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CD? Did you say CD?
In immunology, it is really useful to identify the different types of leukocytes within a sample. But distinguishing leukocytes according to their morphological or functional characteristics is not an easy job.
However, scientists discovered that leukocytes express on their surface a large number of molecules which can be used as markers for their specific identification. The concept of cell immunophenotypic identification was born.
This identification is based on the detection of these membrane markers using monoclonal antibodies followed by analysis in flow cytometry. Since 1982, these markers have been designated according to the cluster of differentiation nomenclature or CD, followed by a number. And the combination of present (CD+) and absent (CD-) markers allows the identification of the state of maturation of the cells.
CD25: a difficult youth
As early as February 1988, a Diaclone monoclonal antibody anti-CD25, the B-B10 clone (now known as Inolimomab or Leukotac) was used in vivo to treat a severe case of acute Graft versus Host disease.
CD25, also known as the interleukin-2 high-affinity receptor alpha chain (IL-2Rα), has been observed on activated B, T lymphocytes and macrophages. Alternative names for the antigen are often used: IDDM10, IL2R, TCGFR, p55 or Tac.
Inhibiting the binding of IL-2 to CD25, anti-CD25 monoclonal antibodies were first developed as therapeutic agents to treat organ transplant rejection (Basilixumab, a Novartis chimeric mouse-human CD25 antibody, 1998 approval) and multiple sclerosis (Daclizumab, a Biogen humanized CD25 antibody, 1997 approval then withdrawn due to safety issues).
But since - as CD25 is constitutively and highly expressed on Treg cells - this marker was rapidly investigated as a potential target for inflammatory, auto-immune and cancer immunotherapies.
In 2013, ADC Therapeutics and Genmab partnered to develop a new antibody format, an Antibody Drug Conjugate (ADC), offering anti-CD25 a second life, this time in the oncology field. Camidanlumab was in the pipeline.
But tackling cancer with a Treg cell depletion tactic - like companies such as Hifibio Therapeutics have promoted since the beginning - was quite a contested therapy approach. So far, clinical trials have proven these strategies wrong. Until recently…
CD25: an oncology boat with the wind in its sails
- Anne Goubier and her team at Tusk Therapeutics made the headlines when they showed that a blockade of IL-2 signalling limits Teff responses. They consequently decided to develop a new version of anti-CD25 antibody that does not block the IL-2 signalling in order to deplete Treg and stimulate Teff at the same time. This promising antibody is going to be tested in cancer. But not by Tusk itself. At the end of 2018, Roche paid €70 million ($81 million) upfront to buy Tusk Therapeutics and in doing so getting complete control on this promising anti-CD25 antibody.
- In 2019, NIH conjugated a near infrared silica-phthalocyanine dye to a monoclonal antibody. This process led to a new cell-specific cancer therapy that locally kills specific cells in the tumour. In this particular context, comparing full length anti-CD25 antibody with its fragment version, it appears that the absence of the Fc portion leads to faster clearance and therefore promotes a superior activated T cell response in tumours.
- January 2020, Alderaan Biotechnology, a preclinical company focused on the development of anti-CD25 monoclonal antibodies for the treatment of cancer, announced that it raised €18.5 million in a Series A financing.
And any recent updates on dysregulated inflammatory response?
In 2019, a Japanese team from Kyoto University and Asahi Kasei Corporation paved the way to a new approach. Apheresis is a technique that makes it possible to take, via a machine, one or more blood components depending on the needs. The team demonstrated that the removal of Tregs from septic patients by apheresis was a good relief solution. However, the removal of cells other than Tregs caused the adverse effect of lowering the immune response against microbes. So, they decided to immobilise an anti-CD25 antibody on a polyethylene fibre to specifically remove Tregs without impacting the other components of PBMCs.
We might agree that it is not a definitive therapeutic solution like those described above for cancer, but this nice creativity - combining immunoaffinity purification with the apheresis technique - must be applauded and encouraged!
Do you have a current project on CD25? Do you want to specifically target Treg?
