NeoTCR-P1, a Novel Neoepitope-Specific Adoptive Cell ......Mar 29, 2019 · NeoTCR-P1 T cells...
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Results Introduction Conclusions Methods 0 200 400 600 800 1000 Polyfunctionality Strenght Index Peptide (nM) 0 10 100 N Peptide (nM) 0 10 100 N 0 200 400 600 800 1000 Polyfunctionality Strenght Index Peptide (nM) 0 10 100 M Peptide (nM) 0 10 100 M neo12 T cells F5 T cells CD4+ CD8+ CD4+ CD8+ NeoTCR-P1, a Novel Neoepitope-Specific Adoptive Cell Therapy, Consists of T Cells with ‘Younger’ Phenotypes that Rapidly Proliferate and Kill Target Cells upon Recognition of Cognate Antigen Barbara Sennino 1 , Andrew Conroy 1 , Bhamini Purandare 1 , Adam Litterman 1 , Kyle Jacoby 1 , Robby Moot 1 , William Lu 1 , Diana Nguyen 1 , Fabrizia Urbinati 1 , Susan Foy 1 , Theresa Hunter 1 , Songming Peng 1 , Olivier Dalmas 1 , Michael Bethune 1 , Tim Park 2 , Alex Franzusoff 1 , Stefanie Mandl 1 1 PACT Pharma, 2 Corporate Drive, South San Francisco, CA 94080, USA. 2 Arcus Biosciences, Inc., 3928 Point Eden Way, Hayward, CA 94545,USA. Abstract: 3758 Figure 1. Total CD4 and CD8 (left panel) subset distribution after laboratory scale manufacturing from blood of healthy donors (black) or patients with cancer (blue) day 13. Final Product CD4 T cell and CD8 T cell phenotype is predominantly Tmsc and Tcm. Healthy donors: n=4 from 3 unique donors; patients: n= 14 from 8 unique donors. Final NeoTCR-P1 product consist mainly of T cells of the “younger” Tscm and Tcm phenotype NeoTCR-P1 T cells rapidly convert to effector cells upon contact with neoantigen NeoTCR-P1 T cells can be manufactured from T cells of both healthy donors or patients with cancer NeoTCR-P1 cells are highly polyfunctional, even when exposed to low concentrations of cognate peptide stimulation Taken together, these ex vivo mechanism-of-action studies demonstrate that PACT NeoTCR-P1 T cells rapidly turn into highly active tumor-killing lymphocytes upon encounter of cognate tumor cells expressing the tumor-exclusive mutated antigen, with the potential to eradicate tumor cells throughout the body. Figure 4. B. Time-lapse microscopy of tumor cell death and T cell proliferation. Neo12 TCR-T cells were co- cultured with K562 tumor cells transfected to express irrelevant peptide-HLA-A2 protein complexes on the surface (left column) or K562-neo12-HLA-A2 expressing cells (right panel). Tumor cells also expressed a variant of green fluorescent protein (GFP or ZsGreen) in a stable and homogeneous manner. Images were collected over 48h and shown here at time 0 (top panels), 24h (middle panels) and 48h (bottom panels). To detect real time apoptosis, a highly-selective phosphatidylserine cyanine fluorescent dye (IncuCyte Annexin V in red) was added to the co-culture. T cells are not labeled in this experiment, but antigen-specific proliferation can be appreciated visually by increased numbers of T cells over the course of 2 days (right column). Figure 3. NeoTCR-P1 T cells expressing neo12 TCR generated from a patient with cancer (melanoma) or a healthy donor showed comparable gene-editing efficiency (% of neoTCR expression), and functional activity as measured by target cell killing, proliferation and cytokine production (IFNγ, IL2 and TNFα) measured in the supernatant using the cytokine bead assay. Mismatched: surrogate tumor target cells expressing MART1-HLA-A2 complex; Positive control: surrogate tumor cells expressing neo12-HLA-A2 complex. No activity was observed with mock control T cells. Figure 5. Graphs showing the percentage of CD4 and CD8 NeoTCR-P1 T cells engineered to either express neo12 or F5 TCR, that secrete 2, 3, 4 or greater than- equal to 5 cytokines (shades of orange) when encountering cognate antigen. T