The efficacy of CD19-specific CAR T cells in the treatment of leukemia/lymphoma relies, at least in part, on the unique properties of the particular CAR and the presence of healthy B cells that enhance the target cell lysis and cytokine secretion through repetitive stimulation. Here, we report to apply the same CAR to target solid tumors, such as ErbB2+ carcinoma. CD19 CAR T cells are redirected towards the ErbB2+ cells by a fusion protein that is composed of the herceptin-derived anti-ErbB2 scFv 4D5 linked to the CD19 exodomain. The CD19-4D5scFv engager enabled CD19 CAR T cells to recognize the ErbB2+ cancer cells and to suppress the ErbB2+ tumor growth. The primary killing capacity by the ErbB2-redirected CD19 CAR T cells was as efficient as by the ErbB2 CAR T cells, however, adding CD19+ B cells furthermore reinforced the activation of the CD19 CAR T cells, thereby improving the anti-tumor activities. The ErbB2-redirected CD19 CAR T cells, moreover, showed a 100-fold superior selectivity in targeting cancer cells versus healthy fibroblasts, which was not the case for the ErbB2 CAR T cells. The data demonstrate that the CD19 CAR T cells can be high-jacked by a CD19-scFv engager protein to attack specifically solid cancer, thereby expanding their application beyond the B cell malignancies.

Background Cell therapeutics are limited by critical issues including antigen escape, antigen heterogeneity, tumor and tumor microenvironment mediated immune suppression and suboptimal T cell expansion, fitness and persistence. We originally created CAR T Engagers (CTEs) to address the issues of antigen escape and heterogeneity. Here we present novel domains added to CTEs to overcome immune suppression and enhance CAR T cell function. Methods Our first generation of functionally enhanced CTEs are built on multi-antigen targeting modules. ALETA-001 is a biologic CTE that binds to CD20 and displays the CD19 extracellular domain (ECD). ALETA-002 is a lentiviral construct that expresses an anti-CD19 CAR domain and a CTE that binds both Her2 and B7-H3. ALETA-001 and -002 CTE protein sequences were further modified to contain additional functional domains including an anti-PD-L1 VHH, a monomeric TGFbetaR2 TRAP, the CD2-binding domain of LFA3, a T cell stimulating cytokine, and an immune system activating cytokine. CTEs that were functionally enhanced (FE) were evaluated for their ability to promote anti-CD19 CAR T-mediated cytotoxicity, to overcome immune suppression and immune escape, and to productively stimulate CAR T cells and engage with endogenous immune cells. Results Functionally enhanced CTE's directed anti-CD19 CAR T cells to attack and kill cancer cells including B cell lymphomas (ALETA-001-FE) and solid tumors (ALETA-002-FE). Further, ALETA-001-FE-1 countered loss of CD19 antigen and provided additional costimulation via LFA3/CD2 engagement. ALETA-001-FE-2 stimulated CAR T expansion via enhanced engagement of common-gamma chain signaling. ALETA-002-FE-1 and ALETA-002-FE-2 mediated potent killing of Her+, B7H3+ and dual+ solid tumor cells. ALETA-002-FE-1 also blocked TGFb signaling with a sub-nM IC50. ALETA-002-FE-2 blocked PD-L1 activity with similar potency. Conclusions Anti-CD19 CAR T cells demonstrate best-in-class expansion, fitness and persistence due to their ability to interact productively with normal CD19+ B cells in circulation and within secondary lymphoid organs that provide T cell nurturing signals. Anti-CD19 CAR T cell use for B cell lymphoma therapy was enhanced by limiting antigen escape and increasing costimulation via LFA3/CD2 binding Anti-CD19 CAR T cells can be redirected to solid tumor antigens by the use of CAR T-secreted CTEs. Here we used multi-antigen targeting, ie. by linking the CD19 ECD to anti-Her2 and anti-B7H3 domains, by providing productive cytokine signaling and by countering immune suppression. Notably, all these functions are incorporated into the CTE, leaving CAR T engineering as separate tool. CTEs carrying multiple domains designed to simultaneously enhance functionality are being designed and evaluated and will be presented.

