Comprehensive clinical testing of investigational candidates is essential for the successful development of both autologous and allogeneic chimeric antigen receptor T cell (CAR-T) therapies. As CAR-T cell programs continue to increase in number, diversity, and complexity, advances in clinical testing strategies are required to improve therapeutic characterization and accelerate development. Human Leukocyte Antigen (HLA) typing, first developed for determining donor-recipient compatibility in hematopoietic stem cell transplants (HSCTs), plays a crucial role in the clinical evaluation of advanced therapies.
CAR-T cells must effectively target and eliminate cancer cells while minimizing off-target effects and avoiding rejection by the host’s immune system—processes closely linked to HLA antigen presentation and T cell recognition. Detailed HLA typing provides insights into how engineered T cells target cancer cells in vivo and interact with the patient’s immune system. This understanding ensures that CAR-T cells perform optimally within the patient’s unique immune context while minimizing the risk of adverse reactions, supporting the development of more effective and safer therapies.
Considerations for HLA Typing in Autologous CAR-T Cell Therapy Development
Within the autologous CAR-T setting, a patient’s own T cells are harvested, modified ex vivo, and subsequently reinfused to target and eliminate cancer cells. While using the patient’s own cells minimizes certain risks, it does not eliminate the potential for adverse effects, such as the risk of graft-versus-host disease (GvHD)1. HLA typing of the engineered CAR-T cells helps predict how these cells will interact with the patient’s immune system once reinfused.
By understanding the HLA profile, researchers can optimize CAR constructs to avoid cross-reactivity with the patient’s own antigens, thereby reducing the risk of unwanted autoimmune reactions. Integrating HLA typing into clinical assessments helps developers precisely tailor CAR-T cell therapies to each patient’s immunological profile, improving both the specificity and safety of the autologous therapy.
Optimizing Allogeneic CAR-T Cell Therapy through HLA Typing
Allogeneic CAR-T therapies are a key focus area in recent years to overcome the limitations associated with autologous approaches. By using T cells from healthy donors as the starting material, these therapies address challenges such as manufacturing failures, complex logistics, extended timelines, and high costs2. The scalability of allogeneic strategies allows for more standardized “off-the-shelf” CAR-T cells, which can be made available for multiple patients, thereby improving clinical accessibility and reducing treatment costs.
Despite these advantages, allogeneic CAR-T therapies face challenges related to graft rejection by the recipient’s immune system and GvHD due to donor-recipient HLA mismatches. Allo-reactive CAR-T cells, which can target and attack recipient tissues, contribute significantly to these complications. Accurate HLA typing is, therefore, essential for donor-recipient matching to minimize immune rejection and ensure that the engineered CAR-T cells function effectively within the recipient’s immune system.
HLA typing is also important in developing universal CAR-T cells, which are engineered to be compatible across multiple HLA types to enhance the feasibility and cost-effectiveness of “off-the-shelf” therapies. Gene editing technologies, such as Transcription Activator-Like Effector Nucleases (TALENs) and CRISPR/Cas9, are employed to modify HLA genes in donor T cells3. By disrupting the expression of specific HLA molecules, the modified T cells are less likely to trigger immune responses in recipients, minimizing the risks associated with HLA mismatches and improving the persistence and efficacy of the engineered T cells in vivo.
Moreover, in clinical trial design, HLA profiling aids in patient selection to maximize the likelihood of clinical success. Stratifying potential trial participants by their HLA profiles can ensure that the engineered CAR-T cells are compatible with a patient’s HLA-peptide complexes. This increases the probability that the therapy will achieve its intended therapeutic effect and contributes to more meaningful trial outcomes.
