Immune Monitoring Automation

We don’t make the biomarker . . .

We make the biomarker better !

 

A.  Cell Mediated Immunity

At ImmunoSite Technologies, LLC (IST) GLP-compliant laboratories, automated cell mediated immunity assays are performed within tight specifications yielding precise and accurate findings with a fast turnaround time. These uniquely developed assays ensure that the data collected on clinical trial patient samples are of the highest quality to support pharma investigations. Additionally, these types of assays have successfully been used to follow drug candidates through all phases of clinical trials over periods as long as 18 to 24 months, and may emerge as a companion diagnostic for therapeutic stratification or monitoring.

B. Automated Phenotypic Evaluations

Cellular analysis using polychromatic flow cytometry is a powerful methodology that has been used in academic and industry settings to phenotypically and functionally characterize normal cells, diseased cells and immune cells responding to treatments like vaccines and biologics. With multi-color flow cytometry, ImmunoSite Technologies (IST) scientists use unique combinations of reagents and preparation techniques to obtain a large amount of information from single-cell evaluations. Using a small quantity of cell-containing sample, they are able to apply specially designed combinations of surface and intracellular phenotyping reagents that bind cells in a highly specific manner in order to analyze and monitor immune function.

IST has the expertise and in-house capabilities to design, develop, validate and run polychromatic flow assays to interrogate cell-mediated immune responses using two-to-twelve colors and even higher parameter flow cytometry.

As part of IST’s flow cytometry based services, samples have been run in various clinical trial settings interrogating cellular immune profiles in infectious diseases, cancer, autoimmunity, allergy/asthma, bone marrow and organ transplantation.

Key Players in Immune Responses

Using unique combinations of surface and intracellular phenotyping reagents, IST researchers specialize in the study of immune cells of interest in both the innate and adaptive arms of immune response.

Flow Panels

Representative polychromatic flow panels used at IST for clinical trial evaluations in Multiple Sclerosis

Key cell types in HIV

IST researchers use polychromatic flow panels such as these for phenotypic evaluation in HIV pathogenesis

Automated and Standardized, Functional, Flow Cytometry-Based, Intracellular Cytokine Assays

Cell-based, functional, immune-response assays are gaining acceptance as surrogate markers of efficacy in the development of vaccines and biologics that impact immune responses. Several functional, cellular, immune-monitoring assays are currently in use in academic settings, and to a limited extent, in pharma settings, for the R&D of vaccines and biologics. These include, for example, intracellular cytokine, cytotoxicity, regulatory capacity, cell signaling capacity, proliferative capacity, apoptotic evaluations, and other assays.

However, the translation of these “difficult to run”, tedious assays into routine clinical trial settings requires that they be standardized and ideally, automated, to ensure reproducibility of the assay from operator to operator, site to site and over time.

Attempts have been made by the field to standardize functional, immune-response assays for use in clinical trial settings (Ref 1-4). However, in all cases the assays have been conducted in manual fashion with inter-site CVs still being unacceptably high; routinely greater than 35%.

IST automated solution strategy schematic

Strategy to Creating Complete Automated Solutions at IST

Automated System, Critical Timing Yield Enhanced Precision and Accuracy

For the past five years, the IST team has worked to automate and standardize functional cell assays in order to achieve enhanced precision and accuracy. By precisely automating an assay process, IST researchers have been able to maintain the critical timing steps associated with running functional, immune-response assays. With a completely automated system, including on-board addition of all reagents and antibodies, as well as on board centrifugation and cell washing, IST researchers are routinely accomplishing CVs equal to or less than 30% under all conditions.

The automation of tedious, multi-step, functional assays such as these has resulted in greatly enhanced assay precision. Properly designed and programmed for automation, immune-response assays such as these can be conducted in a hands-off, operator-free, highly consistent process. The results are highly accurate and precise, and reproducible.

The Biomek liquid handler, with proprietary custom modifications on hardware and software, as well as custom reagent combinations developed by IST, has been used for validation of automated functional intracellular cytokine assays.

IST AutoICS System

Biomek is a registered trademark of Beckman Coulter, Inc.


