Introduction: Monoclonal antibodies as targeted drug carriers combine the specificity of monoclonal antibodies with potent cytotoxic agents to form antibody-drug conjugates (ADCs). These engineered bioconjugates use precise conjugation chemistry, linker design and optimized drug-antibody ratio (DAR) to deliver payloads selectively to diseased cells, minimizing systemic toxicity. Key concepts include antigen targeting, internalization, lysosomal release, cleavable versus non-cleavable linkers, site-specific versus stochastic conjugation, pharmacokinetics, immunogenicity and analytical characterization. B. Pharm students should grasp mechanism-of-action, formulation, stability, regulatory considerations and common toxicities to understand ADC development and clinical use. Now let’s test your knowledge with 30 MCQs on this topic.
Q1. Which component of an ADC is primarily responsible for selective binding to the target cell?
- Monoclonal antibody
- Cytotoxic payload
- Linker
- Drug-antibody ratio
Correct Answer: Monoclonal antibody
Q2. What is the main purpose of the linker in an antibody-drug conjugate?
- Increase immunogenicity
- Join antibody and payload and control payload release
- Change antibody isotype
- Reduce manufacturing cost
Correct Answer: Join antibody and payload and control payload release
Q3. Which type of linker is cleaved inside the lysosome by proteases?
- Non-cleavable thioether linker
- Hydrazone linker
- Peptide (protease-cleavable) linker
- Polyethylene glycol linker
Correct Answer: Peptide (protease-cleavable) linker
Q4. Which conjugation chemistry commonly targets cysteine residues on antibodies?
- NHS ester reacting with lysine
- Maleimide reacting with thiols
- EDC coupling to carboxyl groups
- Click chemistry to glycans only
Correct Answer: Maleimide reacting with thiols
Q5. Drug-antibody ratio (DAR) most directly influences which property of an ADC?
- Antigen specificity
- Potency and pharmacokinetics
- Manufacturing temperature
- Antibody sequence
Correct Answer: Potency and pharmacokinetics
Q6. Which payload class is commonly used in ADCs to disrupt microtubules?
- Topoisomerase inhibitors
- Microtubule inhibitors (e.g., auristatins, maytansinoids)
- Monoclonal antibody fragments
- PEGylated peptides
Correct Answer: Microtubule inhibitors (e.g., auristatins, maytansinoids)
Q7. What is a major advantage of site-specific conjugation over stochastic (random) conjugation?
- Increased heterogeneity
- Improved batch-to-batch consistency and controlled DAR
- Higher immunogenicity
- Eliminates need for purification
Correct Answer: Improved batch-to-batch consistency and controlled DAR
Q8. Which analytical technique is commonly used to determine DAR and detect ADC heterogeneity?
- Size-exclusion chromatography (SEC) only
- Hydrophobic interaction chromatography (HIC) and mass spectrometry
- pH titration
- Light microscopy
Correct Answer: Hydrophobic interaction chromatography (HIC) and mass spectrometry
Q9. The term “bystander effect” in ADC therapy refers to:
- Immune cells attacking non-target tissues
- Released payload killing neighboring antigen-negative cells
- Antibody binding to multiple antigens
- ADC accumulation in the liver only
Correct Answer: Released payload killing neighboring antigen-negative cells
Q10. Which of the following is a clinically approved ADC example?
- Insulin glargine
- Trastuzumab emtansine (T-DM1)
- Rituximab alone
- Adalimumab
Correct Answer: Trastuzumab emtansine (T-DM1)
Q11. Non-cleavable linkers release payloads primarily after:
- pH-dependent diffusion in plasma
- Lysosomal antibody degradation freeing amino acid-linked payload
- Direct extracellular cleavage by proteases
- Spontaneous hydrolysis at body temperature
Correct Answer: Lysosomal antibody degradation freeing amino acid-linked payload
Q12. Which receptor is important for IgG recycling and contributes to long antibody half-life?
- Fc gamma RIIb
- FcRn (neonatal Fc receptor)
- Toll-like receptor 4
- Complement receptor 3
Correct Answer: FcRn (neonatal Fc receptor)
Q13. A common method to assess ADC aggregation is:
- Western blot only
- Size-exclusion chromatography (SEC)
- pH paper testing
- Ion-exchange chromatography exclusively
Correct Answer: Size-exclusion chromatography (SEC)
Q14. Which conjugation approach reduces fragmentation and increases stability by introducing engineered cysteines?
