Il-2 receptor engagement can impact the growth and phenotype of cultured immune cells such as T, NK, or Tumor Infiltrating T cells. By engaging the beta receptor (CD122) with higher affinity, continued proliferation of T cells has been observed. Utilizing generative AI to develop an optimized IL-2 cytokine with enhanced beta receptor (CD122) engagement offers superior performance while maintaining greater than 95% homology to its wild-type counterpart including:
Enhanced CD122 Binding: Increased affinity to the IL-2 beta receptor (CD122) with decreased affinity to IL-2 alpha receptor (CD25), allowing proliferation of effector T cells
allowing proliferation of effector T cells
Improved Output: Maintains growth of CD8+/CD4+ T cells for workflows aimed at extending proliferation of T cells.
Sustained Phenotype: Improved proliferation capacity without increased exhaustion nor more terminally differentiated T cells.
Product Specifications for Recombinant Human IL-2 (CD122-Directed) Protein, CF
Source
E. coli-derived human IL-2 protein Ala21-Thr153 (C145S) with and without an N-terminal Met Proprietary point mutations
Endotoxin level
<0.20 EU per 1 μg of the protein by the LAL method.
N-terminal sequence Analysis
Ala21 & Met-Ala21
Predicted Molecular Mass
15 kDa
SDS-PAGE
15 kDa
Activity
Measured in a cell proliferation assay using NK-92 human natural killer lymphoma cells. The ED50 for this effect is 0.0500-0.500 ng/mL.
Scientific Data Examples for Recombinant Human IL-2 (CD122-Directed) Protein, CF
IL-2 (CD122-Directed) Provides Increased and Sustained CD4+/CD8+ T Cell Proliferation in Side-by-Side Testing with the Standard IL-2 Protein
CD4+ and CD8+ T cells from two different donors were activated with anti-CD3-, anti-CD28-conjugated beads, and grown for 28 days in media containing either 3.6 or 14.3 ng/mL of Animal-free Recombinant Human IL-2 Protein (BT-002-AFL) as the standard IL-2 protein or engineered Recombinant Human IL-2 (CD122-Directed) Protein (BT-002DBR), refreshed every 2-3 days. The average fold expansion of the cells was determined every 2-3 days. The IL-2 (CD122-Directed) Protein supports greater than 6x more CD4+/CD8+ T cell growth than the standard IL-2 protein.
T Cells Expanded with the IL-2 (CD122-Directed) Protein Display Enhanced Expression of Naive Phenotypic Markers and Reduced Expression of T Cell Exhaustion Markers Compared to T Cells Expanded with the Standard IL-2 Protein
CD4+ and CD8+ T cells from three different donors were activated with anti-CD3-, anti-CD28-conjugated beads, and grown in media containing 50 or 200 IU of Animal-free Recombinant Human IL-2 Protein (BT-002-AFL) as the standard IL-2 protein or Recombinant Human IL-2 (CD122-Directed) Protein (BT-002DBR), refreshed every 2-3 days. A) After 28 days, the phenotypes of the cells were compared by flow cytometry using a Brilliant Violet 711™ anti-human CD197 (CCR7) antibody and PE/Dazzle™ 594 anti-human CD45RA antibody to determine the number of CD4+ and CD8+ naive (CD45RA+/CCR7+) Tscm cells. The percentage of Tscm cells was found to be higher when the cells were expanded with the IL-2 (CD122-Directed) Protein at 14.3 ng/mL. B) The number of cells expressing the T cell exhaustion markers, PD-1, LAG-3, and TIM-3 was determined by flow cytometry. The results showed that the percentages of CD4+/CD8+ T cells that were also PD-1, LAG-3, or TIM-3 positive were equivalent or reduced with the IL-2 (CD122-Directed) Protein. These results demonstrate that the IL-2 (CD122-Directed) Protein promotes increased T cell expansion with enhanced expression of naive phenotype markers and equivalent or reduced expression of cell exhaustion markers.
Affinity Measurements and Binding Kinetics of the Interaction between IL-2 R beta and the IL-2 (CD122-Directed) Protein by SPR
Recombinant Human IL 2 R beta Fc Protein (10919-2B) was captured on a Sensor Chip Protein AGL, and binding to Recombinant Human IL-2 (CD122-Directed) Protein (BT-002DBR) was measured at a concentration range between 0.097nM and 200 nM. The double-referenced sensorgram was fit to a 1:1 binding model to determine the binding kinetics and affinity, with an affinity constant of Kd=49.3 nM. Note: The IL-2 (CD122-Directed) Protein was modeled to engage only the IL-2 dimeric beta gamma receptor complex. In comparison, wild type recombinant human IL-2 (BT-002) affinity to Recombinant Human IL 2 R beta Fc Chimera (10919-2B) has a binding constant Kd=544 nm.
Recombinant Human IL-2 (CD122-Directed) Protein Bioactivity.
Recombinant Human IL-2 (CD122-Directed) Protein (Catalog # BT-002DBR) stimulates proliferation of NK-92 human natural killer lymphoma cells. The ED50 for this effect is 0.0500-0.500 ng/mL.
Recombinant Human IL-2 (CD122-Directed) Protein SDS-PAGE.
2 μg/lane of Recombinant Human IL-2 (CD122-Directed) Protein (Catalog # BT-002DBR) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 12‑14 kDa, under reducing conditions.
Formulation, Preparation and Storage
Carrier Free
What does CF mean?
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our
recombinant proteins.
Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant
protein to be stored at a more dilute concentration.
The carrier free version does not contain BSA.
What formulation is right for me?
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or
as an ELISA standard.
In contrast, the carrier free protein is recommended for applications, in which the presence of BSA
could interfere.
BT-002DBR
Formulation
Lyophilized from a 0.2 μm filtered solution in Sodium Acetate with Trehalose.
Reconstitution
Reconstitute the 10 μg size at 100 μg/mL in water. Reconstitute all the other sizes at 500 μg/mL in water.
Shipping
The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage:
Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
12 months from date of receipt, -20 to -70 °C as supplied.
1 month, 2 to 8 °C under sterile conditions after reconstitution.
3 months, -20 to -70 °C under sterile conditions after reconstitution.
Reconstitution Calculator
Background: IL-2
CD122 directed IL-2 has a 7-fold increased binding affinity to the human IL-2 beta receptor while exhibiting significantly reduced affinity for the IL-2 alpha receptor. According to research by Levin (2012), this configuration results in more effective expansion of effector cells and less Treg (1). Our findings show that CD122 directed IL-2 improves immune cell expansion for workflows requiring significant amounts of IL-2. One concern with improving T cell expansion is the possibility of increasing the number of terminally differentiated and exhausted T cells with reduced anti-tumor activity. However, our studies have shown that CD122 directed IL-2 significantly improves T cell expansion without changing T cell phenotype or the expression of exhaustion markers.
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References
Levin, A., Bates, D., Ring, A. et al. (2012) Nature. 484:529.
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