The birth pangs of monoclonal antibody therapeutics: The failure and legacy of Centoxin

This paper examines the development and termination of nebacumab (Centoxin®), a human IgM monoclonal antibody (mAb) drug frequently cited as one of the notable failures of the early biopharmaceutical industry. The non-approval of Centoxin in the United States in 1992 generated major concerns at the time about the future viability of any mAb therapeutics. For Centocor, the biotechnology company that developed Centoxin, the drug posed formidable challenges in terms of safety, clinical efficacy, patient selection, the overall economic costs of health care, as well as financial backing. Indeed, Centocor''s development of the drug brought it to the brink of bankruptcy. This article shows how many of the experiences learned with Centoxin paved the way for the current successes in therapeutic mAb development.     

Monoclonal antibody L6-daunomycin conjugates constructed to release free drug at the lower pH of tumor tissue

Summary Measurements in cancer patients showed that the pH of tumors averages 0.8 unit lower than that of the surrounding normal tissues, confirming published work. Based on this, the anti-carcinoma monoclonal antibody (mAb) L6 was used to prepare immunoconjugates with daunomycin (DM), the drug being released at the acidic pH of the tumor. A direct linking of the aconitic derivative of DM (AcoDM) to mAb L6 led to conjugates that either had a low drug/antibody ratio (<5:1) or precipitated in vitro. In order to increase the drug load and avoid precipitation, several biopolymers were tested as spacers between the drug and the L6. To attach the polymer derivative to the mAb, the former was maleimidized and the mAb was thiolated. The AcoM/mAb ratio obtained was 20, and the mAb retained its highly specific binding to tumor cells. At pH 6 the AcoDM-L6 conjugate was toxic to cultured C-3347 carcinoma cells with an inhibitory concentration (IC₅₀) of 5 µg/ml. The conjugate was less effective than the free DM with an IC₅₀ of 0.2 µg/ml. The L6 alone was not toxic. At a tumor pH of 6.5, 15% of the AcoDM was released. The amount of released drug reached a maximum 24–48 h after exposure to the acidic medium.In vivo localization studies demonstrated a similar tumor uptake of the conjugate and mAb L6 with 18% of the injected dose/g tumor and a maximum uptake in tumor 48 h after injection. Our data indicate that it is possible to construct conjugates based on a pH-sensitive linker that can be targeted successfully to a tumor with release of a portion of the drug at the tumor site, but testing is needed to establish whether such release has anti-tumor activity in vivo and offers an advantage over treatment with unconjugated drug.     

单克隆抗体在抗病毒感染中的研究进展

单克隆抗体(monoclonal antibody, m Ab),指同一种抗原决定簇的细胞克隆所产生的均一性抗体。m Ab已成功应用以诊疗各种疾病,尤其是癌症和免疫性疾病。近些年来,m Ab逐渐用于治疗病毒性疾病,针对急性和慢性病毒感染研发的抗病毒m Ab数量逐渐增长。m Ab的组合疗法可同时靶向病毒多个表位,从而克服病毒免疫逃逸的问题,多价m Ab的设计开发能够更加有效的作用于病毒。本文比较了不同m Ab制备方法的优缺点,总结了m Ab近期在抗病毒感染领域的研究进展,并讨论了m Ab作为治疗病毒性疾病药物的前景。     

Functionalization of liposomes: microscopical methods for preformulative screening

Abstract

The development of smart delivery systems able to deliver and target a drug to the site of action is one of the major challenges in the field of pharmaceutical technology. The surface modification of nanocarriers, such as liposomes, is widely investigated either for increasing the blood circulation time (by pegylation) or for interacting with specific tissues or cells (by conjugation of a selective ligand as a monoclonal antibody, mAb). Microscopical analysis thereby is a useful approach to evaluate the morphology and the size owing to resolution and versatility in defining either surface modification or the architecture and the internal structure of liposomes. This contribution aims to connect the outputs obtained by transmission electron (TEM) and atomic force (AFM) microscopical techniques for identifying the modifications on the liposomal surface. To reach this objective, we prepared liposomes applying two different pegylation technologies and further modifying the surface by mAb conjugation. This work demonstrates the feasibility to apply the combined approach (TEM and AFM analysis) in the evaluation of the efficacy of a surface engineering process.     

