Chimeric Antigen Receptor (CAR) T cells directed to B cell maturation antigen (BCMA) mediate profound responses in patients with multiple myeloma, but most patients do not achieve long-term complete remissions. In addition, recent evidence suggests that high-affinity binding to BCMA can result in on-target, off-tumor activity in the basal ganglia and can lead to fatal Parkinsonian-like disease. Here we develop CAR T cells against multiple myeloma using a binder to targeting transmembrane activator and CAML interactor (TACI) in mono and dual-specific formats with anti-BCMA. These CARs have robust, antigen-specific activity in vitro and in vivo. We also show that TACI RNA expression is limited in the basal ganglia, which may circumvent some of the toxicities recently reported with BCMA CARs. Thus, single-targeting TACI CARs may have a safer toxicity profile, whereas dual-specific BCMA-TACI CAR T cells have potential to avoid the antigen escape that can occur with single-antigen targeting.

Background Genetic engineering of T cells to express anti-CD19 Chimeric Antigen Receptors (CAR-T cells) has been FDA approved for the treatment of refractory/relapsing acute lymphocytic leukemia and diffuse large B cell lymphoma. With more patients receiving treatment with CAR-T cells it has been observed that approximately 10–20% of patients fail to enter remission after therapy,1 and 30–50% of patients who achieve remission with anti-CD19 CAR T cells have disease relapse.2 In prior studies, CAR-binding amphiphile (AMP)-peptides were shown to effectively localize in lymph nodes (LN), where they decorate endogenous antigen-presenting cells (APC) and stimulate CAR signaling to promote potent CAR-T responses against solid tumors.3 In this study, we describe how CD19 mimotope peptides specific for FMC63-based CARs can be modified with AMP technology to enhance peptide accumulation in LNs, enable presentation on APCs to CAR-Ts, and promote activation and effector functionality of CAR-T cells. Methods We performed phage-screening and enrichment for CD19 surrogate peptides recognized by FMC-63-scFv. Surface Plasmon Resonance (SPR) was utilized to evaluate the affinity of the peptides to immobilized FMC-63. AMP versions of peptides were generated. In vitro, human dendritic cells (DCs) were preconditioned with AMP-CD19 or soluble peptides and cocultured with autologous T cells engineered to express CD19 CARs (FMC63-28z and FMC63-41BBz). Markers for activation, proliferation, cytotoxicity, and effector functions were evaluated. In vivo experiments were performed to evaluate the biodistribution of peptides. Luciferase-expressing murine CAR-T cells were engineered to evaluate the expansion and biodistribution of CAR-T cells in combination with AMP or soluble regimens. Results We found surrogate CD19 peptide mimotopes that bind to FMC-63 with different affinities evaluated by ELISA and SPR. Assessment in human autologous DC/CAR-T cell cocultures demonstrated that AMP-CD19 peptides can decorate DCs effectively and promote potent activation (OX40, 41BB, CD69), proliferation, cytokine production (IFNγ, TNFα, and IL2), cytotoxicity (CD107a), and phenotypic enhancement of CD19-specific CAR-T cells. Assessment in vivo showed that AMPs are effectively delivered to LN where endogenous APCs are decorated to promote the activity of murine CAR-T cells. Conclusions In vitro, AMP modification of CAR-binding peptide mimotopes induces activation, cytotoxicity, and effector functions of CAR-T cells. These AMP-peptides effectively accumulate in LN and boost CAR-T activation and expansion in vivo. This platform can potentially be utilized as a mechanism to expand and functionally enhance CAR-T cells in vivo for blood and solid tumors. References Maude SL et al. Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia. N Engl J Med 2018;378:439–448. Park JH et al. Long-term follow-up of CD19 CAR therapy in acute lymphoblastic leukemia. N Engl J Med 2018;378:449–459. Ma L et al. Enhanced CAR–T cell activity against solid tumors by vaccine boosting through the chimeric receptor. Science 2019;365(6449):162–168. Ethics Approval All animal experiments in this study were performed in accordance with the approval of IACUC Protocol CR-0039.

