Targeting Tumor-associated Antigens Research Articles

Abstract B042: Dissection of the T cell infiltrate in mouse pancreatic tumors reveals a diverse tumor-reactive T cell repertoire targeting neoantigens and tumor-associated antigens

Abstract Although pancreatic cancer is generally refractory to immune checkpoint blockade (ICB), recent studies of tumor-infiltrating T cells in human tumor samples demonstrated the presence of in vivo expanded, tumor-reactive T cell receptor (TCR) clonotypes. Here, we explored the T cell repertoire in ICB-refractory murine pancreatic cancer models by combining single-cell transcriptomics with functional TCR characterization. Our initial studies in an orthotopic, transplantable model revealed a remarkable diversity of tumor-reactive TCR clonotypes that could be distinguished from bystander T cells based on gene expression signatures. We identified TCRs exclusively reactive against the tumor of origin, TCRs that also react against an independently derived pancreatic tumor line, and TCRs broadly reactive to tumor cells of diverse tissue origin (e.g. MC38), the latter category being predominant. Subsequent studies were performed in an autochthonous mouse model, in which single focal tumors were induced in the pancreas by means of in vivo electroporation of gene constructs encoding mutant Kras and inducing CRISPR/Cas9-mediated inactivation of tumor suppressor genes. We analyzed the T cell repertoire in tumors with different levels of mutational burden by also inactivating DNA damage repair genes. Interestingly, even in mismatch repair-deficient tumors that featured a high mutational load, the anti-tumor T cell response was primarily directed against recurrent tumor-associated antigens (TAAs) rather than ‘private’ mutanome-encoded neoantigens. Several TAA-reactive TCRs also mediated reactivity against non-transformed cells. Immunopeptidome analysis thus far revealed the identity of 5 cognate antigens targeted by the tumor-reactive TCR repertoire: a mutanome-encoded neoantigen, an epitope encoded by an ectopically expressed endogenous retroviral provirus, and three epitopes derived from TAAs: Lon Peptidase 2 (LONP2), ATPase Na+/K+ Transporting Subunit Beta 3 (ATP1B3) and SEC24B, a component of secretory vesicles. Notably, the overexpression of LONP2 and ATP1B3 was found associated with progression of human cancers, whereas silencing was shown to suppress tumor cell growth. Overexpression of SEC24B in cancers has not been reported, but this protein plays an essential role in the secretory pathway of acinar cells. In summary, in-depth analysis of the natural T cell response in murine pancreatic cancer models revealed a diverse tumor-reactive T cell repertoire that is predominantly directed against TAAs, some of which are also expressed in normal somatic cells. These findings underline the importance of uncovering the antigen-specificity of the natural tumor-reactive TCR repertoire, both in mouse models and human tumor samples, to assess its therapeutic potential and safety with regard to personalized immunotherapy. Citation Format: Stefan Zens, Hannes Kehm, Daniel Baumann, Aline Konrad, Zibo Meng, Arnoud H. de Ru, Julian Mochayedi, Francesca Lucato, Angelika B. Riemer, Isabel Poschke, Michael Volkmar, Peter A. van Veelen, Rienk Offringa. Dissection of the T cell infiltrate in mouse pancreatic tumors reveals a diverse tumor-reactive T cell repertoire targeting neoantigens and tumor-associated antigens [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B042.

Abstract B037: Banana Lectin expressing CAR T cells to target Pancreatic Cancer

Abstract To overcome both the heterogenous and dynamic expression of tumor antigen expression in pancreatic ductal adenocarcinoma (PDAC) and the physical barrier provided by the associated fibrotic stroma, we have developed a novel therapy that recognizes and binds to distinct patterns of aberrant glycosylation specifically expressed on tumor cells and their surrounding stroma. Aberrant glycosylation patterns are a hallmark of cancer and are widely present on tumors including PDAC. We therefore incorporated H84T BanLec as part of a chimeric antigen receptor and co-expressed the construct in T cells and showed that H84T BanLec CAR expressing T-cells can effectively target pancreatic tumors. We hypothesized that our novel lectin CAR would enhance the activity of T cells when co-expressed with conventional scFV – derived CARs targeting tumor associated antigens We compared the activity of dual-expressing H84T/HER.2 CAR-T or H84T/PSMA CAR-T cells against single- expressing CARs targeting PDAC. We determined anti-tumor cytotoxicity by measuring residual GFP+ tumor cells in a sequential killing assay using flow cytometry. We observed superior anti-tumor activity using dual expressing CAR T cells compared to T cells expressing either HER.2- or H84T- or PSMA- CAR alone. Similarly, in 3D multicellular spheroids containing tumor and cancer-supportive stromal cells, an IncuCyte live imaging assay shows dual CAR T cells have greater cytotoxicity against PDAC than single CAR expressing T cells (p= 0.02 H84T/HER.2 vs HER.2 CAR; p=0.00003 H84T/PSMA CAR vs PSMA CAR). These results also translate to improved anti- tumor function of dual CAR T cells in PDAC xenograft in vivo models where H84T/HER.2 CAR T cells significantly reduced the tumor volume burden (p=0.015; H84T/HER.2 average=57.1mm3; HER.2 average=116.1mm3; H84T average=130.3mm3). These findings suggest that H84T BanLec CAR T cells provide a tool to target the aberrant glycosylation patterns of malignant cells and their supporting stroma and may contribute to an effective treatment option for PDAC patients. We are now elucidating the underlying mechanism by which H84T BanLec CARs enhance tumor activity and promote T cell infiltration. Citation Format: Katie McKenna, Ada I Ozcan, David Markovitz, Malcolm K Brenner. Banana Lectin expressing CAR T cells to target Pancreatic Cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B037.

