COMBINED ISOLATION OF MULTI ANTIGEN SPECIFIC T CELL PRODUCTS CONTAINING NAÏVE AND/OR MEMORY VIRUS-SPECIFIC T CELLS AND T CELLS SPECIFIC FOR TUMOR ASSOCIATED AND MINOR HISTOCOMPATIBILITY ANTIGENS
(Abstract release date: 05/19/16)
EHA Library. Jedema I. 06/09/16; 132670; E1121
Disclosure(s): Juno Therapeutics: Lothar Germeroth is Senior Vice President, Managing Director at Juno Therapeutics, which develops Cell therapeutics based on the Streptamer technology.
Assoc. Prof. Inge Jedema
Contributions
Contributions
Abstract
Abstract: E1121
Type: Eposter Presentation
Background
T cell depleted allogeneic stem cell transplantation (aIIoSCT) followed by a donor lymphocyte infusion (DLI) is a potential curative treatment for patients with hematological malignancies. However, between the aIIoSCT and the DLI patients are vulnerable to viral infections and disease relapses. Within a clinical phase I/II study in the EU FP7 program T-control we isolate and adoptively transfer multi-antigen specific T-cell products to prevent these complications. Therefore, we isolate donor T-cells directed against HLA-A1, A2, A24, B7, and/or B8-restricted peptides of CMV, EBV and Adenovirus and HLA-A2-restricted peptides from the tumor-associated antigens (TAA) NY-eso, WT-1, RHAMM, PRAME and proteinase 3, and the minor histocompatibility antigen (MiHA) HA-1h. Due to the relatively high precursor frequency of virus-specific T cells in seropositive donors, memory virus-specific T cells can be easily isolated.
Aims
To investigate the presence and functional activity of the specificities with a very low precursor frequency, e.g. TAA and MiHA specific T cells, and virus specific T cells from seronegative donors.
Methods
HLA-A2, B7, and/or B8-positive CMV seronegative donors (n=5) and EBV seronegative donors (n=2) were selected for isolation of CMV, EBV and AdV-specific T cells. From 4 HLA-A2 positive donors TAA and MiHA specific T cells were isolated. The reversible HLA/peptide streptamer and magnetic nanobead technique (Juno) was used for the isolation out of 1-2*10^9 donor PBMC, using all relevant streptamers according to the HLA-type of the donor. The positive fractions were non-specifically expanded using PHA, IL-2 and allogeneic feeder cells. The presence of the different specificities was assessed 10-14 days after the isolation by tetramer staining. To confirm the presence of the invisible very low frequency T cell specificities, subsequent enrichment and expansion rounds were performed until populations were visible. Specificities that were present in a frequency >1% were clonally expanded and tested for their functional potential.
Results
After 2 or 3 enrichment and expansion rounds, we were able to demonstrate specific T cell populations for 6 CMV specificities from 4 seronegative donors (pp65 A2 NLV (3x), pp65 B7 TPR, 1E-1 A2 VLE, 1E-1 B8 QIK) and 4 EBV specificities from 2 seronegative donors (BMLF-1 A2 GLC, EBNA-3a B7 RPP (2x), BZLF-1 B8 RAK). The CD8/tetramer positive clones from two CMV specificities (pp65 A2 NLV and pp65 B7 TPR) and one EBV specificity (EBNA-3a B7 RPP) were tested for antigen specific reactivity measured by cytokine release (IFN-gamma and GM-CSF) after 24 hours of stimulation with TAP deficient T2 cells and/or allogeneic EBV-LCL exogenously loaded with the relevant peptide. This analysis revealed the presence of clones of high avidity recognizing up to 10^-9M peptide on T2 cells and 10^-8M peptide on allogeneic EBV-LCL. Besides this, all mentioned TAA and MiHA specificities could be isolated from all four HLA-A2 positive donors after 2 to 4 enrichment and expansion rounds, and comprised clones with a range of different functional avidities.
Conclusion
In conclusion, the HLA/peptide streptamer technology allows the simultaneous isolation of multi antigen specific T cell products containing not only highly frequent virus-specific memory T cells, but also EBV and/or CMV specific T cells from the naïve T cell repertoire of seronegative donors, as well as TAA and MiHA specific T cells with differential functional avidities.
Session topic: E-poster
Keyword(s): Cellular therapy, CMV infection, Minor histocompatibility antigen, Tumor antigen
Type: Eposter Presentation
Background
T cell depleted allogeneic stem cell transplantation (aIIoSCT) followed by a donor lymphocyte infusion (DLI) is a potential curative treatment for patients with hematological malignancies. However, between the aIIoSCT and the DLI patients are vulnerable to viral infections and disease relapses. Within a clinical phase I/II study in the EU FP7 program T-control we isolate and adoptively transfer multi-antigen specific T-cell products to prevent these complications. Therefore, we isolate donor T-cells directed against HLA-A1, A2, A24, B7, and/or B8-restricted peptides of CMV, EBV and Adenovirus and HLA-A2-restricted peptides from the tumor-associated antigens (TAA) NY-eso, WT-1, RHAMM, PRAME and proteinase 3, and the minor histocompatibility antigen (MiHA) HA-1h. Due to the relatively high precursor frequency of virus-specific T cells in seropositive donors, memory virus-specific T cells can be easily isolated.