B cell lymphoma therapy has been transformed by CD19-targeting cellular therapeutics that induce high clinical response rates and impressive remissions in relapsed and refractory patients. However, approximately half of all patients who respond to CD19-directed cell therapy relapse, the majority within 6 months. One characteristic of relapse is loss or reduction of CD19 expression on malignant B cells. We designed a unique therapeutic to prevent and reverse relapses due to lost or reduced CD19 expression. This novel biologic, a CAR T Engager, binds CD20 and displays the CD19 extracellular domain. This approach increases the apparent CD19 antigen density on CD19-positive/CD20-positive lymphoma cells, and prevents antigen-loss induced relapse, as CD19 bound to CD20 remains present on the cell surface. We demonstrate that this novel therapeutic prevents and reverses lymphoma relapse in vitro and prevents CD19-negative lymphoma growth and relapse in vivo.

Chimeric Antigen Receptor T cells targeting malignancies expressing CD19 (CAR19) have been widely successful, with products approved to treat B cell lymphomas (NHL) and B cell leukemia. A major limitation of CAR19 therapy is the steep relapse rate within 6 months of treatment, often due to the loss or diminution of tumor cell CD19 expression. ALETA-001 is a CAR-T Engager protein that contains the CD19 extracellular domain (ECD), an anti-CD20 VHH, and an anti-albumin VHH for half-life extension. When combined with CAR19 T cells, ALETA-001 triggers cytotoxicity through CD19 bound to CD20, thus increasing total target antigen density and preventing relapse due to loss of CD19 expression. ALETA-001 will enter Cancer Research UK-sponsored Phase 1/2 clinical trials in CAR19-treated NHL patients next year. We made novel variants of ALETA-001 including CAR-T Engagers with alternative anti-CD20 modules and CAR-T Engagers that target both CD20 and a second B cell cancer antigen. Further, we extended our technology to target Acute Myeloid Leukemia (AML), using novel antigen binding domains and immunomodulatory functional domains. Site-directed mutagenesis was used to make ALETA-001 variants. We mutated the complementary determining regions, CDR2 and/or CDR3, within the anti-CD20 VHH. The novel CAR-T Engager Proteins were evaluated for binding to CD19-negative/CD20-positive lymphoma cells and for cytotoxicity against those cells in the presence of CAR19 T cells. Further, CAR-T Engagers were made that contained novel CD20 binding domains and binding domains to other B cell antigens. In addition, we created a novel CAR-T engager that contains the CD19 ECD and binding domains to two AML antigens, plus an immunomodulatory domain. This novel CAR-T engager is designed to treat CD19-positive Mixed Phenotype Leukemia (MPL) and other forms of AML. We evaluated the series of CDR mutations in the ALETA-001 anti-CD20 VHH. A spectrum of activities was observed, with mutations that retained full CD20 binding and cytotoxic activity to those with partial or no activity. A series of constructs with distinct anti-CD20 binding domains were also evaluated, with some found to be equipotent with ALETA-001. Constructs binding both CD20 and a second B cell tumor antigen were prepared and assayed and found to have potent activity against either or both antigens. Finally, CAR-T Engagers directed to AML antigens were able to bind each antigen individually, and mediated potent toxicity with the addition of CAR19 T cells. We conclude that the CAR-T Engager platform offers robust and modular anti-tumor functionality, featuring potent multi-antigen targeting for diverse indications. Finally, we can incorporate other useful immunomodulatory activities into Engager proteins. Citation Format: Paul D. Rennert, Lihe Su, Lan Wu, Roy R. Lobb, Christine Ambrose. CAR-T engager proteins for the treatment of B cell cancers and acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2759.

