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Background: In early-stage triple-negative breast cancer (TNBC), there is accumulating evidence of a correlation between tumor-infiltrating lymphocytes in tumor tissue and favorable clinical outcomes, with a high CD8+/regulatory T-cell (Treg) ratio after neoadjuvant chemotherapy being predictive of overall survival and associated with pathologic complete response (Ladoire S, et al. Br J Cancer. 2011; Park YH, et al. Nat Commun. 2020). Trilaciclib is a transient inhibitor of cyclin-dependent kinase 4/6 that is administered intravenously prior to chemotherapy. In preclinical studies, trilaciclib has been shown to have immune-enhancing effects by differentially arresting CD8+ T-cell and Treg subsets, which is followed by the faster recovery of CD8+ T cells than Tregs in the tumor microenvironment. Methods: This phase 2, single-arm, open-label study aims to evaluate neoadjuvant, single-dose trilaciclib followed by trilaciclib plus dose-dense anthracycline/cyclophosphamide and taxane in patients with early-stage TNBC (NCT05112536). Patients with previously untreated, non-metastatic, confirmed TNBC and a primary tumor ≥ 1.5 cm of any nodal status receive a single dose of trilaciclib 240 mg/m2 during the lead-in phase, followed by 4 cycles of doxorubicin 60 mg/m2 plus cyclophosphamide 600 mg/m2, and 12 weekly cycles of paclitaxel 80 mg/m2. Trilaciclib 240 mg/m2 is administered prior to the first chemotherapy dose of each cycle. Pembrolizumab 400 mg every 6 weeks starting on day 1, cycle 1, and/or carboplatin AUC 1.5 every week starting on day 1, cycle 5, is allowed per investigator discretion. Tumor biopsies and peripheral blood samples are collected prior to any treatment, 7 days ± 1 day post administration of trilaciclib, and during surgery, with an additional blood sample collection on day 1, cycle 2. The primary objective is to evaluate the immune-based mechanism of action of trilaciclib after a single dose of trilaciclib, as measured by changes in the CD8+/Treg ratio in tumor tissue. Pathologic complete response, safety and tolerability, and additional exploratory immune biomarker endpoints will also be assessed. Results: As of June 3, 2022, 9 patients with early-stage TNBC had been enrolled and 8 patients had received the trilaciclib lead-in dose and initiated doxorubicin/cyclophosphamide. Patients had a median age of 53.0 years, and all had stage II tumors at diagnosis, with 7 having ductal carcinoma. The median number of chemotherapy cycles received was 3 (range 1–6), and all 8 patients received pembrolizumab. Seven patients continue study treatment; 1 patient discontinued due to disease progression. Five patients had an adverse event (AE) related to any study treatment, including 4 patients with ≥ 1 trilaciclib-related AE. There were no grade ≥ 3 treatment-related AEs or serious AEs. On-treatment, post-trilaciclib monotherapy biopsies were available for 4 patients. Following neoadjuvant trilaciclib treatment, the median density of stromal CD8+ T cells increased from 103.1/mm2 at baseline to 229.8/mm2 at day 7. The median CD8+/Treg ratio increased in 2 patients from 1.85 at baseline to 1.90 at day 7. Conclusions: Preliminary analysis of on-treatment tumor biopsies from 4 patients suggests that a single dose of trilaciclib may modulate the immune cell composition in the tumor microenvironment to support antitumor immune responses. The increase in CD8+ T cells following 7-day neoadjuvant treatment with trilaciclib supports previous data suggesting a role in T-cell infiltration. The complete dataset from all patients (estimated enrollment: N ≈ 24) and additional biomarker analyses will be presented. Citation Format: Michael Danso, Joyce O’Shaughnessy, Lisa S. Wang, Kailash Mosalpuria, Sara Hurvitz, Shom Goel, Sarah Ahn, Subing Cao, John S. Yi, Taofik Oyekunle, Amanda Jacobson, Andrew Beelen, Jeremy Force. Trilaciclib induces immune changes within the tumor microenvironment in early-stage triple-negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-06-03.