5024 Background: PSA value is widely used for the monitoring of treatment outcome in mCRPC in the clinical real-world setting. Early PSA changes are not considered in the definition of PSAProg due to the potential for spurious “flare” reactions. We aimed to evaluate the significance of an early PSA increase in mCRPC patients (pts) treated with enzalutamide or abiraterone (Enz/Abi). Methods: We retrospectively evaluated Enz/Abi-treated mCRPC pts from 11 hospitals between 2011-2020. Early PSAProg was defined as a 25% increase in PSA from baseline at 4 (PSAProg4) or 8 (PSAProg8) weeks after treatment initiation. PSA progression at 12 weeks (PSAProg12) was confirmed by a second reading. Uni- and multivariable (MV) Cox regression models were conducted to explore the association of PSAProg and overall survival (OS) in chemotherapy naïve patients treated with Abi or Enz. Interaction tests were conducted to explore differences in the impact of PSA progression on OS in Abi or Enz-treated pts. Results: We analyzed 511 chemotherapy-naïve mCRPC pts treated with Abi (N=391; 76.5%) or Enz (N=120; 23.5%). Median follow-up: 30.2 months. OS was longer in Enz-treated pts (38.1 vs 29m; HR 1.4; p=0.027). 59 (15.1%), 70 (17.9%) and 48 (12.3%) of Abi-treated and 9 (7.5%), 11 (9.2%) and 10 (8.3%) of Enz-treated pts experienced PSAProg4, PSAProg8 and PSAProg12, respectively, although differences were not statistically significant. PSAProg was associated with worse OS at all 3 timepoints only in Abi-treated pts. In Enz-treated pts, PSAProg4 had a large impact on OS, not observed in PSAProg8 or PSAProg12. We observed no significant interaction between agent (Enz/Abi) and PSA progression (Table). Conclusions: PSA progression at 4 weeks after Enz/Abi is significantly associated with shorter OS and may help identify pts not benefitting from Abi/Enz before clinical or radiographic progression. PSA pattern progression and its association with OS might differ depending on the drug used (Enz/Abi). Prospective validation studies are needed.[Table: see text]

High mobility group (HMG)A proteins are nonhistone chromatin proteins that bind to the minor groove of DNA, interact with transcriptional machinery, and facilitate DNA-directed nuclear processes. HMGA1 has been shown to regulate genes involved with systemic inflammatory processes. We hypothesized that HMGA1 is important in the function of mesenchymal stromal cells (MSCs), which are known to modulate inflammatory responses due to sepsis. To study this process, we harvested MSCs from transgenic (Tg) mice expressing a dominant-negative (dn) form of HMGA1 in mesenchymal cells. MSCs harvested from Tg mice contained the dnHMGA1 transgene, and transgene expression did not change endogenous HMGA1 levels. Immunophenotyping of the cells, along with trilineage differentiation revealed no striking differences between Tg and wild-type (WT) MSCs. However, Tg MSCs growth was decreased compared with WT MSCs, although Tg MSCs were more resistant to oxidative stress-induced death and expressed less IL-6. Tg MSCs administered after the onset of Escherichia coli-induced sepsis maintained their ability to improve survival when given in a single dose, in contrast with WT MSCs. This survival benefit of Tg MSCs was associated with less tissue cell death, and also a reduction in tissue neutrophil infiltration and expression of neutrophil chemokines. Finally, Tg MSCs promoted bacterial clearance and enhanced neutrophil phagocytosis, in part through their increased expression of stromal cell-derived factor-1 compared with WT MSCs. Taken together, these data demonstrate that expression of dnHMGA1 in MSCs provides a functional advantage of the cells when administered during bacterial sepsis.