- Glycan remodeling
- THIOMAB/site-directed mutagenesis for engineered cysteines
- N-terminal acetylation
- Lipidation of the antibody
Correct Answer: THIOMAB/site-directed mutagenesis for engineered cysteines
Q15. Immunogenicity against ADCs can lead to:
- Increased therapeutic half-life
- Formation of anti-drug antibodies reducing efficacy and safety
- Guaranteed target engagement
- Complete resistance to all small molecules
Correct Answer: Formation of anti-drug antibodies reducing efficacy and safety
Q16. Which payload mechanism primarily causes DNA double-strand breaks?
- Microtubule stabilization
- DNA-damaging payloads like calicheamicin and pyrrolobenzodiazepines
- ATP synthase inhibition
- Histone acetylation
Correct Answer: DNA-damaging payloads like calicheamicin and pyrrolobenzodiazepines
Q17. What is one common clinical toxicity associated with ADCs containing microtubule inhibitors?
- Hyperglycemia
- Peripheral neuropathy and neutropenia
- Hypotension only
- Increased hair growth
Correct Answer: Peripheral neuropathy and neutropenia
Q18. Which factor most influences ADC tissue penetration?
- Molecular size and tumor microenvironment (e.g., interstitial pressure)
- Patient hair color
- Manufacturing site location
- Choice of dialysis buffer
Correct Answer: Molecular size and tumor microenvironment (e.g., interstitial pressure)
Q19. Hydrazone linkers are sensitive to which physiological condition?
- Neutral pH stability
- Acidic environments (e.g., endosomes/lysosomes)
- High salt only
- Low oxygen tension only
Correct Answer: Acidic environments (e.g., endosomes/lysosomes)
Q20. Which assay is most suitable for quantifying free (unconjugated) payload in ADC formulations?
- ELISA for intact ADC only
- LC-MS/MS for precise small-molecule quantification
- pH meter
- Simple UV absorbance without separation
Correct Answer: LC-MS/MS for precise small-molecule quantification
Q21. Resistance to ADC therapy can arise from:
- Increased antigen expression
- Efficient internalization and lysosomal processing
- Drug efflux pumps and reduced target expression
- Improved linker stability only
Correct Answer: Drug efflux pumps and reduced target expression
Q22. Which regulatory consideration is critical for ADC stability testing?
- Only temperature in summer months
- Forced degradation studies, real-time stability and ICH guidelines
- Ignoring light sensitivity
- Only testing color changes
Correct Answer: Forced degradation studies, real-time stability and ICH guidelines
Q23. Enzymatic site-specific conjugation methods include:
- Sortase A and transglutaminase mediated coupling
- NHS ester random lysine coupling
- Spontaneous disulfide formation only
- Heat-induced aggregation
Correct Answer: Sortase A and transglutaminase mediated coupling
Q24. Which property of the payload affects the potential for a bystander effect?
- Payload hydrophobicity and membrane permeability
- Antibody isotype only
- Storage vial color
- Linker solubility only
Correct Answer: Payload hydrophobicity and membrane permeability
Q25. A high level of ADC aggregation can lead to:
- Enhanced efficacy with no safety concerns
- Increased immunogenicity and altered pharmacokinetics
- Lower molecular weight species only
- Less need for cold chain storage
Correct Answer: Increased immunogenicity and altered pharmacokinetics
Q26. Which statement best describes a non-cleavable linker ADC mechanism?
- Payload is released extracellularly by serum proteases
- Payload remains attached to an amino-acid fragment after antibody degradation inside lysosome
- Payload is released by light activation only
- Payload diffuses out unchanged in plasma
Correct Answer: Payload remains attached to an amino-acid fragment after antibody degradation inside lysosome
Q27. What is the typical optimal average DAR range for many ADCs to balance potency and stability?
- 10–12
- 0.1–0.5
- 2–4
- 20–30
Correct Answer: 2–4
Q28. Which parameter is critical for selecting an antigen target for ADC development?
- Uniform high expression on healthy tissues only
- High tumor-specific expression and internalization upon antibody binding
- Antigen present only in red blood cells
- Low expression in disease tissue
Correct Answer: High tumor-specific expression and internalization upon antibody binding
Q29. Glycan engineering of antibodies for ADCs primarily affects:
- Payload chemical structure
- Fc effector functions and clearance
- DNA sequence of the payload
- Hydrophobicity of the linker only
Correct Answer: Fc effector functions and clearance
Q30. Which preclinical study would best evaluate ADC off-target toxicity in vivo?
- In vitro cell viability assay only
- Species-appropriate toxicology studies in animals including biodistribution and histopathology
- Simple pH stability test
- Antibody sequencing alone
Correct Answer: Species-appropriate toxicology studies in animals including biodistribution and histopathology
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