Antibody-Drug Conjugate development

ADC development form

    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.

    Why choose ProteoGenix for your antibody-drug
    conjugate development project?

    Click chemistry and chemical ADC conjugation
    Flexible conjugation strategies

    Choose between chemical (cysteine and lysine) and click chemistry conjugation methods according to your unique needs

    Dual drug ADC production
    Drug diversity and dual drug ADC development

    Choose the best drug for an optimal therapeutic efficacy or opt for dual drug systems thanks to our click chemistry platform for a synergist treatment

    IP free ADC development
    IP free

    Keep full ownership of the antibodies developed for your ADC applications

    Rapid ADC production
    Accelerated antibody production

    Seep up ADC development and antibody engineering (affinity, click chemistry…) thanks to our highly productive cell line – XtenCHOᵀᴹ

    Extensive bioanalysis
    Extensive bioanalytical capabilities

    Characterize DAR values, drug load distribution, % of free drug and antibody with high accuracy and precision to ensure high success rates during clinical development

    Experts in ADC development
    Solid track record

    Benefit from 20+ years of experience in antibody-drug conjugate (ADC) development a 5 therapeutic ADCs in clinical trials

    ADC complimentary services
    Vast range of complimentary services

    Streamline ADC development thanks to our flexible solutions in antibody discovery (phage display, hybridoma), engineering (affinity, stability), and stable cell line generation

    ProteoGenix' antibody-drug conjugate (ADC) development platform

    ADC preliminary study
    I. Preliminary phase (optional)
    • Antibody discovery (hybridoma or phage display)
    • Antibody affinity maturation
    • Optimal parameter determination: antibody, linker, payload, DAR, payload location and distribution, among others
    Antibody carrier design
    Cleavable or non-cleavable linkers
    Drug payloads for ADCs
    II. Antibody-drug conjugate (ADC) development

    Antibody carrier

    1. Rapid antibody production in XtenCHO™
    2. Antibody engineering for click chemistry conjugation and rapid production in XtenCHO™
    3. Customer-provided antibody

    Cleavable or non-cleavable linker

    1. Off-the-shelf linker (SMCC, MCC, SPDB, Valine-Citrulline)
    2. Custom synthesized linker (alternative peptide linkers, β-glucuronide, β-galactoside, and thioether linkers…)
    3. Customer-provided linker

    Single or dual payload

    1. Off-the-shelf payloads (MMAE, MMAF, DM1, DM4)
    2. Custom synthesized payloads (calicheamicins, PBD dimers, duocarymycins…)
    3. Customer-provided payload

    QC of the antibody by SDS-PAGE

    Antibody conjugation
    III. Antibody conjugation
    • Single or dual payload conjugation
    • Click chemistry (site-specific) or chemical conjugation (cysteine or lysine)
    • Conjugation test with up to 4 different
    • DAR values: 2, 4, 6, or 8
    • Purification of the ADC (removal of free drug)
    • QC by SDS-PAGE and ELISA
    ADC bioanalysis
    IV. Bioanalysis
    • DAR: HIC chromatography
    • DAR distribution: HIC chromatography
    • Site of payload conjugation: RP-HPLC
    • % Free drug: RP-HPLC
    • % Aggregation: SEC-HPLC
    • Endotoxin level detection/removal
    • Binding analysis: ELISA, antibody KD determination (SPR in Biacore systems), FACS
    • Stability: differential scanning calorimetry (DSC)
    • Other QC analysis available upon request: capillary electrophoresis (CE-SDS), mass spectrometry (LC-MS/MS),
    • ion-exchange chromatography (IEX-HPLC)
    ADC product
    Final ADC product
    ADC stable cell lines
    High-productivity stable
    cell line development (optional)

    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
    ADC mechanism of action

    An alternative mechanism of action relies on the release of the payload in the tumor microenvironment. With the use of cleavable linkers, it is possible to trigger the release of the payload via chemical signals, conditions, or enzymes. By releasing the payload in the extracellular matrix, cytotoxic agents are free to diffuse more quickly through solid tumors, thus averting the antigen barrier, and targeting cancer cells with significant mutations in the selected receptor (bystander effect).

    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.

