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CD3 Protein - A hot target for bispecific antibody

T cell bispecific antibodies (TCBs) are molecules that are engineered to include binding sites to the T cell receptor (TCR) and to tumor-associated or tumor-specific antigens within a single entity. One of the most widely studied TCBs in cancer immunotherapy is CD3, a molecule that has the function of stabilizing TCR structure and transmitting activation signals.

Antibodies against CD3 molecules can stimulate or block T cell activation signal transduction. In addition, these antibodies can eliminate effector T cells or induce a regulatory phenotype in T cells thus, providing a new method for the treatment of organ transplant rejection and autoimmune diseases. These CD3 molecules bind to TCR non-covalently to form a TCR/CD3 receptor complex on the surface of the T cell.

There are 4 subtypes of CD3, including CD3δ, CD3ε, CD3γ, and CD3ζ. CD3δ/CD3ε and CD3γ/CD3ε can form the TCR-CD3 complexes through the α/β chain of TCR as heterodimers. CD3-targeted therapeutic antibody usually recognizes the CD3ε in the heterodimer complex to activate the anti-tumor activity of T cells. However, in the process of recombinant expression, CD3ε and CD3δ can randomly form incorrect heterogeneous complexes, which may lead to the loss of bioactivity and high batch-to-batch inconsistency.

To accelerate the research and development of bispecific antibodies, ACROBiosystems has developed a series of homogeneous CD3δ/CD3ε and CD3γ/CD3ε proteins with high bioactivity. These proteins are verified as a 1:1 heterodimer by nonreductive electrophoresis and MALS respectively. In addition, customer data suggests that bispecific antibody products from ACROBiosystems can be used in applications including, but not limited to clinical pharmacokinetics, therapeutic antibody screening, identification, and characterization.

Successively, our superior CD3 product quality helped accelerate the clinical development of a bispecific antibody into a therapeutic. Click here to download CD3 poster.

Product Features

Larger product catalog

5 molecules: CD3ε& CD3δ, CD3ε& CD3γ, CD3ε, CD3δ, CD3γ

Various tags: His, His&Avi, Fc, Llama Fc, Flag

Various species: Human, Mouse, Cynomolgus, Rat, Rabbit

Homogeneous structure: CD3ε& CD3δ and CD3ε& CD3γ are verified as 1:1 heterodimers by non-reductive electrophoresis and MALS

High bioactivity verified by binding with common antibodies such as OKT3, SP34-2, UCHT1 and BCMA×CD3

High batch-to-batch consistency guaranteed by strict quality control

Suitable for different applications including antibody immunotiter detection, biopanning, antibody screening, affinity characterization and clinical pharmacokinetic assay

Product List

TCR-CD3 Complex
CD3E & CD3D
CD3E & CD3G
CD3 epsilon
CD3 delta
CD3 gamma
Bridging ELISA Kits

Bioanalytical assay development service

CD3-targeted bispecific and trispecific antibodies represent a fast-growing field in therapeutic antibody development. However, selecting the appropriate detection reagents for custom bioanalytical assays such as ELISAs is challenging. With an abundant selection of CD3 proteins including labels and tags, companies may struggle in developing a highly sensitive and specific assay for their drug therapy.

Here at ACROBiosystems, we can provide a large product catalog of highly bioactive CD3δ/CD3ε, CD3γ/CD3ε products as well as our expertise and our ISO17025-accedited analytical service platform. Using our analytical service platform, we offer customized services from paired protein screening to ELISA-based assay release method development, validation, and kit development to help accelerate your R&D process!

Case Studies

Bioanalytical assay development service

Immobilized Human CD3E&CD3D Heterodimer Protein, Fc Tag&Fc Tag (Cat. No. CDD-H5255) at 1 μg/mL (100 μL/well) was spiked with different concentrations of bispecific BCMA×CD3 T cell-engaging Antibody. The identified linear range was found to be 0.8-6 ng/mL.


Bioanalytical assay development service

Immobilized Human BCMA, His Tag (Cat. No. BCA-H522y) was bound to a 96-well plate. Increasing concentrations of bispecific T cell Engager (CD3 X BCMA) spiked in 10% human serum and Biotinylated Human CD3E&3D Heterodimer Protein, His,Avitag&Tag Free (Cat. No. CDD-H82W6) at 0.2 μg/mL was added to each well. Detection was performed using HRP-conjugated streptavidin with sensitivity of 15 ng/mL.

Flexible assay designs and report formats for your personalized needs
ACRO proteins are supplied for free
Only samples are required for service completion

Inquiry and order

  • ACROBiosystems scientist will respond within 24 hours of submission.

