Supplementary Figure from Preclinical Efficacy of BCMA-Directed CAR T Cells Incorporating a Novel D Domain Antigen Recognition Domain

Abstract Chimeric antigen receptor (CAR) T-cell therapies directed against B-cell maturation antigen (BCMA) have shown compelling clinical activity and manageable safety in subjects with relapsed and refractory multiple myeloma (RRMM). Prior reported CAR T cells have mostly used antibody fragments such as humanized or murine single-chain variable fragments or camelid heavy-chain antibody fragments as the antigen recognition motif. Herein, we describe the generation and preclinical evaluation of ddBCMA CAR, which uses a novel BCMA binding domain discovered from our D domain phage display libraries and incorporates a 4-1BB costimulatory motif and CD3-zeta T-cell activation domain. Preclinical in vitro studies of ddBCMA CAR T cells cocultured with BCMA-positive cell lines showed highly potent, dose-dependent measures of cytotoxicity, cytokine production, T-cell degranulation, and T-cell proliferation. In each assay, ddBCMA CAR performed as well as the BCMA-directed scFv-based C11D5.3 CAR. Furthermore, ddBCMA CAR T cells demonstrated in vivo tumor suppression in three disseminated BCMA-expressing tumor models in NSG-immunocompromised mice. On the basis of these promising preclinical data, CART-ddBCMA is being studied in a first-in-human phase I clinical study to assess the safety, pharmacokinetics, immunogenicity, efficacy, and duration of effect for patients with RRMM (NCT04155749).

Genetically engineered T cells have demonstrated great promise in the treatment of hematologic malignancies including Acute Lymphocytic Leukemia, Non-Hodgkin’s lymphoma and Multiple Myeloma. However, those successes are often associated with serious dose-limiting adverse events including cytokine release syndrome (CRS) and immune effector-cell associated neurotoxicity syndrome (ICANS), as well as frequent tumor relapse. Specifically, in AML, there is a need for controllable and adaptable cellular therapeutics that can address intra- and inter- patient disease heterogeneity and off-target antigen expression that contribute to these dose-limiting adverse events. Herein, we report a novel T cell therapy, ACLX-002, comprised of a soluble tumor targeting protein (SPRX002) that specifically binds CD123-expressing AML cells and targets those cells for destruction by ex vivo transduced T cells known as Antigen Receptor Complex (ARC) T cells. Functional in vitro studies of ARC-T cells produced from healthy subjects demonstrated SPRX002 dose-dependent cytokine production, T cell proliferation, and cytotoxic activity of co-cultured CD123-expressing cancer cell lines. The cytolytic machinery of the ARC-T cells is only activated when the tri-complex of the ARC-T cell, SPRX002, and CD123-expressing cell is fully formed. ACLX-002 also demonstrated dose- and schedule-dependent in vivo efficacy in both the MOLM14 and MV4-11 disseminated CD123-expressing tumor models in NSG immunocompromised mice, as well as multiple AML patient derived xenograft models. A single dose of 5 x 106 ARC-T cells together with SPRX002 doses of 0.3 mg/kg daily were able to eliminate measurable tumor burden that was similar to a CD123-directed traditional CAR-T that uses the same CD123-binding domain as SPRX002. Furthermore, the in vivo efficacy was not limited by the expression of CD123 on donor T cells, an attribute that is induced by in vitro activation and expansion of ARC-T cells. The data support the paradigm that ARC-T cells can be activated and silenced by controlling the dose and schedule of administered SPRX002, which may improve the safety and effectiveness of T cell therapy. Citation Format: Justin P. Edwards, Liubov Zaritskaya, Jeff Swers, Sinnie Ng, Jenny Mu, Alexandra Witter, Laurene Cheung, Samantha McCullough, David LaFleur, David Hilbert, David Tice. ACLX-002, a novel CD123-targeted universal CAR-T cell therapy for relapsed or refractory acute myeloid leukemia that can be activated and silenced in vivo with soluble protein adapters in a dose dependent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 587.

