Tumor-targeting Antibodies Research Articles

Tumor regression and immunity in combination therapy with anti-CEA chimeric antigen receptor T cells and anti-CEA-IL2 immunocytokine

ABSTRACT Targeted immunotherapy of solid cancers with chimeric antigen receptor (CAR) T cells and immunocytokines are attractive options in that they both rely on the specificity of tumor-targeted antibodies. Since carcinoembryonic antigen (CEA) expression in both colon and breast cancers is correlated with poor prognosis, it was chosen as a model tumor target in immunocompetent CEA transgenic (CEATg) mice. A second-generation anti-CEA CAR derived from CEA-specific antibody T84.66 was used to treat murine MC38 colon or E0771 breast carcinomas transfected with CEA. Anti-CEA CAR vs. mock transduced T cells exhibited a CEA-specific cytotoxic and IFN γ dose response to both CEA transfected cell lines vs. their CEA-negative controls. Anti-CEA CAR vs. mock transduced T cells delayed the median survival of CEA transfected s.c. MC38 or orthotopic E0771 tumor-bearing CEATg mice by 2 days. With the addition of one-day prior cyclophosphamide (CY) lymphodepletion, anti-CEA CAR T cell treatment delayed the median survival of MC38/CEA and E0771/CEA tumor-bearing CEATg mice by ten and 3 days, respectively. Since CAR T cells require IL2 for survival and expansion, anti-CEA-IL2 immunocytokine (ICK) treatment was performed post CAR T cell therapy. Single ICK treatment 1 day after CY plus anti-CEA CAR T cell therapy in the MC38/CEA model, and two ICK treatments every 3 days after CY plus anti-CEA CAR T cell therapy in the E0771/CEA model were ineffective, while four ICK treatments every 3 days after CY plus anti-CEA CAR T cell therapy completely eradicated MC38/CEA tumor growth and induced tumor immunity when the mice were re-challenged with tumor. These studies show the therapeutic potential of anti-CEA CAR T cells combined with ICK to treat CEA-positive tumors. Abbreviations: CAR: Chimeric antigen receptor, CEA: Carcinoembryonic antigen, CEACAM5, ICK: Immunocytokine, CY: Cyclophosphamide, CEATg mouse: transgenic CEA mouse, TDLN: Tumor-draining lymph node

Immune-stimulating antibody conjugates elicit robust myeloid activation and durable antitumor immunity.

Innate pattern recognition receptor agonists, including Toll-like receptors (TLRs), alter the tumor microenvironment and prime adaptive antitumor immunity. However, TLR agonists present toxicities associated with widespread immune activation after systemic administration. To design a TLR-based therapeutic suitable for systemic delivery and capable of safely eliciting tumor-targeted responses, we developed immune-stimulating antibody conjugates (ISACs) comprising a TLR7/8 dual agonist conjugated to tumor-targeting antibodies. Systemically administered human epidermal growth factor receptor 2 (HER2)-targeted ISACs were well tolerated and triggered a localized immune response in the tumor microenvironment that resulted in tumor clearance and immunological memory. Mechanistically, ISACs required tumor antigen recognition, Fcγ-receptor-dependent phagocytosis and TLR-mediated activation to drive tumor killing by myeloid cells and subsequent T-cell-mediated antitumor immunity. ISAC-mediated immunological memory was not limited to the HER2 ISAC target antigen since ISAC-treated mice were protected from rechallenge with the HER2- parental tumor. These results provide a strong rationale for the clinical development of ISACs.

Phase 1 Study of CD19 Targeted 4-1BBL Costimulatory Agonist to Enhance T Cell (Glofitamab Combination) or NK Cell Effector Function (Obinutuzumab Combination) in Relapsed/Refractory B Cell Lymphoma

Phase 1 Study of CD19 Targeted 4-1BBL Costimulatory Agonist to Enhance T Cell (Glofitamab Combination) or NK Cell Effector Function (Obinutuzumab Combination) in Relapsed/Refractory B Cell Lymphoma

Tumor-targeting anti-EGFR x anti-PD1 bispecific antibody inhibits EGFR-overexpressing tumor growth by combining EGFR blockade and immune activation with direct tumor cell killing

