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CAR T Cell Therapy Cytokine Response—ELISA Kits and Multiplex Immunoassays |
Thermo Fisher Scientific offers a vast variety of ELISA and multiplex immunoassays for researching the various facets of cellular therapy research. These sensitive and accurate assays can characterize soluble markers involved with CAR T therapy; including efficacy, potency, and monitoring of T cells.
Cellular immunotherapy or adoptive cell therapy (ACT) involves the use of autologous (self) or allogenic (donor) cells infused into patients for the treatment of cancers. Immune cells that have been utilized or are currently being investigated for adoptive transfer include tumor-infiltrating lymphocytes (TILs), engineered T cell receptor cells (TCR) or chimeric antigen receptor (CAR) T cells, regulatory T cells (Tregs), and other cells such as natural killer (NK) cells. The workflow below highlights five-step process involving CAR generation, functional validation of molecular properties, and testing their efficacy in a variety of mouse models (Figure 1).
Figure 1. CAR T cell workflow.
Learn more about CAR T Cell Therapy Research
See the CAR T Cell and Other Adoptive Cell Therapy Workflows
Cytokines play an important role in CAR T cell therapy, as they are required for in vitro expansion and persistence in delivered T cell therapeutic doses. By incorporating cytokine genes into the vectors that encode CARs during manufacturing, further optimization is achieved. In one such case, the use of IL-2 to improve the effectiveness of autologous TIL therapy in the treatment of metastatic malignant melanoma has been documented [1]. The importance of cytokines in enhancing adoptive cell therapy is also demonstrated by the fact that lymphopenia-induced elevation of homeostatic cytokines, such as IL-7 and IL-15, are key to proliferation and survival of adoptively transferred T cells [2, 3].
Understanding cytokine profiles in context of the tissue microenvironment is also a critical factor to developing effective cellular immunotherapies for solid tumors. In one study, the development of an inverted cytokine receptor (ICR) demonstrated the enhancement of the antitumor potency of T cells in the presence of a rich IL-4 immunosuppressive environment. This type of amplified immune response has also been reported in CAR T cells that secrete IL-12 or IL-18, which help increase half-life of the therapies along with modulating the tumor microenvironments [4, 5, 6].
Cellular immunotherapy can lead to adverse events such as cytokine release syndrome (CRS) or cytokine storms, cardiac and neuro toxicities, and hypotension. These adverse events can be triggered by the adoptively transferred cells and/or other host cells in response to the therapy and is mediated through the release of cytokines. Therefore, the evaluation of serum biomarkers such as pro-inflammatory cytokines in response to cellular immunotherapy can help determine the risk for subsequent toxicity and early intervention. Monitoring cytokines and other factors associated with CAR T therapy include those involved in cytotoxic activity, immunomodulation to support antitumor activity and inflammation (Table 1)[7].
For immune microenvironment | |||
---|---|---|---|
Immune checkpoints | Helps mitigate immunosuppression | Enhances cytotoxic function | Cytokine release syndrome |
LAG-3 | Granzyme B | GM-CSF | GM-CSF |
PD-1 | IFN-gamma | IL-6 | IFN-gamma |
TIM-3 | IL-5 | IL-8 | IL-1 alpha |
IL-8 | IL-12 | IL-1 beta | |
IL-17A | IL-15 | IL-2 | |
MIP-1 | Il-18 | IL-6 | |
TNF-alpha | IL-8 | ||
IL-10 | |||
IL-12 | |||
MCP-1 | |||
MIP-1A | |||
TNF-alpha |
Potency tests such as in vitro IFN-γ production and cytotoxicity assays are used to determine whether CAR T cells have the expected therapeutic efficacy and safety profiles. While these individual potency assays provide important information, they can fall short of predicting clinical efficacy and safety. This could be due to the nature of cellular immunotherapeutic products where single potency test may be insufficient to predict complex features and thus necessitates a more comprehensive analysis with a diverse set of assays.
The use of multiplexed immunoassays to measure cytokines provides an indirect but convenient and comprehensive way to test for potency of CAR T cells. Potency assessments can be measured indirectly using ProcartaPlex panels, which used in combination with cytotoxicity assays show that CAR T cells produced higher cytokine levels than untransduced controls after overnight stimulation with CD19-expressing K562 cells (Figure 4, Tab 4, ProcartaPlex multiplex assays).
