Synthesis and evaluation of antibody-drug conjugates with high drug-to-antibody ratio using dimaleimide-DM1 as a linker- payload.

The notable characteristics of recently emerged Antibody-Drug Conjugates (ADCs) encompass the targeting of Human Epidermal growth factor Receptor 2 (HER2) through monoclonal antibodies (mAbs) and a high ratio of drug to antibody (DAR). The achievements of Kadcyla® (T-DM1) and Enhertu® (T-Dxd) have demonstrated that HER2-targeting antibodies, such as trastuzumab, have shown to be competitive in terms of efficacy and price for development. Furthermore, with the arrival of T-Dxd and Trodelvy®, high-DAR (7-8) ADCs, which differ from the moderate DAR (3-4) ADCs that were formerly regarded as conventional, are being acknowledged for their worth. Following this trend of drug development, we endeavored to develop a high-DAR ADC using a straightforward approach involving the utilization of DM1, a highly potent substance, in combination with the widely recognized trastuzumab. To achieve a high DAR, DM1 was conjugated to reduced cysteine through the simple design and synthesis of various dimaleimide linkers with differing lengths. Using LC and MS analysis, we have demonstrated that our synthesis methodology is uncomplicated and efficacious, yielding trastuzumab-based ADCs that exhibit a remarkable degree of uniformity. These ADCs have been experimentally substantiated to exert an inhibitory effect on cancer cells in vitro, thus affirming their value as noteworthy additions to the realm of ADCs.

Preparation and characterization of antibody-drug conjugates acting on HER2-positive cancer cells.

Two systems of antibody-drug conjugates (ADCs), noncleavable H32-DM1 and cleavable H32-VCMMAE, were developed by using different linkers and drugs attached to the anti-HER2 antibody H32, which is capable of cell internalization. Activated functional groups, including an N-hydroxysuccinimidyl (NHS) ester and a maleimide, were utilized to make the ADCs. Mass spectrometry, hydrophobic interaction chromatography, polyacrylamide gel electrophoresis, and in vitro cell assays were performed to analyze and optimize the ADCs. Several H32-VCMMAE ADCs were established with higher DARs and greater synthetic yields without compromising potency. The anticancer efficacy of H32-DM1 was 2- to 8-fold greater than that of Kadcyla®. The efficacy of H32-VCMMAE was in turn better than that of H32-DM1. The anticancer efficacy of these ADCs against N87, SK-BR-3 and BT474 cells was in the following order: H32-VCMMAE series > H32-DM1 series > Kadcyla®. The optimal DAR for H32-VCMMAE was found to be 6.6, with desirable attributes including good cell penetration, a releasable payload in cancer cells, and high potency. Our results demonstrated the potential of H32-VCMMAE as a good ADC candidate.

Trastuzumab Conjugated Superparamagnetic Iron Oxide Nanoparticles Labeled with 225Ac as a Perspective Tool for Combined α-Radioimmunotherapy and Magnetic Hyperthermia of HER2-Positive Breast Cancer.

It has been proven and confirmed in numerous repeated tests, that the use of a combination of several therapeutic methods gives much better treatment results than in the case of separate therapies. Particularly promising is the combination of ionizing radiation and magnetic hyperthermia in one drug. To achieve this objective, magnetite nanoparticles have been modified in their core with α emitter 225Ac, in an amount affecting only slightly their magnetic properties. By 3-phosphonopropionic acid (CEPA) linker nanoparticles were conjugated covalently with trastuzumab (Herceptin®), a monoclonal antibody that recognizes ovarian and breast cancer cells overexpressing the HER2 receptors. The synthesized bioconjugates were characterized by transmission electron microscopy (TEM), Dynamic Light Scattering (DLS) measurement, thermogravimetric analysis (TGA) and application of 131I-labeled trastuzumab for quantification of the bound biomolecule. The obtained results show that one 225Ac@Fe3O4-CEPA-trastuzumab bioconjugate contains an average of 8-11 molecules of trastuzumab. The labeled nanoparticles almost quantitatively retain 225Ac (>98%) in phosphate-buffered saline (PBS) and physiological salt, and more than 90% of 221Fr and 213Bi over 10 days. In human serum after 10 days, the fraction of 225Ac released from 225Ac@Fe3O4 was still less than 2%, but the retention of 221Fr and 213Bi decreased to 70%. The synthesized 225Ac@Fe3O4-CEPA-trastuzumab bioconjugates have shown a high cytotoxic effect toward SKOV-3 ovarian cancer cells expressing HER2 receptor in-vitro. The in-vivo studies indicate that this bioconjugate exhibits properties suitable for the treatment of cancer cells by intratumoral or post-resection injection. The intravenous injection of the 225Ac@Fe3O4-CEPA-trastuzumab radiobioconjugate is excluded due to its high accumulation in the liver, lungs and spleen. Additionally, the high value of a specific absorption rate (SAR) allows its use in a new very perspective combination of α radionuclide therapy with magnetic hyperthermia.

