e alpha phage display library  (Eureka Therapeutics)

Journal: Science translational medicine

Article Title: GPRC5D is a target for the immunotherapy of multiple myeloma with rationally designed CAR T cells

doi: 10.1126/scitranslmed.aau7746

Figure Lengend Snippet: (A) Human B cell–derived scFv phage display library screening strategy. Positive “hits” were confirmed by scFv binding to NIH-3T3 fibroblasts expressing GPRC5D, but not those expressing an irrelevant protein. Sequencing identified individual clones, which underwent a second validation step of binding to human MM cell lines MM.1S and NCI-H929, but not to the acute myeloid leukemia cell line SET2. scFv clones with the strongest specific binding to the MM cell lines were selected for cloning into a CAR vector. (B) Linear, conformational, and discontinuous epitope binding of a subset of GPRC5D-targeted scFvs assessed by enzyme-linked immunosorbent assay (ELISA)–based technology. (C) GPRC5D-targeted scFvs were cloned into CAR constructs including one of three spacers of varying lengths (S), a CD28 transmembrane (TM) domain, and 4–1BB and CD3ζ signaling domains. (D) Antigen-independent (tonic) signaling of CARs containing the indicated scFvs and spacers. Jurkat Nur77-RFP reporter cells were transduced with 1 of 42 CAR/green fluorescent protein (GFP) bicistronic constructs. Viable GFP+ Jurkat cells (5 × 105) were plated and monitored for RFP expression 11 days after transduction in the absence of target antigen. Expression of both RFP and GFP indicated tonic signaling; expression of GFP alone indicated CAR transduced without tonic signaling. (E) Antigen-dependent versus antigen-independent signaling of candidate CARs. Antigen-dependent signalling was measured after culturing Jurkat Nur77-RFP reporter cells 2:1 with MM.1S cells (expressing endogenous GPRC5D) for 20 hours. Percent CAR T cell signaling represents the proportion of GFP+ (CAR-transduced) cells that are also RFP+ (activated). Data are representative of two experiments.

Article Snippet: Human scFv phage display library screen A human B cell–derived scFv phage display library (E-ALPHA, Eureka Therapeutics) was panned using NIH-3T3 aAPCs stably expressing GPRC5D.

Techniques: Derivative Assay, Library Screening, Binding Assay, Expressing, Sequencing, Clone Assay, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Construct, Transduction

Journal: Science translational medicine

Article Title: GPRC5D is a target for the immunotherapy of multiple myeloma with rationally designed CAR T cells

doi: 10.1126/scitranslmed.aau7746

Figure Lengend Snippet: (A) Cell killing of OPM2–firefly luciferase (ffLuc) MM cells induced by CAR T cells incorporating the indicated scFv after 24 hours of coculture, as indicated by adenosine triphosphate–dependent bioluminescence after addition of luciferin; normalized to tumor cell–alone control (pooled data from two experiments each performed in triplicate, mean ± SEM; P < 0.001). Effectors counted as CAR+ viable cells. (B) Flow cytometry of primary BMMCs from a patient with multiply relapsed MM after overnight coculture with CAR T cells incorporating the indicated scFv at a 1:1 ratio of CAR+ viable T cells to BMMCs. To avoid contribution by T cell expansion or transduction efficiency, percentage of CD3− cells is reported (representative of primary samples from five patients with MM). (C) Cytokines produced by CAR+ viable T cells incorporating the indicated scFv after 1:1 coculture with OPM2 MM cells or alone for 24 hours, measured in the supernatant by multiplex Luminex assay. (D) Proliferation and (E) activation of mock-transduced or GPRC5D(109)-expressing CAR T cells cultured alone, with B-ALL (Nalm6; GPRC5D−), or with MM (OPM2; endogenous GPRC5D+) cells at a 1:1 ratio of CAR+ viable T cells to tumor cells. T cells were stained with CellTrace Violet (CTV) before coculture and stained for CD4, CD8, and CD25 after 72 hours. (D) Proliferation indicated by dilution of CTV fluorescence. (E) Activation indicated by increased CD25 fluorescence. Representative data are from two or more experiments, unless otherwise stated.

Article Snippet: Human scFv phage display library screen A human B cell–derived scFv phage display library (E-ALPHA, Eureka Therapeutics) was panned using NIH-3T3 aAPCs stably expressing GPRC5D.

