Drug-regulated CD33-targeted CAR T cells control AML using clinically optimized rapamycin dosing.

Chimeric antigen receptor (CAR) designs that incorporate pharmacologic control are desirable; however, designs suitable for clinical translation are needed. We designed a fully human, rapamycin-regulated drug product for targeting CD33+ tumors called dimerizaing agent-regulated immunoreceptor complex (DARIC33). T cell products demonstrated target-specific and rapamycin-dependent cytokine release, transcriptional responses, cytotoxicity, and in vivo antileukemic activity in the presence of as little as 1 nM rapamycin. Rapamycin withdrawal paused DARIC33-stimulated T cell effector functions, which were restored following reexposure to rapamycin, demonstrating reversible effector function control. While rapamycin-regulated DARIC33 T cells were highly sensitive to target antigen, CD34+ stem cell colony-forming capacity was not impacted. We benchmarked DARIC33 potency relative to CD19 CAR T cells to estimate a T cell dose for clinical testing. In addition, we integrated in vitro and preclinical in vivo drug concentration thresholds for off-on state transitions, as well as murine and human rapamycin pharmacokinetics, to estimate a clinically applicable rapamycin dosing schedule. A phase I DARIC33 trial has been initiated (PLAT-08, NCT05105152), with initial evidence of rapamycin-regulated T cell activation and antitumor impact. Our findings provide evidence that the DARIC platform exhibits sensitive regulation and potency needed for clinical application to other important immunotherapy targets.

Differential protein-protein interactions underlie signaling mediated by the TCR and a 4-1BB domain-containing CAR.

Cells rely on activity-dependent protein-protein interactions to convey biological signals. For chimeric antigen receptor (CAR) T cells containing a 4-1BB costimulatory domain, receptor engagement is thought to stimulate the formation of protein complexes similar to those stimulated by T cell receptor (TCR)-mediated signaling, but the number and type of protein interaction-mediating binding domains differ between CARs and TCRs. Here, we performed coimmunoprecipitation mass spectrometry analysis of a second-generation, CD19-directed 4-1BB:ζ CAR (referred to as bbζCAR) and identified 128 proteins that increased their coassociation after target engagement. We compared activity-induced TCR and CAR signalosomes by quantitative multiplex coimmunoprecipitation and showed that bbζCAR engagement led to the activation of two modules of protein interactions, one similar to TCR signaling that was more weakly engaged by bbζCAR as compared with the TCR and one composed of TRAF signaling complexes that was not engaged by the TCR. Batch-to-batch and interindividual variations in production of the cytokine IL-2 correlated with differences in the magnitude of protein network activation. Future CAR T cell manufacturing protocols could measure, and eventually control, biological variation by monitoring these signalosome activation markers.

Aptamer-Based Chromatographic Methods for Efficient and Economical Separation of Leukocyte Populations.

The manufacturing process of chimeric antigen receptor T cell therapies includes isolation systems that provide pure T cells. Current magnetic-activated cell sorting and immunoaffinity chromatography methods produce desired cells with high purity and yield but require expensive equipment and reagents and involve time-consuming incubation steps. Here, we demonstrate that aptamers can be employed in a continuous-flow resin platform for both depletion of monocytes and selection of CD8+ T cells from peripheral blood mononuclear cells at low cost with high purity and throughput. Aptamer-mediated cell selection could potentially enable fully synthetic, traceless isolations of leukocyte subsets from a single isolation system.

A stem cell epigenome is associated with primary nonresponse to CD19 CAR T cells in pediatric acute lymphoblastic leukemia.