We developed a strategy to combine conventional targeted therapy with immune checkpoint blockade using a tumor-targeting bispecific antibody (BsAb) to treat solid tumors. The BsAb was designed to simultaneously engage a tumor-associated antigen, epidermal growth factor receptor (EGFR), and programed cell death protein 1 (PD1). In addition to its direct anti-tumor activity via EGFR inhibition, the BsAb mediated efficient antibody-dependent cellular cytotoxicity (ADCC) and activated T cell antitumor im munity through blockade of PD1 from interacting with its counterpart, programed cell death ligand 1 (PDL1). Further, the BsAb exhibited a potent direct tumor cell killing activity in the presence of PBMC, most likely, via activating and, at the same time, physically engaging T cells with tumor cells. Taken together, we here illustrate a new strategy in the design and production of novel BsAbs with enhanced therapeutic efficacy through both direct tumor growth inhibition and T cell activation via tumor-targeted immune checkpoint blockade.

The Neutrophil: The Underdog That Packs a Punch in the Fight against Cancer.

The advent of immunotherapy has had a major impact on the outcome and overall survival in many types of cancer. Current immunotherapeutic strategies typically aim to (re)activate anticancer T cell immunity, although the targeting of macrophage-mediated anticancer innate immunity has also emerged in recent years. Neutrophils, although comprising ≈ 60% of all white blood cells in the circulation, are still largely overlooked in this respect. Nevertheless, neutrophils have evident anticancer activity and can induce phagocytosis, trogocytosis, as well as the direct cytotoxic elimination of cancer cells. Furthermore, therapeutic tumor-targeting monoclonal antibodies trigger anticancer immune responses through all innate Fc-receptor expressing cells, including neutrophils. Indeed, the depletion of neutrophils strongly reduced the efficacy of monoclonal antibody treatment and increased tumor progression in various preclinical studies. In addition, the infusion of neutrophils in murine cancer models reduced tumor progression. However, evidence on the anticancer effects of neutrophils is fragmentary and mostly obtained in in vitro assays or murine models with reports on anticancer neutrophil activity in humans lagging behind. In this review, we aim to give an overview of the available knowledge of anticancer activity by neutrophils. Furthermore, we will describe strategies being explored for the therapeutic activation of anticancer neutrophil activity.

Antibody drug conjugates of cleavable amino-benzoyl-maytansinoids

ADCs based on the natural product maytansine have been successfully employed clinically. In a previous report, ADCs based on hydrophilic non-cell permeable maytansinoids was presented. The authors in this report further explore the maytansine scaffold to develop tubulin inhibitors capable of cell permeation. The research resulted in amino-benzoyl-maytansinoid payloads that were further elaborated with linkers for conjugating to antibodies. This approach was applied to MUC16 tumor targeting antibodies for ovarian cancers. A positive control ADC was evaluated alongside the amino-benzoyl-maytansinoid ADC and the efficacy observed was equivalent while the isotype control ADCs had no effect.

Novel immunotherapy strategies for treatment of neuroendocrine neoplasms.

Neuroendocrine tumors (NETs) and neuroendocrine carcinomas (NECs) are a heterogeneous family of neoplasms. Well-differentiated tumors are often slow growing and characterized by low tumor mutational burden. Poorly differentiated NECs are aggressive, with an increased mutational burden and higher propensity to express PD-L1. While the therapeutic landscape for neuroendocrine neoplasms (NENs) has evolved substantially over the past decade, immunotherapy has been unexplored in NENs until recently. Checkpoint inhibitors such as anti-PD-1 and anti-CTLA-4 agents, bi-specific tumor-targeting antibodies, and chimeric antigen receptor (CAR) T-cell therapy are examples of treatments that have demonstrated efficacy in other cancers and have recently been investigated in NENs. This review examines the immune landscape of NENs in detail, summarizes recent clinical study results, and discusses potential future directions for immunotherapy.

Anti-tumor effects of NK cells and anti-PD-L1 antibody with antibody-dependent cellular cytotoxicity in PD-L1-positive cancer cell lines.