A comparison of DFO and DFO* conjugated to trastuzumab-DM1 for complexing 89Zr - In vitro stability and in vivo microPET/CT imaging studies in NOD/SCID mice with HER2-positive SK-OV-3 human ovarian cancer xenografts.

INTRODUCTION: Desferrioxamine (DFO) is conjugated to antibodies to chelate 89Zr for PET, but DFO forms a hexadentate complex with Zr4+ that exhibits instability contributing to bone uptake of 89Zr, while the cationic charge of the Zr4+-DFO complex may promote normal tissue uptake of the radioimmunoconjugates (RICs). DFO* is a novel chelator that forms a more stable octadentate and neutral complex with 89Zr. Our aim was to compare the in vitro stability of [89Zr]Zr-DFO*-human IgG (hIgG) and [89Zr]Zr-DFO-hIgG RICs, and the in vivo PET imaging properties of the antibody-drug conjugate (ADC), trastuzumab-DM1 (T-DM1), labeled with 89Zr by conjugation to DFO or DFO*. METHODS: SCN-pPhe-DFO and SCN-pPhe-DFO* were reacted with hIgG at a 14.6-fold excess or with T-DM1 at a 4.1-fold or 10-fold excess, respectively, purified and labeled with 89Zr. The number of DFO* introduced was determined by measuring the absorbance at 245/252 nm and the protein concentration was measured at 280 nm. The stability of [89Zr]Zr-DFO*-hIgG was studied in vitro in human plasma, and by challenge with a 385-fold excess (0.1 mM) of DFO or EDTA. An inverse stability study was performed with [89Zr]Zr-DFO-hIgG challenged with 0.1 mM DFO*. The HER2 binding affinity of [89Zr]Zr-DFO*-T-DM1 was measured in a direct (saturation) binding assay using SK-BR-3 human breast cancer cells or SK-OV-3 human ovarian cancer cells. The biodistribution of [89Zr]Zr-DFO*-T-DM1 and [89Zr]Zr-DFO-T-DM1 were compared in non-tumor bearing Balb/c mice and in NOD/SCID mice with s.c. SK-OV-3 xenografts at 96 h post-intravenous injection (p.i.). MicroPET/CT images were obtained at 96 h p.i. of the RICs. RESULTS: hIgG and T-DM1 were conjugated to 4.5-5.3 and 3.1 chelators (DFO or DFO*), respectively, and labeled with 89Zr to a final radiochemical purity of 91-99%. [89Zr]Zr-DFO*-hIgG was stable in vitro in human plasma or to challenge with 0.1 mM EDTA, but incubation with 0.1 mM DFO caused 26.0 ± 2.1% loss of 89Zr after 5 days. In contrast, incubation of [89Zr]Zr-DFO-hIgG with 0.1 mM DFO* resulted in 77.0 ± 3.9% loss of 89Zr after 5 days. [89Zr]Zr-DFO*-T-DM1 retained high affinity binding to HER2 on SK-BR-3 and SK-OV-3 cells with a Kd = 2.2 ± 0.3 nM and 1.9 ± 0.3 nM, respectively, and Bmax = 3.4 ± 0.1 × 105 and 1.1 ± 0.04 × 105 receptors/cell, respectively. Biodistribution studies of [89Zr]Zr-DFO-T-DM1 and [89Zr]Zr-DFO*-T-DM1 in Balb/c and NOD/SCID mice revealed significantly lower uptake in bone, liver, kidneys, and spleen for [89Zr]Zr-DFO*-T-DM1 than [89Zr]Zr-DFO-T-DM1. Uptake of [89Zr]Zr-DFO*-T-DM1 and [89Zr]Zr-DFO-T-DM1 in SK-OV-3 tumors was moderate [5.0 ± 1.8% injected dose/g (%ID/g) and 6.3 ± 0.6%ID/g, respectively; P = 0.18]. Tumors were imaged with both RICs. CONCLUSION: We conclude that DFO* conjugated to T-DM1 provides more stable complexation of 89Zr and therefore, [89Zr]Zr-DFO*-T-DM1 would be more useful than [89Zr]Zr-DFO-T-DM1 to probe the delivery of T-DM1 to tumors by PET, which we previously found is correlated with response to treatment with T-DM1 in mouse tumor xenograft models. ADVANCES IN KNOWLEDGE AND IMPLICATION FOR PATIENT CARE: This study is the first to directly compare the PET imaging properties of [89Zr]Zr-DFO*-T-DM1 and [89Zr]Zr-DFO-T-DM1 in a HER2-overexpressing tumor xenograft mouse model. Our results indicate that [89Zr]Zr-DFO*-T-DM1 provides superior imaging properties due to the greater stability of the [89Zr]Zr-DFO* than [89Zr]Zr-DFO complex.