CD19 chimeric antigen receptor T-cell therapy (CD19-CAR) has changed the treatment landscape and outcomes for patients with pre-B-cell acute lymphoblastic leukemia (B-ALL). Unfortunately, primary nonresponse (PNR), sustained CD19+ disease, and concurrent expansion of CD19-CAR occur in 20% of the patients and is associated with adverse outcomes. Although some failures may be attributable to CD19 loss, mechanisms of CD19-independent, leukemia-intrinsic resistance to CD19-CAR remain poorly understood. We hypothesize that PNR leukemias are distinct compared with primary sensitive (PS) leukemias and that these differences are present before treatment. We used a multiomic approach to investigate this in 14 patients (7 with PNR and 7 with PS) enrolled in the PLAT-02 trial at Seattle Children's Hospital. Long-read PacBio sequencing helped identify 1 PNR in which 47% of CD19 transcripts had exon 2 skipping, but other samples lacked CD19 transcript abnormalities. Epigenetic profiling discovered DNA hypermethylation at genes targeted by polycomb repressive complex 2 (PRC2) in embryonic stem cells. Similarly, assays of transposase-accessible chromatin-sequencing revealed reduced accessibility at these PRC2 target genes, with a gain in accessibility of regions characteristic of hematopoietic stem cells and multilineage progenitors in PNR. Single-cell RNA sequencing and cytometry by time of flight analyses identified leukemic subpopulations expressing multilineage markers and decreased antigen presentation in PNR. We thus describe the association of a stem cell epigenome with primary resistance to CD19-CAR therapy. Future trials incorporating these biomarkers, with the addition of multispecific CAR T cells targeting against leukemic stem cell or myeloid antigens, and/or combined epigenetic therapy to disrupt this distinct stem cell epigenome may improve outcomes of patients with B-ALL.

Locoregional CAR T cells for children with CNS tumors: Clinical procedure and catheter safety.

Central nervous system (CNS) tumors are the most common solid malignancy in the pediatric population. Based on adoptive cellular therapy's clinical success against childhood leukemia and the preclinical efficacy against pediatric CNS tumors, chimeric antigen receptor (CAR) T cells offer hope of improving outcomes for recurrent tumors and universally fatal diseases such as diffuse intrinsic pontine glioma (DIPG). However, a major obstacle for tumors of the brain and spine is ineffective T cell chemotaxis to disease sites. Locoregional CAR T cell delivery via infusion through an intracranial catheter is currently under study in multiple early phase clinical trials. Here, we describe the Seattle Children's single-institution experience including the multidisciplinary process for the preparation of successful, repetitive intracranial T cell infusion for children and the catheter-related safety of our 307 intracranial CAR T cell doses.

Intraventricular B7-H3 CAR T Cells for Diffuse Intrinsic Pontine Glioma: Preliminary First-in-Human Bioactivity and Safety.

Diffuse intrinsic pontine glioma (DIPG) remains a fatal brainstem tumor demanding innovative therapies. As B7-H3 (CD276) is expressed on central nervous system (CNS) tumors, we designed B7-H3-specific chimeric antigen receptor (CAR) T cells, confirmed their preclinical efficacy, and opened BrainChild-03 (NCT04185038), a first-in-human phase I trial administering repeated locoregional B7-H3 CAR T cells to children with recurrent/refractory CNS tumors and DIPG. Here, we report the results of the first three evaluable patients with DIPG (including two who enrolled after progression), who received 40 infusions with no dose-limiting toxicities. One patient had sustained clinical and radiographic improvement through 12 months on study. Patients exhibited correlative evidence of local immune activation and persistent cerebrospinal fluid (CSF) B7-H3 CAR T cells. Targeted mass spectrometry of CSF biospecimens revealed modulation of B7-H3 and critical immune analytes (CD14, CD163, CSF-1, CXCL13, and VCAM-1). Our data suggest the feasibility of repeated intracranial B7-H3 CAR T-cell dosing and that intracranial delivery may induce local immune activation. SIGNIFICANCE: This is the first report of repeatedly dosed intracranial B7-H3 CAR T cells for patients with DIPG and includes preliminary tolerability, the detection of CAR T cells in the CSF, CSF cytokine elevations supporting locoregional immune activation, and the feasibility of serial mass spectrometry from both serum and CSF. This article is highlighted in the In This Issue feature, p. 1.