Tumors are inherently heterogeneous in antigen expression, and escape from immune surveillance due to antigen loss remains one of the limitations of targeted immunotherapy. Despite the clinical use of adoptive therapy with chimeric antigen receptor (CAR)–redirected T cells in lymphoblastic leukemia, treatment failure due to epitope loss occurs. Targeting multiple tumor-associated antigens (TAAs) may thus improve the outcome of CAR-T cell therapies. CARs developed to simultaneously target multiple targets are limited by the large size of each single-chain variable fragment and compromised protein folding when several single chains are linearly assembled. Here, we describe single-domain antibody mimics that function within CAR parameters but form a very compact structure. We show that antibody mimics targeting EGFR and HER2 of the ErbB receptor tyrosine kinase family can be assembled into receptor molecules, which we call antibody mimic receptors (amR). These amR can redirect T cells to recognize two different epitopes of the same antigen or two different TAAs in vitro and in vivo.

Supplementary figures 1-6

Correlation of B7-H3 mRNA expression with AML features in existing datasets.

CD19-T cells show equivalent antitumor activity in a lymphoma tumor model in vivo at a lower dose of CAR-T cells

Clinical details associated with primary AML patient samples

Purpose:CD19-redirected chimeric antigen receptor (CAR.CD19) T cells promote clinical responses in patients with relapsed/refractory B-cell non-Hodgkin lymphomas and chronic lymphocytic leukemia (CLL). However, patients showing sustained clinical responses after CAR.CD19-T treatment show increased infection risk due to compromised B-lymphocyte recovery. Mature B cell–derived malignancies express monoclonal immunoglobulins bearing either κ- or λ-light chains. We initially constructed CAR-T targeting the κ-light-chain (CAR.κ) and established a clinical study with it. After optimizing the CAR molecule, cells developed CAR-T targeting the λ-light chain (CAR.λ) and we explored their antitumor activity.Experimental Design:Using Igλ+ lymphoma cell lines and patient-derived Igλ+ CLL cells, we evaluated the in vitro tumor cytotoxicity and cytokine profiles of CAR.λ. We also assessed the in vivo efficacy of CAR.λ in xenograft Igλ+ lymphoma models including a patient-derived xenograft (PDX) of mantle cell lymphoma, and the effects of λ- or κ-light chain–specific CAR-T on normal B lymphocytes in a humanized murine model.Results:CAR.λ demonstrated antitumor effects against Igλ+ lymphoma cells and patient-derived CLL cells in vitro, and in vivo in xenograft and PDX Igλ+ lymphoma murine models. Antitumor activity of CAR.λ was superimposable to CAR.CD19. Furthermore, we demonstrated in the humanized murine model that λ- or κ-light chain–specific CAR-T cells only depleted the corresponding targeted light chain–expressing normal B cells, while sparing the reciprocal light chain carrying B cells.Conclusions:Adoptive transfer of CAR.λ and CAR.κ-T cells represents a useful and alternative modality to CAR.CD19-T cells in treating mature B-cell malignancies with minimal impact on humoral immunity.See related commentary by Jain and Locke, p. 5736

Supplemental Methods

Purpose:The development of safe and effective chimeric antigen receptor (CAR) T-cell therapy for acute myeloid leukemia (AML) has largely been limited by the concomitant expression of most AML-associated surface antigens on normal myeloid progenitors and by the potential prolonged disruption of normal hematopoiesis by the immunotargeting of these antigens. The purpose of this study was to evaluate B7-homolog 3 (B7-H3) as a potential target for AML-directed CAR T-cell therapy. B7-H3, a coreceptor belonging to the B7 family of immune checkpoint molecules, is overexpressed on the leukemic blasts of a significant subset of patients with AML and may overcome these limitations as a potential target antigen for AML-directed CAR-T therapy.Experimental Design:B7-H3 expression was evaluated on AML cell lines, primary AML blasts, and normal bone marrow progenitor populations. The antileukemia efficacy of B7-H3–specific CAR-T cells (B7-H3.CAR-T) was evaluated using in vitro coculture models and xenograft models of disseminated AML, including patient-derived xenograft models. The potential hematopoietic toxicity of B7-H3.CAR-Ts was evaluated in vitro using colony formation assays and in vivo in a humanized mouse model.Results:B7-H3 is expressed on monocytic AML cell lines and on primary AML blasts from patients with monocytic AML, but is not significantly expressed on normal bone marrow progenitor populations. B7-H3.CAR-Ts exhibit efficient antigen-dependent cytotoxicity in vitro and in xenograft models of AML, and are unlikely to cause unacceptable hematopoietic toxicity.Conclusions:B7-H3 is a promising target for AML-directed CAR-T therapy. B7-H3.CAR-Ts control AML and have a favorable safety profile in preclinical models.

