Antibodies, Volume 10, Issue 2 (June 2021) – 12 articles

Antibody 10E8 is capable of effectively neutralizing HIV through its recognition of the membrane-proximal external region (MPER), and a suitably optimized version of 10E8 might have utility in HIV therapy and prophylaxis. However, 10E8 displays a three-peak profile on size-exclusion chromatography (SEC), complicating its manufacture. Here we show cis-trans conformational isomerization of the Tyr-Pro-Pro (YPP) motif in the heavy chain 3rd complementarity-determining region (CDR H3) of antibody 10E8 to be the mechanistic basis of its multipeak behavior. We observed 10E8 to undergo slow conformational isomerization and delineate a mechanistic explanation for effective comodifiers that were able to resolve its SEC heterogeneity and to allow an evaluation of the critical quality attribute of aggregation. We determined crystal structures of single and double alanine mutants of a key di-proline motif and of a light chain variant, revealing alternative conformations of the CDR H3. We also replicated both multi-peak and delayed SEC behavior with MPER-antibodies 4E10 and VRC42, by introducing a Tyr-Pro (YP) motif into their CDR H3s. Our results show how a conformationally dynamic CDR H3 can provide the requisite structural plasticity needed for a highly hydrophobic paratope to recognize its membrane-proximal epitope. Full article

Disease-specific COVID-19 pediatric comorbidity has not been studied effectively to date. Atopy and food anaphylaxis disease states require improved characterization of SARS-CoV-2 infection risk. To provide the first such characterization, we assessed serum samples of a highly atopic, food anaphylactic, asymptomatic pediatric cohort from across the US during the height of the pandemic. From our biobank, 172 pediatric patient serum samples were characterized specific to atopic, food anaphylactic, and immunologic markers in the US at the beginning of the pandemic, from 1 February to 20 April 2020. Clinical and demographic data were further analyzed in addition to sample analysis for SARS-CoV-2 IgM and IgG ELISA. SARS-CoV-2 antibody results were positive in six patients (4%). Nearly half of the pediatric patients had a history of asthma (49%). Total IgE, total IgG, and IgG1-3 were similar in those positive and negative to SARS-CoV-2. Median total IgG4 in the SARS-CoV-2 positive group was nearly three times (p-value = 0.02) that of the negative group. Atopy controller medications did not confer additional benefit. Our data suggest that food anaphylaxis and highly atopic children are not at increased risk for SARS-CoV-2 seropositivity. This specific population appears either at equal or potentially less risk than the general population. Total and specific IgG4 may be a novel predictor of SARS-CoV-2 infection risk specific to the allergic pediatric population. Full article

The color of a therapeutic monoclonal antibody solution is a critical quality attribute. Consistency of color is typically assessed at time of release and during stability studies against preset criteria for late stage clinical and commercial products. A therapeutic protein solution’s color may be determined by visual inspection or by more quantitative methods as per the different geographical area compendia. The nature and intensity of the color of a therapeutic protein solution is typically determined relative to calibrated standards. This review covers the analytical methodologies used for determining the color of a protein solution and presents an overview of protein variants and impurities known to contribute to colored recombinant therapeutic protein solutions. Full article

Antibody-drug conjugates (ADCs) are biopharmaceutical products where a monoclonal antibody is linked to a biologically active drug (a small molecule) forming a conjugate. Since the approval of first ADC (Gemtuzumab ozogamicin (trade name: Mylotarg)) for the treatment of CD33-positive acute myelogenous leukemia, several ADCs have been developed for the treatment of cancer. The goal of an ADC as a cancer agent is to release the cytotoxic drug to kill the tumor cells without harming the normal or healthy cells. With time, it is being realized that ADCS can also be used to manage or cure other diseases such as inflammatory diseases, atherosclerosis, and bacteremia and some research in this direction is ongoing. The focus of this review is on the clinical pharmacology aspects of ADC development. From the selection of an appropriate antibody to the finished product, the entire process of the development of an ADC is a difficult and challenging task. Clinical pharmacology is one of the most important tools of drug development since this tool helps in finding the optimum dose of a product, thus preserving the safety and efficacy of the product in a patient population. Unlike other small or large molecules where only one moiety and/or metabolite(s) is generally measured for the pharmacokinetic profiling, there are several moieties that need to be measured for characterizing the PK profiles of an ADC. Therefore, knowledge and understanding of clinical pharmacology of ADCs is vital for the selection of a safe and efficacious dose in a patient population. Full article

