Supplementary Figure SF1. Schematic of sipuleucel-T treatment doses (infusions) and serum collection time points in IMPACT and ProACT.

The clinical success of cancer immunotherapy, including engineered T cell therapies, has revolutionized treatment paradigms and patient outcomes. While hematological tumors have benefited most from cell therapy approaches, the treatment of solid tumors remains a challenge in part due to the limited availability of abundant and tumor-specific cell-surface antigens. Peptides derived from intracellular tumor-specific proteins, such as cancer-testis antigens (CTAs), that are presented via HLA (pHLA) enable a therapeutic opportunity to target tumors while sparing normal tissue. Surface-accessible pHLA complexes may be targeted with engineered T cell receptors or TCR mimetic antibodies reformatted to chimeric antigen receptors (CARs). Using MAGE-A4 as a model CTA, we compared engineered human TCR- and CAR-T cells head-to-head to understand how to best deploy these modalities. To this end, we generated fully-human, HLA-A2/MAGE-A4(230-239)-specific TCR and CARs harboring CD28/CD3z or 41BB/CD3z signaling domains. TCR and CAR-T cells demonstrated similar robust on-target reactivity, cytokine release, and target cell lysis in vitro. In vivo, each of these modalities showed potent, dose-dependent anti-tumor efficacy against human xenograft tumors expressing low, endogenous levels (~500 cell-surface copies) of the MAGE-A4 peptide. However, differences emerged when we examined the in vivo kinetics and durability of tumor suppression. MAGE-A4 CD28/z CAR-T demonstrated the most rapid and potent tumor clearance, while the 41BB/z CAR-T showed delayed but ultimately complete efficacy. TCR-T cells induced tumor regressions during the first 2 weeks of treatment, but this response was transient and followed by tumor relapse. These differential responses correlated with early, modest accumulation of CD28/z CAR-T in line with fast tumor clearance. 41BB/z CAR-T showed a remarkable ~800-fold expansion in the tumor versus limited in vivo TCR-T proliferation. Mechanistically, the MAGE-A4 TCR induced strong CD3 proximal signaling associated with a greater induction of T cell dysfunction markers and limited cytotoxic potential in vitro. However, stimulating 41BB signaling pathways in the MAGE-A4 TCR T cells augmented long-term cytotoxicity. These data demonstrate that tumor-specific pHLA complexes can be potently targeted by both TCR and CAR-T cells, and that co-stimulatory signaling is necessary to mediate durable anti-tumor activity. Citation Format: Corinne E. Decker, Jacqueline Idun, Katja Mohrs, Thomas Meagher, Kevin Bray, Iryna Petriv, Jonathon Golas, Timothy Helms, Dharani Ajithdoss, Gavin Thurston, John Lin, Jessica R. Kirshner, David J. DiLillo. TCR-T and CAR-T cells targeting HLA-A2/MAGEA4 demonstrate differential tumor control, reflecting co-stimulatory signaling requirements [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 1780.

