Combination of antibodies with chemotherapeutic agents to attack the heart of the tumor

In recent years there is an increasing number of cytotoxic chemotherapeutic compounds with the ability to rapidly kill dividing cancer cells in preference to non-dividing healthy cells. Nevertheless, the major drawback of chemotherapy is that, in addition to damaging the cancer cells, it also damages healthy tissues, causing side effects sometimes with serious consequences. Pharmaceutical companies face the challenge to search for drug delivery systems that achieve high cytotoxic efficacy against cancer cells while keeping limited systemic toxicity. The next generation of antibody-drug conjugates (ADCs) offers the promise of achieving this objective and increase the therapeutic index significantly [1]. 

To avoid side effects, drugs must be guided and released into the tumor sites through its association with ligands that are overexpressed or selectively expressed in tumor cells. For that purpose, the combination of monoclonal antibodies which selectively recognize cancer cells, together with the power of chemotherapeutic drugs, open up the possibility of using ADCs as a practical application for cancer therapy. Monoclonal antibodies can be linked to a radioisotope (in form of antibody radioimmunoconjugates or RAC), to a highly potent cytotoxic drug (in form of antibody-drug conjugates or ADCs) or protein toxins (known as immunotoxins) [2, 3].

Structure and elements of an antibody-drug conjugate. Chen S, Cao Y (2014) Assembly of Antibody-Drug Conjugates as Potent Immunotherapy. JSM Cell Dev Biol 2(1): 1006.

The production of ADCs faces several issues such as the target cell selection, the nature of the antigen, the structure and stability of the antibody, the linker chemistry and the cytotoxic payload. With the technology of recombinant DNA, phage display and transgenic mice it is now possible to create completely human antibodies that are not immunogenic, thus avoiding immune reactions against foreign antibodies while enhancing the effectiveness of the treatment. Chemotherapeutic drugs include antimetabolites, molecules interfering with microtubule polymerization and structure, as well as DNA damage/alkylating agents. These molecules can kill cells with extremely high potency, and their severe side effects limit the administrable dose as free drugs. They are therefore considered as ideal payload components of ADCs with high therapeutic index [4]. 

The conjugation strategy and chemistry are key factors for the success of ADCs, being their homogeneity one of the main challenges in its design. It is necessary to develop a strategy that allows the reaction of those residues placed on the surface of the antibody through a chemical reactive group present on the linker. Depending on the type of residue used, these strategies can lead to the production of ADCs with variable drug-antibody ratio (DAR). When the DAR is poorly controlled, the efficacy of the ADCs can be dramatically decreased due to an increase in the aggregation possibility, although higher DAR values are beneficial for the overall potency.

Mechanisms od drug delivery and release mediated by ADCs. When the drug contained in the ADC is released, either from the target cell following internalization and degradation of the ADC or within the extracellular space, the drug is then taken up by and kills surrounding or bystander cells which themselves may or may not express the ADC target antigen. Ponziani, S, et al. Antibody-Drug Conjugates: The New Frontier of Chemotherapy. Int. J. Mol. Sci. 2020, 21, 5510.

An ADC is composed of three different components:

1. A monoclonal antibody.

Antibodies used must have the highest affinity and selectivity for the target, recognizing an overexpressed target only at the tumor site to avoid delivering the pharmacological load inappropriately to non-target sites. Furthermore, the antigen against the antibody is directed and should be highly expressed in the target cells   and must be recognized by and bound to the antibody with a reasonable affinity to ensure rapid uptake in the target cell [5]. 

Critical is the design of humanized monoclonal antibody which contains only murine complementary determining regions combined with the human variable region, or fully human antibodies in order to prevent treatment rejection. 

Depending on the antibody used, ADCs can be directed against antigens that may or may not induce internalization through receptor-mediated endocytosis (RME), and can be classified as:

- Internalizing ADCs: ADCs are internalized by the target cells, and the antibody performs a fundamental role as it favors the internalization of the target antigen receptor, a crucial step for most ADCs to be effective. After internalization, drug release into the cytosol can exert its pharmacological effect, killing the cancer cells via molecule specific mechanisms. 

- Non-internalizing ADCs: Here the main pharmacological action relies on the cytotoxic payload exerting a bystander effect upon reaching the target tumor site. Once the ADC reaches the tumor site, proteolytic enzymes or the reducing conditions in the tumor extracellular environment act to liberate the drug payload, which facilitate the entry of drugs into the cells by diffusion, pinocytosis or other mechanisms. This strategy may also allow a bystander effect on non-target cells that are near the main target tumor mass due to diffusion of the released drug.

2. The linker that joints the antibody and the payload.

The linker is the component of the ADC through which the covalent chemical bond between the drug and the antibody is created. Linkers should be chosen based on the mechanism of action of the antibody and limit the potential chemical modification to the drug in order to avoid loss of cytotoxicity. As the drug must only be released at the target site, the linker must be stable enough in a biological environment to avoid unwanted release of the pharmacological molecule. 

We can differentiate two kinds of linkers:

- Cleavable linkers: They can be used in the design of either an internalizing or not internalizing ADC because the release of the payload is required to take place in either the extracellular tumor microenvironment or within the cytosol. Cleavable linkers exploit differential conditions of reducing power or enzymatic degradation that can be present either outside or inside the target cell. 

- Non-cleavable linkers: They must be internalized in order to release their cytotoxic payload as the antibody component needs to be degraded by the cell proteases [6]. ADCs with non cleavable linkers usually cannot exert a bystander effect because upon antibody degradation they are released as fragments of peptides that have a poor ability to permeate cells. They are highly efficient for treatment of tumors that express an antigen at high levels or for hematological tumors [7]. 

The greatest advantage of non-cleavable versus cleavable linkers is their improved plasma stability that results in reduced off-target toxicity while providing greater stability and tolerability. 

3. The payload.

Payloads suitable for an ADC must have: a) Good solubility in aqueous solutions, allowing an easier conjugation to the antibody and ensuring enough solubility to ADC under physiological conditions; b) A significantly higher cytotoxic activity in comparison to standard chemotherapeutic agents; c) Must induce cancer cell death by apoptotic mechanisms; and d) Possess an appropriate functional group to facilitate conjugation to the antibody. 

The most widely used drugs for ADC formulation comprise microtubule targeting agents since rapid cellular proliferation is one of the major discriminating features between cancerous and normal cells and antimitotic agents are in principle less toxic to the normal cells. Microtubule targeting molecules can be grouped in two main categories depending on their mechanism of action: tubulin polymerization promoters (microtubule stabilizers) and inhibitors (microtubule destabilizers). 

The second category of payload is comprised of DNA-damaging drugs, which are more effective than microtubule inhibitors so are better suited for ADCs targeting antigens that are expressed at low levels on tumors. These drugs are capable of killing non-dividing cells through the apoptotic pathway when used in combination with drugs that inhibit DNA repair, also killing target cells at any point of the cell cycle. There are four mechanisms of action exerted by DNA-damaging agents: a) DNA double strand breakage, b) DNA alkylation, c) DNA intercalation, and d) DNA crosslinking.

Other molecules used as payloads in ADCs are those based on different mechanisms of action that include the direct induction of apoptosis, spliceosome and/or RNA polymerase inhibition.

In the present there are almost a thousand of ADCs under investigation at various stages of clinical development for cancer treatment. Undoubtably, the combination of different therapeutic tools enhances our capacity to fight against cancer and other severe diseases. The use of ADCs arises as an important contribution to the future of immune-oncology, aimed to develop effective therapeutic strategies for every type of tumor.