Intracellular Tumor Antigens Research Articles

Abstract 16: CD4+ and CD8+ TCRαβ-deficient bioluminescent reporter T cells for screening and characterization of neoantigen-specific TCRs

Abstract Adoptive antigen-specific T cell therapy is currently comprised of chimeric antigen receptor (CAR) and T cell receptor (TCR) engineered T cells. Clinical results from CAR-T cells have demonstrated promising results in treating leukemias, while TCR-engineered T cells, which have the advantage of recognizing intracellular tumor antigens, is still in early development. Here, we report the development of CD4+ or CD8+ TCRαβ-KO reporter T cell lines for the screening and characterization of transgenic TCRs. A TCRαβ-KO reporter T cell line was first developed by knocking out the endogenous TCR α and β chains in the reporter T cell line using CRISPR/Cas9. Successful knockout was confirmed by phenotypic assays and TCR v chain locus sequencing. We demonstrated that re-introduction of influenza hemagglutinin (HA) peptide-specific TCR (HA1.7) α and β chains into TCRαβ-KO reporter T cell lines results in HA peptide-dependent TCR activation and luciferase reporter expression when HA peptide is presented by a MHCII+ cell line. Similarly, re-introduction of MART-1-specific TCR (DMF5) resulted in MART-1-dependent activation in the presence of MHCI+ cells. The select expression of CD4- or CD8-expressing variants of the TCRαβ-KO reporter T cell line enables the development of transgenic TCRs for both MHCI- and MHCII-restricted tumor antigen targets. Further development of CD4/CD8 double-positive and double-negative cell lines enables screening of low and high affinity TCRs without MHC bias. Together, this TCRαβ-KO platform represents a powerful tool for the screening and characterization of neoantigen-specific TCRs. Citation Format: Mei Cong, Jamison Grailer, Michael Slater, Pete Stecha, Jim Hartnett. CD4+ and CD8+ TCRαβ-deficient bioluminescent reporter T cells for screening and characterization of neoantigen-specific TCRs [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 16.

Abstract 2373: Functional and specific T-cell engagers against a peptide-MHC tumor target

Abstract Accessing peptides from intracellular tumor antigens displayed on MHC class I (pMHC) can expand the T-cell engager (TCE) target pool for solid tumor applications. A challenge limiting TCE development for pMHC targets is the identification of rare, high-affinity, and high-specificity pMHC-binders. In this work, we describe TCEs in a 1 × 1 format with potent tumor-cell killing activity and high specificity for MAGE-A4, a pMHC tumor antigen expressed in multiple cancer types. We identified functional MAGE-A4 x CD3 TCEs with high specificity and affinity to a human MAGE-A4 peptide sequence of 10 amino acids presented on MHC-I (HLA-A:02*01). We strategically selected MAGE-A4-binders with high binding specificity for pMAGE-A4230-239, but not MHC-I or many closely related MHC-restricted peptides. We then paired them with diverse and developable CD3-binders with a range of binding affinities, subunit specificities, and binding kinetic profiles. High-throughput in vitro functional characterization of hundreds of these TCEs enabled identification of molecules with optimal T-cell dependent cellular cytotoxicity in MAGE-A4-expressing tumor cell lines, with comparable tumor-killing activity and cytokine release to a clinical-stage TCE in a 2 × 1 format. Previous studies have highlighted unique challenges associated with pMHC targets, including their potential for cross-reactive binding to pMHCs on healthy tissues, and the absence of pharmacologically relevant species for preclinical toxicity testing. To address these challenges, we have designed and implemented an in vitro and in silico workflow to identify antibody binding to potential off-target peptides. Using this approach, we selected TCEs with several pMHC binding orientations and high specificity and affinity. These TCEs killed tumor cell lines endogenously expressing MAGE-A4 pMHCs and not isogenic cells with MAGE-A4 knocked out. Results demonstrate that our high-resolution, multiparametric antibody discovery capabilities can identify pMHC-binding antibodies with desired functional and specificity attributes. This approach unlocks the discovery and development of optimal TCEs against complex pMHC antigens. Citation Format: Davide Tortora, Peter Bergqvist, Allison Goodman, Ryan Blackler, Nathalie Blamey, Stefania Carrara, Lauren Chong, Gabrielle Conaghan, Cindy-Lee Crichlow, Valentine de Puyraimond, Harveer Dhupar, Patrick Farber, Jessica Fernandes Scortecci, Kate Gibson, Rodrigo Goya, Ahn Lee, Franco Li, Tova Pinsky, Craig Robb, Patrick Rowe, Antonios Samiotakis, Eduardo Solano Salgado, Ping Xiang, Irene Yu, Kelly Bullock, Tara Fernandez, Stephanie K. Masterman, Kush Dalal, Tim Jacobs, Bryan C. Barnhart. Functional and specific T-cell engagers against a peptide-MHC tumor target [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 2373.

