Drug Design_L2 Flashcards
What are Antibody–Drug Conjugates (ADCs)?
(1)Antibody–Drug Conjugates (ADCs) are monoclonal antibodies or antibody fragments attached to biologically active molecules through chemical linkers with labile bonds, the benefit of applying the ADCs is the targeted antigens expressed exclusively or in extremely low abundance on the tumour cell surface
(2) Deliver highly cytotoxic agents directly to tumour cells without affecting other dividing cells in the body
while bound to the antibody, the chemotherapeutic ‘‘payload’’ no longer circulates systemically and is therefore well tolerated by healthy cells or tissues.
How is the antibody nature utilised by the ADCs?
(1) antibodies bind to the receptor of the cell specifically, ADCs are designed to directly target and kill cancer cells, and so the antibody has to be able to recognise and bind to its corresponding antigen localized on the tumour cell.
(2) the cell internalises the receptor-antibody complex. Once bound to the antigen, the entire antigen–ADC complex is then internalized through receptor-mediated endocytosis. The internalization process proceeds with the formation of a clathrin-coated early endosome containing the ADC–antigen complex.
(3) attached pay-loader released/digested inside the lysozyme. Once inside the lysosome, the ADC is degraded and free cytotoxic payload released into the cell, leading to cell death. The mechanism of cell death will depend on the type of cytotoxic payload, the antibosy and linker are digested inside the endosome and the receptors are recylced to the cell surface membrane, payload molecule is released to cause cell death.
(4) act either inside the nucleus or in the cytosol
What are the limitations of ADCs? What strategies can be applied for improvement?
(1) An ADC binds to an antigen on the surface of a cancer cell and then internalises, after which the highly cytotoxic payload molecule is released, typically by lysosomal cleavage, the ADC molecules kill the tumour cells, at the same time, somatic cells are killed as thwy often contain the same receptor as the cancer cells, moreover, the selection pressure posed on the tumour cells promotes the faster gene mutation.
(2) ADC needs to retain the selectivity of the original monoclonal antibody while being able to release the attached cytotoxic payload in concentrations high enough to kill the targeted tumour cells. Three key components to an ADC: the antibody, the linker and the payload, each of which requires optimisation.
What properties should the payloaders have in order to lead to sufficient cell death?
(1) cytoxic enough in low concentration, since the concentration applied to the cell does not mean the concentration reaching the cell, additionally, overloading the antibodies in the bloodstream can induce problems
challenges in designing ADCs?
(1) circulation of ADCs into the bloodstream: prevent/minimise the pre-mature release of payloader from the antibodies and the off-target toxicity
(2) binding: the mADC must retain high affinity to binding to the tumour cells
(3) inefficient internalisation due to limited target antigen level may prevent the cytokinetic from reaching the threshold concentration within the cell
(4) excessive binding to the FcRn can reduce the amount of toxic payload released into the cell
(5) ADC needs to release the payloader correctly in its active form
(6) potency of the payloader must be sufficient to kill the cell in the low concentration
What are the two major types of the linker present in the ADC cells?
(1) cleavable
(2) non-cleavable
What criteria should be based on for the tumour antigen selection?
(1) Requires substantial expression by tumour cells but limited expression by cells in normal tissues
(2) Limited number of antigens on the tumour cell surface (ranging from approximately 5,000– 106 antigens per cell) and the average drug-to-antibody ratio (DAR) of most current clinical-stage ADCs is limited to 3.5–4, the amount of the drug delivered by ADCs into tumour cells is low. However, the antibody based treatment is more effective in the bloodstream but not for the solid cancers.
(3) highly expressed on cancer cells and altogether absent on normal cells— is extremely rare, if not nonexistent, the cancer cell expression is higher than that of the normal cells, therefore, the side effects imposed on the normal counterparts are relatively small.
What criteria should be based on for the antigen-drug linker selection?
(1) Need to consider stability and drug release
(2) Stability typically refers to retention of drug by the antibody either ex vivo in buffers, plasma, or blood or in vivo after administration
(3) Antibody stability should also be considered upon conjugation with the drugs due to faster clearance: antobodies are more prone to degradation with more drug molecule attached, DAR is a good choice for the antibodies as it not only supports the drug transport and recognition but also facilitates in killing cancer cells.
