Emerging Role of Antibody Drug Conjugates (ADCs) in Therapeutics: An Appraisal

Antibody drug conjugates intends to pursue the monoclonal antibodies (mAbs) as the potent source of delivering cytotoxic drugs to more specific site which binds selectively to the antigen expressing tumor cells. Inspite of the facts, various other safety profile must be considered while designing and optimizing ADC such as selecting congruous target antigen and method of conjugation. Each and every component of the ADC i.e antibody, linker and the drug should be optimized to the extent of desirable targeted therapy which will ameliorate as well as enhance tolerability. The past decade had witnessed advances in newer cancer treatments with extremely selective small molecules targeting the specific genetic abnormality causing the disease. The approach of traditional cytotoxic agents in the treatment of cancer, unlike the target specificity, they affect both healthy as well as cancer cells. In order to build a powerful and more specific cytotoxic agent with target oriented mAb’s designing attributes would lead to pertinent and potential breakthrough in cancer treatments. Therefore ADC’s were developed with the intention that antibody would target the specific antigen of the tumor wherein the drug attached to it would induce its cytotoxicity. Developement of new techniques and methods in implementing new generation ADC’s in the past decades incorporated non-immunogenic monoclonal antibodies comprising linkers having equitable stability and distinctly potent cytotoxic agents. Newer challenges although remain but comprehensive clinical accomplishment is generating increased interest in this therapeutic class of drugs.


Introduction
Bioconjugation strategies offer promising therapeutic benefits by synergy of those of the individual components [1][2]. The bioconjugation method approaches towards numerous medical necessities hence it plays an important role in the emerging therapeutic field. Simply bioconjugates are the covalent crosslinking of a semisynthetic/synthetic chemotypes such as drugs, oligomers, peptides etc to a biomolecule like macromolecular proteins, polysaccharides or nucleotides [2][3]. The bioconjugates are attributed towards transpiring class of medicines and are gaining more importance in the biopharmaceutical field across the globe. Bioconjugates like Antibody Drug Conjugates (ADCs) in targeted oncology research is found to improve the durability of anti-tumor responses in cancer patients [4][5][6][7][8][9][10][11][12][13].
Promising preliminary data of the Antibody Drug Conjugates in the research pipeline and in clinical trial stages help to move forward cancer therapy from traditional chemotherapy to target anti-cancer treatment modalities [7][8][14][15][16][17][18][19]. The increased growth of ADC's were proportionally higher as corroborated in the last two decades. The clinical approval of Adcetris (brentuximab vedotin) in 2011 and Kadcyla (ado-trastuzumab emtansine) in 2013 has sparked development of novel ADCs [14]. In addition, towards the improvement of safe and effective immuno-oncology drugs like ADCs, a plethora of payloads with specific molecular mechanisms of cytotoxic activity have been considered during past years [8,12]. The progress of preclinical and clinical research of ADCs driven by the higher ADCs exposure levels, development of site-specific conjugation biotechnology, combined with improvements in linker chemistries [12].
The majority of ADCs in clinical pipeline are conjugation of IgG antibodies with bioactive tubulin-targeting antimitotic agents like derivatives of maytansine, auristatins etc [14]. Maytansinoids and auristatins bind with the vinca binding domain of tubulin and arrest the microtubule dynamics in the G2/M phase [20][21]. Although, emerging clinical experience with ADCs containing DNA-targeting payloads, explore the pivotal requirements needed for successful ADC design [14]. In order to evaluate the impact of different mechanism-of-action payloads on various immune cells more exploration of ADC research is needed.
This review article intends to exemplify the proposed challenges, mechanism of action, therapeutic efficacy and safety level parameters which are in association with the judicious use of ADC's thereby enhancing the improvement in the upcoming treatment methods in medical science. This developmental process which targets the payloads would be prospective enough to bridge the gap in future findings.

Study Methodology
More than 50 relevant articles from indexed journals were extensively reviewed based on the keywords-Bioconjugation, Antibody Drug Conjugation, site-specific conjugation, ADCs cytotoxic payload, etc. from PubMed central and Google Scholar with special emphasis on articles published after 2014. Recent developments in ADCs design, ADCs in the research pipeline, various targeting antibodies were carefully studied and comprehensively noted down to come to the novel findings.

Antibody Drug Conjugate and Its Design
Previously, anti-cancer agent conjugated with chimeric or murine antibody fails to address enough localization in the target cancerous cells thereby leading to immunogenicity [22][23]. In order to avoid off-target toxicity, the antibody should be designed in such a way where the antibody is selective and have better binding affinity to that of target antigen with high expression at the tumor site but low expression on normal tissue [23][24][25][26][27][28]. The knowledge arises from the recent development of chemistry and biology is able to address the designing problem of ADCs [24].