Preclinical evaluation of a novel CAR-T therapy utilizing a scFv antibody highly specific to MAGE-A4p230-239/HLA-A∗02:01 complex

Despite the revolutionary success of chimeric antigen receptor (CAR)-T therapy for hematological malignancies, successful CAR-T therapies for solid tumors remain limited. One major obstacle is the scarcity of tumor-specific cell surface molecules. One potential solution to overcome this barrier is to utilize antibodies that recognize peptide/MHC complexes in a T-cell receptor (TCR)-like fashion, allowing CAR-T cells to recognize intracellular tumor antigens. This study reports a highly specific scFv antibody against the MAGE-A4p230-239/HLA-A*02:01 complex (MAGE-A4 pMHC), screened from a human scFv phage display library. Indeed, retroviral vectors encoding CAR, utilizing this scFv antibody as a recognition component, efficiently recognized and lysed MAGA-A4+ tumor cells in an HLA-A*02:01-restricted manner. Additionally, the adoptive transfer of T cells modified by the CAR-containing glucocorticoid-induced TNFR-related receptor (GITR) intracellular domain (ICD), but not CD28 or 4-1BB ICD, significantly suppressed the growth of MAGE-A4+ HLA-A*02:01+ tumors in an immunocompromised mouse model. Of note, a comprehensive analysis revealed that a broad range of amino acid sequences of the MAGE-A4p230-239 peptide were critical for the recognition of MAGE-A4 pMHC by these CAR-T cells, and no cross-reactivity to analogous peptides was observed. Thus, MAGE-A4-targeted CAR-T therapy using this scFv antibody may be a promising and safe treatment for solid tumors.

DNA-delivered monoclonal antibodies targeting the p53 R175H mutant epitope inhibit tumor development in mice

The tumor suppressor p53 is the most common mutated gene in cancer, with the R175H as the most frequent p53 missense mutant. However, there are currently no approved targeted therapies or immunotherapies against mutant p53. Here, we characterized and investigated a monoclonal antibody (mAb) that recognizes the mutant p53-R175H for its affinity, specificity, and activity against tumor cells in vitro. We then delivered DNA plasmids expressing the anti-R175H mAb or a bispecific antibody (BsAb) into mice to evaluate their therapeutic effects. Our results showed that the anti-R175H mAb specifically bound to the p53-R175H antigen with a high affinity and recognized the human mutant p53-R175H antigen expressed on HEK293T or MC38 cells, with no cross-reactivity with wild-type p53. In cultured cells, the anti-R175H mAb showed higher cytotoxicity than the control but did not induce antibody-dependent cellular cytotoxicity. We made a recombinant MC38 mouse cell line (MC38-p53-R175H) that overexpressed the human p53-R175H after knocking out the endogenous mutant p53 alleles. In vivo, administration of the anti-R175H mAb plasmid elicited a robust anti-tumor effect against MC38-p53-R175H in mice. The administration of the anti-R175H BsAb plasmid showed no therapeutic effects, yet potent anti-tumor activity was observed in combination with the anti-PD-1 antibody. These results indicate that targeting specific mutant epitopes using DNA-delivered mAbs or BsAbs presents a form of improved natural immunity derived from tumor-infiltrating B cells and plasma cells against intracellular tumor antigens.

Abstract 2752: Targeting intracellular tumor antigens using fully human TCR mimic antibodies derived from HLA transgenic RenMiceTM

Abstract Therapeutic antibodies have ushered in a new age of cancer immunotherapy. Historically, these therapies have targeted a limited subset of soluble and cell surface tumor-associated antigens (TAAs). T cell receptors (TCRs) on cytotoxic CD8+ T cells recognize peptide antigens bound to major histocompatibility class I (MHC-I) proteins, called HLA-A/B/C in humans. By this pathway, antigen is regularly sampled from the intracellular peptidome, processed, and presented to cytotoxic T cells. Expanding TCR-based recognition of soluble, intracellular TAAs presented on the surface of malignant cells by this mechanism is a propitious therapeutic strategy. Here we describe a novel platform for generating T cell receptor mimic (TCRm) antibodies using our humanized immunoglobulin (RenMabTM) mice engineered to express HLA. TCRm antibodies have the same binding properties as endogenous TCRs and recognize processed, HLA-bound peptides including intracellular tumor-associated antigens, viral oncoproteins, and cancer-testis antigen (CTA). TCRm antibodies bind peptide-HLA with high specificity and up to nanomolar affinity. Our optimized immunization protocols and high-throughput screening methods allow for one-step TCRm antibody generation. TCRm antibodies can also be used to assemble bispecific T cell engaging antibodies (BiTEs) to enhance tumor targeting of cytotoxic T cells. Biocytogen’s TCRm antibodies are a flexible and powerful tool for cancer immunotherapy. By enabling TCR-mediated recognition of an unrestricted repertoire of cancer neoantigens, TCRm antibodies may find broad clinical application. Citation Format: Jun Du, Taolin Liu, Wanbo Tang, Yue Zhang, Limin Zhao, Xin Jiao, Chao Sun, Pengfei Du, Yuqi Zhang, Baihong Liu, Qingcong Lin, Yi Yang. Targeting intracellular tumor antigens using fully human TCR mimic antibodies derived from HLA transgenic RenMiceTM [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 2752.

Posttranslational modifications induce autoantibodies with risk prediction capability in patients with small cell lung cancer.

Small cell lung cancer (SCLC) elicits the generation of autoantibodies that result in unique paraneoplastic neurological syndromes. The mechanistic basis for the formation of such autoantibodies is largely unknown but is key to understanding their etiology. We developed a high-dimensional technique that enables detection of autoantibodies in complex with native antigens directly from patient plasma. Here, we used our platform to screen 1009 human plasma samples for 3600 autoantibody-antigen complexes, finding that plasma from patients with SCLC harbors, on average, fourfold higher disease-specific autoantibody signals compared with plasma from patients with other cancers. Across three independent SCLC cohorts, we identified a set of common but previously unknown autoantibodies that are produced in response to both intracellular and extracellular tumor antigens. We further characterized several disease-specific posttranslational modifications within extracellular proteins targeted by these autoantibodies, including citrullination, isoaspartylation, and cancer-specific glycosylation. Because most patients with SCLC have metastatic disease at diagnosis, we queried whether these autoantibodies could be used for SCLC early detection. We created a risk prediction model using five autoantibodies with an average area under the curve of 0.84 for the three cohorts that improved to 0.96 by incorporating cigarette smoke consumption in pack years. Together, our findings provide an innovative approach to identify circulating autoantibodies in SCLC with mechanistic insight into disease-specific immunogenicity and clinical utility.