Murine CAR19 Tregs suppress acute graft-versus-host disease and maintain graft-versus-tumor responses.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) efficacy is complicated by graft-versus-host disease (GVHD), a leading cause of morbidity and mortality. Regulatory T cells (Tregs) have shown efficacy in preventing GVHD. However, high Treg doses are often required, necessitating substantial ex vivo or in vivo expansion that may diminish suppressor function. To enhance in vivo suppressor function, murine Tregs were transduced to express an anti-human CD19 chimeric antigen receptor (hCAR19) and infused into lethally irradiated, hCD19-transgenic recipients for allo-HSCT. Compared with recipients receiving control transduced Tregs, those receiving hCAR19 Tregs had a marked decrease in acute GVHD lethality. Recipient hCD19 B cells and murine hCD19 TBL12-luciferase (TBL12luc) lymphoma cells were both cleared by allogeneic hCAR19 Tregs, which was indicative of graft-versus-tumor (GVT) maintenance and potentiation. Mechanistically, hCAR19 Tregs killed syngeneic hCD19+ but not hCD19- murine TBL12luc cells in vitro in a perforin-dependent, granzyme B-independent manner. Importantly, cyclophosphamide-treated, hCD19-transgenic mice given hCAR19 cytotoxic T lymphocytes without allo-HSCT experienced rapid lethality due to systemic toxicity that has been associated with proinflammatory cytokine release; in contrast, hCAR19 Treg suppressor function enabled avoidance of this severe complication. In conclusion, hCAR19 Tregs are a potentially novel and effective strategy to suppress GVHD without loss of GVT responses.

FGFR4-Targeted Chimeric Antigen Receptors Combined with Anti-Myeloid Polypharmacy Effectively Treat Orthotopic Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue cancer in children. Treatment outcomes, particularly for relapsed/refractory or metastatic disease, have not improved in decades. The current lack of novel therapies and low tumor mutational burden suggest that chimeric antigen receptor (CAR) T-cell therapy could be a promising approach to treating RMS. Previous work identified FGF receptor 4 (FGFR4, CD334) as being specifically upregulated in RMS, making it a candidate target for CAR T cells. We tested the feasibility of an FGFR4-targeted CAR for treating RMS using an NSG mouse with RH30 orthotopic (intramuscular) tumors. The first barrier we noted was that RMS tumors produce a collagen-rich stroma, replete with immunosuppressive myeloid cells, when T-cell therapy is initiated. This stromal response is not seen in tumor-only xenografts. When scFV-based binders were selected from phage display, CARs targeting FGFR4 were not effective until our screening approach was refined to identify binders to the membrane-proximal domain of FGFR4. Having improved the CAR, we devised a pharmacologic strategy to augment CAR T-cell activity by inhibiting the myeloid component of the T-cell-induced tumor stroma. The combined treatment of mice with anti-myeloid polypharmacy (targeting CSF1R, IDO1, iNOS, TGFbeta, PDL1, MIF, and myeloid misdifferentiation) allowed FGFR4 CAR T cells to successfully clear orthotopic RMS tumors, demonstrating that RMS tumors, even with very low copy-number targets, can be targeted by CAR T cells upon reversal of an immunosuppressive microenvironment.

Discovery of a Transferrin Receptor 1-Binding Aptamer and Its Application in Cancer Cell Depletion for Adoptive T-Cell Therapy Manufacturing.