T cells engineered to express chimeric antigen receptors (CARs) targeting CD19 have produced impressive outcomes for the treatment of B cell malignancies, but different products vary in kinetics, persistence, and toxicity profiles based on the co-stimulatory domains included in the CAR. In this study, we performed transcriptional profiling of bulk CAR T cell populations and single cells to characterize the transcriptional states of human T cells transduced with CD3 zeta, 4-1BB-CD3 zeta(BB zeta), or CD28-CD3 zeta (28 zeta) co-stimulatory domains at rest and after activation by triggering their CAR or their endogenous T cell receptor (TCR). We identified a transcriptional signature common across CARs with the CD3z signaling domain, as well as a distinct program associated with the 4-1BB co-stimulatory domain at rest and after activation. CAR T cells bearing BB zeta had increased expression of human leukocyte antigen (HLA) class II genes, ENPP2, and interleukin (IL)-21 axis genes, and decreased PD1 compared to 28 zeta CAR T cells. Similar to previous studies, we also found BB zeta CAR CD8 T cells to be enriched in a central memory cell phenotype and fatty acid metabolism genes. Our data uncovered transcriptional signatures related to costimulatory domains and demonstrated that signaling domains included in CARs uniquely shape the transcriptional programs of T cells.

One of the most promising candidates for Chimeric Antigen Receptor (CAR) T cell therapy beyond CAR19 is treatment of multiple myeloma with CAR T cells targeting B cell maturation antigen (BCMA). However, current reports of BCMA CAR in the clinic have a median progression free survival of 11.8 months, suggesting that targeting BCMA alone may not be sufficient. Evidence suggests patients treated with BCMA-targeted therapies may be thwarted by BCMA negative relapse. We have developed a novel second generation CAR T cell against another multiple myeloma target: transmembrane activator and CAML interactor (TACI). Mice were immunized against TACI and we designed a new scFv targeting TACI based on the resulting antibodies. Anti-TACI CAR T cells are cytotoxic in vitro and in vivo against multiple myeloma. To overcome single antigen loss, we designed bispecific tandem scFv CAR T cells targeting BCMA and TACI. These two antigens share a natural ligand, A Proliferation-Inducing Ligand (APRIL), which has also been used to design a dual-targeting CAR called TriPRIL. We show that these dual targeting CARs, based on tandem scFv or natural ligand design, have similar efficacy against wild type multiple myeloma models. However, this changes in the context of single antigen loss. We have characterized the proliferative (population doublings) and activation capability (CD69) of these CARs, as well as their memory (CCR7, CD45RA) and exhaustion phenotype (PD-1, TIM-3, LAG-3), with long term exposure to single antigen. Our studies show that sensitivity to antigen density differs between the tandem bispecifics and the natural ligand CAR. These data provide insight into how structural differences between dual-targeting CAR T cells affects their function.

Tissue colonization (homing) by blood-borne cells critically hinges on the ability of the cells to adhere to vascular endothelium with sufficient strength to overcome prevailing hemodynamic shear stress. These adhesive interactions are most effectively engendered via binding of the endothelial lectin E-selectin (CD62E) to its cognate ligand, sialyl Lewis-X (sLeX), displayed on circulating cells. Although chimeric antigen receptor (CAR) T-cell immunotherapy holds promise for treatment of various hematologic and non-hematologic malignancies, there is essentially no information regarding the efficiency of CAR T-cell homing. Accordingly, we performed integrated biochemical studies and adhesion assays to examine the capacity of human CAR T-cells to engage E-selectin. Our data indicate that CAR T-cells do not express sLeX and do not bind E-selectin. However, enforced sLeX display can be achieved on human CAR T-cells by surface fucosylation, with resultant robust E-selectin binding under hemodynamic shear. Importantly, following intravascular administration into mice, fucosylated human CAR-T cells infiltrate marrow with 10-fold higher efficiency than do unfucosylated cells. Collectively, these findings indicate that custom installation of sLeX programs tissue colonization of vascularly administered human CAR T-cells, offering a readily translatable strategy to augment tissue delivery, thereby lowering the pertinent cell dosing and attendant cell production burden, for CAR T-cell immunotherapy applications.