BackgroundAlthough programmed cell death-1/programmed death-ligand 1 (PD-L1) inhibitors show remarkable antitumor activity, a large portion of patients with cancer, even those with high PD-L1-expressing tumors, do not respond to their effects. Most PD-L1 inhibitors contain modified fragment crystallizable region (Fc) receptor binding sites to prevent antibody-dependent cellular cytotoxicity (ADCC) against PD-L1-expressing non-tumor cells. However, natural killer (NK) cells have specific antitumor activity in the presence of tumor-targeting antibody through ADCC, which could enhance NK cell-induced cytotoxicity. We evaluated the antitumor efficacy of ADCC via anti-PD-L1 monoclonal antibodies (mAbs) and NK cells against several PD-L1-positive cancer cell lines.MethodsVarious cancer cell lines were used as target cell lines. Surface PD-L1 expression was analyzed by flow cytometry. IMC-001 and anti-hPD-L1-hIgG1 were tested as anti-PD-L1 mAbs with ADCC and atezolizumab as an anti-PD-L1 mAb without ADCC. NK cell cytotoxicity was measured by 51Cr-release assay and CD107a degranulation assay. Also, live cell imaging was performed to evaluate cytotoxicity in a single-cell level. NK-92-CD16 (CD16-transduced NK-92 cell line) and peripheral blood mononuclear cells from healthy donors, respectively, were used as an effector cell. FcγRIIIa (CD16a)-V158F genotyping was performed for healthy donors.ResultsWe demonstrated that the cytotoxicity of NK-92-CD16 cells toward PD-L1-positive cancer cell lines was significantly enhanced in the presence of anti-PD-L1 mAb with ADCC. We also noted a significant increase in primary human NK cell cytotoxicity against PD-L1-positive human cancer cells when cocultured with anti-PD-L1 mAb with ADCC. Moreover, NK cells expressing a FCGR3A high-affinity genotype displayed higher anti-PD-L1 mAb-mediated ADCC lysis of tumor cells than donors with a low-affinity genotype.ConclusionThese results suggest that NK cells induce an ADCC response in combination with anti-PD-L1 mAbs, which helps promote ADCC antitumor activity against PD-L1-positive tumors. This study provides support for NK cell immunotherapy against high PD-L1-expressing tumors in combination with ADCC through anti-PD-L1 mAbs.

Abstract PR07: GD2 is a macrophage checkpoint molecule and combined GD2/CD47 blockade results in synergistic effects and tumor clearance in xenograft models of neuroblastoma and osteosarcoma

Abstract The use of anti-GD2 antibodies for neuroblastoma (NBL) has resulted in enhanced survival, but many patients still relapse and ultimately die of their disease. Additionally, despite expression of GD2 on osteosarcoma (OS), anti-GD2 antibodies have not proven widely effective in that disease. Enhancing the efficacy of anti-GD2 antibodies could result in improved patient outcomes. CD47 is the dominant “Don’t Eat Me” signal expressed by cancer cells to inhibit macrophage phagocytosis. Blocking CD47 with antibodies leads to phagocytosis of tumor cells. A recent trial of anti-CD47 combined with rituximab (anti-CD20) resulted in a high complete response rate in patients with B-cell lymphoma. Using the same rationale, that disinhibiting a macrophage in the presence of a tumor-targeting antibody would result in enhanced antitumor activity, we studied the combination of CD47 blockade with dinutuximab, an FDA-approved antibody targeting GD2. We found a striking synergy between dinutuximab and anti-CD47 in vivo. This combination led to the complete clearance of both orthotopic and metastatic models of NBL. Additionally, the combination significantly delayed the growth of OS xenografts, resulting in enhanced survival, whereas single-agent anti-GD2 or anti-CD47 had no antitumor activity. Finally, in a murine model of metastatic pulmonary OS, the combination of anti-GD2/CD47 led to a near elimination of all metastatic burden. To understand the biologic basis for the synergistic effects observed, we studied both the effects of GD2 crosslinking on tumor cells and the effects of GD2 blockade on macrophages. When NBL cells are treated with dinutuximab in vitro, approximately 50% of the cells die, and those that survive upregulate surface calreticulin, an important prophagocytic (“Eat Me”) signal that stimulates macrophages to remove dying cells. Therefore, GD2 ligation results in cellular processes that drive macrophages to phagocytose tumors. We measured in vitro phagocytosis of NBL cells and observed a synergistic effect in the presence of anti-GD2/CD47 compared to the single agents. However, when we combined anti-CD47 with a tumor-specific antibody recognizing B7-H3, there was no synergy. We therefore hypothesized that GD2 itself is inhibitory to macrophages. Indeed, we produced an anti-GD2 Fab and, simply by blocking GD2, we observed increased phagocytosis of tumor cells. Further, we have identified the ligand for GD2, a molecule expressed on macrophages known to inhibit phagocytosis. Therefore, GD2 is a macrophage checkpoint capable of suppressing tumor cell phagocytosis. In summary, we have identified a novel combination of anti-GD2 and anti-CD47 antibodies that is highly effective in xenograft models of NBL and OS and will soon be tested in children. Additionally, we have shown that GD2 itself is a macrophage checkpoint or “Don’t Eat Me” signal. This finding may explain the high levels of GD2 expressed on NBL and OS as a mechanism for immune evasion. This abstract is also being presented as Poster A73. Citation Format: Johanna Theruvath, Benjie Smith, Miles H. Linde, Elena Sotillo, Sabine Heitzeneder, Kristopher Marjon, Aidan Tousley, Jake Lattin, Allison Banuelos, Shaurya Dhingra, Surya Murty, Crystal L. Mackall, Robbie G. Majzner. GD2 is a macrophage checkpoint molecule and combined GD2/CD47 blockade results in synergistic effects and tumor clearance in xenograft models of neuroblastoma and osteosarcoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr PR07.

