Quantification for Antibody-Conjugated Drug in Trastuzumab Emtansine and Application to In Vitro Linker Stability and In Vivo Pharmacokinetic Study in Rat Using an Immuno-Affinity Capture Liquid Chromatography-Mass Spectrometric Method

Trastuzumab emtansine (T-DM1, brand name: Kadcyla®) is the first FDA-approved antibody-drug conjugate (ADC) for metastatic human epidermal growth factor receptor 2 positive (HER2+) breast cancer. It consists of three components: trastuzumab, an anti-HER2 monoclonal antibody, maytansinoid (DM1) as a cytotoxic drug, and maleimidomethyl cyclohexane-1-carboxylate (MCC) as a linker. In particular, the MCC linker is known as a non-cleavable linker and has a feature of being conjugated to DM1 by a covalent thioether bond. In this study, we developed an immuno-affinity capture liquid chromatography-mass spectrometric (LC-MS/MS) assay for quantifying the antibody-conjugated drug (acDrug) component of T-DM1. To quantify acDrug, desulfurated DM1 was prepared using a chemical desulfuration pretreatment and quantified as an acDrug. A quadratic regression (weighted 1/concentration), with equation y = ax2 + bx + c, was used to fit the calibration curves over the concentration range of 17.09~1709.44 ng/mL for the acDrug of T-DM1. The quantification run met the in-house acceptance criteria of ±25% accuracy and precision values for the quality control (QC) samples. In conclusion, an immuno-affinity capture LC-MS/MS assay was successfully developed to quantify acDrug of T-DM1 and applied to evaluate in vitro plasma linker stability and preclinical pharmacokinetic (PK) study in rats. This assay could be helpful when applied to other ADCs with the same linker-cytotoxic drug platform.

Health professionals’ attitude and associated factors toward cytotoxic drug handling in University of Gondar specialized hospital: Institution-based cross-sectional study

Background Studies have shown that cytotoxic drugs are dangerous to health care workers. Health care professionals’ attitude to cytotoxic drugs is vital to apply safety protocols in the prevention of cytotoxicity. The current study aimed to assess health professionals’ desirable attitude and associated factors toward cytotoxic drugs handling in the University of Gondar Specialized Hospital. Methods An institution-based cross-sectional study was conducted on 412 health professionals from June to August 2019. Simple random sampling was used to select participants and a self-administered questionnaire was used to collect data. Epi Info and SPSS 20 were used for data entry and analysis, respectively. Variables with a p value < 0.05 were declared as determinants. Results The number of health professionals included was approximately 412. The mean age of participants was 29.9 ± 5.4 years. The proportion of participants with desirable attitude toward cytotoxic drugs handling was 224 (54.4%) (95% confidence interval: 51.95–56.85). Male (adjusted odds ratio = 1.69, 95% confidence interval:[1.08–2.65]), work experience of 5–8 years (adjusted odds ratio = 1.92, 95% confidence interval: [1.10–3.34]), weekly working hours of 44–55 (adjusted odds ratio = 2.25, 95% confidence interval: [1.02–4.96]), medium work stress (adjusted odds ratio = 1.67, 95% confidence interval: [1.01–2.69]), and good practice of cytotoxic drug handling (adjusted odds ratio = 1.67, 95% confidence interval: [1.04–2.67]) were significantly associated with the attitude of health professionals. Conclusion A significant proportion of health care workers did not have desirable attitude to handle cytotoxic drugs. Thus, strategies are suggested to improve the positive attitude of health professionals to cytotoxic drug handling. Female health professionals, those who had work experience of less than 2 years and those with higher work stress demand special attention.

The cytotoxic conjugate of highly internalizing tetravalent antibody for targeting FGFR1-overproducing cancer cells

Background Antibody drug conjugates (ADCs) represent one of the most promising approaches in the current immuno-oncology research. The precise delivery of cytotoxic drugs to the cancer cells using ADCs specific for tumor-associated antigens enables sparing the healthy cells and thereby reduces unwanted side effects. Overexpression of fibroblast growth factor receptor 1 (FGFR1) has been demonstrated in numerous tumors and thereby constitutes a convenient molecular target for selective cancer treatment. We have recently engineered tetravalent anti-FGFR1 antibody, T-Fc, and have demonstrated that it displays extremely efficient internalization into FGFR1 producing cells, a feature highly desirable in the ADC approach. We have revealed that T-Fc mediates clustering of FGFR1, largely enhancing the uptake of FGFR1-T-Fc complexes by induction of clathrin-independent endocytic routes. The aim of this study was to obtain highly internalizing cytotoxic conjugate of the T-Fc for specific delivery of drugs into FGFR1-positive cancer cells. Methods Conjugation of the T-Fc to a cytotoxic payload, vcMMAE, was carried out via maleimide chemistry, yielding the T-Fc-vcMMAE. The specific binding of the T-Fc-vcMMAE conjugate to FGFR1 was confirmed in vitro with BLI technique. Confocal microscopy and flow cytometry were applied to determine FGFR1-dependence of the T-Fc-vcMMAE internalization. Western blot analyses of FGFR1-dependent signaling were conducted to assess the impact of the T-Fc-vcMMAE on FGFR1 activation and initiation of downstream signaling cascades. Finally, using FGFR1-negative and FGFR1-possitive cell lines, the cytotoxic potential of the T-Fc-vcMMAE was evaluated. Results We have performed the efficient conjugation of the tetravalent engineered antibody with a cytotoxic drug and generated FGFR1-specific ADC molecule, T-Fc-vcMMAE. We have demonstrated that T-Fc-vcMMAE conjugate exhibits high selectivity and affinity for FGFR1, similarly to T-Fc. Furthermore, we have shown that T-Fc constitutes an effective drug delivery vehicle as T-Fc-vcMMAE was efficiently and selectively internalized by FGFR1-producing cells leading to their death. Interestingly, we show that the efficiency of the uptake of T-Fc-vcMMAE corresponds well with the cytotoxicity of the conjugate, but doesn’t correlate with the FGFR1expression level. Conclusion Our results show that T-Fc-vcMMAE fulfills the key criteria for the successful cytotoxic drug carrier in a targeted approach against FGFR1-positive cancer cells. Furthermore, our data implicate that not solely expression level of the receptor, but rather its cellular trafficking should be taken into account for selection of suitable molecular targets and cancer models for successful ADC approach.