Supplementary Figures Legends

The immunosuppressive microenvironment of solid tumours reduces the antitumour activity of chimeric antigen receptor T cells (CAR-T cells). Here, we show that the release-through the implantation of a hyaluronic acid hydrogel-of CAR-T cells targeting the human chondroitin sulfate proteoglycan 4, polymer nanoparticles encapsulating the cytokine interleukin-15 and platelets conjugated with the checkpoint inhibitor programmed death-ligand 1 into the tumour cavity of mice with a resected subcutaneous melanoma tumour inhibits the local recurrence of the tumour as well as the growth of distant tumours, through the abscopal effect. The hydrogel, which functions as a reservoir, facilitates the enhanced distribution of the CAR-T cells within the surgical bed, and the inflammatory microenvironment triggers platelet activation and the subsequent release of platelet-derived microparticles. The post-surgery local delivery of combination immunotherapy through a biocompatible hydrogel reservoir could represent a translational route for preventing the recurrence of cancers with resectable tumours.

Intravenous administration of trilaciclib transiently arrests cyclin-dependent kinase 4/6-dependent cells in the G1 phase of the cell cycle during chemotherapy exposure. In an open-label, phase 2 study in patients with metastatic triple-negative breast cancer (TNBC; NCT02978716), administering trilaciclib prior to gemcitabine plus carboplatin improved overall survival and resulted in enriched T-cell diversity and decreased clonality in peripheral blood compared with administering chemotherapy alone. This phase 2, single-arm, neoadjuvant study (NCT05112536) aims to determine the immune mechanism of action of trilaciclib and its role in modulating antitumor immune responses in patients with TNBC. Previously untreated patients with early-stage TNBC (N = 24) received single-dose trilaciclib monotherapy during the lead-in phase. After 7 days, patients received systemic therapy with 4 cycles of dose-dense doxorubicin plus cyclophosphamide and 12 weekly cycles of paclitaxel, with trilaciclib administered prior to the first dose of systemic therapy per cycle. Pembrolizumab from cycle 1 and/or carboplatin from cycle 5 was permitted per investigator discretion. Patients underwent curative surgery 3-5 weeks after the last dose of systemic therapy. Peripheral blood samples were collected at baseline, 7 (±1) days post trilaciclib monotherapy prior to chemotherapy, and pre dose on cycle 2, day 1. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples and cryopreserved for immune profiling via cytometry by time-of-flight (CyTOF), and for functional analysis via intracellular cytokine staining (ICS) and dimensionality reduction. A preliminary analysis of PBMCs from 7 patients showed a decrease in peripheral myeloid-derived suppressor cells (MDSCs), monocytes, and classical dendritic cells following single-dose trilaciclib monotherapy. There were nominal changes to the frequency of peripheral CD4+ and CD8+ T cells. In 4/7 patients, trilaciclib monotherapy resulted in increased proportions of activated and polyfunctional CD4+ and CD8+ T cells producing IFNγ, TNFα, and IL-2 cytokines. Furthermore, trilaciclib enhanced the frequency of CD8+ T cells producing granzyme B and perforin. The decrease in MDSCs and increase in functional T cells following trilaciclib monotherapy supports findings from an immune analysis of data from the phase 2 study of trilaciclib in patients with metastatic TNBC. Overall, these data suggest that the transient G1 arrest of peripheral immune cells by single-dose trilaciclib monotherapy favorably modulates immune responses by promoting the generation of polyfunctional T cells and limiting the number of MDSCs in circulation. Enrollment was completed in August 2022. The complete dataset from all 24 patients will be presented. Citation Format: Sarah Ahn, Subing Cao, Amanda Jacobson, Melissa A. Russo, John S. Yi. Single-dose trilaciclib monotherapy potentiates antitumor immunity in the neoadjuvant setting of triple-negative breast cancer by modulating the composition and effector function of peripheral immune cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6674.