Aims
To investigate the presence and functional activity of the specificities with a very low precursor frequency, e.g. TAA and MiHA specific T cells, and virus specific T cells from seronegative donors.
Methods
HLA-A2, B7, and/or B8-positive CMV seronegative donors (n=5) and EBV seronegative donors (n=2) were selected for isolation of CMV, EBV and AdV-specific T cells. From 4 HLA-A2 positive donors TAA and MiHA specific T cells were isolated. The reversible HLA/peptide streptamer and magnetic nanobead technique (Juno) was used for the isolation out of 1-2*10^9 donor PBMC, using all relevant streptamers according to the HLA-type of the donor. The positive fractions were non-specifically expanded using PHA, IL-2 and allogeneic feeder cells. The presence of the different specificities was assessed 10-14 days after the isolation by tetramer staining. To confirm the presence of the invisible very low frequency T cell specificities, subsequent enrichment and expansion rounds were performed until populations were visible. Specificities that were present in a frequency >1% were clonally expanded and tested for their functional potential.
Results
After 2 or 3 enrichment and expansion rounds, we were able to demonstrate specific T cell populations for 6 CMV specificities from 4 seronegative donors (pp65 A2 NLV (3x), pp65 B7 TPR, 1E-1 A2 VLE, 1E-1 B8 QIK) and 4 EBV specificities from 2 seronegative donors (BMLF-1 A2 GLC, EBNA-3a B7 RPP (2x), BZLF-1 B8 RAK). The CD8/tetramer positive clones from two CMV specificities (pp65 A2 NLV and pp65 B7 TPR) and one EBV specificity (EBNA-3a B7 RPP) were tested for antigen specific reactivity measured by cytokine release (IFN-gamma and GM-CSF) after 24 hours of stimulation with TAP deficient T2 cells and/or allogeneic EBV-LCL exogenously loaded with the relevant peptide. This analysis revealed the presence of clones of high avidity recognizing up to 10^-9M peptide on T2 cells and 10^-8M peptide on allogeneic EBV-LCL. Besides this, all mentioned TAA and MiHA specificities could be isolated from all four HLA-A2 positive donors after 2 to 4 enrichment and expansion rounds, and comprised clones with a range of different functional avidities.
Conclusion
In conclusion, the HLA/peptide streptamer technology allows the simultaneous isolation of multi antigen specific T cell products containing not only highly frequent virus-specific memory T cells, but also EBV and/or CMV specific T cells from the naïve T cell repertoire of seronegative donors, as well as TAA and MiHA specific T cells with differential functional avidities.
Session topic: E-poster
Keyword(s): Cellular therapy, CMV infection, Minor histocompatibility antigen, Tumor antigen
Abstract: E1121
Type: Eposter Presentation
Background
T cell depleted allogeneic stem cell transplantation (aIIoSCT) followed by a donor lymphocyte infusion (DLI) is a potential curative treatment for patients with hematological malignancies. However, between the aIIoSCT and the DLI patients are vulnerable to viral infections and disease relapses. Within a clinical phase I/II study in the EU FP7 program T-control we isolate and adoptively transfer multi-antigen specific T-cell products to prevent these complications. Therefore, we isolate donor T-cells directed against HLA-A1, A2, A24, B7, and/or B8-restricted peptides of CMV, EBV and Adenovirus and HLA-A2-restricted peptides from the tumor-associated antigens (TAA) NY-eso, WT-1, RHAMM, PRAME and proteinase 3, and the minor histocompatibility antigen (MiHA) HA-1h. Due to the relatively high precursor frequency of virus-specific T cells in seropositive donors, memory virus-specific T cells can be easily isolated.
Aims
To investigate the presence and functional activity of the specificities with a very low precursor frequency, e.g. TAA and MiHA specific T cells, and virus specific T cells from seronegative donors.
Methods
HLA-A2, B7, and/or B8-positive CMV seronegative donors (n=5) and EBV seronegative donors (n=2) were selected for isolation of CMV, EBV and AdV-specific T cells. From 4 HLA-A2 positive donors TAA and MiHA specific T cells were isolated. The reversible HLA/peptide streptamer and magnetic nanobead technique (Juno) was used for the isolation out of 1-2*10^9 donor PBMC, using all relevant streptamers according to the HLA-type of the donor. The positive fractions were non-specifically expanded using PHA, IL-2 and allogeneic feeder cells. The presence of the different specificities was assessed 10-14 days after the isolation by tetramer staining. To confirm the presence of the invisible very low frequency T cell specificities, subsequent enrichment and expansion rounds were performed until populations were visible. Specificities that were present in a frequency >1% were clonally expanded and tested for their functional potential.
