Things You Need to Know About Therapeutic Monoclonal Antibodies

What are therapeutic monoclonal antibodies?

A therapeutic monoclonal antibody (mAb) is a homogenous collection of antibodies used to treat an illness. They are selected for their affinity to a target antigen. Depending on the mechanism of treatment, the target may be a plasma protein, infectious organism, cell surface antigen, or IgG receptor. The region of the antibody responsible for affinity to a target is the complementarity-determining region (CDR). Monoclonal antibodies may also promote therapeutic response by recruiting other immune cells.

What are monoclonal antibodies?

Monoclonal antibodies (mAbs) are antibodies produced by a clonal population of B cells and bind to a single target antigen. These differ from polyclonal antibodies (pAbs), which are heterogeneous mix that recognize and bind to multiple epitopes of a single antigen.They are employed in immunotherapies in one of several ways: Stimulate the patient’s immune system, Modulate or inhibit a biochemical disease pathway to deliver a therapeutic radionuclide or drug to a target cell type. Therapeutic mAbs exhibit much higher specificity than small molecule drugs, which minimizes adverse side effects, especially when highly toxic drug substances must be delivered.

Monoclonal antibody therapeutics have been rapidly increasing their market share over the last decade, and in 2019, six out of the ten top-selling drugs were mAbs. Humira therapy, a rheumatoid arthritis drug, sales topped $19 billion, while Keytruda therapy, a cancer drug which forms part of multiple immunotherapy strategies, had sales of >$11 billion [1]. In 2020, there are approximately 100 therapeutic mAbs on the market in the US, with many more in development and trials.

It can be a long and costly journey to find an effective therapeutic; a study carried out on 63 drugs and biologic agents approved by the US Food and Drug Administration between 2009 and 2018 found that the estimated median capitalized research and development cost per product was $985 million [2].

There are many factors to think about from the basic research behind selecting a target antigen to the various antibody generation methods available, characterization strategies, and commercial production. This article introduces key topics in therapeutic mAb development.

How are monoclonal antibodies produced? 

Monoclonal antibodies are developed in bulk by a clonal population of hybridoma cells. Hybridoma production starts with injecting a mouse with an antigen that provokes an immune response. The next step is to harvest the B cells that produce antibodies binding to the antigen. These B cells are then fused with a myeloma cell (immortal B cell cancer cell) from a cell line that has lost the ability to produce immunoglobulins, but retains the ability to multiply indefinitely. The resulting hybridoma cell line has the longevity and reproductivity of the myeloma and the antibody-producing ability of the B cell. Monoclonal antibody development occurs when hybridomas are propagated in cell culture to produce large quantities of antibody molecules that all bind to the same antigen.

Since the hybridoma technique was introduced by Köhler and Milstein in 1975 [3], monoclonal antibody generation technologies have developed to include methods such as the use of transgenic animals to generate fully human mAbs [4,5] antibody phage display [6] and single B cell isolation followed by clonal production [7].

Newer technologies use molecular biology techniques to amplify antibody genes by PCR and recombinant technology for production in either bacterial or mammalian expression systems.

Why is so much effort going into therapeutic monoclonal antibody development?

Monoclonal antibody development provides a number of advantages for therapeutic purposes. For example, therapeutic mAbs exhibit much higher specificity than small molecule drugs. This has the advantage of minimizing adverse side effects, especially when highly toxic drug substances must be delivered.

Monoclonal antibody development therapeutics have been rapidly increasing their market share over the last decade, and in 2019, six out of the ten top-selling drugs were mAbs. There are approximately 100 therapeutic mAbs on the market in the US, with many more in development and trials.

It can be a long and costly journey to find an effective therapeutic; a study carried out on 63 drugs and biologic agents approved by the US Food and Drug Administration between 2009 and 2018 found that the estimated median capitalized research and development cost per product was $985 million [2].

There are many factors to think about from the basic research behind selecting a target antigen to the various antibody generation methods available, characterization strategies, and commercial production. This article introduces key topics in therapeutic mAb development.

What are the applications of monoclonal antibodies?

Monoclonal antibody development has had revolutionary success in the generation of therapeutic drugs for treatment of conditions such as cancer, asthma, Crohn’s disease, psoriasis, and many other infectious and inflammatory diseases.

However, monoclonal antibodies are also useful as a tools in a vast number of medical applications. For instance, test kits use monoclonal antibodies to detect hormones in the diagnosis of ovulation, pregnancy, or menopause. Monoclonal antibodies are also used to detect a safe and appropriate match for blood transfusions or organ transplantation.

How are humanized monoclonal antibodies produced?

When monoclonal antibody development is intended for downstream therapeutic purposes, the best course of action is often to create a humanized monoclonal antibody. A humanized monoclonal antibody consists of a human antibody combined with a small part of a murine monoclonal antibody. The mouse or rat part of the antibody binds the antigen while the humanized part helps the molecule evade the body’s immune system.