Along with the overall potency of CAR T therapies, the expression of T cell exhaustion markers is an important variable that can be characterized via immunoassays. Solid tumors have dense microenvironments which can reduce CAR T efficacy, and by monitoring the amount of cytokines released, the overall effect of the therapeutics can be assessed. Several studies report how immunosuppressive cytokines such as TGF-B can rapidly increase the exhaustion of CAR T cells [8]. This type of research is important for designing therapies that deliver the optimal amount of cytokines without adverse effects or premature exhaustion. In one such instance, while IL-2 is noted as a strong inducer for T cell proliferation, it also increases the rate of exhaustion. Substitution with IL-15 or IL-7 showed improved survival in pre-clinical models, which was measured though secretion of cytokines and chemokines using ProcartaPlex multiplex immunoassays [9].
ELISA kits can be used to detect and measure a variety of markers associated with cell therapy research ranging from cytokines for inflammation, to growth factors, to immune checkpoints. These kits can be used with different forms of biological sources, and enable researchers to study critical targets that are involved in cancer treatments using CAR T.
Invitrogen ELISA kits for popular targets such as IL-2, IFN alpha, TNF etc. are listed in Table 2. Comparison of IFN gamma Human ELISA Kit with other commercial alternative is shown in Figure 2.
Search cytokine ELISA kits Search growth factor ELISA kits Search immuno-oncology ELISA kits
Learn more about ELISA kits and components
Figure 2. Representative data using Invitrogen Human IFN-gamma ELISA and competitor ELISA. Human serum samples were tested using Invitrogen IFN gamma Human ELISA Kit, and compared to a competitor ELISA. Data shows a correlation factor of R2=0.9083.
ProQuantum immunoassays are highly sensitive, high performance kits for protein detection that require no specialized instruments. Utilizing proximity-based amplification technology, these assays combine analyte specific high-affinity antibody-antigen binding with signal detection and amplification capabilities of qPCR to achieve a simple yet powerful next-generation protein quantitation platform. ProQuantum assays can be used to detect target protein targets with small sample volumes, which can be beneficial when using limited quantities of samples for cell therapy research.
Invitrogen ProQuantum immunoassay kits for popular targets such as IL-10, TNF alpha, Eotaxin etc. are listed in Table 3. Standard curve of Human IL-2 using IL-2 Human ProQuantum Immunoassay Kit is shown in Figure 3.
Find cytokine ProQuantum assays Find growth factor ProQuantum assays Find cancer ProQuantum assays
Learn more about how the ProQuantum immunoassays work
Read BioProbes Journal article: Introducing ProQuantum High-Sensitivity Immunoassays—The new generation of target-specific protein quantitation
Figure 3. Representative standard curve of Human IL-2. The standard curve for IL-2 using IL-2 Human ProQuantum Immunoassay Kit shows a broad dynamic range (0.0128–5,000 pg/mL) of IL-2 protein.
Invitrogen ProcartaPlex immunoassay panels allow for the simultaneous measurement and tracking of various soluble targets involved in cell therapy research. The potency of donor CAR T cells can be determined by measuring their in vitro cytotoxicity. For example, cytokines produced after overnight stimulation with CD19-expressing K562 cells were detected using the ProcartaPlex Human Cytokine & Chemokine Panel 1A, 34plex(Figure 4). CAR T cells produced higher cytokine signals than untransduced controls, albeit with differences among the three donors’ CAR T cells in expression levels of different markers (Figure 4).
Select one of our preconfigured panels described below (Table 4) or use the Panel Configurator to customize a panel best suited to your CAR T cell therapy research needs including immunity and inflammation, T cell monitoring or regulation, or researching growth factors, chemokines, or cytokines.
ProcartaPlex Panel Configurator
Learn more about ProcartaPlex multiplex immunoassays
Figure 4. Potency assessment of CAR T cells after overnight co-culture with CD19-expressing K562 cells. Indirect cytotoxicity measurement was performed using the ProcartaPlex Human Cytokine & Chemokine Panel 1A, 34plex. The following donors were used in this study: donor A, 39-year-old male (Cat. No. LP101-1/8, Lot. No. A5708, ID No. 9745); donor B, 39-year-old male (Cat. No. LP101-1/4, Lot. No. 3018208, ID No. 12990); donor C, 43-year-old female (Cat. No. LP101-1/4, Lot. No. 3018209, ID No. 12117).
