INTRODUCTION:

Though drug is considered as magic bullet but drug discovery was never magical, the idea of monoclonal antibody (mAb) technology by Kohler and Milstein was since 1975 ¹. After intensive efforts, clinically approved drugs and monoclonal antibodies (mAbs) worked together; this has modest activities and is immunogenic. These studies promoted the investigation of several key parameters that influenced activity and tolerability. These includes the antigen targets, the use of non- immunogenic mAb carriers, incorporation of highly potent drug, novel conditionally stable linker technologies, specific method used to attach drugs to mAbs and as a result of these investigations, new agents with pronounced clinical activities have been developed which are commonly considered as antibody drug conjugates (ADCs)^1,2.

ADCs are an example of Bioconjugation, where antibodies can be engineered to carry a biologically active drug and deposit it in the target cell. Bioconjugation involves the linking of two or more molecules to form a novel complex having the combined properties of its individual components.

Natural or synthetic compounds with their individual activities can be chemically combined to create unique substances possessing carefully engineered characteristics³.Here, the monoclonal antibodies (mAbs) are empowered through conjugation with cytotoxic drugs by chemical linkers with labile bonds and making it capable of antigen-specific delivery of highly potent cytotoxic drugs to tumor cells. The synergistic effect is noted by combining the unique targeting ability of mAbs with the cancer-killing ability of cytotoxic drugs. This allows sensitive discrimination between healthy and diseased tissue, with fewer toxic side effects than traditional chemotherapies.

Advances in coupling antibodies to cytotoxic drugs permit greater control of drug pharmacokinetics and significantly improve delivery to target tissue. Potent new anticancer drugs can now be used to target cancers while minimizing exposure of healthy tissue^4,5.One approach to improving selectivity is to identify therapeutic targets with altered levels of expression on malignant versus normal cells and direct therapy against those targets.

ADCs requires unique supporting infrastructure to meet bio/ pharmaceutical industry standards for safe, effective and reliable manufacturing. These highly potent biopharmaceuticals present a series of unique manufacturing challenges driven by their potency and raw material supply chain. Facility design must take into consideration these manufacturing challenges to insure employee safety and robust process performance.⁶

HISTORY:

The first success in this field was when Gemtuzumab Ozogamicin, an antibody-drug conjugate that is composed of the potent DNA minor groove binder Calicheamicin linked covalently to an anti-CD33 mAb,^{7, 8}was approved in 2000 by the United States Food and Drug Administration for the treatment of patients with relapsed acute myeloid leukemia⁹. Other antibody-drug conjugates currently in development use a number of different cytotoxic drugs and linkage systems. Following to its achievement in the development of monoclonal antibodies (mAbs) for the treatment of cancer, such as Rituximab for the treatment of non-Hodgkin’s B-cell lymphoma and Trastuzumab for breast cancer, ¹⁰ have proven that mAbs in form of ADCs can be a valuable weapon in the battle against cancer.

With the recent approvals of Brentuximab Vedotin (Adcetris; Seattle Genetics/ Millennium Pharmaceuticals) and ado-Trastuzumab Emtansine (Kadcyla (T-DM1); Roche/Genentech), antibody– drug conjugates (ADCs) are a drug class with a robust pipeline and a flurry of deal-making among drug discovery companies. Owing to improved technology and appropriate targeting, the clinical application of ADCs is accelerating rapidly^11,
12.There has been a continuous increase in the count of ADCs which are currently undergoing clinical trials for the treatment of a variety of cancers. At least 17 ADCs entered the clinic in 2011 and 2012, up from just 8 in 2009 and 2010^{13, 14}.

Many others are making their way through preclinical development. This increased attention to ADCs has been triggered by advances in linker chemistries. Modern linker chemistries with enhanced stability have been developed to ensure that ADCs remain intact until they reach their target cells¹⁴.

Refinements in linker technology combined with greater knowledge of targets have led to the emergence of second-generation ADCs. These newer agents possess improved stability in the bloodstream, thereby allowing for the appropriate delivery of the cytotoxic to the target cell.

WHAT ARE ADCS?

An ADC contains two parts: a monoclonal antibody and a small amount of a highly potent cytotoxic drug, linked to the antibody. Like a key sliding into a lock, the ADC’s antibody binds with a particular receptor on the target cell’s surface. Then the linkage breaks, and the ADC releases a lethal toxin into the cell.