Results
After 2 or 3 enrichment and expansion rounds, we were able to demonstrate specific T cell populations for 6 CMV specificities from 4 seronegative donors (pp65 A2 NLV (3x), pp65 B7 TPR, 1E-1 A2 VLE, 1E-1 B8 QIK) and 4 EBV specificities from 2 seronegative donors (BMLF-1 A2 GLC, EBNA-3a B7 RPP (2x), BZLF-1 B8 RAK). The CD8/tetramer positive clones from two CMV specificities (pp65 A2 NLV and pp65 B7 TPR) and one EBV specificity (EBNA-3a B7 RPP) were tested for antigen specific reactivity measured by cytokine release (IFN-gamma and GM-CSF) after 24 hours of stimulation with TAP deficient T2 cells and/or allogeneic EBV-LCL exogenously loaded with the relevant peptide. This analysis revealed the presence of clones of high avidity recognizing up to 10^-9M peptide on T2 cells and 10^-8M peptide on allogeneic EBV-LCL. Besides this, all mentioned TAA and MiHA specificities could be isolated from all four HLA-A2 positive donors after 2 to 4 enrichment and expansion rounds, and comprised clones with a range of different functional avidities.
Conclusion
In conclusion, the HLA/peptide streptamer technology allows the simultaneous isolation of multi antigen specific T cell products containing not only highly frequent virus-specific memory T cells, but also EBV and/or CMV specific T cells from the naïve T cell repertoire of seronegative donors, as well as TAA and MiHA specific T cells with differential functional avidities.
Session topic: E-poster
Keyword(s): Cellular therapy, CMV infection, Minor histocompatibility antigen, Tumor antigen
Type: Eposter Presentation
Background
T cell depleted allogeneic stem cell transplantation (aIIoSCT) followed by a donor lymphocyte infusion (DLI) is a potential curative treatment for patients with hematological malignancies. However, between the aIIoSCT and the DLI patients are vulnerable to viral infections and disease relapses. Within a clinical phase I/II study in the EU FP7 program T-control we isolate and adoptively transfer multi-antigen specific T-cell products to prevent these complications. Therefore, we isolate donor T-cells directed against HLA-A1, A2, A24, B7, and/or B8-restricted peptides of CMV, EBV and Adenovirus and HLA-A2-restricted peptides from the tumor-associated antigens (TAA) NY-eso, WT-1, RHAMM, PRAME and proteinase 3, and the minor histocompatibility antigen (MiHA) HA-1h. Due to the relatively high precursor frequency of virus-specific T cells in seropositive donors, memory virus-specific T cells can be easily isolated.
Aims
To investigate the presence and functional activity of the specificities with a very low precursor frequency, e.g. TAA and MiHA specific T cells, and virus specific T cells from seronegative donors.
Methods
HLA-A2, B7, and/or B8-positive CMV seronegative donors (n=5) and EBV seronegative donors (n=2) were selected for isolation of CMV, EBV and AdV-specific T cells. From 4 HLA-A2 positive donors TAA and MiHA specific T cells were isolated. The reversible HLA/peptide streptamer and magnetic nanobead technique (Juno) was used for the isolation out of 1-2*10^9 donor PBMC, using all relevant streptamers according to the HLA-type of the donor. The positive fractions were non-specifically expanded using PHA, IL-2 and allogeneic feeder cells. The presence of the different specificities was assessed 10-14 days after the isolation by tetramer staining. To confirm the presence of the invisible very low frequency T cell specificities, subsequent enrichment and expansion rounds were performed until populations were visible. Specificities that were present in a frequency >1% were clonally expanded and tested for their functional potential.
Results
After 2 or 3 enrichment and expansion rounds, we were able to demonstrate specific T cell populations for 6 CMV specificities from 4 seronegative donors (pp65 A2 NLV (3x), pp65 B7 TPR, 1E-1 A2 VLE, 1E-1 B8 QIK) and 4 EBV specificities from 2 seronegative donors (BMLF-1 A2 GLC, EBNA-3a B7 RPP (2x), BZLF-1 B8 RAK). The CD8/tetramer positive clones from two CMV specificities (pp65 A2 NLV and pp65 B7 TPR) and one EBV specificity (EBNA-3a B7 RPP) were tested for antigen specific reactivity measured by cytokine release (IFN-gamma and GM-CSF) after 24 hours of stimulation with TAP deficient T2 cells and/or allogeneic EBV-LCL exogenously loaded with the relevant peptide. This analysis revealed the presence of clones of high avidity recognizing up to 10^-9M peptide on T2 cells and 10^-8M peptide on allogeneic EBV-LCL. Besides this, all mentioned TAA and MiHA specificities could be isolated from all four HLA-A2 positive donors after 2 to 4 enrichment and expansion rounds, and comprised clones with a range of different functional avidities.
Conclusion
In conclusion, the HLA/peptide streptamer technology allows the simultaneous isolation of multi antigen specific T cell products containing not only highly frequent virus-specific memory T cells, but also EBV and/or CMV specific T cells from the naïve T cell repertoire of seronegative donors, as well as TAA and MiHA specific T cells with differential functional avidities.
Session topic: E-poster
Keyword(s): Cellular therapy, CMV infection, Minor histocompatibility antigen, Tumor antigen
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