September 23, 2023

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A brief guide to cell and gene therapy analysis strategies

A brief guide to cell and gene therapy analysis strategies


A brief guide to cell and gene therapy analysis strategies.  The field of Cellular and Gene Therapy (CGT) is developing rapidly, and the number of cell and gene therapies developed in the past five years has greatly increased.

The application of CGT is expected to bring significant therapeutic effects to people suffering from various diseases (from ophthalmological diseases to cancer).


In cell therapy, cells from a donor (allogeneic therapy) or from a patient (autotherapy) are proliferated in vitro and then introduced into the patient. In gene therapy, the genetic material of somatic cells can be modified in vivo or in vitro.

A functional copy of the mutant gene can be inserted, or a new protein related to the treatment method can be introduced into the patient’s genome. For example, genetic diseases may involve genetic mutations in key metabolic enzyme genes, leading to non-functional and toxic accumulation of enzyme substrates in the body.

Gene therapy will use viral vectors or lipid nanoparticles to introduce functional genes into the body. Genetic modification can allow the production of replacement enzymes, which has significant advantages over alternative methods that require regular administration of exogenous enzyme replacement therapy.

Current research is also aimed at developing more sophisticated editing (“gene editing”) therapies for gene mutations in cells.


Compared with non-viral methods, the use of viral vectors to transduce host cells has a relatively high efficiency. However, the challenge of immunogenicity and cytotoxicity has led to an increase in the use of non-viral gene delivery vehicles in in vitro and in vivo treatment procedures.

In addition to safety advantages, non-viral methods also have the advantages of being easier to prepare, capable of transferring larger genes, and significantly improving transfection efficiency. Non-viral genetic modification is being explored as a potential treatment for HIV, β-thalassemia and other diseases.


Case: CGT in Oncology

The field of oncology provides examples of CGT treatment. Historically, cancer treatment is based on surgery, chemotherapy, and radiation therapy. In the past two decades, targeted therapies—mainly monoclonal antibodies—have become the gold standard for many cancer treatments.

Recently, immunotherapy that uses the patient’s immune system to attack tumors has become another powerful tool in cancer treatment options. Then there is the emerging immunotherapy that we are talking about today, which is a treatment based on cell therapy. Cell-based therapy may involve stem cell transplantation or adoptive cell therapy (ACT), including the collection and use of the patient’s own immune cells to treat cancer.


The advantages of autologous therapy include low immunogenicity and avoidance of graft-versus-host disease. On the contrary, the main disadvantages are the length of time required for cell expansion, the variability of donor starting materials, and the logistical challenges of large-scale production of individualized treatments.


Nevertheless, challenges such as shortage of raw materials, invasive assay methods, cell heterogeneity and purity have driven the search for alternatives to allogeneic cells and gene therapies. To meet these challenges, new methods for creating cell therapy suitable for allogeneic methods are emerging.

Mesenchymal Stem Cells (MSCs) are derived from induced pluripotent stem cells (iPSCs) and have significant improvements compared with tissue-derived cells. For cancer therapy, scientists are trying to turn the Chimeric Antigen Receptor T-Cell Immunotherapy (CAR-T) method into allogeneic therapy by silencing the protein that mediates the immune response between the host and the graft. Some examples of treatment are the use of CAR-T cells and silent T cell receptors or conversion from T cells to natural killer (NK) cells that are less immunogenic.


Due to its targeted and personalized characteristics, in vitro gene therapy usually has higher safety. However, cell recovery and in vitro manipulation can be an expensive and complicated process. In vivo gene therapy may be more direct; but it faces challenges such as toxicity or inducing immune responses. The most common clinically observed toxicity of gene therapy in vivo is hepatotoxicity and cytokine release syndrome (CRS). In addition, genome editing technology is being explored as a strategy for advancing CAR scientific engineering. Compared with traditional CAR-T cells, CRISPR/Cas9-edited CAR-T cells show enhanced efficacy and delayed differentiation and failure.



Design suitable detection and analysis methods for CGT

As cell and gene therapy products use unique delivery and treatment mechanisms, it is necessary to develop non-traditional and comprehensive bioanalytical testing methods and strategies to prove the safety and effectiveness of CGT.