Diaclone has developed several anti-CD25 monoclonal antibodies for your research:
Clone B-B10: anti-IL2Ra, a biologically active mAb
Clone B-F2: anti-IL2Ra
Clone B-G3: anti-IL2Ra
CD25 ELISA Pair:
Clone B-G3: Capture antibody
The most common and potentially severe toxicity seen across trials using immunotherapies such as monoclonal antibodies, bispecific antibodies (Bi-specific T-cell engagers or BiTEs) and adoptive T-cell therapies (e.g. CAR-modified T-cell) is CRS.
But what does CRS mean?
CRS stands for Cytokine Release Syndrome. Cytokine Release Syndrome is a clinical syndrome resulting from generalized immune activation correlating with marked elevations of serum inflammatory markers and cytokines. The first clinical signs of CRS are fevers, myalgias, and fatigue. But starting from fever with or without constitutional symptoms (CRS grade #1), symptoms can rapidly evolve to hypoxia requiring O2 (grade #3) and finally death (grade #5).
But is the probability of a CRS event really alarming?
The risk of CRS is influenced by factors related to the type of therapy, underlying disease, and characteristics of the patient. Treatment with most conventional monoclonal antibodies carries a relatively low risk of CRS, whereas CAR-T therapies carry a significantly higher risk of CRS incidence.
With regard to recently approved CAR-T therapies, Yescarta was studied in 107 adults with large B cell lymphoma and ninety-four percent of treated patients developed CRS and 12% developed grade ≥3 CRS. Kymriah was tested in 68 paediatric and young adult patients with relapsed/refractory ALL and seventy-nine percent developed CRS with 49% developing grade ≥3 CRS!
But what can we do when CRS occurs?
Corticosteroids may be necessary in some cases, but in high doses have proven to have a detrimental effect on CARTs.
Extracorporeal blood purification techniques, such as high-volume hemofiltration, cascade hemofiltration, plasma exchange, and coupled plasma filtration adsorption, have also been reported as a potential alternative to treat CRS syndrome. But they remain time consuming and labour extensive solutions.
Since the initial observation that tocilizumab, an antibody against the IL-6 receptor, rapidly reversed severe CRS, the drug remains at the forefront of CRS treatment.
For the future, many CARTs in development incorporate suicide targets as methods to mitigate toxicity. Another approach is manufacturing CARTs that can be regulated without actually killing them, for example requiring infusion of an additional agent for activation.
But diagnosis is better than treatment.
Early prediction is necessary as the side effects appear rapidly - within 1 to 2 weeks - following CAR-T infusion. The elevation of the signature set of 24 different cytokines is then an interesting and crucial measure to monitor in patients.
The main cytokines implicated in the pathogenesis of CRS include effector cytokines released from activated T cells, but also cytokines mainly secreted by monocytes and macrophages. IFN-γ and sgp130 levels, for example, rise early and their elevation is different for severe versus non-severe CRS.
But we can also mention sIL6R, interleukin-6 (IL-6), interleukin-10 (IL-10), interferon INF-α, chemokines that are chemotactic for monocytes/macrophages (MCP1, MIP1α, MIP1β), granulocyte-macrophage colony-stimulating factor (GM-CSF), but also TNF-α, IL-1b, IL1RA, IL-2, sIL2Rα, C-reactive protein (CRP) and IL-8. Other biomarkers of endothelial cell activation, such as Angiopoietin-2 and von Willebrand factor, have also been recently described to predict CRS severity.
In order to effectively monitor the risk linked to immunotherapy, Diaclone has developed a large panel of standard recombinant cytokines and monoclonal antibodies targeting specific cytokines engaged in Cytokine Release Syndrome, as well as ELISA and DIAplex kits. As a first evaluation test, and for 7 of the targeted set of cytokines, Diaclone DIAplex is a particularly useful tool for providing a rapid response and with a reduced sample volume requirement starting from only a few µl.
They are sometimes qualified as antagonist, sometimes as agonist. Sometimes named inductive, sometimes repressive, sometimes blocking, or sometimes neutralizing. But they are always called antibodies. Have you ever guessed what it was about? Yes, that’s them, the biologically active antibodies!
Depending on the nature of the antigen, a specific antibody can demonstrate different biological functions:
- if the target is a cell surface marker for instance, the desired effect of the mAb may involve proliferation (induction/activation effect through a signaling cascade ), inhibition, cell maturation or even the killing of the target cell. Cell depletion will then occur through the recruitment of immune mediators with the Fc portion of the mAb to trigger antibody-dependent cellular cytotoxicity (ADCC), antibody-mediated phagocytosis cytotoxicity (ADCP) or complement-dependent cytotoxicity (CDC).