Safety and immunotoxicity assessment of immunomodulatory monoclonal antibodies

Most therapeutic monoclonal antibodies (mAbs) licensed for human use or in clinical development are indicated for treatment of patients with cancer and inflammatory/autoimmune disease and as such, are designed to directly interact with the immune system. A major hurdle for the development and early clinical investigation of many of these immunomodulatory mAbs is their inherent risk for adverse immune-mediated drug reactions in humans such as infusion reactions, cytokine storms, immunosuppression and autoimmunity. A thorough understanding of the immunopharmacology of a mAb in humans and animals is required to both anticipate the clinical risk of adverse immunotoxicological events and to select a safe starting dose for first-in-human (FIH) clinical studies. This review summarizes the most common adverse immunotoxicological events occurring in humans with immunomodulatory mAbs and outlines non-clinical strategies to define their immunopharmacology and assess their immunotoxic potential, as well as reduce the risk of immunotoxicity through rational mAb design. Tests to assess the relative risk of mAb candidates for cytokine release syndrome, innate immune system (dendritic cell) activation and immunogenicity in humans are also described. The importance of selecting a relevant and sensitive toxicity species for human safety assessment in which the immunopharmacology of the mAb is similar to that expected in humans is highlighted, as is the importance of understanding the limitations of the species selected for human safety assessment and supplementation of in vivo safety assessment with appropriate in vitro human assays. A tiered approach to assess effects on immune status, immune function and risk of infection and cancer, governed by the mechanism of action and structural features of the mAb, is described. Finally, the use of immunopharmacology and immunotoxicity data in determining a minimum anticipated biologic effect Level (MABEL) and in the selection of safe human starting dose is discussed.Key words: monoclonal antibodies, non-clinical testing, immunopharmacology, immunotoxicity, cytokine release, immunosuppression, autoimmunity, hypersensitivity, immunogenicity, anti-drug antibody, MABEL     

Platform development for expression and purification of stable isotope labeled monoclonal antibodies in Escherichia coli

The widespread use of monoclonal antibodies (mAbs) as a platform for therapeutic drug development in the pharmaceutical industry has led to an increased interest in robust experimental approaches for assessment of mAb structure, stability and dynamics. The ability to enrich proteins with stable isotopes is a prerequisite for the in-depth application of many structural and biophysical methods, including nuclear magnetic resonance (NMR), small angle neutron scattering, neutron reflectometry, and quantitative mass spectrometry. While mAbs can typically be produced with very high yields using mammalian cell expression, stable isotope labeling using cell culture is expensive and often impractical. The most common and cost-efficient approach to label proteins is to express proteins in Escherichia coli grown in minimal media; however, such methods for mAbs have not been reported to date. Here we present, for the first time, the expression and purification of a stable isotope labeled mAb from a genetically engineered E. coli strain capable of forming disulfide bonds in its cytoplasm. It is shown using two-dimensional NMR spectral fingerprinting that the unlabeled mAb and the mAb singly or triply labeled with ¹³C, ¹⁵N, ²H are well folded, with only minor structural differences relative to the mammalian cell-produced mAb that are attributed to the lack of glycosylation in the Fc domain. This advancement of an E. coli-based mAb expression platform will facilitate the production of mAbs for in-depth structural characterization, including the high resolution investigation of mechanisms of action.     

Development of a stable low-dose aglycosylated antibody formulation to minimize protein loss during intravenous administration

The physical and chemical integrity of a biopharmaceutical must be maintained not only during long-term storage but also during administration. Specifically for the intravenous (i.v.) delivery of a protein drug, loss of stability can occur when the protein formulation is compounded with i.v. bag diluents, thus modifying the original composition of the drug product. Here we present the challenges associated with the delivery of a low-dose, highly potent monoclonal antibody (mAb) via the i.v. route. Through parallel in-use stability studies and conventional formulation development, a drug product was developed in which adsorptive losses and critical oxidative degradation pathways were effectively controlled. This development approach enabled the i.v. administration of clinical doses in the range of 0.1 to 0.5 mg total protein, while ensuring liquid drug product storage stability under refrigerated conditions.     