Murine mAbs produced from hybridomas can have high specificity, but do not always trigger human effector functions (e.g., complement systems). They are recognized by the patient’s immune system as foreign proteins, stimulating the production of human anti-mouse antibodies (the HAMA response) [8] and resulting in a short serum half-life, which may limit their therapeutic use and cause possible allergic reactions.

The creation of transgenic mice strains engineered to produce human antibodies instead of mouse antibodies was a breakthrough in the therapeutic monoclonal antibody development. The mice are produced by introducing gene segments of human immunoglobulin loci into the germlines of mice deficient in mouse antibody production due to targeted gene deletions. When immunized, these animals generate antigen-specific, fully human monoclonal antibodies. Human and humanized mAbs are the dominant modalities in the field of therapeutic mAbs; human, humanized, chimeric, and murine antibodies, respectively account for 51, 34.7, 12.5, and 2.8% of all mAbs in clinical use [10].

Combinatorial library screening with phage display

Antibody phage display libraries [11] can be created either by using a complete immune repertoire of hybridomas or B cells from naïve or immunized animals, or constructed synthetically. To create the phage display library, DNA sequences encoding antibody fragments are spliced into a coat protein gene of a filamentous phage, causing the phage to express or "display" the resulting fusion proteins on its surface. The phenotype (specificity and sensitivity) of the peptide sequence displayed on the surface is linked to the genotype sequence of the engineered phage genome. The combinatorial libraries that result from this process provide a means of identifying target-binding antibody fragments from a library of millions of different fragments without having to screen each one individually. These antibody fragments can be further manipulated using genetic engineering techniques to yield complete antibodies for functional evaluation. To enrich for the desired antibody molecules from clones present at one in a billion or less, affinity selection by “panning” proteins that bind to a particular antigen, known as bio-panning, is essential. 

Phage eluted in the final step of panning can be used to infect a suitable bacterial host. The phagemids can be collected and the relevant DNA sequence excised and sequenced to identify the interacting proteins or protein fragments. The DNA sequence can be further genetically manipulated to produce the desired monoclonal antibody. Once optimized, the sequence is cloned into expression vectors for production.

Large antibodies can be difficult to express. The discovery of smaller recombinant antibody fragments has advanced antibody phage display technology. Examples of more easily expressed smaller fragments include variable domain (Fv), single-chain variable domain (scFv), diabodies (bivalent scFvs), camelid and shark antibody fragments, and fragment antigen binding (Fab) regions.

Antibody phage display is a versatile in vitro selection technology used to discover high-affinity antibodies specific to a wide variety of antigens. including toxic and nonimmunogenic antigens. Repeated rounds of antigen-guided selection allow highly specific fully human-derived mAbs to be isolated from large immunoglobulin gene repertoires displayed on the surface of bacteriophages.

Antibodies from human cells

Single B cell antibody technologies use the robust response of the human immune system to generate fully human mAbs. These methods only require a few cells, which can be rapidly and efficiently isolated from either peripheral blood mononuclear cells (PBMCs) or lymphoid tissues. Fluorescence-activated cell sorting (FACS), or flow cytometry, is widely utilized to identify specific B cells based on their expression of cell surface markers, and a process called antigen baiting using antigen-coated magnetic beads and fluorescence-conjugated antigens is used to select antigen-specific cells.

After single B cells are isolated, expressed immunoglobulin transcripts are amplified using reverse transcription polymerase chain reaction (RT-PCR), and then cloned and expressed in mammalian cell lines.

Single B cell isolation and cloning can be used to rapidly develop immunotherapies for infectious diseases. Human mAbs have been generated by the single B cell method for bacterial, parasitic, viral, and autoimmune diseases.

Methods for screening therapeutic monoclonal antibody candidates

Antibodies that bind to the target are identified by via ligand binding assays. These assays are performed early in the development and lifecycle management phases to determine the presence and nature of ligand–receptor complexes formed. They must be sufficiently high-throughput and accurate enough to eliminate poor candidates early in the drug development process.

The following methods can be used for candidate screening:

• Multiplex enzyme-linked immunosorbent assay (ELISA)—a solid-phase type of enzyme immunoassay (EIA) used to detect the presence of a ligand in a liquid sample
• Surface plasmon resonance (SPR)—an electrochemical technology that produces data on affinity, specificity, and binding kinetics, without the need for labels. Binding is analyzed in real time
• Western blotting—a widely used analytical technique based on gel electrophoresis and antibody binding
• Cell-based microarrays—a high-throughput method for the rapid screening and detection of single cells secreting antibodies with the desired reactivity profiles
  
  

During the screening stage, “hits” that bind specifically to the target are identified and ranked according to binding strength. The nature of the mAb–target interaction is then investigated.