Tumor Cell Specific Antigens:

A variety of cell surface antigens expressed on hematologic malignancies and epithelial tumors are being evaluated as ADC targets. Cancer cells do not display novel immunogenic antigens because such behavior would lead to rapid clearance by the immune system. However, cancer cells occasionally express normal cell surface antigens at levels that are distinguishable from their healthy normal cellular counterparts.ADC target antigens are typically highly expressed on the surface of cancer cells compared to normal cells.

These antigens include over-expressed B-cell surface proteins in Non-Hodgkin’s Lymphoma (NHL) such as CD19, CD20, CD21, CD22, CD40, CD72, CD79b and CD180, extending to the T-cell proteins CD25 and CD30 of the immune system. Moreover, proteins that are over expressed on carcinoma cells, including the Human Epidermal Growth Factor Receptor 2 (HER2); Prostate-Specific Membrane Antigen (PSMA) and Cryptic Family Protein 1 B (Cripto) are also antigens. These tumour-associated antigens have been studied as potential treatments for the following oncology indications: Leukaemia, Lymphoma and Multiple Myeloma¹².

Cytotoxic drugs in the development of ADCs:

Readily available and clinically approved drugs used in early ADC development such as Doxorubicin, Methotrexate^{16,
17, 18, 19,} have relatively low potencies, so access to solid tumors by macromolecules was inefficient and accumulation of the cytotoxic drug in target cells was poor. The low clinical activity exhibited by these early ADCs prompted the development of ADCs employing much more potent cytotoxic drugs, which, though too toxic to use in an untargeted manner, have sufficient potency to be used in a more targeted manner. The majority of highly potent cytotoxic agents used in current ADCs are Auristatins, Maytansinoids, or Calicheamicins¹⁴.

Main categories of highly potent cytotoxic anticancer agents are generally used in the development of ADCs. That are-

a. Microtubule disrupting agents-Microtubules play important roles in the cell cycle. Cells are unable to divide in case of malfunctioning of microtubule. They bind to tubulin and inhibit of polymerization, causing cell cycle arrest and subsequent apoptosis of the target cell Eg. Auristatins, Maytansinoids, Taxol.

b. DNA minor groove disruptors - Kill cells at any point. These agents exert their cytotoxic effects by binding to DNA in the double-helix minor groove. For example, binding of Calicheamicin analogs induces double-strand breaks in DNA, resulting in cell death. These molecules are very potent, but their lack of selectivity has prevented their use as single-agent therapeutics. Eg. Duocarmycin and Calicheamicin analogs.

c. Topoisomerase II inhibitors- Topoisomerases are one of the popular targets for chemotherapy treatments. They block the ligation step of the cell cycle and generate single and double stranded breaks which harm the integrity of the genome. Introduction of such breaks then leads to apoptosis and cell death.Eg. Doxorubicins and Camptothecins^{20, 21}

d. The other emerging drug payloads include the sequence selective DNA alkylating agents called Pyrrolo Benzo Diazepines(PBDs) ^{22, 23.}

All the above cytotoxic drugs have shown to possess in vitro potency against various tumour cell lines in the 10^-9-to 10^-11 M range compared to first generation ADCs using doxorubicin (IC50 = 10^-7 M)^22,
23.

Methods for conjugation of the cytotoxic drug to the mAb^22-24:

(A) Lysine conjugation-

It occurs when the mAb attaches to the linker to generate the new linker-modified mAb. This linker modified mAb facilitates a conjugation reaction with the cytotoxic drug to generate the ADC.

(B) Cysteine conjugation

This approach involves the inter-chain partial reduction of the disulfide bonds to generate Cysteine Sulfhydryl (Cys-SH) groups on the mAb. These Cys-SH groups allow for a single step conjugation to the linker-drug to produce the heterogeneous ADC.

In both conjugation processes, it is important to control the molar ratios of drug to antibody (DAR) to optimize the in vivo pharmacokinetics, efficacy and safety profiles of the ADC. Most linkers have a short half-life; for example, the hydrazine linker in BR96-doxorubicin has an in vivo half-life of 43 hours in blood compared to the naked BR96 mAb half-life of several days and/or weeks and therefore chemical modification is required to prolong linker stability and aid solubility. The use of non-reducible hydrophilic linkers and/or spacers such as bis-Maleimido-Trioxyethylene Glycol (BMPEO) in antibody-linker-drug combinations, has contributed to the biological stability to bypass multi-drug resistance (MDR) of ADCs^{25, 26}.