Background B cell lymphoma therapy has been transformed by CD19-targeting cellular therapeutics that induce high clinical response rates and impressive remissions in relapsed and refractory patients. However, approximately half of all patients who respond to CD19-directed cell therapy relapse, the majority within six months. One characteristic of relapse is loss or reduction of CD19 expression on malignant B cells. We designed a novel biologic, a CAR T Engager, that binds CD20 and displays the CD19 extracellular domain. This approach increases the apparent CD19 antigen density on CD19-positive/CD20-positive lymphoma cells, and prevents antigen-loss induced relapse, as CD19 bound to CD20 remains present on the cell surface. We demonstrate that this novel therapeutic prevents and reverses lymphoma relapse in vitro and prevents CD19-negative lymphoma growth and relapse in vivo. Methods The CTE biologic has three functional domains: a modified CD19 ECD, an anti-CD20 binding domain and an anti-albumin binding domain. The protein, termed a CAR-19-CD20 T cell Engager (CTE-19.20), binds to CD20 and displays the CD19 ECD and increases CD19 antigen density on target lymphoma cells regardless of their level of CD19 expression. Results CTE-19.20 proteins potently triggered CD19-negative lymphoma cell death in the presence of CAR-19 T cells in vitro and prevented antigen-loss relapse in an in vitro model of lymphoma escape from CAR-19 therapy. Using in vivo modeling we show that CTE-19.20 protein given alongside CAR-19 T cells prevented CD19-negative lymphoma expansion, eliminated disease, and significantly impacted survival. CTE-19.20 was readily expressed and secreted by transfected mammalian cells, was efficiently purified and demonstrated favorable biophysical properties. Conclusions Patient relapse from CAR-19 therapy occurs rapidly, often within the first months following therapy. The kinetics of relapse offer the potential to intervene using CTE-19.20 protein, using several different clinical designs. In the first instance we are developing this molecule with the goal of treating patients who have already received a CAR-19 therapy, by evaluating their clinical response through the first few months post CAR infusion. Diverse biomarkers can be used to track response and risk of relapse. PET imaging and ctDNA analyses have emerged as sensitive means of tracking lymphoma and leukemia regression. This is an optimal and straightforward approach to productive and sustained activation of CAR-19 T cells, using a potent CD19-anti-CD20 bridging protein with an extended half-life.

Adoptive T cell therapies directed to the B cell malignancy antigen CD19 (CAR19 T cells) have transformed the care of otherwise refractory, last-line leukemia and lymphoma patients. The overall response rates achieved are very high, routinely above 60%, and a substantial number of responding patients have durable responses that can last many years. However, 50% of responders relapse within 6 months, and outcomes for these relapsed patients are poor. Relapses occur most often in patients whose initial CAR19 T cell expansion is suboptimal, and whose tumor cells reduce or lose expression of the target antigen, CD19.

Solid tumors display pronounced antigen heterogeneity and clinical studies have shown that antigen escape from therapy occurs rapidly, limiting the persistence and efficacy of CAR T cells. Here we present dual and triple-antigen binding proteins that bridge CAR T cells to multiple antigens, allowing a simultaneous attack on tumor antigens by a single CAR T antibody domain. We call these CAR T Engager proteins. CAR T Engager proteins can be encoded into lentiviral vectors for secretion from CAR T cells, can be encoded into oncolytic viral vectors for secretion from transduced tumor cells, or can be engineered as biologics for injection.CAR T Engagers contain a protein target for a CAR T cell, eg. an anti-CD19 CAR T cell. We have previously presented a Her2-binding CAR T Engager protein with potent in vivo activity against solid tumors. We used this CAR T Engager as the basis for building dual and triple antigen binding proteins. Specifically, we mapped antigen expression for Her2-positive solid tumors, Her2-positive metastases, and primary CNS tumors. Our analysis identified expression patterns of two and three antigens that would essentially saturate the cellular composition of specific solid tumors, greatly reducing the chance of antigen escape from therapy. We created the corresponding CAR T Engagers and have developed single, dual and triple antigen expression cells lines to model the activity and potency of these novel proteins, administered alongside CAR T cells.CAR T cells plus dual antigen CAR T Engagers that recognize and target Her2 and B7H3 demonstrate potent cytotoxicity against either antigen alone, and synergistic potency (2 pM) if both antigens are expressed. Similarly, triple antigen CART Engagers show single antigen binding and potent cytotoxicity which is enhanced when multiple antigens are expressed on a target cell. All of the cytotoxicity is mediated through one CAR domain expressed on the primary T cells. T cells can be pre-loaded with multi-antigen CAR T Engagers and retain cytotoxic activity. Because the underlying CAR is an anti-CD19 CAR, cell persistence and fitness is further enhanced in the presence of normal B cells.The CAR T Engager platform is a robust and modular solution for the multi-antigen targeting of solid tumors. Diverse antigens can be readily targeted for diverse indications. Examples of other functional modalities that can be added will be presented.We thank Cancer Research UK for their ongoing support.