Tumor-targeted CD28 bispecific antibodies enhance the antitumor efficacy of PD-1 immunotherapy.

Monoclonal antibodies that block the programmed cell death 1 (PD-1) checkpoint have revolutionized cancer immunotherapy. However, many major tumor types remain unresponsive to anti-PD-1 therapy, and even among responsive tumor types, most of the patients do not develop durable antitumor immunity. It has been shown that bispecific antibodies activate T cells by cross-linking the TCR/CD3 complex with a tumor-specific antigen (TSA). The class of TSAxCD3 bispecific antibodies have generated exciting results in early clinical trials. We have recently described another class of "costimulatory bispecifics" that cross-link a TSA to CD28 (TSAxCD28) and cooperate with TSAxCD3 bispecifics. Here, we demonstrate that these TSAxCD28 bispecifics (one specific for prostate cancer and the other for epithelial tumors) can also synergize with the broader anti-PD-1 approach and endow responsiveness-as well as long-term immune memory-against tumors that otherwise do not respond to anti-PD-1 alone. Unlike CD28 superagonists, which broadly activate T cells and induce cytokine storm, TSAxCD28 bispecifics display little or no toxicity when used alone or in combination with a PD-1 blocker in genetically humanized immunocompetent mouse models or in primates and thus may provide a well-tolerated and "off the shelf" combination approach with PD-1 immunotherapy that can markedly enhance antitumor efficacy.

Targeting macrophage checkpoint inhibitor SIRPα for anticancer therapy.

The CD47/signal regulatory protein α (Cd47/SIRPα)interaction provides a macrophage immune checkpoint pathway that plays a critical role in cancer immune evasion across multiple cancers. Here, we report the engineering of a humanized anti-SIRPα monoclonal antibody (1H9) for antibody target cancer therapy. 1H9 has broad activity across a wide range of SIRPα variants. Binding of 1H9 to SIRPα blocks its interaction with CD47, thereby promoting macrophage-mediated phagocytosis of cancer cells. Preclinical studies in vitro and in vivo demonstrate that 1H9 synergizes with other therapeutic antibodies to promote phagocytosis of tumor cells and inhibit tumor growth in both syngeneic and xenograft tumor models, leading to survival benefit. Thus, 1H9 can potentially act as a universal agent to enhance therapeutic efficacy when used in combination with most tumor-targeting antibodies. We report a comparison of anti-SIRPα and anti-CD47 antibodies in CD47/SIRPα double-humanized mice and found that 1H9 exhibits a substantially reduced antigen sink effect due to the limited tissue distribution of SIRPα expression. Toxicokinetic studies in nonhuman primates show that 1H9 is well tolerated, with no treatment-related adverse effects noted. These data highlight the clinical potential of 1H9 as a pan-therapeutic with the desired properties when used in combination with tumor-targeting antibodies.

Correlation of clinical activity of NEO201 mAb with the expression of NK activation markers and levels of soluble factors.

