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3768 rennert

Paul D. Rennert
Paul D. Rennert

1) CAR T cells specific for CD19 have shown remarkable success in treating blood cancers but have had limited effect on solid tumors which lack the CD19 antigen. 2) The IMPACT technology uses fusion proteins containing the extracellular domain of CD19 linked to single-chain antibodies targeting other antigens to bridge CAR19 T cells to solid tumors. 3) Experiments showed CAR19 T cells redirected by a CD19-anti-HER2 fusion protein were cytotoxic against HER2-positive ovarian cancer cells in vitro, demonstrating the IMPACT technology can redirect CAR T cells to new tumor targets through antigen bridging.

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CAR T cells specific for CD19 can be redirected to kill CD19 negative tumors 1 - Introduction Remarkable progress has been made in the treatment of relapsed/refractory Acute Lymphocytic Leukemia and Non- Hodgkin Lymphoma with CAR-CD19 T cells. In contrast, progress against CD19-negative hematological cancers and solid tumors has been limited. Intensive efforts to optimize cellular therapeutics for better ef鍖cacy include provision of cytokine support and countering immuno-suppression. However, lack of suf鍖cient antigen is a signi鍖cant additional hurdle that CAR-T therapeutics for solid tumors must overcome. We present a novel strategy to utilize CD19 for sustained antigen presentation in order to promote cellular therapeutic expansion, ef鍖cacy and persistence. The strategy, called IMPACTTM (Integrated Modules oPtimize Adoptive Cell Therapy), employs a methodology that is modular in design and can be applied to diverse antigens and tumor types, yet retains the well-established advantages of CAR T cells directed to CD19. 2 - Technology Overview IMPACTTM fusion proteins are created by cloning the extracellular domain (ECD) of a CAR T cell target protein (e.g. CD19) to an scFv that recognizes a second target protein. The system is modular: diverse ECD-scFv fusion proteins have been designed and expressed. In one iteration, this fusion protein is puri鍖ed and utlized in conjunction with an exising CAR T cell, e.g. a CD19-anti-Her2 fusion protein is puri鍖ed and added to a culture of CAR19 T cells and Her2+ tumor cells, creating a bridge that triggers CAR19 T cell cytotoxic activity. In another iteration, the CAR19 T cells expresses the CD19- anti-Her2 fusion protein, and this expression is suf鍖cient to create the "cytotoxic bridge" as shown in Figure 1. The normal B cell pools ensures expansion and persistance of the CAR19 T cells in vivo. Figure 1. Bridging CAR19 T cells to Her2+ tumor cells Christine Ambrose1, Lihe Su1, Lan Wu1, Fay Dufort1, Roy R. Lobb1, Andreas Hombach2, Hinrich Abken2, Paul D. Rennert1. 1: Aleta Biotherapeutics, Natick, MA USA; 2: University of Cologne, Cologne, Germany #3768 Tumor cell CD19+ B cell CAR19 T cell anti-CD19 scFv CD19 ECD Her2anti-Her2 scFv 3 - Puri鍖ed IMPACTTM fusion proteins We cloned the human CD19 ECD (two IgC domains) and the scFv derived from the anti-Her2 mAb trastuzumab into a lentiviral expression system for transfection then puri鍖cation by af鍖nity chromatography and SEC (Lake Pharma). Mono- meric protein was used in our experiments; this monomer is stable in solution. The fusion protein carries a C-terminal His tag. Control proteins (CD19 ECD-His and CD22 ECD 1-3 trastuzumab scFv) were also created. Table 1. Af鍖nities of fusion proteins for target molecules, determined by ELISA analyses biotin-Her2 His-scFv CD19 ECD Figure 2. FACs