Glioblastoma (GBM) is a devastating disease with an extremely poor prognosis. Immune therapy with T cells engineered to express chimeric antigen receptors (CARs), represents a promising alternative to conventional therapy, but responses have been limited to date due to heterogeneous target antigen expression and the emergence of immune escape following treatment with CAR T cells directed at a single target. This was observed in clinical studies with CART-EGFRvIII, wherein EGFRvIII-targeted T cells successfully localized to the brain tumor microenvironment, but ultimately failed to prevent disease progression—with post-treatment specimens demonstrating high levels of wild-type EGFR despite reduced expression of EGFRvIII. To help address this therapeutic barrier, we developed a novel CAR construct engineered for local delivery of bispecific T-cell engagers (BiTEs) that target residual tumor. Specifically, EGFRvIII-targeted CAR T cells were engineered to secrete BiTEs against wild-type EGFR, which is not expressed in normal brain but frequently amplified in GBM, recurrent disease and other cancers. Our results demonstrate that human T cells are efficiently transduced with the dual CART.BiTE transgene, and that modified cells successfully secrete biologically active EGFR-specific BiTEs that not only redirect CAR T cells but also recruit and activate untransduced bystander T cells against wild-type EGFR. Recapitulating clinical data, EGFRvIII CAR T cells were unable to completely treat tumors with heterogenous EGFRvIII expression, leading to outgrowth of EGFRvIII-negative, EGFR-positive recurrence. Conversely, CART.BiTE cells mediated potent antitumor efficacy in the setting of heterogeneous tumors and EGFRvIII antigen loss. In these models, infusion of CART.BiTE cells yielded extended survival and cured mice with even late-stage, established intracerebral tumors, including those derived from glioma cell lines and patient-derived glioma neurosphere cultures. Unlike CAR T cells directly targeting EGFR, which caused on-target toxicity against human skin grafts in vivo, secreted BiTE-EGFR was locally effective at exceedingly low concentrations that did not mediate immune responses against healthy human tissue. These results demonstrate that CARs and BiTEs can be combined strategically to mitigate the impact of antigen heterogeneity in GBM, and also provide a unique T-cell-based delivery method for BiTEs to tumors in the brain. Citation Format: Bryan D. Choi, Xiaoling Yu, Ana P. Castano, Amanda A. Bouffard, William T. Curry, Bob S. Carter, Marcela V. Maus. BiTE-armored CARs overcome antigen escape in EGFRvIII-targeted therapy for glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-066.

Citation for pulished version (APA): Schmidts, A., Ormhøj, M., Choi, B. D., Taylor, A. O., Bouffard, A. A., Scarfò, I., Larson, R. C., Frigault, M. J., Gallagher, K., Castano, A. P., Riley, L. S., Cabral, M. L., Boroughs, A. C., Velasco Cárdenas, R. M. H., Schamel, W., Zhou, J., Mackay, S., Tai, Y. T., Anderson, K. C., & Maus, M. V. (2019). Rational design of a trimeric Aprilbased CAR-binding domain enables efficient targeting of multiple myeloma. Blood Advances, 3(21), 3248-3260. https://doi.org/10.1182/bloodadvances.2019000703

Despite remarkable success in the treatment of hematological malignancies, CAR T-cell therapies for solid tumors have floundered, in large part due to local immune suppression and the effects of prolonged stimulation leading to T-cell dysfunction and exhaustion. One mechanism by which gliomas and other cancers can hamper CAR T cells is through surface expression of inhibitory ligands such as programmed cell death ligand 1 (PD-L1). Using the CRIPSR-Cas9 system, we created universal CAR T cells resistant to PD-1 inhibition through multiplexed gene disruption of endogenous T-cell receptor (TRAC), beta-2 microglobulin (B2M) and PD-1 (PDCD1). Triple gene-edited CAR T cells demonstrated enhanced activity in preclinical glioma models. Prolonged survival in mice bearing intracranial tumors was achieved after intracerebral, but not intravenous administration. CRISPR-Cas9 gene-editing not only provides a potential source of allogeneic, universal donor cells, but also enables simultaneous disruption of checkpoint signaling that otherwise impedes maximal antitumor functionality.