Techniques: Luciferase, Flow Cytometry, Transduction, Produced, Multiplex Assay, Luminex, Activation Assay, Expressing, Cell Culture, Staining, Fluorescence

Journal: Science translational medicine

Article Title: GPRC5D is a target for the immunotherapy of multiple myeloma with rationally designed CAR T cells

doi: 10.1126/scitranslmed.aau7746

Figure Lengend Snippet: (A to C) Mice were injected with 3 × 106 human CAR+ viable T cells expressing a CAR containing a human/murine cross-reactive anti-GPRC5D scFv (clone 122) 14 days after injection of OPM2; nontreated mice and mice injected with mock-transduced T cells or T cells expressing a CAR containing scFv clone 109 (which recognizes only human GPRC5D) served as controls. (A) Body mass, (B) body temperature, and (C) BLI of OPM2-ffLuc cells. (D to E) Three cynomolgus monkeys were injected with autologous cynomolgus T cells modified to express a CAR containing a human/cynomolgus cross-reactive anti-GPRC5D scFv clone 108 (1 × 107 CAR+caspase− T cells/kg) and 1 × 107 cGPRC5D+caspase− autologous aAPC T cells/kg on day 4 (D4) and then euthanized for pathologic evaluation at 21 days (full protocol in fig. S14). (D) Body temperature, (E) body mass, and (F) pathologic investigation of skin, lung, and small intestine (representative images). All abnormal pathologic findings are summarized in Table 1.

Article Snippet: Human scFv phage display library screen A human B cell–derived scFv phage display library (E-ALPHA, Eureka Therapeutics) was panned using NIH-3T3 aAPCs stably expressing GPRC5D.

Techniques: Injection, Expressing, Modification

Journal: Science translational medicine

Article Title: GPRC5D is a target for the immunotherapy of multiple myeloma with rationally designed CAR T cells

doi: 10.1126/scitranslmed.aau7746

Figure Lengend Snippet: (A) Binding of HEK293 cells transiently expressing a library of human GPCRs with cytoplasmic GFP to cocultured HEK293 cells transiently expressing anti-GPRC5D scFv clone 109, a long spacer, and cytoplasmic mCherry (both cell types in suspension), quantified by automated flow cytometric analysis. Prespecified threshold for significance (dashed line): Z score = 3, P < 0.0027. (B) Binding of anti-GPRC5D scFv clone 109-mIgG2a Fc chimeric antibody to HEK293 cells expressing the indicated cell surface proteins. Confirmation of binding to potential off-target proteins and nonspecific binders identified in a microarray screen of >4400 transmembrane proteins is shown. ZsGreen1, transfection control; Isotype, irrelevant scFv-mIgG2a Fc, negative control; CTLA-4/CD86 interaction, positive control. (C) Evaluation of GPRC5D(109) CAR activation by potential off-target proteins PCDH1A and FCGR2A. Jurkat Nur77-RFP activation reporter cells expressing a bicistronic plasmid containing a GPRC5D(109) CAR and GFP were cocultured with K562 cells expressing the indicated antigens, GPRC5D (positive control), or BCMA (negative control). Activation is determined as %RFP+GFP+/total GFP+ cells.

Article Snippet: Human scFv phage display library screen A human B cell–derived scFv phage display library (E-ALPHA, Eureka Therapeutics) was panned using NIH-3T3 aAPCs stably expressing GPRC5D.

Techniques: Binding Assay, Expressing, Microarray, Transfection, Negative Control, Positive Control, Activation Assay, Plasmid Preparation

Journal: Science translational medicine

Article Title: GPRC5D is a target for the immunotherapy of multiple myeloma with rationally designed CAR T cells