When administered prior to chemotherapy, intravenous trilaciclib transiently arrests cyclin-dependent kinase 4/6-dependent cells in the G1 phase of the cell cycle, which has been shown to augment antitumor immunity. In an open-label, phase 2 trial in patients with metastatic triple-negative breast cancer (NCT02978716), administering trilaciclib prior to gemcitabine plus carboplatin improved overall survival, potentially through protection and direct activation of immune function. To determine the impact of trilaciclib on effector T-cell function, we conducted in vitro studies to assess the kinetics of cytotoxic function and generation of polyfunctional T cells. Peripheral blood mononuclear cells (PBMCs) or naïve CD4+ and CD8+ T cells were purified from 6 healthy human donors and activated with CD2/3/28 beads. On days 0, 1, and 3 post activation, 100 nM trilaciclib was added to the cells. To visualize phenotypic and functional changes, T cells that had been activated for 3, 7, or 14 days with or without trilaciclib were restimulated with phorbol myristate acetate (PMA) and ionomycin. Dimension-reduction analysis of flow cytometric data was used to identify polyfunctional subpopulations of CD4+ and CD8+ T cells. T-cell clusters with polyfunctional profiles were identified based on the coproduction of IFNγ, TNFα, and IL-2 cytokines. Subsets of polyfunctional clusters were defined according to other immune phenotypic profiles, including T-cell exhaustion (PD-1 and LAG-3), degranulation (granzyme B), proliferation (Ki-67), and transcription factors (TOX, TCF-1, and T-bet). For both CD4+ and CD8+ T cells, 3 polyfunctional subpopulations were identified based on differential expression of granzyme B, PD-1, and TOX. When CD4+ and CD8+ T cells were activated in the presence of trilaciclib and restimulated with PMA and ionomycin, there was an increase in the frequency of polyfunctional T-cell subsets producing granzyme B. This was compensated by a decrease in granzyme Blow polyfunctional cells, which was associated with increased PD-1high and TOXhigh expression. Clustering analysis of trilaciclib-treated PBMCs also revealed a reduction in proliferating CD8+ T cells with undetectable effector cytokine function based on a decrease in the frequency of CD8+ T cells expressing TOXhigh and Ki-67high. Transient G1 cell cycle arrest in T cells may limit T-cell hyperactivation, which has been shown to induce immunopathological responses. The increased generation of polyfunctional CD8+ T cells is associated with enhanced effector T-cell responses and memory T-cell differentiation. In combination with previous data showing increased differentiation of naïve T cells into memory T cells upon exposure to trilaciclib, these data support a beneficial role for trilaciclib in enhancing antitumor T-cell responses. Citation Format: Sarah Ahn, Subing Cao, Melissa A. Russo, John S. Yi. Transient G1 cell cycle arrest with trilaciclib enhances the generation of polyfunctional CD4+ and CD8+ T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 703.

Purpose: The development of safe and effective chimeric antigen receptor (CAR) T-cell therapy for acute myeloid leukemia (AML) has largely been limited by the concomitant expression of most AML-associated surface antigens on normal myeloid progenitors and by the potential prolonged disruption of normal hematopoiesis by the immunotargeting of these antigens. The purpose of this study was to evaluate B7-homolog 3 (B7-H3) as a potential target for AML-directed CAR T-cell therapy. B7-H3, a coreceptor belonging to the B7 family of immune checkpoint molecules, is overexpressed on the leukemic blasts of a significant subset of patients with AML and may overcome these limitations as a potential target antigen for AML-directed CAR-T therapy. Experimental Design: B7-H3 expression was evaluated on AML cell lines, primary AML blasts, and normal bone marrow progenitor populations. The antileukemia efficacy of B7-H3–specific CAR-T cells (B7-H3.CAR-T) was evaluated using in vitro coculture models and xenograft models of disseminated AML, including patient-derived xenograft models. The potential hematopoietic toxicity of B7-H3.CAR-Ts was evaluated in vitro using colony formation assays and in vivo in a humanized mouse model. Results: B7-H3 is expressed on monocytic AML cell lines and on primary AML blasts from patients with monocytic AML, but is not significantly expressed on normal bone marrow progenitor populations. B7-H3.CAR-Ts exhibit efficient antigen-dependent cytotoxicity in vitro and in xenograft models of AML, and are unlikely to cause unacceptable hematopoietic toxicity. Conclusions: B7-H3 is a promising target for AML-directed CAR-T therapy. B7-H3.CAR-Ts control AML and have a favorable safety profile in preclinical models.

