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Written by | T
cells genetically modified to express TCR that recognize tumor-associated antigens are helpful for cancer immunotherapy, but usually because of the risk of cross-reactivity with off-target peptides on healthy cells, and natural TCRs with high affinity for their own tumor antigens are extremely rare, and synthetic TCRs may introduce more unpredictable cross-reactions, resulting in more serious consequences 【1, 2】
。 Therefore, the development of platforms that can simultaneously engineer TCR and detect cross-reactivity based on its activity, i.
e.
, antigen-induced signaling, will bring great benefits
.
Recently, Sai T.
Reddy's team from ETH Zurich published a paper in the journal Immunity titled High-throughput T cell receptor engineering by functional screening identifies candidates with Enhanced potency and specificity article, they developed TCR-Engine, a method that combines genome editing, computational design, and deep sequencing to design the functional activity and specificity of TCR on the surface of human T cell lines at high throughput
。 They successfully designed synthetic TCRs using this approach to improve potency and specificity against clinically relevant tumor-associated antigens, and validated their translational potential
through multiple in vitro and in vivo evaluations of safety and efficacy.
Thus, TCR-Engine represents a valuable technique for designing safe and effective synthetic TCRs
for immunotherapeutic applications.
The researchers developed TCR-receiving T cell (TnT) lines through five sequential steps of CRISPR-Cas9 genome editing to knock out endogenous TCR expression and introduce additional functional components, Finally, TCR-mediated activation can be detected by nuclear factor (NFAT) of activated T cell response elements associated with surface expression of GFP reporter or early T cell activation marker CD69, followed by clonal verification
by FACS, cell expansion, and Sanger sequencing 。 The resulting TnT cell line constructively expresses Cas9 nucleases, human CD8, and mRuby, NFAT-GFP reporter genes with TCR signaling, and lacks expression of CD4, endogenous TCR, and Fas
.
In contrast to traditional TCR engineering platforms, TnTs cells allow TCR to be assessed across multiple parameters such as CD3 expression, binding to peptide-MHC polymers, and activation of responses
to antigen presentation.
The authors selected MART-1-specific TCRDMF4 and MAGE A3-specific TCRA3 for mutation analysis
.
Following FACS, deep sequencing was performed to identify TCR variants enriched in the selection step, with TCRA3 variants S3G, P4L, M6L, and A7V showing the greatest increase in binding and activation, while for TCRDMF4 variants, only modest improvement
relative to wild type was observed.
Subsequently, the authors designed a combined library using deep sequencing enrichment data obtained from CDR3b DMS to apply TCR-Engine to TCR A3, and the resulting cells exhibited strong NFAT-GFP expression after co-culture with MAGE-A3-expressing EJM myeloma cells, indicating the presence of an enhanced TCRA3 variant
。 In fact, a total of 195 unique variants showed more than 2-fold enrichment in SEL 3A, 29 potential candidates were selected based on scoring and 14 were further selected for further characterization, and all 14 selected TCR A3 synthetic variants showed enhancedMAGE-A3 peptide-MHC binding capacity
.
Next, the authors wanted to know about the potential off-target effects of the TCR-Engine TCRA3 variant, by scanning the library of single-substituted peptides against the MAGE-A3168-176 peptide (EVDPIGHLY) antigen and engineering TCR with previous bacteriophages A comparison of off-target conditions of a3a showed that the number of off-target predicted by TnT-TCRa3a was significantly reduced
.
The authors further characterize the activity of selected synthetic TCR A3 variants in primary human CD8+ T cells by applying the CRISPR-Cas9 genome editing method (transgenic TCR integration that knocks out endogenous TCRs and targets the TRAC locus).
。 The results showed that: 1) the B-LCL line expressing the high-frequency HLA class I allele did not show alloreactivity; 2) lack of cross-reactivity to beating cardiomyocytes expressing high levels of actin; 3) showed effective tumor cell killing effect; 4) Significant antitumor activity
was shown in human cell line-derived xenograft mouse models.
Overall, this work describes the development and application of TCR-Engine, which can achieve TCR engineering and cross-reactivity analysis at the same time, which will greatly accelerate the characterization and development of therapeutic TCR, improving efficacy and safety
.
Platemaker: Eleven
1.
Linette, G.
P.
, Stadtmauer, E.
A.
, Maus, M.
V.
, Rapoport, A.
P.
, Levine, B.
L.
, Emery, L.
, Litzky, L.
, Bagg, A.
, Carreno, B.
M.
, Cimino, P.
J.
, et al.
(2013).
Cardiovascular toxicity and titin cross-reactivity of affinity-enhanced T cells in myeloma and melanoma.
Blood 122, 863–871.
2.
Morgan, R.
A.
, Chinnasamy, N.
, Abate-Daga, D.
, Gros, A.
, Robbins, P.
F.
, Zheng, Z.
, Dudley, M.
E.
, Feldman, S.
A.
, Yang, J.
C.
, Sherry, R.
M.
, et al.
(2013).
Cancer regression and neurological toxicity following anti-MAGE-A3 TCR gene therapy.
J.
Immunother.
