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European Biopharmaceutical Review

Next-Generation ADCs

Antibody drug conjugates (ADCs) are a growing class of chemotherapeutic agents for the treatment of cancer, combining the tumour-targeting properties of antibodies with the cellkilling properties of potent cytotoxic drugs. In the drive to develop safe and effective ADCs, there is an increasing focus on reducing the risk of failure in the clinic at an early stage. Although both antibodies and cytotoxic drugs are often used independently to treat cancer, the covalent attachment of a drug to a targeting antibody allows selective delivery of the drug to the tumour site, as well as minimising systemic toxic sideeffects from exposure of healthy tissue to the drug.

The usual starting point for the development of an ADC is an antibody that is specifi c to the target of interest. The fi rst recombinant antibody approved for clinical use, in 1986, was muromonab- CD3 (OKT3), a murine anti-CD3 IgG2. Since then, human-murine chimeric antibodies – such as rituximab (Rituxan®) and infl iximab (Remicade®) – have been introduced, and antibodies have become fi rmly established as therapeutic agents; there are currently four antibodies among the top fi ve best-selling biopharmaceutical products.

Anti-Drug Antibodies

However, murine and chimeric antibodies are considered to be foreign objects by the human immune system and, therefore, patients can generate antibodies against them (1-5). These anti-drug antibodies can lead to side-effects or neutralise the activity of the therapeutic antibody, rendering it less effective. To try to reduce the risk of patients developing anti-drug antibodies, many therapeutic antibodies have undergone a process of humanisation. A range of different methods have been used to make the antibodies more human. These include humanisation by modifi cation of the non-binding portion of the chimeric antibody; inserting the binding regions of the mouse antibody into a human antibody scaffold (for example, alemtuzumab) (6,7); or by producing fully human antibodies using phage display (for example, adalimumab) or in transgenic mice (8). However, although humanised and fully human antibodies are generally less immunogenic than chimeric antibodies, they can still produce an immune response in patients.

So why do humanised or fully human antibodies continue to generate immune responses in patients? It turns out that the concept of generating these antibodies had not accounted for the fundamental drivers of a long-lived, memory-based immune response within individuals (9). Humanised or fully human antibodies contain amino acid sequences that deviate from those derived from the human germ line, and thus will not have been previously encountered by the patient’s immune system. Furthermore, the antibodies are designed to target a particular disease site where, in the case of ADCs being used to treat cancer, the objective is to destroy cells, which can lead to signifi cant infl ammation. The infl ammatory cell signalling molecules, such as cytokines, can act as adjuvants to assist in the development of immunogenicity.

Effective Deimmunisation

To minimise the risk of anti-drug antibodies being generated, it is essential to remove the triggers that elicit such responses. Within the immunology community, it is generally accepted that the drivers for an immune response are helper T cell epitopes, which are small peptide sequences within a protein sequence that activate helper CD4+ T cells. These activated T cells, in turn, instruct B cells to produce anti-drug antibodies (10,11). The consequences of such responses can range from the transient appearance of antibodies with minimal clinical signifi cance, to persistent antibodies with severe, lifethreatening effects when associated with recombinant human proteins (12-14).

It is now becoming clear that removal of these sequences, or ‘deimmunisation’, has made antibodies immunologically ‘silent’, allowing them to evade the human immune system. Many patients have now been treated with deimmunised antibodies and no signifi cant immunogenicity has been reported.

Payload Considerations

Having generated an antibody that can avoid the immune system, what about the attachment and nature of the cytotoxic payload needed to create an ADC? As a hybrid molecule consisting of an antibody (a large biological entity) and a cytotoxic payload (usually a small chemical entity), building the ideal ADC requires careful consideration in terms of payload selection, site and extent of loading – referred to as the drug-to-antibody ratio (DAR) – and the method of attaching the payload to the antibody. Due to the potent cytotoxic effects of these payloads, ADCs must be particularly stable until they reach the tumour site, in order to avoid systemic circulation of free drug that could produce side-effects or competitive inhibition of binding of the ADC to the tumour by antibody without its payload attached, which would reduce effi cacy.

Two major classes of cytotoxic drugs, currently used as payloads to be delivered to the tumour in an ADC, are tubulin polymerisation inhibitors (for example, maytansines and auristatins) and DNA-damaging agents (for instance, calicheamicins and duocarmycins). Most of these payloads are so potent that they cause too much damage to healthy cells to be given to patients as standalone chemotherapeutic drugs and need to be targeted to the tumour.