Importance of HLA Typing in CAR-T Development
- Minimizes safety risks in CAR-T therapies
- Optimizes CAR constructs for better therapeutic efficacy
- Enhances donor-recipient matching in allogeneic therapies
- Facilitates development of universal CAR-T cells
Advancements in HLA Typing Technologies
HLA typing was initially performed using serological methods, hybridization techniques, and restriction fragment length polymorphism (RFLP) analysis4. Modern HLA typing uses DNA sequencing technologies to provide more detailed genetic information (Table 1). Real-time polymerase chain reaction (qPCR) is widely used for HLA typing where primers targeting HLA alleles amplify and quantify specific DNA sequences. This method can detect low frequency alleles and provides quantitative real-time data on HLA allele frequencies and gene copy numbers.
Higher-resolution allele-based methods, such as dideoxy-based Sanger sequencing, are commonly used in the clinical setting due to faster speed and better cost-effectiveness. This well-established technique can precisely determine nucleotide sequences with high accuracy. Next-generation sequencing (NGS) technologies offer even higher resolution, throughput, and accuracy compared to Sanger sequencing. NGS enables the sequencing of both coding and noncoding regions of entire genes and allows for simultaneous analysis of multiple HLA loci and other genomic regions. This provides a more comprehensive view of the HLA polymorphisms beyond the traditionally examined exons.
Single-molecule sequencing platforms represent the latest advancements in HLA typing. These third-generation technologies produce long reads that can cover entire HLA regions. They can detect complex structural and copy number variations and resolve phase ambiguity across single nucleotide polymorphisms (SNPs), which are challenging to address with current HLA typing methods4.
Table 1. Sequencing-Based HLA Typing Technologies
| Technology | Benefits |
|---|---|
qPCR | • Quantitative real-time HLA data • Able to detect low-abundance HLA alleles |
Sanger Sequencing | • High accuracy and reliability • Cost-effective • Well-established and validated |
Next Generation Sequencing (NGS) | • Higher resolution and throughput • Simultaneous analysis of multiple HLA loci • Detailed view of HLA polymorphisms • Low cost |
Single-Molecule Sequencing | • Detects complex structural and copy number variations • Covers entire HLA regions • Useful for resolving phase ambiguity across various genetic variations |
In addition to molecular techniques, complementary technologies such as immunohistochemistry (IHC), proteomics, and flow cytometry broaden clinical evaluation and expand the relevance of HLA profiling for CAR-T cell therapy. Resolving HLA genotype ambiguities often necessitates additional testing across different platforms to ensure precise matching between patients and potential donors4.
Combining these advanced HLA testing methods allows for more precise identification of HLA-restricted epitopes and helps in understanding how tumor-specific antigens are presented to T cells with higher accuracy and resolution. This is particularly important to ensure optimal donor-recipient matching and compatibility in allogeneic therapies, contributing to improved therapeutic efficacy and safety.
Future Outlook
The emergence of new therapeutic targets and the diversification of CAR-T cell products will continue to drive advancements in clinical testing programs across the cell therapy space. Integrating both novel and established HLA technologies as part of a comprehensive testing framework will be critical to accelerating the development of new candidates and streamlining the path to regulatory approval.
References
- Kawamoto H, Masuda K. Trends in cell medicine: from autologous cells to allogeneic universal-use cells for adoptive T-cell therapies. Int Immunol. 2024;36(2):65-73. doi:10.1093/intimm/dxad051
- Lonez C, Breman E. Allogeneic CAR-T Therapy Technologies: Has the Promise Been Met? Cells. 2024;13(2):146. Published 2024 Jan 12. doi:10.3390/cells13020146
- Chen X, Tan B, Xing H, et al. Allogeneic CAR-T cells with HLA-A/B and TRAC disruption exhibit promising antitumor capacity against B cell malignancies. Cancer Immunol Immunother. 2024;73(1):13. Published 2024 Jan 17. doi:10.1007/s00262-023-03586-1
- Geo JA, Ameen R, Al Shemmari S, Thomas J. Advancements in HLA Typing Techniques and Their Impact on Transplantation Medicine. Med Princ Pract. 2024;33(3):215-231. doi:10.1159/000538176