INTRODUCTION: Immune therapy with T cells engineered to express chimeric antigen receptors (CARs) represents a promising therapy for patients with glioblastoma (GBM). However, clinical responses have been limited due to heterogeneous target antigen expression and outgrowth of tumors lacking the antigen targeted by CAR T cells directed against a single target. In clinical studies with CART-EGFRvIII, EGFRvIII-targeted T cells successfully localized to the brain tumor microenvironment, but ultimately failed to prevent disease progression with post-treatment specimens demonstrating high levels of wild-type EGFR despite reduced expression of EGFRvIII. METHODS: We developed a novel bicistronic CAR construct engineered for local delivery of bispecific T-cell engagers (BiTEs) that target residual tumor. Specifically, EGFRvIII-targeted CAR T cells were engineered to secrete BiTEs against wild-type EGFR, which is frequently amplified and overexpressed in GBM. RESULTS: Human T cells were efficiently transduced with the dual CART.BiTE transgene. These modified cells secreted biologically active EGFR-specific BiTEs that not only redirected CAR T cells but also recruited and activated untransduced bystander T cells against wild-type EGFR. Recapitulating clinical data, EGFRvIII CAR T cells were unable to completely treat tumors with heterogenous EGFRvIII expression, leading to outgrowth of EGFRvIII-negative, EGFR-positive GBM. Conversely, CART.BiTE cells cured mice even in the setting of antigen-loss, against heterogeneous and well-established intracerebral tumors in mice. Unlike CAR T cells directly targeting EGFR, which caused toxicity in human skin grafts in vivo, secreted BiTE-EGFR was both locally effective and did not result in toxicity against grafted human skin. CONCLUSION: This is the first instance in which CARs and BiTEs have been combined into a single platform of immune therapy. Our results demonstrate that CARs and BiTEs can be combined strategically to mitigate antigen heterogeneity in GBM and also provide a unique T-cell-based delivery method for BiTEs to tumors in the brain.

e16530 Background: Changes in PSA are widely used as a biomarker for the monitoring of treatment outcome in Metastatic Castration-Resistant Prostate Cancer (mCRPC) in the clinical real-world setting. Early PSA changes (before 12 weeks) are not considered in the definition of PSA Progression (PSAProg) due to the potential for spurious “flare” reactions. We aimed to evaluate the significance of an early PSA increase in Abiraterone/Enzalutamide (Abi/Enz)-treated mCRPC patients (pts). Methods: We retrospectively evaluated Abi/Enz-treated mCRPC pts from 11 hospitals between 2011-2018. Early PSAProg was defined as a 25% increase in PSA from baseline at 4 (PSAProg4) or 8 (PSAProg8) weeks after treatment initiation. PSA progression at 12 weeks (PSAProg12) was confirmed by a second reading. Uni- and multivariable (MV) Cox regression models were conducted to explore the association of PSAProg and overall (OS) and radiographic progression-free (rPFS) survival. Sensitivity (Se), specificity (Sp) and predictive values (PPV, NPV) for the association of early PSAProg with PSAProg12 were calculated. Results: We analyzed 581 mCRPC pts; median follow-up: 19.1 months. 96 (17.1%); 105 (21.6%) and 85 (16.9%) pts had PSAprog at 4, 8 and 12 wks. PSAProg4 and PSAProg8 were significantly associated with confirmed PSAProg12. 55.3% of pts with PSAProg4 and 66.7% of pts with PSAProg8 had a confirmed PSAProg12. Only 9% of pts with no PSA prog at 4 wks and 4.1% of pts with no PSAProg8 had a confirmed PSAProg12. PSAProg4 had Se: 56.6%, Sp: 90.5%, PPV: 55.2%, NPV: 91% for the detection of PSAProg12. PSAProg8 had Se: 81.9%, Sp: 91.2%, PPV: 66.7%, NPV: 95.9% for the detection of PSAProg12. PSAprog at 4, 8 and 12 wks was significantly associated with OS and rPFS in uni- and MV Cox models (Table). Conclusions: Early PSAProg after Abi/Enz is significantly associated with both confirmed PSA Prog at 12 wks and outcome, and may help identify pts not benefitting from Abi/Enz before clinical or radiographic progression. Prospective validation studies are needed. [Table: see text]