The CGT bioanalysis package must be able to determine whether the therapeutic protein is present and acting at the site of action, sometimes systemic. Since many gene therapies are delivered via vectors (usually viruses), the presence and appearance of anti-carrier antibodies should also be monitored.

Special consideration should also be given to the pre-existing and urgent immunogenicity of therapeutic proteins, especially in patient populations that have undergone extensive pretreatment. In addition, the host immune system may recognize the newly expressed protein as a foreign protein of the patient and trigger an immune response.



Methods of detecting therapeutic protein levels

Creating the pharmacokinetics (PK) profile of CGT is a complex task. The pharmacokinetics or exposure bioanalysis strategies of therapeutic proteins do not follow the normal pharmacokinetic models used for traditional small and macromolecular drugs.

For example, the translation product of a transgene may only be expressed at the site of action, so bioanalysis strategies must be able to detect the expressed protein at the site of action or throughout the body, depending on the specific treatment method.

In addition, because protein is expressed constitutively (gene expression is not affected by time, location, or environment, and has no temporal and spatial specificity), exposure does not follow typical metabolic stages. Instead, monitor the continuous expression of the exposed functional replacement protein or enzyme.

Adsorption, distribution and metabolism also have the unique characteristics of traditional biological agents and small molecules.


Determining the ideal matrix for identifying changes in therapeutic protein levels is an important part of the bioanalytical procedure. This may require monitoring of expressed proteins in a variety of matrices (such as serum and cerebrospinal fluid), and may even require monitoring of protein levels in human tissues from biopsies of accessible parts such as skin or muscle.

With the advent of precision medicine, these operations must also be minimally invasive to the relevant patients, especially in cases where repeated biopsies are required. For gene therapy that causes enzyme expression, the instability of the protein in the collected matrix may require special consideration for sample handling, including rapid collection at the clinical site, reducing freezing and thawing, adding stable excipients to the collection tube, and minimizing thawing Time and thaw on ice.

Therefore, compared to more standard pharmacokinetic testing of antibody therapeutics in patient serum samples, the importance of investigating the impact of upstream processing steps before the sample reaches the bioanalytical laboratory may increase.


In the case of gene therapy for rare diseases, the enrolled patients may have almost no protein expression, and the protein may be many orders of magnitude higher after treatment. This requires the use of highly sensitive methods, sometimes with a sensitivity of up to pg/mL, and a wide dynamic range to fully characterize the patient’s response to treatment.

The preferred method for protein PK determination is immunoassay. The immunoassay is simple in design, has ready-made reagents, and can be easily adapted to automation to reduce operation changes over time and achieve high-throughput analysis. Immunoassay design needs to consider whether antibody pairs can correctly distinguish between truncated proteins or other non-functional proteins that may exist.

Enzyme-linked immunosorbent assay (ELISA), mesoscale discovery electrochemiluminescence (MSD-ECL) and Luminex assay (this method can Methods such as rapid detection and quantitative analysis of cytokines, chemokines and growth factors at the same time are available. When designing protein PK methods to maximize sensitivity and dynamic range, ultra-sensitive immunoassay platforms can be evaluated.


Many gene therapies involve expressing the dose of protein components with similar endogenous counterparts, which increases the complexity of protein PK immunoassay design. The presence of endogenous substances complicates the analysis design: the reference must be prepared in a matrix from which endogenous proteins have been removed, which is a laborious and variable process, or the reference must be in a buffer/analytical diluent preparation.

A common strategy is to use a standard curve prepared in a buffer/analytical diluent and the corresponding recombinant protein, produced by itself or obtained from a commercial source. In order to confirm the applicability of recombinant protein as a reference material, it is recommended to conduct specific testing and verification experiments.

The detection design strategy should also ensure that the capture and detection antibodies used can properly detect recombinant proteins and endogenous proteins. The precision assessment of endogenous quality control samples (QC) should be performed to ensure that the test has acceptable precision for recombination and endogenous materials.