Antagonizing vasoactive intestinal peptide (VIP) receptors with Muc16CD-directed armored CAR T cells for pancreatic cancer.

2558 Background: Chimeric antigen receptor (CAR) T therapies for pancreatic ductal adenocarcinoma (PDAC) still face significant immunosuppressive obstacles in the tumor microenvironment (TME). We hypothesize that an optimal CAR T cell for PDAC combines targeting an ideal tumor-associated antigen (TAA) and overcomes unique immunosuppression in the PDAC TME. The retained ectodomain of Muc16 (Muc16CD) is a known TAA in ovarian cancer but has yet to be explored in pancreatic cancer. Vasoactive intestinal peptide (VIP) is an emerging checkpoint pathway for T cell function, which expresses VIP receptors (VIPR) and is abundantly expressed by PDAC. In this work, we present a novel armored CAR T cell that targets Muc16CD and antagonizes VIPRs (CAR/VIPRa) to overcome the immunosuppressive PDAC TME. Methods: Patient expression data was assessed based on data generated by the TCGA Research Network. Primary human T cells from healthy donors or PDAC patients were retrovirally transduced to express Muc16CD-directed CARs with or without secretion of novel, potent VIPR antagonist peptides. In vivo, PDAC PDX tumors were engrafted into SCID/Beige mice and treated with CAR T cells. Results: PDAC tumors have significantly increased expression of Muc16 compared to normal pancreas tissue and patients with high Muc16 expression have a significantly decreased overall survival. PDAC patient-derived tumors show robust expression of both Muc16CD and VIP. CAR/VIPRa T cells reveal that VIPR antagonism metabolically reprograms CAR T cells and drives a memory-rich product. CAR/VIPRa T cells are less activated and less exhausted by the manufacturing process, which lends to better viability and a metabolically quiescent phenotype at baseline. These distinct features allow CAR/VIPRa T cells, when antigen-stimulated, to have enhanced activation and expansion with repeated stimulation. To investigate clinical relevance, CAR/VIPRa T cells manufactured from PDAC patient blood are also significantly enriched for memory phenotypes. In vivo, CAR/VIPRa T cells have enhanced expansion, phenotype, infiltration, and persistence, which ultimately reduces PDAC tumor burden. In a patient-derived PDAC preclinical mouse model where CAR T is typically ineffective, CAR/VIPRa T cells significantly reduce tumor burden. Conclusions: This work demonstrates Muc16CD as a clinically relevant TAA target for CAR T therapy in PDAC. Furthermore, antagonizing the previously undescribed VIP checkpoint pathway in CAR T cells produces enhanced phenotypic and functional profiles. Collectively, this data demonstrates that novel CAR/VIPRa T cells create an advantageous cellular therapy product capable of treating PDAC. The long-term goal of this work is translating CAR/VIPRa T cells for the treatment of PDAC and expanding these preclinical findings of cellular therapies for other VIP-abundant tumors.

Abstract LB344: A novel approach to limit off-tumor toxicity of CAR-T cells targeting solid cancers

Abstract T cells genetically engineered to express a chimeric antigen receptor (CAR) specific for the molecule CD19 have achieved significant gains in the treatment of chemotherapy-resistant leukemia and lymphoma. Despite evidence of long-lived cures in patients with metastatic solid cancer after receiving tumor-specific T cells, no CAR-T cell treatment is currently approved for use against any solid cancer. A large majority of solid cancers express one of many tumor-associated antigens (TAAs) that are vulnerable to CAR T cell recognition. However, in nearly all cases those same TAAs are present in healthy tissues. Since CAR T cells cannot differentiate between cancer and normal cells, CAR T cell recognition of TAAs on healthy tissues provokes dose-limiting ‘on-target off-tumor’ toxicities at sites of endogenous expression (lung, intestine). Thus, there remains a pressing need to develop strategies to mitigate off-tumor toxicity of CAR T cells. To this end, we develop two immunocompetent models of CAR T cell transfer targeting TAAs abundant in metastatic colorectal cancer and pancreatic cancer. TAA-specific CAR T cells affected tumor shrinkage along with infiltrating and damaging sites of endogenous expression (primarily lung, pancreas, & colon). In both of our models, off-tumor toxicities were antigen-dependent and resulted in tissue damage, systemic inflammation, and host death that was titratable to CAR-T cell dose. TAA-specific CAR T cells infiltrated sites of endogenous expression such as the intestine, pancreas, and lungs. Notably, the respiratory system was the site of greatest pathology in both of our models, with massive CAR T cell accumulation and tissue destruction that resulted in systemic wasting and rapid weight loss. Remarkably, we found that the presence of an antigen-bearing tumor in the liver, but not other sites, greatly exacerbated CAR-T cell pulmonary infiltration, tissue destruction, and host mortality.The liver is the most common site of metastasis in patients with colorectal and pancreatic adenocarcinoma and the only site of metastasis in one-third of colorectal cancers. Our models indicated that TAA-specific CAR-T cells given for the treatment of liver metastasis result in dose-limiting pulmonary toxicity due to the presence of the TAA in the healthy lung. To address this translational barrier, we designed and validated a multiparameter genetic reprogramming targeting T cell trafficking and tissue residency introduced with the CAR to imbue CAR-T cells with liver tropism. In multiple models, CAR T cells programmed with liver-specific tropism augmented CAR T cell liver trafficking, decreased lung infiltration and toxicity, and effectively treated TAA+ liver metastasis. We aim to initiate a pilot clinical trial using tissue resident-programmed CAR T cells to treat colorectal liver metastasis. Citation Format: Alex Bartlett, Matthew Mcwhorter, Camille Collier, Chelsea Jenkins, Robert Eil. A novel approach to limit off-tumor toxicity of CAR-T cells targeting solid cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB344.