The clinical manufacturing of chimeric antigen receptor (CAR) T cells includes cell selection, activation, gene transduction, and expansion. While the method of T-cell selection varies across companies, current methods do not actively eliminate the cancer cells in the patient's apheresis product from the healthy immune cells. Alarmingly, it has been found that transduction of a single leukemic B cell with the CAR gene can confer resistance to CAR T-cell therapy and lead to treatment failure. In this study, we report the identification of a novel high-affinity DNA aptamer, termed tJBA8.1, that binds transferrin receptor 1 (TfR1), a receptor broadly upregulated by cancer cells. Using competition assays, high resolution cryo-EM, and de novo model building of the aptamer into the resulting electron density, we reveal that tJBA8.1 shares a binding site on TfR1 with holo-transferrin, the natural ligand of TfR1. We use tJBA8.1 to effectively deplete B lymphoma cells spiked into peripheral blood mononuclear cells with minimal impact on the healthy immune cell composition. Lastly, we present opportunities for affinity improvement of tJBA8.1. As TfR1 expression is broadly upregulated in many cancers, including difficult-to-treat T-cell leukemias and lymphomas, our work provides a facile, universal, and inexpensive approach for comprehensively removing cancerous cells from patient apheresis products for safe manufacturing of adoptive T-cell therapies.

Modified Manufacturing Process Modulates CD19CAR T-cell Engraftment Fitness and Leukemia-Free Survival in Pediatric and Young Adult Subjects.

T cells modified to express a chimeric antigen receptor (CAR) targeting CD19 can induce potent and sustained responses in children with relapsed/refractory acute lymphoblastic leukemia (ALL). The durability of remission is related to the length of time the CAR T cells persist. Efforts to understand differences in persistence have focused on the CAR construct, in particular the costimulatory signaling module of the chimeric receptor. We previously reported a robust intent-to-treat product manufacturing success rate and remission induction rate in children and young adults with recurrent/refractory B-ALL using the SCRI-CAR19v1 product, a second-generation CD19-specific CAR with 4-1BB costimulation coexpressed with the EGFRt cell-surface tag (NCT02028455). Following completion of the phase I study, two changes to CAR T-cell manufacturing were introduced: switching the T-cell activation reagent and omitting midculture EGFRt immunomagnetic selection. We tested the modified manufacturing process and resulting product, designated SCRI-CAR19v2, in a cohort of 21 subjects on the phase II arm of the trial. Here, we describe the unanticipated enhancement in product performance resulting in prolonged persistence and B-cell aplasia and improved leukemia-free survival with SCRI-CAR19v2 as compared with SCRI-CAR19v1.

B7-H3 Specific CAR T Cells for the Naturally Occurring, Spontaneous Canine Sarcoma Model.

One obstacle for human solid tumor immunotherapy research is the lack of clinically relevant animal models. In this study, we sought to establish a chimeric antigen receptor (CAR) T-cell treatment model for naturally occurring canine sarcomas as a model for human CAR T-cell therapy. Canine CARs specific for B7-H3 were constructed using a single-chain variable fragment derived from the human B7-H3-specific antibody MGA271, which we confirmed to be cross-reactive with canine B7-H3. After refining activation, transduction, and expansion methods, we confirmed target killing in a tumor spheroid three-dimensional assay. We designed a B7-H3 canine CAR T-cell and achieved consistently high levels of transduction efficacy, expansion, and in vitro tumor killing. Safety of the CAR T cells were confirmed in two purposely bred healthy canine subjects following lymphodepletion by cyclophosphamide and fludarabine. Immune response, clinical parameters, and manifestation were closely monitored after treatments and were shown to resemble that of humans. No severe adverse events were observed. In summary, we demonstrated that similar to human cancers, B7-H3 can serve as a target for canine solid tumors. We successfully generated highly functional canine B7-H3-specific CAR T-cell products using a production protocol that closely models human CAR T-cell production procedure. The treatment regimen that we designed was confirmed to be safe in vivo. Our research provides a promising direction to establish in vitro and in vivo models for immunotherapy for canine and human solid tumor treatment.

Off-the-shelf, steroid-resistant, IL13Rα2-specific CAR T cells for treatment of glioblastoma.