MECHANISM OF ACTION:

In the process of ADCs, an anticancer drug is coupled to an antibody that specifically targets a certain tumor marker (e.g. a protein that is only to be found in or on tumor cells). Antibodies track these proteins down in the body and attach themselves to the surface of cancer cells. The biochemical reaction between the antibody and the target protein (antigen) triggers a signal in the tumor cell, which then absorbs or internalizes the antibody together with the cytotoxin. After the ADC is internalized, the cytotoxic drug is released and kills the cancer. Due to this targeting, ideally the drug has lower side effects and gives a wider therapeutic window than other chemotherapeutic agents^{27, 28}.

Mechanism of ADC activity:

ADCs bind to antigen expressing cells following tumor localization and get internalized into endosomes/ lysosomes. Here biologically active drug gets released following enzymatic cleavage ormAb catabolism. The drug which is released enters the cytoplasm and there binds to its molecular target (typically DNA or tubulin). This results in cell cycle arrest and apoptosis. The drug may also diffuse out of or be released from dying cells and if it is membrane permeable, it can enter cells (antigen positive or negative) in close proximity and mediate by stander killing.The theory behind the mechanism of action of ADCs involves the processes given in Table 2.

TYPES OF ADCS:

Upon binding of the ADC, many antigens undergo rapid internalization along with the bound ADC through receptor-mediated endocytosis. The efficiency of internalization can vary between an ADC and the unconjugated antibody; in some cases both species internalize at the same rate, whereas in other cases the ADC internalizes more efficiently. The rate and extent of internalization are important because it influences uptake of the drug as well as release of the drug in tumor and normal cells.

Non-Internalizing ADCs:

To be effective, non-internalizing ADCs needed to remain intact in the circulation and yet selectively release active drug at the tumor site.

Typically these ADCs utilized Peptidyl linkers designed to be cleaved by enzymes such as Cathepsins and matrix Metalloproteinase expressed in the tumor, or linkers that would release drug by hydrolysis at the slightly acidic pH observed in many solid tumors. For the most part, these non-internalizing ADCs did not show significant antigen-specific activity and did not improve the therapeutic index (maximum tolerated dose/active dose) relative to that of the free drug.

Internalizing ADCs:

The use of mAb’s that internalize following antigen binding has resulted in the design of linkers that are stable in circulation and efficiently release active drug following antigen specific binding, internalization and trafficking to endosomes/ lysosomes. Internalizing ADCs have demonstrated impressive preclinical and clinical activity^{29- 31}.

Typical ADC Characteristics and challenges^{1,
5, 17, 32-34}:

· ADCs are designed to be stable in circulation and to effect intracellular drug release following antigen-specific binding and internalization of the ADC.

· Broader therapeutic window (efficacy at low dosages).

· High therapeutic activity.

· Targeted delivery of potent drugs.

· Reduced undesirable side-effects of chemotherapy.

· Address unmet needs in cancer therapies.

· The dissociation of modern linker chemistries is driven by enzymatic degradation in the target cell, whereas early generation dissociation was a pH - dependent process.

· Targeted therapeutic binding specifically to the target antigen.

· Highly potent agents can be delivered selectively to tumor cells.

· Wide therapeutic index.

· Prolonged circulation half-life; conjugate remains stable in circulation.

· Decreased adverse effects.

· The efficacy of an ADC is largely dependent upon how many copies of the target are expressed on the tumor cells and how fast the ADC is being transported into the tumor cell. In general, rather high copy numbers (> 1, 00,000 per cell) are required to import enough toxins to kill the cell. Despite advances in ADC engineering, only a fraction of the total injected antibody dose can be effectively delivered to the target tumor. Hence the cytotoxic agent bound to the antibody needs to be highly potent so that any ADCs that do reach their target cells have the maximum killing potential³⁵.