Abstract 2357: T cell receptor beta chain-directed Tri-specific antibody molecules retarget V-beta T cell subsets to tumors to promote potent and durable antitumor activity

Abstract Background We have previously shown that novel bifunctional “STAR” antibody-fusion molecules comprising germline β chain TCR-targeting antibodies fused to costimulatory cytokines co-engage the TCR and T cell cytokine receptors, thereby promoting activation and expansion of subsets of T cells expressing distinct germline variable β (Vβ) chain TCRs. Here we describe a new class of T cell engager (TriSTAR) that utilizes a tri-specific format comprising the STAR TCR β chain antibody-IL-2 fusion with an additional Fab arm targeting tumor-associated antigens (TAA). By design, TriSTAR molecules promote the activation and expansion of Vβ T cell subsets that adopt a novel memory phenotype and are subsequently re-targeted to tumors expressing specific TAAs. TriSTAR molecules induce potent and durable anti-tumor activity which in refractory murine solid tumor models appear to be superior to established anti-CD3 T cell engager (TCE) formats. Methods Using the gp75 (Tryp1) melanoma antigen as a model TAA, gp75-targeting TriSTARs were tested in vivo in the B16F10 syngeneic mouse tumor model, compared with a conventional gp75-targeting CD3 bispecific TCE. Cured mice were re-challenged with tumors to assess the potential of TriSTARs to induce immunological memory responses. The pharmacodynamics and pharmacokinetics of gp75-targeting TriSTARs and anti-CD3 TCEs were assessed in blood and tumor using flow cytometry, NanoString and other immunoassays. This work was further extended to testing human TAA-targeting TriSTARs using in vitro human cytotoxicity assays and a transgenic MC38 model. Results In the B16F10 murine melanoma model, gp75-targeting TriSTARs induced potent anti-tumor responses that were superior to control constructs and gp75-targeting CD3 TCEs. The superior anti-tumor activity of the gp75-targeting TriSTARs was attributed to a significant increase and accumulation of CD8+, CD25+ and Granzyme B+ T cells in tumors that was due to the expansion of the target Vβ T cell subset. Further immune profiling by flow cytometry and NanoString in tumors and blood isolated from treated B16F10 mice highlighted significant remodeling of tumor-infiltrating lymphocytes (TILs) and other immunological changes associated with immune activation. These observations with murine gp75-targeting TriSTAR molecules were broadly confirmed with TriSTARs targeting human TAAs in both human in vitro studies and human TAA transgenic mouse tumor models. Conclusions Vβ TCR-targeting TriSTAR molecules represent a new class of T cell engager with the potential to deliver improved anti-tumor activity compared with existing anti-CD3 T cell engagers in immunologically “cold” solid tumors with low levels of T cell infiltration and antigen presentation and may therefore offer a new therapeutic approach for the treatment of patients with refractory solid tumors. Citation Format: Madan Katragadda, Jacques Moisan, Gurkan Guntas, Manuel Sequeria, Roya Servattalab, Jessica Lowry, Robert Ruidera, Kent Nybakken, Erik Gerson, Ke Liu, Kevin Chin, Zhen Su, Andrew Bayliffe. T cell receptor beta chain-directed Tri-specific antibody molecules retarget V-beta T cell subsets to tumors to promote potent and durable antitumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2357.

Targeting colorectal cancer cells using AND-gated adaptor RevCAR T-cells.

Despite the success of chimeric antigen receptor (CAR) T-cells especially for treating hematological malignancies, critical drawbacks, such as "on-target, off-tumor" toxicities, need to be addressed to improve safety in translating to clinical application. This is especially true, when targeting tumor-associated antigens (TAAs) that are not exclusively expressed by solid tumors but also on hea9lthy tissues. To improve the safety profile, we developed switchable adaptor CAR systems including the RevCAR system. RevCAR T-cells are activated by cross-linking of bifunctional adaptor molecules termed target modules (RevTM). In a further development, we established a Dual-RevCAR system for an AND-gated combinatorial targeting by splitting the stimulatory and co-stimulatory signals of the RevCAR T-cells on two individual CARs. Examples of common markers for colorectal cancer (CRC) are the carcinoembryonic antigen (CEA) and the epithelial cell adhesion molecule (EpCAM), while these antigens are also expressed by healthy cells. Here we describe four novel structurally different RevTMs for targeting of CEA and EpCAM. All anti-CEA and anti-EpCAM RevTMs were validated and the simultaneous targeting of CEA+ and EpCAM+ cancer cells redirected specific in vitro and in vivo killing by Dual-RevCAR T-cells. In summary, we describe the development of CEA and EpCAM specific adaptor RevTMs for monospecific and AND-gated targeting of CRC cells via the RevCAR platform as an improved approach to increase tumor specificity and safety of CAR T-cell therapies.

Bispecific antibodies revolutionizing breast cancer treatment: a comprehensive overview.

