Antibody-Drug Conjugates Carbon-14 Radiolabeling ...

Antibody-Drug Conjugates: Carbon-14 Labeling Requirements Published in Drug Discovery & Development

http://www.dddmag.com/articles/2013/05/antibody-drug-conjugates-carbon-14labeling-requirements Sean L. Kitson, Investigator; Thomas S. Moody, Head of Biocatalysis and Isotope Chemistry; David Rozzell, Biocatalysis Consultant; Jill Caswell, Molecular Biologist, Department of Biocatalysis and Isotope Chemistry; Almac, Craigavon, U.K. The next blockbuster drug revenue streams are likely to come from a class of targeted therapies geared towards the treatment and management of chronic human disease states—especially cancer—and involve the use of antibody-drug conjugates (ADCs).1 A wide range of monoclonal antibodies (mAb) have already been approved by the FDA and includes six engineered monoclonal antibodies Rituxan (rituximab), Herceptin (trastuzumab), Campath (alemtuzumab), Avastin (bevacizumab), Erbitux (cetuximab), and Vectibix (panitumumab). Two radionuclide-conjugated monoclonal antibodies Zevalin (ibritumomab tiuxetan) and Bexxar (tositumomab; iodine-131 tositumomab) have also been approved by the FDA.2 The promise of ADCs is to deliver a therapeutic or cytotoxic drug directly to tumor cells,

while

limiting

damage

to

healthy

ones.

By

comparison

traditional

chemotherapeutics destroy tissue indiscriminately. The first FDA approved ADC was Mylotarg (gemtuzumab ozogamicin) and was initially developed by Wyeth in 2000 for the treatment of acute myeloid leukemia. After a decade, it was withdrawn from the market due to an undesirable efficacy and safety profile. Mylotarg targeted the CD33 receptor which turned out to be nonselective for tumor cells.3 Additionally, the linker used to attach the drug to the antibody was unstable and the drug was released prior to reaching its target.4 Since this failure, huge strides have been made in the science of properly binding antibodies to drugs. By using linkers with an acceptable biological half-life, researchers ensure that ADCs can reach the targeted cell with limited side effects.5

In 2011, the U.S. Food and Drug Administration (FDA) approved Seattle Genetics’ Adcetris (brentuximab vedotin) to treat Hodgkin’s lymphoma and systematic anaplastic large-cell lymphoma.6 This ADC made about $34.5 million in the first quarter of 2012 and revenues continue to bolster. Use of ADCs is anticipated to increase nearly 50% over the next decade just in the oncology market. Roche and Genentech alone have over 25 ADCs in their pipeline, including nine in clinical trials.7 The first potential blockbuster ADC reached the market in February 2013 when the Kadcyla (trastuzumab emtansine, T-DM1) was approved for the treatment of metastatic breast cancer.8 Kadcyla combines the breast cancer treatment antibody Herceptin (trastuzumab) with the cytotoxic payload mertansine (DM1), a drug licensed from ImmunoGen. The projected sales for the treatment are in the region of $2 to $5 billion per year.7 The continued success of the ADC technology platform is due to the collaborations within biopharma and the promising clinical trials.9 These collective results indicate a commitment towards targeted therapeutic delivery using humanized monoclonal antibody technologies.10 To date, most of the drugs utilized in the development of ADCs use auristatins (Seattle Genetics) or maytansine (ImmunoGen)11 but other emerging

payloads

pyrrolobenzodiazepines

are

in

(PBDs)

development for

the

next

from

Spirogen

generation

of

such

as

antibody-drug

conjugates.12 ADC mechanism-of-action A typical ADC consists of a monoclonal antibody which is capable of binding to the surface of tumor cell-specific antigens.13 These can include proteins on the surface of the B and T cells of the immune system, such as CD20 and CD22, the human epidermal growth factor receptor 2 (HER2), or prostate-specific membrane antigen (PSMA).14 This specific antibody is attached to a cytotoxic drug via a cleavable linker.15 The drug is designed to induce tumor cell death by causing irreversible DNA damage or interfering with cell division.16

The mechanism-of-action of ADCs is the ability of the antibody to recognize and bind with a specific antigen. This binding initiates a cascade of events (Stages 1 to 5) via the process called endocytosis whereby the cell’s lysosomal enzymes release the cytotoxic drug to kill tumor cells (Figure 1).