Product Name | Size | Cat. No. |
---|---|---|
ProcartaPlex Human Cytokine/Chemokine/Growth Factor Panel 1, 45plex Target list [bead region]: | 96 tests | EPX450-12171-901 |
ProcartaPlex Human Cytokine & Chemokine Panel 1A, 34Plex Target list [bead region]: | 96 tests | EPX340-12167-901 |
ProcartaPlex Human Immuno-Oncology Checkpoint Panel 1, 14plex Target list [bead region]: | 96 tests | EPX14A-15803-901 |
ProcartaPlex Human Immuno-Oncology Checkpoint Panel 2, 14plex Target list [bead region]: | 96 tests | EPX140-15815-901 |
ProcartaPlex Human Immuno-Oncology Checkpoint Panel 3, 9plex Target list [bead region]: | 96 tests | EPX090-15820-901 |
ProcartaPlex Human Immune Checkpoint Panel, 37plex Target list [bead region]: | 96 tests | EPX370-15846-901 |
ProcartaPlex Human Th1/Th2/Th9/Th17 Cytokine Panel, 18plex Target list [bead region]: | 96 tests | EPX180-12165-901 |
ProcartaPlex Human Th9/Th17/Th22 Cytokine Panel 1, 16plex Target list [bead region]: | 96 tests | EPX160-12175-901 |
QuantiGene RNA gene expression assays provide a fast and high-throughput solution for multiplexed gene expression quantitation, with simultaneous measurement of up to 80 genes of interest in a single well of a 96- or 384-well plate. The QuantiGene Plex assay is based on hybridization and incorporates branched DNA (bDNA) technology, which uses signal amplification rather than target amplification for direct measurement of RNA transcripts. The assay is run on the Luminex platform, has a simple workflow and does not require RNA purification. These features allow the user to merge the QuantiGene workflow for gene expression profiling with the ProcartaPlex workflow for protein quantitation (Figure 5) using the same sample.
Learn more about QuantiGene RNA Assays for Gene Expression Profiling
Figure 5. Combined workflow for QuantiGene gene expression and ProcartaPlex protein quantitation assays.
Cellular immunotherapy or adoptive cell therapy (ACT) involves the use of autologous (self) or allogenic (donor) cells infused into patients for the treatment of cancers. Immune cells that have been utilized or are currently being investigated for adoptive transfer include tumor-infiltrating lymphocytes (TILs), engineered T cell receptor cells (TCR) or chimeric antigen receptor (CAR) T cells, regulatory T cells (Tregs), and other cells such as natural killer (NK) cells. The workflow below highlights five-step process involving CAR generation, functional validation of molecular properties, and testing their efficacy in a variety of mouse models (Figure 1).
Figure 1. CAR T cell workflow.
Learn more about CAR T Cell Therapy Research
See the CAR T Cell and Other Adoptive Cell Therapy Workflows
Cytokines play an important role in CAR T cell therapy, as they are required for in vitro expansion and persistence in delivered T cell therapeutic doses. By incorporating cytokine genes into the vectors that encode CARs during manufacturing, further optimization is achieved. In one such case, the use of IL-2 to improve the effectiveness of autologous TIL therapy in the treatment of metastatic malignant melanoma has been documented [1]. The importance of cytokines in enhancing adoptive cell therapy is also demonstrated by the fact that lymphopenia-induced elevation of homeostatic cytokines, such as IL-7 and IL-15, are key to proliferation and survival of adoptively transferred T cells [2, 3].
Understanding cytokine profiles in context of the tissue microenvironment is also a critical factor to developing effective cellular immunotherapies for solid tumors. In one study, the development of an inverted cytokine receptor (ICR) demonstrated the enhancement of the antitumor potency of T cells in the presence of a rich IL-4 immunosuppressive environment. This type of amplified immune response has also been reported in CAR T cells that secrete IL-12 or IL-18, which help increase half-life of the therapies along with modulating the tumor microenvironments [4, 5, 6].