The Linker is Key

The clinically established approach to conjugating cytotoxic drugs to antibodies is to use linkers that are reactive towards either the amino side chains of lysine residues, as in the case of ado-trastuzumab emtansine (Kadcyla®), or to the thiol side chains created from reducing interchain disulfi de bonds, as is the case with brentuximab vedotin (Adcetris®). However, both approaches have been shown to have limitations. Conjugation to lysines, of which there are more than 80, cannot be precisely controlled, leading to a heterogeneous mixture of ADCs (15) with different DARs, most of which exceed a DAR of four – commonly accepted as the ideal DAR for an ADC (16).

Selective attachment of cytotoxic drugs to the antibody via cysteine residues is often used as an alternative to conjugation to lysines as there are far fewer cysteine residues in an antibody. An intact IgG1 has four interchain disulfi de bonds that can be reduced to release eight free cysteine thiols, which can serve as sites for conjugation. The reaction between the antibody and the cytotoxic drug reagent yields an ADC comprising a mixture of species, with DARs ranging between zero and eight (15,17). The DAR is again diffi cult to control, although the number of ADC variants in the mixture is fewer than when conjugation is to lysines. The structural integrity of the antibody is perturbed as the disulfi de bond remains broken after conjugation when the established maleimide linker

Additionally, the maleimide linker used to attach the cytotoxic drug to the antibody is inherently unstable, and there is a risk that the drug may be released into the circulation, potentially producing sideeffects. Furthermore, instead of being cleared quickly, the cytotoxic drug is capable of attaching to serum albumin, so it recirculates (18,19). Moreover, the antibody without the attached payload can bind to the tumour, thereby blocking binding of intact ADC and reducing its effi cacy.

Re-Engineering Approaches

An alternative method to overcome a heterogeneous mixture of ADC species with wide DAR distribution is to use antibodies with engineered cysteine residues. Antibodies have been produced with an engineered and unpaired cysteine residue on each heavy or light chain, specifi cally designed for conjugation of a cytotoxic payload (18-21). Each of the cysteine residues provides a site for conjugation generating an ADC with a DAR of close to two (20,21). While this approach can be used to produce more homogeneous products, it does not address the issue of the instability of maleimide linkers in vivo, and the possible release of the cytotoxic drug.

Another re-engineering approach is to incorporate non-natural amino acids in the antibody as sites for conjugation. These non-natural amino acids have functionalities that enable specific conjugation chemistries that avoid native amino acids. Conjugation to non-natural amino acids within ADCs is a promising alternative to produce homogeneous ADCs, but this approach has major limitations.

Re-engineering antibodies to incorporate non-natural amino acids is complex and costly, and may introduce undesired immune responses in patients (22). In addition, building an ADC from a novel re-engineered antibody, even for the purpose of reducing DAR distribution, adds to the complexity of an already intricate hybrid molecule. Regulatory agencies may require additional evidence of the safety of the antibody due to the inclusion of non-natural amino acids.

Targeting Native Disulfides


One approach that addresses or avoids the issues discussed above targets the native disulfi des in a native antibody, using chemistry that specifically re-bridges the reduced disulfi de bonds during the process of conjugating the cytotoxic drug, thereby leaving the protein structurally intact. This approach is well established for the attachment of polyethylene glycol to therapeutic proteins in order to enhance their pharmacokinetic properties while retaining their activity (23,24). It has now been used successfully to attach a cytotoxic drug to the four interchain disulfi des of a monoclonal antibody therapy.

The generation of ADCs predominantly with a DAR of four using this method has been confirmed with many of the established payloads. Furthermore, the structural nature of the linker and its re-bridging of the two cysteines confer exceptional stability to both the antibody and the ADC, relative to other chemistries that conjugate to cysteine thiols. These benefits considerably reduce the risk of failure associated with the current technologies in the ADC field.

In conclusion, with two recent approvals – and many more in latestage clinical development – ADCs are rapidly emerging as the next generation of antibody therapeutics. As we learn more about them, it appears clear that key features for success in the clinic – and achieving regulatory approval – will be the ability to control the conjugation process in order to provide an ADC with an optimal DAR for efficacy and safety, and to ensure that the risk of toxicity is reduced by having a non-immunogenic antibody to which the cytotoxic drug remains attached until it reaches its target cancer cell.

