What is the workflow of TCR-T cell therapy for tumor treatment?

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What is the workflow of TCR-T cell therapy for tumor treatment?

Overview

In order to isolate therapeutic TCR, antigen-specific T cells must first be isolated from the blood of patients or healthy blood donors, and amplified with specific peptide antigens and cytokines (such as IL-2 and IL-15) in vitro.

This process requires prior identification of specific tumor-associated peptide targets that can be safely targeted to the patient. After selecting the target antigen, different methods can be used to screen for TCRs with the required high affinity and tumor specificity.

Preclinical safety testing is also necessary to ensure minimal off-target effects and cross-reactivity of isolated high-affinity TCRs.

Viral vectors are usually used to genetically modify autologous patient T cells to express validated therapeutic TCRs, and then return them to the patient’s body.

Identify the target antigen

The melanoma antigen 1 ( MART-1 ) recognized by T cells is the first tumor-associated antigen targeted in clinical trials of TCR-T.

Adoptive transfer of MART-1-specific TCR-T cells in 15 patients achieved a persistent presence at the level of more than 10% of peripheral blood lymphocytes for at least 2 months after infusion, and showed beneficial effects, including The tumor subsided.

In addition to anti-tumor effects, some patients also show targeted toxicity to normal melanocytes, causing vision or hearing problems, but these problems are largely resolved by steroid therapy.

After this breakthrough, TCR-T therapies for a variety of tumor antigens have been developed, including MAGE-A3, MAGE-A4, GD2, mesothelin, gp100, MART1, AFP, CEA, NY-ESO-1, and TCR-T therapy derived from HPV and EBV viral peptides.

Among them, NY-ESO-1 has been proven to be one of the most promising targets of TCR-T cells, and it has been successful in the treatment of synovial sarcoma, with an objective effective rate of 67%.

The ideal TCR-T target antigen shows the following characteristics:

(1) the ability to induce an immune response;

(2) it is related to driving tumor phenotypes ( such as oncogenes ) to reduce the risk of antigen loss and tumor immune evasion;

(3) Expression on tumor stem cells to promote permanent tumor eradication.

Identification method of tumor-associated antigen

High-resolution mass spectrometry ( MS ) has proven to be the most powerful high-throughput method to facilitate the direct identification of HLA-I binding peptides from tumor cells.

In this method, HLA-I/peptide complexes are separated from tumor tissues or cell lines by immunoprecipitation ( IP ), and then washed thoroughly and applied with acidic elution buffer, from HLA-I molecules and antibodies for IP Separation of bound peptide antigens.

This strategy allows each tumor sample to identify thousands of verified peptide targets and has been used to identify glioblastoma ( GB ), melanoma, renal cell carcinoma ( RCC ) and colorectal cancer ( CRC ), etc. HLA-I ligand.

Identification method of tumor neoantigen

Although MS-based techniques can be used to identify neoantigens, they are more difficult to identify due to their relatively low abundance and the limited sensitivity of MS, especially for tumor samples of limited size.

However, the development of next-generation sequencing technology helps to identify and locate such tumor targets. Whole-exome DNA sequencing, combined with computational prediction algorithms, allows identification of specific genetic changes in cancer cells.

These changes can produce mutant peptides and can be displayed on tumor HLA-I molecules.

All somatic mutant genes can be analyzed by computer to predict potential high-affinity epitopes that may bind to individual HLA-I molecules of the patient and be recognized by T cells.

With the use of large MS eluted peptide databases, HLA-I peptide binding prediction algorithms are constantly updated and improved, and other prediction algorithms try to consider biological variables related to the complexity of intracellular processes.

Another frequently used method is tumor RNA sequencing, which allows the selection of neoantigens with the highest transcriptional expression. It is worth noting that although these prediction methods generally show very good accuracy in identifying presented and highly immunogenic neoantigens, they usually predict a higher number of neoantigen targets than the actual number of real targets1 To 2 orders of magnitude.

Discovering new antigens through trogocytosis is a new method that has emerged in recent years.

Trogocytosis is a biological phenomenon that occurs in the process of cell binding, during which cells share and transfer membranes and membrane-related proteins. Li et al. found that T cell membrane proteins specifically transfer to tumor target cells, and these target cells present homologous HLA-I/peptide complexes.

Using these T cell-target cell interactions, they created a new antigen discovery system by incubating T cells expressing labeled orphan TCR with homologous target cells.

By transferring fluorescent labels from T cells to target cells, this method can isolate these target cells and sequence the homologous TCR ligands, thereby establishing a new antigen library.

Isolation of tumor-specific T cells and TCR

Using HLA-I multimers, single-cell TCR sequencing, or antigen-negative humanized mice, tumor-reactive T cells and TCRs can be identified and identified from autologous, allogeneic or heterogeneous cell banks.

Using the HLA-I multimer method, antigen-specific CD8+ T cells can be directly separated by multimer staining and flow cytometry sorting.

Before isolating the paired full-length TCR sequences, these polyclonal T cells were subjected to homologous peptide recognition and anti-tumor function tests.

