Antibody, Site, Linker & Payload Combinations to Optimize ADCs
The therapeutic window of an ADC highly depends on its (monoclonal) antibody component that determines the destiny of cytotoxic payload conjugated to it. Therefore, the antibody should have high target specificity and low cross-reactivity in order to limit undesired toxicity. As such clones are determined, it is critical to establish their homogeneity. This requires robust analytical methods (liquid chromatography/mass spectrometry) for characterization of the antibody. Otherwise, the heterogenous antibody profile can render the ADC prone to failure.
Another challenge in ADC development is the immunogenicity against the antibody. First-generation ADCs utilized antibodies of mouse origin which caused their failure because they were rejected by the human immune system. Later generations have chimeric, humanized, or even human antibodies instead to solve this problem. Apart from avoiding immune cells, the antibody should have enough stability to have long circulating life.
Chemical properties of the antibody is also a barrier in ADC development as they decide how many and which type of linkers can be conjugated to the antibody and to which part. Combinations of these parameters also significantly influence the stability of ADC.
In order to deliver cytotoxic payloads into tumor cells, antibody-antigen interaction should lead to efficient internalization of ADC. In the same context, having good retention after binding to target cells would serve to that end.
In some cases, the antibody binding to its target receptor may induce signaling and produce cytotoxic effect itself through mechanisms such as antibody-dependent cell-mediated cytotoxicity (ADCC), as it happens in the case of trastuzumab emtansine (Kadcyla). Such independent functioning of an antibody is not always considered beneficial for ADC efficacy, since it may reduce internalization of ADC into tumor cells. Such two-pronged killing (antibody and payload) is also a cause for concern since it may be too toxic for healthy cells.
Lastly, antibody isotype selection needs careful planning. Subclasses of IgG - IgG1, IgG2, or IgG4 (mostly IgG1) - is used in current ADC development. IgG3 is not employed as it has fast plasma clearance. The isotype selection defines the difficulty of conjugating linkers to antibody backbone. Antibody isotype also determines the potential immune effector functions such as ADCC.
In an ideal case, a target antigen should be highly expressed in the tumor (with homogeneous expression across the tumor cells), while having minimal or no expression in normal tissues. As an example, HER2 receptor, which is targeted by trastuzumab emtansine (T-DM1), have 100-fold higher expression in the tumor cells than the healthy cells. Accordingly, T-DM1 provides the greatest benefit to the patients with the highest expression of HER2.
Then, it should be internalized efficiently by receptor-mediated endocytosis and not recycled back to the surface. Non-internalized ADCs exert toxicity on neighboring cells. Also, its expression should not get downregulated. In this context, epitope on the target protein also can be important. For example, it was reported that different epitopes of the HER-2 receptor show differences in terms of internalization and degradation of antibody-antigen complex.
Antigens expressed on the surface of the tumor cells are preferred, as they are easier targets for circulating ADC. However, this may not be always possible or in some cases internalization may be hindered due to different reasons such as the high interstitial tumor pressure. For those cases, ADC could be targeted against antigens in the tumor microenvironment.
Shedding of the antigen could be a significant problem as free antigens wandering within the circulation will bind the ADC antibody and compromise its efficacy. Therefore, the target antigen should have minimal shedding.
In ADC design, one of the questions is that how many cytotoxic drugs will be conjugated to each antibody. In other words, what will be the drug‐to‐antibody ratio (DAR)? If DAR is lower than optimal, that will limit the efficacy of the ADC. However, when DAR is too high, ADC becomes unstable. Altered pharmacokinetic properties of ADC in that case reduces half-life and increases systemic toxicity. The optimal DAR depends on other ADC components as it ranges between 2 and 4 in clinically approved ADC systems.
Payload should be highly potent in terms of cytotoxicity because research shows that at best 0.01% of injected monoclonal antibody (or ADC) binds to target tumor cells. Moreover, optimal DAR also limits the amount of payload that is delivered into tumor cells. As a result, ADC payloads should have IC50 in nanomolar and even picomolar range. Consistently, classical chemotherapy drugs failed to show clinical benefit under ADC framework.
