ANTIBODY-DRUG CONJUGATE DEVELOPMENT

Enhance the homogeneity, potency, and safety of your antibody-drug conjugates (ADCs) thanks to ProteoGenix’s flexible and diversified platform. Drawing from 20+ years of experience in ADC development, our platform allies the high productivity of the XtenCHOᵀᴹ cell line with the high efficiency of click chemistry for antibody conjugation and robust bioanalytical methods. Obtain high-quality ADC products with up to 90% of antibodies conjugated with the selected DAR.

Main components and mechanism
of action of ADCs

ADCs consist of the following components:

• Antibody carrier: monoclonal antibody, bispecific antibody, antibody fragment
• Linker: cleavable or non-cleavable linker
• Payload: small drugs, toxins, antibiotics, or enzymes, among others

These complex molecules combine the specificity of immunotherapies with the potency of cytotoxic agents. In the past decades, ADCs have found great success in the treatment of cancer, particularly, hematological conditions. With the continued improvement of linker chemistry and conjugation methods, ADCs will soon be viable tools to fight a larger number of malignancies.

Given the structure of ADCs, they are designed to target membrane-bound receptors that are abundant, specific to cancer cells, and efficiently recycled after cellular internalization. The major mechanism of action of ADCs involves the following steps:

• Biding of the antibody carrier to the specific membrane-bound receptor
• Internalization of the ADC complex by receptor-mediated clathrin-dependent endocytosis
• Maturation of the endosome and fusion with the lysosome
• Lysosomal degradation of the ADC and release of the cytotoxic payload

Key principles of ADC development

Common Antibody Conjugation Strategies Used In Adc Development

Multiple strategies have been developed to conjugate linker-payload pairs to antibody carriers. The efficiency of these methodologies directly influences the load and distribution of payloads along the antibody backbone and the heterogeneity of the resulting ADC.

• Chemical conjugation: it is the most commonly used conjugation method and relies on exposed and reactive functional residues on the surface of the antibody carrier. The two most common residues used for this conjugation strategy are cysteine (reactive with thiol groups inserted on the payload) and lysine (amine groups). Of the two reactive residues, cysteine was proven to allow superior control over DAR and drug load distribution properties due to the lower occurrence of accessible residues on the antibody surface. The unique advantage of this method is that it does not require antibody engineering prior to drug conjugation.
• Enzymatic conjugation: enzymes can modify an antibody in a site or sequence-specific manner. Most enzymatic-based antibody conjugation strategies are based on the use of sortase A. This enzyme cleaves threonine-glycine bonds and attaches an oligoglycine molecule at the site. Microbial transglutaminase has also been successfully used to attach payloads to glycans on the Fc fragment of the antibody carrier.
• Site-specific conjugation: a number of site-specific conjugation methods have been developed in the past years as a way to improve the homogeneity of final ADC products. These methods rely on the engineering of specific residues along the antibody backbone allowing a stricter control of DAR and drug load distribution.
• Click chemistry conjugation: click chemistry is defined as an efficient and high-yield process making use of inexpensive reagents and requiring only mild conditions. Both payload-linker pairs and the antibody backbone are engineered to carry a specific, self-reacting tag. Once these components are put in contact, the reaction occurs naturally and quickly. Click chemistry allows very precise control over the DAR and drug distribution in the antibody backbone, significantly increasing the homogeneity of ADC products.

Selecting the best linker for a specific ADC ultimately depends on several factors including the abundance of the target antigen, toxicity of the payload, and nature of the tumor (solid versus hematological tumor). For instance, cleavable linkers are more suitable when targeting low abundance target and solid tumors, given that these linkers are able to release their warheads on the tumor microenvironment allowing the rapid diffusion of the small drugs without the need for internalization.

Comprehensive bioanalysis of ADCs

Key Properties Of Adcs

What makes the bioanalysis of ADCs so challenging is the heterogeneous nature of these products – particularly relevant when conventional chemical conjugation methods (cysteine and lysine) are used. Heterogeneity is known to influence a number of ADC properties such as stability and therapeutic effectiveness, for this reason, extensive analysis of ADC products is essential to ascertain their success in later stages of development.

The major properties of ADCs and common bioanalytical methods used for their study include:

• Drug-to-antibody ratio (DAR): ultraviolet-visible (UV/Vis) spectroscopy, cathepsin-B enzyme-based digestion method, hydrophobic interaction chromatography (HIC), reversed-phase high-performance liquid chromatography (RP-HPLC), and mass spectrometry (MS)
• Drug load distribution: capillary electrophoresis (CE), HIC, RP-HPLC, and MS
• Unconjugated antibody: CE, ELISA HIC, RP-HPLC, and MS
• Degradation/biotransformation: LC-MS/MS (can be preceded by enrichment and proteolytic degradation steps to increase analytical resolution
• Free drug content: LC-MS/MS
• Charge variants: ion-exchange chromatography (IEX) and imaged capillary isoelectric focusing (iCIEF)
• Stability and aggregation profile: size-exclusion chromatography (SEC), LC-MS, and CE
• Post-translational modifications: LC-MS/MS
• Pharmacokinetics: using animal models, the pharmacokinetics (studies the fate of the ADC in an organism) can be estimated by measuring the serum after treatment and quantifying: total and conjugated antibodies; free and conjugated drug; and biotransformation pathways (LC-MS coupled with protein digestion)

Of all the properties mentioned above, DAR and drug load distribution remain the most important. For their bioanalysis, the most widely used methods during early development include UV/Vis, HIC, and RP-HPLC, or LC-MS.