Immunologic rejection of triple-negative breast cancer (TNBC) is rare. Focused Ultrasound (FUS) promotes anti-tumor immunity by inducing tissue destruction and inflammation. We hypothesized that thermally-ablative FUS (tFUS) would increase the immune response to TNBC. However, TNBC is commonly accompanied by the expansion of immunosuppressive myeloid cells. By implanting a metastasizing TNBC cell line, 4T1, into BALB/c mice, we showed that a combinatorial therapy of tFUS and Gemcitabine (GEM), a myeloablative chemotherapy, was able to control primary tumor growth and reduce mortality. This response is dependent on T cell-mediated immunity. While this treatment remarkably resulted in some cures, the effects were not durable in every case; suggesting immunosuppression may be re-established. We hypothesize GEM is synergizing with FUS to immunologically control tumor growth by either depleting myeloid derived suppressor cells (MDSCs) or inducing tumor cell death. To discriminate between these possibilities, we will use a Ly6G-specific 1A8 antibody to deplete the MDSC population. In parallel, we are interrogating the ability of GEM to induce immunogenic cell death in this model. Current studies are focused on understanding alterations and the role of CD4 and CD8 T cells in tumor control after FUS+GEM therapy. Finally, RNAseq analysis has been performed on treated samples to develop hypotheses to explain the effectiveness of dual therapy. Notably, similar therapeutic approaches are now ongoing as a clinical trial at our institution. We expect our studies to reveal the mechanistic basis in which FUS and GEM are eliciting an immunological response resulting in primary tumor growth control and an overall survival advantage. Supported by grants from NIH (R01 164985 101 GB10846 41017)

Due to the recent rise in immunotherapy research to treat Glioblastoma, immunocompetent mouse models have become increasingly crucial for the study of novel immunotherapies against brain tumors. However, the kinetics of the immune response against the most prevalent immunocompetent GBM models, GL261 and CT2A, have not been well studied, nor have they been compared adequately in the literature. In this study, we compared the immune response in these models using flow cytometry and immunohistochemistry. We investigated several factors that influence the immune response, such as kinetics, tumor size, and transfection status. We hypothesize that these factors influence the immune response enough to warrant consideration while studying new immunotherapeutic approaches for treating GBM. Our results will aid in the preclinical studies of immunotherapy for GBM by allowing scientists to make informed decisions about their chosen models. Citation Format: Breanna R. Noffsinger, Alexandra Witter, Natasha Shebani, Aizhen Xiao, Qing Zhong, Tajie Harris, Benjamin Purow. The kinetics of the anti-glioblastoma immune response in immunocompetent mouse models is influenced by neglected factors [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO056.

Glioblastoma (GB) poses formidable challenges to systemic immunotherapy approaches owing to the paucity of immune infiltration and presence of the blood brain/tumor barriers (BBB/BTB). We hypothesize that BBB/BTB disruption (BBB/BTB-D) with focused ultrasound (FUS) and microbubbles (MB) increases immune infiltration in GB. As a prelude to rational combination of FUS with ITx, we herein investigate the impact of localized BBB/BTB-D on innate and adaptive immune responses in an orthotopic murine GB model. Mice with GL261 gliomas received i.v. MB and underwent FUS BBB/BTB-D (1.1 MHz, 0.5 Hz pulse repetition frequency, 10 ms bursts, 0.4–0.6 MPa). Brains, meninges, and peripheral lymphoid organs were excised and examined by flow cytometry 1–2 weeks following FUS. The number of dendritic cells (DC) was significantly elevated in GL261 tumors and draining cervical LN in response to sonication. CD86 + DC frequency was also upregulated with 0.6 MPa FUS, suggesting increased maturity. While FUS did not significantly alter CD8 + T cell frequency across evaluated organs, these cells upregulated checkpoint molecules at 1 week post-FUS, suggesting increased activation. By 2 weeks post-FUS, we noted emergence of adaptive resistance mechanisms, including upregulation of TIGIT on CD4 + T cells and CD155 on non-immune tumor and stromal cells. FUS BBB/BTB-D exerts mild, transient inflammatory effects in gliomas—suggesting that its combination with adjunct therapeutic strategies targeting adaptive resistance may improve outcomes. The potential for FUS-mediated BBB/BTB-D to modify immunological signatures is a timely and important consideration for ongoing clinical trials investigating this regimen in GB.