Resistance mechanisms that cancer cells develop against cytotoxic drugs should also be considered as a preclinical challenge. These mechanisms may include increased expression of efflux pumps that remove drugs from cells and altered microtubule composition among others. Exploration of the susceptibility of the payload to drug resistance is crucial. Appropriate drugs with minimal susceptibility should be chosen.
Hydrophobicity/Hydrophilicity of drugs is another issue in ADC design. More lipophilic drugs tend to pass through the cell membrane and kill neighboring cells. This phenomenon is named as bystander effect. It may confer advantage for the treatment of certain solid tumors as their cells have heterogeneous expression of ADC target protein. However, in other cases bystander effect may cause off-target toxicity.
The other challenges in finding an appropriate drug are its stability in blood, solubility in water, and possession of chemical functionalities to conjugate it to a linker molecule.
It is obvious that finding appropriate drugs that meet these criteria is challenging. Currently, two main classes of cytotoxic drugs are used in ADC development. Microtubule inhibitors block assembly of tubulins and cause cell cycle arrest at mitotic phase. Auristatins and Maytansinoids are microtubule inhibitors that are used in three FDA approved ADCs (Adcetris, Kadcyla, and Polivy). The other class includes DNA damaging drugs. Calicheamicin, a drug used in two FDA approved ADCs (Besponsa and Mylotarg), binds DNA’s minor groove and induces double-strand DNA breaks and rapid cell death. Since they could function independently from cell cycle progression, DNA damaging drugs could be used against cancer stem cells which have lower proliferation rate.
Linker connects cytotoxic payload to monoclonal antibody, hence its properties play a significant role in pharmacokinetics and therapeutic window of ADC. The major preclinical challenge to the ADC efficacy and therapeutic index is high deconjugation rate. Ideally, it should have enough stability that allows ADC to circulate in blood without releasing the payload and causing systemic toxicity. On the other hand, they should release payload once they are inside the target cell. The two types of linkers used in ADC structure are non-cleavable and cleavable.
Non-cleavable linkers provide more stability to ADC and the release of payload happens by lysosomal degradation of the antibody. Even after degradation, payload is released as attached to the linker and the terminal amino acid residue of the antibody. Thus non-cleavable linkers are optimal for drugs that preserve their potency when bearing the moieties left from the degradation. For example, Trastuzumab emtansine (Kadcyla) has non-cleavable thioether linker its maytansinoid-based DM1 drug and antibody. Conversely, MMAE used in the Brentuximab vedotin (Adcetris) is a protein-based cytotoxic drug and has optimal potency in its unconjugated form hence the linker used with this drug is cleavable.
Cleavable linkers release their payload when they meet certain physiological stimulus present in their target site. For example, acid-sensitive linkers (e.g. hydrazone-based linker of Gemtuzumab ozogamicin/Mylotarg) are cleaved in the low pH environment of lysosomes/endosomes. However, these linkers have shown a certain level of plasma instability. As another example, valine-citrulline linker in Brentuximab vedotin is a protease-sensitive linker that is cleaved by cathepsins in lysosomes. Lastly, disulfide linkers used in the design of some ADCs are cleaved by high glutathione concentration found in tumor cells.
Linker’s hydrophilicity/hydrophobicity also affects ADC therapeutic index from many aspects. One of them is bystander effect, that, as it is mentioned above, may be beneficial against solid tumors. Depending on whether you want to enhance or reduce this effect, linker design may change. Non-cleavable linkers that stay with the payload together with a charged amino acid may prevent payload’s passage through membranes thus lowering bystander effect. This effect may be enhanced by using non-polar, more hydrophobic linkers. On the other hand, increased hydrophobicity is associated with high plasma clearance. Moreover, as the linker-drug molecule gets more hydrophobic, it becomes more prone to the work of MDR1 efflux pumps. Therefore, the question of which non-cleavable linker to select depends on which type of cancer cell you are targeting (i.e. does it have a drug-resistance mechanism), whether it is a solid tumor, and which drug you are using as a payload. Cleavable linkers allow drugs to leave alone thus here drug’s chemical properties determine its final destiny.
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