In order to evaluate the parallelism of recombination and endogenous materials, endogenous QC materials were prepared from samples of healthy individuals and analyzed at multiple dilutions to determine which dilutions gave consistent feedback (between dilutions) result. When the endogenous protein level is low compared to the calibration range, the calibrator protein can be added to the endogenous QC test to assess the accuracy of the full quantitative range of the calibration curve in the matrix.

Alternatively, a QC sample can be prepared by adding recombinant protein to the assay buffer to evaluate the accuracy of the calibration curve. In this case, additional endogenous QC samples can be added to represent actual research samples.


In addition to determining the presence of the protein, the function of the protein must also be evaluated in the context of gene therapy applications. The gold standard for determining the efficacy of a drug is the clinical result, but a comprehensive biological analysis package should also contain the mechanism of action of the therapeutic agent. In the case of many gene therapies, this is achieved through the use of alternative PK/PD (pharmacodynamic) functional analysis, such as enzyme activity analysis or biological analysis.

When paired with protein quantitative analysis, alternative functional analysis can also provide information, because functional analysis may have greater analytical variability and smaller dynamic range. These factors reduce the accuracy of the data generated and may reduce the ability to detect subtle differences between treatment groups.

In addition, enzyme activity is generally more susceptible to instability in patient samples than protein levels. Enzymatic testing can be validated using methods similar to the endogenous QC testing strategy described previously, and recombinase or endogenous QC mixtures should be used to monitor potential changes in daily test performance due to environmental conditions.


For cell therapies like CAR-T, options for quantifying the number of treated cells in the body include flow cytometry and quantitative polymerase chain reaction (qPCR). Recently, digital PCR (dPCR), including droplet digital PCR (ddPCR), has been used in patient samples to quantitatively assess CAR-T levels with high sensitivity and accuracy. These methods allow assessment of the durability of CAR-T therapy over time. Although dPCR and ddPCR report absolute values ​​and do not rely on calibrators or standards for quantification, the lack of standard CAR-T reference materials complicates cell-based treatment analysis.


The use of detection based on qPCR and ddPCR technology has been successfully used to monitor the biodistribution and virus shedding in CGT therapy. These tests allow the detection and quantification of virus insertion and integration in target organisms or patient tissues, resulting in the copy number variation (VCN) of the vector, which is the average integrated copy of the target gene in each diploid cell number. These integrations carry the risk of abnormal gene inactivation or unnecessary gene expression.

In order to safely monitor the integration of viruses in research, integration site analysis (ISA) is commonly used by next-generation sequencing (“Next-generation” sequencing technology, NGS). ISA can not only confirm the presence of target genes in target cells or tissues, but also identify the location of integration. Together, these strategies can monitor the number and location of therapeutic agents driven by lentivirus and AAV vectors.


Assess safety and immunogenicity

Detection of introduced or newly expressed proteins or cells is only one part of the overall biological analysis of CGT. Depending on the specific product and its application, safety and immunogenicity also need to be evaluated as part of clinical bioanalysis.


For CAR-T cell therapy, biomarker-based methods are important for monitoring safety risks. For example, patients receiving CAR-T cell therapy are at risk of cytokine release syndrome (CRS), which is an overreaction of the immune system. C-reactive protein and inflammatory cytokine analysis can monitor the development of CRS. These tests can also determine the risk of CRS in CAR-T patients.


Evaluation of immunogenicity is also essential for the CGT program. In addition to standard anti-drug antibody (ADA) immunogenicity screening, pre-existing antibodies against the vector and expressed protein must be characterized before the start of treatment and throughout the research process. The viral vectors commonly used for CGT are adeno-associated virus (AAV), adenovirus (AdV) and lentivirus (LV).

Because these viruses are widespread in nature, the immunogenicity of the patient to the viral vector must be evaluated before the patient is enrolled to ensure that the therapeutic drug can be delivered to the target site and the therapeutic effect is ensured. Many CGT studies include viral vector immunogenicity screening as part of its inclusion in the screening process, so patients with high levels of pre-existing antibodies may be excluded from the study, or their dose may be adjusted or empty capsids may be supplemented to ensure targeted delivery .