Anti-tumor effect of aquaporin 3 monoclonal antibody on syngeneic mouse tumor model

Aquaporin-3 (AQP3), a water channel protein, has been found to be involved in cancer progression via water and small molecule transport function. However, drug development targeting AQP3 has not yet begun.Here, we showed that a recently established anti-AQP3 monoclonal antibody (mAb) suppresses tumor growth in allograft mouse colorectal tumor models produced using CT26 or MC38 cancer cells. Administration of the anti-AQP3 mAb to BALB/c mice with transplanted CT26 cells increased the M1/M2 ratio of tumor-associated macrophages (TAM) and improved the mitochondrial function of T cells in the tumor microenvironment (TME). Administration of anti-AQP3 mAb also restored the TAM-induced decrease in T cell proliferation. Macrophage depletion in wild-type mice counteracted the antitumor effect of anti-AQP3 mAb in the mouse tumor model, suggesting that one of the primary targets of anti-AQP3 mAb is macrophages. In in vitro studies using mice bone marrow monocytes and human monocyte THP-1 cells, anti-AQP3 mAb attenuated carcinoma cell-mediated polarization of monocytes into M2-like TAMs.These data suggest that anti-AQP3 mAb suppresses tumor growth by attenuating immunosuppressive M2-like TAMs, which in turn maintains the antitumor function of T cells in the TME. Thus, the anti-AQP3 mAb is a potential cancer therapy that functions by targeting TAMs.     

A monoclonal antibody against annexin A2 targets stem and progenitor cell fractions in tumors

The involvement of cancer stem cells (CSCs) in driving tumor dormancy and drug resistance is well established. Most therapeutic regimens however are ineffective in targeting these regenerative populations. We report the development and evaluation of a monoclonal antibody, mAb150, which targets the metastasis associated antigen, Annexin A2 (AnxA2) through recognition of a N-terminal epitope. Treatment with mAb150 potentiated re-entry of CSCs into the cell cycle that perturbed tumor dormancy and facilitated targeting of CSCs as was validated by in vitro and in vivo assays. Epigenetic potentiation further improved mAb150 efficacy in achieving total tumor regression by targeting regenerative populations to achieve tumor regression, specifically in high-grade serous ovarian adenocarcinoma.     

鲨源单域抗体的研究进展

单克隆抗体是重要的生物大分子,在免疫检测、体外诊断以及药物开发等领域获得广泛的应用。但是单克隆抗体的分子量大、结构复杂等固有属性正日益成为制约其进一步发展的关键因素。因此,当前迫切需要开发单克隆抗体的替代品。鲨源单域抗体即鲨源新抗原受体可变区(Variabledomainofimmunoglobulinnew antigenreceptor,VNAR),是基于鲨总目鱼类天然存在的新抗原受体(Immunoglobulinnewantigenreceptor,IgNAR)并通过基因工程技术获得的抗原结合域,其分子量仅为12kDa,是目前已知脊椎动物中尺寸最小的抗原结合域。鲨源单域抗体具有分子量小、亲和力高、稳定性强、溶解度好、组织穿透性强以及可识别隐藏抗原表位等优点,在免疫试剂以及药物开发等领域受到广泛的关注。文中综述了鲨源单域抗体的结构及功能特性、制备及人源化改造技术、亲和力成熟策略以及应用领域,并系统性分析了鲨源单域抗体的优缺点,最后对其发展前景进行了展望。     