Background Antibody-mediated delivery of agonists into the tumor microenvironment to selectively activate intracellular targets in immune cells (effector cells) relative to tumor cells (target cells) is not well understood. Here we develop a method using a fluorescent biosensor to quantitatively assess the number of antibodies binding to a cell and frequency of internalized antibodies per cell in both tumor and immune cells in co-cultures by utilizing quantitative spectral flow cytometry. Methods A hIgG1, anti-HER2 monoclonal antibody (HER2-mAb) or isotype-matched control antibody (Iso-mAb) were conjugated with a fluorescent biosensor consisting of Alexa Fluor 568 (AF568) and Alexa Fluor 647 (AF647) separated by a cathepsin B-cleavable peptide linker. AF568 fluorescence is quenched until the biosensor is cleaved inside cells, while AF647 is fluorescent pre- and post-cleavage. Quantitative flow cytometry calibration curves were prepared to calculate the number of fluorophore molecules per cell and antibody molecules per cell using the measured median fluorescent intensity (MFI). Murine effector cells, RAW264.7 and RAW309 (macrophage) and MutuDC1940 (dendritic), were plated in 96-well plates 24 hours prior to the addition of target cells expressing variable levels of HER2: JIMT-1, N87, and SKBR3, pre-loaded with pHrodo green cell tracker. Target cells and biosensor-conjugated mAb were added simultaneously to effector cells at 37 °C for 0, 1, 4, or 24 hours. At each designated time point, cells were stained with CD11b-BV785, and flow cytometry was conducted with at least three independent runs. Flow gates were placed on live single cells of either pHrodo green positive tumor cells, CD11b-BV785 positive immune cells, or double-positive cells. The AF568 and AF647 MFI values were converted to the number of fluorophore molecules or antibodies bound per cell allowing for the number of antibody molecules and% internalized antibody per cell to be determined. Results Target cells expressing HER2 were required for appreciable HER2-mAb binding and internalization into effector cells. Antibody binding on effector cells correlated with increased HER2 expression across the three tumor cell lines. Importantly, the HER2-mAb-biosensor showed rapid and increased internalization into immune cells with 40-60% of antibody internalized by 1 hour in contrast to <10% internalization into tumor cells. Conclusions This in vitro quantitative flow cytometry allowed determination of both the number of antibodies and the% internalized antibodies per cell, important parameters for modeling delinvery of antibody-mediated immunostimulatory agonists. The demonstrated methodology can be applied to in vivo models to optimize therapeutic agonist delivery.

Odronextamab is a fully-human IgG4-based CD20xCD3 bispecific antibody that binds to CD3 on T cells and CD20 on B cells, triggering T-cell-mediated cytotoxicity independent of T-cell-receptor recognition. Adequate safety, tolerability, and encouraging durable complete responses have been observed in an ongoing first-in-human (FIH) study of odronextamab in patients with relapsed/refractory (R/R) B-cell non-Hodgkin lymphoma (B-NHL; NCT02290951). We retrospectively evaluated the pharmacokinetic, pharmacodynamic, and antitumor characteristics of odronextamab in a series of in vitro/in vivo preclinical experiments, to assess their translational value to inform dose escalation for the FIH study. Half-maximal effective concentration values from in vitro cytokine release assays (range: 0.05-0.08 mg/L) provided a reasonable estimate of odronextamab concentrations in patients associated with cytokine release at a 0.5 mg dose (maximum serum concentration: 0.081 mg/L) on week 1/day 1, which could therefore be used to determine the week 1 clinical dose. Odronextamab concentrations resulting in 100% inhibition of tumor growth in a Raji xenograft tumor mouse model (1-10 mg/L) were useful to predict efficacious concentrations in patients and inform dose-escalation strategy. Although predicted human pharmacokinetic parameters derived from monkey data overestimated projected odronextamab exposure, they provided a conservative estimate for FIH starting doses. With step-up dosing, the highest-tested weekly odronextamab dose in patients (320 mg) exceeded the 1 mg/kg single dose in monkeys without step-up dosing. In conclusion, combination of odronextamab in vitro cytokine data, efficacious concentration data from mouse tumor models, and pharmacokinetic evaluations in monkeys has translational value to inform odronextamab FIH study design in patients with R/R B-NHL.

Bispecific antibodies engineered to recruit T cells as a means to kill tumor cells are a promising new class of therapeutics in the field of oncology. A challenge for this class of agents is the requirement for cell surface expressed tumor specific targets, as a large number of tumor-specific antigens are only expressed intracellularly. One strategy to access these targets is to take advantage of the natural processing and presentation of intracellular antigens in the context of the major histocompatibility complex (MHC). However, this requires the development of therapeutics that can be active against targets with a very low surface density. Here, we describe the generation and characterization of Flexbodies, a novel format bispecific antibody engineered to target MHC-peptide tumor neoantigens. We demonstrate that the orientation of the targeting arms and the valency of these reagents allow specific T-cell activation and potent cytotoxic activity against cell lines expressing endogenous level of peptide antigens. This bispecific format has the potential to become an important new tool to expand the range of tumors able to be specifically targeted with t-cell redirecting biologics. Citation Format: Lauric Haber, ryan McKay, jennifer Finney, stephen castaneda, kristen spitler, sahar Rizvi, aidan Hwang, kevin Bray, willy Ramos, pamela Krueger, frank delfino, Craig Meagher, tammy huang, yang shen, william olson, john Lin, eric smith. FlexBodies: Targeting intracellular tumor antigens with T cell redirecting bispecific antibodies [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 1084.