e15002 Background: NEO-201 is a humanized IgG1 mAb that targets tumor-associated variants of CEACAM-5/6. NEO201 exerts anti-tumor activity by (NK)-mediated ADCC, CDC, and by enhancing NK cell cytotoxicity through blockade of CEACAM5-CEACAM1 interaction. The first in human phase I clinical trial is ongoing. Neutropenia caused DLT and was observed at 2mg/kg (DL 2). At DL2, 2/6 patients with colorectal cancers had stable disease. In the present study we evaluated the correlation between response, NK status, and profiles of soluble factors. Methods: This is a classic 3+3 dose escalation. NEO-201 is administered intravenously every 2 weeks, with 4 dose levels planned (DL1 = 1mg/kg, DL2 = 2mg/kg, DL3 = 4mg/kg and DL4 = 6mg/kg). So far, 3 patients received DL1 and 6 patients DL2. Safety is evaluated according to CTCAEv5.0, and response according to RECISTv1.1. Biological samples are collected at baseline, at 4, 24 and 72 hours after the first dose, and before C1D15 dose to understand NEO-201 pharmacokinetics (PK), effects on immune profile and correlation with treatment toxicity and response. CD56+/CD16+ NK cells were evaluated for modulation of NKG2D, CD107a, NKp46 (activation markers), and CEACAM1 (inhibitory marker) by flow cytometry. Soluble factors (cytokines, sMICA and sCEACAM5) were evaluated by ELISA. Results: Among the 3 patients achieving radiological SD, one (DL1) had clinical progression (PD) without radiological progression after 2 cycles due to mucous producing disease, a second patient (DL2) went off study after 2 cycles for drug unrelated issues, and the third patient (DL2) has stable disease (SD) for 6 months without significant toxicity. All other patients had radiologic PD after 2 cycles. Interestingly, baseline CD56+/CD16+ NK cells from the two patients with SD showed an increase in NKG2D, CD107a and NKp46, and a low expression of CEACAM1. They also had low serum levels of sMICA, sCEACAM5 and IL-6. On contrary, CD56+/CD16+ NK cells from patients with PD had low expression of NKG2D and CD107a, high expression of CEACAM1, and high levels of sMICA. Conclusions: High expression of activating markers and low expression of CEACAM1 on CD56+/CD16+ NK cells, as well as low levels of sMICA and sCEACAM5 correlate with clinical response to NEO-201. Thus, the activity of NK cells may serve as predictors for efficacy of tumor-targeting antibody therapy. Further correlation of these biomarkers with PK and CEACAM1/5/6 expression in patients’ tissue samples will provide further support for optimizing the use of NEO201. Clinical trial information: NCT03476681 .

Multimodal fluorescence-guided surgery of colorectal peritoneal metastases, a phase I/II clinical trial.

TPS4119 Background: Successful treatment of patients with colorectal peritoneal carcinomatosis highly depends on complete surgical tumor resection of all tumor. Oncological outcomes can potentially be improved by intraoperative imaging using a tumor-targeting antibody conjugated to a fluorophore and a radiotracer. This enables preoperative radionuclide imaging, real-time intraoperative fluorescence imaging and gamma detection. In this study we investigate the feasibility, accuracy and safety of CEA-targeted preoperative SPECT/CT and intraoperative fluorescence imaging in patients with colorectal PC. Methods: In this phase I/II single arm protein dose escalation study patients with peritoneal metastases of colorectal origin who are scheduled for cytoreductive surgery and HIPEC will receive an intravenous injection of the CEA-targeting tracer 111In-DOTA-labetuzumab-IRDye800CW. The first 15 patients will receive a single dose of 2,10 or 50 mg 6 to 7 days prior to surgery. Four to five days after injection SPECT/CT imaging of the thorax and abdomen is performed to determine intra-abdominal tumor load and detect extra-abdominal metatases. At day 6/7 after injection, standard cytoreductive surgical resection extended with real-time near-infrared fluorescence imaging and radio guidance is performed. After surgery, the peritoneal cavity will be re-examined for residual disease with fluorescence imaging. Resected specimens are analyzed microscopically, immunohistochemically (CEA and H&E) and by gamma counting. Blood samples are drawn for farmacokinetics and safety analysis at 180 minutes, 4 days, 6 days and 3 weeks after tracer injection. In the phase II dose expansion cohort, 14 more patients will receive the optimal dose as determined in the phase I trial. The primary objectives of the trial are to assess the safety, feasibility and accuracy of preoperative SPECT/CT and intraoperative fluorescence imaging after administration of 111In- labetuzumab-IRDye800CW in patients with peritoneal carcinomatosis of colorectal origin who will undergo cytoreductive surgery and HIPEC. The secondary objectives are to assess whether additional malignant lesions can be visualized by fluorescence imaging after cytoreductive surgery, to assess the intensity of fluorescence in malignant and non-malignant tissue, to assess the correlation between localization of the dual-labeled antibody and CEA expression in tumor and healthy tissue and to determine blood concentrations of the dual labelled antibody at several time points in patients. Clinical trial information: NCT03699332 .