doi: 10.1126/scitranslmed.aau7746

Figure Lengend Snippet: NSG mice were injected intravenously with OPM2-ffLuc cells to establish a bone marrow–tropic MM xenograft and then treated with a single intravenous injection of CAR T cells at the indicated time and dose. (A) Survival of mice treated 14 days after OPM2 injection with 3 × 106 4-1BB–containing CAR+ viable T cells incorporating the indicated anti-GPRC5D scFv clones (n = 8 per arm). (B) Survival of mice treated at 21 days after OPM2 injection with 3 × 106 CAR+ viable T cells gene-modified to express a bicistronic construct encoding extGLuc and a CAR incorporating scFv CD19(SJ25C1) or GPRC5D(109) and either a 4-1BB or CD28 costimulatory domain (n = 5 per arm). (C) Tumor burden (d-luciferin BLI of OPM-ffLuc) of mice from (B). (D) CAR T cell homing (coelenterazine BLI of extGLuc CAR T cells) of mice from (B) performed on day 7 after CAR T cell treatment. (E and F) Dose response of GPRC5Dand BCMA-targeted CAR+ viable T cells, administered 14 days after OPM2 injection (n = 8 mice per arm). (E) Tumor burden as assessed by BLI of OPM-ffLuc and (F) survival. (G) NSG mice were injected with a mixed population of unmanipulated OPM2WT (75%) + GFP/ffLuc+ CRISPR-mediated OPM2BCMA-KO (25%). On day 8 and day 16, mice were injected with the indicated CAR+ viable T cells. BLI monitors only OPM2BCMA-KO, which are the only cells to express GFP/ffLuc (n = 5 mice per arm, representative of two experiments). Note that, in the two BLI images of mice with the highest tumor burden, a higher minimum color scale threshold was used compared to the shown scale to remove BLI scatter. All P values shown are relative to mock-transduced (A, E, and F) or irrelevantly targeted (B) CAR T cells. Average myeloma distribution and percent weight change were assessed by two-way analysis of variance (ANOVA). P ≤ 0.05 is considered significant. The log-rank (Mantel-Cox) test was used to calculate statistical significance of survival experiments, with P values adjusted for multiple comparisons via Benjamini-Hochberg correction.

Article Snippet: Human scFv phage display library screen A human B cell–derived scFv phage display library (E-ALPHA, Eureka Therapeutics) was panned using NIH-3T3 aAPCs stably expressing GPRC5D.

Techniques: Injection, Clone Assay, Modification, Construct, CRISPR

Journal: Science translational medicine

Article Title: GPRC5D is a target for the immunotherapy of multiple myeloma with rationally designed CAR T cells

doi: 10.1126/scitranslmed.aau7746

Figure Lengend Snippet: (A) Human B cell–derived scFv phage display library screening strategy. Positive “hits” were confirmed by scFv binding to NIH-3T3 fibroblasts expressing GPRC5D, but not those expressing an irrelevant protein. Sequencing identified individual clones, which underwent a second validation step of binding to human MM cell lines MM.1S and NCI-H929, but not to the acute myeloid leukemia cell line SET2. scFv clones with the strongest specific binding to the MM cell lines were selected for cloning into a CAR vector. (B) Linear, conformational, and discontinuous epitope binding of a subset of GPRC5D-targeted scFvs assessed by enzyme-linked immunosorbent assay (ELISA)–based technology. (C) GPRC5D-targeted scFvs were cloned into CAR constructs including one of three spacers of varying lengths (S), a CD28 transmembrane (TM) domain, and 4–1BB and CD3ζ signaling domains. (D) Antigen-independent (tonic) signaling of CARs containing the indicated scFvs and spacers. Jurkat Nur77-RFP reporter cells were transduced with 1 of 42 CAR/green fluorescent protein (GFP) bicistronic constructs. Viable GFP+ Jurkat cells (5 × 105) were plated and monitored for RFP expression 11 days after transduction in the absence of target antigen. Expression of both RFP and GFP indicated tonic signaling; expression of GFP alone indicated CAR transduced without tonic signaling. (E) Antigen-dependent versus antigen-independent signaling of candidate CARs. Antigen-dependent signalling was measured after culturing Jurkat Nur77-RFP reporter cells 2:1 with MM.1S cells (expressing endogenous GPRC5D) for 20 hours. Percent CAR T cell signaling represents the proportion of GFP+ (CAR-transduced) cells that are also RFP+ (activated). Data are representative of two experiments.

Article Snippet: A human B cell–derived scFv phage display library (E-ALPHA, Eureka Therapeutics) was panned using NIH-3T3 aAPCs stably expressing GPRC5D.

