Multiple Tumor-associated Antigens Research Articles

Light-Controllable Binary Switch Activation of CAR T Cells.

Major challenges to chimeric antigen receptor (CAR) T cell therapies include uncontrolled immune activity, off‐tumor toxicities and tumor heterogeneity. To overcome these challenges, we engineered CARs directed against small molecules. By conjugating the same small molecule to distinct tumor‐targeting antibodies, we show that small molecule specific‐CAR T cells can be redirected to different tumor antigens. Such binary switches allow control over the degree of CAR T cell activity and enables simultaneous targeting of multiple tumor‐associated antigens. We also demonstrate that ultraviolet light‐sensitive caging of small molecules blocks CAR T cell activation. Exposure to ultraviolet light, uncaged small molecules and restored CAR T cell‐mediated killing. Together, our data demonstrate that a light‐sensitive caging system enables an additional level of control over tumor cell killing, which could improve the therapeutic index of CAR T cell therapies.

Abstract 1737: Understanding the geometry and valency of bispecific antibodies in the optimization of tumor-dependent activation of 4-1BB

Abstract 4-1BB is a TNF family receptor expressed on the surface of tumor-infiltrating T cells. Activation of 4-1BB enhances the activation, metabolism and function of tumor-infiltrating T cells and promotes tumor regression. Several anti-4-1BB antibodies have entered the clinic and have suffered from liver toxicity or lack of activity. To address the clinical liabilities with 4-1BB targeting, we designed bispecific 4-1BB x tumor associated antigen (TAA) antibodies to selectively activate T cells within the tumor microenvironment. To understand the impact of antibody format on potency of T cell activation, we generated a panel of 4-1BBxTAA bispecific antibodies in different formats with the AzymetricTM and EFECTTM platforms. We compared potency and maximal activity of these constructs in co-culture assays with tumor cells and T or Jurkat reporter cells, in comparison to urelumab as a clinical benchmark. The AzymetricTM platform allowed the production and evaluation of unique antibody formats from which an extensive structure activity relationship analysis was performed. We also examined the accessible epitope space by sampling the possible paratope binding conformations of the molecule. From this we determined the geometric limits of antigen engagement. Bispecific antibody constructs utilizing monovalent 4-1BB targeting showed inferior activity than urelumab in co-culture assays with TAAhi tumor cells. Activity greater than urelumab was seen with constructs containing two 4-1BB arms with one or two TAA binding scFv on the C-terminus of the Fc. Bivalent 4-1BB antibodies with an anti-TAA scFv fused to the N-terminus of the 4-1BB Fab showed decreased activity, potentially due to reduced ability to engage 4-1BB in a bivalent manner. Critically, the activity seen was transferable across multiple TAAs. The activity of the 4-1BB bispecific constructs was dependent on the expression level of the TAA. Without the presence of tumor cells, or with constructs where the TAA scFv was replaced by an irrelevant scFv, minimal activity was seen. The modelling of the space accessible to the 4-1BB paratopes showed that the constructs with which we saw the most activity had a distance between 4-1BB and TAA binding paratopes similar to that seen between T and APC membranes as part of an immune synapse, suggesting that this may be a key characteristic for the development of conditional T cell agonist bispecifics. This work represents an investigation of 4-1BBxTAA formats which would be difficult to make without an efficient bispecific technology. We identified a series of 4-1BBxTAA bispecific agonist antibody formats which were transferable between multiple TAAs. These formats also allowed optimization of activity and selectivity to promote maximal therapeutic index and efficacy, key factors which are potentially able to contribute to improved clinical outcomes. Citation Format: Anna von Rossum, Harsh Pratap, Lisa Newhook, Elizabeth C. Halvorsen, Lee Freiburger, Renee Duan, Charles Stevens, Dimitri Tcaciuc, Gesa Volkers, Duncan Browman, Brandon Clavette, David Mills, Thomas Spreter, Daniel T. Patton. Understanding the geometry and valency of bispecific antibodies in the optimization of tumor-dependent activation of 4-1BB [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 1737.

Abstract 2712: Donor selection strategies to optimize humanized mouse models for the evaluation of T cell engagers

Abstract The success of early cancer immunotherapies has led to the development of novel therapeutic approaches including multi-specific antibodies engaging innate and adaptive effector cells. To evaluate the antitumor potency of these multi-specific cell engagers, we have established a protocol to select human PBMCs from donors of a commercial frozen source by their potency to produce inflammatory cytokines and the capacity to engraft into NOD-Scid IL2Rgammanull (NSG)mice. The selected human PBMCs were used to reconstitute NSG mice, or as donors of effector cells that upon in vitro expansion were adoptively transferred to NSG mice for humanization. We have employed these humanization strategies to evaluate T cell engagers, which are typically multi-specific antibodies directed against the T cell and one or multiple tumor-associated antigen (TAA), whose therapeutic strategy is to 1) engage T cells, 2) activate the T cells and 3) engage tumor cells, and thus induce tumor cell killing. We will present representative data from several humanization approaches using liquid and solid orthotopic tumor models to assess the effectiveness of multi-specific T cell engagers in mediating antitumor responses. Thus, pre-clinical evaluations rely on the development of optimized tumor bearing humanized models that mirror key properties of a human setting, to assess the therapeutic potential of anti-tumor multi-specific biologics. Citation Format: Liqing Cheng, Jane Chen, Qunyan Yu, Natalia Malkova, Virna F. Cortez-Retamozo, Fangxian Sun, Sukhvinder Sidhu. Donor selection strategies to optimize humanized mouse models for the evaluation of T cell engagers [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 2712.