Despite the enormous promise of T cell therapies, the isolation and study of human T cell receptors (TCRs) of dedicated specificity remains a major challenge. To overcome this limitation, we generated mice with a genetically humanized system of T cell immunity. We used VelociGene technology to replace the murine TCRαβ variable regions, along with regions encoding the extracellular domains of co-receptors CD4 and CD8, and major histocompatibility complex (MHC) class I and II, with corresponding human sequences. The resulting “VelociT” mice have normal myeloid and lymphoid immune cell populations, including thymic and peripheral αβ T cell subsets comparable with wild-type mice. VelociT mice expressed a diverse TCR repertoire, mounted functional T cell responses to lymphocytic choriomeningitis virus infection, and could develop experimental autoimmune encephalomyelitis. Immunization of VelociT mice with human tumor-associated peptide antigens generated robust, antigen-specific responses and led to identification of a TCR against tumor antigen New York esophageal squamous cell carcinoma-1 with potent antitumor activity. These studies demonstrate that VelociT mice mount clinically relevant T cell responses to both MHC-I– and MHC-II–restricted antigens, providing a powerful new model for analyzing T cell function in human disease. Moreover, VelociT mice are a new platform for de novo discovery of therapeutic human TCRs.

CD3-engaging bispecific antibodies (bsAbs) and chimeric antigen receptor (CAR) T cells are potent therapeutic approaches for redirecting patient T cells to recognize and kill tumors. Here we describe a fully human bsAb (REGN5458) that binds to B-cell maturation antigen (BCMA) and CD3, and compare its antitumor activities vs those of anti-BCMA CAR T cells to identify differences in efficacy and mechanism of action. In vitro, BCMAxCD3 bsAb efficiently induced polyclonal T-cell killing of primary human plasma cells and multiple myeloma (MM) cell lines expressing a range of BCMA cell surface densities. In vivo, BCMAxCD3 bsAb suppressed the growth of human MM tumors in murine xenogeneic models and showed potent combinatorial efficacy with programmed cell death protein 1 blockade. BCMAxCD3 bsAb administration to cynomolgus monkeys was well tolerated, resulting in the depletion of BCMA+ cells and mild inflammatory responses characterized by transient increases in C-reactive protein and serum cytokines. The antitumor efficacy of BCMAxCD3 bsAb was compared with BCMA-specific CAR T cells containing a BCMA-binding single-chain variable fragment derived from REGN5458. Both BCMAxCD3 bsAb and anti-BCMA CAR T cells showed similar targeted cytotoxicity of MM cell lines and primary MM cells in vitro. In head-to-head in vivo studies, BCMAxCD3 bsAb rapidly cleared established systemic MM tumors, whereas CAR T cells cleared tumors with slower kinetics. Thus, using the same BCMA-binding domain, these results suggest that BCMAxCD3 bsAb rapidly exerts its therapeutic effects by engaging T cells already in place at the tumor site, whereas anti-BCMA CAR T cells require time to traffic to the tumor site, activate, and numerically expand before exerting antitumor effects.

Improving therapies for multiple myeloma (MM) remains a high medical need because of the significant morbidity and mortality of the disease. Targeted immunotherapies represent a promising opportunity to fill this clinical need. B cell maturation antigen (BCMA) is an attractive cell-surface target for MM due to its consistent expression on MM patient malignant plasma cells and expression limited in normal tissue primarily to plasma cells. Redirection of a patient's T cells to recognize tumors by CD3-binding bispecific molecules or through the generation of chimeric antigen receptor (CAR) T cells, has shown preliminary evidence of clinical activity. Bispecific antibodies concurrently engage a tumor antigen on cancer cells and the CD3 signaling machinery on T cells, bringing the tumor cell and T cell into proximity and facilitating T cell activation and tumor cell killing. By contrast, CAR T cell therapy involves re-infusion of the patient's own T cells after ex vivo engineering to express CARs targeting tumor antigens and triggering T cell signaling.