binding of CD19-anti-Her2 scFv and CD22- anti-Her2 scFv fusion proteins to SKOV-3 ovarian cancer cells 4 - Construction and characterization of CAR19 T cells The scFv from anti-CD19 mAb FMC63 was cloned in frame with a FLAG-tagged linker, the CD28 transmembrane sequence and the cytoplasmic domains of CD28, 4-1BB and CD3龍. The FLAG tag was encoded to facilitate detection of the CAR by FACs analysis following transduction into primary human T cells (Figure 3). The CAR19 T cells were tested for cytotoxic activity in a cell killing assay using B cell lymphoma lines (ATCC). One example is shown in Figure 3. anti-CD19 mAb FMC63 biotinylated Her2 protein CD19-anti- Her2 0.4nM 3.15nM CD22-anti- Her2 1.92nM CD19 5.1nM SKOV-3 mAb-PE anti-His-PE CD19-anti- Her2 2nM 9nM CD22-anti- Her2 2.2nM 1nM FMC63-PE anti-His-PE His-scFv Her2 CD19 ECD Tumor cell anti-CD19- coated well ≒Her-2---biotin anti-CD22-PE anti-His-PE Figure 3. Expression of the CD19 CAR and cytotoxicity against a CD19+ B cell lymphoma line 5 - Fusion protein mediated cytotoxicity The ability of the fusion proteins to bridge to CAR19 cells and mediate killing of Her2+ SKOV-3 tumor cells was evaluated. First we performed a titration. Figure 4. Cytotoxicity induced by titration of the IMPACTTM CD19-anti-Her2 scFv fusion protein with CAR19 T cells and SKOV3 tumor cells Next, 1ug/ml of protein or was added either to the CAR19 T cells, or to the SKOV-3 tumor cells, then incubated for 30' on ice, before mixing with the cognate cell partner. In the control experiment both cell types and the protein were added simultaneously. a) CAR expression pro鍖le (anti-Flag, FACs) b) Induction of IFN粒 (ELISA) c) Cytotoxicity assayed using GFP+ Raji cells a cb curve 鍖tting 10 : 1 10 : 1 5 : 1 EC50 (cytotoxicity) 9pM 11.6pM CAR19 : SKOV-3 at 10 : 1 CAR19 : SKOV-3 at 5 : 1 RESULTS RESULTS RESULTS KEY POINTS ≒ activity at very low concentration ≒ no inactivation in the presence of excess fusion protein up to 15ug/ml Figure 5. Cytotoxicity following different orders of addition of fusion protein to cells 6 CAR19 with the IMPACTTM fusion protein encoded as an integrated gene The development candidates are being constructed as integrated genes (i-genes) using lentiviral vectors and packaging systems. A prototype schematic is shown here: SvFv and bispeci鍖c scFv CD19 fusion protein constructs are being optimized. Our current programs use scFvs to CD20, BCMA, CLL-1, ROR-1 and Her2. Transduction of primary human T cells with the viral particles yielded cell surface CAR expression and detectible fusion protein (< 5ng/ml). These cells were placed into culture with SKOV-3 tumor cells in order to assess cytotoxicity. Figure 6. CAR19 T cells secreting an IMPACTTM fusion protein (CAR130) demonstrate redirected cytotoxicity In Closing: We conclude that IMPACTTM fusion proteins mediate redirected tumor cell killing at very low concentrations, are not 'shut-down' by the soluble protein, and can be successfully secreted from CAR T cells. The 鍖rst in vivo study using puri鍖ed fusion proteins, CAR19 cells and SKOV-3 tumors is in progress. fusion protein controls fusion protein controls fusion protein control 42: CD19-anti-Her2 scFv 28: CD19 RESULTS: The order of addition had no impact on cytotoxicity induced by the fusion protein FMC63 CAREF1a promoter CD19-anti-Her2 scFvT2A FMC63 CAR T2A + CD19-anti-Her2 scFv RESULTS: The CAR19 secreting the CD19-anti-Her2- scFv fusion protein speci鍖cally mediated killing of Her2+ SKOV-3 cells 130: IMPACT cells UTD: control cells expansion, persistence CAR19 : SKOV-3 at 10 : 1