cells were co-cultured with target cells pulsed with no peptide, 10 nM or 100 nM specific peptide, or with target cells constitutively expressing peptide-HLA on their surface (N=neo12 HLA-A2 cells; M=MART1 HLA-A2 cells). Secreted cytokine levels were assessed after 24 hours of co-culture using IsoPlexis single cell secretome analysis. Cells secreting 2 or more cytokines are considered polyfunctional. The efficiency of gene editing of neo12 TCR or F5 neoTCR into the total T cell population was ~40%. Figure 6. Polyfunctional strength index (PSI) is defined as the number of T cells secreting greater than 2 effector molecules per cell (polyfunctional T cells in Fig. 5), multiplied by mean fluorescence intensity (MFI) of the proteins secreted by those cells. For both neo12 and F5 TCR-T cell products, the polyfunctional T cell responses were strongly driven by secretion of effector proteins (in green), including granzyme B, IFNγ, MIP1α, perforin, TNFα, TNFβ. The secretion of the following molecules from target-activated TCR-T cells were also detected: Stimulatory category included IL8 (in blue); Regulatory category included sCD137, sCD40L (in pink); Chemo-attractive category included MIP-1b (in purple). NeoTCR-P1 T cells rapidly convert to effector cells on antigen exposure NeoTCR-P1 T cells are mainly of the “younger” Tscm & Tcm phenotype Specific killing of antigen-expressing surrogate tumor target cells and antigen-specific proliferation of NeoTCR-P1 T cells Baseline (t=0) After co-culture (1h interval between images) NeoAg tumor cells PACT NeoTCR T cells (neo12 T cells) Non-edited T cells Target cells expressing neo12 pHLA Control target cells pHLA (irrelevant peptide) Live tumor target cells Apoptotic cells (Annexin V staining) Neo12 T cells are not labeled A B Edited T cells: CD8 and CD4 T cells from healthy donors or patients with cancer were precision genome engineered (Jacoby et al. Abstract: 4858) to express the neo12 TCR or the F5 TCR. Co-Culture assay: NeoTCR-P1 T cells were co-cultured with K562 cells expressing HLA-A02 pulsed with different concentrations of peptides (0-1000 nM) or with K562 cells constitutively expressing peptide-HLA complex at a final Product to Target (P:T) ratio of 4:1. Cytokine secretion was measured in the cell supernatant at 24h using the BD Cytokine Bead Array (CBA) Human Th1/Th2 Cytokine Kit II. Target cell killing and T cell proliferation were evaluated at 48h and 72h, respectively. IsoPlexis single cell secretome analysis: Polyfunctional cytokine secretion of NeoTCR-P1 T cells was assessed by single cell secretome analysis using the IsoPlexis platform. This approach allows for the simultaneous analysis of 32 secreted proteins on a single cell basis. Briefly, NeoTCR-P1 T cells were stimulated for 24 h and then loaded onto an single-cell barcode chip containing ~12000 microchambers prepatterned with a 32-plex, antibody array. T cells were imaged to identify single-cell locations and incubated for additional 16 h. Single-cell cytokine signals were then captured and digitized with a microarray scanner. Polyfunctional T cells were defined as cells co- secreting at least 2 proteins from the pre-specified panel per cell. Furthermore, the Polyfunctional Strength Index (PSI) of each sample was computed using a pre-specified formula, defined as the percentage of polyfunctional cells, multiplied by mean fluorescence intensity (MFI) of the proteins secreted by those cells. CD4+ CD8+ 0 20 40 60 80 100 CD4 and CD8 % of CD3+ T cells Healthy Patient Tn Tmsc Tcm Ttm Tem Teff 0 10 20 30 CD4 Subsets % of CD3+ T cells Tn Tmsc Tcm Ttm Tem Teff 0 20 40 60 80 CD8 Subsets % of CD3+ T cells CD45RA CD62L CD27 CD95 + + + - + + + + - + + + - - - + - - - + + - +/- + CD45RA CD62L CD27 CD95 + + + - + + + + - + + + - - - + - - - + + - +/- + Comparable antigen-specific activity of NeoTCR-P1 T cells derived from patients with cancer or healthy donors Healthy Patient 0 20 40 60 80 100 % TCR Edited cells 0.01 0.1 1 10 100 1000 0 20 40 60 80 100 % kill Mismatched Positive control 0.01 0.1 1 10 100 1000 0 20 40 60 80 100 % dividing cells Mismatched Positive control Healthy donor neo12 T cells Patient neo12 T cells Mock IFN γ IL2 TNF α 0.00 0.02 0.04 0.06 0.08 Amount (pg/edited T cells) Peptide (nM) Peptide (nM) % neoTCR expression Killing Proliferation Cytokine production (at 100 nM peptide) PACT Pharma is developing NeoTCR-P1, a personalized adoptive T cell therapy, which is composed of apheresis-derived, patient- autologous, CD8 and CD4 T cells that have been precision genome engineered to express one autologous T cell receptor targeting a neoepitope presented exclusively on the surface of the patient’s tumor cells (neoTCR) (Peng et al. Abstract: 3714; Jacoby et al, Abstract: 4858) Upon reinfusion of a defined dose into the patient, NeoTCR-P1 cells are anticipated to traffic to tissues harboring tumor cells presenting the neoE peptide in the context of the autologous cognate HLA receptor. Recognition of the cognate neoE-HLA complexes will trigger T cell proliferation and secretion of effector molecules from the engineered T cells. To demonstrate these activities, ex vivo mechanism-of-action studies were performed by generating NeoTCR-P1 T cells derived from the blood of healthy donors or patients with cancer. T cells were engineered to express two model TCRs: neo12, a neoTCR isolated from a melanoma patient’s PBMCs using the imPACT technology, and F5 TCR, a clinically validated TCR against the tumor antigen MART1. Phenotypic analysis was performed to characterize the T cell subset distribution of the NeoTCR-P1 final cell product. Antigen-specific activity was characterized by measuring target- specific killing, proliferation and cytokine production. 0 10 20 30 40 Polyfunctionality (% of Sample) Peptide (nM) 0 10 100 N Peptide (nM) 0 10 100 N 0 10 20 30 40 Polyfunctionality (% of Sample) Peptide (nM) 0 10 100 M Peptide (nM) 0 10 100 M neo12 T cells F5 T cells CD4+ CD8+ CD4+ CD8+ Figure 4. A. NeoTCR-P1 T cells expressing mCherry (red) and neo12 TCR were co-cultured with tumor cells expressing ZsGreen and the specific neoantigen (neo12) and HLA-A02 complex. At baseline, edited (red) and non-edited T cells (grey) were round and smaller in size than tumor cells (green). After encountering antigen- expressing tumor cells, neoTCR T cells became elongated, formed immunological synapses and killed the target tumor cell. The non-edited T cells did not show any cytotoxic activity. Images were taken at 1h intervals. CD4 and CD8 NeoTCR-P1 T cells are polyfunctional NeoTCR-P1 polyfunctional responses are strongly driven by proteins associated with effector function Tmsc Tcm Ttm/Tem Teff neo12 T cells + K562 HLA-A02 (- control) neo12 T cells + K562 HLA-A02 + neo12 % of CD8 T cells 24 48 72 0 24 48 72 0 24 48 72 0 24 48 72 0 0 20 40 60 80 Figure 2. NeoTCR-P1 T cells expressing the neo12 TCR were co-cultured with tumor cells pulsed with cognate peptide (K562 neo12 peptide-HLA-A2 displaying tumor cells, red circle) for up to 72h. No changes in phenotype were observed when NeoTCR-P1 T cells were co-cultured with tumor cells alone (K562 HLA-A2 displaying, negative control tumor cells, black triangles). The 0h time point was T cells alone. Effector Stimulatory Chemo-attractive Regulatory 2 proteins 3 proteins 4 proteins 5+ proteins Day 0, 0:00 Day 0, 0:00 Day 1, 0:00 Day 1, 0:00 Day 2, 18:00 Day 2, 18:00
Transcript of NeoTCR-P1, a Novel Neoepitope-Specific Adoptive Cell ......Mar 29, 2019 · NeoTCR-P1 T cells...