Successful CAR T cell therapy for the treatment of solid tumors requires exemplary CAR T cell expansion, persistence and fitness, and the ability to target tumor antigens safely. Here we address this constellation of critical attributes for successful cellular therapy by using integrated technologies that simplify development and derisk clinical translation. We have developed a CAR-CD19 T cell that secretes a CD19-anti-Her2 bridging protein. This cell therapy strategy exploits the ability of CD19-targeting CAR T cells to interact with CD19 on normal B cells to drive expansion, persistence and fitness. The secreted bridging protein potently binds to Her2-positive tumor cells, mediating CAR-CD19 T cell cytotoxicity in vitro and in vivo . Because of its short half-life, the secreted bridging protein will selectively accumulate at the site of highest antigen expression, ie. at the tumor. Bridging proteins that bind to multiple different tumor antigens have been created. Therefore, antigen-bridging CAR-CD19 T cells incorporate critical attributes for successful solid tumor cell therapy. This platform can be exploited to attack tumor antigens on any cancer.

Refractory acute myeloid leukemia (AML) remains an incurable malignancy despite the clinical use of novel targeted therapies, new antibody-based therapies, and cellular therapeutics. Here, we describe the preclinical development of a novel cell therapy that targets the antigen CLEC12A with a biparatopic bridging protein. Bridging proteins are designed as "CAR-T cell engagers," with a CAR-targeted protein fused to antigen binding domains derived from antibodies. Here, we created a CD19-antiCLEC12A bridging protein that binds to CAR19 T cells and to the antigen CLEC12A. Biparatopic targeting increases the potency of bridging protein-mediated cytotoxicity by CAR19 T cells. Using CAR19 T cells that secrete the bridging protein we demonstrate potent activity against aggressive leukemic cell lines in vivo. This CAR-engager platform is facile and modular, as illustrated by activity of a dual-antigen bridging protein targeting CLEC12A and CD33, designed to counter tumor heterogeneity and antigen escape, and created without the need for extensive CAR T-cell genetic engineering. CAR19 T cells provide an optimal cell therapy platform with well-understood inherent persistence and fitness characteristics.