BACKGROUND: Wide-spread application of chimeric antigen receptor (CAR) T cell therapy for cancer is limited by the current use of autologous CAR T cells necessitating the manufacture of individualized therapeutic products for each patient. To address this challenge, we have generated an off-the-shelf, allogeneic CAR T cell product for the treatment of glioblastoma (GBM), and present here the feasibility, safety, and therapeutic potential of this approach. METHODS: We generated for clinical use a healthy-donor derived IL13Rα2-targeted CAR+ (IL13-zetakine+) cytolytic T-lymphocyte (CTL) product genetically engineered using zinc finger nucleases (ZFNs) to permanently disrupt the glucocorticoid receptor (GR) (GRm13Z40-2) and endow resistance to glucocorticoid treatment. In a phase I safety and feasibility trial we evaluated these allogeneic GRm13Z40-2 T cells in combination with intracranial administration of recombinant human IL-2 (rhIL-2; aldesleukin) in six patients with unresectable recurrent GBM that were maintained on systemic dexamethasone (4-12 mg/day). RESULTS: The GRm13Z40-2 product displayed dexamethasone-resistant effector activity without evidence for in vitro alloreactivity. Intracranial administration of GRm13Z40-2 in four doses of 108 cells over a two-week period with aldesleukin (9 infusions ranging from 2500-5000 IU) was well tolerated, with indications of transient tumor reduction and/or tumor necrosis at the site of T cell infusion in four of the six treated research subjects. Antibody reactivity against GRm13Z40-2 cells was detected in the serum of only one of the four tested subjects. CONCLUSIONS: This first-in-human experience establishes a foundation for future adoptive therapy studies using off-the-shelf, zinc-finger modified, and/or glucocorticoid resistant CAR T cells.

Hematopoietic Cell Transplantation after CD19 Chimeric Antigen Receptor T Cell-Induced Acute Lymphoblastic Lymphoma Remission Confers a Leukemia-Free Survival Advantage.

Consolidative hematopoietic cell transplantation (HCT) after CD19 chimeric antigen receptor (CAR) T cell therapy is frequently performed for patients with refractory/ relapsed B cell acute lymphoblastic leukemia (B-ALL). However, there is controversy regarding the role of HCT following remission attainment. We evaluated the effect of consolidative HCT on leukemia-free survival (LFS) in pediatric and young adult subjects following CD19 CAR T cell induced remission. We evaluated the effect of consolidative HCT on LFS in pediatric and young adult subjects treated with a 41BB-CD19 CAR T cell product on a phase 1/2 trial, Pediatric and Young Adult Leukemia Adoptive Therapy (PLAT)-02 (ClinicalTrials.gov identifier NCT02028455), using a time-dependent Cox proportional hazards statistical model. Fifty of 64 subjects enrolled in PLAT-02 phase 1 and early phase 2 were evaluated, excluding 14 subjects who did not achieve remission, relapsed, or died before day 63 post-CAR T cell therapy. An improved LFS (P = .01) was observed in subjects who underwent consolidative HCT after CAR T cell therapy versus watchful waiting. Consolidative HCT improved LFS specifically in subjects who had no prior history of HCT, with a trend toward significance (P = .09). This benefit was not evident when restricted to the cohort of 34 subjects with a history of prior HCT (P = .45). However, for subjects who had CAR T cell functional persistence of 63 days or less, inclusive of those with a history of prior HCT, HCT significantly improved LFS outcomes (P = .01). These data support the use of consolidative HCT following CD19 CAR T cell-induced remission for patients with no prior history of HCT and those with short functional CAR T cell persistence.

Arming Immune Cell Therapeutics with Polymeric Prodrugs.