Cytokines that stimulate T cell proliferation, such as interleukin (IL)-15, have been explored as a means of boosting the antitumor activity of chimeric antigen receptor (CAR) T cells. However, constitutive cytokine signaling in T cells and activation of bystander cells may cause toxicity. IL-23 is a two-subunit cytokine known to promote proliferation of memory T cells and T helper type 17 cells. We found that, upon T cell antigen receptor (TCR) stimulation, T cells upregulated the IL-23 receptor and the IL-23α p19 subunit, but not the p40 subunit. We engineered expression of the p40 subunit in T cells (p40-Td cells) and obtained selective proliferative activity in activated T cells via autocrine IL-23 signaling. In comparison to CAR T cells, p40-Td CAR T cells showed improved antitumor capacity in vitro, with increased granzyme B and decreased PD-1 expression. In two xenograft and two syngeneic solid tumor mouse models, p40-Td CAR T cells showed superior efficacy in comparison to CAR T cells and attenuated side effects in comparison to CAR T cells expressing IL-18 or IL-15.

Millions of COVID-19 patients have succumbed to respiratory and systemic inflammation. Hyperstimulation of toll-like receptor (TLR) signaling has been shown to be a key driver of immunopathology following infection by viruses. We found that severely ill COVID-19 patients in the Intensive Care Unit (ICU) display hallmarks of such hyper-stimulation with abundant agonists of nucleic acid sensing TLRs present in their blood and lungs. These nucleic acid-containing Damage and Pathogen Associated Molecular Patterns (DAMPs/PAMPs) can be depleted using nucleic acid-binding microfibers to limit the patient samples’ ability to hyperactivate such innate immune receptors. Single-cell RNA-sequencing revealed that CD16+ monocytes from deceased but not recovered ICU patients exhibit a TLR-tolerant phenotype and a deficient anti-viral response after ex vivo TLR stimulation. Plasma proteomics confirmed such myeloid hyperactivation and revealed DAMP/PAMP carrier consumption in deceased patients. Treatment of these COVID-19 patient samples with MnO nanoparticles effectively neutralizes TLR activation by the abundant nucleic acid-containing DAMPs/PAMPs present in their lungs and blood. Finally, MnO nanoscavenger treatment limits the ability of DAMPs/PAMPs to induce TLR tolerance in monocytes. Thus, treatment with microfiber- or nanoparticle-based DAMP/PAMP scavengers may prove useful for limiting SARS-CoV-2 induced hyperinflammation, preventing monocytic TLR tolerance and improving outcomes in severely ill COVID-19 patients.

Tumors are inherently heterogeneous in antigen expression, and escape from immune surveillance due to antigen loss remains one of the limitations of targeted immunotherapy. Despite the clinical use of adoptive therapy with chimeric antigen receptor (CAR)–redirected T cells in lymphoblastic leukemia, treatment failure due to epitope loss occurs. Targeting multiple tumor-associated antigens (TAAs) may thus improve the outcome of CAR-T cell therapies. CARs developed to simultaneously target multiple targets are limited by the large size of each single-chain variable fragment and compromised protein folding when several single chains are linearly assembled. Here, we describe single-domain antibody mimics that function within CAR parameters but form a very compact structure. We show that antibody mimics targeting EGFR and HER2 of the ErbB receptor tyrosine kinase family can be assembled into receptor molecules, which we call antibody mimic receptors (amR). These amR can redirect T cells to recognize two different epitopes of the same antigen or two different TAAs in vitro and in vivo.

Purpose: CD19-redirected chimeric antigen receptor (CAR.CD19) T cells promote clinical responses in patients with relapsed/refractory B-cell non-Hodgkin lymphomas and chronic lymphocytic leukemia (CLL). However, patients showing sustained clinical responses after CAR.CD19-T treatment show increased infection risk due to compromised B-lymphocyte recovery. Mature B cell–derived malignancies express monoclonal immunoglobulins bearing either κ- or λ-light chains. We initially constructed CAR-T targeting the κ-light-chain (CAR.κ) and established a clinical study with it. After optimizing the CAR molecule, cells developed CAR-T targeting the λ-light chain (CAR.λ) and we explored their antitumor activity. Experimental Design: Using Igλ+ lymphoma cell lines and patient-derived Igλ+ CLL cells, we evaluated the in vitro tumor cytotoxicity and cytokine profiles of CAR.λ. We also assessed the in vivo efficacy of CAR.λ in xenograft Igλ+ lymphoma models including a patient-derived xenograft (PDX) of mantle cell lymphoma, and the effects of λ- or κ-light chain–specific CAR-T on normal B lymphocytes in a humanized murine model. Results: CAR.λ demonstrated antitumor effects against Igλ+ lymphoma cells and patient-derived CLL cells in vitro, and in vivo in xenograft and PDX Igλ+ lymphoma murine models. Antitumor activity of CAR.λ was superimposable to CAR.CD19. Furthermore, we demonstrated in the humanized murine model that λ- or κ-light chain–specific CAR-T cells only depleted the corresponding targeted light chain–expressing normal B cells, while sparing the reciprocal light chain carrying B cells. Conclusions: Adoptive transfer of CAR.λ and CAR.κ-T cells represents a useful and alternative modality to CAR.CD19-T cells in treating mature B-cell malignancies with minimal impact on humoral immunity. See related commentary by Jain and Locke, p. 5736