Sipuleucel-T (Provenge®), indicated for the treatment of asymptomatic or minimally symptomatic metastatic castration resistant prostate cancer, is the first FDA-approved Active Cellular Immunotherapy (ACI). Here we describe the development of a therapeutic ACI for the treatment of renal, lung, colon, and cervical cancer. Like PROVENGE®, this new ACI utilizes a recombinant antigen consisting of carbonic anhydrase IX (CA9) linked to GM-CSF (CA9:GM-CSF). In these studies, we investigated the effects of incorporating selective TLR9 agonists on in vitro measures of ACI potency. Initially, employing a panel of proprietary TLR9 agonists, we compared the phenotype of antigen presenting cells (APC) following culture of PBMC from normal healthy donors with either CA9:GM-CSF or CA9:GM-CSF plus TLR9 agonist. While antigen derived GM-CSF activity matured APC, characterized by elevated cell surface expression of CD40, CD54, CD80 and CD86, proprietary agonists for TLR9 further enhanced expression of CD40, CD80, and CD86. By extension, stimulation via TLR9 elicited increased costimulatory capacity of CD14+ large APC in allogeneic mixed lymphocyte response assays. Elevated levels of MCP1-3 in culture supernatant were consistent with APC activation, and notably, viability of CD14+ large APC was unaffected following TLR9 stimulation within the ACI product. Interestingly, using an HLA class II restricted CA9-specific T cell hybridoma reporter assay, staggering the addition of CA9:GM-CSF prior to TLR9 agonist was important to maximize antigen uptake and peptide presentation. Collectively, antigen derived GM-CSF activity is robust at activating APC and certain TLR9 agonists strengthen this effect. Next, as determined by cell surface expression of activation markers (CD27, CD38, CD40, CD54, CD86, IgD), the potential for TLR9 agonists to activate B cells was examined. Relative to cultures supplemented with only CA9:GM-CSF, TLR9 agonists were sufficient to produce a generalized B cell activation pattern consisting of enhanced cell surface expression of CD38, CD40, CD86, and CD54; while expression of CD27 was decreased. Expression of IgD remained unchanged. Associated with the prominent pattern of B cell activation was a marked increase in the accumulation of proinflammatory Type-1 like growth factors (IFN-α, IFN-γ, CXCL9, CXCL10, CCL3, and CCL4) in response to each TLR9 agonist. Despite the degree of B cell activation, this phenotype did not correlate with cellular activation of any T cell subset; suggesting the inflammatory signature resulting from TLR9 stimulation is primarily driven by B cells and/or APC in the ACI setting. Importantly, IL-10 levels were significantly enhanced following TLR9 stimulation, raising the possibility for enhanced regulatory cell activity, but changes in the frequency of regulatory CD4+ T cells were not observed. However, continuing studies are aimed at examining whether regulatory B cell numbers may be enhanced. Thus, in the ACI product, TLR9 agonists potentiate B cell activation and represent powerful polarizers of predominantly Type 1-like cytokine responses. Overall, agonists of TLR9 are compatible with the ACI platform for potentially enhancing antigen specific immunotherapy of cancer. In current experiments, the capacity of TLR9 agonists to enhance cross-priming and generate cytotoxic T cell responses is under investigation. Citation Format: Jason Chinn, Crystal Cummings, Felecia Wagener, Shaarwari Sridhar, Kien Khuu-Duong, Xinhui Ge, Karen Yoshino, Sam Li, Lisa Martel, Chris Ramsborg, Ken Brasel, James Trager, Craig Meagher. Combining selective toll-like receptor 9 (TLR9) agonists and GM-CSF activity for potentiating cellular activation in active cell immunotherapy (ACI). [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B18.