ICS T-Cell Functional Analysis: Cocktails, Samples, Parameters and Steps

AutoICS method outline

AutoICS Method

The following five-color custom antibody cocktails were developed by the IST team to evaluate T helper (TH1/TH2) and T inflammatory (TH17) responses:

  • TH1-CD3/4/8/IFNg/TNFa
  • TH1-CD3/4/8/IFNg/IL-2
  • TH2-CD3/4/8/IL-4/IL-10
  • TH17-CD3/4/8/CCR6/IL-17

The following samples were analyzed:

  • Fresh whole blood stimulated with PMA/Ionomycin or CD3/CD28 or Staphylococcal Enterotoxin B
  • 24 hr whole blood stimulated with PMA/Ionomycin or CD3/CD28 or Staphylococcal Enterotoxin B
  • Freshly isolated Peripheral blood mononuclear cells stimulated with PMA/Ionomycin or CD3/CD28 or Staphylococcal Enterotoxin B
  • Frozen and thawed Peripheral blood mononuclear cells stimulated with PMA/Ionomycin or CD3/CD28 or Staphylococcal Enterotoxin B

Validation parameters evaluated:

  • Accuracy-Correlation to manual gold standard
  • Intra-assay precision
  • Inter-assay precision
  • Specificity using un-stimulated samples as well as isotype controls
  • Linearity
  • Limit of detection

Automated Assay Steps and Timings
Results: Histograms

The following are representative histogram profiles for automated staining of samples for various intracellular cytokines.

IFNgTNFa with AutoICS

Intracellular staining for IFNg and TNFa in CD4 T cells

IL4-IL10 from AutoICS

Intracellular staining for IL-4 and IL-10 in CD4 T cells Intracellular staining of IL-17 in CD4 T cells



Representative Accuracy Results

The following CVs show the correlation of manual, gold-standard, intracellular cytokine assays to automated run assays achieved by the IST team.

The AutoICS System at IST

IST AutoICS SystemThe fully automated intracellular cytokine staining system designed at ImmunoSite Technologies incorporates a liquid handler robot, plate-based centrifuge, automated tube barcode reader, custom reagent racks, and optimized performance reagents.

AutoICS Accuracy- IL2

Intracellular IL2 cytokine detection in stimulated whole blood.  Accuracy of the automated ICS system.

R2 = 0.9258

AutoICS Accuracy– IL4

Intracellular IL4 cytokine detection in stimulated whole blood.  Accuracy of the automated ICS system.

R2 = 0.9462

AutoICS Accuracy– IL17

Intracellular IL17 cytokine detection in stimulated whole blood.  Accuracy of the automated ICS system.

R2 = 0.9169

AutoICS Accuracy– IFNg

Intracellular IFNg cytokine detection in stimulated whole blood.  Accuracy of the automated ICS system.

R2 = 0.9493

AutoICS Accuracy– TNFa

Intracellular TNFa cytokine detection in stimulated whole blood.  Accuracy of the automated ICS system.

R2 = 0.9719

Assay Performance Ranges

AutoICS Performance Criteria

Compiled data for automated platform interrogating TH1/TH2/TH17 responses

Compiled data from validation studies performed with the automated intracellular cytokine staining (AutoICS) platform at ImmunoSite technologies.  The three assay performance ranges shown here are for TH1, TH2, and TH17.

IST has proven unique in its capability to develop and validate a completely automated, hands-off platform capable of running intracellular cytokine assays with accuracies that mirror manual gold standard results. IST developed assays deliver improved precisions of 30-50% over manually run assays. As a result, the IST team can double the throughput of manually run assays while maintaining critical timing and precision.

References:

Ref 1: Genetic Engineering and Biotechnology News Feb 1 2010 (Vol. 30, No. 3) Cryopreserved PBMCs and Cell-Based Assays-Preserving Fundamental Characteristics for Immune Monitoring and Drug Development

Ref 2: http://www.beckmancoulter.com/literature/Bioresearch/P-10201A.pdf

Ref 3: BMC Immunology 2005, 6:13doi:10.1186/1471-2172-6-13 Standardization of cytokine flow cytometry assays

Ref 4: Nature Protocols: 10.1038/nprot.2007.65The Journal of Immunology, 2007, 179, 2627 -2633 Functional T Cell Responses to Tumor Antigens in Breast Cancer Patients Have a Distinct Phenotype and Cytokine Signature
Cytotoxicity: A Window Into Cellular Immune Function
Cytotoxic effector lymphocytes (CTL) are the primary cells of the acquired immune response for defense against viral infections and tumors. The accurate analysis of such an effector population is critical to understanding the correlates of protection for diseases involving cellular immunity. Once validated, these signatures can be routinely used as surrogate markers of efficacy in preventative and therapeutic strategies involving immune response.

At IST, multiparametric flow cytometry has enabled the accurate identification and evaluation of such finely targeted lymphocyte subpopulations. IST research teams have evaluated the functional capacity of effector T cells using 5-color, 7-paramater flow cytometry by interrogating a combination of phenotypic and functional surface and intracellular markers.