Background: CAR-T cells have led to a revolution in the treatment of advanced hematologic malignancies. Since these cells target antigens that are expressed on the cellular surface, it is imperative that there is near ubiquitous tumor expression with minimal expression vital human tissues. Finding targets with these characteristics in myeloid malignancies has been challenging. Typical markers expressed on the surface of AML are also expressed on essential innate immune effector cells (e.g. neutrophils) which, if targeted, could lead to prolonged absence of this immune arm, which is not survivable or replaceable. Current approaches rely on the use of CAR-T cells against common myeloid targets (e.g. CD123, CD33) as an ablative strategy with a planned allogeneic stem cell transplant rescue to eradicate the CAR-T cells afterwards. These solutions have resulted in significant toxicity with several deaths resulting from CD123-targeted CAR-T cells. Another approach has involved gene editing donor progenitor cells to delete CD33, repopulation of the marrow with these CD33 negative cells, and then treatment with CD33-targeted CAR-T cells. (Kim, Cell 2018). However, this approach is challenging, costly, and genomic editing of stem cells remains a concern. CD70 is an immune checkpoint found on antigen presenting cells and activated T cells. Multiple studies have shown a strong degree of expression on AML blasts and leukemic stem cells, with minimal normal tissue expression (Perna, Cell 2017, Riether J Exp Med 2017). A Phase 1 study of a CD70 targeted antibody drug conjugate in combination with azacitidine (which has been shown to increase CD70 expression on leukemic stem cells) for untreated AML patients has shown impressive results (Blood 2018 132:2680, Blood 2017 130:2652). Based on these findings, we explored CD70-targeting CARs for the treatment of AML. Methods: Based on our success with a trimeric ligand-based CAR of another TNFα family member, APRIL, for multiple myeloma (Schmidt Blood 2018 132:2059), we generated monomeric and trimeric second-generation ligand-based CAR constructs to target CD70 on AML. In vitro effector function was compared by cytotoxic potency and cytokine production. In vivo anti-tumor efficiency was assessed in a xenograft mouse model of AML. Effect of surface CD70 expression on AML cell lines after co-culture with azacitidine was assessed. Results: CAR T cell manufacturing of both constructs was accomplished successfully (transduction efficiency 70-93%) from three different healthy donors with no apparent fratricide. CD70 CARs were efficacious in in vitro cytotoxicity assays targeting an AML cell line Molm13. Unexpectedly, monomeric CD70 targeted CAR-T cells were superior to trimeric in cytotoxicity assays and, thus, were carried forward for in vivo assays. Next, we treated NSG mice that had been engrafted with Molm13 and demonstrated a substantial dose-dependent therapeutic effect with prolonged survival of CAR treated mice compared to those treated with untransduced T-cells (UTD). Treated mice demonstrated a CAR-T robust expansion in the peripheral blood assessed by flow cytometry that was commensurate with individual animal treatment responses. Bone marrow from these mice revealed substantially reduced CD70 in all groups. Preliminary in vitro co-culture of AML cells with azacitidine showed increased CD70 expression. Conclusion: CD70 based CAR-T targeting of AML is effective in vitro and in vivo. Combination treatment with azacitidine may increase target antigen expression and lead to synergistic activity and represents a viable therapeutic strategy that warrants further investigation. Treatment of AML engrafted NSG mice with CD70 CAR-T cells in conjunction with azacitidine is ongoing. Disclosures Frigault: Xenetic: Consultancy; Novartis: Consultancy; Juno/Celgene: Consultancy; Foundation Medicine: Consultancy; Incyte: Consultancy; Nkarta: Consultancy; Kite/Gilead: Honoraria. Maus:INFO PENDING: Other: INFO PENDING.