Background Treatment of solid tumors with cell therapeutics will require optimal T cell persistence, fitness, and trafficking. Heterogeneous solid tumors will also have to be attacked through multiple antigens simultaneously in order to prevent resistance linked to loss of antigen expression. Here we use chimeric antigen receptor (CAR) T cells that secrete bridging proteins that act as CAR-T engagers to create an optimal platform for attacking solid tumors in the CNS. Methods Lentiviral vectors encoding an anti-CD19 CAR and secreted bridging proteins were created. The bridging proteins contained the CD19 extracellular domain, which is the target for the CAR, and anti-tumor antigen binding domains derived from antibodies (scFv and llama VH). The resulting anti-CD19 CAR T cells secrete the bridging proteins. These candidate cell therapeutics were evaluated for antigen binding and induction of antigen-specific cytotoxicity. An anti-CD19 CAR that secretes a CD19-anti-Her2 bridging protein has moved into development. Using the CD19-anti-Her2 bridging protein as a core module, we have begun evaluating a series of multi-antigen bridging proteins. Results CAR-CD19 T cells that secrete bridging proteins have potent cytotoxic activity against single- and multi-antigen-positive cells. ALETA-002 is the lead candidate lentiviral vector construct encoding the anti-CD19 CAR domain and the CD19-anti-Her2 bridging protein, and has entered a GMP viral particle development campaign. This therapeutic will be systemically administered to Her2-positive breast cancer patients who are relapsing with CNS metastases. Next, multi-antigen bridging proteins encoding an anti-Her2 scFv and anti-B7H3, anti-B7H6 or anti-IL13Ra2 llama VH were assayed for potency. Lead candidates for development for the treatment of primary CNS malignancies were identified and are being manufactured at pilot-scale in 4-plasmid lentivirus production runs. Conclusions The use of anti-CD19 CAR T cells that can expand off of the normal CD19-positive B cell pool enables tumor-antigen independent persistence, fitness and robust trafficking into the CNS. The use of small, modular bridging proteins allows us to leverage anti-CD19 CAR T cells and use these to attack solid tumor antigens that are present on CNS resident cancers and on CNS metastatic lesions. Novel cell therapeutics for the treatment of Her2-positive CNS metastases and heterogeneous primary CNS malignancies including glioblastoma and the pediatric gliomas have been developed.

The B-cell surface protein CD19 is present throughout the cell life cycle and is uniformly expressed in leukemias, making it a target for chimeric antigen receptor engineered immune cell therapy. Identifying the sequence dependence of the binding of CD19 to antibodies empowers fundamental study and more tailored development of CD19-targeted therapeutics. To identify the antibody-binding epitopes on CD19, we screened a comprehensive single-site saturation mutation library of the human CD19 extracellular domain to identify mutations detrimental to binding FMC63-the dominant CD19 antibody used in chimeric antigen receptor development-as well as 4G7-2E3 and 3B10, which have been used in various types of CD19 research and development. All three antibodies had partially overlapping, yet distinct, epitopes near the published epitope of antibody B43. The FMC63 conformational epitope spans spatially adjacent, but genetically distant, loops in exons 3 and 4. The 3B10 epitope is a linear peptide sequence that binds CD19 with 440 pM affinity. Along with their primary goal of epitope mapping, the mutational tolerance data also empowered additional CD19 variant design and analysis. A designed CD19 variant with all N-linked glycosylation sites removed successfully bound antibody in the yeast display context, which provides a lead for aglycosylated applications. Screening for thermally stable variants identified mutations to guide further CD19 stabilization for fusion protein applications and revealed evolutionary affinity-stability trade-offs. These fundamental insights into CD19 sequence-function relationships enhance our understanding of antibody-mediated CD19-targeted therapeutics.