Breast cancer (BCa) is known as a complex and prevalent disease requiring the development of novel anticancer therapeutic approaches. Bispecific antibodies (BsAbs) have emerged as a favorable strategy for BCa treatment due to their unique ability to target two different antigens simultaneously. By targeting tumor-associated antigens (TAAs) on cancer cells, engaging immune effector cells, or blocking critical signaling pathways, BsAbs offer enhanced tumor specificity and immune system involvement, improving anti-cancer activity. Preclinical and clinical studies have demonstrated the potential of BsAbs in BCa. For example, BsAbs targeting human epidermal growth factor receptor 2 (HER2) have shown the ability to redirect immune cells to HER2-positive BCa cells, resulting in effective tumor cell killing. Moreover, targeting the PD-1/PD-L1 pathway by BsAbs has demonstrated promising outcomes in overcoming immunosuppression and enhancing immune-mediated tumor clearance. Combining BsAbs with existing therapeutic approaches, such as chemotherapy, targeted therapies, or immune checkpoint inhibitors (ICIs), has also revealed synergistic effects in preclinical models and early clinical trials, emphasizing the usefulness and potential of BsAbs in BCa treatment. This review summarizes the latest evidence about BsAbs in treating BCa and the challenges and opportunities of their use in BCa.

Oncogenic viral antigens for engineered T cell immunotherapy: Challenges and opportunities

AbstractViruses that cause malignancies such as hepatocellular carcinoma and cervical cancer are the cause of approximately 20% of all human cancers. In recent years, engineered T cell immunotherapy targeting tumor‐associated antigens (TAAs) has had some success against virus‐associated cancer, although these treatments are associated with side effects. TAA‐specific‐modified T cells may kill cancer cells but they also react with and damage healthy tissue. During an oncogenic virus infection, viral DNA integrates into the host genome, leading to the expression of viral‐specific antigens in the tumor in a restricted and durable manner. The cross‐reactive side‐effects of conventional TAA‐specific engineered T cell treatment can be avoided by creating engineered T cells that target oncogenic viral antigens. To chart a course for the discovery of additional viral‐specific antigens and their combination with immune checkpoint inhibition therapies, this review summarizes the development, preclinical research, and clinical application of oncogenic viral antigen–specific T cell immunotherapy. This review also addresses challenges such as virus mutation and diverse integration, which can result in the loss of the target.

Novel Peptide-Based Plectin-1 CAR T-Cells Target Specifically Pancreatic Cancer Cells

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is one of the most devastating cancers, and therapies to treat this malignancy have not evolved rapidly to fulfill the need of affected patients. PDAC, like many solid malignancies, is considered a “cold immunological tumor” where the lack of targetable tumor-associated antigens has hampered the development of successful immunotherapies. Despite newer treatment approaches, PDAC patients with advanced disease have a dismal prognosis. Therefore, there is an urgent need to develop new therapies. Plectin-1 is a high molecular weight protein (∼500 kDa) that participates in cytoskeleton network organization linking intermediate filaments to microtubules and microfilaments. Plectin-1 is aberrantly expressed in the PDAC cellular membrane, contributes to PDAC pathogenesis, and confers a poor prognosis. Plectin-1-targeting peptides (PTP) in mice and humans have been successfully explored for their use in PDAC imaging and targeted payload delivery (small molecules, radioisotopes, and chemotherapy). We sought to take advantage of the specific binding of PTP and develop a peptide-based plectin-1 chimeric antigen receptor (CAR), and test T-cell transduced with this construct in vitro against PDAC cell lines. Methods: Using in silico tools, we optimized different pepto-CAR constructs and selected one that showed the highest levels of cytotoxicity against plectin-1-expressing PDAC cell lines. A myc tag was incorporated in the hinge region for flow cytometry or western blot detection. The intracellular signaling domain of these CAR sequences included 4-1BB and the zeta chain of the CD3 molecule ( Figure 1A). Plasmids containing the CAR sequence were packaged on lentivirus particles using HEK293T cells. Purified plectin-1 CAR lentiviruses were used to transduce T-cells derived from human peripheral blood T-cells obtained and purified from healthy donor whole blood. The expanded CAR T-cells were used for cytotoxic experiments against different cell lines. Results: Expression of plectin-1 CAR was confirmed by flow cytometry using an anti-myc tag antibody ( Figure 1B). Plectin-1 expression was analyzed in different pancreatic cancer cell lines, and the BxPC-3 cells showed the highest expression level ( Figure 1B). Using a luciferase-based assay, we plated 50,000 BxPC-3 cells per well in duplicates with different effector-to-target (E:T) ratios. After 24 hours, luciferin was added, and the plate was read using a luminometer. As a negative control for effector cells, we use T-cells without a CAR ( Figure 1B), and for the target cells, we use the plectin-1 negative lung cancer cell line A549 ( Figure 1B). Plectin-1 CAR T-cells effectively killed BxPC-3 cells at different E:T ratios in a specific manner compared with controls using unmodified T-cells. Furthermore, no cytotoxicity was observed against plectin-1 negative A549 cells ( Figure 1A). Conclusion: Our results show that a novel peptide-based CAR construct design against plectin-1 can induce specific cytotoxicity on plectin-1 expressing PDAC cell line BxPC-3. Furthermore, these data demonstrate the feasibility of using a CAR with a peptide-based antigen binding domain instead of the traditional antibody-derived scFv. This novel peptide-based CAR design offers alternatives for cellular therapy bioengineering. Moreover, our results can significantly impact immune-oncology therapeutic applications, particularly in challenging diseases like pancreatic cancer. Figure 1 . A) In the above section, the Plectin-1 CAR construct is depicted. Cytotoxicity of unmodified T-cells and Plectin-1 CAR T-cells against BxPC-3 and A549 is shown below. B) Expression of Plectin-1 CAR in unmodified T-cells and Plectin-1 CAR T-cells is shown on the left. The expression of plectin-1 antigen on the surface of BxPC-3 cells and A549 cells is shown on the right. PTP: plectin-1 targeting peptide.