  • Call us at: +1 800-810-0816 or email at services@acrobiosystems.com for consultation.

Data

1:1 heterodimer verified by nonreductive electrophoresis and MALS

ACROBiosystems has developed a series of homogeneous CD3 proteins with good bioactivity, which are verified as 1:1 heterodimer by nonreductive electrophoresis and MALS.


Fig.1 Human CD3E&CD3D Heterodimer Protein (Cat. No. CDD-H52Wa) on SDS-PAGE under reducing (R) and non-reducing(NR)condition and the purity of the protein is greater than 95%. The purity of the protein is more than 85% and around 80-90 kDa verified by SEC-MALS.

High binding ability and low batch-to-batch differences

These verified authentic 1:1 heterodimer proteins show high bioactivity in different applications. The products are homogenous with strict quality control to guarantee the minimal batch-to-batch differences.


Fig.2 Immobilized Biotinylated Human CD3E&CD3D Heterodimer Protein, His,Avitag&Tag Free (Cat. No. CDD-H82W6) at 1 μg/mL (100 μL/well) on Streptavidin (Cat. No. STN-N5116) precoated (0.5 μg/well) plate, can bind Monoclonal Anti-Human CD3 Antibody, Mouse IgG2a (Cat. No. CDE-M120a) with a linear range of 0.2-6 ng/mL (QC tested), and batch differences EC50<0.0001 μg/mL.

Designed for different applications of drug development

These products are suitable for various applications at different stages of drug development, such as antibody immunotiter detection, biopanning, antibody screening, affinity characterization and clinical pharmacokinetic assays.

Application 1: Early Bispecific Antibody Discovery & Molecular Screening
Early Bispecific Antibody Discovery & Molecular Screening

Immobilized Human BCMA, Mouse IgG2a Fc Tag (Cat. No. BCA-H5253) at 1 μg/mL, add increasing concentrations of Bispecific T cell Engager (CD3×BCMA) and then add Biotinylated Human CD3 epsilon, His,Avitag (Cat. No. CDE-H82E1) at 0.2 μg/mL. Detection was performed using HRP-conjugated Streptavidin (Cat. No. STN-NH913) with sensitivity of 6.23 ng/mL (Routinely tested).

Early Bispecific Antibody Discovery & Molecular Screening

Bispecific T-cell Engager (CD3×DLL3) captured on Protein A Chip can bind Human CD3 epsilon, His Tag (Cat. No. CDE-H5223) with an affinity constant of 0.843 nM as determined in a SPR assay (Biacore 8K).


Application 2: Drug Process Development & Manufacturing QC
Drug Process Development & Manufacturing QC

Immobilized Human CD20 Full Length Protein, His Tag (Cat. No. CD0-H52H3) at 2 μg/mL, add increasing concentrations of Glofitamab and then add HRP-Human CD3E&CD3D Heterodimer Protein, His Tag&Tag Free (Cat. No. CDD-HR2W3) at 1 μg/mL. Detection was performed using HRP-conjugated Streptavidin (Cat. No. STN-NH913) with sensitivity of 40.30 ng/mL (Routinely tested).

Drug Process Development & Manufacturing QC

Immobilized Human CD20 Full Length Protein, His Tag (Cat. No. CD0-H52H3) at 2 μg/mL, add increasing concentrations of Glofitamab and then add Biotinylated Human CD3 epsilon, His,Avitag (Cat. No. CDE-H82E1) at 0.2 μg/mL. Detection was performed using HRP-conjugated Streptavidin (Acro, Cat. No. STN-NH913) with sensitivity of 41.71 ng/mL (Routinely tested).

Drug Process Development & Manufacturing QC

Immobilized Human BCMA, Mouse IgG2a Fc Tag (Cat. No. BCA-H5253) at 1 μg/mL, add increasing concentrations of Teclistamab and then add Biotinylated Human CD3E&CD3G Heterodimer Protein, His,Avitag&Tag Free (Cat. No. CDG-H82W3) at 1 μg/mL. Detection was performed using HRP-conjugated Streptavidin (Cat. No. STN-NH913) with sensitivity of 20.12 ng/mL (Routinely tested).

Drug Process Development & Manufacturing QC

Immobilized Human DLL3, His Tag (Cat. No. DL3-H52H4) at 0.2 μg/mL, add increasing concentrations of Tarlatamab and then add Biotinylated Human CD3 epsilon, His,Avitag (Cat. No. CDE-H82E1) at 0.2 μg/mL. Detection was performed using HRP-conjugated Streptavidin (Cat. No. STN-NH913) with sensitivity of 8.21 ng/mL (Routinely tested).