References

1. Matzinger P, The danger model. A renewed sense of self, Science 296: pp301-305, 2002
2. Matzinger P, Friendly and dangerous signals: Is the tissue in control? Nat Immunol 8: pp11-13, 2007
3. Gneiss C et al, Interferon-􀁠 antibodies have a higher affinity in patients with neutralizing antibodies compared to patients with non-neutralizing antibodies, J Neuroimmunol 174(1-2): pp174-179, 2006
4. Haraoui B, Cameron L, Ouellet M and White B, Anti-infliximab antibodies in patients with rheumatoid arthritis who require higher doses of infliximab to achieve or maintain a clinical response, J Rheumatol 33(1): pp31-36, 2006
5. Balint E et al, Therapy-induced antibodies against the antiviral and antiproliferative effects of interferons in patients with chronic hepatitis C virus infection, Acta Microbiol Immunol Hung 51(3): pp359-369, 2004
6. Jones PT et al, Replacing the complementarity-determining regions in a human antibody with those from a mouse, Nature 321: pp522-525, 1986
7. Verhoeyen M, Milstein C and Winter G, Reshaping human antibodies: Grafting an antilysozyme activity, Science 239: pp1,534-1,536, 1988
8. McCafferty J, Griffiths AD, Winter G and Chiswell DJ, Nature, 348: pp552-554, 1990
9. Schellekens H and Casadevall N, Immunogenicity of recombinant human proteins: Causes and consequences, J Neurol 251(Suppl 2): ppII4-II9, 2004
10. Baker MP and Jones TD, Identification and removal of immunogenicity in therapeutic proteins, Current Opinion in Drug Discovery & Development 10(2): pp219-227, 2007
11. Baker MP, Reynolds HM, Lumicisi B and Bryson CJ, Self/Nonself 1(4): pp1-9, 2010
12. Li J et al, Thrombocytopenia caused by the development of antibodies to thrombopoietin, Blood 98(12): pp3,241- 3,248, 2001
13. Schonholzer C et al, High prevalence in Switzerland of pure red-cell aplasia due to antierythropoietin antibodies in chronic dialysis patients: Report of five cases, Nephrol Dial Transplant 19(8): pp2,121-2,125, 2004
14. Basser RL et al, Development of pancytopenia with neutralizing antibodies to thrombopoietin after multicycle chemotherapy supported by megakaryocyte growth and development factor, Blood 99(7): pp2,599-2,602, 2002
15. Hamblett KJ et al, Effects of drug loading on the antitumor activity of a monoclonal antibody drugconjugate, Clin Cancer Res 10: pp7,063- 7,070, 2004
16. Alley SC, Okeley NM and Senter PD, Antibody-drug conjugates: targeted drug delivery for cancer, Curr Opin Chem Biol 14: pp529-537, 2010
17. Doronina SO et al, Development of potent monoclonal antibody auristatin conjugates for cancer therapy, Nat Biotechnol 21: pp778-774, 2003
18. Shen BQ et al, Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates, Nat Biotechnol 30: pp184-189, 2012 19. Wang L et al, Structural characterization of the maytansinoidmonoclonal antibody immunoconjugate, huN901-DM1, by mass spectrometry, Protein Sci 14: pp2,436-2,446, 2005
20. Junutula JR et al, Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index, Nat Biotechnol 26: pp925-932, 2008
21. Junutula JR et al, Engineered thiotrastuzumab- DM1 conjugate with an improved therapeutic index to target human epidermal growth factor receptor 2-positive breast cancer, Clin Cancer Res 16: pp4,769-4,778, 2010
22. Axup JY et al, Synthesis of site-specific antibody-drug conjugates using unnatural amino acids. Proc Nat Acad Sci USA 109: pp16,101-16,106, 2012
23. Khalili H et al, Comparative binding of disulfide-bridged PEG-Fabs, Bioconjug Chem 23: pp2,262-2,277, 2012
24. Balan S et al, Site-specific PEGylation of protein disulfide bonds using a threecarbon bridge, Bioconjug Chem 18: pp61-76, 2007



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Neil Butt is Vice President of Business Development at PolyTherics. He has a background in molecular biology and immunology, obtaining his PhD in Molecular Microbiology at the University of Southampton. Following several years in postdoctoral positions and, later, in biotech R&D, he moved into marketing and business development at PA Consulting Group. Neil joined Antitope in 2006 to lead the business development group, and took his current role when Antitope and PolyTherics merged in 2013.
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