Purpose Due to the recent rise in immunotherapy research to treat glioblastoma (GBM), immunocompetent mouse models have become increasingly crucial. However, the character and kinetics of the immune response against the most prevalent immunocompetent GBM models, GL261 and CT2A, have not been well studied, nor has the impact of commonly-used marker proteins and foreign antigens. Methods In this study, we compared the immune response in these models using flow cytometry and immunohistochemistry as well as investigated several factors that influence the immune response, including kinetics, tumor size, and expression of commonly-used marker proteins and foreign antigens. We hypothesize that these factors influence the immune response enough to warrant consideration when studying new immunotherapeutic approaches for GBM. Results CT2A-Luc, but not GL261-Luc2, drastically increased the number of T cells in the brain compared with wild-type controls, and significantly altered CT2A’s responsiveness to anti-PD-1 antibody therapy. Additionally, a larger cell inoculum size in the GL261 model increased the T cell response’s magnitude at day 28 post-injection. CT2A and GL261 models both stimulate a peak T cell immune response at day 21 post-injection. Conclusions Our results suggest that the impact of foreign proteins like luciferase on the intracranial immune response is dependent upon the model, with CT2A being more sensitive to added markers. In particular, luciferase expression in CT2A could lead to meaningful misinterpretations of results from immune checkpoint inhibitor (ICI) studies.

BackgroundTriple-negative breast cancer (TNBC) remains recalcitrant to most targeted therapy approaches. However, recent clinical studies suggest that inducing tumor damage can render TNBC responsive to immunotherapy. We therefore tested a strategy for immune sensitization of murine TNBC (4T1 tumors) through combination of focused ultrasound (FUS) thermal ablation and a chemotherapy, gemcitabine (GEM), known to attenuate myeloid-derived suppressor cells (MDSCs).MethodsWe applied a sparse-scan thermally ablative FUS regimen at the tumor site in combination with systemically administered GEM. We used flow cytometry analysis to investigate the roles of monotherapy and combinatorial therapy in mediating local and systemic immunity. We also tested this combination in Rag1−/−mice or T cell-depleted wild-type mice to determine the essentiality of adaptive immunity. Further, we layered Programmed cell death protein 1 (PD-1) blockade onto this combination to evaluate its impact on tumor outgrowth and survival.ResultsThe immune-modulatory effect of FUS monotherapy was insufficient to promote a robust T cell response against 4T1, consistent with the dominant MDSC-driven immunosuppression evident in this model. The combination of FUS+GEM significantly constrained primary TNBC tumor outgrowth and extended overall survival of mice. Tumor control correlated with increased circulating antigen-experienced T cells and was entirely dependent on T cell-mediated immunity. The ability of FUS+GEM to control primary tumor outgrowth was moderately enhanced by either neoadjuvant or adjuvant treatment with anti-PD-1.ConclusionThermally ablative FUS in combination with GEM restricts primary tumor outgrowth, improves survival and enhances immunogenicity in a murine metastatic TNBC model. This treatment strategy promises a novel option for potentiating the role of FUS in immunotherapy of metastatic TNBC and is worthy of future clinical evaluation.Trial registration numbersNCT03237572andNCT04116320.

Metastatic breast cancer (BrCa) is incurable, with a 5 year survival of only 22%. While immunotherapies are capable of generating durable responses in many forms of cancer, immune rejection of BrCa is very rare. Indeed, immunotherapies (e.g. checkpoint blockade) for BrCa are limited by poor functional CD8+ T cell infiltration and immunosuppression in the tumor microenvironment. Thus, adjunct strategies that render BrCa tumors responsive to immunotherapies are desperately needed. Focused ultrasound (FUS) mediates noninvasive acoustic energy deposition into tumors, conferring localized thermal and mechanical damage to targeted tissue. Here, we tested the hypothesis that FUS thermal ablation (TA) can serve as an auto-vaccine for treatment of BrCa with immunotherapy. Murine mammary carcinoma (4T1-HA) tumors were partially ablated with a 3 MHz ultrasound-guided FUS system. In tumor-draining lymph nodes, 1 week after FUS, we observed a ~3-fold increase in mature dendritic cells (CD11c-hi/CD86+), as well as an increasing trend in M1 macrophage representation by flow cytometry. While FUS did not confer changes in intratumoral CD8+ and CD4+ T cells, likely due to an overwhelming immunosuppressive burden imposed by myeloid derived suppressor cells (MDSCs), infiltrating T cells did express PD1. Therefore, we developed a combination treatment approach of FUS + gemcitabine and/or anti-PD1. This combination treatment elicited an ~50% reduction in 4T1-HA tumor growth at 5 days post-FUS ablation, suggesting that partial TA of BrCa tumors with FUS may represent a promising immunoadjuvant approach. Taken together, these results suggest immunoadjuvants abrogating immunosuppression may enable FUS to stimulate anti-tumor immunity in BrCa.