Application 3: Clinical Pharmacokinetics (PK) Analysis
Clinical Pharmacokinetics (PK) Analysis

Immobilized Human BCMA, His Tag (Cat. No. BCA-H522y) at 2 μg/mL, add increasing concentrations of Bispecific T cell Engager (CD3×BCMA) in 10% human serum and then add Biotinylated Human CD3E&CD3D Heterodimer Protein, His,Avitag&Tag Free (Cat. No. CDD-H82W6) at 0.2 μg/mL. Detection was performed using HRP-conjugated streptavidin with sensitivity of 15 ng/mL.

Total Solutions for the Development of Bispecific Antibodies

FAQ

Q

How is the high biological activity of the recombinant CD3 proteins demonstrated? What representative antibodies are used by ACROBiosystems as positive controls?

The biological activity of CD3 recombinant proteins is validated through their binding capability to well-characterized CD3 antibodies. ACROBiosystems uses classic CD3 antibodies such as OKT3, SP34-2, and UCHT1, as well as BCMA×CD3 bispecific antibodies, for binding activity verification. These reference antibodies cover different epitope specificities, enabling a comprehensive assessment of the correct conformation and functional activity of the CD3 recombinant proteins. This provides R&D personnel with highly reliable quality assurance for immune titer determination and blocking assays. Notably, recent studies in 2026 have shown that novel high-affinity CD3 antibodies (e.g., 4B1) exhibit even superior affinity to the TCR-CD3 complex compared to the classic OKT3 antibody, further highlighting the critical role of highquality CD3 recombinant proteins in the screening of next-generation antibodies.
Q

What is CD3 protein, and what role does it play in T-cell activation?

CD3 is the signaling component of the T-cell receptor complex. It does not directly recognize antigens but transmits activation signals into T cells after the T-cell receptor binds an antigen. The TCR-CD3 complex is expressed on the surface of T lymphocytes and contains CD3 epsilon, CD3 delta, CD3 gamma and CD3 zeta subunits. These subunits associate with the antigen-recognizing TCR chains and initiate intracellular signaling required for T-cell development, activation and adaptive immune responses.
Q

What measures are implemented to ensure the lot-to-lot consistency of CD3 proteins during long-term, multi-phase drug discovery campaigns?

Long-term biologics discovery programs require reagents that deliver consistent performance across multiple stages of drug development.ACROBiosystems enforces stringent lot-to-lot quality baselines. Each production batch undergoes comprehensive analytical characterization, including SEC-MALS to verify the expected molecular mass and oligomeric state, and ELISA / SPR-based binding analysis to confirm consistent binding activity relative to qualified reference standards.Furthermore, our formulation development includes optimized cryoprotectants that maintain structural integrity and biological activity across multiple freeze-thaw cycles. These quality measures help minimize lot-related assay variability and improve data comparability throughout long-term screening and characterization campaigns.
Q

How can I mitigate the risk of tag interference or steric hindrance when immobilizing CD3 proteins in SPR/BLI kinetic assays?

Random chemical biotinylation may modify lysine residues within or near antibody-binding epitopes on the CD3 complex, potentially affecting binding activity or introducing assay variability. To prevent steric hindrance, we recommend utilizing AviTag™ site-specific biotinylated CD3 heterodimers. This enzymatic biotinylation occurs exclusively at a single lysine residue within the specific peptide tag, far removed from the native extracellular domains of CD3ε/δ. This ensures uniform orientation upon streptavidin sensor chip immobilization, maximizing active binding sites and yielding highly reproducible kinetic data. Alternatively, Tag-Free CD3 heterodimers can be paired with immobilized therapeutic candidates to evaluate binding dynamics without any structural artifact interference.
Q

How do I choose between human and cynomolgus CD3 proteins when validating cross-species reactivity for bispecific T-cell engagers (TCEs)?

For translational studies, evaluating cross-reactivity is critical as many anti-CD3 clones (e.g., SP34-2) bind both human and non-human primate (NHP) CD3 complexes, while others (e.g., OKT3) are human-specific. ACROBiosystems offers both Human and Cynomolgus CD3ε/CD3δ heterodimers designed with identical 1:1 stoichiometry. When executing screening pipelines, pairing these authentic heterodimers enablesthat the affinity kinetics measured via SPR accurately reflect the target molecule's true binding profile across species, minimizing the risk of unexpected toxicity or loss of potency during in vivo NHP safety assessments.
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