Nanobody-based CAR T cells targeting intracellular tumor antigens

Chimeric antigen receptor (CAR) T-cell immunotherapy has become one of the research hotspots in the treatment of malignant tumors nowadays. However, the available tumor surface antigens are limited in number. Most tumor-associated antigens are intracellular molecules that can’t be targeted by conventional CAR T cells. As the major histocompatibility complex (MHC)/peptide complex is a presentation form of intracellular proteins on the surface of tumor cells, here, we chose the Glypican-3 (GPC3) oncoprotein and Wilms tumor 1 (WT1) oncoprotein as examples to explore whether nanobody (Nb)-based T cell receptor (TCR)-like CAR T cells could kill tumor cells by targeting the MHC/peptide complexes. Using the immune nanobody phage display library, we developed human leukocyte antigen (HLA)-A2/GPC3- and HLA-A2/WT1-specific nanobodies for the first time and then incorporated these nanobodies in two TCR-like CARs, targeting HLA-A2/GPC3 and HLA-A2/WT1 respectively. These TCR-like Nb CAR-redirected T cells could selectively recognize and lyse MHC/peptide complex-expressing tumor cells in vitro assays and subcutaneous mouse tumor models. This study offers a possible strategy for targeting intracellular antigens and widening the application of CAR T-cell therapy.

USA EXPERIENCE: IN-HOUSE PREPARATION: PROSPECTS AND PROBLEMS

Adoptive Cellular Therapy (ACT) is transitioning from experimental to standard of care. Limited specificities of chimeric antigen receptor T cells (CAR-T) are licensed drugs, with commercial products selling for ∼400,000 USD. Tumor infiltrating lymphocytes (TIL) can target multiple cell surface and intracellular tumor antigens. TIL have not been commercialized, due to complex logistics and cost. Our objective is to leverage a single standardized platform (Miltenyi Prodigy) for in-house ACT. We currently manufacture CD19 CAR-T under an FDA IND and are developing a virtually identical TIL protocol minus the lentiviral vector. TIL are manufactured from malignant pleural effusions rich in immune infiltrates. CD4+ and CD8+ cells are enriched and activated with anti-CD3/CD28 beads. CAR-T are transduced, and both CAR-T and TIL are expanded (IL-7/IL-15) for 5 days. The product is infused fresh, avoiding losses associated with cryopreservation and thawing. Developing and validating release tests (absence of replication competent virus, vector copy number, chimeric antigen receptor expression, endotoxin testing) posed an initial challenge. Having completed assay development, our manufacturing process, including release testing, can be performed for less than 1/10 the cost of commercial CAR-T. We expect that the platform that we have validated will be easily transitioned to new chimeric antigen receptor designs and specificities and will likewise be adaptable to TIL manufacture for the wide variety of cancers that metastasize to the pleura of peritoneum. Standardized in-house ACT manufacture may greatly reduce the cost of cellular immunotherapies, making it more widely available to patients.

Therapeutic Potential of Intrabodies for Cancer Immunotherapy: Current Status and Future Directions.

Tumor cells are characterized by overexpressed tumor-associated antigens or mutated neoantigens, which are expressed on the cell surface or intracellularly. One strategy of cancer immunotherapy is to target cell-surface-expressed tumor-associated antigens (TAAs) with therapeutic antibodies. For targeting TAAs or neoantigens, adoptive T-cell therapies with activated autologous T cells from cancer patients transduced with novel recombinant TCRs or chimeric antigen receptors have been successfully applied. Many TAAs and most neoantigens are expressed in the cytoplasm or nucleus of tumor cells. As alternative to adoptive T-cell therapy, the mRNA of intracellular tumor antigens can be depleted by RNAi, the corresponding genes or proteins deleted by CRISPR-Cas or inactivated by kinase inhibitors or by intrabodies, respectively. Intrabodies are suitable to knockdown TAAs and neoantigens without off-target effects. RNA sequencing and proteome analysis of single tumor cells combined with computational methods is bringing forward the identification of new neoantigens for the selection of anti-cancer intrabodies, which can be easily performed using phage display antibody repertoires. For specifically delivering intrabodies into tumor cells, the usage of new capsid-modified adeno-associated viruses and lipid nanoparticles coupled with specific ligands to cell surface receptors can be used and might bring cancer intrabodies into the clinic.

Abstract 1084: FlexBodies: Targeting intracellular tumor antigens with T cell redirecting bispecific antibodies

Abstract 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.