ICOS and OX40 tandem co-stimulation enhances CAR T-cell cytotoxicity and promotes T-cell persistence phenotype.

Chimeric Antigen Receptor (CAR) T-cell therapies have emerged as an effective and potentially curative immunotherapy for patients with relapsed or refractory malignancies. Treatment with CD19 CAR T-cells has shown unprecedented results in hematological malignancies, including heavily refractory leukemia, lymphoma, and myeloma cases. Despite these encouraging results, CAR T-cell therapy faces limitations, including the lack of long-term responses in nearly 50-70% of the treated patients and low efficacy in solid tumors. Among other reasons, these restrictions are related to the lack of targetable tumor-associated antigens, limitations on the CAR design and interactions with the tumor microenvironment (TME), as well as short-term CAR T-cell persistence. Because of these reasons, we developed and tested a chimeric antigen receptor (CAR) construct with an anti-ROR1 single-chain variable-fragment cassette connected to CD3ζ by second and third-generation intracellular signaling domains including 4-1BB, CD28/4-1BB, ICOS/4-1BB or ICOS/OX40. We observed that after several successive tumor-cell in vitro challenges, ROR1.ICOS.OX40ζ continued to proliferate, produce pro-inflammatory cytokines, and induce cytotoxicity against ROR1+ cell lines in vitro with enhanced potency. Additionally, in vivo ROR1.ICOS.OX40ζ T-cells showed anti-lymphoma activity, a long-lasting central memory phenotype, improved overall survival, and evidence of long-term CAR T-cell persistence. We conclude that anti-ROR1 CAR T-cells that are activated by ICOS.OX40 tandem co-stimulation show in vitro and in vivo enhanced targeted cytotoxicity associated with a phenotype that promotes T-cell persistence.

Abstract 815: Improved tolerability and tumor specific delivery of a therapeutic bispecific T cell engager using a pH-sensitive nanoparticle platform

Abstract Bispecific antibodies are an emerging class of therapeutics for immune-oncology applications. T cell engagers (TCEs) target tumor-associated antigens (TAA) to eradicate cancer cells. TCEs for solid tumors have demonstrated encouraging preclinical efficacy but development has been challenging with dose-limiting toxicities due to on-target/off-tumor effect. To overcome the issue, we have developed ON-BOARD, an ultra-pH sensitive nanoparticle platform for masked and targeted delivery of payloads to the acidic tumor microenvironment (TME). Herein we report efficacious masked delivery of a TCE to tumors in mice using ON-BOARD demonstrating significantly improved tolerability and potential for clinical translation. TCEs were encapsulated in ON-BOARD nanoparticles and characterized for particle properties. The formulations were assessed in vitro under neutral or acid-activated conditions in TDCC assays. In vivo studies were performed in mice bearing “immune desert” pancreatic cancer. ON-BOARD tumor localization was measured by fluorescence while unencapsulated TCE and ON-BOARD/TCE pharmacokinetics was evaluated. PD studies evaluated immune-phenotype changes in tumors and draining lymph nodes, and systemic cytokine levels. Efficacy studies were performed in tumor-bearing mice comparing unencapsulated TCE to ON-BOARD-TCE as monotherapy and in combination with anti-4-1BB agonist therapy. ON-BOARD encapsulated TCEs have high encapsulation efficiency (>88%) in uniformly distributed stable particles (Dh<100nm). pH-specific and target-specific killing were confirmed in vitro in TDCC killing assays using a human gastric cancer cell line and a genetically engineered mouse pancreatic cancer cell line with 20 to 200-fold activation window between the acid-activated and intact formulations. In vivo, fluorescently labeled ON-BOARD nanoparticles localized to tumors in mice while ON-BOARD/TCE formulations demonstrated minimal payload leakage and mitigated payload degradation. ON-BOARD/TCE formulations showed favorable immunophenotype changes in tumors including increased CD8+, CD8+/T reg ratio and PD-1+Ki67+CD8+. ON-BOARD encapsulation also significantly improved TCE tolerability showing reduced body weight loss nadir (3% vs 17%) and reduced systemic proinflammatory cytokine response (IL-6, TNF, IFNγ, etc). When dosed in combination with anti-4-1BB agonist therapy in tumor-bearing animals, unencapsulated TCE induced 100% treatment-related animal death while ON-BOARD/TCE prevented animal death with minimum body weight loss. Furthermore, ON-BOARD/TCE with a4-1BB combo therapy showed 61% tumor growth inhibition in the “immune desert” pancreatic cancer model. The ON-BOARD platform demonstrated potential as an effective tool for masked tumor-specific delivery and improved tolerability of bispecific TCEs for cancer therapy. Citation Format: Qingtai Su, Stephen Gutowski, Gaurav Bharadwaj, Austin Burcham, Bhargavi Allu, Irina Kalashnikova, Zirong Chen, Ruolan Han, Jason B. Miller, Tian Zhao. Improved tolerability and tumor specific delivery of a therapeutic bispecific T cell engager using a pH-sensitive nanoparticle platform [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 815.