Clinical pharmacology strategies in supporting drug development and approval of antibody–drug conjugates in oncology

Antibody–drug conjugates (ADCs) are important molecular entities in the treatment of cancer. These conjugates combine the target specificity of monoclonal antibodies with the potent anti-cancer activity of small-molecule therapeutics. The complex structure of ADCs poses unique challenges to characterize the drug’s pharmacokinetics (PKs) and pharmacodynamics (PDs) since it requires a quantitative understanding of the PK and PD properties of multiple different molecular species (e.g., ADC conjugate, total antibody and unconjugated cytotoxic drug). As a result, clinical pharmacology strategy of an ADC is rather unique and dependent on the linker/cytotoxic drug technology, heterogeneity of the ADC, PK and safety/efficacy profile of the specific ADC in clinical development. In this review, we summarize the clinical pharmacology strategies in supporting development and approval of ADCs using the approved ADCs as specific examples to illustrate the customized approach to clinical pharmacology assessments in their clinical development.

Incorporation of Unnatural Amino Acid into Antibody Fragment for Creating a Stable Antibody Drug Conjugate

The traditional chemotherapy drug has been used as a standard cancer treatment, however it has resulted a modest survival benefit and damaged non-cancerous cells. Thus, the novel strategies which can improve selectivity and specificity in chemotherapy are urgently needed. Antibody drug conjugate (ADC) combines monoclonal antibody and cytotoxic drug is a potential regimen as targeted therapy. However, the heterogeneous mixtures has been observed using the current ADC methods. Here, we develop the strategy for generation a stable ADC utilising modified single chain antibody fragment (scFv) containing azide group for click chemistry reaction with alkyne containing cytotoxic drug. This research focused on targeting prostate cancer as a model disease utilising targeting prostate specific membrane antigen (PSMA) receptor which is overexpressed in all prostate cancer stages. The unnatural amino acid para-azido phenyl alanine (pAzF) has been successfully incorporated into anti-PSMA J591 scFv and specifically bound and internalised into PSMA positive cancer cells. This mutant scFv were also successfully conjugated into a linker containing cyclo-alkyne, DBCO-PEG4-DBCO as a model for creating ADC through copper-free click chemistry reaction. This bioconjugation method is promising as a versatile strategy for generating a stable ADC to improve therapeutic efficacy in cancer treatment.

A Model of Chemotherapy-Induced Tumor Dormancy: Doxorubicin Retention and Degradation Defines Resumed Growth in Vivo

Background. Cancer recurrence after adjuvant chemotherapy with long periods of remission is common. After cessation of the therapy, the dormant cells may repopulate, but the signals that control the tumor to exit dormancy are not completely understood. We hypothesized that tissue bound cytotoxic drug indwelling in a body for years might somehow contribute to dormancy or recurrence of a tumor. The purpose of this study was to design a model demonstrating that viable cells implanted in mice can repopulate or be suppressed, depending on the cytotoxic preload conditions.Methods. A two-step dormancy/recurrence (TSDR) murine model was designed, which mimics the extrusion of taken up drug from tumor cells. The viable cells preloaded with drug were implanted into mice. The survival rates of these mice were then used as criteria to demonstrate the relationship between resumed growth and drug cellular efflux/viability. The drug internalization patterns following their exposure to doxorubicin (dox) or degraded dox (dox-dgr) were investigated by exploring flow cytometry, spectral analysis, high performance liquid chromatography and confocal microscopy. Antiproliferative and myelotoxic capacity was evaluated by hematological nadir induced by the iv injected drug. Results. The viable SL2 lymphoma cells exposed to 10 µg/ml of dox for 30 min and injected into syngeneic DBA/2 mice were made unable to recure. Exposure of cells to lower dox concentrations (0.01 – 1.0 µg/ml) resulted in tumor recurrence, similar to that which was observed during implantation of an untreated tumor. Dox-dgr kept at 37 °C for 365 days lost its tumoricidal and antiproliferative capacity and displayed a loss of selectivity of nuclear fluorescence.Conclusions. Our TSDR model is a rapid convenient tool to study in vivo behavior of cells preloaded with cytotoxic drug. This approach focuses on mechanisms of tumor cells exiting dormancy, in relation to cytotoxic drug efflux. Multiple modifications of our TSDR model are possible, including nude mice models. As an example, we used one-year body temperature exposed dox to demonstrate its inability to retain sufficient cytotoxic capacity.