Abstract 2750: Bioluminescent bioassays for the discovery and development of molecular and cellular T-cell redirecting cancer therapy

Abstract Redirected T cell therapies represent a new paradigm for cancer treatment. Two main approaches for T-cell-based therapies involve molecular T cell redirection by CD3 bispecific molecules such as bispecific T-cell engagers (BiTE) and cellular T cell redirection by genetic modification of T cells with chimeric antigen receptors (CAR) or transgenic T cell receptors (TCR). BiTEs redirect the cytotoxic activity of endogenous polyclonal T cells by simultaneously engaging CD3 on T cells and tumor antigens on target cells. Hitherto, BiTE potency studies have relied on primary cells, which measure target cell killing through redirected T cell cytotoxicity (RTCC) or cytokine release. However, these primary cell-based assays suffer from high donor-to-donor variability, as well as lengthy and hard to implement protocols. We have recently developed a new NanoBiT-based assay platform-based RTCC assay and cytokine immunoassays that are simple, sensitive and can quantitatively measure the potency of CD3 bispecific antibodies or BiTEs and similar biologics that induce T Cell-dependent target cell killing and cytokine production. In this assay, preactivated, Thaw-and-Use cytotoxic T cells and target cells stably expressing HaloTag-HiBiT are co-incubated with a BiTE, which results in lysis of the target cells and release of HiBiT proteins. These HiBiT proteins then bind to LgBiT in the detection reagent and form functional NanoLuc Luciferase to generate luminescence. The assay is homogenous, highly sensitive, and has a robust assay window. Furthermore, BiTE-induced cytokine production (e.g IL-2 and IFN gamma) from the activated cytotoxic T cells can be quantitatively measured in the homogenous NanoBiT Immunoassays. Use of CAR-T has demonstrated promising results in treating leukemia, while the development of TCR-engineered T cells that can recognize intracellular tumor antigens, is still in early stages. To facilitate the screening and characterization of new transgenic TCRs, we used CRISPR/Cas9 to develop two TCRab-null reporter T cell lines, which are CD4+ or CD8+. Reintroduction of peptide-specific TCR a and b chains into TCRab-null reporter T cell lines results in peptide-dependent TCR activation and luciferase reporter expression. The select expression of CD4 or CD8 in the TCRab-null reporter T cell lines can enable the development of transgenic TCRs for both MHCI- and MHCII-restricted tumor antigen targets. Together, these bioluminescent bioassays represent a new set of tools for the discovery and development of T cell-based immunotherapies. Citation Format: Julia Gilden, Jamison Grailer, Michael Slater, Pete Stecha, Jim Hartnett, Dan Lazar, Gopal Krishnan, Frank Fan, Mei Cong, Zhijie Jey Cheng. Bioluminescent bioassays for the discovery and development of molecular and cellular T-cell redirecting cancer therapy [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 2750.

A Sampling of Highlights from the Literature

T cells expressing WT137–45-specific TCRs kill AML cells (by BruceBlaus via Wikimedia Commons)T cells engineered to express T-cell receptors (TCR) targeting intracellular tumor antigens have potential for treating cancer. Two studies identify HLA-A*02:01–restricted TCRs specific for Wilms’ tumor antigen 1 (WT1) peptide WT137–45 as candidates for clinical translation. Ruggiero et al. show that T cells engineered to express such TCRs significantly reduce tumor growth in xenograft models of acute myeloid leukemia (AML), acute lymphoblastic leukemia, and glioblastoma. Lahman et al. show that generation of WT137–45 is immunoproteasome independent, which is important because AML recurrence in one patient they treated with T cells expressing an HLA-A*02:01–restricted, WT126–134-specific TCR occurred because of loss of immunoproteasome processing of WT1.Ruggiero E, …, Bonini C. Sci Trans Med 2022 February 9;14. DOI:10.1126/scitranslmed.abg8027.Lahman MC, …, Greenberg PD. Sci Trans Med 2022 February 9;14. DOI:10.1126/scitranslmed.abg8070.PERK signaling promotes M2 macrophage immunosuppressive function (by Noahtsmith via Wikimedia Commons)Intratumoral macrophage phenotype is determined by the signals the cells receive from the tumor microenvironment. Raines et al. identify new molecular pathways that promote the activation and immunosuppressive function of M2 macrophages. They find that tumor-associated macrophages from patients with lung carcinoma are characterized by increased activity of protein kinase RNA-like ER kinase (PERK) signaling. This promotes an immunosuppressive phenotype in macrophages through metabolic reprograming and histone demethylation. Inhibition of PERK signaling reduces growth of B16-F10 tumors. This is associated with reduced numbers and immunosuppressive activity of intratumoral macrophages, suggesting PERK signaling inhibition as a potential therapeutic approach to target macrophages.Raines LN, …, Huang SCC. Nature Immunol 2022;23:431–445.CRISPR technology allows for characterization of T-cell functional regulators (by Clipart Panda via Clipart Panda)Identifying regulators of cytokine production upon cellular activation has had its limitations. Using novel CRISPR screens, Schmidt et al. identify regulators of IL2 and IFNγ production by T cells. Distinct cytokine profiles are identified and characterize T cells during various states of activation. The data highlight screening that can be performed to determine how T cells can be reprogramed and could be used to better design immunotherapy for the treatment of cancer.Schmidt R, …, Marson A. Science 2022 February 4;375. DOI:10.1126/science.abj4008.Prediction modeling could be used to identify patients who will respond to immune checkpoint blockade (by Eva K. via Wikimedia Commons)Identifying markers of therapy response and patient outcome is an unmet clinical need. Patil et al. find that plasma cells in tumoral tertiary lymphoid structures are a biomarker of overall survival in patients treated with immune checkpoint blockade (ICB), an effect independent of tumor CD8+ T cells and PD-L1 expression. Asrir et al. identify tumor-associated high endothelial venules as key players for lymphocyte entry into tumors and associated with survival and response to combination ICB. These two studies highlight key immune mechanisms and biomarkers that could be potentially used to improve treatment strategies for patients with cancer.Asrir A, …, Girard JP. Cancer Cell 2022 March 14;40:318–334.e9.Patil NS, …, Shames DS. Cancer Cell 2022 March 14;40:289–300.e4.CAR T cells are being developed for many types of cancer (from Fig. 7B of Kantari-Mimoun et al., Cancer Immunol Res 2021)Patient data following chimeric-antigen receptor (CAR) T-cell therapy are critical to ongoing clinical development. Through longitudinal analysis of two patients with chronic lymphocytic leukemia who achieved lasting complete remission after CD19-specific CAR T-cell therapy in 2010, Melenhorst et al. find that CAR T cells persist for more than a decade, with a population of highly activated CD4+ CAR T cells dominating in both patients. Majzner et al. report that three of four patients with H3K27M-mutant diffuse midline glioma who received GD2-specific CAR T cells intravenously and then intracerebroventricularly had clinical and radiographic improvement. These studies provide new insights that will inform continued CAR T-cell development for hematological malignancies and solid tumors.Melenhorst JJ, …, June CH. Nature 2022;602:503–509.Majzner RG, …, Monje M. Nature 2022 February 7;14. DOI:10.1038/s41586-022-04489-4.The mycobiome facilitates pancreatic tumor progression (Pancreas, in Opera Omnia via Wellcome Collection)T helper 2 cells (TH2) and innate lymphoid cells 2 (ILC2) infiltrate pancreatic ductal adenocarcinomas (PDAC) and secrete cytokines that promote tumorigenesis. Alam et al. identify KrasG12D-regulated IL33 as a mediator of TH2 and ILC2 recruitment in mouse models of PDAC. Fungal species found in the gut and tumor microenvironment induce tumor cell IL33 secretion by activating the dectin-1 receptor and downstream Src–Syk–CARD9 pathway. Anti-fungal treatment reduces TH2 and ILC2 infiltration and reduces tumor growth, highlighting a novel mechanism by which the PDAC tumor mycobiome can regulate the pro-tumorigenic inflammatory response and suggesting a potential therapeutic strategy.Alam A, …, Dey P. Cancer Cell 2022 February 14;40:153–167.E11.

