Monoclonal antibody therapeutics and immunogenicity

Background

The introduction of monoclonal antibody drugs has had major impact in medicine, leading to extraordinary progress in the treatment of a wide variety of diseases, particularly malignancies and chronic inflammatory and autoimmune diseases.

Content

This chapter provides a general overview of monoclonal antibody drugs approved for clinical use, including the basic biology of antibodies, production, pharmacologic characteristics, nomenclature, and various clinical indications. Because of the widespread use of tumor necrosis factor (TNF) antagonists in clinical medicine, we focus on the clinical use of this class of monoclonal antibody drugs to explain the mechanisms that elicit immunogenicity and to describe methodologies for drug monitoring and immunogenicity testing in the clinical laboratory. Finally, interference of monoclonal antibodies with clinical laboratory testing is reviewed since this is an emerging issue increasingly faced by laboratories as the number of patients treated with these drugs is on the rise.

Monoclonal antibody therapeutics (MAT) were initially introduced in transplantation medicine ^, following the approval of the first monoclonal antibody drug, muromonab-CD3, by the United States Food and Drug Administration (FDA) in 1985 as an antirejection agent in renal transplant patients. Subsequently in the 1990s several other monoclonal antibodies were approved for clinical use such as rituximab in 1997, followed by trastuzumab and infliximab a year later. All three of these MAT achieved remarkable success, ranking among the top best-selling drugs ever since. The implementation of MAT led to unprecedented progress in the treatment of a wide variety of diseases, including malignancies and chronic inflammatory and autoimmune diseases. MAT are used in oncology, rheumatology, gastroenterology, neurology, and many other areas of medicine. In recent years there have been dramatic advancements in cancer immunotherapy with the introduction of antibody drugs targeting checkpoint inhibitors cytotoxic T lymphocyte-associated 4 (CTLA-4) and programmed cell death-1 (PD-1).

As of the end of 2019, there were 100 unique monoclonal antibody, antibody fragment, or Fc fusion-based drugs, and 20 biosimilars approved by the FDA for the treatment of various diseases. These numbers are expected to increase dramatically in coming years since many more are currently in development.

Generation of monoclonal antibody therapeutics

Development of the hybridoma technology in 1975 allowed for production of monoclonal antibodies in vitro, using a hybridoma cell obtained by fusing an immortal myeloma cell (plasma cell-derived tumor cell) and a splenic normal B-cell derived plasma cell. This technology allowed for in vitro production of large amounts of MAT against a wide range of targets, including soluble and cell surface proteins. Early MAT products contained large amounts of nonhuman (i.e., mouse) proteins due to the use of mouse spleen derived plasma cells, which was responsible for the development of adverse effects due to the generation of human anti-mouse antibodies. Clinical experience with the first MAT, murine anti-CD3 muromonab, indicated poor pharmacokinetics and high immunogenicity, making treatment ineffective, which eventually resulted in withdrawal of the drug.

Using recombinant DNA technologies and better understanding of antibody structure-function correlation allowed for the development of chimeric (>65% human on average), humanized (>80% human), and fully human (>95% human) antibodies. Chimeric antibodies contain murine sequences in the variable region since they are initially developed as mouse antibodies. The rest of the molecule is replaced with human sequences, which reduces, but does not eliminate immunogenicity, and improves effector functions due to the presence of the human Fc part. The first chimeric antibody, abciximab, a human-mouse Fab fragment, was approved in the US in 1994, followed by rituximab in 1997, the first chimeric full-length antibody. The next phase of technology development allowed for the exchange of rodent sequences almost entirely to human sequences throughout the molecule, by grafting of the rodent CDR onto human IgG. The first humanized antibody, daclizumab, an antibody to IL-2 receptor α subunit, was approved in the United States in 1997 for the treatment of transplant rejection (withdrawn in 2009). Generation of fully human antibodies became possible with the development of phage-display technology, and transgenic mouse platforms. Phage display platform has become a commodity since the technology patents expired recently. It allows for designing and manipulating the repertoire of antibody genes used as antibody sources, followed by in vitro selection. The first antibody to reach the US market developed by this technology was the tumor necrosis factor (TNF) antagonist adalimumab, approved in 2002. In contrast to phage display platform, transgenic animal technology allows for in vivo selection process and requires less optimization and shorter timelines to reach clinical development. Antibody-producing plasma cells isolated from the spleen of immunized, genetically modified mice that express fully human monoclonal antibodies but are unable to produce mouse antibodies, are used to create hybridomas, which are used for subsequent large-scale production. MAT produced by transgenic technologies include panitumumab, approved in 2006 for oncology, and golimumab, a TNF antagonist approved in 2009.