36, 133–151.
cells genetically modified to express TCR that recognize tumor-associated antigens are helpful for cancer immunotherapy, but usually because of the risk of cross-reactivity with off-target peptides on healthy cells, and natural TCRs with high affinity for their own tumor antigens are extremely rare, and synthetic TCRs may introduce more unpredictable cross-reactions, resulting in more serious consequences 【1, 2】
。 Therefore, the development of platforms that can simultaneously engineer TCR and detect cross-reactivity based on its activity, i.
e.
, antigen-induced signaling, will bring great benefits
.
Recently, Sai T.
Reddy's team from ETH Zurich published a paper in the journal Immunity titled High-throughput T cell receptor engineering by functional screening identifies candidates with Enhanced potency and specificity article, they developed TCR-Engine, a method that combines genome editing, computational design, and deep sequencing to design the functional activity and specificity of TCR on the surface of human T cell lines at high throughput
。 They successfully designed synthetic TCRs using this approach to improve potency and specificity against clinically relevant tumor-associated antigens, and validated their translational potential
through multiple in vitro and in vivo evaluations of safety and efficacy.
Thus, TCR-Engine represents a valuable technique for designing safe and effective synthetic TCRs
for immunotherapeutic applications.
The researchers developed TCR-receiving T cell (TnT) lines through five sequential steps of CRISPR-Cas9 genome editing to knock out endogenous TCR expression and introduce additional functional components, Finally, TCR-mediated activation can be detected by nuclear factor (NFAT) of activated T cell response elements associated with surface expression of GFP reporter or early T cell activation marker CD69, followed by clonal verification
by FACS, cell expansion, and Sanger sequencing 。 The resulting TnT cell line constructively expresses Cas9 nucleases, human CD8, and mRuby, NFAT-GFP reporter genes with TCR signaling, and lacks expression of CD4, endogenous TCR, and Fas
.
In contrast to traditional TCR engineering platforms, TnTs cells allow TCR to be assessed across multiple parameters such as CD3 expression, binding to peptide-MHC polymers, and activation of responses
to antigen presentation.
The authors selected MART-1-specific TCRDMF4 and MAGE A3-specific TCRA3 for mutation analysis
.
Following FACS, deep sequencing was performed to identify TCR variants enriched in the selection step, with TCRA3 variants S3G, P4L, M6L, and A7V showing the greatest increase in binding and activation, while for TCRDMF4 variants, only modest improvement
relative to wild type was observed.
Subsequently, the authors designed a combined library using deep sequencing enrichment data obtained from CDR3b DMS to apply TCR-Engine to TCR A3, and the resulting cells exhibited strong NFAT-GFP expression after co-culture with MAGE-A3-expressing EJM myeloma cells, indicating the presence of an enhanced TCRA3 variant
。 In fact, a total of 195 unique variants showed more than 2-fold enrichment in SEL 3A, 29 potential candidates were selected based on scoring and 14 were further selected for further characterization, and all 14 selected TCR A3 synthetic variants showed enhancedMAGE-A3 peptide-MHC binding capacity
.
Next, the authors wanted to know about the potential off-target effects of the TCR-Engine TCRA3 variant, by scanning the library of single-substituted peptides against the MAGE-A3168-176 peptide (EVDPIGHLY) antigen and engineering TCR with previous bacteriophages A comparison of off-target conditions of a3a showed that the number of off-target predicted by TnT-TCRa3a was significantly reduced
.
The authors further characterize the activity of selected synthetic TCR A3 variants in primary human CD8+ T cells by applying the CRISPR-Cas9 genome editing method (transgenic TCR integration that knocks out endogenous TCRs and targets the TRAC locus).
。 The results showed that: 1) the B-LCL line expressing the high-frequency HLA class I allele did not show alloreactivity; 2) lack of cross-reactivity to beating cardiomyocytes expressing high levels of actin; 3) showed effective tumor cell killing effect; 4) Significant antitumor activity
was shown in human cell line-derived xenograft mouse models.
Overall, this work describes the development and application of TCR-Engine, which can achieve TCR engineering and cross-reactivity analysis at the same time, which will greatly accelerate the characterization and development of therapeutic TCR, improving efficacy and safety
.
Original link:
https://doi.
org/10.
1016/j.
immuni.
2022.
09.
004
Platemaker: Eleven
References
1.
Linette, G.
P.
, Stadtmauer, E.
A.
, Maus, M.
V.
, Rapoport, A.
P.
, Levine, B.
L.
, Emery, L.
, Litzky, L.
, Bagg, A.
, Carreno, B.
M.
, Cimino, P.
J.
, et al.
(2013).
Cardiovascular toxicity and titin cross-reactivity of affinity-enhanced T cells in myeloma and melanoma.
Blood 122, 863–871.
2.
Morgan, R.
A.
, Chinnasamy, N.
, Abate-Daga, D.
, Gros, A.
, Robbins, P.
F.
, Zheng, Z.
, Dudley, M.
E.
, Feldman, S.
A.
, Yang, J.
C.
, Sherry, R.
M.
, et al.
(2013).
Cancer regression and neurological toxicity following anti-MAGE-A3 TCR gene therapy.
J.
Immunother.
36, 133–151.
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