Cell-based therapies are emerging as potent agents against cancer and other diseases, but are uniquely uncontrolled "living drugs". For example, chimeric antigen receptor (CAR) T cells can effectively target hematologic malignancies yet pose a risk for toxic hyperactivation. Future cell-based therapies, including CAR T cells, could be improved by incorporating specific and reversible control systems. However, clinically suitable ON- and OFF-switches engineered from non-immunogenic human polypeptide sequences and regulated by non-immunosuppressive FDA-approved drugs are needed. Here we report the engineering of a robust lenalidomide-responsive degron tag, which we then used to construct a degradable CAR affording reversible OFF-switch functional control at clinically relevant lenalidomide doses. Thalidomide, lenalidomide, and pomalidomide are effective and clinically approved therapies for multiple myeloma, subtypes of non-Hodgkin lymphoma, and myelodysplastic syndrome with chromosome 5q deletion. These drugs exert therapeutic properties by acting as molecular glue, bridging interactions between the CRL4CRBN ubiquitin ligase and disease-relevant proteins that are subsequently ubiquitinated and degraded by the proteasome. A set of Cys2-His2 (C2H2) zinc fingers have emerged as degron motifs mediating drug-dependent interactions with the CRL4CRBN ubiquitin ligase. We hypothesized that these small, modular, human polypeptide domains could be further engineered and repurposed as tags to induce drug-dependent depletion of engineered proteins. By systematically shuffling the subdomains of all known zinc finger degrons and functionally screening this hybrid zinc finger library, we engineered "super-degron" tags that are more efficiently degraded at lower drug concentrations than any C2H2 zinc finger in the human proteome. We then incorporated one of these super-degrons (SD01) into a second-generation CAR targeting CD19 (FMC63-4-1BB-CD3z), thereby constructing a degradable CAR (Figure 1A). In a Jurkat T cell model, addition of lenalidomide induced rapid and near-complete depletion of the degradable CAR (Figure 1B/C). When co-cultured with target cells expressing CD19, therapeutically relevant lenalidomide concentrations robustly inhibited T cell activation (Figure 1D). Indeed, the IC50 for inhibition of IL2 secretion and CD69 expression were 2 and 22 nM lenalidomide, respectively, whereas in myeloma patients the plasma concentration of lenalidomide following one 25 mg oral dose decays from ~1000 to 10 nM over the course of 24 hours (Connarn et al, CPDD, 2017). In primary T cells, 100 nM lenalidomide suppressed degradable CAR effector functions including tumor cell killing and cytokine release (Figure 1E). Using pomalidomide, which has a longer in vivo half-life, we demonstrated robust and reversible depletion of the degradable CAR in T cells engrafted in NSG mice. Together, these findings demonstrate reversible OFF-switch control of degradable CARs at clinically relevant lenalidomide concentrations. Experiments are underway to determine whether, in the context of tumor clearance in NSG mice, degradable CAR T cell effector functions can be paused and subsequently released with short-term administration of lenalidomide. Whereas the current management of CAR T cell hyperactivation syndromes consists of supportive care, tocilizumab, and/or high-dose corticosteroids, we propose that cytokine release and CAR-related encephalopathy syndromes may be more easily diagnosed and managed with degradable CARs. The super-degron tags presented here are generalizable and clinically suitable tools to achieve chemical genetic control of diverse genetically engineered cell therapies. Disclosures Jan: Broad Institute: Other: Contributor to a patent filing on this technology that is held by the Broad Institute.. Sievers:Broad Institute: Other: Contributor to a patent filing on this technology that is held by the Broad Institute.. Maus:Broad Institute: Other: Contributor to a patent filing on this technology that is held by the Broad Institute.. Ebert:Broad Institute: Other: Contributor to a patent filing on this technology that is held by the Broad Institute.; Deerfield: Research Funding; Celgene: Research Funding.