Despite their clinical success, chimeric antigen receptor (CAR)-T cell therapies for B cell malignancies are limited by lengthy, costly and labor-intensive ex vivo manufacturing procedures that might lead to cell products with heterogeneous composition. Here we describe an implantable Multifunctional Alginate Scaffold for T Cell Engineering and Release (MASTER) that streamlines in vivo CAR-T cell manufacturing and reduces processing time to a single day. When seeded with human peripheral blood mononuclear cells and CD19-encoding retroviral particles, MASTER provides the appropriate interface for viral vector-mediated gene transfer and, after subcutaneous implantation, mediates the release of functional CAR-T cells in mice. We further demonstrate that in vivo-generated CAR-T cells enter the bloodstream and control distal tumor growth in a mouse xenograft model of lymphoma, showing greater persistence than conventional CAR-T cells. MASTER promises to transform CAR-T cell therapy by fast-tracking manufacture and potentially reducing the complexity and resources needed for provision of this type of therapy.

Retinoblastoma (RB) is a pediatric retinal tumor that overexpresses the ganglioside GD2. Although it is treatable in patients with early diagnosis, patients may lose one or two eyes. We generated GD2-specific chimeric antigen receptor T lymphocytes (GD2.CAR-Ts) and locally delivered them to mice with an in situ grafting RB. We showed that, when used in combination with the local release of interleukin-15 and an injectable hydrogel, GD2.CAR-Ts successfully eliminate RB tumor cells without impairing mouse vision. Han and colleagues develop a CAR T cell directed at GD2, a molecule overexpressed in retinoblastoma, which showed efficacy against murine retinoblastoma when combined with IL-15 in a hydrogel delivery system.

Summary Chimeric antigen receptor (CAR) T cell costimulation mediated by CD28 and 4-1BB is essential for CAR-T cell-induced tumor regression. However, CD28 and 4-1BB differentially modulate kinetics, metabolism and persistence of CAR-T cells, and the mechanisms governing these differences are not fully understood. We found that LCK recruited into the synapse of CD28-encoding CAR by co-receptors causes antigen-independent CAR-CD3ζ phosphorylation and increased antigen-dependent T cell activation. In contrast, the synapse formed by 4-1BB-encoding CAR recruits the THEMIS-SHP1 phosphatase complex that attenuates CAR-CD3ζ phosphorylation. We further demonstrated that the CAR synapse can be engineered to recruit either LCK to enhance the kinetics of tumor killing of 4-1BB CAR-T cells or SHP1 to tune down cytokine release of CD28 CAR-T cells.

The cell surface expression of B7-H3 across multiple tumor subtypes and its restricted expression in normal tissues make it an attractive target for cancer immunotherapy. We generated chimeric antigen receptor (CAR) T cells targeting B7-H3 (B7-H3.CAR-Ts), and tested them in pancreatic ductal adenocarcinoma (PDAC), ovarian cancer (OC) and neuroblastoma (NB) models in vitro and in vivo. The results showed that B7-H3.CAR-Ts effectively controlled the growth of PDAC, OC and NB tumor cells in vitro and in orthotopic, metastatic and patient-derived xenograft (PDX) mouse models. In addition, we found that 4-1BB costimulation promotes lower PD1 expression in B7-H3.CAR-Ts, and thus have superior antitumor activity when targeting PD-L1 constitutively expressing tumor cells. Finally, taking the advantage of the cross-reactivity of B7-H3.CAR with murine B7-H3 (mB7-H3), we investigated the antitumor efficacy and safety of B7-H3.CAR-Ts in a syngeneic pancreatic orthotopic tumor model in immunocompetent mouse. The mB7-H3.CAR-Ts significantly controlled the tumor growth in immunocompetent mice tumor model without decreasing the hematopoietic or immune cell numbers, and no tissue damage in any analyzed organs was observed, these findings further support the safety and efficacy of B7-H3.CAR-Ts for clinical application.