In one published study, for example, the IST team obtained samples of peripheral blood mononuclear cells from healthy donors. The samples were subjected to restricted polyclonal stimulation using Staphylococcal enteretoxin B, or peptide specific stimulation, for varying times ranging from 6 to 72 hours. The T Cell populations were then analyzed using combinations of markers differentiating naïve, effector, memory, activated and proliferating subpopulations. In addition, functional evaluation of the cells was analyzed utilizing intracellular cytokine, granzyme B, perforin and degranulation, as assessed by activation-induced CD 107 expression.

The IST team was able to find that there is a complex profile of effector T Cells that varies with the donor and time post stimulation. The functional capacity of effector T Cells is especially dependent on the “intrinsic” state of the donor PBMC population with granzyme B upregulation being the most striking feature of the response. Such profiles, when correlated with disease outcome, could enable the targeted identification of effector subpopulations associated with therapeutic success, thus leading to the development of “surrogate profiles” of efficacy.
Functional Polyspectral Flow Assays
Functional flow cytometry based assays routinely performed by IST include:

  • Intracellular Cytokines
  • TH1: IL2/IFNg/TNFa
  • TH2: IL-4/IL10
  • TH17: IL17/IL-21
  • DC1/DC2: IFNa/IL-12
  • Cytotoxicity
  • Proliferation
  • Cell Signaling Assays
  • Functional Treg Cell Assays
  • Mixed Lymphocyte Reactions

C.  ELISPOT Assay: Detection of Antigen-Specific Cells

Elispot Method Graphic

Cytokine Elispot Assay Method

Evaluation of functional capacity of immune cells is critical to understanding several disease conditions and the efficacy of therapeutic strategies directed towards them. Immune cells are either responsible for prevention/therapy, for example in infectious diseases like HIV; or responsible for the disease condition itself, for example, autoimmune disease conditions. There are several assays that have been used for functional interrogation of immune cells of interest in these diseases. The ELISPOT is one such assay.

ELISPOT is a sensitive technique that allows detection of low frequency antigen specific cells in fresh and cryopreserved PBMCs. This study has been carried out for qualification of the ELISPOT assay in the context of interrogating the frequency of autoimmune T cell responses in cryopreserved PBMCs to myelin antigens.
Qualification of Manual ELISPOT Assay for Antigen Specific Reactivity
ELISPOT is an accepted technique used by IST researchers to detect immune cells and their frequency. Due to the high sensitivity of the assay, analyses of rare cell populations have been successfully carried out, including antigen-specific cells.

A typical IST research project was designed to evaluate and qualify the response of cryopreserved PBMCs to specific antigens of interest for potential downstream use in clinical trial settings. The response was assessed as IFNg secretion detected ELISPOT.
Experimental Outline and Representative Results:

  • Polyclonal activation is determined using CD3/CD28 stimulation
  • Antigen specific activation is determined using tetanus peptides
  • Auto antigen specific reactivity is determined using 2 doses of myelin peptides for stimulation in patients with Multiple Sclerosis

ELISPOT Intra-Assay and Inter-Assay Precision

Elispot precision data IFNg

Intra-Assay and Inter-Assay Precision of IFNg Cytokine Elispot


ELISPOT Assay scanned plate profile of positive responding donor for antigen specific and polyclonal stimulation run on two separate occasions to evaluate intra-assay and inter-assay precision.

Elispot intra-assay precision IFNg

Intra-Assay Precision of IFNg Elispot Assay

Elispot inter-assay precision IFNg

Inter-Assay Precision of IFNg Elispot Assay

ELISPOT Assay Conclusions

Percent CV values ranged from 12% to 47% for intra and inter assay evaluations. These data provide target values for precision of a manual ELISPOT assay for low responders.

The automation of the various stages of the ELISPOT assay will benefit greatly enabling standardization of the assay, thus improving its utility as a relevant functional cell assay for interrogating immune cell activity in the context of disease and therapy.
EIA Cell-Free Responses
EIA assays routinely performed by IST include:

  • Multiplexed Immunoassays
  • Neutralizing Antibody Assays

D.  IST Custom Designed Assay Development
As part its cell mediated immune phenotyping services, IST offers custom design services that range from design, development, verification and validation of multicolor phenotyping cocktails to development of a relevant quality control suite of reagents. IST is also able to develop and standardize assay protocols for the sample and/or conditions of interest.

Typical IST cell assay development steps are outlined in the following illustration. The timing of each step varies depending upon parameters, but IST is poised to complete the steps and entire process in highly accelerated time frames to meet critical client needs.

IST functional assay design process

Schematic of the IST process of designing new functional assays