(4) Stability of drug upon release
classfiy the cleavable linkers?
(1) Cleavable linkers utilise the differences in conditions between the bloodstream and the cytoplasm within tumour cells
(2) The change in environment once the ADC–antigen complex has internalized triggers cleavage of the linker and release of the active payload
(3) Cleavable linkers are divided into three main sub-categories: (1) Acid-labile (e.g.hydrazones), (2) Reducible (e.g.disulphides) and (3) Enzyme-cleavable (e.g., peptides, linkers include glucuronide-triggered linker/Val-Cit linker).
define the meaning of acid-labile linker
(1) Acid labile hydrazone linkers remain stable in neutral pH and utilize the low pH within the endosome/lysosomes to cleave the conjugation and release the drug from the ADC
(2) Clinical studies indicating that acid-cleavable linkers are associated with non-specific release of the payload, which can lead to systemic toxicities
define the meaning of reducible linker
(1) The principle behind this type of linker is the ability to respond to a difference in reduction potential between the intracellular compartment of a tumour cellversusthe blood based on glutathione concentration gradients, the environemnt inside the cancer cells has strong reducing power to enable the reduction of disulphide, as a result of the glutathione
(2) Disulfides are stable at physiologic pH but are susceptible to nucleophilic attack from thiols, such as glutathiol
(3) Abundance of thiol molecules within tumors, especially generated during stress like hypoxia, for thiols are involved in survival and growth of tumor cells
define the meaning of enzyme-cleavable linker
(1) This type of linker takes advantage of hydrolytic enzymes capable of recognising and cleaving particular peptide sequences contained within linkers, thus ensuring that the ADC only undergoes cleavage in the lysosomal environment and not in the plasma
(2) Enzyme-cleavable linker is based on the β-glucuronide moiety. The enzyme β-glucuronidase, which can release payloads from β-glucuronide-containing linkers, is present in lysosomes and is over-expressed in some tumour cell types. Hopefully, the tumour cells are specifically targeted. An important characteristic of a β-glucuronide linker is its hydrophilic properties, which can potentially reduce aggregation during conjugation compared with constructs containing dipeptide-based or other linker types
What is the mode of ADC action for ADCs with cleavage linker?
(1) binding to a specific Ag, followed by internalization of the ADC-Ag complex according to a clathrin dependent mechanism
(2) transfer into lysosomes
(3) lysosomal cleavage of the linker between the trigger and the spacer (if present), which then degrades to release the free drug
(4) binding of the drug (MMAE, MMAF, DM1, DM4) to tubulin or translocation of the drug (calicheamicin) into the nucleus
(5) Alternatively to 4, transfer of the drug to the cytosol
(6) diffusion of the drug to neighboring cancer cells to achieve bystander killing effect (all drugs except MMAF), it is most desirable if the neighbouring cell is a cancer cell and vice versa.
(7) MMAF, because of its charged character, cannot cross the membrane and therefore does not exhibit a bystander killing effect
(8) all the previous steps lead to cell death
define the meaning of non-cleavable linkers?
(1) Drug usually remains attached to mAB, the covalent linkage is not disruptable by pH, reducing strength and the enzyme cleavage
(2) Non-cleavable linkers engage lysosomal degradation only.
(3) Increased plasma stability
(4) Lower risk of systemic toxicity due to premature release of the payload
(5) Better therapeutic window, with improved stability and tolerability
(6) for example, the thioester bond is one of the non-cleavable linkers: -S-CH3-CH3-CO-
What is the mode of ADC action for ADCs with non-cleavage linker?
(1) ADC recognises a specific Ag, followed by internalization of the complex ADC-Ag through a clathrin-dependent mechanism
(2) transfer into lysosome
(3) complete digestion of the mAb to release the active corresponding metabolite amino-acid-NCL-drug
(4) transfer to tubulin or transfer to cytoplasm, becuase of the linker protein, the drug cannot be transported to the nucleus and acts in the nucleus
(5) Impossible to achieve bystander effect, therefore transferring to tubulin.
(6) all the previous steps lead to cell death of the Ag-positive cancer cell.
