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Bispecific antibody CD3-CD28 for T cell expansion

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bsAb CD3-CD28

Bispecific antibody against human CD3 and human CD28 for T cell expansion

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100 µg

2 x 100 µg

4 x 100 µg

bsab-tex-1
+-

T cell-activating monoclonal scFv antibody against human CD3 and CD28

T cell activation by Anti-hCD3-CD28
T cell activation by Anti-hCD3-CD28

InvivoGen offers a bispecific antibody (bsAb) that binds to both CD3 and CD28 at the cell surface of human T cells.  It is designed to activate and expand enriched T cell populations or resting T cells from PBMCs.

Description

bsAb CD3-CD28 is a fusion protein dimer comprising tandem single-chain variable fragments (scFv)2 from two monoclonal antibodies (mAbs). These mAbs were derived from the clones OKT3 and 15E8 targeting the human CD3 and CD28 receptors, respectively. It mimics the natural cross-linking of these molecules by APCs in vivo and delivers the necessary signal 1 and signal 2 to the T cells for their activation and expansion.

More details More details

 

This so-called (scFv)2-(scFv)2-Fc molecule belongs to the family of IgG-like antibodies. It features an IgG1 Fc fragment, which contributes to increased solubility, serum half-life, as well as facilitated purification. The ability of bsAb CD3-CD28 to activate and expand enriched CD4+ T cells in the presence of recombinant human IL-2 has been validated by imaging and flow cytometry (see figures).

Key features

  • Strong activation and expansion of enriched T cells and T cells from PBMC
  • Simple, rapid, and cost-effective
  • Increased solubility and serum half-life
  • No additional material, magnetic beads, feeder cells, or medium needed
  • Compatible with transfection or transduction methods

Applications

  • In vitro T cell activation and expansion
  • Cancer immunotherapy studies
  • Understanding T cell responses to bacterial and viral infections
  • Development of CAR-T cells

 

References:

1. Brameshuber, M. et al. 2018. Monomeric TCRs drive T cell antigen recognition. Nat Immunol 19, 487-496.
2. Esensten, J.H. et al. 2016. CD28 Costimulation: From Mechanism to Therapy. Immunity 44, 973-988.

Figures

Activated morphology of human T Cells
Activated morphology of human T Cells

Activated morphology of human T Cells. Morphology of unstimulated (left) and activated human T cells (right) after 48 h of stimulation with bsAb CD3-CD28 in the presence of recombinant human IL-2.

Human T cell expansion
Human T cell expansion

Human T cell expansion. Isolated human T cells were expanded over 25 days with bsAb CD3-CD28 in RPMI medium supplemented with recombinant human IL-2. Viable cells were counted on days 2, 4, 7, 9, 11, 14, and 18 (mean ± SEM in 8 experiments with 2 donors).

 

CD25 & CD69 cell surface staining after T cell activation
CD25 & CD69 cell surface staining after T cell activation

CD25 & CD69 cell surface staining after T cell activation. Isolated human T cells were activated for 48 h using bsAb CD3-CD28 in RPMI medium supplemented with recombinant human IL-2. The negative control T cells were not activated. Cells were fluorescently stained using CD25-APC-Cy7 and CD69-BV 421 and subsequently analyzed using flow cytometry.

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Specifications

Target: Human CD3 (hCD3) and human CD28 (hCD28) 

Clone: OKT3 (Anti-CD3 ) and 15E8 (Anti-CD28) 

Capacity: 100 µg is sufficient to expand up to 1 x 108 T cells

Source: CHO (Chinese hamster ovary) cells 

Formulation: Lyophilized from a 0.2 μm filtered phosphate buffer solution (pH 7.4) containing 5% saccharose  

Purity: Purified by affinity chromatography with >95% purity (SDS-PAGE)  

Quality Control:  

  • The complete sequence of the bispecific antibody has been verified.
  • The absence of bacterial contamination (e.g. lipoproteins and endotoxin) has been confirmed using HEK-Blue™  TLR cellular assays.
  • Biological activity (activation and expansion of T cell populations) has been confirmed using cellular assays.

 

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Contents

bsAb CD3-CD28 is provided azide-free and lyophilized. It is available in three pack sizes:

  • bsab-tex-1: 100 µg
  • bsab-tex-2: 200 µg (2 x 100 µg)
  • bsab-tex-4: 400 µg (4 x 100 µg)

 

Room temperature Product is shipped at room temperature

Store Upon receipt, the product should be stored at -20°C. 

stability Lyophilized products are stable for at least 1 year. Reconstituted antibody is stable for 1 year when aliquoted and stored at -20°C.

Alert Avoid repeated freeze-thaw cycles.

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Details

T cell activation

Critical to almost all functions of the adaptive immune response is the activation of T cells, such as CD8+ cytotoxic T cells and CD4+ helper T cells. The activation of T cells requires two signals:

  • Signal 1: TCR (CD3) engagement with the MHC:peptide complexes on antigen-presenting cells (APCs) [1] and
  • Signal 2: A co-stimulatory signal delivered notably by the interaction between the T cell CD28 receptor and CD80 (B7.1) or CD86 (B7.2) on APCs [2].

Importantly, both signals are required for T cell proliferation, cytokine production (e.g. IL-2, IFN-γ), and cellular functions.

T cell-based immunotherapy is an effective strategy to treat a wide range of cancers. Such therapies rely on the ex-vivo activation and expansion of naturally occurring tumor-specific T cells that are otherwise only retrieved in small numbers from the patient's blood or tissues. Importantly, the inherent T cell immune‑modulating functions need to be preserved. A promising approach for the activation of T cells is the use of bispecific antibodies. These unique molecules bring together the specificities of two antibodies and can bind simultaneously to two separate and unique antigens or epitopes such as CD3 and CD28.

 

References:

1. Brameshuber, M. et al. 2018. Monomeric TCRs drive T cell antigen recognition. Nat Immunol 19, 487-496.
2. Esensten, J.H. et al. 2016. CD28 Costimulation: From Mechanism to Therapy. Immunity 44, 973-988.

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