The importance of defining critical quality attributes early in the development process

A critical quality attribute (CQA) is defined as “a physical, chemical, biological, or microbiological property or characteristic that should be within an appropriate limit, range, or distribution to ensure the desired product quality [12].” CQAs are defined at the start of development and guarantee the safety and efficacy of the final product. As additional product knowledge is gained from ongoing bioanalytical characterization, animal studies, and clinical experience, the CQAs can be refined. Ultimately, the objective is to link CQAs to clinical performance.

It is often difficult to evaluate the large number of critical quality attributes that relate to safety and efficacy, so CQAs can be grouped into categories to simplify the assessment approach. Example critical quality attribute categories [13] are:

• Product-specific variants: size, charge, aggregation, glycosylation, sequence variation, N/C terminal processing, isomerization
• Process-related impurities: host cell protein, protein A leachate, DNA, raw materials
• Obligatory CQAs: composition and strength (pH, excipients, quantity/concentration, osmolarity, adventitious agents, other particulates

A systematic, scientifically based risk ranking and filtering approach allows a thorough understanding of quality attributes and an assignment of criticality for their impact on drug safety and efficacy. 

The right development and characterization strategy, combined with well-defined CQAs, can identify the monoclonal antibodies that are most likely to be successful drugs early in the process and ensure the production of safe and consistent therapeutics. 

In recent years, increasing attention has been paid to developability assessment, with thorough evaluations of mAb lead candidates at an early stage reducing delays during late-stage development.

Key assays for functional characterization of therapeutic monoclonal antibodies

Extensive biophysical, biochemical, biological, immunochemical, and immunological mAb characterization must be carefully conducted. Use of robust methods for the characterization of therapeutic monoclonal antibodies results in shorter development times and ensures the production of safe, consistent, and stable therapeutic mAbs that meet strict regulatory standards.

Antibody affinity assays

In general, as affinity increases, tissue and tumor penetration decreases due to binding site barrier effects [14]. Therefore, although a high affinity can increase the likelihood of a mAb successfully binding to a target, it is important to determine an optimal affinity for the mAb to be of therapeutic use. This is done via cycles of affinity maturation and screening variants with different affinities.

The measurement of affinity to target is done using the equilibrium dissociation constant, KD. KD is a ratio of K_off to K_on between the antibody and its antigen. The lower the KD value, the higher the affinity.

There are multiple methods for determining KD. For example, there are gel-shift assays, pull-down assays, equilibrium dialysis, surface plasmon, isothermal titration calorimetry, spectroscop, and analytical ultracentrifugation.

Bioassays for antibody characterization

Choose an appropriate bioassay method to characterize your mAb based on the disease indication and mode of action of the therapeutic. Popular bioassay methods include:

• Immune checkpoint reporter-based bioassays provide a simple, pathway-specific, cell-based option to measure antibody function
• Surrogate reporter assays reflect the mode of action of pathway-specific therapeutic antibodies and can indicate the stability of a mAb
• Tumor growth/inhibition assays arethree-dimensional cellular assays adapted to test the cytotoxic activity of a panel of monoclonal antibodies directed to various antigenic determinants on the surface of mouse mammary tumor cells
• Antibody-dependent cellular cytotoxicity (ADCC) assays allow evaluation of a mAb’s ability to kill cancer cells through natural killer (NK) cell–mediated cell lysis. Dead cells are quantified using either flow cytometry, fluorophore release, or a label-free assay

Analytical methods for structural characterization of therapeutic mAbs

As well as confirming the primary amino acid sequence, molecular characterization of a mAb must involve analysis of the secondary and tertiary structure of the protein, as well as any post-translational modifications.

Some of the most popular techniques for protein structure analysis are:

• Chromatography—size exclusion (SEC), ion exchange (IEC) and reversed-phase liquid chromatography (LC) are used to determine molecular weight distributions and assess aggregate ratios
• Liquid chromatography coupled to mass spectrometry (LC-MS)—a powerful analytical technique for characterizing mAbs, providing information down to the level of individual amino acids
• High-performance liquid chromatography (HPLC) or ultra-HPLC (UHPLC) with inline mass spectrometry (MS)—for peptide mapping can covers 70–80% of the mAb amino acid sequence; this is usually sufficient for preclinical development characterization
• Hydrogen-deuterium exchange mass spectrometry (HDX-MS)—an increasingly common way to investigate the tertiary structure of a mAb, as it is much faster than traditional X-ray crystallography, requires less material, and can tolerate impurities

Assessing monoclonal antibody stability

To be safe and effective, mAbs must be stable, and not form dimers or aggregates.