Cellular immunotherapy can lead to adverse events such as cytokine release syndrome (CRS) or cytokine storms, cardiac and neuro toxicities, and hypotension. These adverse events can be triggered by the adoptively transferred cells and/or other host cells in response to the therapy and is mediated through the release of cytokines. Therefore, the evaluation of serum biomarkers such as pro-inflammatory cytokines in response to cellular immunotherapy can help determine the risk for subsequent toxicity and early intervention. Monitoring cytokines and other factors associated with CAR T therapy include those involved in cytotoxic activity, immunomodulation to support antitumor activity and inflammation (Table 1)[7].
For immune microenvironment | |||
---|---|---|---|
Immune checkpoints | Helps mitigate immunosuppression | Enhances cytotoxic function | Cytokine release syndrome |
LAG-3 | Granzyme B | GM-CSF | GM-CSF |
PD-1 | IFN-gamma | IL-6 | IFN-gamma |
TIM-3 | IL-5 | IL-8 | IL-1 alpha |
IL-8 | IL-12 | IL-1 beta | |
IL-17A | IL-15 | IL-2 | |
MIP-1 | Il-18 | IL-6 | |
TNF-alpha | IL-8 | ||
IL-10 | |||
IL-12 | |||
MCP-1 | |||
MIP-1A | |||
TNF-alpha |
Potency tests such as in vitro IFN-γ production and cytotoxicity assays are used to determine whether CAR T cells have the expected therapeutic efficacy and safety profiles. While these individual potency assays provide important information, they can fall short of predicting clinical efficacy and safety. This could be due to the nature of cellular immunotherapeutic products where single potency test may be insufficient to predict complex features and thus necessitates a more comprehensive analysis with a diverse set of assays.
The use of multiplexed immunoassays to measure cytokines provides an indirect but convenient and comprehensive way to test for potency of CAR T cells. Potency assessments can be measured indirectly using ProcartaPlex panels, which used in combination with cytotoxicity assays show that CAR T cells produced higher cytokine levels than untransduced controls after overnight stimulation with CD19-expressing K562 cells (Figure 4, Tab 4, ProcartaPlex multiplex assays).
Along with the overall potency of CAR T therapies, the expression of T cell exhaustion markers is an important variable that can be characterized via immunoassays. Solid tumors have dense microenvironments which can reduce CAR T efficacy, and by monitoring the amount of cytokines released, the overall effect of the therapeutics can be assessed. Several studies report how immunosuppressive cytokines such as TGF-B can rapidly increase the exhaustion of CAR T cells [8]. This type of research is important for designing therapies that deliver the optimal amount of cytokines without adverse effects or premature exhaustion. In one such instance, while IL-2 is noted as a strong inducer for T cell proliferation, it also increases the rate of exhaustion. Substitution with IL-15 or IL-7 showed improved survival in pre-clinical models, which was measured though secretion of cytokines and chemokines using ProcartaPlex multiplex immunoassays [9].
ELISA kits can be used to detect and measure a variety of markers associated with cell therapy research ranging from cytokines for inflammation, to growth factors, to immune checkpoints. These kits can be used with different forms of biological sources, and enable researchers to study critical targets that are involved in cancer treatments using CAR T.
Invitrogen ELISA kits for popular targets such as IL-2, IFN alpha, TNF etc. are listed in Table 2. Comparison of IFN gamma Human ELISA Kit with other commercial alternative is shown in Figure 2.
Search cytokine ELISA kits Search growth factor ELISA kits Search immuno-oncology ELISA kits
Learn more about ELISA kits and components
Figure 2. Representative data using Invitrogen Human IFN-gamma ELISA and competitor ELISA. Human serum samples were tested using Invitrogen IFN gamma Human ELISA Kit, and compared to a competitor ELISA. Data shows a correlation factor of R2=0.9083.
ProQuantum immunoassays are highly sensitive, high performance kits for protein detection that require no specialized instruments. Utilizing proximity-based amplification technology, these assays combine analyte specific high-affinity antibody-antigen binding with signal detection and amplification capabilities of qPCR to achieve a simple yet powerful next-generation protein quantitation platform. ProQuantum assays can be used to detect target protein targets with small sample volumes, which can be beneficial when using limited quantities of samples for cell therapy research.