AbstractPurpose: T cells engineered to express a chimeric antigen receptor (CAR) against CD19 have recently been FDA approved for the treatment of relapsed or refractory large B-cell lymphoma. Despite the success and curative potential of CD19 CAR T cells, several reports describing disease relapse due to antigen loss are now emerging. Experimental Design: We developed a novel CAR construct directed against CD79b, a critical receptor for successful B-cell development that remains highly expressed in several subtypes of B-cell lymphoma, including mantle cell lymphoma (MCL). We tested CAR T cells directed against CD79b alone or in combination with CD19 targeting in a single construct, against cell line- and patient-derived xenograft models. Results: We demonstrate CAR79b antigen-specific recognition and cytotoxicity against a panel of cell lines and patient-derived xenograft models of MCL. Importantly, we show that downregulation of CD19 does not influence surface expression of CD79b and that anti-CD79b CAR T cells alone or arranged in a dual-targeting format with a CD19 single-chain variable fragment (scFv) are able to recognize and eliminate CD19+, CD19−, and mixed CD19+/CD19−B-cell lymphoma. Conclusions: Our findings demonstrate that CAR T cells targeting CD79b alone or in combination have promise for treating and preventing CD19 antigen escape in B-cell lymphomas.

Chimeric antigen receptor (CAR)-T-cell therapy for solid tumors is limited due to heterogeneous target antigen expression and outgrowth of tumors lacking the antigen targeted by CAR-T cells directed against single antigens. Here, we developed a bicistronic construct to drive expression of a CAR specific for EGFRvIII, a glioblastoma-specific tumor antigen, and a bispecific T-cell engager (BiTE) against EGFR, an antigen frequently overexpressed in glioblastoma but also expressed in normal tissues. CART.BiTE cells secreted EGFR-specific BiTEs that redirect CAR-T cells and recruit untransduced bystander T cells against wild-type EGFR. EGFRvIII-specific CAR-T cells were unable to completely treat tumors with heterogenous EGFRvIII expression, leading to outgrowth of EGFRvIII-negative, EGFR-positive glioblastoma. However, CART.BiTE cells eliminated heterogenous tumors in mouse models of glioblastoma. BiTE-EGFR was locally effective but was not detected systemically after intracranial delivery of CART.BiTE cells. Unlike EGFR-specific CAR-T cells, CART.BiTE cells did not result in toxicity against human skin grafts in vivo.

Chimeric antigen receptor (CAR) T cells (CARTs) have shown tremendous potential for the treatment of certain B-cell malignancies, including patients with relapsed/refractory multiple myeloma (MM). Targeting the B-cell maturation antigen (BCMA) has produced the most promising results for CART therapy of MM to date, but not all remissions are sustained. Emergence of BCMA escape variants has been reported under the selective pressure of monospecific anti-BCMA CART treatment. Thus, there is a clinical need for continuous improvement of CART therapies for MM. Here, we show that a novel trimeric APRIL (a proliferation-inducing ligand)-based CAR efficiently targets both BCMA(+) and BCMA(-) MM. Modeled after the natural ligand-receptor pair, APRIL-based CARs allow for bispecific targeting of the MM-associated antigens BCMA and transmembrane activator and CAML interactor (TACI). However, natural ligands as CAR antigen-binding domains may require further engineering to promote optimal binding and multimerization to adequately trigger T-cell activation. We found that using a trimeric rather than a monomeric APRIL format as the antigen-binding domain enhanced binding to BCMA and TACI and CART activity against MM in vitro and in vivo. Dual-specific, trimeric APRIL-based CAR are a promising therapeutic approach for MM with potential for preventing and treating BCMA escape.