    BEST LINKER CHEMISTRIES USED FOR ADC DEVELOPMENT

    Chemical linkers act as the interface between drugs and antibodies in ADCs. Linkers consist of antibody-binding and a payload-binding domain. Their properties heavily influence the stability, safety, aggregation profile, therapeutic widow, and mechanism of action of ADCs.

    Linkers are broadly classified as cleavable or non-cleavable. A list of the most commonly used linkers can be found in the table below.

    Linker chemistry Subclass Groups Examples FDA or EMA approved ADCs
    Cleavable Acid
    cleavable
    linkers
    Hydrazone-
    disulfides
    4-(4′-acetylphenoxy) butanoic
    acid (acetyl butyrate)
    linker [AcBut linker]
    Gemtuzumab ozogamicin;
    Inotuzumab ozogamicin
    PEGylated PEG8- and
    triazole-containing
    PABC-peptide-mc
    linker [CL2A linker]
    Sacituzumab govitecan
    Enzyme
    cleavable
    linkers
    Peptide linkers Valine-Citrulline
    [Val-Cit linker]
    Valine-Alanine
    [Val-Ala linker]
    Brentuximab vedotin;
    Polatuzumab vedotin;
    Loncastuximab tesirine
    Noncleavable
    linkers
    Thioether
    linkers
    Succinimidyl-4-(N-
    maleimidomethyl)
    cyclohexane-1-carboxylate
    linker [SMCC linker]
    Maleimidocaproyl linker
    [MC linker]
    Belantamab mafodotin;
    Ado- trastuzumab
    emtansine

    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.

    MAJOR CYTOTOXIC PAYLOADS OF ADCS

    Suitable payloads for ADCs are typically defined as efficient at killing cancer cells at the nanomolar and picomolar range. They also must be fairly soluble to avoid excessive aggregation, non-immunogenic, and possess reactive sites available for conjugation with a linker.

    The major cytotoxic payloads belong to two families: tubulin inhibitors (maytansinoids, auristatins, or taxol derivates) and DNA-modifying agents (mainly calicheamicins). Most of these agents are also too toxic for system administration in a mono-therapeutic regimen. Tubulin inhibitors have an extensive and solid track record as payloads of ADCs. They act during the cell cycle, causing cell death via mitotic arrest. Most ADCs in the clinic carry tubulin inhibitors as payloads. In contrast, DNA-modifying agents can act independently of the cell growth cycle, causing the cleavage of the DNA molecule and leading to cell death by apoptosis.

    More recently, alternative payloads such as camptothecin derivates and pyrrolobenzodiazepines (PBD) dimers have been gaining ground over more conventional drug classes.

    Payload class Mechanism of action Examples FDA or EMA approved ADCs
    Auristatins Tubulin
    polymerase
    inhibitor
    Monomethyl auristatin E
    (MMAE) and Monomethyl
    auristatin F (MMAF)
    Brentuximab vedotin;
    Polatuzumab vedotin;
    Enfortumab vedotin;
    Belantamab mafodotin;
    Tisotumab vedotin
    Calicheamicins DNA cleavage Calicheamicin and derivates Gemtuzumab ozogamicin;
    Inotuzumab ozogamicin
    Camptothecin Topoisomerase inhibitor exatecan derivate (Dxd) and
    irinotecan derivate (SN-38)
    Trastuzumab deruxtecan;
    Sacituzumab govitecan
    Maytansines Tubulin depolymerization Maytansinoids (DM1 and DM4) Trastuzumab emtansine
    Pyrrolobenzodiazepines
    (PBD) dimers
    DNA minor groove
    cross-linker
    PBD derivates Loncastuximab tesirine
    Protein toxins Several Pseudomonas exotoxin (PE)
    and diphtheria toxin (DT)
    Moxetumomab pasudotox
    Duocarymycins DNA minor groove
    alkylating agent
    CC-1065 and duocarmycin SA None so far
    Amatoxins RNA polymerase II inhibitor α-Amanitin None so far
    Antibiotics Several 4-dimethylamino
    piperidino-hydroxybenzoxazino
    rifamycin (dmDNA31)
    None so far
    Enzymes Several β-glucuronidase, urease,
    among others
    None so far

    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