Figure 1. Schematic representation of the ADC internalization process

ADCs in clinical development The commercial pipeline of antibody-based therapeutics now totals nearly 350 candidates. There are currently 20 ADCs at varying stages of clinical development; Table 1 shows a snapshot of ADCs in Phase 1 and above.17 ADC (Target Antigen ) Inotuzumab ozogamicin (CD22) Gemtuzumab ozogamicin (CD33) Lorvotuzumab mertansine (CD56)

[Monoclonal Antibody]–[Linker]–[Drug Class]

Oncology Indications

[Hz IgG4] – [Hydrazone] – [Calicheamicin]

NHL

Pfizer

III

[Hz IgG4] – [Hydrazone] – [Calicheamicin]

Relapsed AML Solid Tumours, MM Breast Cancer, Melanoma NHL

Pfizer

III

Immunogen

II

Celldex Therapeutics

II

Sanofi

II

Progenics

II

Genentech/ Roche Genentech/ Roche Biotest

II

[Hz IgG1] – [SPP] – [Maytansine DM1]

Glembatumumab vedotin (GPNMB)

[Hu IgG2] – [Valine-citrulline] –[Auristatin MMAE]

SAR-3419 (CD19) PSMA ADC (PSMA) RG7593/DCDT2980S (CD22) RG-7596 (CD79b) BT-062 (CD138) SGN-75 (CD70) BAY 79-4620 (CA-IX) Milatuzumab-doxorubicin (CD74) AGS-5ME (SLC44A4)

[Hz IgG1] – [SPDB] – [Maytansine DM4]

BAY 94-9343 (Mesothelin) ASG-22ME (Nectin-4) Labestuzumab-SN-38 (CD66e/CEACAM5)

[Hu IgG1] – [Valine-citrulline] – [Auristatin MMAE] [Hz IgG1] – [Valine-citrulline] –[Auristatin MMAE]

Prostate Cancer NHL

[Hz IgG1] – [Valine-citrulline] – [Auristatin MMAE]

NHL

[Ch IgG4] – [SPDB] – [Maytansine DM4]

MM

[Hz IgG1] – [Maleimidocaproyl]–[Auristatin MMAF]

NHL, RCC

[Hu IgG1] – [Valine-citrulline] – [Auristatin MMAE]

Solid Tumours MM

[Hz IgG1] – [Hydrazone] – [Doxorubicin] [Hu IgG2] – [Valine-citrulline] – [Auristatin MMAE]

[Hu IgG1] – [SPDB] – [Maytansine DM4] [Hu IgG1] – [Valine-citrulline] – [Auristatin MMAE] [Hu IgG1] – [Lysine] – [CPT-11 SN38]

Pancreatic, Prostate Cancer Solid Tumours Soild Tumours CRC

Developer

Clinical Phase

II II I

Seattle Genetics Bayer

I

Immunomedics

I

Astellas

I

Bayer

I

Astellas

I

Immunomedics

I

Abbreviations - Ch: chimeric; Hz: humanized; Hu: fully human; MMAE: monomethyl auristatin E; MMAF: monomethyl auristatin F; NHL: non-Hodgkin Lymphoma; PSMA: Prostate-Specific Membrane Antigen; RCC: Renal Cell Carcinoma; GPMNB: Glycoprotein NMB; AML: Acute Myelogenous Leukemia; MM: Multiple Myeloma; CRC: Colorectal Carcinoma. Source: www.clinicaltrials.gov.December 2012

Table 1. Shows a snapshot of ADCs in Phase 1 and above

Choosing the right antibody-drug linker A critical factor in the manufacture of ADCs is choosing the right linker to attach the antibody to the drug. Today, linker technology is the main focus for biopharma in the development of

ADC platforms to maximize the killing of cancer cells.