Engineered immune cells are an exciting therapeutic modality, which survey and attack tumors. Backpacking strategies exploit cell targeting capabilities for delivery of drugs to combat tumors and their immune-suppressive environments. Here, a new platform for arming cell therapeutics through dual receptor and polymeric prodrug engineering is developed. Macrophage and T cell therapeutics are engineered to express a bioorthogonal single chain variable fragment receptor. The receptor binds a fluorescein ligand that directs cell loading with ligand-tagged polymeric prodrugs, termed "drugamers." The fluorescein ligand facilitates stable binding of drugamer to engineered macrophages over 10 days with 80% surface retention. Drugamers also incorporate prodrug monomers of the phosphoinositide-3-kinase inhibitor, PI-103. The extended release of PI-103 from the drugamer sustains antiproliferative activity against a glioblastoma cell line compared to the parent drug. The versatility and modularity of this cell arming system is demonstrated by loading T cells with a second fluorescein-drugamer. This drugamer incorporates a small molecule estrogen analog, CMP8, which stabilizes a degron-tagged transgene to provide temporal regulation of protein activity in engineered T cells. These results demonstrate that this bioorthogonal receptor and drugamer system can be used to arm multiple immune cell classes with both antitumor and transgene-activating small molecule prodrugs.

Locoregional infusion of HER2-specific CAR T cells in children and young adults with recurrent or refractory CNS tumors: an interim analysis.

Locoregional delivery of chimeric antigen receptor (CAR) T cells has resulted in objective responses in adults with glioblastoma, but the feasibility and tolerability of this approach is yet to be evaluated for pediatric central nervous system (CNS) tumors. Here we show that engineering of a medium-length CAR spacer enhances the therapeutic efficacy of human erb-b2 receptor tyrosine kinase 2 (HER2)-specific CAR T cells in an orthotopic xenograft medulloblastoma model. We translated these findings into BrainChild-01 ( NCT03500991 ), an ongoing phase 1 clinical trial at Seattle Children's evaluating repetitive locoregional dosing of these HER2-specific CAR T cells to children and young adults with recurrent/refractory CNS tumors, including diffuse midline glioma. Primary objectives are assessing feasibility, safety and tolerability; secondary objectives include assessing CAR T cell distribution and disease response. In the outpatient setting, patients receive infusions via CNS catheter into either the tumor cavity or the ventricular system. The initial three patients experienced no dose-limiting toxicity and exhibited clinical, as well as correlative laboratory, evidence of local CNS immune activation, including high concentrations of CXCL10 and CCL2 in the cerebrospinal fluid. This interim report supports the feasibility of generating HER2-specific CAR T cells for repeated dosing regimens and suggests that their repeated intra-CNS delivery might be well tolerated and activate a localized immune response in pediatric and young adult patients.

Rationally Designed Transgene-Encoded Cell-Surface Polypeptide Tag for Multiplexed Programming of CAR T-cell Synthetic Outputs.

Synthetic immunology, as exemplified by chimeric antigen receptor (CAR) T-cell immunotherapy, has transformed the treatment of relapsed/refractory B cell-lineage malignancies. However, there are substantial barriers-including limited tumor homing, lack of retention of function within a suppressive tumor microenvironment, and antigen heterogeneity/escape-to using this technology to effectively treat solid tumors. A multiplexed engineering approach is needed to equip effector T cells with synthetic countermeasures to overcome these barriers. This, in turn, necessitates combinatorial use of lentiviruses because of the limited payload size of current lentiviral vectors. Accordingly, there is a need for cell-surface human molecular constructs that mark multi-vector cotransduced T cells, to enable their purification ex vivo and their tracking in vivo. To this end, we engineered a cell surface-localizing polypeptide tag based on human HER2, designated HER2t, that was truncated in its extracellular and intracellular domains to eliminate ligand binding and signaling, respectively, and retained the membrane-proximal binding epitope of the HER2-specific mAb trastuzumab. We linked HER2t to CAR coexpression in lentivirally transduced T cells and showed that co-transduction with a second lentivirus expressing our previously described EGFRt tag linked to a second CAR efficiently generated bispecific dual-CAR T cells. Using the same approach, we generated T cells expressing a CAR and a second module, a chimeric cytokine receptor. The HER2txEGFRt multiplexing strategy is now being deployed for the manufacture of CD19xCD22 bispecific CAR T-cell products for the treatment of acute lymphoblastic leukemia (NCT03330691).

Optimized serum stability and specificity of an αvß6 integrin-binding peptide for tumor targeting.