Introduction CAR T cells that recognize the antigen CD19 (CAR19s) have achieved remarkable success in otherwise untreatable B cell malignancies including refractory and relapsed ALL and NHL. However, clinical data from diverse CAR19 trials, and real-world experience with the approved CAR19 therapeutics (tisagenlecleucel and axicabtagene ciloleucel), highlight a critical issue, that of patient relapse due to the loss of expression of the target antigen (CD19) or the antigenic epitope. Antigen loss relapse rate of up to 50% have been reported across indications (adult ALL, pediatric ALL, adult NHL) irrespective of the specific CAR19 used. Attempts to treat patients who have relapsed from CAR19 treatment include provision of a CAR T cell to a second antigen, for example CD22. Such attempts have met with limited success, further, many patients cannot tolerate a second regimen of apheresis, consolidation, lymphodepletion and CAR T infusion. Importantly, many of the patients relapsing with CD19-negative malignancies still have detectable levels of CAR19 T cells in circulation, since the CAR19s persist in the presence of normal B cells being produced by the bone marrow (these B cells are CD19-positive). Therefore, a technology that reactivates the patient-resident CAR19s to attack the relapsing tumor cell would be a highly attractive alternative to subsequent CAR T therapy. Here we present this technology and illustrate its' ability to prevent relapses and importantly, to reverse relapses in vivo. Experimental Procedures A stabilized form of the CD19 extracellular domain (ECD) was cloned in frame with an anti-CD20 scFv and an anti-albumin VHH, to create a monomeric CD19-ECD-anti-CD20 bridging protein with extended circulating half-life characteristics. The protein was purified from a mammalian cell expression system. Protein stability, binding affinities, and cytotoxic activity were analyzed in vitro. We utilized CD19-positive, CD20-positive and double positive cell lines to assess single and dual antigen activity. We utilized patient derived CD20-positive/CD19-negative cells to demonstrate translational relevance. Finally, we used single and dual flank in vivo models to assess the potency of the bridging protein in the relapse setting and in the prevention setting. Results and Discussion The CD19-anti-CD20 bridging protein was shown to be expressed at high levels, readily purified and highly stable (no aggregation or clipping, thermostable, and stable in media/serum at 37oC for extended periods). The purified bridging protein directed CAR19 cytotoxicity against CD19-negative/CD20-positive cells with superb potency (IC50 = 23pM = 1.6 ng/ml). CAR19 T cells that were previously activated by a CD19-positive tumor cell could subsequently be activated by a CD19-negative tumor cell in the presence of the CD19-anti-CD20 bridging protein. In vitro, a CAR19 T cells found and eliminated CD19-negative cells "hidden" in a population of dual-positive cells in a mixing experiment but only if the bridging protein was present, otherwise, the CD19-negative cells invariably escaped from CAR19 T cells. The activity of the CD19-anti-CD20 bridging protein extended to CD19-negative/CD20-positive patient-derived cells tested in vitro. In vivo, using a dual flank model, CAR19 T cells plus the injected bridging protein controlled both CD19-positive/CD20-positive and CD19-negative/CD20-positive tumors, while CAR19 alone did not impact the latter tumor. In a relapse setting the growth of a mixture of CD19-positive and CD19-negative cells was merely delayed by CAR19 T cells alone but was eradicated when CAR19 cells were given along with the CD19-anti-CD20 bridging protein injected systemically. Importantly, CAR19 cells that had "lost" control over the mixed population could be restimulated to eliminate the CD19-negative population when the CD19-anti-CD20 bridging protein was added after those cells have begun to escape the initial (CAR19-only) treatment in vivo. These results have led to the identification of a development candidate for the treatment of CD19-negative relapse from CAR19 treatment. The GMP production campaign is underway. The first-in-human trial will enroll patients relapsing from CAR19 therapy with CD19-negative malignancy, in whom CAR19 T cells are shown to still be present. Disclosures Rennert: Aleta Biotherapeutics: Employment, Equity Ownership. Su:Aleta Biotherapeutics: Employment. Dufort:Aleta Biotherapeutics: Employment. Birt:Aleta Biotherapeutics: Employment. Sanford:Aleta Biotherapeutics: Employment. Wu:Aleta Biotherapeutics: Employment. Ambrose:Aleta Biotherapeutics: Employment. Lobb:Aleta Biotherapeutics: Consultancy, Equity Ownership.