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3768 rennert

  • 1. CAR T cells specific for CD19 can be redirected to kill CD19 negative tumors 1 - Introduction Remarkable progress has been made in the treatment of relapsed/refractory Acute Lymphocytic Leukemia and Non- Hodgkin Lymphoma with CAR-CD19 T cells. In contrast, progress against CD19-negative hematological cancers and solid tumors has been limited. Intensive efforts to optimize cellular therapeutics for better ef鍖cacy include provision of cytokine support and countering immuno-suppression. However, lack of suf鍖cient antigen is a signi鍖cant additional hurdle that CAR-T therapeutics for solid tumors must overcome. We present a novel strategy to utilize CD19 for sustained antigen presentation in order to promote cellular therapeutic expansion, ef鍖cacy and persistence. The strategy, called IMPACTTM (Integrated Modules oPtimize Adoptive Cell Therapy), employs a methodology that is modular in design and can be applied to diverse antigens and tumor types, yet retains the well-established advantages of CAR T cells directed to CD19. 2 - Technology Overview IMPACTTM fusion proteins are created by cloning the extracellular domain (ECD) of a CAR T cell target protein (e.g. CD19) to an scFv that recognizes a second target protein. The system is modular: diverse ECD-scFv fusion proteins have been designed and expressed. In one iteration, this fusion protein is puri鍖ed and utlized in conjunction with an exising CAR T cell, e.g. a CD19-anti-Her2 fusion protein is puri鍖ed and added to a culture of CAR19 T cells and Her2+ tumor cells, creating a bridge that triggers CAR19 T cell cytotoxic activity. In another iteration, the CAR19 T cells expresses the CD19- anti-Her2 fusion protein, and this expression is suf鍖cient to create the "cytotoxic bridge" as shown in Figure 1. The normal B cell pools ensures expansion and persistance of the CAR19 T cells in vivo. Figure 1. Bridging CAR19 T cells to Her2+ tumor cells Christine Ambrose1, Lihe Su1, Lan Wu1, Fay Dufort1, Roy R. Lobb1, Andreas Hombach2, Hinrich Abken2, Paul D. Rennert1. 1: Aleta Biotherapeutics, Natick, MA USA; 2: University of Cologne, Cologne, Germany #3768 Tumor cell CD19+ B cell CAR19 T cell anti-CD19 scFv CD19 ECD Her2anti-Her2 scFv 3 - Puri鍖ed IMPACTTM fusion proteins We cloned the human CD19 ECD (two IgC domains) and the scFv derived from the anti-Her2 mAb trastuzumab into a lentiviral expression system for transfection then puri鍖cation by af鍖nity chromatography and SEC (Lake Pharma). Mono- meric protein was used in our experiments; this monomer is stable in solution. The fusion protein carries a C-terminal His tag. Control proteins (CD19 ECD-His and CD22 ECD 1-3 trastuzumab scFv) were also created. Table 1. Af鍖nities of fusion proteins for target molecules, determined by ELISA analyses biotin-Her2 His-scFv CD19 ECD Figure 2. FACs binding of CD19-anti-Her2 scFv and CD22- anti-Her2 scFv fusion proteins to SKOV-3 ovarian cancer cells 4 - Construction and characterization of CAR19 T cells The scFv from anti-CD19 mAb FMC63 was cloned in frame with a FLAG-tagged linker, the CD28 transmembrane sequence and the cytoplasmic domains of CD28, 4-1BB and CD3龍. The FLAG tag was encoded to facilitate detection of the CAR by FACs analysis following transduction into primary human T cells (Figure 3). The CAR19 T cells were tested for cytotoxic activity in a cell killing assay using B cell lymphoma lines (ATCC). One example is shown in Figure 3. anti-CD19 mAb FMC63 biotinylated Her2 protein CD19-anti- Her2 0.4nM 3.15nM CD22-anti- Her2 1.92nM CD19 5.1nM SKOV-3 mAb-PE anti-His-PE CD19-anti- Her2 2nM 9nM CD22-anti- Her2 2.2nM 1nM FMC63-PE anti-His-PE His-scFv Her2 CD19 ECD Tumor cell anti-CD19- coated well ≒Her-2---biotin anti-CD22-PE anti-His-PE Figure 3. Expression of the CD19 CAR and cytotoxicity against a CD19+ B cell lymphoma line 5 - Fusion protein mediated cytotoxicity The ability of the fusion proteins to bridge to CAR19 cells and mediate killing of Her2+ SKOV-3 tumor cells was evaluated. First we performed a titration. Figure 4. Cytotoxicity induced by titration of the IMPACTTM CD19-anti-Her2 scFv fusion protein with CAR19 T cells and SKOV3 tumor cells Next, 1ug/ml of protein or was added either to the CAR19 T cells, or to the SKOV-3 tumor cells, then incubated for 30' on ice, before mixing with the cognate cell partner. In the control experiment both cell types and the protein were added simultaneously. a) CAR expression pro鍖le (anti-Flag, FACs) b) Induction of IFN粒 (ELISA) c) Cytotoxicity assayed using GFP+ Raji cells a cb curve 鍖tting 10 : 1 10 : 1 5 : 1 EC50 (cytotoxicity) 9pM 11.6pM CAR19 : SKOV-3 at 10 : 1 CAR19 : SKOV-3 at 5 : 1 RESULTS RESULTS RESULTS KEY POINTS ≒ activity at very low concentration ≒ no inactivation in the presence of excess fusion protein up to 15ug/ml Figure 5. Cytotoxicity following different orders of addition of fusion protein to cells 6 CAR19 with the IMPACTTM fusion protein encoded as an integrated gene The development candidates are being constructed as integrated genes (i-genes) using lentiviral vectors and packaging systems. A prototype schematic is shown here: SvFv and bispeci鍖c scFv CD19 fusion protein constructs are being optimized. Our current programs use scFvs to CD20, BCMA, CLL-1, ROR-1 and Her2. Transduction of primary human T cells with the viral particles yielded cell surface CAR expression and detectible fusion protein (< 5ng/ml). These cells were placed into culture with SKOV-3 tumor cells in order to assess cytotoxicity. Figure 6. CAR19 T cells secreting an IMPACTTM fusion protein (CAR130) demonstrate redirected cytotoxicity In Closing: We conclude that IMPACTTM fusion proteins mediate redirected tumor cell killing at very low concentrations, are not 'shut-down' by the soluble protein, and can be successfully secreted from CAR T cells. The 鍖rst in vivo study using puri鍖ed fusion proteins, CAR19 cells and SKOV-3 tumors is in progress. fusion protein controls fusion protein controls fusion protein control 42: CD19-anti-Her2 scFv 28: CD19 RESULTS: The order of addition had no impact on cytotoxicity induced by the fusion protein FMC63 CAREF1a promoter CD19-anti-Her2 scFvT2A FMC63 CAR T2A + CD19-anti-Her2 scFv RESULTS: The CAR19 secreting the CD19-anti-Her2- scFv fusion protein speci鍖cally mediated killing of Her2+ SKOV-3 cells 130: IMPACT cells UTD: control cells expansion, persistence CAR19 : SKOV-3 at 10 : 1