The integrin αvß6 is an antigen expressed at low levels in healthy tissue but upregulated during tumorigenesis, which makes it a promising target for cancer imaging and therapy. A20FMDV2 is a 20-mer peptide derived from the foot-and-mouth disease virus that exhibits nanomolar and selective affinity for αvß6 versus other integrins. Despite this selectivity, A20FMDV2 has had limited success in imaging and treating αvß6+ tumors in vivo because of its poor serum stability. Here, we explore the cyclization and modification of the A20FMDV2 peptide to improve its serum stability without sacrificing its affinity and specificity for αvß6. Using cysteine amino acid substitutions and cyclization by perfluoroarylation with decafluorobiphenyl, we synthesized six cyclized A20FMDV2 variants and discovered that two retained binding to αvß6 with modestly improved serum stability. Further d-amino acid substitutions and C-terminal sequence optimization outside the cyclized region greatly prolonged peptide serum stability without reducing binding affinity. While the cyclized A20FMDV2 variants exhibited increased nonspecific integrin binding compared with the original peptide, additional modifications with the non-natural amino acids citrulline, hydroxyproline, and d-alanine were found to restore binding specificity, with some modifications leading to greater αvß6 integrin selectivity than the original A20FMDV2 peptide. The peptide modifications detailed herein greatly improve the potential of utilizing A20FMDV2 to target αvß6 in vivo, expanding opportunities for cancer targeting and therapy.

CD28 Co-Stimulus Achieves Superior CAR T Cell Effector Function against Solid Tumors Than 4-1BB Co-Stimulus.

Spacer or co-stimulatory components in chimeric antigen receptor (CAR) design influence CAR T cell effector function. Few preclinical mouse models optimally support CAR candidate pre-selection for clinical development. Here we use a model in which murine CAR T cells can be exploited with human tumor xenografts. This mouse-in-mouse approach avoids limitations caused by species-specific factors crucial for CAR T cell survival, trafficking and function. We compared trafficking, expansion and tumor control for T cells expressing different CAR construct designs targeting two antigens (L1CAM or HER2), structurally identical except for spacer (long or short) or co-stimulatory (4-1BB or CD28) domains to be evaluated. Using monoclonal, murine-derived L1CAM-specific CAR T cells in Rag-/- mice harboring established xenografted tumors from a human neuroblastoma cell line revealed a clear superiority in CAR T cell trafficking using CD28 co-stimulation. L1CAM-targeting short spacer-CD28/ζ CAR T cells expanded the most at the tumor site and induced initial tumor regression. Treating patient-derived neuroblastoma xenografts with human L1CAM-targeting CAR T cells confirmed the superiority of CD28 co-stimulus. CD28 superiority was also demonstrated with HER2-specific CAR T cells (targeting ovarian carcinoma xenografts). Our findings encourage incorporating CD28 signaling into CAR design for adoptive T cell treatment of solid tumors.

Genetically engineered macrophages persist in solid tumors and locally deliver therapeutic proteins to activate immune responses.