Aggregation can dramatically influence the bioactivity of mAbs and is generally irreversible. Aggregates also have the potential to cause side effects and increase the elimination rate due to their high immunogenicity [15]. 

Aggregate formation is often product- or process-related; for example, on-column aggregate formation is influenced by the resin type and on the resin’s interactions with the particular mAb in production.

There are several methods to perform protein aggregate analysis:

• High-performance size exclusion chromatography—a modern high throughput method based on ultra-performance liquid chromatography (UPLC) that can detect mAb aggregates in a matter of minutes
• Analytical ultracentrifugation—a simple technique that measures the sedimentation rate of a mAb in a solution, providing information about its molecular mass and shape
• Differential scanning calorimetry—a well-established technique for biomolecular stability studies based on forced thermal denaturation of biomolecules in solution
• Dynamic light scattering—a convenient but relatively low-resolution method that measures the size of particles present from the scattered light in a solution
• Asymmetric flow field-flow fractionation—a gentle characterization method that provides Information about molar mass, polydispersity, size, shape/conformation, or density

Choice of cell line impacts transition from development to production

Therapeutic mAbs require a mammalian expression system that provides the cell machinery required to glycosylate, fold, orient, and covalently bind antibody peptide chains to produce the complete, biologically functional molecule. Lower organism expression platforms can be used if the product is a mAb fragment or where glycosylation is simpler, or not required. 

Antibody genes of interest are introduced into a suitable expression vector and transfected into cell lines for antibody expression and secretion. Expression vectors are designed to maximize mAb expression and ensure cell line stabilization through host cell codon optimization and the addition of highly efficient transcription, secretion, selection, and integration elements.

Chinese hamster ovary (CHO) cells are a popular host for producing therapeutic mAbs as they are suited to high-yield production of recombinant proteins and are good at performing the post-translational modifications required to produce the correct high-level conformation of an active mAb.

Mammalian cell lines have been adapted to suspension culture and engineered for enhanced functions (e.g., introducing glycosylation pathways and resistance to apoptosis). These newer cell lines support high cell densities and product high mAb titers, so are suitable for antibody production in large scale-fed batches, perfusion systems, or continuous culture techniques. Commercially developed CHO cell lines with enhanced stability are used in many current mAb therapeutic expression platforms.

The ability to produce transient CHO-derived monoclonal antibodies early during biotherapeutic development is highly desirable, as products will closely mimic the final CQAs of the mAb when manufactured at bioproduction scales.

Cell culture media impacts antibody characteristics

Conformational and post-translational modification changes and impurities can be caused by the cell media used during the mAb development and production process. Glycosylation pattern, charge variants, aggregates, and low-molecular-weight species may be significantly altered during culture media optimization. To develop a mAb with the desired quality, the analysis of CQAs by appropriate analytical methods throughout process development is critical.

Glycosylation, the enzyme-triggered addition of glycans to the mAb’s protein backbone, is one of the most important critical attributes to assess when characterizing a product and maintaining its quality and safety, as it affects the physicochemical and pharmacological properties of a mAb. The correct glycosylation pattern is required to produce a functionally active protein, but unwanted glycosylation modifications commonly occur during the production of mAbs via cell culture and other stages of mAb production (e.g., the purification, formulation, and storage processes). Modification of the glycan profile can be achieved by supplementing or modifying media. 

The commercial manufacture of mAbs has transitioned to low-protein, serum-free, chemically defined, and animal-free media, which has removed a source of expression variability and the risks of using animal-derived products.

Several media supplements such as plant and yeast digests (peptones and hydrolysates), surfactants (e.g., Gibco Pluronic F-68 non-ionic surfactant), DNA methyltransferase (azacytidine), and histone deacetylase (sodium butyrate and valproic acid) inhibitors have been found to support cell viability and enhance mAb expression.

The future of therapeutic monoclonal antibodies

As our understanding of the molecular mechanisms underlying disease grows, opportunities for the development of new mAb-based drugs increase. In the last decade the focus has been on producing mAbs for cancer immunotherapy, inflammatory diseases, and autoimmune diseases, but they are becoming increasingly important in treatments and prophylaxis for infectious diseases such as fungal diseases, Ebola, and the novel coronavirus SARS-CoV-2. In the future, mAbs could be used to target multidrug-resistant pathogens and may help prevent the emergence of antimicrobial resistance.

The therapeutic antibody field is also exploring the use of new modality antibodies such as bispecific and trispecific antibodies that recognize multiple epitopes on the same antigen, single-domain antibodies that can more easily penetrate tissues, and antibody-drug conjugates for targeting chemotherapy agents to specific cell types. Some bispecific antibodies and antibody-drug conjugates are already on the market, with several more in development.

One thing is certain, therapeutic monoclonal antibodies are going to continue to increase in importance for many years to come.
 

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