Abstract 6357: Generation of a mutant SIRPa fusion protein with highly-improved affinity and favorable safety profile

Abstract Background/Purpose: CD47 has been validated to be expressed on various tumor types including acute myeloid leukemia, myelodysplastic syndrome and other hematologic or solid tumors. As primary ‘don’t eat me’ signal, high expression of CD47 on tumor cells interacts with SIRPα negatively regulating the phagocytosis level. However, poor safety profile as well as insufficient combinatory effect remain to be the issues limiting the clinical outcomes of CD47-targeting molecules. Herein, we used rational design strategies to generate SCR9168 with potent affinity improvement, and maintained favorable safety profiles in different animal species, including cynomolgus monkeys. Procedures: Mutagenesis was introduced to the residues critical for CD47/SIRPα binding interface. Affinity was measured by SPR analysis. In vitro activities were determined by biochemical- or cell-based binding, blocking and antibody-dependent cellular phagocytosis (ADCP) assays. The efficacy in vivo was assessed with the OE19 xenograft model. Pharmacokinetics (PK) and safety were monitored in both mice and cynomolgus monkeys. Results: The affinity of SCR9168 achieves 178 pM for human CD47 which is nearly 50-fold increase in comparison with wild-type SIRPα. Enhanced affinity has also been confirmed in binding to OE19 or DLD1 cell lines, as well as in blocking human SIRPα binding to Raji cells. SCR9168 remains strong binding potency to monkey, mouse or rat CD47 which allows the PK and safety assessment using these animal species. The combined effects of SCR9168 and multiple therapeutic antibodies targeting tumor-associated antigens (TAAs) have been evaluated with ADCP assays using human monocyte-derived macrophages. SCR9168 markedly increases the ratio of phagocytic cells combining with antibodies, such as cetuximab. However, due to the inert function of Fc fragment, no phagocytosis of red blood cells or platelets was caused after the incubation in vitro. In addition, SCR9168 treatment in combination with trastuzumab leads to significantly improved suppression of tumor growth in a dose-dependent manner. Moreover, administration of two doses at day 1 and day 11 in cynomolgus monkeys results in no toxicity events related to SCR9168 based on the data from hematological analysis. Conclusion: SCR9168 demonstrates the best-in-class potential among SIRPα mutein molecules. It elicits improved and dose-dependent efficacy in phagocytosis or tumor suppression combining with therapeutic antibodies, such as trastuzumab or cetuximab. Favorable safety profile with no phagocytosis of RBC or platelets in vitro as well as no hematological toxicity observation in cynomolgus monkeys allows broader dose range exploration in early clinical phase. SCR9168 is currently in development stage and IND enabling will be expected in the end of 2023. Citation Format: Yingying Hu, Yayuan Fu, Lei Liu, Wenjing Li, Liting Xue, Zhiyong Yu, Yun Zhang, Meijuan Gao, Yixin Tan, Fudong Wang, Yadan Wu, Jie Li, Zhenzhen Li, Feng Zhou, Wenqing Yang, Zhuoxiao Cao, Renhong Tang. Generation of a mutant SIRPa fusion protein with highly-improved affinity and favorable safety profile [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 6357.

PRAME and CTCFL-reactive TCRs for the treatment of ovarian cancer.

Recurrent disease emerges in the majority of patients with ovarian cancer (OVCA). Adoptive T-cell therapies with T-cell receptors (TCRs) targeting tumor-associated antigens (TAAs) are considered promising solutions for less-immunogenic 'cold' ovarian tumors. In order to treat a broader patient population, more TCRs targeting peptides derived from different TAAs binding in various HLA class I molecules are essential. By performing a differential gene expression analysis using mRNA-seq datasets, PRAME, CTCFL and CLDN6 were selected as strictly tumor-specific TAAs, with high expression in ovarian cancer and at least 20-fold lower expression in all healthy tissues of risk. In primary OVCA patient samples and cell lines we confirmed expression and identified naturally expressed TAA-derived peptides in the HLA class I ligandome. Subsequently, high-avidity T-cell clones recognizing these peptides were isolated from the allo-HLA T-cell repertoire of healthy individuals. Three PRAME TCRs and one CTCFL TCR of the most promising T-cell clones were sequenced, and transferred to CD8+ T cells. The PRAME TCR-T cells demonstrated potent and specific antitumor reactivity in vitro and in vivo. The CTCFL TCR-T cells efficiently recognized primary patient-derived OVCA cells, and OVCA cell lines treated with demethylating agent 5-aza-2'-deoxycytidine (DAC). The identified PRAME and CTCFL TCRs are promising candidates for the treatment of patients with ovarian cancer, and are an essential addition to the currently used HLA-A*02:01 restricted PRAME TCRs. Our selection of differentially expressed genes, naturally expressed TAA peptides and potent TCRs can improve and broaden the use of T-cell therapies for patients with ovarian cancer or other PRAME or CTCFL expressing cancers.

A tumor-associated autoantibody panel for the detection of non-small cell lung cancer.