Murine models of Autologous Cellular Immunotherapy (ACI) have been useful tools for understanding the mechanisms by which human immunotherapy may work. Sipuleucel-T is an FDA approved therapeutic for the treatment of asymptomatic or minimally symptomatic, metastatic castration-resistant metastatic prostate cancer; Phase 3 studies demonstrated a statistically significant prolongation in overall survival when compared to a control group. Sipuleucel-T is manufactured by activating peripheral blood mononuclear cells (PBMC), including antigen presenting cells, with a recombinant protein fusion of prostatic acid phosphatase and granulocyte macrophage colony-stimulating factor (GM-CSF). We have previously reported a method for the use of cultured PBMC in a pre-clinical setting to elicit a T cell response against a tumor associated antigen, and to protect mice from tumors expressing that antigen. The model antigen we used was human Carbonic Anhydrase IX (hCA9). Mouse PBMCs were cultured with the antigen fused to murine GM-CSF (hCA9-GM) or with GM-CSF alone. Mice were immunized 3 times with these cells at 2 week intervals. Satellite mice were then harvested for evaluation of in vitro T cell response (antigen recall assay) and the remaining mice were inoculated with hCA9-expressing tumor cells, and tumor growth followed over time. Immunized mice mounted a robust T cell response in vitro to hCA9 in a dose dependent manner, a result that correlated with protection from tumor challenge in mice immunized with PBMC cultured in hCA9-GM. Herein, we investigate the cellular requirements for protection against tumor challenge and ability of a selective Toll-like receptor (TLR) 9 agonist to enhance the potency of this cellular immunotherapy. To investigate the importance of T cells for anti-tumor responses, we demonstrate that selective depletion of either CD4 or CD8 T cells results in a diminution or loss of protection toward tumor challenge; indicating T cells are necessary in vivo to prevent tumor growth. Next, to stimulate antigen specific T cell activity in the ACI therapy, relative to hCA9 antigen alone, cells were cultured with hCA9-GM antigen in combination with the TLR9 agonist CpG1826. Addition of CpG1826 resulted in significant accumulation of pro-inflammatory cytokines (IL-1b, IL-6, IL-10, TNFa, KC and IFNg) in the culture medium, and to higher surface expression of activation and co-stimulatory markers (CD25, CD69, CD80, CD86, and MHC Class II) on cultured cells. Moreover, inclusion of CpG1826 to cultures enhanced protection against tumor cell challenge. Interestingly, this enhancement in potency was not associated with increased antigen specific T cell responses, as measured in the in vitro antigen recall assay. In conclusion, we show that, in mice, unfractionated white blood cells collected from peripheral blood can be harnessed to mount effective anti-tumor immune responses and that TLR9 agonists complement this approach. Furthermore, this active immunotherapy triggers a balanced immune response relying on the activity of both CD4 and CD8 T cells. Future studies will be aimed at evaluating the role of various PBMC subsets (T cell, B cell and myeloid cell) in protecting mice from tumor cell challenge. Citation Format: Kenneth A. Brasel, Craig Meagher, Marykay Ligocki, Lauren Cerretti, Felecia Wagener, Sam Li, James Trager. Protective immunizations using cultured peripheral blood cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B71.

Immunotherapies are coming to the forefront as a treatment paradigm in cancer with multiple US FDA approvals in recent years and a better understanding of their therapeutic mode of action. The control of tumor growth by the immune system is orchestrated by a complex array of cellular interactions and molecular pathways, both in the immune cells as well as the tumor. Although research over the past three decades has elucidated many aspects of tumor immunosurveillance, given the inherent complexity of the immune cell phenotypes and function, high-throughput molecular profiling ('omics') approaches have now become essential to support the discovery and development of new therapies. Technologies, such as DNA and protein microarrays, deep sequencing, mass spectrometry, as well as the computational methods for their analyses, are advancing the contributions of systems biology towards the development and mechanistic understanding of cancer immunotherapies. In this review, the authors illustrate this through some recently reported studies.