Assessing localized conformational stability of antibody-drug conjugate by protein conformation assay.

Antibody-drug conjugates (ADCs) are a class of attractive therapeutic agents to fight cancer with conjugation of potent chemical agents on target-selective antibodies. The conceptually elegant approach has encountered mounting practical challenges in combining the mAb and potent drug while maintaining the conformational and physiochemical stability of the bioconjugates. The attachment of hydrophobic drug-linker with antibody could potentially alter the antibody conformational scaffold, locally or globally. Here we propose to use a protein conformation assay (PCA) to measure the higher-order structure of antibodies upon drug-linker conjugation. The PCA analysis provides insights into the formation of partially unfolded ADCs, which may correlate with protein stability and aggregation propensity. To further elucidate the cause of the unfolding events, in-depth peptide mapping combined with the PCA conformational footprints were performed on a commercial ADC trastuzumab emtansine in this study. The locally altered conformational hot-spots observed in PCA matched with conjugation sites with high occupancy rate identified in peptide mapping. In summary, by combining PCA and in-depth peptide mapping, a snapshot of ADC structural conformation and stability profile could be obtained and provide a swift and convenient measurement of the 'fitness' of ADC to facilitate payload selection, conjugation process development and early predictive developability assessment.

Repurposing of Cetuximab in antibody-directed chemotherapy-loaded nanoparticles in EGFR therapy-resistant pancreatic tumours.

The anti-Epidermal Growth Factor Receptor (EGFR) antibody Cetuximab (CTX) has demonstrated limited anti-cancer efficacy in cells overexpressing EGFR due to activating mutations in RAS in solid tumours, such as pancreatic cancer. The utilisation of antibodies as targeting components of antibody-drug conjugates, such as trastuzumab emtansine (Kadcyla), demonstrates that antibodies may be repurposed to direct therapeutic agents to antibody-resistant cancers. Here we investigated the use of CTX as a targeting agent for camptothecin (CPT)-loaded polymeric nanoparticles (NPs) directed against KRAS mutant CTX-resistant cancer cells. CPT was encapsulated within poly(lactic-co-glycolic acid) (PLGA) NPs using the solvent evaporation method. CTX conjugation improved NP binding and delivery of CPT to CTX-resistant cancer cell lines. CTX successfully targeted CPT-loaded NPs to mutant KRAS PANC-1 tumours in vivo and reduced tumour growth. This study highlights that CTX can be repurposed as a targeting agent against CTX-resistant cancers and that antibody repositioning may be applicable to other antibodies restricted by resistance.

Middle-Down Multi-Attribute Analysis of Antibody-Drug Conjugates with Electron Transfer Dissociation.