Gene therapy serves as a promising therapy in the pipeline for treatment of epidermolysis bullosa (EB). However, with great promise, the risk of autoimmunity must be considered. While EB is a group of inherited blistering disorders caused by mutations in various skin proteins, autoimmune blistering diseases (AIBD) have a similar clinical phenotype and are caused by autoantibodies targeting skin antigens. Often, AIBD and EB have the same protein targeted through antibody or mutation, respectively. Moreover, EB patients are also reported to carry anti-skin antibodies of questionable pathogenicity. It has been speculated that activation of autoimmunity is both a consequence and cause of further skin deterioration in EB due to a state of chronic inflammation. Herein, we review the factors that facilitate the initiation of autoimmune and inflammatory responses to help understand the pathogenesis and therapeutic implications of the overlap between EB and AIBD. These may also help explain whether corrections of highly immunogenic portions of protein through gene therapy confers a greater risk towards developing AIBD. Full article

Monoclonal antibodies (mAbs) have demonstrated tremendous effects on the treatment of various disease indications and remain the fastest growing class of therapeutics. Production of recombinant antibodies is performed using mammalian expression systems to facilitate native antibody folding and post-translational modifications. Generally, mAb expression systems utilize co-transfection of heavy chain (hc) and light chain (lc) genes encoded on separate plasmids. In this study, we examine the production of two FDA-approved antibodies using a bidirectional (BiDi) vector encoding both hc and lc with mirrored promoter and enhancer elements on a single plasmid, by analysing the individual hc and lc mRNA expression levels and subsequent quantification of fully-folded IgGs on the protein level. From the assessment of different promoter combinations, we have developed a generic expression vector comprised of mirrored enhanced CMV (eCMV) promoters showing comparable mAb yields to a two-plasmid reference. This study paves the way to facilitate small-scale mAb production by transient cell transfection with a single vector in a cost- and time-efficient manner. Full article

Chimeric antigen receptor (CAR)-T cells are one of the most exciting areas of immunotherapy to date. Clinically available CAR-T cells are used to treat advanced haematological B-cell malignancies with complete remission achieved at around 30–40%. Unfortunately, CAR-T cell success rates are even less impressive when considering a solid tumour. Reasons for this include the paucity of tumour specific targets and greater degree of co-expression on normal tissues. However, there is accumulating evidence that considerable competition for nutrients such as carbohydrates and amino acids within the tumour microenvironment (TME) coupled with immunosuppression result in mitochondrial dysfunction, exhaustion, and subsequent CAR-T cell depletion. In this review, we will examine research avenues being pursued to dissect the various mechanisms contributing to the immunosuppressive TME and outline in vitro strategies currently under investigation that focus on boosting the metabolic program of CAR-T cells as a mechanism to overcome the immunosuppressive TME. Various in vitro and in vivo techniques boost oxidative phosphorylation and mitochondrial fitness in CAR-T cells, resulting in an enhanced central memory T cell compartment and increased anti-tumoural immunity. These include intracellular metabolic enhancers and extracellular in vitro culture optimisation pre-infusion. It is likely that the next generation of CAR-T products will incorporate these elements of metabolic manipulation in CAR-T cell design and manufacture. Given the importance of immunometabolism and T cell function, it is critical that we identify ways to metabolically armour CAR-T cells to overcome the hostile TME and increase clinical efficacy. Full article

ZIP8 is a newly identified metal transporter. In human patients, mutations in ZIP8 result in severe manganese deficiency, suggesting a critical role for ZIP8 in regulating systemic manganese homeostasis. In mice, the deletion of ZIP8 recapitulates the symptoms of patients with ZIP8 mutations. However, further studies using mouse models to examine ZIP8′s function were hindered by the lack of suitable antibodies to detect endogenous ZIP8 protein. In this study, we report the design, generation, and validation of a polyclonal antibody against mouse ZIP8. We have demonstrated that the newly generated antibody can be reliably used in immunoblotting analysis to detect endogenous ZIP8 protein in mouse tissues. The successful generation and validation of anti-mouse ZIP8 antibody provide opportunities to further examine the function and regulation of this metal transporter. In addition, our study may provide valuable insights into the future development of antibodies targeting polytopic membrane proteins. Full article