Invitrogen ProQuantum immunoassay kits for popular targets such as IL-10, TNF alpha, Eotaxin etc. are listed in Table 3. Standard curve of Human IL-2 using IL-2 Human ProQuantum Immunoassay Kit is shown in Figure 3.
Find cytokine ProQuantum assays Find growth factor ProQuantum assays Find cancer ProQuantum assays
Learn more about how the ProQuantum immunoassays work
Read BioProbes Journal article: Introducing ProQuantum High-Sensitivity Immunoassays—The new generation of target-specific protein quantitation
Figure 3. Representative standard curve of Human IL-2. The standard curve for IL-2 using IL-2 Human ProQuantum Immunoassay Kit shows a broad dynamic range (0.0128–5,000 pg/mL) of IL-2 protein.
Invitrogen ProcartaPlex immunoassay panels allow for the simultaneous measurement and tracking of various soluble targets involved in cell therapy research. The potency of donor CAR T cells can be determined by measuring their in vitro cytotoxicity. For example, cytokines produced after overnight stimulation with CD19-expressing K562 cells were detected using the ProcartaPlex Human Cytokine & Chemokine Panel 1A, 34plex(Figure 4). CAR T cells produced higher cytokine signals than untransduced controls, albeit with differences among the three donors’ CAR T cells in expression levels of different markers (Figure 4).
Select one of our preconfigured panels described below (Table 4) or use the Panel Configurator to customize a panel best suited to your CAR T cell therapy research needs including immunity and inflammation, T cell monitoring or regulation, or researching growth factors, chemokines, or cytokines.
ProcartaPlex Panel Configurator
Learn more about ProcartaPlex multiplex immunoassays
Figure 4. Potency assessment of CAR T cells after overnight co-culture with CD19-expressing K562 cells. Indirect cytotoxicity measurement was performed using the ProcartaPlex Human Cytokine & Chemokine Panel 1A, 34plex. The following donors were used in this study: donor A, 39-year-old male (Cat. No. LP101-1/8, Lot. No. A5708, ID No. 9745); donor B, 39-year-old male (Cat. No. LP101-1/4, Lot. No. 3018208, ID No. 12990); donor C, 43-year-old female (Cat. No. LP101-1/4, Lot. No. 3018209, ID No. 12117).
Product Name | Size | Cat. No. |
---|---|---|
ProcartaPlex Human Cytokine/Chemokine/Growth Factor Panel 1, 45plex Target list [bead region]: | 96 tests | EPX450-12171-901 |
ProcartaPlex Human Cytokine & Chemokine Panel 1A, 34Plex Target list [bead region]: | 96 tests | EPX340-12167-901 |
ProcartaPlex Human Immuno-Oncology Checkpoint Panel 1, 14plex Target list [bead region]: | 96 tests | EPX14A-15803-901 |
ProcartaPlex Human Immuno-Oncology Checkpoint Panel 2, 14plex Target list [bead region]: | 96 tests | EPX140-15815-901 |
ProcartaPlex Human Immuno-Oncology Checkpoint Panel 3, 9plex Target list [bead region]: | 96 tests | EPX090-15820-901 |
ProcartaPlex Human Immune Checkpoint Panel, 37plex Target list [bead region]: | 96 tests | EPX370-15846-901 |
ProcartaPlex Human Th1/Th2/Th9/Th17 Cytokine Panel, 18plex Target list [bead region]: | 96 tests | EPX180-12165-901 |
ProcartaPlex Human Th9/Th17/Th22 Cytokine Panel 1, 16plex Target list [bead region]: | 96 tests | EPX160-12175-901 |
QuantiGene RNA gene expression assays provide a fast and high-throughput solution for multiplexed gene expression quantitation, with simultaneous measurement of up to 80 genes of interest in a single well of a 96- or 384-well plate. The QuantiGene Plex assay is based on hybridization and incorporates branched DNA (bDNA) technology, which uses signal amplification rather than target amplification for direct measurement of RNA transcripts. The assay is run on the Luminex platform, has a simple workflow and does not require RNA purification. These features allow the user to merge the QuantiGene workflow for gene expression profiling with the ProcartaPlex workflow for protein quantitation (Figure 5) using the same sample.
Learn more about QuantiGene RNA Assays for Gene Expression Profiling
Figure 5. Combined workflow for QuantiGene gene expression and ProcartaPlex protein quantitation assays.
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