NK cells have been shown to either promote or protect from autoimmune diseases. Several studies have examined the role of receptors preferentially expressed by NK cells in the spontaneous disease of NOD mice or the direct role of NK cells in acute induced disease models of diabetes. Yet, the role of NK cells in spontaneous diabetes has not been directly addressed. Here, we used the NOD.NK1.1 congenic mouse model to examine the role of NK cells in spontaneous diabetes. Significant numbers of NK cells were only seen in the pancreas of mice with disease. Pancreatic NK cells displayed an activated surface phenotype and proliferated more than NK cells from other tissues in the diseased mice. Nonetheless, depletion of NK cells had no effect on dendritic cell maturation or T cell proliferation. In spontaneous disease, the deletion of NK cells had no significant impact on disease onset. NK cells were also not required to promote disease induced by adoptively transferred pathogenic CD4(+) T cells. Thus, NK cells are not required for spontaneous autoimmune diabetes in NOD mice.

Sjögren’s Syndrome (SS) is a human autoimmune disease characterized by immune-mediated destruction of the lacrimal and salivary glands. In this study, we show that the Aire-deficient mouse represents a new tool to investigate autoimmune dacryoadenitis and keratoconjunctivitis sicca, features of SS. Previous work in the Aire-deficient mouse suggested a role for α-fodrin, a ubiquitous Ag, in the disease process. Using an unbiased biochemical approach, however, we have identified a novel lacrimal gland autoantigen, odorant binding protein 1a, targeted by the autoimmune response. This novel autoantigen is expressed in the thymus in an Aire-dependent manner. The results from our study suggest that defects in central tolerance may contribute to SS and provide a new and clinically relevant model to investigate the pathogenic mechanisms in lacrimal gland autoimmunity and associated ocular surface sequelae.

OBJECTIVE The progressive infiltration of pancreatic islets by lymphocytes is mandatory for development of autoimmune type 1 diabetes. This inflammatory process is mediated by several mediators that are potential therapeutic targets to arrest development of type 1 diabetes. In this study, we investigate the role of one of these mediators, interleukin-16 (IL-16), in the pathogenesis of type 1 diabetes in NOD mice. RESEARCH DESIGN AND METHODS At different stages of progression of type 1 diabetes, we characterized IL-16 in islets using GEArray technology and immunoblot analysis and also quantitated IL-16 activity in cell migration assays. IL-16 expression was localized in islets by immunofluorescence and confocal imaging. In vivo neutralization studies were performed to assess the role of IL-16 in the pathogenesis of type 1 diabetes. RESULTS The increased expression of IL-16 in islets correlated with the development of invasive insulitis. IL-16 immunoreactivity was found in islet infiltrating T-cells, B-cells, NK-cells, and dendritic cells, and within an insulitic lesion, IL-16 was derived from infiltrating cells. CD4+ and CD8+ T-cells as well as B220+ B-cells were identified as sources of secreted IL-16. Blockade of IL-16 in vivo protected against type 1 diabetes by interfering with recruitment of CD4+ T-cells to the pancreas, and this protection required the activity of the chemokine CCL4. CONCLUSIONS IL-16 production by leukocytes in islets augments the severity of insulitis during the onset of type 1 diabetes. IL-16 and CCL4 appear to function as counterregulatory proteins during disease development. Neutralization of IL-16 may represent a novel therapy for the prevention of type 1 diabetes.

Autoimmune pancreatitis (AIP) is a heterogeneous autoimmune disease in humans characterized by a progressive lymphocytic and plasmacytic infiltrate in the exocrine pancreas. In this study, we report that regulatory T cell-deficient NOD.CD28KO mice spontaneously develop AIP that closely resembles the human disease. NOD mouse AIP was associated with severe periductal and parenchymal inflammation of the exocrine pancreas by CD4(+) T cells, CD8(+) T cells, and B cells. Spleen CD4(+) T cells were found to be both necessary and sufficient for the development of AIP. Autoantibodies and autoreactive T cells from affected mice recognized a similar to 50-kDa protein identified as pancreatic amylase. Importantly, administration of tolerogenic amylase-coupled fixed spleen cells significantly ameliorated disease severity, suggesting that this protein functions as a key autoantigen. The establishment and characterization of this spontaneous pancreatic amylase-specific AIP in regulatory T cell-deficient NOD.CD28KO mice provides an excellent model for the study of disease pathogenesis and development of new therapies for human autoimmune pancreatitis.