- if the target is a soluble molecule such as plasma protein (TNF, VEGF…) or a drug, the binding may trigger a blocking effect. When bound to the mAb, these drugs are not able to interact with their normal targets anymore. Of course, blocking effect can also occur via the cell surface receptor like the well-known immune checkpoint inhibitors (targeting CTLA-4, LAG3, PD1 and PDL1).
- If the target is an infectious organism, the desired function of antibodies may be neutralization of the foreign host, so disabling the virus, bacteria or other. In fact, most licensed vaccines teach the body how to make neutralizing antibodies.
Cell proliferation induced by IL-6 and Neutralization by anti-IL-6 antibody
(a) Recombinant human IL-6 stimulates proliferation in XG1 cell line in a dose-dependent manner. The ED50 is typically 30-40 pg/mL.
(b) Proliferation elicited by rhIL-6 (1ng/ml) is neutralized by increasing concentrations of human IL-6 monoclonal antibody. The ND50 is 0.07nM .
Unfortunately, many difficulties can appear during the process of raising biologically functional antibodies. We can name:
- epitope masking by variable loops or multimerization event,
- the presence of cryptic binding domains,
- the very high binding affinity of the target for its receptor, which makes the generation of inhibitory antibodies extremely challenging,
- the very narrow window for neutralizing antibodies to act before the establishment of a virus infection and its genome integration into the cell host.
Either from a classical hybridoma approach or by phage display technique, DIACLONE has over the years learned to tackle all these obstacles to offer you the best possible antibody references. Thanks to its bioassay platform, all our references are highly validated via proliferation, cytotoxicity, neutralization or various cell functionality tests.
KD is the equilibrium dissociation constant, a calculated ratio of koff/kon, between the antibody and its antigen. The association constant (kon) is used to characterize how quickly the antibody binds to its target. The dissociation constant (koff) is used to measure how quickly an antibody dissociates from its target.
KD and affinity are inversely related. A high affinity interaction is characterized by a low KD, a fast recognizing (high Kon) and a strong stability of formed complexes (low Koff).
10-4 to 10-6 Micromolar (μM)
10-7 to 10-9 Nanomolar (nM)
10-10 to 10-12 Picomolar (pM)
10-13 to 10-15 Femtomolar (fM)
At Diaclone, we measure using the SPR (Surface Plasmon Resonance) technology and the Octet instrument. This technique can help you to rank your antibodies but also to better understand the performance of your antibodies.
For example, this ELISA development showed that the pair worked only when mAb1 is coated (Fig.1)
Fig. 1 : mAb1-mAb2 pairing evaluation
The determination of KD and association/dissociation profiles of antibodies by Octet permitted us to understand why the pair works only in one way.
Fig. 2 : Profiles of association dissociation of mAb1 (top) and mAb2 (bottom)
The mAb2 has a quick dissociation time (Fig. 2), so, when it’s coated and after all the washing steps, the antigen doesn’t stay on mAb2. Using this antibody as revelation avoids this dissociation which is linked to accumulated washing steps.
Octet analysis can also help to validate the accessibility of tags (HIS, GST…), to study the interaction receptor-ligand and the potential inhibition of interaction with an antibody, and to perform epitope binning (also known as epitope mapping or pairing).
The advantage of Octet compared to the highly popular Biacore technique is the lower price, the rapid execution of experiments and the small amount of proteins required.
Whatever the interaction to study, Octet is a valuable tool.
Use Diaclone’s expertise to further characterize your antibody
The Octet technology is fully integrated into our custom monoclonal antibody development so that it can be implemented to further study monoclonal antibody candidates.
A large range of custom services, dedicated to your needs:
· Biological activity: agonist or antagonist, secretion enhancer, blocking signal transduction, cellular growth activation or inhibition,….or any new model to design
· Effector activity: ADCC, ADCP or CDC
· Antibody applications: ELISA, Western Blotting, Flow cytometry…
· Antibody labeling
Contact us to discover how Diaclone can support your activity - email@example.com
A little bit of history ...