Underdeveloped linkers can be cleaved before entering the tumor cell, reducing the potency of the treatment and releasing the drug into systemic circulation. ADCs are constructed through the reaction of drugs with amino acids such as lysine and cysteine on the antibody. Most linkers have a short half-life compared to the antibody and therefore chemical modification is needed to prolong linker stability and aid solubility. This can be circumvented by attaching PEGlyated derivatives to the linker to help to optimize the antibody-linker-drug combination. Linker technology can fall into two areas: cleavable linkers (peptide, hydrazone, or disulfide) or noncleavable linkers (thioethers).18 This can be further extended into the area of site specific protein modification and has a critical role to play in many aspects of drug development including protein PEGylation, antibody-drug conjugates, bi-specific therapeutics and molecular imaging agents based on the thioester and hydrazone or oxime linker technology.19 Carbon-14 labeling of ADCs Biopharmaceutical companies need a reliable supply of isotopically labeled active pharmaceutical ingredients (APIs). The isotope of choice is carbon-14 to produce API for Phase 0 to Phase 3, ADME, mass balance, and micro-dosing studies. For human studies, this necessitates procedures to perform synthesis and repurifications under cGMP conditions, in compliance with ICH Q7A Section 19 guidelines (single batches for investigational drugs). These carbon-14 labeled compounds provide vital information for chemistry, manufacturing and controls.20 Carbon-14 labeling of ADCs (Figure 2) can be executed on the chemical linker, incorporated into the drug payload, or a combination of the two. Various labeling positions can be considered which may include heterocycles, aromatics, chiral compounds, peptides and proteins including steroids. Extending too the modification of carbohydrates, polysaccharides, and glycoconjugates, including PEGylated materials, biopolymers, cytotoxic, and highly potent compounds.21

Figure 2. ADC carbon-14 labeling sites The manufacture of a radiolabeled ADC starts with the activation of the carbon-14 labeled linker and allows the attachment of the drug payload. The other end of the linker is able to facilitate a bio-conjugation with a monoclonal antibody usually via a surface lysine residue on the antibody. The carbon-14 labeled ADC undergoes a purification process using a system such as the Sartoflow Slice 200 benchtop crossflow system. The critical functions of this system are to remove unbound linker and drug and to use hydrophobic interaction chromatography purification to obtain finished ADC without free drug. The quality of the final product can be determined using size-exclusion chromatography analysis. The future of ADCs The emergence of antibody-drug conjugates (ADCs) delivering drug payloads to their target guided by a specific

antibody provides

a golden opportunity for

biopharmaceutical companies and contract manufacturers.22-23 The continued interest in the manufacture of ADCs and the associated regulatory issues surrounding this therapeutic class of drug will bring many issues regarding the ADME-Tox profile of each ADC. Focus must firmly remain on the efficacy and pharmacokinetics profile of the ADC on the human body regarding the stability of the linker between the cytotoxic drug and the antibody. Carbon-14 labeling of the linker

and/or the drug will be able to identify potential metabolites and free drug in the systemic circulation in various animal models. These preclinical studies can lead onto human microdosing AME clinical studies using carbon-14 labeled ADCs towards FDA approval. About the Authors Dr. Kitson is an investigator at Almac and has more than 10 years’ experience in the synthesis of carbon-14 radiolabeled compounds. He is also the editor-in-chief of Current Radiopharmaceuticals and a scientific committee member of

the

International Isotope Society (UK Group). He is a recipient of the 2006 Wiley Journal of Labeled Compounds and Radiopharmaceuticals award for radiochemistry. Dr. Moody is Head of Biocatalysis and Isotope Chemistry at Almac with over 13 years’ of extensive academic and industry experience. He is a technical expert in chiral chemistry and biocatalysis and a leader in the field of hydrolase, oxidoreductase, and transferase enzymes. He also holds the position of honorary lecturer at Queen’s University, Belfast and the recipient of the 2012 BMI technology award. Dr. Rozzell has more than 25 years’ of experience working in the field of biocatalysis and enzyme technology. In late 2008 he founded Sustainable Chemistry Solutions to promote green chemistry through the development and implementation of biocatalysis and other technologies in chemical manufacturing. Previously, he was the founder and CEO of BioCatalytics, Inc., a company specialized in the development and commercialization of new enzymes products for the pharmaceutical industry. Dr. Caswell is a molecular biologist at Almac with over 12 years’ of extensive industry experience working in the area of enzyme evolution. She has extensive experience in the cloning and development of therapeutic monoclonal antibodies and has multiple accreditations in antibody research. She was a recipient of the 2012 BMI technology award.

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