The targeted delivery of drugs by means of linking them to antibodies (Abs) to form antibody–drug conjugates (ADCs) has become an important approach in oncology and could potentially be used in other therapeutic areas. Targeted therapy is aimed at improving clinical efficacy while minimizing adverse reactions. The nonclinical safety assessment of ADCs presents several unique challenges involving the need to examine a complex molecule, each component of which can contribute to the effects observed, in appropriate animal models. Some considerations for the nonclinical safety evaluation of ADCs based on a literature review of ADCs in clinical development (currently or previously) are discussed. Full article

Lymphatic filariasis (LF) is a debilitating parasitic disease caused by filarial parasites and it is prevalent across the underprivileged population throughout the globe. The inadequate efficacy of the existing treatment options has provoked the conception of alternative strategies, among which immunotherapy is steadily emerging as a promising option. Herein, we demonstrate the efficacy of an antibody-based immunotherapeutic approach in an experimental model of filariasis, i.e., Wistar rat infected with Setaria cervi (a model filarial parasite). The polyclonal antibodies were raised against filarial surface antigen bestrophin protein (FSAg) in mice using the purified Wuchereria bancrofti FSAg. The adoptive transfer of anti-FSAg antibody-containing serum resulted in the significant reduction of parasite burden in filaria-infected rats. Intriguingly, anti-FSAg sera-treated animals also displayed a reduction in the level of proinflammatory cytokines as compared to the infected but untreated group. Furthermore, our in silico immunoinformatics data revealed eight B-cell epitopes and several T-cell epitopes in FSAg and these epitopes were linked to form a refined antigen in silico. The immune simulation suggested IgM and IgG1 as the predominant immunoglobulins induced in response to FSAg. Taken together, our experimental and simulation data collectively indicated a therapeutic potential of anti-FSAg sera against LF. Full article

Recombinant protein-based biotherapeutics drugs have transformed clinical pipelines of the biopharmaceutical industry since the launch of recombinant insulin nearly four decades ago. These biologic drugs are structurally more complex than small molecules, and yet share a similar principle for rational drug discovery and development: That is to start with a pre-defined target and follow with the functional modulation with a therapeutic agent. Despite these tremendous successes, this “one target one drug” paradigm has been challenged by complex disease mechanisms that involve multiple pathways and demand new therapeutic routes. A rapidly evolving wave of multispecific biotherapeutics is coming into focus. These new therapeutic drugs are able to engage two or more protein targets via distinct binding interfaces with or without the chemical conjugation to large or small molecules. They possess the potential to not only address disease intricacy but also exploit new therapeutic mechanisms and assess undruggable targets for conventional monospecific biologics. This review focuses on the recent advances in molecular design and applications of major classes of multispecific biotherapeutics drugs, which include immune cells engagers, antibody-drug conjugates, multispecific tetherbodies, biologic matchmakers, and small-scaffold multispecific modalities. Challenges posed by the multispecific biotherapeutics drugs and their future outlooks are also discussed. Full article

Chemokine (C-X-C motif) ligand 4 (CXCL4) is a biomarker of unfavorable prognosis in Systemic Sclerosis (SSc), a potentially severe autoimmune condition, characterized by vasculitis, fibrosis and interferon (IFN)-I-signature. We recently reported that autoantibodies to CXCL4 circulate in SSc patients and correlate with IFN-α. Here, we used shorter versions of CXCL4 and CXCL4-L1, the CXCL4 non-allelic variant, to search for autoantibodies exclusively reacting to one or the other CXCL4 form. Moreover, to address whether anti-CXCL4/CXCL4-L1 antibodies were present before SSc onset and predicted SSc-progression, we longitudinally studied two VEDOSS (Very Early Diagnosis of Systemic Sclerosis) patient cohorts, separating SSc-progressors from SSc-non-progressors. We found that anti-CXCL4-specific autoantibodies were present in both SSc and VEDOSS patients (both SSc-progressors and SSc-non-progressors). Anti-CXCL4-L1-specific autoantibodies were especially detected in long-standing SSc (lsSSc). Anti-CXCL4/CXCL4-L1 antibodies correlated with IFN-α and with specific SSc-skin features but only in lsSSc and not in early SSc (eaSSc) or VEDOSS. Thus, a broader antibody response, with reactivity spreading to CXCL4-L1, is characteristic of lsSSc. The early anti-CXCL4 autoantibody response seems qualitatively different from, and likely less pathogenic than, that observed in advanced SSc. Lastly, we confirm that anti-CXCL4 autoantibodies are SSc-biomarkers and uncover that also CXCL4-L1 becomes an autoantigen in lsSSc. Full article