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Therapeutic Monoclonal Antibody Fluorescent Labeling Tools for Imaging and Flow Cytometry

Abstract


Therapeutic monoclonal antibodies (mAbs) represent a promising class of biopharmaceutical tools revolutionizing the treatment landscape of immunological disorders, reversal of drug effects, and cancer therapy. Since the late 1990s to 2018, the total number of approved therapeutic mAbs by the US FDA reached 64, with eleven being approved in 2017. This growing interest in therapeutic mAb technologies has sparked a noteworthy need to enhance and expand the range of available tools for diagnostic and therapeutic evaluation of mAbs in pre-clinical models. To satisfy these demands, scientists at AAT Bioquest have developed a plethora of ReadiLink™ conjugation technologies to assist researchers in developing custom-labeled fluorescent mAb conjugates for use in imaging and flow cytometric target identification and validation studies, as well as, determining viable lead candidates.

Keywords: Antibody Conjugation, Flow Cytometry, Therapeutic Monoclonal Antibodies, CD4 Antibody Conjugates

 

Introduction


In recent decades, there has been an intensifying interest in therapeutic monoclonal antibodies (mAbs) as a form of immunotherapy to treat autoimmune diseases, neurological disorders, cancer, cardiovascular disease, infection, organ transplantation, and so on. Since the 1986 commercialization of the first therapeutic mAb, Orthoclone OKT3 an immunosuppressive agent used in liver and kidney transplants, therapeutic mAbs and their derivatives (e.g. Fc-fusion proteins, antibody fragments, and antibody-drug conjugates) have grown to become a dominant class of tools within the biopharmaceutical sector and academia. Today, a reported 570 therapeutic mAbs have been studied in clinical trials, and approximately 79 therapeutic mAbs have been approved by the United States Food and Drug Administration (US FDA) for commercialization.

Why Therapeutic mAbs


The usefulness of therapeutic mAbs is three fold. First, is their monovalent affinity, which is their ability to recognize and target a specific antigen. This high level of specificity for a single target antigen improves consistency among experiments, and reduces cross-reactivity and the adverse effects that accompany it. Second, therapeutic mAbs have the capacity to elicit various mechanisms of action to promote a natural immune response, which enables them to be applied to a wide range of therapeutic targets. Third, improvements in genetic engineering and recombinant manufacturing technologies have made industrial scale manufacturing of therapeutic mAbs possible and cost-efficient.

Mechanisms of Action of Therapeutic mAbs


The underlying objective of therapeutic mAb immunotherapy is to stimulate and restore the patient's natural immune system functions, or to utilize the antibody as a drug delivery carrier (more commonly known as missile therapy). Natural immune system functions include neutralization, blocking, signaling, antibody-dependent cell mediated cytotoxic (ADCC) activity, or complement-dependent cytotoxic (CDC) activity (Table 1).
 

Table 1. Mechanisms of therapeutic mAbs

Mechanism (Mode)
Principle
Therapeutic mAb (Target)
Neutralization Therapeutic mAbs bind to a target ligand or cell surface receptor, thereby blocking that target's signaling pathway. The suppression of the signal through that particular antigen can result in loss of cellular activity, inhibition of proliferation, the activation of pro-apoptotic pathways, or the cell being re-sensitized to cytotoxic agents.Ibalizumab (CD3)
Muromonab-CD3 (CD3)
Bevacizumab (VEGF)
Infliximab (TNF)
Daclizumab (CD25)
ADCC activity ADDC is initiated when the Fv binding domain of a therapeutic mAb binds to an antigen expressed on the surface of a target cell. The mAb subsequently recruits immune effector cells (e.g. Natural Killer cells and macrophages) to lyse the target cell.Rituximab (CD20)
Obinutuzumab (CD20)
Trastuzumab (HER-2)
CDC activity CDC occurs when C1q binds to the mAb-antigen complex. This activates a cascade of complement proteins resulting in the formation of a membrane attack complex (MAC) and ultimately the target cell lysis.Alemtuzumab (CD52)
Panitumumab (EGFR)
Catumaxomab (CD3, EpCAM)
Drug Delivery Carrier Therapeutic mAbs are conjugated to toxins, drugs, radioisotopes, or cytokines. This facilitates the direct delivery of cytotoxic agents to target cells at higher concentrations, without damaging healthy cells.Gemtuzumab ozogamicin (CD33)

Significance of Fluorescent of Therapeutic mAbs


Before a mAb can be considered for therapeutic use it is funneled through a rigorous drug discovery process. During the initial drug discovery phase – target identification, validation and selection of lead candidates – it is often advantageous to fluorescently modify mAbs to facilitate detection in imaging and flow cytometric analysis. For in vivo target-specific cancer imaging, where low background and high target-to-noise ratios are critical (e.g. tumor targeting or whole-animal bio-distribution studies) near-infrared (NIR) and infrared (IR) fluorescence modification of mAbs is preferred. Within the NIR to IR spectrum (700 to 1000 nm) both light scattering and autofluorescence are drastically reduced due to the minimal absorption of biological molecules in this region.