Using a highly sensitive PCR-based single-cell TCR analysis method (TCR-SCAN), a TCR with high affinity and specificity can be obtained.

Another method utilizes a humanized mouse TCR gene bank, which does not cause clonal deletion or tolerance of T cells produced in humans.

To this end, Li et al. used the entire human TCRα/β gene locus and the chimeric HLA-A2 transgene to construct a transgenic mouse to achieve the isolation of human TCR against human TAA.

The single-cell sequencing method represents a more promising method for high-throughput isolation of tumor-specific TCR-encoding genes.

Using RNA decoy libraries that target each individual V and J element in the TCRα and TCRβ locus, TCR-encoding genomic elements can be selectively separated from the sheared genomic DNA ( gDNA ) fragments for subsequent paired-end deep sequencing.

This makes it possible to identify antigen-specific TCRs from human materials or oligoclonal T cell populations from humanized TCR mice.

Naive T cells can also be used as a source of TCR for TCR-T therapy. TAA and neoantigen-specific T cells can be derived and expanded from low-frequency precursors in the peripheral blood of cancer patients, and can be re-infused or used as a source of antigen-specific TCR.

Since cancer patients usually exhibit immunosuppression or dominant T cell tolerance, the original sequence of HLA-I matched healthy donors also represents a reliable source, because it has a huge diversity of TCR sequences, theoretically T cells have Any antigen specificity, including tumor neoantigens.

A high-throughput technology platform has been developed to find the original sequence in order to quickly and effectively identify rare but therapeutically valuable TCRs for personalized adoptive T cell therapy.

Cloning of TCR

Most TCR-based gene therapy methods rely on the use of viral vectors to transduce T cells in vitro. The first vector used for gene therapy is adenovirus. However, since they cannot integrate the transgene into the host genome, TCR expression is lost during T cell proliferation. In addition, the immune genetic characteristics of adenovirus also limit its application as a gene therapy vector. In contrast, retroviruses show greater promise as gene transfer vectors because they can infect a variety of cells and have the ability to insert transgenes into the host genome, thereby enabling stable expression of ectopic TCRα/β chains.

Retroviral vectors derived from γ-retroviruses such as mouse leukemia virus ( MLV ) have been widely used for gene transfer into human T cells. This method has been used to deliver a variety of genes, including suicide genes, TCR and CARs. The main disadvantage is that they cannot transduce non-proliferative target cells, which precludes the use of quiescent T cells in TCR-T therapy. In addition, retroviral insertion mutations may cause potential side effects.

Recently, lentiviral vectors ( LV ) have gained more attention as gene transfer vectors because they can deliver genes to dividing and non-dividing cells. Various techniques, such as Golden Gate cloning and LR cloning, are commonly used to construct vectors for inserting TCRα/β genes.

Adeno-associated virus (AAV) is another widely used viral vector. Compared with adenovirus vectors, AAV has lower immunogenicity and wider cell tropism, so it has been widely used in tumor gene therapy.

In order to promote the integration of transgenes, a self-complementary AAV vector ( scAAV ) was developed to make AAV independent of the host cell’s complementary chain synthesis.

The efficacy of scAAV in preclinical models is better than that of traditional AAV.

At the same time, some non-viral gene editing methods have also been developed. mRNA electroporation has been shown to achieve transient TCR and CAR expression, thereby minimizing the risk of persistent viral components.

Clinical data shows that mRNA-modified TCR-T and CAR T cells are both feasible and safe, and there is no obvious evidence of non-targeted toxicity to normal tissues.

However, the lack of sustained TCR expression may limit efficacy, requiring repeated infusions.

In addition, the non-viral Sleeping Beauty retrotransposon system has also been used for the transduction of TCR and CARs.

Gene editing can specifically and efficiently insert large gene fragments into target cells through homology directed repair ( HDR ).

TCR-T cells developed using CRISPR/CAS9 have been shown to specifically recognize tumor antigens in vitro and induce anti-tumor responses in vivo.

TCR verification method

After TCR cloning, extensive preclinical verification is required to prove the specificity and safety of engineered TCR-T cells.

Validation includes evaluating the affinity of TCR by titrating homologous peptide antigens, and measuring the killing effect of a group of HLA-I matched tumor cell lines.

If no such tumor cell line exists, the target cell can transduce the relevant antigen and the relevant HLA-I molecule.

Neoantigens can also be expressed in autologous antigen presenting cells to assess the antigen reactivity of TCR.

Safety testing includes testing the candidate TCR-T’s ability to recognize HLA-I matching primary tissues to ensure that no normal tissues are used as targets, which may cause non-targeted toxicity.

In at least two clinical trials of TCR-T cell therapy, cross-reactions to normal brain cells and heart cells have occurred, which led to the death of patients.

The results of these trials underscore the importance of conducting extensive safety trials before TCR enters clinical trials.

References:

1.Evolution of CD8+ T Cell Receptor(TCR) Engineered Therapies for the Treatment of Cancer. Cells. 2021 Sep;10(9): 2379.