· Production of nearly homogeneous preparations (i.e., single chemical species) during the synthesis of antibody conjugates is one of the important goal as drug-loading stoichiometry and homogeneity are also important determinants of the safety and efficacy of antibody conjugates. It is important to avoid both under conjugated antibodies. Highly conjugated species can have markedly decreased circulating half-life and impaired binding to the target protein which decreases the potency and efficacy of the antibody conjugate. Therefore, for most ADCs, on an average of 3 to 4 drug molecules are linked per antibody molecule as it minimizes the percentage of unconjugated antibody, maintains the circulating half-life near that of the naked antibody, preserves antibody binding to the target protein and delivers sufficient numbers of cytotoxic molecules to the target cell to be lethal.

· Specialized facilities are required in the Chemistry Manufacturing Control (CMC) for ADCs to handle proteins, potent cytotoxic drugs and also to provide assurance in quality regarding stability and batch-to-batch consistency⁵.

Disadvantages:

· Requires that the tumor be tested for expression of the antigen.

· Molecular target may have some normal tissue expression, potentially leading to toxicity.

· Toxic payload may have some premature release.

· Antibody conjugate may not reach the target cells in sufficient concentration to be lethal.

· Antigen expression could be heterogeneous, especially in solid tumors degree of tumor selectivity to approved anticancer drugs and thus improve their therapeutic index.

Table 2: Stage wise mechanism of action of ADCs

Stage 1- Binding	The MAb component of the ADC binds to the target antigen on the surface of the tumour cell to produce an ADC-antigen (ADC-CDX) complex, which is engulfed into a clathrin-coated vesicle;
Stage 2- Clathrin-Mediated Endocytosis	This binding then initiates a cascade of events, involving the internalization of the ADC-antigen clathrin coated vesicle into the tumour cell. Consequently, the vesicle loses its coat and enables the ADC-antigen complex to fuse with an early sorting endosome, to initiate the release of the antigen from the ADC. At this stage, the antigen may be recycled back to the cell membrane. Furthermore, the early endosome converts to a late endosome containing the ADC;
Stage 3- Degradation	The internalized ADC is transported through the late endosome pathway to the intracellular compartment of a lysosome, where it is degraded to release the cytotoxic drug. The cleavable linkers rely on processes inside the cell to liberate the cytotoxic drug such as reduction of disulfide bonds mediated by glutathione (GSH) in the cytoplasm, exposure to acidic conditions (pH ~4) in the lysosome, or cleavage by specific proteases within the cell. Conversely, non-cleavable linkers require catabolic degradation of the Mab, to release the cytotoxic drug retaining the linker and amino acid (lysine) residue, by which it was attached to the MAb;
Stage 4- Release	The cytotoxic drug enters the cytoplasm, where it binds to its molecular target • Route A - calicheamicin based drugs interact with the minor groove of DNA • Route B – auristatins and maytansinoids disrupt the microtubules. Subsequently, the cytotoxic drug may also pass through the cell membrane and enter other cells in close proximity thereby mediating a bystander killing effect;
Stage 5 - Cell Death	The interaction of the cytotoxic drug with DNA and microtubules initiates a chain of events leading to apoptosis.

ANALYTICAL METHODS:

In order to release the ADC certain analytical methods must be implemented to verify and identify the type of mAb and cytotoxic drug to be used in its manufacture. These analytical techniques are used for the release of the ADC and may include protein mass spectrometry and capillary electrophoresis ^{6, 20, 22, 27, 35}.

a) Peptide Mapping and Sequencing –As an analytical tool used to determine the molecular weight of the ADC.

b) Multi-NMR and FTIR spectroscopy techniques- The structure of the linker-drug combination can further be determined.

c) X-Ray crystallography- Used to assess the peptide or antibody structure.

d) UV methods- Used to evaluate drug to antibody ratio (DAR).

e) Size-Exclusion Chromatography (SEC) techniques- Used to determine fragmentation and aggregate patterns during the synthesis of the ADC.

f) ELISA- used to assess the antigen binding and biological activity of the mAbs against, in vitro cell-based assays and in vivo studies.

A critical factor is to develop robust analytical methods to determine the level of free cytotoxic drug. In addition, chemical impurities obtained during the synthesis which include the impurity profile from host cell proteins must also be identified³².