As of the end of 2019, there were 100 unique monoclonal antibody-based drugs approved in the United States ( Table 98.1 ). Eighty-six of these are monoclonal antibodies or antibody fragments, 12 are Fc fusion proteins or peptides, and 2 are engineered chimeric antigen receptor T-cell (CAR T) therapies. Of the 86 monoclonal antibody drugs 4 are murine, 10 are chimeric, 40 humanized (human-murine, human-rat, human-camelid), and 32 are fully human. Most of these drugs are full-length antibodies, the most common isotype being IgG1, which occurs in 61 (71%) of approved antibodies; 8 are IgG2 (9%), 2 are IgG2/IgG4 hybrid (2%), and 13 are IgG4 (15%). Additional structures include camelid, and single chain Fv fragments. 11 out of the 12 approved Fc fusion drugs contain IgG1, and one is fused to IgG4 Fc. Engineering the Fc part is becoming more common in order to modulate effector functions, such as using glycoengineering to enhance effector function. As an example, afucosylation of IgG1 Fc in benralizumab facilitates binding to FcγRIII on natural killer (NK) cells, enhancing ADCC-mediated apoptosis of eosinophils and basophils, which is beneficial for the treatment of severe eosinophilic asthma. The Fc part can also be modified to silence effector functions such as in abatacept or eculizumab. Another approach for improving pharmacokinetics is to incorporate mutations to increase neonatal Fc receptor (FcRn) binding, such as the YTE mutation in some antibodies in development. Fusing IgG Fc with other proteins or peptides improves their stability and half-life. This approach is used in 12 currently approved MAT including TNF receptor, enzymes (phosphatase), peptides (glucagon-like), and clotting factors (VIII, IX). In addition to the full-length antibodies, five of the MAT are Fab antibody fragments. Some Fabs are fused to stabilizer molecules to improve pharmacokinetics in the absence of the Fc part, such as certolizumab pegol, which is PEGylated. Another innovative approach takes advantage of bispecific monoclonal antibodies designed as single-chain variable fragments of two different antibodies combined, such as in blinatumomab (approved in 2014 for acute leukemia), which is a bi-specific T-cell engager, connecting T cells via CD3 with tumor cells expressing CD19, facilitating immunologic synapse formation and tumor cell killing. Most of the approved MAT are nonconjugated intact IgG. However, ten of the MAT used in oncology are conjugated with drug molecules such as antimitotic agents, enzymes, or radioisotopes, which provide additional therapeutic benefits aimed at killing the tumor cell.

As a major breakthrough for cancer immunotherapy, two CAR T-cell therapy agents were approved in 2017: tisagenlecleucel for advanced leukemia and axicabtagene ciloleucel for lymphoma. Chimeric antigen receptor (CAR) is engineered as a single-chain variable fragment molecule fused to signaling domains, which is then expressed on host T cells, grafting monoclonal antibody specificity to the T cells. When injected back to the body, engineered host T cells expressing CAR receptors will recognize the target antigen on the cancer cells, such as CD19 on leukemic or lymphoma cells, and will induce tumor cell killing.

Pharmacokinetic/pharmacodynamic properties

MAT are administered parenterally by intravenous, subcutaneous, or intramuscular routes. The intravenous route has the advantage of offering systemic delivery, the ability to deliver high volumes, and most importantly, assures complete bioavailability. However, limitations include inconvenience and adverse effects due to infusion reactions. Subcutaneous and intramuscular delivery offers lower bioavailability (24 to 95%) but allows self-administration and decreased rate of infusion-related adverse events. Although MAT distribute to most tissues to varying degrees, due to their large molecular weight (typically ~150 kDa) they are not able to cross the blood brain barrier and cannot enter the central nervous system.