In 1983, two laboratories on opposite sides of the world described almost simultaneously what would later be called the ELISpot: Cecil Cervinsky in Gothenburg, Sweden and Jonathon D. Sedgwick in Perth, Australia. This “invention” was first dedicated to enumerate frequency of B hybridroma cells secreting an antigen specific immunoglobulin and then, a few years later, to measure the frequency of T lymphocytes secreting a specific lymphokine.
At that time, Monoclonal Antibodies were rare, the ELISA was still a very new technique and the specificity of most polyclonal antibodies was highly questionable, but with the proximity of the team of Dr Outcherlony at Gothenburg University, Cecil Cervinsky had the idea to imagine an “Antibody forming cell ELISA assay” in a gel matrix.
Since the first description, key advances have included the first use of membrane bottomed plate (nitrocellulose and then PVDF) that enables increased sensitivity, improvement in substrates and application of computer-assisted spot counting technologies (1993) and the commercialization of these technologies by numerous instrumentation companies.
The assay was called by a range of names including spot-ELISA, ELISA-spot, ELISA-immunospot and, of course, ELISpot ultimately chosen by the scientific community.
Diaclone’s ELISpot ...
ELISpot is now the method of choice in clinic for monitoring Interferon-gamma secretion in cancer immunotherapy and viral vaccine trials.
The Diaclone IFNg ELISpot assay is used in several clinical studies and published in Clinical Cancer Research by Dosset et al.,Godet et al. and Teixeira, L. et al.*
Diaclone ELISpot Kits and ELISpot Sets
Diaclone ELISpots (Enzyme-Linked Immunospot Assays) are highly specific immunoassays for the analysis of cytokine and other soluble molecule production and secretion from T-cells at a single cell level in conditions closely comparable to the in-vivo environment with minimal cell manipulation.
Utilising sandwich immuno-enzyme technology, Diaclone ELISpot and Dual ELISpot assays can detect both secreted cytokines and measure the frequency of single cells that simultaneously produce multiple cytokines or other effector molecules.
- ELISpot Kits: Pre-coated PVDF plate(s), Detection antibody, Alkaline Phosphatase conjugate, BSA and BCIP/NBT ready- to-use substrate buffer.
- ELISpot Sets: Capture and Detection antibody, Alkaline Phosphatase conjugate, BSA and BCIP/NBT ready-to-use substrate buffer.
- ELISpot Pair: Capture and Detection Antibodies.
- Dual ELISpot Sets: Capture and Detection antibodies for two analytes, Alkaline phosphatase conjugate, blocking reagent, Ready-to-use BCIP/NBT substrate buffer, Peroxydase conjugate, BSA, Ready-to-use AEC substrate buffer.
- Dual Fluorospot Sets: Capture antibody for two analytes, FITC-conjugated Detection antibody for cytokine 1, anti-FITC antibody green fluorescence conjugate, biotinylated detection antibody for cytokine 2, streptavidin-phycoerythrin conjugate, BSA.
Valuable Investigative Tool
ELISpot techniques are amongst the most-sensitive methods available (up to 400x more sensitive than conventional ELISA) for cytokine research and benefit from a technically easy performance, rapid detection time and no requirement for expensive equipment or analysis software.
- Highly Sensitive assay can allow the detection of a single cell out of 100,000
- Fast procedure following cell stimulation
- Accuracy and reliability are guaranteed as all our reagents have been validated according the ISO 9001:2000 quality systems
- Reagents generate well focused, defined and easy to analyse ‘spots’
- No cross reactivity with other human cytokines tested
- Extensive kit range across different species with high specificity and affinity for a number of different cytokines and soluble molecules
- Mono and Dual cytokine analysis available using both enzymatic and fluorescent detection systems
- ELISpot reagents available in a number of formats from whole pre-coated kits for easy analysis and increased throughput to matched Antibody Pairs a great tool for developing research
The cell adhesion molecules (CAMs) family includes more than 50 proteins with four main groups: immunoglobulin (Ig)-like CAMs, cadherins, selectins, and integrins.
Many cellular functions are directly linked to cell adhesion such as signal transduction, cellular communication and recognition, embryogenesis, inflammatory and immune responses, apoptosis and some of them also act as viral receptors (Cohen MB, Am J Clin Pathol. 1997, 107(1):56-63).