Immunoavidity-Based Capture of Tumor Exosomes Using Poly(amidoamine) Dendrimer Surfaces.

Tumor-derived blood-circulating exosomes have potential as a biomarker to greatly improve cancer treatment. However, effective isolation of exosomes remains a tremendous technical challenge. This study presents a novel nanostructured polymer surface for highly effective capture of exosomes through strong avidity. Various surface configurations, consisting of multivalent dendrimers, PEG, and tumor-targeting antibodies, were tested using exosomes isolated from tumor cell lines. We found that a dual layer dendrimer configuration exhibited the highest efficiency in capturing cultured exosomes spiked into human serum. Importantly, the optimized surface captured a > 4-fold greater amount of tumor exosomes from head and neck cancer patient plasma samples than that from healthy donors. Nanomechanical analysis using atomic force microscopy also revealed that the enhancement was attributed to multivalent binding (avidity) and augmented short-range adhesion mediated by dendrimers. Our results support that the dendrimer surface detects tumor exosomes at high sensitivity and specificity, demonstrating its potential as a new cancer liquid biopsy platform.

Ex Vivo Assessment of Tumor-Targeting Fluorescent Tracers for Image-Guided Surgery.

Image-guided surgery can aid in achieving complete tumor resection. The development and assessment of tumor-targeted imaging probes for near-infrared fluorescence image-guided surgery relies mainly on preclinical models, but the translation to clinical use remains challenging. In the current study, we introduce and evaluate the application of a dual-labelled tumor-targeting antibody for ex vivo incubation of freshly resected human tumor specimens and assessed the tumor-to-adjacent tissue ratio of the detectable signals. Immediately after surgical resection, peritoneal tumors of colorectal origin were placed in cold medium. Subsequently, tumors were incubated with 111In-DOTA-hMN-14-IRDye800CW, an anti-carcinoembryonic antigen (CEA) antibody with a fluorescent and radioactive label. Tumors were then washed, fixed, and analyzed for the presence and location of tumor cells, CEA expression, fluorescence, and radioactivity. Twenty-six of 29 tumor samples obtained from 10 patients contained malignant cells. Overall, fluorescence intensity was higher in tumor areas compared to adjacent non-tumor tissue parts (p < 0.001). The average fluorescence tumor-to-background ratio was 11.8 ± 9.1:1. A similar ratio was found in the autoradiographic analyses. Incubation with a non-specific control antibody confirmed that tumor targeting of our tracer was CEA-specific. Our results demonstrate the feasibility of this tracer for multimodal image-guided surgery. Furthermore, this ex vivo incubation method may help to bridge the gap between preclinical research and clinical application of new agents for radioactive, near infrared fluorescence or multimodal imaging studies.

Abstract A01: Treatment with Imprime PGG, a soluble yeast β-glucan PAMP, induces trafficking of activated Ly6Ghigh cells to secondary lymphoid organs and tumor and facilitates immune activation within the tumor microenvironment