Lung cancer is the second most frequent malignancy and the leading cause of cancer-associated death worldwide. Compared with patients diagnosed at advanced disease stages, early detection of lung cancer significantly improved the 5-year survival rate from 3.3% to 48.8%, which highlights the importance of early detection. Although multiple technologies have been applied to the screening and early diagnosis of lung cancer so far, some limitations still exist so they could not fully suit the needs for clinical application. Evidence show that autoantibodies targeting tumor-associated antigens(TAAs) could be found in the sera of early-stage patients, and they are of great value in diagnosis. Methods, we identified and screened TAAs in early-stage non-small cell lung cancer(NSCLC) samples using the serological analysis of recombinant cDNA expression libraries(SEREX). We measured the levels of the 36 autoantibodies targeting TAAs obtained by preliminary screening via liquid chip technique in the training set(332 serum samples from early-stage NSCLC patients, 167 samples from patients with benign lung lesions, and 208 samples from patients with no obvious abnormalities in lungs), and established a binary logistic regression model based on the levels of 8 autoantibodies to distinguish NSCLC samples. Results, We validated the diagnostic efficacy of this model in an independent test set(163 serum samples from early-stage NSCLC patients, and 183 samples from patients with benign lung lesions), the model performed well in distinguishing NSCLC samples with an AUC of 0.8194. After joining the levels of 4 serum tumor markers into its independent variables, the final model reached an AUC of 0.8568, this was better than just using the 8 autoantibodies (AUC:0.8194) or the 4 serum tumor markers alone(AUC: 0.6948). In conclusion, we screened and identified a set of autoantibodies in the sera of early-stage NSCLC patients through SEREX and liquid chip technique. Based on the levels of 8 autoantibodies, we established a binary logistic regression model that could diagnose early-stage NSCLC with high sensitivity and specificity, and the 4 conventional serum tumor markers were also suggested to be effective supplements for the 8 autoantibodies in the early diagnosis of NSCLC.

CAR-T cell therapy in triple-negative breast cancer: Hunting the invisible devil.

Triple-negative breast cancer (TNBC) is known as the most intricate and hard-to-treat subtype of breast cancer. TNBC cells do not express the well-known estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2) expressed by other breast cancer subtypes. This phenomenon leaves no room for novel treatment approaches including endocrine and HER2-specific antibody therapies. To date, surgery, radiotherapy, and systemic chemotherapy remain the principal therapy options for TNBC treatment. However, in numerous cases, these approaches either result in minimal clinical benefit or are nonfunctional, resulting in disease recurrence and poor prognosis. Nowadays, chimeric antigen receptor T cell (CAR-T) therapy is becoming more established as an option for the treatment of various types of hematologic malignancies. CAR-Ts are genetically engineered T lymphocytes that employ the body's immune system mechanisms to selectively recognize cancer cells expressing tumor-associated antigens (TAAs) of interest and efficiently eliminate them. However, despite the clinical triumph of CAR-T therapy in hematologic neoplasms, CAR-T therapy of solid tumors, including TNBC, has been much more challenging. In this review, we will discuss the success of CAR-T therapy in hematological neoplasms and its caveats in solid tumors, and then we summarize the potential CAR-T targetable TAAs in TNBC studied in different investigational stages.

Combined targeting of soluble latent TGF-ß and a solid tumor-associated antigen with adapter CAR T cells

ABSTRACT Solid tumors consist of malignant and nonmalignant cells that together create the local tumor microenvironment (TME). Additionally, the TME is characterized by the expression of numerous soluble factors such as TGF-β. TGF-β plays an important role in the TME by suppressing T cell effector function and promoting tumor invasiveness. Up to now CAR T cells exclusively target tumor-associated antigens (TAA) located on the cell membrane. Thus, strategies to exploit soluble antigens as CAR targets within the TME are needed. This study demonstrates a novel approach using Adapter CAR (AdCAR) T cells for the detection of soluble latent TGF-β within the TME of a pancreatic tumor model. We show that AdCARs in combination with the respective adapter can be used to sense soluble tumor-derived latent TGF-β, both in vitro and in vivo. Sensing of the soluble antigen induced cellular activation and effector cytokine production in AdCAR T cells. Moreover, we evaluated AdCAR T cells for the combined targeting of soluble latent TGF-β and tumor cell killing by targeting CD66c as TAA in vivo. In sum, our study broadens the spectrum of targetable moieties for AdCAR T cells by soluble latent TGF-β.

Dissecting Immunotherapy Strategies for Small Cell Lung Cancer: Antibodies, Ionizing Radiation and CAR-T.

Small cell lung cancer (SCLC) is a highly aggressive malignancy that accounts for about 14% of all lung cancers. Platinum-based chemotherapy has been the only available treatment for a long time, until the introduction of immune checkpoint inhibitors (ICIs) recently changed first-line standard of care and shed light on the pivotal role of the immune system. Despite improved survival in a subset of patients, a lot of them still do not benefit from first-line chemo-immunotherapy, and several studies are investigating whether different combination strategies (with both systemic and local treatments, such as radiotherapy) may improve patient outcomes. Moreover, research of biomarkers that may be used to predict patients' outcomes is ongoing. In addition to ICIs, immunotherapy offers other different strategies, including naked monoclonal antibodies targeting tumor associated antigens, conjugated antibody, bispecific antibodies and cellular therapies. In this review, we summarize the main evidence available about the use of immunotherapy in SCLC, the rationale behind combination strategies and the studies that are currently ongoing in this setting, in order to give the reader a clear and complete view of this rapidly expanding topic.

NK Cell-Based Immunotherapy in Colorectal Cancer.

Human Natural Killer (NK) cells are all round players in immunity thanks to their powerful and immediate response against transformed cells and the ability to modulate the subsequent adaptive immune response. The potential of immunotherapies based on NK cell involvement has been initially revealed in the hematological setting but has inspired the design of different immune tools to also be applied against solid tumors, including colorectal cancer (CRC). Indeed, despite cancer prevention screening plans, surgery, and chemotherapy strategies, CRC is one of the most widespread cancers and with the highest mortality rate. Therefore, further efficient and complementary immune-based therapies are in urgent need. In this review, we gathered the most recent advances in NK cell-based immunotherapies aimed at fighting CRC, in particular, the use of monoclonal antibodies targeting tumor-associated antigens (TAAs), immune checkpoint blockade, and adoptive NK cell therapy, including NK cells modified with chimeric antigen receptor (CAR-NK).