The l -isoaspartyl protein carboxyl methyltransferase (PCM) repairs protein damage resulting from spontaneous conversion of aspartyl or asparaginyl residues to isoaspartate and increases long-term stationary-phase survival of Escherichia coli under stress. In the course of studies intended to examine PCM function in metabolically inactive cells, we identified pcm as a gene whose mutation influences the formation of ofloxacin-tolerant persisters. Specifically, a Δ pcm mutant produced persisters for an extended period in stationary phase, and a Δ glpD mutation drastically increased persisters in a Δ pcm background, reaching 23% of viable cells. The high-persister double mutant showed much higher competitive fitness than the pcm mutant in competition with wild type during long-term stationary phase, suggesting a link between persistence and the mitigation of unrepaired protein damage. We hypothesized that reduced metabolism in the high-persister strain might retard protein damage but observed no gross differences in metabolism relative to wild-type or single-mutant strains. However, methylglyoxal, which accumulates in glpD mutants, also increased fitness, suggesting a possible mechanism. High-level persister formation in the Δ pcm Δ glpD mutant was dependent on guanosine pentaphosphate [(p)ppGpp] and polyphosphate. In contrast, persister formation in the Δ pcm mutant was (p)ppGpp independent and thus may occur by a distinct pathway. We also observed an increase in conformationally unstable proteins in the high-persister strain and discuss this as a possible trigger for persistence as a response to unrepaired protein damage. IMPORTANCE Protein damage is an important factor in the survival and function of cells and organisms. One specific form of protein damage, the formation of the abnormal amino acid isoaspartate, can be repaired by a nearly universally conserved enzyme, PCM. PCM-directed repair is associated with stress survival and longevity in bacteria, insects, worms, plants, mice, and humans, but much remains to be learned about the specific effects of protein damage and repair. This paper identifies an unexpected connection between isoaspartyl protein damage and persisters, subpopulations in bacterial cultures showing increased tolerance to antibiotics. In the absence of PCM, the persister population in Escherichia coli bacteria increased, especially if the metabolic gene glpD was also mutated. High levels of persisters in pcm glpD double mutants correlated with increased fitness of the bacteria in a competition assay, and the fitness was dependent on the signal molecule (p)ppGpp; this may represent an alternative pathway for responding to protein damage.

Chimeric antigen receptor T (CAR-T) cell therapy has produced impressive clinical responses in patients with B-cell malignancies. Critical to the success of CAR-T cell therapies was the achievement of robust gene transfer in T cells mediated by viral vectors such as gamma-retroviral vectors. However, current methodologies of retroviral gene transfer rely on spinoculation and the use of retronectin, which may limit the implementation of cost-effective CAR-T cell therapies. Herein, we describe a low cost, tunable, macroporous, alginate scaffold that transduces T cells with retroviral vectors under static condition. CAR-T cells produced by macroporous scaffold-mediated viral transduction exhibited >60% CAR expression, retained effector phenotype, expanded to clinically relevant cell numbers and eradicated CD19(+) lymphoma tumor in vivo. Efficient transduction was dependent on scaffold macroporosity. Taken together, our data shows that macroporous alginate scaffolds serve as an attractive alternative to current transduction protocols and have high potential for clinical translation to genetically modify T cells for adoptive cellular therapy.

Chimeric antigen receptor (CAR)-redirected T lymphocytes (CAR T cells) show modest therapeutic efficacy in solid tumors. The desmoplastic structure of the tumor and the immunosuppressive tumor microenvironment usually account for the reduced efficacy of CAR T cells in solid tumors. Mild hyperthermia of the tumor reduces its compact structure and interstitial fluid pressure (IFP), increases blood perfusion, releases antigens and promotes the recruitment of endogenous immune cells. Therefore, the combination of mild hyperthermia with the adoptive transfer of CAR T cells could potentially increase the therapeutic index of these cells in solid tumors. We found that the chondroitin sulfate proteoglycan-4 (CSPG4)-specific CAR T cells infused in Nod scid gamma (NSG) mice engrafted with the human melanoma WM115 cell line had superior antitumor activity after photothermal ablation of the tumor. Our findings suggest that photothermal therapy facilitates the accumulation and effector function of CAR T cells within the solid tumor.