general state alteration. This model may represent a ready accessible tool to screen neuroprotective strategies in the context of peripheral neuropathies induced by MMAE-ADCs.

Synthesis of Prostate-Specific Membrane Antigen-Targeted Bimodal Conjugates of Cytotoxic Agents and Antiandrogens and Their Comparative Assessment with Monoconjugates.

Prostate cancer is the second most common cancer among men. We designed and synthesized new ligands targeting prostate-specific membrane antigen and suitable for bimodal conjugates with diagnostic and therapeutic agents. In vitro studies of the affinity of the synthesized compounds to the protein target have been carried out. Based on these ligands, a series of bimodal conjugates with a combination of different mitosis inhibitors and antiandrogenic drugs were synthesized. The cytotoxicity of the compounds obtained in vitro was investigated on three different cell lines. The efficacy of the two obtained conjugates was evaluated in vivo in xenograft models of prostate cancer. These compounds have been shown to be highly effective in inhibiting the growth of PSMA-expressing tumors.

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] αVß3 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 αVß3 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 15 min 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.

Pharmacokinetics and Pharmacodynamics of Antibody-Drug Conjugates Administered via Subcutaneous and Intratumoral Routes.

We hypothesize that different routes of administration may lead to altered pharmacokinetics/pharmacodynamics (PK/PD) behavior of antibody-drug conjugates (ADCs) and may help to improve their therapeutic index. To evaluate this hypothesis, here we performed PK/PD evaluation for an ADC administered via subcutaneous (SC) and intratumoral (IT) routes. Trastuzumab-vc-MMAE was used as the model ADC, and NCI-N87 tumor-bearing xenografts were used as the animal model. The PK of multiple ADC analytes in plasma and tumors, and the in vivo efficacy of ADC, after IV, SC, and IT administration were evaluated. A semi-mechanistic PK/PD model was developed to characterize all the PK/PD data simultaneously. In addition, local toxicity of SC-administered ADC was investigated in immunocompetent and immunodeficient mice. Intratumoral administration was found to significantly increase tumor exposure and anti-tumor activity of ADC. The PK/PD model suggested that the IT route may provide the same efficacy as the IV route at an increased dosing interval and reduced dose level. SC administration of ADC led to local toxicity and reduced efficacy, suggesting difficulty in switching from IV to SC route for some ADCs. As such, this manuscript provides unprecedented insight into the PK/PD behavior of ADCs after IT and SC administration and paves the way for clinical evaluation of these routes.