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NeoTCR-P1, a Novel Neoepitope-Specific Adoptive Cell Therapy, Consists of T Cells with ‘Younger’ Phenotypes that Rapidly Proliferate and Kill Target Cells upon Recognition of Cognate Antigen
Barbara Sennino1, Andrew Conroy1, Bhamini Purandare1, Adam Litterman1, Kyle Jacoby1, Robby Moot1, William Lu1, Diana Nguyen1, Fabrizia Urbinati1, Susan Foy1, Theresa Hunter1, Songming Peng1, Olivier Dalmas1, Michael Bethune1, Tim Park2, Alex Franzusoff1, Stefanie Mandl1
1PACT Pharma, 2 Corporate Drive, South San Francisco, CA 94080, USA. 2Arcus Biosciences, Inc., 3928 Point Eden Way, Hayward, CA 94545,USA. Abstract: 3758
Figure 1. Total CD4 and CD8 (left panel) subset distribution after laboratory scale manufacturing from blood ofhealthy donors (black) or patients with cancer (blue) day 13. Final Product CD4 T cell and CD8 T cell phenotype ispredominantly Tmsc and Tcm. Healthy donors: n=4 from 3 unique donors; patients: n= 14 from 8 unique donors.
Final NeoTCR-P1 product consist mainly of T cells of the “younger” Tscm and Tcmphenotype
NeoTCR-P1 T cells rapidly convert to effector cells upon contact with neoantigen NeoTCR-P1 T cells can be manufactured from T cells of both healthy donors or patients
with cancer NeoTCR-P1 cells are highly polyfunctional, even when exposed to low concentrations of
cognate peptide stimulation
Taken together, these ex vivo mechanism-of-action studies demonstrate that PACTNeoTCR-P1 T cells rapidly turn into highly active tumor-killing lymphocytes uponencounter of cognate tumor cells expressing the tumor-exclusive mutated antigen, withthe potential to eradicate tumor cells throughout the body.
Figure 4. B. Time-lapse microscopy of tumor cell death and T cell proliferation. Neo12 TCR-T cells were co-cultured with K562 tumor cells transfected to express irrelevant peptide-HLA-A2 protein complexes on thesurface (left column) or K562-neo12-HLA-A2 expressing cells (right panel). Tumor cells also expressed a variant ofgreen fluorescent protein (GFP or ZsGreen) in a stable and homogeneous manner. Images were collected over48h and shown here at time 0 (top panels), 24h (middle panels) and 48h (bottom panels). To detect real timeapoptosis, a highly-selective phosphatidylserine cyanine fluorescent dye (IncuCyte Annexin V in red) was addedto the co-culture. T cells are not labeled in this experiment, but antigen-specific proliferation can be appreciatedvisually by increased numbers of T cells over the course of 2 days (right column).
Figure 3. NeoTCR-P1 T cells expressing neo12 TCR generated from a patient with cancer (melanoma) or a healthydonor showed comparable gene-editing efficiency (% of neoTCR expression), and functional activity as measured bytarget cell killing, proliferation and cytokine production (IFNγ, IL2 and TNFα) measured in the supernatant using thecytokine bead assay. Mismatched: surrogate tumor target cells expressing MART1-HLA-A2 complex; Positive control:surrogate tumor cells expressing neo12-HLA-A2 complex. No activity was observed with mock control T cells.
Figure 5. Graphs showing the percentage of CD4 and CD8 NeoTCR-P1 T cells engineered to either express neo12 or F5TCR, that secrete 2, 3, 4 or greater than- equal to 5 cytokines (shades of orange) when encountering cognate antigen. Tcells were co-cultured with target cells pulsed with no peptide, 10 nM or 100 nM specific peptide, or with target cellsconstitutively expressing peptide-HLA on their surface (N=neo12 HLA-A2 cells; M=MART1 HLA-A2 cells). Secreted cytokinelevels were assessed after 24 hours of co-culture using IsoPlexis single cell secretome analysis. Cells secreting 2 or morecytokines are considered polyfunctional. The efficiency of gene editing of neo12 TCR or F5 neoTCR into the total T cellpopulation was ~40%.
Figure 6. Polyfunctional strength index (PSI) is defined as the number of T cells secreting greater than 2 effectormolecules per cell (polyfunctional T cells in Fig. 5), multiplied by mean fluorescence intensity (MFI) of the proteinssecreted by those cells. For both neo12 and F5 TCR-T cell products, the polyfunctional T cell responses were stronglydriven by secretion of effector proteins (in green), including granzyme B, IFNγ, MIP1α, perforin, TNFα, TNFβ. Thesecretion of the following molecules from target-activated TCR-T cells were also detected: Stimulatory category includedIL8 (in blue); Regulatory category included sCD137, sCD40L (in pink); Chemo-attractive category included MIP-1b (inpurple).