LINKER TECHNOLOGY:

A stable link between the antibody and cytotoxic (anti-cancer) agent is a crucial aspect of an ADC. Cleavable and non-cleavable types of linkers have been proven to be safe in preclinical and clinical trials. The copy number and heterogeneity of antigen expression must be considered in the selection of drug and linker. This is particularly important for antigens expressed heterogeneously within a tumor where ADCs with local bystander activity may be particularly desirable. In addition to the mechanism of drug release, the site of conjugation, the potency of the drug and the average number of drug molecules per antibody need to be considered in the selection of the linker.Linkers that are

short spacers that covalently couple the drug to the antibody protein must be stable in circulation. Inside of the cell, most linkers are labile; however, some are stable, requiring degradation of the antibody and linker to release the cytotoxic agent^{3, 5,
35}.

Linkers can be broadly classified by their mechanism of drug release^37-40. These are as follows:

Cleavable linkers:

They release drug by hydrolysis or enzymatic cleavage following internalization. They are catalyzed by enzymes in the cancer cell where it releases the cytotoxic agent. The difference is that the cytotoxic payload delivered via a cleavable linker can escape from the targeted cell and, in a process called “bystander killing,” attack neighboring cancer cells. Currently used linkers most frequently react with lysine side chains or Sulfhydrylsin the hinge regions of the antibody. Linkers in clinical use include acid-labile Hydrazone linkers that are degraded under the low pH conditions found in lysosomes ⁵. Disulfide-based linkers are selectively cleaved in the cytosol in the reductive intracellular milieu. Eg. Disulfides, Hydrazones or Peptides.

Noncleavable linkers:

They release drug via degradation of the mAb in lysosomes following antigen-specific internalization. It keeps the drug within the cell. As a result, the entire antibody, linker and cytotoxic (anti-cancer) agent enter the targeted cancer cell where the antibody is degraded to the level of an amino acid. The resulting complex – amino acid, linker and cytotoxic agent – now becomes the active drug. Noncleavable linkers like Thioethers release the small-molecule drug after degradation of the antibody in the lysosome, and peptide linkers, such as Citrulline-Valine, are stable in circulation and degraded by lysosomal proteases in cells.

Another type of cleavable linker, currently in development, adds an extra molecule between the cytotoxic drug and the cleavage site^38-41. This linker technology allows researchers to create ADCs with more flexibility without worrying about changing cleavage kinetics. Researchers are also developing a new method of peptide cleavage based on Edman degradation, a method of sequencing amino acids in a peptide. Future direction in the development of ADCs also include the development of site-specific conjugation (TDCs) to further improve stability and therapeutic index and α emitting immunoconjugates and antibody-conjugated nanoparticles.

More recently, linkers with polyethylene glycol spacers have been developed in an effort to increase the solubility of the conjugate. Linkers can influence the circulating half-life and safety of conjugates by minimizing the release of the drug molecule in circulation and optimizing the delivery of the conjugate to the target tissue. Often during the drug development process, investigators will test several linkers in safety and efficacy assays to select the best candidate conjugate⁵.

CONCLUSION:

I keep wondering whether there are more mutant proteins on the cell surface that could be used as ADC targets. The biggest question is whether this technology can be applied beyond oncology to broader indications. In general, when selective targeting by antibodies is feasible and cell death of the target is the therapeutic goal, ADCs could be a viable treatment strategy. Certainly, further investigation in additional oncology indications will be a first step, as demonstrated by several data presented recently by Seattle Genetics of Brentuximab Vedotin at the American Society of Hematology’s third annual meeting. Taken together, outstanding results in the clinic together with significant investment by various innovative and well-capitalized companies points to a bright future for the ADCs. Researchers face pressure both inside and outside the laboratory. Inside, researchers have to find the right combination of drugs and a stable conjugate to use as a linker, as well as antibodies that flag cancer cells while leaving healthy cells alone. Outside the laboratory, researchers must identify the subgroup of cancer patients most likely to respond favorably. The next few years will likely bring impressive advances in the various pieces of ADC technology: the antibodies, the drugs, the linkers that bind them together and new found targets for the ADCs to attack.

However, further insight into the optimal design characteristics of effective ADCs will be best gained as additional clinical data become available. There are currently over 25 ADCs in clinical development and evolving clinical data from these trials will provide critical insight into the design of next generation ADCs.