Glioblastoma (GB) is the most common and malignant brain tumor. Despite standard treatment with surgery, radiation and chemotherapy, its diffuse nature and proclivity for recurrence renders it largely intractable. Immunotherapy (ITx) approaches (e.g. anti-PD1) may hold promise for treating GB; however, the blood-brain (BBB) and blood-tumor (BTB) barriers hinder delivery of systemically administered ITx drugs. A potential approach to enhancing ITx delivery is MRI-guided focused ultrasound (FUS), a non-invasive technique that, when combined with concomitant systemic injection of microbubbles (MB), can transiently disrupt the BBB/BTB and mechanically perturb the tumor microenvironment. Here, we investigate whether localized BBB/BTB disruption with FUS+MB enhances anti-tumor immune responses and inhibits tumor growth, as a prelude to eventual combination with immune checkpoint blockade. One week after FUS+MB (peak negative acoustic pressure=0.6 MPa) treatment of a murine glioma model stably transfected with luciferase (GL261-luc2), CD86 mean fluorescence intensity on dendritic cells (DC) increased ~3-fold in deep cervical lymph nodes, intratumoral CD4+ T cells doubled, and intratumoral CD8+ T cells increased by ~17% (by flow cytometry). Serial bioluminescence imaging of tumors revealed significant reduction in total photon flux as early as 6 days following FUS+MB (p=0.0106), indicating tumor growth inhibition. We conclude that FUS+MB can promote DC maturity and potentially mediate adaptive immunity against glioma, independent of drug delivery. Ongoing studies entail combining FUS+MB with anti-PD-1 delivery to evaluate whether an allied treatment approach can promote an even more robust anti-glioma response.

Abstract Extracellular superoxide dismutase (ecSOD) is an enzyme associated with the extracellular matrix that plays a protective role during reactive oxygen species mediated inflammatory responses. Previously, we reported that neutrophils are essential for protection during infection with Listeria monocytogenes (LM), and that high ecSOD activity is detrimental to host resistance during infection, while lack of ecSOD activity is beneficial. Furthermore, using depletion studies, it was determined that neutrophils from ecSOD KO mice are more protective than neutrophils from ecSOD expressing mice during LM infection. To understand how ecSOD modulates neutrophil function during LM infection, bacterial uptake and phagosomal escape were measured. A higher percentage of neutrophils from the spleen and liver of ecSOD KO mice associated with and allowed for phagosomal escape of LM. However, using MFI as an indicator of the relative number of bacteria per neutrophil, we observed that the ratio of cell associated to cytosolic bacteria was higher in ecSOD KO neutrophils in comparison to neutrophils with ecSOD activity. These data suggest that the absence of ecSOD activity enhances neutrophil association with LM and simultaneously limits LM escape from the phagosome. Future studies utilizing neutrophils obtained from other organs as well as from infected mice should help conclusively determine how the lack of ecSOD activity enhances the protective capabilities of neutrophils.

Neutrophils have historically been characterized as first responder cells vital to host survival because of their ability to contain and eliminate bacterial and fungal pathogens. However, recent studies have shown that neutrophils participate in both protective and detrimental responses to a diverse array of inflammatory and infectious diseases. Although the contribution of neutrophils to extracellular infections has been investigated for decades, their specific role during intracellular bacterial infections has only recently been appreciated. During infection with the Gram-positive intracellular pathogen Listeria monocytogenes, neutrophils are recruited from the bone marrow to sites of infection where they use novel bacterial-sensing pathways leading to phagocytosis and production of bactericidal factors. This review summarizes the requirement of neutrophils during L. monocytogenes infection by examining both neutrophil trafficking and function during primary and secondary infection.