NeoTCR-P1 T cells rapidly convert to effector cells on antigen exposure
NeoTCR-P1 T cells are mainly of the “younger” Tscm & Tcm phenotype
Specific killing of antigen-expressing surrogate tumor target cells and antigen-specific proliferation of NeoTCR-P1 T cells
Baseline (t=0) After co-culture (1h interval between images)
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Co-Culture assay: NeoTCR-P1 T cells were co-cultured with K562cells expressing HLA-A02 pulsed with different concentrations ofpeptides (0-1000 nM) or with K562 cells constitutively expressingpeptide-HLA complex at a final Product to Target (P:T) ratio of 4:1.Cytokine secretion was measured in the cell supernatant at 24husing the BD Cytokine Bead Array (CBA) Human Th1/Th2 CytokineKit II. Target cell killing and T cell proliferation were evaluated at 48hand 72h, respectively.
IsoPlexis single cell secretome analysis: Polyfunctional cytokinesecretion of NeoTCR-P1 T cells was assessed by single cell secretomeanalysis using the IsoPlexis platform. This approach allows for thesimultaneous analysis of 32 secreted proteins on a single cell basis.Briefly, NeoTCR-P1 T cells were stimulated for 24 h and then loadedonto an single-cell barcode chip containing ~12000 microchambersprepatterned with a 32-plex, antibody array. T cells were imaged toidentify single-cell locations and incubated for additional 16 h.Single-cell cytokine signals were then captured and digitized with amicroarray scanner. Polyfunctional T cells were defined as cells co-secreting at least 2 proteins from the pre-specified panel per cell.Furthermore, the Polyfunctional Strength Index (PSI) of each samplewas computed using a pre-specified formula, defined as thepercentage of polyfunctional cells, multiplied by mean fluorescenceintensity (MFI) of the proteins secreted by those cells.
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PACT Pharma is developing NeoTCR-P1, a personalized adoptiveT cell therapy, which is composed of apheresis-derived, patient-autologous, CD8 and CD4 T cells that have been precision genomeengineered to express one autologous T cell receptor targeting aneoepitope presented exclusively on the surface of the patient’stumor cells (neoTCR) (Peng et al. Abstract: 3714; Jacoby et al,Abstract: 4858)
Upon reinfusion of a defined dose into the patient, NeoTCR-P1cells are anticipated to traffic to tissues harboring tumor cellspresenting the neoE peptide in the context of the autologouscognate HLA receptor. Recognition of the cognate neoE-HLAcomplexes will trigger T cell proliferation and secretion of effectormolecules from the engineered T cells.
To demonstrate these activities, ex vivo mechanism-of-actionstudies were performed by generating NeoTCR-P1 T cells derivedfrom the blood of healthy donors or patients with cancer. T cellswere engineered to express two model TCRs: neo12, a neoTCRisolated from a melanoma patient’s PBMCs using the imPACTtechnology, and F5 TCR, a clinically validated TCR against the tumorantigen MART1. Phenotypic analysis was performed to characterizethe T cell subset distribution of the NeoTCR-P1 final cell product.Antigen-specific activity was characterized by measuring target-specific killing, proliferation and cytokine production.
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Figure 4. A. NeoTCR-P1 T cells expressing mCherry (red) and neo12 TCR were co-cultured with tumor cellsexpressing ZsGreen and the specific neoantigen (neo12) and HLA-A02 complex. At baseline, edited (red) andnon-edited T cells (grey) were round and smaller in size than tumor cells (green). After encountering antigen-expressing tumor cells, neoTCR T cells became elongated, formed immunological synapses and killed the targettumor cell. The non-edited T cells did not show any cytotoxic activity. Images were taken at 1h intervals.
CD4 and CD8 NeoTCR-P1 T cells are polyfunctional
NeoTCR-P1 polyfunctional responses are strongly driven by proteins associated with effector function
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Figure 2. NeoTCR-P1 T cells expressing the neo12 TCR were co-cultured with tumor cells pulsed with cognate peptide(K562 neo12 peptide-HLA-A2 displaying tumor cells, red circle) for up to 72h. No changes in phenotype wereobserved when NeoTCR-P1 T cells were co-cultured with tumor cells alone (K562 HLA-A2 displaying, negative controltumor cells, black triangles). The 0h time point was T cells alone.
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