Techniques: Derivative Assay, Library Screening, Binding Assay, Expressing, Sequencing, Clone Assay, Biomarker Discovery, Cloning, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Construct, Transduction

Journal: Science translational medicine

Article Title: GPRC5D is a target for the immunotherapy of multiple myeloma with rationally designed CAR T cells

doi: 10.1126/scitranslmed.aau7746

Figure Lengend Snippet: (A) Cell killing of OPM2–firefly luciferase (ffLuc) MM cells induced by CAR T cells incorporating the indicated scFv after 24 hours of coculture, as indicated by adenosine triphosphate–dependent bioluminescence after addition of luciferin; normalized to tumor cell–alone control (pooled data from two experiments each performed in triplicate, mean ± SEM; P < 0.001). Effectors counted as CAR+ viable cells. (B) Flow cytometry of primary BMMCs from a patient with multiply relapsed MM after overnight coculture with CAR T cells incorporating the indicated scFv at a 1:1 ratio of CAR+ viable T cells to BMMCs. To avoid contribution by T cell expansion or transduction efficiency, percentage of CD3− cells is reported (representative of primary samples from five patients with MM). (C) Cytokines produced by CAR+ viable T cells incorporating the indicated scFv after 1:1 coculture with OPM2 MM cells or alone for 24 hours, measured in the supernatant by multiplex Luminex assay. (D) Proliferation and (E) activation of mock-transduced or GPRC5D(109)-expressing CAR T cells cultured alone, with B-ALL (Nalm6; GPRC5D−), or with MM (OPM2; endogenous GPRC5D+) cells at a 1:1 ratio of CAR+ viable T cells to tumor cells. T cells were stained with CellTrace Violet (CTV) before coculture and stained for CD4, CD8, and CD25 after 72 hours. (D) Proliferation indicated by dilution of CTV fluorescence. (E) Activation indicated by increased CD25 fluorescence. Representative data are from two or more experiments, unless otherwise stated.

Article Snippet: A human B cell–derived scFv phage display library (E-ALPHA, Eureka Therapeutics) was panned using NIH-3T3 aAPCs stably expressing GPRC5D.

Techniques: Luciferase, Control, Flow Cytometry, Transduction, Produced, Multiplex Assay, Luminex, Activation Assay, Expressing, Cell Culture, Staining, Fluorescence

Journal: Science translational medicine

Article Title: GPRC5D is a target for the immunotherapy of multiple myeloma with rationally designed CAR T cells

doi: 10.1126/scitranslmed.aau7746

Figure Lengend Snippet: (A to C) Mice were injected with 3 × 106 human CAR+ viable T cells expressing a CAR containing a human/murine cross-reactive anti-GPRC5D scFv (clone 122) 14 days after injection of OPM2; nontreated mice and mice injected with mock-transduced T cells or T cells expressing a CAR containing scFv clone 109 (which recognizes only human GPRC5D) served as controls. (A) Body mass, (B) body temperature, and (C) BLI of OPM2-ffLuc cells. (D to E) Three cynomolgus monkeys were injected with autologous cynomolgus T cells modified to express a CAR containing a human/cynomolgus cross-reactive anti-GPRC5D scFv clone 108 (1 × 107 CAR+caspase− T cells/kg) and 1 × 107 cGPRC5D+caspase− autologous aAPC T cells/kg on day 4 (D4) and then euthanized for pathologic evaluation at 21 days (full protocol in fig. S14). (D) Body temperature, (E) body mass, and (F) pathologic investigation of skin, lung, and small intestine (representative images). All abnormal pathologic findings are summarized in Table 1.

Article Snippet: A human B cell–derived scFv phage display library (E-ALPHA, Eureka Therapeutics) was panned using NIH-3T3 aAPCs stably expressing GPRC5D.

Techniques: Injection, Expressing, Modification

Journal: Science translational medicine

Article Title: GPRC5D is a target for the immunotherapy of multiple myeloma with rationally designed CAR T cells