MAT are eliminated from circulation through various mechanisms: antigen-specific target-mediated disposition and elimination through endothelial cells of the reticuloendothelial system. Target-mediated disposition involves antigen recognition and binding, typically to a membrane bound target, and subsequent clearance by endocytosis and lysosomal degradation. Since the number of antigenic targets is limited, this process is saturable and nonlinear. Elimination through the reticuloendothelial system, however, is non-saturable and linear, and it involves both free and bound drug. These two processes typically occur in parallel, for MAT targeting a membrane-bound antigen. In contrast to small molecule drugs, the liver and kidneys are not considered essential in the elimination of MAT under normal physiologic conditions, but clearance may be affected in the context of kidney injury leading to disruption of endothelial lining of the glomeruli (such as glomerulonephritis). Other routes affecting MAT clearance include loss of the drug through the gastrointestinal tract in protein losing enteropathy in patients with inflammatory bowel disease (IBD).

Since MAT are typically IgG molecules, their expected half-lives are quite long. This is explained by FcRn mediated uptake and endosomal recycling, a process that substantially extends the half-life of IgG ( Fig. 98.2 ). The binding affinity of FcRn for IgG is negligible at physiologic pH, but when IgG is internalized and enters the endosomes, in the acidic environment of endocytic vacuoles (pH 6.5) it binds to FcRn with high affinity, which rescues IgG from degradation, recycling it through transcytosis. ^, The poor pharmacokinetics of mouse antibody-based drugs observed in earlier trials can be explained by their low affinity to human FcRn, which makes the recycling process less efficient.

Of interest, high interindividual variability has been observed in pharmacokinetics of several MAT drugs. Based on multiple clinical trials, the most commonly identified covariates on MAT pharmacokinetics include body weight/surface area, gender, antidrug antibodies (ADA), creatinine clearance, age, disease severity, and inflammation markers such as C-reactive protein. Other factors influencing inter-individual variability include influence of disease elements such as proteinuria and protein losing enteropathy as mentioned above, or injury to blood-brain barrier, and influence of co-administered drugs. Better understanding of pharmacokinetics and patient-specific covariates paves the way toward personalized MAT therapy, with the goal of increasing efficacy in a cost-effective way, while decreasing toxicity.

Nomenclature

Nomenclature of MAT is currently standardized by international guidelines adopted by the American Medical Association’s United States Adopted Names Council. All monoclonal antibody product names end in the suffix –mab ( Fig. 98.3 ). The nomenclature includes a distinct starting prefix to create a unique name, which is followed by an infix representing the target or disease. Infixes indicating the target class, or disease the antibody is used to treat for include tu-/t- for tumor, li-/l- for immunomodulator, vi- for viral, ba- for bacterial, etc. Since the source of the antibody has important safety consideration in that it may induce immunogenicity in patients, a second infix included indicates the source of the antibody (the species on which the Ig sequence of the antibody is based): o for mouse, a for rat, u for human, i for primate. In addition, the degree of presence and origin of nonhuman sequences is further indicated by additional letters in the source infix such as –xi- indicating chimeric (~65% human), –zu- indicating humanized (~80% human), and -u- indicating fully human (>95% human) antibody. For engineered fusion proteins, suffix –cept indicates the presence of receptor molecules as part of the recombinant Fc fusion (such as etanercept for TNF receptor Ig fusion).