A Novel PD-L1-Containing MSLN Targeting Vaccine for Lung Cancer Immunotherapy.

Therapeutic tumor vaccines have become an important breakthrough in the treatment of various solid tumors including lung cancer. Dendritic cells (DCs)-based tumor vaccines targeting tumor-associated antigens (TAAs) play a key role in immunotherapy and immunoprevention. However, the weak immunogenicity of TAAs and low immune response rates are a major challenge faced in the application of therapeutic tumor vaccines. Here, we tested whether targeting an attractive target Mesothelin (MSLN) and PD-L1 immune checkpoint molecule to DCs in vivo would elicit therapeutic antitumor cytotoxic T lymphocyte (CTL) response. We generated specific MSLN fragment combined with PD-L1 and GM-CSF peptide immunogen (MSLN-PDL1-GMCSF) based on the novel anti-PD-L1 vaccination strategy we recently developed for the cancer treatment and prevention. We found that DCs loaded with MSLN-PDL1-GMCSF vaccine elicited much stronger endogenous anti-PD-L1 antibody and T cell responses in immunized mice and that antigen specific CTLs had cytolytic activities against tumor cells expressing both MSLN and PD-L1. We demonstrated that vaccination with MSLN-PDL1-GMCSF potently inhibited the tumor growth of MSLN+ and PD-L1+ lung cancer cells, exhibiting a significant therapeutic anti-tumor potential. Furthermore, PD-1 blockade further improved the synergistic antitumor therapeutic efficacy of MSLN-PDL1-GMCSF vaccine in immunized mice. In summary, our data demonstrated for the first time that this PD-L1-containing MSLN therapeutic vaccine can induce persistent anti-PD-L1 antibody and CTL responses, providing an effective immunotherapeutic strategy for lung cancer immunotherapy by combining MSLN-PDL1-GMCSF vaccine and PD-1 blockade.

Abstract 5568: Revitalization of affinity-tuned CAR T cells via antigen-dependent release of interleukin-12

Abstract Introduction: The application of chimeric antigen receptor (CAR) T cells in solid tumors has met many challenges, arising from the paucity of effective yet safe targets and T cell dysfunction within the immunosuppressive milieu. Clinical trials over the years have reported severe on-target off-tumor toxicities associated with CAR T cell therapies targeting tumor-associated antigens (TAAs). Here, we propose a strategy to target TAAs effectively and safely: 1) improve tumor-targeting specificity by affinity-tuning; 2) armor T cells with antigen-dependent expression of interleukin-12 (IL-12) to compensate loss of CAR T cell activity caused by affinity-tuning. In this system, IL-12 expression is tightly regulated by the nuclear factor of the activated T cells promoter (NFAT) to limit IL-12 to the environ of activated affinity-tuned CAR T cells within the tumor. We demonstrate here the feasibility of this strategy using epithelial cell adhesion molecule (EpCAM) as a model TAA. Methods: Affinity variants were generated by alanine scanning mutagenesis in the complementary-determining region 3 (CDR3) of an EpCAM antibody (UBS54). The affinity of CAR variants was determined by flow cytometry-based saturation binding assay. On-target off-tumor cytotoxicity of CAR T cells was examined against human primary normal epithelial cells. In vivo anti-tumor efficacy was evaluated in mouse models of gastric cancer with cell line-derived (SNU-638 and MKN-45) and patient-derived xenografts. Results: Substitution of alanine into the UBS54 CDR3 led to identification of a tyrosine (6th residue in heavy chain CDR3) as a hot spot for affinity tuning. Among several more subtle amino acid substitutions for Tyr-6, we found that CAR molecule with valine substituent (Y6V) possesses 10 µM affinity toward EpCAM, rendering CAR T cells to be selective to EpCAM-high tumors while being not reactive to primary normal epithelial cells in vitro. In gastric cancer mouse models, compared to UBS54 CAR T cells that mediated rapid tumor remission, Y6V CAR T cells produced mainly partial responses. To revitalize affinity-tuned CAR T cells, we further engineered CAR T cells to release IL-12 under NFAT. We found that both the activity of the NFAT promoter and the level of IL-12 release were tightly regulated by the antigen density of targets when inducible IL-12 is combined with 10 µM affinity Y6V CAR; however, such dependence is lost when 1 µM affinity UBS54 was examined. In mouse models of gastric cancer, inducible lL-12 armored Y6V CAR T cells produced enhanced anti-tumor responses without elevating systemic exposure to IL-12, evidenced by low levels of IL-12 in mouse sera (below 100 pg/ml). Conclusions: Combination of affinity-tuned CAR with inducible expression of IL-12 is a promising strategy to develop CAR T cell therapy against TAAs with the potential for reduced on-target off-tumor toxicity and tumor resistance. Citation Format: Yanping Yang, Huan Yang, Yago Alcaina, Jaclyn E. McCloskey, Janusz Puc, Alyssa Birt, Yogindra Vedvyas, Juan Gonzalez-Valdivieso, Irene M. Min, Eric von Hofe, Moonsoo M. Jin. Revitalization of affinity-tuned CAR T cells via antigen-dependent release of interleukin-12 [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 5568.