List of Abbreviations:

ADC	Antibody Drug Conjugation
mAb	Monoclonal Antibody
NHL	Non-Hodgkin’s Lymphoma
HER	Human Epidermal Growth Factor Receptor
PSMA	Prostate-Specific Membrane Antigen
PBDs	PyrroloBenzoDiazepines
Cys-SH	Cysteine Sulfhydryl
MDR	Multi-drug resistance
SEC	Size-Exclusion Chromatography
NMR	Nuclear Magnetic Resonance
FTIR	Fourier Transform Infra Red
DAR	Drug to antibody ratio

REFERENCES:

1. Kohler, G.; Milstein, C. Continuous cultures of fused cells secreting antibody of predefined specificity, Nature, 1975, 256, 495–497

2. Chu YW, Polson A, Antibody-drug conjugates for the treatment of B-cell non-Hodgkin's lymphoma and leukemia, Future Oncology, 2013 Mar; 9(3):355-68. doi: 10.2217/fon.12.189

3. Greg T. Hermanson, BioconjugateTechniques- 2ndedition, Pierce Biotechnology,Thermo Fisher Scientiﬁc,Rockford, Illinois, USA, Elsevier

4. Rohrer Thomas ; Antibody drug conjugates: Potent weapons for the oncology arsenal, ChimicaOggi, 2009, vol. 27, 5, pp. 56-60; ISSN 0392-839X

5. Beverly A. Teicher1 and Ravi V.J. Chari, Antibody Conjugate Therapeutics: Challenges and Potential, American Association for Cancer Research, Clinical Cancer Research, October 15, 2011 17; 6389, doi: 10.1158/1078-0432.CCR-11-1417

6. Thomas Rohrer, Consideration for the safe and effective manufacturing of antibody drug conjugates, Chimica Oggi/ Chemistry Today - vol. 30 n. 5, September/October 2012

7. Sievers EL, Larson RA, Stadtmauer EA, et al., Efficacy and safety of Gemtuzumab Ozogamicin in patients with CD33-positive acute myeloid leukemia in first relapse, J Clinical Oncology 2001;19(13):3244 –54

8. Kevin J. Hamblett, Peter D. Senter, Dana F. Chace, Michael M. C. Sun, Joel Lenox, Charles G. Cerveny, Kim M. Kissler, Starr X. Bernhardt, Anastasia K. Kopcha, Roger F. Zabinski, Damon L. Meyer, and Joseph A. Francisco, Effects of Drug Loading on the Antitumor Activity of a Monoclonal Antibody Drug Conjugate, Clinical Cancer Research, October 15, 2004 10; 7063,doi: 10.1158/1078-0432.CCR-04-0789

9. Bross PF, Beitz J, Chen G, et al., Approval summary: Gemtuzumab Ozogamicin in relapsed acute myeloid leukemia, Clinical Cancer Research 2001; 7(6):1490–6.

10. Carter P., Improving the efficacy of antibody-based cancer therapies, Natures Review-Cancer, 2001;1(2):118 –29

11. Rachel S. Zolot, Satarupa Basu and Ryan P. Million, Antibody-drug conjugates; Nature Reviews, Drug Discovery News and Analysis, From the Analyst’s Couch; Volume 12, April 2013, 259

12. ADC Review: Brentuximab Vedotin Shows 42% Objective Response Rate in Patients with Relapsed or Refractory Diffuse Large B-cell Lymphoma, Journal of Antibody-drug Conjugates, December 10, 2013.

13. Asher Mullard, News And Analysis; Maturing Antibody–Drug Conjugate Pipeline Hits 30; Nature Reviews, Drug Discovery Volume 12, May 2013, 329

14. Eric L. Sievers and Peter D. Senter, Antibody-Drug Conjugates in Cancer Therapy, Annual Review of Medicine, October 3, 2012, 64:15–29, doi 10.1146/annurev-med-050311-201823

15. Junutula J.R., Raab H., Clark S., Bhakta S., Leipold D.D., Weir S.,et al., Nat. Biotechnol., 26(8), 925-928 (2008).

16. Smyth, M.J.; Pietersz, G.A.; McKenzie, I.F. The mode of action of Methotrexate-monoclonal antibody conjugates, Immunology Cell Biology, 1987, 65, 189–200

17. Ghose, T.; Ferrone, S.; Blair, A.H.; Kralovec, Y.; Temponi, M.; Singh, M.; Mammen, M. Regression of human Melanoma Xenografts in nude mice injected with methotrexate linked to monoclonal antibody to human high molecular weight-melanoma associated antigen, Cancer Immunology and Immunotherapeutics, 1991, 34, 90–96.