CD19-targeted chimeric antigen receptor (CAR) T-cells (CAR19s) show remarkable efficacy in the treatment of relapsed/refractory acute lymphocytic leukemia and Non-Hodgkin's lymphoma. However, the use of CAR T-cell therapy against CD19-negative hematological cancers and solid tumors has been challenging. We propose CD19-fusion proteins (CD19-FPs) to leverage the benefits of CAR19s while retargeting this validated cellular therapy to alternative tumor antigens. We demonstrate the ability of a fusion of CD19 extracellular domain (ECD) and a human epidermal growth factor receptor 2 (HER2) single-chain antibody fragment to retarget CAR19s to kill HER2+ CD19- tumor cells. To enhance the modularity of this technology, we engineered a more robust CD19 ECD via deep mutational scanning with yeast display and flow cytometric selections for improved protease resistance and anti-CD19 antibody binding. These enhanced CD19 ECDs significantly increase, and in some cases recover, fusion protein expression while maintaining target antigen affinity. Importantly, CD19-FPs retarget CAR19s to kill tumor cells expressing multiple distinct antigens, including HER2, CD20, EGFR, BCMA, and Clec12A as N- or C-terminal fusions and linked to both antibody fragments and fibronectin ligands. This study provides fundamental insights into CD19 sequence-function relationships and defines a flexible and modular platform to retarget CAR19s to any tumor antigen.

Introduction: CAR T-cells targeting CD19 (CAR19s) can eradicate B cell leukemias and lymphomas. The effectiveness of CAR19s is linked to their robust expansion properties but also their long-term persistence. Persistence is maintained by normal CD19+ B cells: a non-tumor dependent, self-renewing source of antigen. In this manner, CAR19s are quite unique. We have re-engineered CAR19s to secrete a wide variety of retargeting fusion proteins (FPs) by encoding expression cassettes downstream of the CAR sequence in lentiviral vectors. By hijacking CAR19s, we utilize their inherent persistence properties. By designing multispecific FP, we directly counter the clinically critical issues of tumor heterogeneity and antigen loss. Experimental Procedures: A lentiviral vector with an MCSV promoter was used to express the CAR19 construct and FPs. FPs and multispecific-FPs were designed to encode the extracellular domain of the CD19 protein, followed by one or two scFv sequences, separated from the CAR sequence by a P2A cleavage site, a design termed IMPACTtm (Integrated Modular Proteins for Adoptive Cell Therapy). The FPs therefore consist of the CD19 extracellular domain linked to one or more scFvs. The FPs redirect CAR19 T-cell cytotoxic activity to any tumor antigen of interest by coating that antigen with CD19 via the scFv. Further, multiple antigens can be coated with CD19 by encoding multiple scFv in the FP. Hijacked CAR19s therefore serve as a platform for targeting diverse antigens. Results: Here we describe one example in detail, focusing on Her2+ solid tumors. The CD19/anti-Her2 FP was highly potent in cytotoxicity assays targeting Her2+/CD19- solid tumor cell lines. The concentration of fusion protein required to reduce tumor cell number by 50% was 10 pM (0.7 ng/ml). Primary donor T-cells transduced with the CAR19 - CD19/anti-Her2 FP lentiviral vector secreted > 20 ng/ml of FP in cell culture. Cytotoxic activity of this redirected CAR19 against Her2+ SKOV3 tumor cells was demonstrated in vitro and in vivo. CD19-mediated persistence was demonstrated in serial restimulation assays. A bispecific FP containing CD19 linked to anti-Her2 and anti-EGFR scFv had specific activity against both antigens with a potency of 0.75 pM. For each antigen, the potent cytotoxicity was specifically mediated by the secreted fusion protein. Additional program examples of multispecific targeting for diverse hematologic and solid tumor types will be shown. Conclusions: The IMPACT platform addresses critical issues in cell therapy including CAR persistence, antigen escape and antigen heterogeneity, and provides important solutions for treating both hematologic and solid tumors. The potency of redirected cytotoxicity supports clinical development of CAR19/IMPACT programs, four of which are now ready for IND enabling studies. Citation Format: Paul Rennert, Fay Dufort, Lihe Su, Lan Wu, Alyssa Birt, Christine Ambrose, Roy Lobb. Hijacking CAR19 T-cells for use in targeting diverse hematopoietic and solid tumors [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A040.