Workflow of TCR-T cell therapy

overview 

To isolate therapeutic TCR, antigen-specific T cells must first be isolated from the blood of a patient or healthy donor and amplified in vitro with specific peptide antigens as well as cytokines such as IL-2 and IL-15
.
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 TCRs
with the desired 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 commonly used to genetically modify autologous patient T cells to express validated therapeutic TCR before being transfused back into the patient
.

Identify the target antigen

Melanoma antigen 1 (MART-1) recognized by T cells is the first tumor-associated antigen
targeted in TCR-T clinical trials.
Adoptive metastasis of MART-1-specific TCR-T cells in 15 patients achieved persistent presence at the level of more than 10% peripheral blood lymphocytes for at least 2 months after infusion and showed beneficial effects, including tumor regression
.
In addition to anti-tumor effects, some patients show targeted toxicity to normal melanocytes, causing vision or hearing problems, but these problems are largely resolved
with steroid therapy.

Since this breakthrough, TCR-T therapies have been developed against multiple tumor antigens, including TCR-T therapies
targeting MAGE-A3, MAGE-A4, GD2, mesothelin, gp100, MART1, AFP, CEA, NY-ESO-1, and viral peptides derived from HPV and EBV.
Among them, NY-ESO-1 has proven to be one of the most promising targets for TCR-T cells, with success 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) correlated with driving tumor phenotypes (such as oncogenes) to reduce the risk of antigen loss and tumor immune evasion; and (3) expression on cancer stem cells to promote permanent tumor eradication
.

Methods for identifying tumor-associated antigens

High-resolution mass spectrometry (MS) has proven to be the most powerful high-throughput method
to facilitate direct recognition of HLA-I-binding peptides from tumor cells.
In this method, HLA-I/peptide complexes are isolated from tumor tissues or cell lines by immunoprecipitation (IP), followed by a thorough wash and application of acidic elution buffer to isolate binding peptide antigens
from HLA-I molecules and antibodies used for IP.
This strategy allows each tumor sample to identify thousands of validated peptide targets and has been used to identify HLA-I ligands for glioblastoma (GB), melanoma, renal cell carcinoma (RCC), and colorectal cancer (CRC), among others.

Methods for identifying tumor neoantigens

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 has helped to identify and locate such tumor targets
.
Whole exome DNA sequencing, combined with computational prediction algorithms, allows the identification of specific genetic alterations in cancer cells that can produce mutant peptides and be able to be presented on
tumor HLA-I molecules.

All somatic mutant genes can be computed to predict potential high-affinity epitopes that may bind to the patient's individual HLA-I molecule for recognition by T cells
.
With the use of large MS-eluting peptide databases, HLA-I peptide binding prediction algorithms are constantly being updated and improved, and other prediction algorithms attempt to account for 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 typically predict the number of neoantigen targets by 1 to 2 orders of magnitude
higher than the actual number of real targets.

The discovery of neoantigens through trogocytosis is a new method that has emerged in recent years
.
Trogocytosis is a biological phenomenon that occurs during cell binding, during which cells share and transfer membrane and membrane-associated proteins
.
Li et al.
found that T cell membrane proteins specifically metastasize to tumor target cells, which present homologous HLA-I/peptide complexes
.
Using these T cell-target cell interactions, they created the Neoantigen Discovery System
by co-incubating T cells expressing labeled orphan TCRs with homologous target cells.
By transferring fluorescent labeling from T cells to target cells, this method enables isolation of these target cells and sequencing of homologous TCR ligands, resulting in the establishment of a neoantigen library
.

Isolation of tumor-specific T cells and TCRs

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

Antigen-specific CD8+ T cells
can be directly isolated by polymer staining and flow cytometry sorting using the HLA-I polymer method.
These polyclonal T cells were tested
for homologous peptide recognition and anti-tumor function prior to isolation of pairs, full-length TCR sequences.
The highly sensitive PCR-based single-cell TCR analysis method (TCR-SCAN) can obtain TCR
with high affinity and specificity.