Antibody Selection for Antibody Drug Conjugates
Various points including affinity, specificity, pharmacokinetics (PK) should be considered before selecting the mAbs [22]. Currently researchers like to select human or humanized IgG1 mAbs with a few chimerics in the development of ADCs as it show acceptable PK properties [29]. Brentuximab vedotin is one of the approved ADC is effective in Anaplastic Large Cell Lymphoma and Hodgkin's lymphoma, It comprises a chimeric IgG1 (cAC10) against CD30 receptors on cancer cells. T-DM1 is an ADC that contains humanized anti-HER2 IgG1 antibody trastuzumab. [23] Comparing in vivo efficacy between IgG1 and IgG2 conjugates was identified for anti-CD70 which is an IgG isotype while conjugating with the payload monomethyl auristatin phenylalanine (MMAF). [30] In addition, IgG2 and IgG4 (hinge stabilized) are also used in clinical development i.e., AGS-16M8F (anti-ENPP3 IgG2-MMAF) and inotuzumab ozogamicin (anti-CD22 IgG4-calicheamicin) [26,27]. Therefore, the isotype of the antibody plays a key role in substantiating the stability factor of the ADC pertaining to the strategy of conjugation and payload attachment.

Cytotoxic Payloads Delivery
As very small amount of the injected dose gets localized to the tumor site, payloads should active in the picomolar or better potencies range, i.e. cytotoxic payload should be highly potent to the tumor cells [22]. Scientists generally favour to choose natural product or derivatives of natural product as ADC payload [31].
In order to get more insight into the mechanism of action of cytotoxic payload there are mainly two types of mechanism: (1  soil microorganism M. echinospora calichensis. [35][36][37] Streptomyces sp. Bacteria. [39] African shrub Maytenus ovatus and subsequently in the soil microorganism Nocardia sp. [42][43][44]. MOA Inhibit tubulin polymerization.Metaphase arrest.The third amino acid (dolaisoleucine) was the most important for cytotoxicity. [33,34] Generation of highly-reactive 1,4-benzenoid diradical, thatcauses cleavage of DNA double strand and finally cell death. [37] Duocarmycins derivativesbind to AT-rich regions of the DNA minor groove, which results in irreversible alkylation, and ultimately lead to cell death. [40] Bind to the same site on tubulin as the vinca alkaloids. [45][46] Remarks Various conjugated antibody for this payloads areanti-CD79b The clinically successful antibody drug conjugates currently under evaluations are briefly enumerated:
Doxorubicin binds to topoisomerase, via intercalation it inhibit DNA replication and poses cellular cytotoxicity. For CD74-positive multiple myeloma, milatuzumab-doxorubicin conjugate is presently in Phase I/II clinical trials [48].
The pyrrolbenzodiazepines containing ADC's (SGN-CD33A and SGN-CD70A) are some of the evident payloads which binds covalently to discontinuous sequences in the subsidiary groove of DNA manifesting the potential chemotherapeutic activity and they are isolated from Streptomycin species. These PBD-containing ADCs are also under clinical pipeline [22].
In order to explore novel cytotoxic payloads, researches are being investigated worldwide. Identification of novel antitumor chemotype leads to future development of ADCs.

Conclusion
The multiple linker payloads screening on multiple sites of an antibody is very challenging. There has been a growing interest to screen ADCs with the best combination of site and payload. Previous mentioned combinatorial problem may be addressed by high-throughput methodologies. Very recently Puthenveetil et al. [49] published an article, in which they introduce a method to screen multiple linker payloads, sites or their combinations in-parallel in 96-well plate. The authors reported solid-phase, site-specific conjugation methodology of linker-payloads to antibodies and Fab fragment. Interestingly the purification of ADCs with typical size-exclusion or hydrophobic interaction chromatography is also avoided by this novel method.
Very recently a novel ADC ASG-15ME (Product name, AGS15E, conjugation of SLITRK6-specific human gamma 2 antibodies (Igγ2) with MMAE via a protease-cleavable linker) is developed by Morrison et al [50]. The proposed ADC were found to be effective for the treatment of advanced urothelial cancer as suggested by the evident in vitro and in vivo cytotoxic activity after conjugation to Monomethyl Auristatin E and this AGS15E entered into the Clinical Trials Phase I. SLITRK6, belongs to the transmembrane SLITRK proteins family that having conserved leucine-rich repeat domains, is a bladder tumor antigen containing an open reading frame gene of 841 amino acids and. High expression of SLITRK6 in advanced transitional cell bladder cancer and in addition of a lesser expression in lung, breast and glioblastoma epithelial tumors were first time reportedby Morrison et al. Waiting for the success ofASG-15ME in all phases of clinical trials that may leads to clinical development of ASG-15ME as a new treatment for advanced bladder cancer.
On the other hand, a new type of stable, pharmacokinetically safe antibody drug conjugate was designed in the lab of Yusuke Ogitani with having excellent anti-tumor activity [51] In rats and cynomolgus monkey the designed payload DS-8201a has got admissible safety profile. It contains novel topoisomerase I inhibitior namely exatecan mesylate (DX-8951f) as a payload with antibody that targeting epidermal growth factor receptor HER2. Belonging to the family of transmembrane receptor, HER2 is highly over expressed in breast, bladder, cervical, colorectal, esophageal, gastric, head and neck, liver, lung, ovarian and salivary gland cancer cell lines. Generally the drug-to-antibody ratio (DAR) of ADCs ranges from 2 to 4. The rapid clearance of the ADC is observed in vivo if the DAR is increased and also off-target toxicity results due to high exposure of the released drug in plasma. All though 8 molecules of THE DRUG is conjugated with each antibody in case of DS-8201a, but interestingly the above mention problems were not observed. Above all, DS-8201a is promising to answer T-DM1 refractory breast cancer, IHC 1+ and 2+/FISH negative patients and HER2-positive patients.