Abstract Imprime PGG (Imprime), an intravenously administered, soluble, yeast β-1,3/1,6 glucan, is currently in clinical development with tumor-targeting, antiangiogenic and checkpoint inhibitor antibodies. The fundamental mechanistic rationale for these different therapeutic combinations is that Imprime, a pathogen-associated molecular pattern (PAMP), primes innate immune effector functions to drive a coordinated anticancer immune response. Previous preclinical mechanistic work has shown that Imprime activates the functionalities of monocytes, macrophages, and dendritic cells (cells that principally influence the TME), eliciting a tumor microenvironment (TME) that activates T cell-dependent antitumor immunity. Studies have shown that mature neutrophils and immature myeloid-derived suppressor cells (PMN-MDSC) accumulate, like other myeloid cells, in the tumor and play a role in establishing the immunosuppressive TME and impair T-cell immunity. Herein, we provide evidence that Imprime treatment reorients neutrophil function, enlisting these cells in anticancer immunity. To date, exploration of the effect of Imprime on neutrophils has evaluated direct neutrophil-mediated tumor killing. In this study, we further explored the impact of Imprime treatment on mature neutrophils and immature PMN-MDSC (i.e., the Ly6Ghigh population) in both tumor-free and tumor-bearing mice. In tumor-free C57BL/6 mice, Imprime mobilized Ly6Ghigh cells into the blood, spleen, and skin draining lymph nodes (sdLNs). The increase of these cells in the sdLN coincided with increased levels of CCL2, CCL3, CCL4, CXCL1 and CXCL10, chemokines important for the mobilization of myeloid cells. In the H1299 lung cancer xenograft model, the addition of Imprime to bevacizumab enhanced CD86 expression on Ly6Ghigh cells in both spleen and tumor tissues when compared to bevacizumab alone, indicating that these cells could play a role in T-cell activation. We therefore assessed the effect of Imprime in MC-38, syngeneic tumor model, shown to be sensitive to Imprime in combination with immune checkpoint inhibitor antiboides (i.e., anti-PD1 and PD-L1). Consistent with our earlier findings, Imprime and Imprime + anti-PD-1 treatments significantly increased the percentage of Ly6Ghigh cells in the spleen and the tumor compared to the vehicle alone. The Ly6Ghigh cells in both the spleen and TME showed increased expression of CD86, MHCII and PD-L1. The enriched PMN-MDSC (~60-70% Ly6Ghigh) from the spleens of Imprime-treated mice were significantly less suppressive to CD3/CD28-mediated proliferation of CD3+ splenocytes. Collectively, these data show that neutrophils have a role in Imprime-mediated remodeling of the immunosuppressive TME, driving a more conducive environment to T-cell immunity. The effects of Imprime on peripheral and tumor-associated neutrophils in cancer patients are currently being explored in phase 2 clinical trials employing the combination of Imprime and anti-PD1 antibody, pembrolizumab. Citation Format: Adria L. Bykowski Jonas, Anissa S.H. Chan, Nadine C. Ottoson, Xiaohong Qui, Keith Gordon, Mark Uhlik, Jeremy Graff, Nandita Bose. Treatment with Imprime PGG, a soluble yeast β-glucan PAMP, induces trafficking of activated Ly6Ghigh cells to secondary lymphoid organs and tumor and facilitates immune activation within the tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A01.

Abstract B21: Synergistic antitumor immunity observed with combination FRα-targeting antibody-drug conjugate plus anti-PD-1 therapy is CD8+ cell dependent

Abstract Background: Immune checkpoint blockade, specifically targeting PD-1/PD-L1, is a promising approach for cancer therapy. However, for some common cancers, such as epithelial ovarian cancer (EOC), PD1/PDL1 blockade as monotherapy has a low response rate. In these less immunogenic cancer types, focus has turned to combining checkpoint inhibitors with cancer therapies that elicit immunologic cell death (ICD). Antibody-drug conjugates (ADCs), comprising a tumor-targeting antibody armed with highly potent small-molecule payloads, are a clinically validated anticancer therapy. Mirvetuximab soravtansine is an ADC comprised of a folate receptor alpha (FRα)-binding antibody linked to the potent tubulin-disrupting maytansinoid DM4 via a cleavable disulfide sulfo-SPDB linker and is currently being evaluated in combination with pembrolizumab (anti-PD1 antibody) in a phase 1b study (FORWARDII, NCT02606305). Here we report preclinical studies that provide a mechanistic understanding for the antitumor activity of combination therapy consisting of PD-1/PD-L1 blockade and an FRα targeting disulfide linked DM4 ADC. Methods: A chimeric anti-mouse FRα antibody (rmFR1-12) was generated and its specificity for murine FRα was assessed by ELISA and FACS. rmFR1-12 was conjugated to DM4 via a s-SPDB linker to generate rmFR-1-12-s-SPDB-DM4 ADC, which was quality checked using standard analytical methods. In vitro assays were used to assess cytotoxicity against FRα-positive cell lines, as well as the ability of rmFR1-12-s-SPDB-DM4 to induce ICD and engage cells of the immune system. In vivo studies performed in albino C57/B6 mice evaluated the antitumor activity of rmFR1-12-s-SPDB-DM4, anti-PD-1 or the combination therapy against ID8 tumors, a translationally relevant murine model of EOC. Results: rmFR1-12 binding was specific for FRα. The rmFR1-12-s-SPDB-DM4 ADC was cytotoxic against FRα-positive cell lines and induced ICD as measured by upregulation of ICD markers. In vivo studies using syngeneic ID8 tumors demonstrated that rmFR1-12-s-SPDB-DM4 or anti-PD-1 monotherapy treatment resulted in modest antitumor activity. In contrast, combination treatment demonstrated synergistic antitumor activity, inducing complete tumor regressions and increased survival over control or monotherapy regimens. Depletion of CD8+ cells abrogated the antitumor activity of the combination, whereas depletion of CD4+ cells had no impact. Finally, combination-treated mice generated a robust memory response when rechallenged with ID8 cells. Conclusion: Anti-FRα-s-SPDB-DM4 induces ICD in FRα-positive cell lines and demonstrates synergistic antitumor activity when combined with anti-PD1 in a murine EOC model. The antitumor immunity is dependent upon CD8+ cells. These findings support the clinical assessment of mirvetuximab soravtansine and pembrolizumab. Citation Format: L. Cristina Gavrilescu, Yulius Setiady, Ling Dong, Sharon Chicklas, Luke Harris, Jan Pinkas, Richard Gregory, Joe Ponte. Synergistic antitumor immunity observed with combination FRα-targeting antibody-drug conjugate plus anti-PD-1 therapy is CD8+ cell dependent [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B21.