CRISPR-based gene disruption and integration of high-avidity, WT1-specific T cell receptors improve antitumor T cell function.

T cell receptor (TCR)-based therapy has the potential to induce durable clinical responses in patients with cancer by targeting intracellular tumor antigens with high sensitivity and by promoting T cell survival. However, the need for TCRs specific for shared oncogenic antigens and the need for manufacturing protocols able to redirect T cell specificity while preserving T cell fitness remain limiting factors. By longitudinal monitoring of T cell functionality and dynamics in 15 healthy donors, we isolated 19 TCRs specific for Wilms' tumor antigen 1 (WT1), which is overexpressed by several tumor types. TCRs recognized several peptides restricted by common human leukocyte antigen (HLA) alleles and displayed a wide range of functional avidities. We selected five high-avidity HLA-A*02:01-restricted TCRs, three that were specific to the less explored immunodominant WT137-45 and two that were specific to the noncanonical WT1-78-64 epitopes, both naturally processed by primary acute myeloid leukemia (AML) blasts. With CRISPR-Cas9 genome editing tools, we combined TCR-targeted integration into the TCR α constant (TRAC) locus with TCR β constant (TRBC) knockout, thus avoiding TCRαβ mispairing and maximizing TCR expression and function. The engineered lymphocytes were enriched in memory stem T cells. A unique WT137-45-specific TCR showed antigen-specific responses and efficiently killed AML blasts, acute lymphoblastic leukemia blasts, and glioblastoma cells in vitro and in vivo in the absence of off-tumor toxicity. T cells engineered to express this receptor are being advanced into clinical development for AML immunotherapy and represent a candidate therapy for other WT1-expressing tumors.

Irreversible Electroporation: An Emerging Immunomodulatory Therapy on Solid Tumors.

Irreversible electroporation (IRE), a novel non-thermal ablation technique, is utilized to ablate unresectable solid tumors and demonstrates favorable safety and efficacy in the clinic. IRE applies electric pulses to alter the cell transmembrane voltage and causes nanometer-sized membrane defects or pores in the cells, which leads to loss of cell homeostasis and ultimately results in cell death. The major drawbacks of IRE are incomplete ablation and susceptibility to recurrence, which limit its clinical application. Recent studies have shown that IRE promotes the massive release of intracellular concealed tumor antigens that become an “in-situ tumor vaccine,” inducing a potential antitumor immune response to kill residual tumor cells after ablation and inhibiting local recurrence and distant metastasis. Therefore, IRE can be regarded as a potential immunomodulatory therapy, and combined with immunotherapy, it can exhibit synergistic treatment effects on malignant tumors, which provides broad application prospects for tumor treatment. This work reviewed the current status of the clinical efficacy of IRE in tumor treatment, summarized the characteristics of local and systemic immune responses induced by IRE in tumor-bearing organisms, and analyzed the specific mechanisms of the IRE-induced immune response. Moreover, we reviewed the current research progress of IRE combined with immunotherapy in the treatment of solid tumors. Based on the findings, we present deficiencies of current preclinical studies of animal models and analyze possible reasons and solutions. We also propose possible demands for clinical research. This review aimed to provide theoretical and practical guidance for the combination of IRE with immunotherapy in the treatment of malignant tumors.

Intravital molecular imaging reveals that ROS-caspase-3-GSDME-induced cell punching enhances humoral immunotherapy targeting intracellular tumor antigens.