18. Elias, D.J.; Kline, L.E.; Robbins, B.A.; Johnson, H.C., Jr.; Pekny, K.; Benz, M.; Robb, J.A.; Walker, L.E.; Kosty, M.; Dillman, R.O., Monoclonal antibody KS1/4-Methotrexate immunoconjugate studies in non-small cell lung carcinoma. American Journal of Respiratory Critical Care Medicines, 1994, 150, 1114–1122

19. Petersen, B.H.; DeHerdt, S.V.; Schneck, D.W.; Bumol, T.F., The human immune response to KS1/4-desacetylvinblastine (ly256787) and KS1/4-desacetylvinblastine hydrazide (LY203728) in single and multiple dose clinical studies. Cancer Research, 1991, 51, 2286–2290

20. Schrappe, M.; Bumol, T.F.; Apelgren, L.D.; Briggs, S.L.; Koppel, G.A.; Markowitz, D.D.; Mueller, B.M.; Reisfeld, R.A., Long-term growth suppression of human GliomaXenografts by chemoimmunoconjugates of 4-Desacetylvinblastine-3-carboxyhydrazide and monoclonal antibody 9.2.27. Cancer Research, 1992, 52, 3838–3844.

21. Yang, H.M.; Reisfeld, R.A., Doxorubicin conjugated with a monoclonal antibody directed to a human melanoma-associated proteoglycan suppresses the growth of established tumor Xenografts in nude mice. Proc. Natl. Acad. Sci. USA 1988, 85, 1189–1193.

22. A.M. Wu, P.D. Senter, Nature Biotechnology, 23, pp. 1137-1146 (2005).

23. M.J. Sagnau, P.W. Howard, Bio-organic and Medicinal Chemistry Letters, 10, pp. 2083-2086.- http://bio.lonza.com- Accessed on November 2013

24. JeetKalia and Ronald T. Raines, Advances in Bioconjugation, Current Organic Chemistry 2010 January; 14(2): 138–147.

25. Sanderson RJ, Hering MA, James SF et al., In vivo drug-linker stability of an anti-CD30 Dipeptide linked Auristatinimmuno conjugate, Clinical Cancer Research, 2005, 11:843–52

26. Senter P, Sievers EL., The discovery and development of BrentuximabVedotin for use in relapsed Hodgkin lymphoma and systemic anaplastic large cell lymphoma, Nature Biotechnology, 2012, 30:631–37

27. Teicher B.A., Chari R.V.J., Clinical Cancer Research, 17(20), 6389-6397(2011)

28. Sean L Kitson, Derek J Quinn, Thomas S Moody, David Speed, William Watters, David Rozzell, Antibody-Drug Conjugates (ADCs) – Biotherapeutic bullets., Monographic supplement series CROs/CMOs-ChimicaOggi- Chemistry Today - vol. 31(4) July/August 2013

29. Blanc, V.; Bousseau, A.; Caron, A.; Carrez, C.; Lutz, R.J.; Lambert, J.M., SAR3419: An anti- CD19-maytansinoid immunoconjugate for the treatment of B-cell malignancies. Clinical Cancer Research,2011, 17, 6448–6458

30. Lewis Phillips, G.D.; Li, G.; Dugger, D.L.; Crocker, L.M.; Parsons, K.L.; Mai, E.; Blattler, W.A.; Lambert, J.M.; Chari, R.V.; Lutz, R.J.; et al. Targeting HER2-positive breast cancer with Trastuzumab-DM1, an Antibody-Cytotoxic Drug Conjugate, Cancer Research, 2008, 68, 9280–9290.

31. Petrul, H.M.; Schatz, C.A.; Kopitz, C.C.; Adnane, L.; McCabe, T.J.; Trail, P.; Ha, S.; Chang, Y.S.; Voznesensky, A.; Ranges, G.; et al, Therapeutic mechanism and efficacy of the antibody drug conjugate BAY 79-4620 targeting human Carbonic anhydrase 9, Mol. Cancer Therapeutics, 2012, 11, 340–349.