Labeling mAbs for use as imaging reagents, however, presents its own unique challenges. For example, conjugation methods utilizing amine-reactive dyes, such as FITC succinimidyl esters, require large starting volumes (milligrams) of mAbs. Once labeled, conjugates must be purified via spin column or gel filtration media, which reduce conjugation yields to ?50 – 60%. More importantly the degree of labeling (DOL), which refers to the number of dye molecules per antibody, must be optimized to preserve the antibody's immunoreactivity and reproducibility in conjugation results. Under-labeled (DOL < 2) and over-labeled mAbs (DOL > 6) can experience a reduction in their fluorescence intensities. The latter is due to the conjugation of multiple fluorophore to a mAb and its effect on the fluorescence quantum yield (a factor that contributes to dye brightness). This effect is a result of self-quenching between neighboring dyes and between dyes and the mAb.

To address the aforementioned concerns, AAT Bioquest has developed an arsenal of ReadiLink™ Rapid Antibody Labeling Kits for microscale labeling (50 to 100 µg) of therapeutic mAbs for use in imaging and flow cytometry. Utilizing a streamlined labeling protocol in combination with amine-reactive fluorophores, ReadiLink™ kits produces mAb conjugates in two simplified steps at optimal DOL values. To assess the fluorescence brightness, a comparative analysis between CD4-iFluor® 488 conjugates prepared with ReadiLink™ Rapid iFluor® 488 Antibody Labeling Kit and commercially purchased CD4-Alexa Fluor® 488 was performed by lymphocyte immunophenotyping.

 

Materials and Methods


Cells


Cryopreserved human peripheral blood mononuclear cells (PBMCs) were obtained from iXCells Biotechnology (San Diego, CA). Cryopreserved PBMCs were thawed in a 37°C water bath with continuous agitation until completely melted, and then placed on ice for 2 minutes. Each 100 µL of thawed cell suspension was diluted with 9 mL Blood Cell Culture medium (iXCells Biotechnology) supplemented with 1 mL 10% fetal bovine serum. Cells were centrifuged (1000 rpm) at room temperature for 5 minutes and washed a second time with 10 mL of medium. Cell viability was quantified by trypan blue dye exclusion from AAT Bioquest (Sunnyvale, CA), counted on a hemocytometer and resuspended in medium required for lymphocyte immunophenotyping.

Antibodies


CD4 antibody was obtained from AAT Bioquest (Sunnyvale, CA). Alexa Fluor® 488 conjugated CD4 antibody was obtained from Thermo Fisher (Waltham, MA). ReadiLink™ Rapid iFluor® 488 Antibody Labeling Kit from AAT Bioquest (Sunnyvale, CA) were used to prepare CD4 conjugates. Antibodies were dialyzed against 1X PBS (pH 7.2 – 7.4) to remove impurities. CD4 working solution was prepared by adding 100 µL PBS with 10 µL reaction buffer (Component B, ReadiLink Kit) to 100 µg of purified CD4. Working solution was added directly to vial of iFluor® 488 (Component A, ReadiLink Kit), vortexed and incubated at room temperature for 30-60 minutes. Free dye was quenched by adding 10 µL of TQ™-Dyed Quencher Buffer (Component C, ReadiLink Kit) into the conjugation reaction mixture and incubated at room temperature for 10 minutes.

 

Results


Improved stain index with CD4-iFluor® 488 conjugated by ReadiLink™ technology


Stain Index (SI) measurements were taken to assess the fluorescence brightness of CD4-iFluor® 488 conjugates prepared using ReadiLink™ Rapid iFluor® 488 Antibody Labeling Kit and commercially available CD4-Alexa Fluor® 488. This index provides a practical definition of conjugate brightness and enables side-by-side comparison of CD4- iFluor® 488 and CD4-Alexa Fluor® 488. Flow cytometry analysis shows both CD4-iFluor® 488 and CD4-Alexa Fluor® 488 conjugates provide distinct discrimination of positive and negative populations. The separation between respective populations at conjugate saturation provides a measurable parameter for determining conjugate brightness. Using this model, CD4-iFluor® 488 conjugates prepared using ReadiLink™ technology had a 16% improvement in the fluorescence brightness versus CD4-Alexa Fluor® 488 conjugates.


Flow cytometric analysis of Alexa Fluor® 488 or iFluor® 488 anti-human CD4 on human lymphocytes. PBMC cells were stained with 0.5 µg Alexa Fluor® 488 anti-human CD4 or 0.5 µg iFluor® 488 anti-human CD4 in each test. Flow cytometric analysis was performed on a ACEA flow cytometry system.

 

Discussion


The key motivator for this experiment was to determine if ReadiLink™ conjugation technology had to the potential to produce commercial grade fluorescent mAb conjugates for flow cytometry. As previously stated, this experiment showed CD4- iFluor® 488 conjugates had a 16% improvement in fluorescence compared to commercial grade CD4-Alexa Fluor® 488 conjugates. Thus, for producing custom mAb conjugates for flow cytometry applications, ReadiLink™ conjugation technology is a competitive method.

Another quality to note is the dramatic increase in conjugation yield using ReadiLink™ labeling technologies. By replacing the spin column or gel filtration media (two common purification techniques used in conjugation methods) with a novel TQ™-Dyed Quencher Buffer (Component C, ReadiLink Kit), 100% of the conjugate is retained.

 

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