Tumor antigens (TAs)-induced humoral immune responses or TAs-specific antibodies have great application prospects for tumor therapy. However, more than half of TAs are intracellular antigens (intra-Ags) that are hardly recognized by antibodies. It is worthy to develop immunotherapeutic strategies for targeting intra-Ags. Methods: We used the far-red fluorescent protein tfRFP as an intracellular antigen to immunize mice and generated a liver metastasis model by injecting tfRFP-expressing B16 melanoma cells (tfRFP-B16) via the spleen. Intravital molecular imaging and atomic force microscopy were performed to visualize the formation of tfRFP antigen-antibody complexes (also known as immune complexes) and punched holes in cell membranes. Results: The results showed that the tfRFP-elicited immune responses inhibited the metastasis of tfRFP-expressing melanoma cells in the liver. In the circulating tfRFP-B16 tumor cells, elevated reactive oxygen species (ROS) induced slight caspase-3 activation, a probable key factor in the cleavage of gasdermin E (GSDME) proteins and punching of holes in the tumor cell membrane. Increased tumor cell membrane permeability led to the release of intra-Ag tfRFP and binding with anti-tfRFP antibodies. The formation of tfRFP antigen-antibody complexes on the membranes of tfRFP-B16 cells activated complement components to form membrane attack complexes to further destroy the cell membrane. Neutrophils were rapidly recruited, and F4/80+ macrophages phagocytized the dying tumor cells. Conclusion: The process of circulating tumor cell elimination in the tfRFP-immunized mice was triggered through the ROS-caspase-3-GSDME pathway to form intra-Ag-antibody immune complexes, which were involved in the activation of the complement system, as well as the recruitment of neutrophils and F4/80+ macrophages. An intra-Ag-elicited humoral immune response is a potent strategy for eliminating liver metastasis, which is unaffected by the liver immune tolerogenic status.

Abstract 1658: Gemcitabine impacts expression of antigen presentation proteins by pancreatic cancer cells

Abstract Background: Pancreatic adenocarcinoma is one of the deadliest type of cancers with a 5-year overall survival rate of 10%. A standard chemotherapy approach for pancreatic cancer is treatment with gemcitabine. Gemcitabine is also considered a potential immunomodulatory agent, capable of increasing human leukocyte antigen (HLA) class I. HLA class I is a heterodimer of a highly polymorphic alpha heavy chain (HLA-A, -B, or -C) and the light chain beta-2-microglobulin (β2m) that is loaded with an endogenous antigen in the endoplasmic reticulum. The pool of antigens available for HLA class I loading is selected by the transporter associated with antigen processing protein (TAP). However, HLA class I peptide loading can occur independently of TAP, causing the HLA class I molecules to present a different antigen pool to cytotoxic T cells. Because many cancers downregulate HLA class I and prevent intracellular tumor antigens from being presented to cytotoxic T cells, understanding more about gemcitabine's capability to increase HLA class I expression is of great importance. Methods: Alterations in HLA class I and TAP protein levels were monitored via western blot analysis in a pancreatic cancer cell line (S2-013) in both a time and dose-dependent manner. The time points and dosages which solicited the largest increase in HLA class I expression were also examined by flow cytometry. Results: Administration of gemcitabine to S2-013 pancreatic cancer cells increased total HLA class I α heavy chain protein, with maximal increases of 141% (HLA-A) and 323% HLA-B/C. Flow cytometry revealed a 3-fold increase in HLA-A2 surface expression post-gemcitabine treatment and a maximal 2-fold increase in HLA-B/C expression at the corresponding gemcitabine concentration, but differing time point. Both western blot and flow cytometry analyses indicate HLA-B/C generally has its largest increase in expression at an earlier time point than HLA-A. In addition, high concentrations of gemcitabine that promoted HLA-A,B,C surface expression were associated with a large decrease in TAP total protein. Conclusion: In summary, gemcitabine demonstrates an ability to stimulate expression of HLA class I in pancreatic cancer cells, with the rate dependent on the HLA class I allele. Because gemcitabine treatment increases HLA class I surface expression with a simultaneous decrease in TAP expression, our findings indicate that gemcitabine may induce TAP-independent peptide loading of HLA class I. These results suggest the potential for gemcitabine treatment as a priming mechanism to enhance immunotherapy efficacy and potentially alter vaccination strategies in pancreatic cancer. Citation Format: Alaina C. Larson, Shelby M. Knoche, Joyce C. Solheim. Gemcitabine impacts expression of antigen presentation proteins by pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1658.

Abstract 334: Novel bioluminescent bioassays for the discovery and development of molecular and cellular T cell-redirecting cancer therapy