32. Sarah Pope, Miksinski, Marjorie Shapiro (USFDA), Regulatory Considerations for Antibody Drug Conjugates, presentation at CDER, AAPS, October 18, 2012

33. Trail, P.A.; Bianchi, A.B., Monoclonal antibody drug conjugates in the treatment of cancer, Current Opinion Immunology, 1999, 11, 584–588

34. Dubowchik, G.M.; Walker, M.A., Receptor-mediated and enzyme-dependent targeting of cytotoxic anticancer drugs. Pharmacology Therapeutics, 1999, 83, 67–123.

35. Antibody Drug Conjugates, White Paper documents by QPS - November 2012- http://www.qps.com/qpson_antibody.php- Accessed on January 2014

36. United States Patent, Number: 5,010,176, Apr. 23, 1991

37. Polson AG, Calemine-Fenaux J, Chan P, Chang W, Christensen E, Clark S, de Sauvage FJ, et al., Antibody-drug conjugates for the treatment of non-Hodgkin's lymphoma: target and linker-drug selection, Cancer Research, 2009 Mar 15; 69(6):2358-64. doi: 10.1158/0008-5472.CAN-08-2250. Epub, 2009 Mar

38. Czuczman M S, Horwitz S M, Pinter-Brown LC, Shustov AR, Younes A., Introduction: Antibody-Drug Conjugates in Lymphoma, January 31, 2014

39. Bednar R A., Reactivity and pH dependence of thiol conjugation to N-ethylmaleimide: Detection of a conformational change in Chalconeisomerase, Biochemistry 1990;2 9:3684–3690, PubMed: 2340265

40. Chalker J M, Bernardes G J, Lin Y A, Davis B G, Chemical modification of proteins at cysteine: Opportunities in Chemistry and Biology. Chemistry- Asian Journal, 2009;4:630–640, PubMed: 19235822

41. Mc Caldon P, Argos P., Oligopeptide biases in protein sequences and their use in predicting protein coding regions in nucleotide sequences, Proteins 1988;4:99–122, PubMed: 3227018

Received on 16.05.2014 Modified on 20.06.2014

Research J. Pharm. and Tech. 7(8): August 2014 Page 931-937

Sr. No.	Component of ADC	Characteristics and Role
1	Monoclonal Antibody	· There are currently 9 unconjugated mAbs approved by the FDA as cancer therapeutics. These mAbs include 2 Chimeric, 4 Humanized and 3 Fully Human monoclonal antibodies · Unconjugated monoclonal antibodies (mAbs) selectively recognize antigens that are preferentially expressed on or near tumor cells and exert their cytotoxic effects through mechanisms such as cell signaling, antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis and complement-dependent cytotoxicity. · Maintains characteristics when linked to the requisite number of cytotoxic molecules via linker · Targeted at a well-characterized antigen · Targeted at an antigen found only on target cells · Targeted at an antigen that is not down regulated on Ab binding · Minimal non-specific binding
2	Linker	· The linker that connects the cytotoxic drug to the mAb is a key determinant of ADC activity. · These linkers covalently couple the cytotoxic drug to the antibody, producing an ADC that should be relatively stable in circulation. · Stability of the linker is important because it prevents damage to non-target tissue through spontaneous release of the cytotoxic drug while maximizing tumor drug exposure. · Upon internalization, however, the linkers should facilitate efficient drug release. · Intracellular conditions such as the low-pH environment in lysosomes or the reducing environment of the cytosol can destabilize acid-labile hydrazine linkers or disulfide-based linkers, respectively, resulting in drug release. · Does not alter the Ab characteristics (pharmacokinetics) · Ensures that the cytotoxic agent is functional once at target site
3	Cytotoxic agent	· For application of the highly potent cytotoxic compounds in antibody conjugates, the analogs used must have sufficient water solubility and prolonged stability in aqueous formulations and in plasma, because antibody conjugates may be in circulation for several days. · They must have a functional group that is nsuitable for conjugation with a linker and must not bereadily susceptible to lysosomal enzyme degradation. · Consistent with the potent nature of the drug component ofADCs, these agents are often scheduled like cytotoxic chemotherapy in clinical regimens, with dosing once every thrice weeks. · They are non-immunogenic and Non-toxic (dormant or inactive) during circulation in the blood.