Antibody-mediated delivery of viral epitopes to tumors harnesses CMV-specific T cells for cancer therapy.

Several cancer immunotherapy approaches, such as immune checkpoint blockade and adoptive T-cell therapy, boost T-cell activity against the tumor, but these strategies are not effective in the absence of T cells specific for displayed tumor antigens. Here we outline an immunotherapy in which endogenous T cells specific for a noncancer antigen are retargeted to attack tumors. The approach relies on the use of antibody-peptide epitope conjugates (APECs) to deliver suitable antigens to the tumor surface for presention by HLA-I. To retarget cytomegalovirus (CMV)-specific CD8+ T cells against tumors, we used APECs containing CMV-derived epitopes conjugated to tumor-targeting antibodies via metalloprotease-sensitive linkers. These APECs redirect pre-existing CMV immunity against tumor cells in vitro and in mouse cancer models. In vitro, APECs activated specifically CMV-reactive effector T cells whereas a bispecific T-cell engager activated both effector and regulatory T cells. Our approach may provide an effective alternative in cancers that are not amenable to checkpoint inhibitors or other immunotherapies.

Targeted Alpha Therapy with Thorium-227.

Targeted alpha therapy (TAT) can deliver high localized burden of radiation selectively to cancer cells as well as the tumor microenvironment, while minimizing toxicity to normal surrounding cell. Radium-223 (223Ra), the first-in-class α-emitter approved for bone metastatic castration-resistant prostate cancer has shown the ability to prolong patient survival. Targeted Thorium-227 (227Th) conjugates represent a new class of therapeutic radiopharmaceuticals for TAT. They are comprised of the α-emitter 227Th complexed to a chelator conjugated to a tumor-targeting monoclonal antibody. In this review, the authors will focus out interest on this therapeutic agent. In recent studies 227Th-labeled radioimmunoconjugates showed a relevant stability both in serum and vivo conditions with a significant antigen-dependent inhibition of cell growth. Unlike 223Ra, the parent radionuclide 227Th can form highly stable chelator complexes and is therefore amenable to targeted radioimmunotherapy. The authors discuss the future potential role of 227Th TAT in the treatment of several solid as well as hematologic malignancies.

An antibody-supermolecule conjugate for tumor-specific targeting of tumoricidal methylated β-cyclodextrin-threaded polyrotaxanes.

We previously found that acid-labile polyrotaxane containing methylated β-cyclodextrin (Me-PRX) induces endoplasmic reticulum (ER) stress-related autophagy and autophagic cell death. Me-PRX-induced autophagic cell death occurs even in apoptosis-resistant cells; tumor-targeted Me-PRX delivery could thus be an effective cancer treatment approach. In this study, antibody-supermolecule conjugates, consisting of a tumor-specific antibody and Me-PRX, were designed to achieve a tumor-specific delivery of Me-PRX. Trastuzumab, a monoclonal antibody against HER2 expressed in various malignant tumors, was selected as a tumor-targeting antibody, and phenyl maleimide group-modified Me-PRX (Mal-Me-PRX) was conjugated to the cysteine residue of the reduced Trastuzumab to obtain a Trastuzumab-Me-PRX conjugate (Tras-Me-PRX). The cellular association of Tras-Me-PRX to HER2-expressing tumor cells was remarkably greater than that of unmodified Me-PRX. Moreover, Tras-Me-PRX effectively reduced the viability of HER2-expressing tumor cells at a lower concentration compared to the unmodified Me-PRX. In conclusion, antibody-Me-PRX conjugates are regarded as a new class of antibody-drug conjugates that would contribute to the chemotherapy of cancers.