Listeria monocytogenes is a Gram-positive intracellular pathogen that causes spontaneous abortion in pregnant women, as well as septicemia, meningitis, and gastroenteritis, primarily in immunocompromised individuals. Although L. monocytogenes can usually be effectively treated with antibiotics, there is still around a 25% mortality rate with individuals who develop clinical listeriosis. Neutrophils are innate immune cells required for the clearance of pathogenic organisms, including L. monocytogenes The diverse roles of neutrophils during both infectious and noninfectious inflammation have recently gained much attention. However, the impact of reactive oxygen species, and the enzymes that control their production, on neutrophil recruitment and function is not well understood. Using congenic mice with varying levels of extracellular superoxide dismutase (ecSOD) activity, we have recently shown that the presence of ecSOD decreases clearance of L. monocytogenes while increasing the recruitment of neutrophils that are not protective in the liver. The data presented here show that ecSOD activity does not lead to a cell-intrinsic increase in neutrophil-homing potential or a decrease in protection against L. monocytogenes Instead, ecSOD activity enhances the production of neutrophil-attracting factors and protects hyaluronic acid (HA) from damage. Furthermore, neutrophils from the livers of ecSOD-expressing mice have decreased intracellular and surface-bound myeloperoxidase, are less capable of killing phagocytosed L. monocytogenes, and have decreased oxidative burst. Collectively, our data reveal that ecSOD activity modulates neutrophil recruitment and function in a cell-extrinsic fashion, highlighting the importance of the enzyme in protecting tissues from oxidative damage.

Extracellular superoxide dismutase (ecSOD) regulates extracellular concentrations of reactive oxygen species (ROS) to protect tissues during infection and inflammation. Using three groups of mice with varying levels of ecSOD activity, we have previously shown that ecSOD activity enhances neutrophil recruitment to the liver, yet inhibits the innate immune response against Listeria monocytogenes (LM) leading to increased host susceptibility. However, it is unclear whether ecSOD activity affects neutrophil recruitment and function in a cell-intrinsic manner or by modulating the extracellular environment. Using adoptive transfer experiments, we observed that ecSOD activity does not affect neutrophil recruitment or function in a cell-intrinsic manner. Additionally, we determined that ecSOD activity protects the extracellular matrix (ECM) and leads to an increase in phenotypically immature neutrophils in the bone marrow and liver. Collectively, our data suggest that ecSOD activity inhibits degradation of the ECM and promotes egress of immature neutrophils out of the bone marrow and into the liver where they provide inadequate protection against LM. These studies highlight the potential therapeutic value of ecSOD inhibitors to enhance immune responses during bacterial infections.

Abstract The IL-23/IL-17 axis contributes to autoimmune disorders, but is protective against certain pathogens, particularly extracellular bacteria. We have previously shown that IL-23 provides resistance against the intracellular bacterium, Listeria monocytogenes (LM) by mediating the recruitment of neutrophils to the liver, and inflammatory monocytes to the spleen. However, it is not known whether IL-23 can impact the function of phagocytic cells including inflammatory monocytes, neutrophils, and macrophages, during LM infection. Phagocytic cells express receptors for IL-23 and IL-17A suggesting the activity of these cells could be regulated by IL-23 or IL-17A. Surprisingly, phagocytic cells from mice lacking IL-23 (IL-23p19 knockout) had equivalent phagocytic potential and ROS production compared to C57BL/6 mice. Additionally, exogenous stimulation with rIL-23 or rIL-17A did not induce or enhance the production of pro-inflammatory mediators from splenocytes isolated from C57BL/6 mice. Therefore, IL-23 does not impact the function of phagocytic cells either by a direct or an indirect mechanism during LM infection. Collectively, our data reveal that the enhanced susceptibility of IL-23p19 knockout mice is not due to functional impairment of phagocytic cells; instead it is caused by the inefficient recruitment of neutrophils to the liver, and inflammatory monocytes to the spleen, resulting in a reduction in the overall levels of TNF-α and nitric oxide during LM infection.