mAbs: A business perspective

The twenty two monoclonal antibodies (mAbs) currently marketed in the U.S. have captured almost half of the top-20 U.S. therapeutic biotechnology sales for 2007. Eight of these products have annual sales each of more than $1 B, were developed in the relatively short average period of six years, qualified for FDA programs designed to accelerate drug approval, and their cost has been reimbursed liberally by payers. With growth of the product class driven primarily by advancements in protein engineering and the low probability of generic threats, mAbs are now the largest class of biological therapies under development. The high cost of these drugs and the lack of generic competition conflict with a financially stressed health system, setting reimbursement by payers as the major limiting factor to growth. Advances in mAb engineering are likely to result in more effective mAb drugs and an expansion of the therapeutic indications covered by the class. The parallel development of biomarkers for identifying the patient subpopulations most likely to respond to treatment may lead to a more cost-effective use of these drugs. To achieve the success of the current top-tier mAbs, companies developing new mAb products must adapt to a significantly more challenging commercial environment.Key words: autoimmune, biosimilars, buy and bill, comparative trials, drug approval, monoclonal, oncology, reimbursement     

Inhibition of beta-secretase in vivo via antibody binding to unique loops (D and F) of BACE1

β-Secretase (BACE1) is an attractive drug target for Alzheimer disease. However, the design of clinical useful inhibitors targeting its active site has been extremely challenging. To identify alternative drug targeting sites we have generated a panel of BACE1 monoclonal antibodies (mAbs) that interfere with BACE1 activity in various assays and determined their binding epitopes. mAb 1A11 inhibited BACE1 in vitro using a large APP sequence based substrate (IC(50) ～0.76 nm), in primary neurons (EC(50) ～1.8 nm), and in mouse brain after stereotactic injection. Paradoxically, mAb 1A11 increased BACE1 activity in vitro when a short synthetic peptide was used as substrate, indicating that mAb 1A11 does not occupy the active-site. Epitope mapping revealed that mAb 1A11 binds to adjacent loops D and F, which together with nearby helix A, distinguishes BACE1 from other aspartyl proteases. Interestingly, mutagenesis of loop F and helix A decreased or increased BACE1 activity, identifying them as enzymatic regulatory elements and as potential alternative sites for inhibitor design. In contrast, mAb 5G7 was a potent BACE1 inhibitor in cell-free enzymatic assays (IC(50) ～0.47 nm) but displayed no inhibitory effect in primary neurons. Its epitope, a surface helix 299-312, is inaccessible in membrane-anchored BACE1. Remarkably, mutagenesis of helix 299-312 strongly reduced BACE1 ectodomain shedding, suggesting that this helix plays a role in BACE1 cellular biology. In conclusion, this study generated highly selective and potent BACE1 inhibitory mAbs, which recognize unique structural and functional elements in BACE1, and uncovered interesting alternative sites on BACE1 that could become targets for drug development.     

Safety and immunotoxicity assessment of immunomodulatory monoclonal antibodies

Most therapeutic monoclonal antibodies (mAbs) licensed for human use or in clinical development are indicated for treatment of patients with cancer and inflammatory/autoimmune disease, and as such are designed to directly interact with the immune system. A major hurdle for the development and early clinical investigation of many of these immunomodulatory mAbs is their inherent risk for adverse immune-mediated drug reactions in humans such as infusion reactions, cytokine storms, immunosuppression and autoimmunity. A thorough understanding of the immunopharmacology of a mAb in humans and animals is required to both anticipate the clinical risk of adverse immunotoxicological events and to select a safe starting dose for first-in-human (FIH) clinical studies. This review summarizes the most common adverse immunotoxicological events occurring in humans with immunomodulatory mAbs and outlines non-clinical strategies to define their immunopharmacology and assess their immunotoxic potential, as well as reduce the risk of immunotoxicity through rational mAb design. Tests to assess the relative risk of mAb candidates for cytokine release syndrome, innate immune system (dendritic cell) activation and immunogenicity in humans are also described. The importance of selecting a relevant and sensitive toxicity species for human safety assessment in which the immunopharmacology of the mAb is similar to that expected in humans is highlighted, as is the importance of understanding the limitations of the species selected for human safety assessment and supplementation of in vivo safety assessment with appropriate in vitro human assays. A tiered approach to assess effects on immune status, immune function and risk of infection and cancer, governed by the mechanism of action and structural features of the mAb, is described. Finally, the use of immunopharmacology and immunotoxicity data in determining a minimum anticipated biologic effect Level (MABEL) and in the selection of safe human starting dose is discussed.     