Abstract Redirected T cell therapies represent a new paradigm for cancer treatment. Two main approaches for T-cell-based therapies involve molecular T cell redirection by CD3 bispecific molecules such as bispecific T-cell engagers (BiTE) and cellular T cell redirection by genetic modification of T cells with chimeric antigen receptors (CAR) or transgenic T cell receptors (TCR). BiTEs redirect the cytotoxic activity of endogenous polyclonal T cells by simultaneously engaging CD3 on T cells and tumor antigens on target cells. Hitherto, BiTE potency studies have relied on primary cells, which measure target cell killing through redirected T cell cytotoxicity (RTCC) or cytokine release. However, these primary cell-based assays suffer from high donor-to-donor variability, as well as lengthy and hard to implement protocols. We have recently developed a new RTCC assay and cytokine immunoassays that are simple, sensitive and can quantitatively measure the potency of BiTEs and similar biologics. In this assay, preactivated, Thaw-and-Use cytotoxic T cells and target cells stably expressing HaloTag-HiBiT are co-incubated with a BiTE, which results in lysis of the target cells and release of HiBiT proteins. These HiBiT proteins then bind to LgBiT in the detection reagent and form functional NanoLuc Luciferase to generate luminescence. The assay is homogenous, highly sensitive, and has a robust assay window. Furthermore, BiTE-induced cytokine production (e.g IL-2 and IFN-γ) from the activated cytotoxic T cells can be quantitatively measured in the homogenous NanoBiT Immunoassays. Use of CAR-T has demonstrated promising results in treating leukemia, while the development of TCR-engineered T cells that can recognize intracellular tumor antigens, is still in early stages. To facilitate the screening and characterization of new transgenic TCRs, we used CRISPR/Cas9 to develop two TCRαβ-null reporter T cell lines, which are CD4+ or CD8+. Reintroduction of peptide-specific TCR α and β chains into TCRαβ-null reporter T cell lines results in peptide-dependent TCR activation and luciferase reporter expression. The select expression of CD4 or CD8 in the TCRαβ-null reporter T cell lines can enable the development of transgenic TCRs for both MHCI- and MHCII-restricted tumor antigen targets. Together, these bioluminescent bioassays represent a new set of tools for the discovery and development of T cell-based immunotherapies. Citation Format: Julia Gilden, Jamison Grailer, Michael Slater, Pete Stecha, Jim Hartnett, Dan Lazar, Vanessa Ott, Frank Fan, Mei Cong, Zhijie Jey Cheng. Novel bioluminescent bioassays for the discovery and development of molecular and cellular T cell-redirecting cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 334.

Abstract 2179: Novel bioluminescent bioassays for the discovery and development of molecular and cellular T cell redirecting cancer therapy

Abstract Redirected T cell therapies represent a new paradigm for cancer treatment. Two main approaches for T-cell-based therapies involve molecular T cell redirection by CD3 bispecific molecules such as bispecific T-cell engagers (BiTE) and cellular T cell redirection by genetic modification of T cells with chimeric antigen receptors (CAR) or transgenic T cell receptors (TCR). BiTEs redirect the cytotoxic activity of endogenous polyclonal T cells by simultaneously engaging CD3 on T cells and tumor antigens on target cells. Hitherto, BiTE potency studies have relied on primary cells, which measure target cell killing through redirected T cell cytotoxicity (RTCC) or cytokine release. However, these primary cell-based assays suffer from high donor-to-donor variability, as well as lengthy and hard to implement protocols. We have recently developed a new RTCC assay and cytokine immunoassays that are simple, sensitive and can quantitatively measure the potency of BiTEs and similar biologics. In this assay, preactivated, Thaw-and-Use cytotoxic T cells and target cells stably expressing HaloTag-HiBiT are co-incubated with a BiTE, which results in lysis of the target cells and release of HiBiT proteins. These HiBiT proteins then bind to LgBiT in the detection reagent and form functional NanoLuc Luciferase to generate luminescence. The assay is homogenous, highly sensitive, and has a robust assay window. Furthermore, BiTE-induced cytokine production (e.g IL-2 and IFN-γ) from the activated cytotoxic T cells can be quantitatively measured in the homogenous NanoBiT Immunoassays. Use of CAR-T has demonstrated promising results in treating leukemia, while the development of TCR-engineered T cells that can recognize intracellular tumor antigens, is still in early stages. To facilitate the screening and characterization of new transgenic TCRs, we used CRISPR/Cas9 to develop two TCRαβ-null reporter T cell lines, which are CD4+ or CD8+. Reintroduction of peptide-specific TCR α and β chains into TCRαβ-null reporter T cell lines results in peptide-dependent TCR activation and luciferase reporter expression. The select expression of CD4 or CD8 in the TCRαβ-null reporter T cell lines can enable the development of transgenic TCRs for both MHCI- and MHCII-restricted tumor antigen targets. Together, these bioluminescent bioassays represent a new set of tools for the discovery and development of T cell-based immunotherapies. Citation Format: Julia K. Gilden, Jamison Grailer, Michael Slater, Pete Stecha, Jim Hartnett, Dan Lazar, Frank Fan, Mei Cong, Zhijie Jey Cheng. Novel bioluminescent bioassays for the discovery and development of molecular and cellular T cell redirecting cancer therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2179.

Bi- and Tri-Specific T Cell Engager-Armed Oncolytic Viruses: Next-Generation Cancer Immunotherapy.

Oncolytic viruses (OVs) are potent anti-cancer biologics with a bright future, having substantial evidence of efficacy in patients with cancer. Bi- and tri-specific antibodies targeting tumor antigens and capable of activating T cell receptor signaling have also shown great promise in cancer immunotherapy. In a cutting-edge strategy, investigators have incorporated the two independent anti-cancer modalities, transforming them into bi- or tri-specific T cell engager (BiTE or TriTE)-armed OVs for targeted immunotherapy. Since 2014, multiple research teams have studied this combinatorial strategy, and it showed substantial efficacy in various tumor models. Here, we first provide a brief overview of the current status of oncolytic virotherapy and the use of multi-specific antibodies for cancer immunotherapy. We then summarize progress on BiTE and TriTE antibodies as a novel class of cancer therapeutics in preclinical and clinical studies, followed by a discussion of BiTE- or TriTE-armed OVs for cancer therapy in translational models. In addition, T cell receptor mimics (TCRm) have been developed into BiTEs and are expected to greatly expand the application of BiTEs and BiTE-armed OVs for the effective targeting of intracellular tumor antigens. Future applications of such innovative combination strategies are emerging as precision cancer immunotherapies.