doi: 10.1126/scitranslmed.aau7746

Figure Lengend Snippet: (A) Binding of HEK293 cells transiently expressing a library of human GPCRs with cytoplasmic GFP to cocultured HEK293 cells transiently expressing anti-GPRC5D scFv clone 109, a long spacer, and cytoplasmic mCherry (both cell types in suspension), quantified by automated flow cytometric analysis. Prespecified threshold for significance (dashed line): Z score = 3, P < 0.0027. (B) Binding of anti-GPRC5D scFv clone 109-mIgG2a Fc chimeric antibody to HEK293 cells expressing the indicated cell surface proteins. Confirmation of binding to potential off-target proteins and nonspecific binders identified in a microarray screen of >4400 transmembrane proteins is shown. ZsGreen1, transfection control; Isotype, irrelevant scFv-mIgG2a Fc, negative control; CTLA-4/CD86 interaction, positive control. (C) Evaluation of GPRC5D(109) CAR activation by potential off-target proteins PCDH1A and FCGR2A. Jurkat Nur77-RFP activation reporter cells expressing a bicistronic plasmid containing a GPRC5D(109) CAR and GFP were cocultured with K562 cells expressing the indicated antigens, GPRC5D (positive control), or BCMA (negative control). Activation is determined as %RFP+GFP+/total GFP+ cells.

Article Snippet: A human B cell–derived scFv phage display library (E-ALPHA, Eureka Therapeutics) was panned using NIH-3T3 aAPCs stably expressing GPRC5D.

Techniques: Binding Assay, Expressing, Suspension, Microarray, Transfection, Control, Negative Control, Positive Control, Activation Assay, Plasmid Preparation

Journal: Science translational medicine

Article Title: GPRC5D is a target for the immunotherapy of multiple myeloma with rationally designed CAR T cells

doi: 10.1126/scitranslmed.aau7746

Figure Lengend Snippet: NSG mice were injected intravenously with OPM2-ffLuc cells to establish a bone marrow–tropic MM xenograft and then treated with a single intravenous injection of CAR T cells at the indicated time and dose. (A) Survival of mice treated 14 days after OPM2 injection with 3 × 106 4-1BB–containing CAR+ viable T cells incorporating the indicated anti-GPRC5D scFv clones (n = 8 per arm). (B) Survival of mice treated at 21 days after OPM2 injection with 3 × 106 CAR+ viable T cells gene-modified to express a bicistronic construct encoding extGLuc and a CAR incorporating scFv CD19(SJ25C1) or GPRC5D(109) and either a 4-1BB or CD28 costimulatory domain (n = 5 per arm). (C) Tumor burden (d-luciferin BLI of OPM-ffLuc) of mice from (B). (D) CAR T cell homing (coelenterazine BLI of extGLuc CAR T cells) of mice from (B) performed on day 7 after CAR T cell treatment. (E and F) Dose response of GPRC5Dand BCMA-targeted CAR+ viable T cells, administered 14 days after OPM2 injection (n = 8 mice per arm). (E) Tumor burden as assessed by BLI of OPM-ffLuc and (F) survival. (G) NSG mice were injected with a mixed population of unmanipulated OPM2WT (75%) + GFP/ffLuc+ CRISPR-mediated OPM2BCMA-KO (25%). On day 8 and day 16, mice were injected with the indicated CAR+ viable T cells. BLI monitors only OPM2BCMA-KO, which are the only cells to express GFP/ffLuc (n = 5 mice per arm, representative of two experiments). Note that, in the two BLI images of mice with the highest tumor burden, a higher minimum color scale threshold was used compared to the shown scale to remove BLI scatter. All P values shown are relative to mock-transduced (A, E, and F) or irrelevantly targeted (B) CAR T cells. Average myeloma distribution and percent weight change were assessed by two-way analysis of variance (ANOVA). P ≤ 0.05 is considered significant. The log-rank (Mantel-Cox) test was used to calculate statistical significance of survival experiments, with P values adjusted for multiple comparisons via Benjamini-Hochberg correction.

Article Snippet: A human B cell–derived scFv phage display library (E-ALPHA, Eureka Therapeutics) was panned using NIH-3T3 aAPCs stably expressing GPRC5D.

Techniques: Injection, Clone Assay, Modification, Construct, CRISPR

Journal: Science translational medicine

Article Title: GPRC5D is a target for the immunotherapy of multiple myeloma with rationally designed CAR T cells

doi: 10.1126/scitranslmed.aau7746

Figure Lengend Snippet: Summary of pathologic findings from cynomolgus injected with human/cynomolgus cross-reactive anti-GPRC5D(scFv clone 108) CAR T cells.

Article Snippet: A human B cell–derived scFv phage display library (E-ALPHA, Eureka Therapeutics) was panned using NIH-3T3 aAPCs stably expressing GPRC5D.

Techniques: Injection