BACKGROUND: Though currently approved immunotherapies, including chimeric antigen receptor T cells and checkpoint blockade antibodies, have been successfully used to treat hematological and some solid tumor cancers, many solid tumors remain resistant to these modes of treatment. In solid tumors, the development of effective antitumor immune responses is hampered by restricted immune cell infiltration and an immunosuppressive tumor microenvironment (TME). An immunotherapy that infiltrates and persists in the solid TME, while providing local, stable levels of therapeutic to activate or reinvigorate antitumor immunity could overcome these challenges faced by current immunotherapies. METHODS: Using lentivirus-driven engineering, we programmed human and murine macrophages to express therapeutic payloads, including Interleukin (IL)-12. In vitro coculture studies were used to evaluate the effect of genetically engineered macrophages (GEMs) secreting IL-12 on T cells and on the GEMs themselves. The effects of IL-12 GEMs on gene expression profiles within the TME and tumor burden were evaluated in syngeneic mouse models of glioblastoma and melanoma and in human tumor slices isolated from patients with advanced gastrointestinal malignancies. RESULTS: Here, we present a cellular immunotherapy platform using lentivirus-driven genetic engineering of human and mouse macrophages to constitutively express proteins, including secreted cytokines and full-length checkpoint antibodies, as well as cytoplasmic and surface proteins that overcomes these barriers. GEMs traffic to, persist in, and express lentiviral payloads in xenograft mouse models of glioblastoma, and express a non-signaling truncated CD19 surface protein for elimination. IL-12-secreting GEMs activated T cells and induced interferon-gamma (IFNγ) in vitro and slowed tumor growth resulting in extended survival in vivo. In a syngeneic glioblastoma model, IFNγ signaling cascades were also observed in mice treated with mouse bone-marrow-derived GEMs secreting murine IL-12. These findings were reproduced in ex vivo tumor slices comprised of intact MEs. In this setting, IL-12 GEMs induced tumor cell death, chemokines and IFNγ-stimulated genes and proteins. CONCLUSIONS: Our data demonstrate that GEMs can precisely deliver titratable doses of therapeutic proteins to the TME to improve safety, tissue penetrance, targeted delivery and pharmacokinetics.

Biomaterials in Chimeric Antigen Receptor T-Cell Process Development.

Chimeric antigen receptor (CAR) T-cell therapy has transformed the cancer treatment landscape, utilizing ex vivo modified autologous T cells to treat relapsed or refractory B-cell leukemias and lymphomas. However, the therapy's broader impact has been limited, in part, by a complicated, lengthy, and expensive production process. Accordingly, as CAR T-cell therapies are further advanced to treat other cancers, continual innovation in cell manufacturing will be critical to their successful clinical implementation. In this Account, we describe our research efforts using biomaterials to improve the three fundamental steps in CAR T-cell manufacturing: (1) isolation, (2) activation, and (3) genetic modification.Recognizing that clinical T-cell isolation reagents have high cost and supply constraints, we developed a synthetic DNA aptamer and complementary reversal agent technology that isolates label-free CD8+ T cells with high purity and yield from peripheral blood mononuclear cells. Encouragingly, CAR T cells manufactured from both antibody- and aptamer-isolated T cells were comparable in therapeutic potency. Discovery and design of other T-cell specific aptamers and corresponding reversal reagents could fully realize the potential of this approach, enabling inexpensive isolation of multiple distinct T-cell populations in a single isolation step.Current ex vivo T-cell activation materials do not accurately mimic in situ T-cell activation by antigen presenting cells (APCs). They cause unequal CD4+ and CD8+ T-cell expansion, necessitating separate production of CD4+ and CD8+ CAR T cells for therapies that call for balanced infusion compositions. To address these shortcomings, we designed a panel of biodegradable cell-templated silica microparticles with supported lipid bilayers that display stimulatory ligands for T-cell activation. High membrane fluidity, elongated shape, and rough surface topography, all properties of endogenous APCs, were found to be favorable parameters for activation, promoting unbiased and efficient CD4/CD8 T-cell expansion while not terminally differentiating the cells.Viral and electroporation-based gene delivery systems have various drawbacks. Viral vectors are expensive and have limited cargo sizes, whereas electroporation is highly cytotoxic. Thus, low-cost nonviral platforms that transfect T cells with low cytotoxicity and high efficiency are needed for CAR gene delivery. Our group thus synthesized a panel of cationic polymers with different architectures and evaluated their T-cell transfection ability. We identified a comb-shaped polymer formulation that transfected primary T cells with low cytotoxicity, although transfection efficiency was low compared to conventional methods. Analysis of intracellular and extracellular barriers to transfection revealed low uptake of polyplexes and high endosomal pH in T cells, alluding to biological and polymer properties that could be further improved.These innovations represent just a few recent developments in the biomaterials field for addressing CAR T-cell production needs. Together, these technologies and their future advancement will pave the way for economical and straightforward CAR T-cell manufacturing.