Antigens and diseases targeted by monoclonal antibody therapeutics

MAT were developed against a number of unique antigen targets. Some antigens show overlap between clinical areas, for example, TNF antagonists are used in rheumatology, as well as gastroenterology for the treatment of Crohn disease (CD). Some antigens are more frequently targeted than others (100 drugs for 61 targets), with TNF and CD20 being the most popular with 6 drugs approved for each, followed by HER2 and VEGF with 5 drugs, and CD19, EGFR, IL-17RA, IL-23 p19, PD-1, and PD-L1 with three antibodies for each. The majority of approved MAT are directed against targets in oncology (35%). Other areas include rheumatology (11%), dermatology (7%), blood disorders (7%), gastroenterology (6%), autoinflammatory and other immunologic disease (6%), allergy and asthma (4%), infectious disease (4%), neurologic disease including multiple sclerosis (4%), bone disorders (4%), cardiovascular disease (3%), ophthalmology (3%), transplant and systemic autoimmune disease (2% each), and type II diabetes (1%).

Antigenic targets for MAT are frequently cell membrane bound receptors such as GPIIb/IIIa, integrins, EGFR, CD20, CD52, CTLA-4, PD-1, PD-L1, or soluble targets including growth factors (VEGF), cytokines (TNF, IL-6, IL-17, IL-12/23), complement (C5), or IgE. While most MAT are designed to recognize human targets, a few of them that are used to treat or prevent infectious diseases bind to microbial protein targets (RSV-F protein, Bacillus anthracis PA toxin, Clostridium difficile toxin).

MAT targets for oncology can be classified into three different categories. The first category includes tumor-specific antigens, adhesion molecules serving as “postal addresses” for which killing mechanisms can be targeted. Tumor cell killing can be achieved by activation of immunologic mechanisms following antibody binding to tumor cells (e.g., ADCC or CDC), or by conjugating the antibody to another molecule mediating the killing such as toxins or radioactive isotopes. The second target category overlaps with the first, since it includes tumor cell receptors, which are targeted by MAT with the goal to block ligand binding and signal transduction, for example Her2. The third category includes immunotherapy agents such as checkpoint inhibitors designed to block co-inhibitory signals and thus directly stimulate tumor-specific T-cell responses, or CAR-T cells that express single chain chimeric antigen receptors specific to tumor antigen and are used to induce tumor cell killing.

MAT targeting cytokines include antibodies to pro-inflammatory cytokines IL-1, IL-6, IL-17, and TNF, which play crucial roles in the pathogenesis of numerous chronic inflammatory and autoimmune diseases. These MAT typically block the biological effect by either neutralizing the cytokine or blocking the cytokine receptor that mediates signaling. Due to their pleiotropic biological effects, which include playing a crucial role in host defense against pathogens, systemic pro-inflammatory cytokine blockade may interfere with host defense against infections, which is an unwanted adverse effect.

Biosimilars

Patent expiration of biological drugs created the opportunity for manufacturers to develop biosimilar products, which are highly similar to and have no clinically meaningful differences from existing FDA-approved reference products. An abbreviated pathway for approval was created by Congress through the Biologics Price Competition and Innovation Act of 2009, assuring a shorter and less costly development program with the goal of providing more treatment options, lowering cost through competition, and increasing patient access to treatment. Similarity is determined by extensive structural and functional analysis, comparison of purity, chemical identity, and bioactivity; minor differences are however acceptable (e.g., stabilizer, buffer). For approval of a biosimilar, the manufacturer must demonstrate that there are no clinically meaningful differences compared to the reference product in pharmacokinetics, clinical efficacy, and safety. Assessment of clinical immunogenicity is important during biosimilar development. Due to the complexity of the manufacturing processes for MAT, involving recombinant DNA technology and production by living cells, it cannot be assumed that biosimilars are completely identical to the reference drug (in contrast to small molecule generic drugs, which have chemically identical structure to the reference drug).

Since 2015, 20 biosimilars to MAT have been approved in the US, 18 of which are monoclonal antibodies, along with 2 Fc fusions ( Table 98.3 ). The majority of them are TNF antagonists: four are biosimilars for infliximab, five for adalimumab, and two for etanercept. Biosimilars are expected to show similar immunogenicity profile compared to the reference drug, which is verified during clinical trials. However, due to the variety of assay platforms used across clinical laboratories, internal validation to verify assay performance for the biosimilar compared to the reference drug should be performed before implementing testing for biosimilar drug or ADA to biosimilars.