Target-specific NMR detection of protein–ligand interactions with antibody-relayed <Superscript>15</Superscript>N-group selective STD

Fragment-based drug design has been successfully applied to challenging targets where the detection of the weak protein–ligand interactions is a key element. ¹H saturation transfer difference (STD) NMR spectroscopy is a powerful technique for this work but it requires pure homogeneous proteins as targets. Monoclonal antibody (mAb)-relayed ¹⁵N-GS STD spectroscopy has been developed to resolve the problem of protein mixtures and impure proteins. A ¹⁵N-labelled target-specific mAb is selectively irradiated and the saturation is relayed through the target to the ligand. Tests on the anti-Gal-1 mAb/Gal-1/lactose system showed that the approach is experimentally feasible in a reasonable time frame. This method allows detection and identification of binding molecules directly from a protein mixture in a multicomponent system.     

Engineered domain-based assays to identify individual antibodies in oligoclonal combinations targeting the same protein

Quantitation of individual monoclonal antibodies (mAbs) within a combined antibody drug product is required for preclinical and clinical drug development. We have developed two antitoxins, XOMA 3B and XOMA 3E, each consisting of three mAbs that neutralize type B and type E botulinum neurotoxin (BoNT/B and BoNT/E) to treat serotype B and E botulism. To develop mAb-specific binding assays for each antitoxin, we mapped the epitopes of the six mAbs. Each mAb bound an epitope on either the BoNT light chain (LC) or translocation domain (H_N). Epitope mapping data were used to design LC-H_N domains with orthogonal mutations to make them specific for only one mAb in either XOMA 3B or XOMA 3E. Mutant LC-H_N domains were cloned, expressed, and purified from Escherichia coli. Each mAb bound only to its specific domain with affinity comparable to the binding to holotoxin. Further engineering of domains allowed construction of enzyme-linked immunosorbent assays (ELISAs) that could characterize the integrity, binding affinity, and identity of each of the six mAbs in XOMA 3B and 3E without interference from the three BoNT/A mAbs in XOMA 3AB. Such antigen engineering is a general method allowing quantitation and characterization of individual mAbs in a mAb cocktail that bind the same protein.     

A novel approach to monitor clearance of host cell proteins associated with monoclonal antibodies

Co‐purification of a subset of host cell proteins (HCPs) with monoclonal antibodies (mAbs) during the capture of mAbs on Protein A affinity chromatography is primarily caused by interactions of HCPs with the mAbs. To date, there is limited information about the identity of those HCPs due to the difficulty in detecting low abundance HCPs in the presence of a large amount of the mAb. Here, an approach is presented that allows identification of HCPs that specifically associate with the mAb, while avoiding interference from the mAb itself. This approach involves immobilization of purified mAb onto chromatography resin via cross‐linking, followed by incubation with HCPs obtained from supernatant of non‐mAb producer cells that are representative of the expression systems used in mAb manufacturing. The HCPs that bind to the mAb are recovered and identified using mass spectrometry. This approach has not only allowed a comprehensive comparison of HCP subpopulations that associate with different mAbs, but also enabled monitoring of the effects of a variety of wash modifiers on the dissociation of individual HCP–mAb interactions. The dissociation of HCPs that associated with the mAb was monitored by enzyme‐linked immunosorbent assay and mass spectrometry. This approach can be utilized as a screening tool to assist the development of effective and targeted wash steps in Protein A chromatography that ensures not only reduction of HCP levels copurified with the mAb but also removal of specific HCPs that may have a potential impact on mAb structural stability and patient safety. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1114–1124, 2014     

Collaborative enhancement of antibody binding to distinct PECAM-1 epitopes modulates endothelial targeting