Antibody-drug conjugates (ADCs) are designed to combine the target specificity of monoclonal antibodies and potent cytotoxin drugs to achieve better therapeutic outcomes. Comprehensive evaluation of the quality attributes of ADCs is critical for drug development but remains challenging due to heterogeneity of the construct. Currently, peptide mapping with reversed-phase liquid chromatography (RPLC) coupled to mass spectrometry (MS) is the predominant approach to characterize ADCs. However, it is suboptimal for sequence characterization and quantification of ADCs because it lacks a comprehensive view of coexisting variants and suffers from varying ionization effects of drug-conjugated peptides compared to unconjugated counterparts. Here, we present the first middle-down RPLC-MS analysis of both cysteine (Adcetris; BV) and lysine (Kadcyla; T-DM1) conjugated ADCs at the subunit level (∼25 kDa) with electron transfer dissociation (ETD). We successfully achieved high-resolution separation of subunit isomers arising from different drug conjugation and subsequently localized the conjugation sites. Moreover, we obtained a comprehensive overview of the microvariants associated with each subunits and characterized them such as oxidized variants with different sites. Furthermore, we observed relatively high levels of conjugation near complementarity-determining regions (CDRs) from the heavy chain but no drug conjugation near CDRs of light chain (Lc) from lysine conjugated T-DM1. Based on the extracted ion chromatograms, we accurately measured average drug to antibody ratio (DAR) values and relative occupancy of drug-conjugated subunits. Overall, the middle-down MS approach enables the evaluation of multiple quality attributes including DAR, positional isomers, conjugation sites, occupancy, and microvariants, which potentially opens up a new avenue to characterize ADCs.

Conjugation of Emtansine Onto Trastuzumab Promotes Aggregation of the Antibody-Drug Conjugate by Reducing Repulsive Electrostatic Interactions and Increasing Hydrophobic Interactions.

The impact of drug conjugation on intra- and intermolecular interactions of trastuzumab (TmAb) was determined by comparing the conformational and colloidal stabilities of TmAb and trastuzumab emtansine (T-DM1). In low ionic strength formulations, drug conjugation to native lysine residues of TmAb significantly reduced the repulsive electrostatic interactions between T-DM1 molecules. When these electrostatic interactions were screened in solutions with high ionic strength, intermolecular interactions between T-DM1 molecules were found to be more attractive than those between TmAb molecules. Drug conjugation lowered the colloidal stability of T-DM1 compared to TmAb, making T-DM1 more susceptible to agitation-induced aggregation. The presence of polysorbate-20 in the formulations inhibited aggregation of TmAb and T-DM1 induced by the hydrophobic air-water interface. Furthermore, the effect of increased hydrophobic interactions between T-DM1 molecules was studied by monitoring aggregation in TmAb and T-DM1 solutions that were incubated at 4°C, 25°C, and 50°C. Conjugating DM1 to TmAb increased the hydrophobicity of the molecule, and faster aggregation of T-DM1 at 50°C could be attributed to a temperature-dependent increase in hydrophobic interactions between T-DM1 molecules.

Reductive Desulfuration as an Important Tool in Detection of Small Molecule Modifications to Payload of Antibody Drug Conjugates.

A high resolution accurate mass LC-MS method was developed to facilitate the characterization of a subset of antibody drug conjugate (ADC) biotherapeutics, where the payload is linked to the antibody by a thioether bond. Desulfuration of the thioether linker was optimized for release of the payload to take advantage of the high resolution and high mass accuracy of the Orbitrap to characterize metabolism of the payload. Two model ADCs, trastuzumab emtansine (T-DM1) and SigmaMAb dansyl-cadavarine-SMCC (SigmaMAb ADC mimic) were selected for optimization of the desulfuration reaction as a function of reaction time, pH, organic solvent, and chaotropic reagents (urea, guanidine HCl) by monitoring the yield of released desulfurated DM1 from T-DM1 and desulfurated dansyl-cadaverine-SMCC from SigmaMAb ADC mimic, respectively. The optimized desulfuration technique was successfully applied to enable characterization of the ADC following its incubation in hepatocytes, liver microsomes, and buffers, as illustrated by the identification of a hydrolyzed thiosuccinimide ring of SigmaMAb ADC mimic following incubation in buffer for 48 h. The results from this study demonstrate that the chemical cleavage of thioether bond by desulfuration is simple, efficient, and specific. This technique is useful in characterization of metabolism on the payload of ADC to provide guidance for improvement of its biopharmaceutical profile. This is the first report on characterization of modification to payload of ADC following desulfuration.