How To Optimize The Therapeutic Efficacy Of Adcs

Due to the complexity and inherent heterogeneity of ADC products, it can be challenging to ensure their success during clinical development. However, focusing on optimizing a number of key properties is known to significantly increase their odds of receiving marketing approval:

• Antibody affinity: once a highly specific target has been chosen, an antibody with optimal affinity should be produced. One would assume that the higher the affinity, the better the antibody-drug conjugate. However, this is rarely true. For instance, high-affinity antibodies can have a positive effect on the internalization rate but limit solid tumor penetration due to the site-barrier effect. Thus, optimal antibody affinity has to be determined by taking into account the therapeutic application.
• Linker stability: the linker is a crucial component of an ADC as it covalently tethers the antibody to the payload. Properties of a good linker include:
  • Stability in blood circulation to prevent the off-target payload release and limit toxic side effects
  • Fast release of the payload within or in the extracellular matrix of cancer cells
• DAR: DAR is the average number of drug molecules attached to an antibody in an ADC product. According to many studies, an average DAR of 4 is considered optimal for anti-cancer therapies. However, this may vary according to the target and the disease. For instance, higher DAR values can be considered when targeting tumor-associated antigens with low-expression or slow internalization kinetics. But since these ADCs have a high propensity for aggregation and fast clearance, the extensive characterization of their pharmacokinetic profile is vital to ensure their success in the clinic.
• Drug load distribution: unlike DAR, drug loading is the distribution of drugs per antibody in an ADC product, often represented by an interval of values. The broader the interval, the more heterogeneous the ADC product. Highly heterogeneous drug loadings can induce modifications in terms of toxicity and/or therapeutic index of the ADC product. Moreover, it makes it harder to characterize its fate in the organism given that different ADC species will undergo different degradation pathways. Narrowing the drug load distribution in a given ADC product is thus key to ensuring a better therapeutic effect.
• Aggregation: high DAR values and hydrophobicity contribute to a higher aggregation of ADCs. In turn, aggregation decreases the therapeutic potency of these biopharmaceuticals. For this reason, the use of lower DAR, conjugation with more soluble payloads, and PEGylation of ADCs are only a few of the strategies that can be used to mitigate aggregation.
• Free drug: reducing the percentage of free drug in a given ADC product is vital to limit the toxicity of these complex therapeutics. High percentages of free drug can be caused by poor linker chemistry and, thus, can be mitigated by engineering linkers for higher stability in circulation.

Successes and trends
in therapeutic ADC development

Major Targets Of Therapeutic Adcs

Dozens of ADCs have reached the clinic since the approval of Mylotarg (gemtuzumab ozogamicin) in 2020. The current and future major cancer targets of these immunotherapeutics include:

• HER2 – also known as receptor tyrosine-protein kinase erbB-2, CD340 (cluster of differentiation 340), proto-oncogene Neu, or ERBB2 protein. Found in 30% of breast cancer patients and linked with poor prognosis and aggressive forms of the disease.
• CD22 – it is a transmembrane glycoprotein associated with B cell malignancies such as non-Hodgkin lymphoma, and acute lymphoblastic leukemia, among others.
• Tissue factor (TF) – frequently expressed by cancer cells and is known to promote tumor growth, angiogenesis, metastasis, and thrombosis.
• Hepatocyte growth factor receptor (HGFR) or c-Met – a membrane receptor with tyrosine kinase activity. Amplification of c-Met receptors has been reported in different types of cancer, often leading to cancer cell transformation, tumor progression, and treatment resistance.
• Folate receptor alpha (FRα) – a glycosyl-phosphatidylinositol (GPI) membrane-bound glycoprotein overexpressed in several types of cancer cells with a putative role in cancer progression.

Major Targets Of Therapeutic Adcs

The improvement of linker chemistry and conjugation strategies has been promoting the development of ADCs with different structures or mechanisms of action. Multiple trends are expected to mark the next generation of these complex biopharmaceuticals including:

• Dual-targeting (bispecific antibody carrier) or dual-drug systems (two different payloads)
• Probody drug conjugates (only activated by specific enzymes)
• Shifting of chemical and enzymatic conjugation towards less heterogeneity-prone methods such as site-specific and click chemistry
• Creation of fast-releasing linkers for the efficient and safe targeting of solid tumors