Targeting Intracellular WT1 in AML Utilizing a T Cell Bispecific Antibody Construct: Augmenting Efficacy through Combination with Lenalidomide

Antibody-based immunotherapy represents a promising strategy to target chemo-resistant leukemic cells. However, current antibody-based approaches are restricted to cell lineage surface antigens. Targeting intracellular antigens enables to enlarge the number of suitable tumor-associated target antigens with a more restricted expression profile. In this study we evaluated a 2+1 T Cell Bispecific (TCB) antibody for immunotherapy of acute myeloid leukemia (AML). The T cell receptor (TCR)-like TCB targets the intracellular tumor antigen Wilms tumor 1 (WT1) by bivalent recognition of the peptide RMFPNAPYL in the context of human leukocyte antigen allele A*02 (HLA-A2). Complementary binding to CD3ε recruits T cells irrespective of their TCR-specificity. We further analyzed enhancement of TCB-mediated T cell cytotoxicity through combination with the immune-modulatory drug lenalidomide. WT1 expression levels in cancer cell lines and primary AML patient samples at different time points during course of the disease were determined by quantitative real-time PCR, western blot and immunohistochemical staining. WT1-TCB-mediated cytotoxicity was analyzed by co-cultivation of WT1-expressing HLA-A2+ cancer cell lines with T cells from healthy donors. Specific lysis was assessed by flow cytometry. TCR downstream signaling was measured by co-cultivation of primary AML cells with NFAT Luciferase Reporter Jurkat cells. WT1-TCB-mediated cytotoxicity against primary AML cells and combination with 10 μM lenalidomide was evaluated in our pre-established feeder layer-based ex vivo long-term culture system. For in vivo testing, NSG mice (NOD.Cg-Prkdcscid-Il2rgtm1Wjl/SzJ) were humanized with human HLA-A2+ CD34+ cord blood cells. After successful engraftment and development of human T cells, WT1-expressing HLA-A2+ SKM-1 tumor cells were subcutaneously inoculated followed by weekly administration of the WT1-TCB. In accordance with previous reports, we observed WT1 expression in 79% (n=38) of cancer cell lines and in 92% (n=65) of AML patient samples at the time of initial diagnosis. Moreover, WT1 expression levels correlated with the percentage of AML blasts: no significant WT1 expression was observed at time of CR (n=26), whereas WT1 was expressed again at time of relapse (n=21). WT1-TCBs elicited antibody-mediated T cell cytotoxicity against peptide-pulsed T2 cells and AML cell lines in a WT1 and HLA-restricted manner. Equally, TCR downstream signaling was observed in a WT1-restrictive manner by co-cultivation of primary AML cells with NFAT Luciferase Reporter Jurkat cells. WT1-TCBs further mediated specific lysis of primary AML cells upon addition of allogenic T cells from healthy donors (mean specific lysis: 67±6% after 13-14 days; ±SEM; n=18). Correspondingly, up-regulation of T cell activation and surrogate exhaustion markers was observed (MFI fold change CD69: 9.3±1.5, PD-1: 5.1±0.7, TIM-3: 4.7±0.6; ±SEM; n=22). WT1-TCBs also mediated killing of primary AML cells in an autologous setting (mean specific lysis: 38±13% after 13-14 days; ±SEM; n=5). In comparison with WT1RMF-specific T cells, only bivalent binding by WT1-TCB induced efficient lysis of primary AML cells. Interestingly, combination of WT1-TCB with lenalidomide further enhanced antibody-mediated T-cell cytotoxicity against primary AML cells (mean specific lysis on day 3-4: 32±10% vs 59±9%; p=0.0017; ±SEM; n=13). This was accompanied by an increased secretion of the proinflammatory cytokines IL-2, IFN-γ and TNF-α and promoted the differentiation of naïve T cells towards a memory phenotype characterized by a downregulation of CD45RA. Furthermore, WT1-TCB-treated humanized mice bearing SKM-1 tumors showed a dose dependent and significant reduction in tumor growth resulting in tumor control. TCR-like TCBs targeting intracellular tumor antigens are a promising tool for cancer immunotherapy. Notably, the 2+1 TCB molecular format for bivalent binding facilitates potent in vitro, ex vivo and in vivo killing of AML cell lines and primary AML samples which present low numbers of the RMF peptide-MHC complex on the cell surface validating WT1-TCB as a promising therapeutic agent for the treatment of AML. Our results further indicate that the combinatorial approach with lenalidomide leads to increased TCB-mediated T cell cytotoxicity. Disclosures Klein: Roche: Employment, Equity Ownership, Patents & Royalties. Xu:Roche: Employment, Equity Ownership, Patents & Royalties. Heitmüller:Roche: Employment. Hanisch:Roche: Employment, Equity Ownership, Patents & Royalties. Sam:Roche: Employment, Equity Ownership, Patents & Royalties. Pulko:Roche: Employment, Equity Ownership, Patents & Royalties. Schönle:Roche: Employment, Equity Ownership, Patents & Royalties. Challier:Roche: Employment, Equity Ownership, Patents & Royalties. Carpy:Roche: Employment, Equity Ownership, Patents & Royalties. Lichtenegger:Roche: Employment. Umana:Roche: Employment, Equity Ownership, Patents & Royalties. Subklewe:Roche: Consultancy, Research Funding; Miltenyi: Research Funding; Oxford Biotherapeutics: Research Funding; Morphosys: Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; AMGEN: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Janssen: Consultancy.