Another approach utilizes the TCR gene bank of humanized mice, which does not occur in the absence or tolerance
of T cell clones produced in humans.
To this end, Li et al.
constructed transgenic mice using whole human TCR α/β loci and chimeric HLA-A2 transgenes to achieve the isolation
of human TCR against human TAA.

Single-cell sequencing methods represent a more promising method
for high-throughput isolation of tumor-specific TCR-coding genes.
Using RNA decoy libraries targeting each individual V and J element within the TCRα and TCRβ loci, TCR-encoded genomic elements can be selectively isolated from sheared genomic DNA (gDNA) fragments for subsequent paired-end deep sequencing
.
This makes it possible to
identify antigen-specific TCR from human materials or oligoclonal T cell populations of humanized TCR mice.

Naïve T cells can also be used as a TCR source
for TCR-T therapy.
TAA and neoantigen-specific T cells can be derived and amplified from low-frequency precursors in the peripheral blood of cancer patients and can be reinfused or used as a source of
antigen-specific TCR.
Since cancer patients often exhibit immunosuppression or dominant T cell tolerance, the original sequence of an HLA-I-matched healthy donor also represents a reliable source because of its enormous diversity of TCR sequences, with T cells theoretically having any antigen specificity, including tumor neoantigens
.
High-throughput technology platforms have been developed to find raw sequences in order to quickly and efficiently identify rare but therapeutically valuable TCRs
for personalized adoptive T cell therapy.

Cloning of TCR

Most TCR-based gene therapy approaches rely on in vitro transduction of T cells with viral vectors, with the earliest vector used for gene therapy being adenovirus
.
However, since they cannot integrate transgenes into the host genome, TCR expression is lost
during T cell proliferation.
In addition, the immunogenetic nature of adenovirus also limits its use
as a gene therapy vector.
In contrast, retroviruses show greater promise as gene transfer vectors because they can infect a wide variety of cells and have the ability to insert transgenes into the host genome, allowing for stable expression
of ectopic TCRα/β strands.

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, TCRs, and CARs
.
The main drawback 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 (LVs) have gained more attention as gene transfer vectors because they can deliver genes into 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 adenoviral vectors, AAV has lower immunogenicity and wider cytotropism, so it has been widely used
in tumor gene therapy.
To promote transgene integration, self-complementary AAV vectors (scAAVs) have been developed to make AAVs independent of complementary strand synthesis in host cells, and scAAV is more effective than traditional AAV
in preclinical models.

At the same time, some non-viral gene editing methods have also been developed
.
mRNA electroporation has been shown to enable transient TCR and CAR expression, minimizing the risk of persisting viral components
.
Clinical data suggest that mRNA-modified TCR-T and CAR T cells are both feasible and safe, with no clear evidence of non-targeted toxicity
to normal tissues.
However, the lack of sustained TCR expression may limit efficacy and require repeated infusions
.
In addition, non-viral Sleeping Beauty retrotransposon systems have also been used for transduction
of TCRs and CARs.

Gene editing uses homologous directed repair (HDR) to specifically and efficiently insert large gene fragments into target cells
.
TCR-T cells developed using CRISPR/CAS9 have been shown to specifically recognize tumor antigens in vitro and induce generative anti-tumor responses
in vivo.

Verification method of TCR

After TCR cloning, extensive preclinical validation is required to demonstrate the specificity and safety
of engineered TCR-T cells.
Validation includes assessing the affinity of TCR by titrating homologous peptide antigens, as well as measuring the killing effect
on a set of HLA-I-matched tumor cell lines.
If no such tumor cell line exists, target cells can transduce to express the relevant antigen and the associated HLA-I molecule.

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

Safety testing involves testing candidate TCR-T's ability to recognize HLA-I-matched primary tissues to ensure that no normal tissue is being targeted, producing non-targeted toxicity
that can result.
In at least two TCR-T cell therapy clinical trials, cross-reactivity with normal brain cells and heart cells has occurred, resulting in the death
of patients.
These trial results underscore the importance of extensive safety testing of
TCRs before they enter clinical trials.

References:

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

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