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In vitro–differentiated Th1/Th17/Treg cells

CD4+ helper T cells (Th cells) serve as mediators of cellular immunity and play a critical role in activating other immune cells, such as B cells and cytotoxic T cells, as well as in regulating immune responses.

Antibody-Dependent Cell-Mediated Cytotoxicity Assay (ADCC)

Antibodies, as integral components of the immune system, play a crucial role in defending against disease. Antibody-dependent cell-mediated cytotoxicity (ADCC) is one of the mechanisms by which antibodies exert their effector functions: when IgG antibodies specifically bind via their Fab fragments to antigenic epitopes on the surface of target cells—such as virus-infected cells and tumor cells—the Fc portion of the antibody can engage Fc receptors on effector cells, including natural killer (NK) cells, monocytes–macrophages, and neutrophils, thereby triggering the effector cells’ cytotoxic activity and directly killing the target cells. The ability to elicit ADCC against target cells is an important functional criterion for antibody candidates that are directed against cancer-associated antigens.

Antibody-dependent cellular phagocytosis

Antibody-dependent cellular cytotoxicity (ADCC) is one of the mechanisms by which antibody-based therapies exert their antitumor and other therapeutic effects. Currently, therapeutic strategies aimed at enhancing macrophage responses to therapeutic antibodies have garnered significant attention from researchers, including the identification of novel targets and the development of antibodies with enhanced functionality.

Complement-dependent cytotoxicity (CDC)

Complement is a group of heat-labile, enzymatically active proteins found in human and vertebrate serum and tissue fluids, comprising more than 30 soluble and membrane-bound proteins. Complement-dependent cytotoxicity (CDC) refers to the lytic effect on target cells resulting from the formation of a membrane attack complex after complement is activated by specific antibodies that bind to corresponding antigens on the cell membrane via the classical pathway of complement activation. Initially, antibodies bind to complement component C1q, which then triggers the sequential activation of C2 through C9 to form the membrane attack complex, ultimately leading to lysis of the target cell.

Cytokine Release Syndrome Risk Assessment (CRS)

Cytokine release syndrome (CRS) refers to a hyperactive immune response that occurs following infection with pathogenic microorganisms, leading to the rapid activation of numerous immune cells and the massive release of multiple cytokines—including TNF-α, IL-1, IL-6, IL-12, IFN-α, IFN-β, and IFN-γ—within a short period. This results in a severe systemic inflammatory response syndrome. The excessive production of these cytokines can damage tissues and organs, thereby giving rise to a wide range of clinical manifestations. Currently, the standard approach is to closely monitor and target the specific cytokines that trigger the cytokine storm.

Flow Cytometry-Based Cell Characterization Experiments (FACS)

The targets of antibody drugs are primarily disease-associated antigens or specific receptor molecules on the cell surface. Competitive binding between ligands and antibodies is assessed by using flow cytometry to determine the population of antigen-positive cells. By employing antigen-presenting cells in these assays, the spatial conformation of surface antigens more closely resembles their in vivo configuration, thereby yielding results that better reflect physiological conditions.

RCL detection

In CAR-T cell manufacturing, most retroviruses and lentiviruses used for cellular gene therapy are engineered as replication-deficient viral vectors and are co-transfected using multiple plasmids to express the lentivirus or γ-retrovirus. During production, homologous recombination may occur between the deleted vector backbone and wild-type viral sequences, leading to the generation of replication-competent virus (RCL), which poses a serious risk to human health. Therefore, RCL testing is one of the most critical components of safety assessment.

I. Introduction to RCL Testing

In CAR-T cell manufacturing, most retroviruses and lentiviruses used for cellular gene therapy are engineered as replication-deficient viral vectors and are co-transfected using multiple plasmids to express the lentivirus or γ-retrovirus. During production, homologous recombination may occur between the deleted vector backbone and wild-type viral sequences, leading to the generation of replication-competent virus (RCL), which poses a serious risk to human health. Therefore, RCL detection is one of the most critical components of safety testing.

The FDA-recommended RCL assay involves culturing and amplifying any RCL that may be present in viral vectors using susceptible cells, followed by detection at the end of the culture period.

Amplification phase: Incubate the sample to be tested with HIV-1–susceptible cell lines that support robust viral replication (typically C8166), passage the cells at least five times, and culture for a minimum of three weeks.

Incubation period: Collect the culture supernatant after 3 weeks, inoculate it into C8166 cells, and culture for 7 days before assaying for RCL markers.

RCL induction period culture

RCL Standard Operating Procedure

Detection method:

A: The P24 ELISA method is suitable for detecting RCL formed by recombination between viral genomes during vector preparation.

B: PERT method: After RCL amplification, the PERT assay can also be used to measure reverse transcriptase activity. A drawback of this method is the presence of high background in certain cell types.

C: qPCR method: Real-time quantitative PCR (qPCR) is used to quantify the VSV-G gene sequence during the replication of pseudotyped VSV-G virus, or to detect the psi-gag sequence generated by recombination between the vector plasmid and the packaging plasmid.

II. Analysis of RCL Detection Methods

In its 2020 guidance on RCR/RCL testing, the U.S. FDA states that RCR/RCL assays encompass four methods: indicator cell culture, ELISA (for p24 protein detection), PCR/Q-PCR (using psi-gag or VSV-G–targeted polymerase chain reactions), and PERT (product-enhanced reverse transcription assay).

The indicator cell culture assay for replication-competent lentiviral RCL requires 28 days, making it time-consuming; in contrast, Q-PCR assays based on the VSV-G sequence and PCR assays based on the gag sequence offer advantages such as shorter turnaround times, higher sensitivity, and strong reproducibility. Currently, many CAR-T product applicants employ Q-PCR/PCR methods to directly quantify VSV-G or psi-gag sequences in the final product as a rapid release testing approach.

Shanghai Heyou Sheng Biotechnology Co., Ltd. has leveraged market demand and its technological platform strengths to accumulate extensive development experience in methods such as ELISA for p24 protein detection, PCR targeting the Psi-gag sequence region, and VSV-G–based polymerase chain reaction. We welcome inquiries from all potential customers.

National Toll-Free Consultation Hotline: 021-36696819

1. ELISA method (p24 protein assay)

A 96-well plate pre-coated with HIV-1 core antibodies is incubated with the substrate TMB in the presence of peroxidase, resulting in a color change from blue to yellow upon acidification. The optical density (OD) is then measured at 450 nm using a microplate reader.

b) Quantitative analysis with high sensitivity

A standard curve is generated using reference standards, and the p24 protein concentration in the samples is calculated; RCL assays are also performed. The P24 ELISA method achieves a sensitivity of 3 pg/mL.

c) Suitable for multiple sample types

The P24 assay can detect a variety of sample types, including terminal cells, concentrated vectors, and serum or plasma. This platform employs stringent cutoff criteria to ensure the accuracy, validity, and reproducibility of test results.