Antibodies to platelet endothelial cell adhesion molecule-1 (PECAM-1) facilitate targeted drug delivery to endothelial cells by "vascular immunotargeting." To define the targeting quantitatively, we investigated the endothelial binding of monoclonal antibodies (mAbs) to extracellular epitopes of PECAM-1. Surprisingly, we have found in human and mouse cell culture models that the endothelial binding of PECAM-directed mAbs and scFv therapeutic fusion protein is increased by co-administration of a paired mAb directed to an adjacent, yet distinct PECAM-1 epitope. This results in significant enhancement of functional activity of a PECAM-1-targeted scFv-thrombomodulin fusion protein generating therapeutic activated Protein C. The "collaborative enhancement" of mAb binding is affirmed in vivo, as manifested by enhanced pulmonary accumulation of intravenously administered radiolabeled PECAM-1 mAb when co-injected with an unlabeled paired mAb in mice. This is the first demonstration of a positive modulatory effect of endothelial binding and vascular immunotargeting provided by the simultaneous binding a paired mAb to adjacent distinct epitopes. The "collaborative enhancement" phenomenon provides a novel paradigm for optimizing the endothelial-targeted delivery of therapeutic agents.     

A strategy to accelerate protein production from a pool of clones in Chinese hamster ovary cells for toxicology studies

In the biopharmaceutical industry, a clonally derived cell line is typically used to generate material for investigational new drug (IND)‐enabling toxicology studies. The same cell line is then used to generate material for clinical studies. If a pool of clones can be used to produce material for IND‐enabling toxicology studies (Pool for Tox (PFT) strategy) during the time a lead clone is being selected for clinical material production, the toxicology studies can be accelerated significantly (approximately 4 months at Genentech), leading to a potential acceleration of 4 months for the IND submission. We explored the feasibility of the PFT strategy with three antibodies—mAb1, mAb2, and mAb3—at the 2 L scale. For each antibody, two lead cell lines were identified that generated material with similar product quality to the material generated from the associated pool. For two antibody molecules, mAb1 and mAb2, the material generated by the lead cell lines from 2 L bioreactors was tested in an accelerated stability study and was shown to have stability comparable to the material generated by the associated pool. Additionally, we used this approach for two antibody molecules, mAb4 and mAb5, at Tox and GMP production. The materials from the Tox batch at 400 L scale and three GMP batches at 2000 L scale have comparable product quality attributes for both molecules. Our results demonstrate the feasibility of using a pool of clonally derived cell lines to generate material of similar product quality and stability for use in IND‐enabling toxicology studies as was derived from the final production clone, which enabled significant acceleration of timelines into clinical development. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1449–1455, 2017     

Challenges and opportunities for the future of monoclonal antibody development: Improving safety assessment and reducing animal use

The market for biotherapeutic monoclonal antibodies (mAbs) is large and is growing rapidly. However, attrition poses a significant challenge for the development of mAbs, and for biopharmaceuticals in general, with large associated costs in resource and animal use. Termination of candidate mAbs may occur due to poor translation from preclinical models to human safety. It is critical that the industry addresses this problem to maintain productivity. Though attrition poses a significant challenge for pharmaceuticals in general, there are specific challenges related to the development of antibody-based products. Due to species specificity, non-human primates (NHP) are frequently the only pharmacologically relevant species for nonclinical safety and toxicology testing for the majority of antibody-based products, and therefore, as more mAbs are developed, increased NHP use is anticipated. The integration of new and emerging in vitro and in silico technologies, e.g., cell- and tissue-based approaches, systems pharmacology and modeling, have the potential to improve the human safety prediction and the therapeutic mAb development process, while reducing and refining animal use simultaneously. In 2014, to engage in open discussion about the challenges and opportunities for the future of mAb development, a workshop was held with over 60 regulators and experts in drug development, mechanistic toxicology and emerging technologies to discuss this issue. The workshop used industry case-studies to discuss the value of the in vivo studies and identify opportunities for in vitro technologies in human safety assessment. From these and continuing discussions it is clear that there are opportunities to improve safety assessment in mAb development using non-animal technologies, potentially reducing future attrition, and there is a shared desire to reduce animal use through minimised study design and reduced numbers of studies.