The metastatic dissemination of tumor cells is the leading cause of morbidity and mortality in patients with cancer since it designates the transition from a localized, potentially curable to a generalized, usually incurable disease (Makrilia N, Cancer Invest. 2009, 27(10)).
Across the years, it has become evident that the adhesion properties of neoplastic cells play a pivotal role in the development and progression of cancer. (Okegawa T, Acta Biochim Pol. 2004;51(2):445-57)
(Windisch R, Cancers 2019, 11(3), 311)
Changes in the expression or function of CAMs have been associated with alterations in the adhesive or signalling status of tumor cells, allowing them to acquire a more motile and invasive phenotype prognostic biomarkers or as potential therapeutic targets in malignancies.
Additionally, many of CAMs can be cleaved and released by proteolytic cleavage activity, and their soluble forms were found to be increased in serum levels of cancer patients. Even if elevated levels of soluble CAM are also observed in bacterial and viral infections or in acute inflammation, some of them have been identified to be interesting prognostic markers of cancer progression, such as EpCAM, described to be upregulated in colorectal cancer with clinical relevance (Han S, Ebiomedicine 2017; 20:61–69).
Diaclone has been interested for many years in the adhesion molecules and can provide antibodies against all of the selectin and integrin families, most of IgSF CAM family and against EpCAM, H-CAM, M-CAM, and BL-CAM.
Knowing that the soluble form levels could become an innovative tool of cancer monitoring, Diaclone has also developed ELISA kits for measuring serum levels of a wide range of sCAMs.
Diaclone continues its implication in innovative health technology with its participation in the development of IL-1RAP CAR T cells (Warda W Cancer Res February 1 2019 79 (3) 663-675) and in a CD123 CAR T cells project (both in collaboration with the INSERM U1098 research team in Besançon, France).
Diaclone is renowned as a specialist in antibody development - in particular CD markers and membrane expressed proteins. Diaclone’s competence in cell biology and antibody development coupled with its new molecular biology platform and the antibody engineering capabilities of the mAbexperts team (www.mabexperts.com) can provide:
A case study:
Diaclone, with over 30 years of experience and expertise in immunology products, offers a vast catalogue of mAbs and ELISA kits for use in research and diagnostic applications. A large number of Diaclone’s products are cytokine related and specifically Interleukins (IL).
The development of the anti-IL-8 antibody was initiated in 1991, according to Köhler and Milstein’s technique (Nature-1975, 256, p495-7); six fusions were carried out giving only one specific anti-IL-8 mAb, the clone B-K8, which is still used in Diaclone’s IL-8 ELISA kit paired with a rabbit polyclonal. The aim of this project was to replace the polyclonal.
At the beginning of 2019, a new project of anti-IL-8 mAb development was initiated with a new IL-8 mouse immunization. One part of the cells from the immunized animals was unsuccessfully fused using the classical mAb method and the second was processed by the Diaclone Phage Display Platform. The phage display method (Smith, Science-1985, vol. 228, n°4705, p1315-17) is an alternative way of developing mAbs using molecular biology. The platform is capable of obtaining a strong Fab library of IgG sequences from spleen cells coming from immunized mice.
The library obtained was screened in biopanning steps with IL-8 to select very specific clones. One hundred selected Fabs were tested in ELISA to keep the best candidates. Ten of them were selected and evaluated in pairs with B-K8. Finally, we obtained four good substitutes to the polyclonal anti-IL-8. These four Fabs were sequenced and reformatted by molecular biology in full IgG to be further validated in the IL-8 Elisa kit. As a result of this project, phage display technology has demonstrated its interest, notably in allowing mAb development which was particularly difficult to obtain with a classical fusion.
Case Study Poster available
It has been demonstrated that T helper cells can differentiate into IL-17 producing cells independent of Th1 or Th2 pathways establishing Th17 cells as a unique T-cell lineage challenging the classical two pathway theory.
This new T helper cell lineage is defined by its ability to produce IL-17A, IL-17F and IL-17A/F.
Diaclone has developed a large range of ELISA and ELISpot kits for the detection of Human IL-17A, IL-17F and IL-17A/F.
Read more about this product range