For example, cell-based testing is often used to assess the presence of neutralizing antibodies in AAV delivery vectors. The immunogenicity of the virus must also be monitored during the study to determine whether adverse events or low efficacy are due to emergency immunogenicity.


Viral immunogenicity assays, which monitor B cell-mediated immunogenicity, usually have methods similar to ADA immunogenicity assays; they are usually in the form of bridging or sandwich immunoassays. Challenges in designing virus immunogenicity assays include reagent acquisition and virus strain cross-reaction. Reagents are usually commercially available, but non-standard virus strains and positive control antibodies with sufficient sensitivity and specificity may require custom production because they may be difficult to obtain.


In addition, Nab detection requires a viral vector with a reporter gene insert (ie, luciferase). It should be pointed out that low transfection efficiency will lead to highly empty capsids and subsequent poor expression of reporter genes, which will have a serious negative impact on the accuracy and sensitivity of AAV-based Nab detection. Sponsors should investigate the availability of reagents early in the drug development process, and start signing contracts or producing high-quality reagents as early as possible. The presence of pre-existing AAV antibodies in populations caused by environmental exposure to related viruses can complicate test development. These complications may require extensive pre-screening of negative individuals to determine cut-off points and/or the preparation of negative control pools.


The second B cell-mediated immunogenicity of CGT involves antibodies directed against the expressed protein. Patients in the CGT trial may receive a lot of pretreatment, especially if there are approved enzyme or protein replacement products on the market. The immunogenicity needs to be evaluated at the time of enrollment and throughout the study. People with a high incidence of pre-existing antibodies may need alternative statistical strategies to detect and characterize the immunogenicity of treatment, such as determining the fold change of ADA titer after treatment.


Protein immunogenicity analysis methods usually include standard immunoassay bridging or sandwich analysis formats. Some of the challenges of method development include obtaining sufficient quantities of high-quality protein reserves, labeling non-antibody proteins, and obtaining sufficiently sensitive positive control antibodies. In addition, protein interference and protein binding partner interference may be more difficult to engineer outside of the analysis, so non-traditional dissociation or target consumption methods such as heat treatment or immunoprecipitation sample processing may be required.


ELISPOT (enzyme-linked immunospot) test can monitor T cell immune response, detect specific cytokines or antigen-specific antibodies, and the frequency of low-frequency secreting cells. It is best to use this sensitive detection technology for detection. This assay can detect the secretion of cytokines or effector molecules at the single cell level and is more sensitive than ELISA or intracellular staining techniques. Although it requires the isolation of PBMC or other cell subpopulations, it can be automated to achieve high-throughput screening. This becomes useful in gene therapy, for example, when AAV-based gene transfer anti-capsid T cell responses can eliminate transduced cells and need to be monitored.


Flow cytometry monitors the cell dynamics of injected cells in adoptive cell therapy (such as CAR-T cells). This is important for assessing the associated expansion and persistence of injected cells after antigen exposure in the body. Flow cytometry can also perform immunophenotyping analysis on a variety of cell types that are frequently monitored in cell therapy. One such example is the T cell lymphocyte phenotype panel, which can monitor regulatory markers, activation, memory, or proliferation.


In the case of cell therapy, immunogenicity assessment considers ADAs, whether pre-existing or emerging after treatment, targeting key expressed proteins on the surface of CAR-T cells. When designing a biological analysis method, the research team must decide on the specific target protein used for ADA development on CAR-T cells, and consider whether to use the expressed, soluble protein or cell junction protein for detection development and verification.



Sum up:

Although there are many commonalities between the analysis types between the CGT program and other macromolecular biological analysis, the unique characteristics of cell and gene therapy require attention to various biological analysis challenges, including analysis sensitivity, dynamic range, analyte stability, and pre-existing antibodies , The heterogeneous performance of endogenous and recombinant materials, cell products and vectors (virus or LNP), and the need to integrate multiple analysis platforms.

Therefore, the early assessment of analytical requirements and technical capabilities is very important to ensure the production of bioanalytical packages that can accurately characterize the exposure, mechanism of action, efficacy, and safety of therapeutic products.


(source:internet, reference only)

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