Abstract 525: Novel DARPin multi-specific T-cell engager with an improved therapeutic window to overcome dose limiting toxicities in AML therapies

Abstract Purpose: The medical need due to high mortality in acute myeloid leukemia (AML) remains high, and the treatment of relapsed or refractory AML continues to be therapeutically challenging. MYLOTARG, the only approved anti-CD33 antibody drug conjugate (ADC), has provided proof-of-concept for targeted immunotherapies in AML. Currently, a plethora of ADCs and T-cell engager (TCE) therapies have entered clinical development in AML, but those therapies are often accompanied by dose limiting toxicities, preventing dose escalation to desired anti-tumor efficacy. The biggest challenges seem to be limited target specificity and hyperstimulation of the immune system leading to e.g. myelotoxicities and cytokine release syndrome, respectively. Therefore, more selective therapies are needed to allow for robust anti-tumor activity with a more acceptable safety profile. Experimental design: To address the selectivity challenge, we have generated multi-specific T-cell engaging DARPin® molecules, targeting two different tumor associated antigens (TAAs) with optimized affinity for their targets. In order to find the right target combination, the optimal affinity to increase tumor specificity via avidity, as well as the best molecular architecture, we took advantage of our unique modular DARPin® platform and screened 1000s of combinations of multi-specific DARPin® molecules, binding simultaneously to multiple TAAs in conjunction with our CD3-binding DARPin® molecule. Results: We constructed multi-specific TCEs targeting two different AML antigens with optimized affinity leading to a substantial avidity gain when both targets are co-expressed on tumor cells. The avidity gain resulted in strongly enhanced in vitro potency as shown by activation of both CD8+ and CD4+ T cells and subsequent killing of AML tumor cells, with bioactivities in the range of established TCE benchmark formats (e.g. BiTE® and DART®). In contrast, in an ex vivo whole blood assay the multi-specific DARPin® constructs induced profoundly less cytokine release as compared to benchmark molecules indicating an improved therapeutic window. Finally, we also demonstrated tumor regression in PMBC humanized mouse models bearing MOLM-13 tumors, using both half-life extended (HLE) and non-HLE lead constructs. In conclusion, we have generated TCEs based on multi-specific DARPin® constructs with high potency, selectivity and ultimately with the potential for an improved therapeutic window for the treatment of AML. Citation Format: Nina Reschke, Thamar Looser, Jennifer Krieg, Matteo Bianchi, Patricia Schildknecht, Nicole Bassler, Yvonne Gruebler, Sebastian Grimm, Laura Jeanbart, Tanja Hospodarsch, Alexandra Neculcea, Daniel Steiner, Bernd Schlereth, Christian Reichen. Novel DARPin multi-specific T-cell engager with an improved therapeutic window to overcome dose limiting toxicities in AML therapies [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 525.

Allogeneic Vδ1 gamma delta T cells engineered with glypican-3 (GPC3)-specific CAR expressing soluble IL-15 have enhanced antitumor efficacy against hepatocellular carcinoma in preclinical models.

e14511 Background: Autologous αβ chimeric antigen receptor (CAR) T cell therapy has shown promising clinical results in hematologic malignancies but limited success in solid tumors. Allogeneic αβ T cell therapy may overcome several challenges faced by autologous therapy but carries the risk of graft-versus-host disease (GvHD) and does not readily recognize multiple tumor-associated antigens. Gamma delta (γδ) T cells are highly cytolytic effectors that can recognize and kill tumor cells in an MHC-unrestricted manner without causing GvHD. The Vδ1 subset is preferentially localized in peripheral tissue and is critical for tumor immunosurveillance. Engineering Vδ1 T cells with CARs can further enhance antitumor activity and represents an attractive and safe approach to treating solid tumors. However, their clinical use has been hindered by the limited number of circulating Vδ1 T cells. Here, we describe the development of the first allogeneic Vδ1 T cells that have been expanded from healthy donor PBMCs and genetically modified to secrete IL-15 (sIL15) and express a CAR targeting glypican-3 (GPC3), a rational target for hepatocellular carcinoma (HCC). Methods: Vδ1 T cells in healthy donor PBMCs were activated by a Vδ1-specific monoclonal antibody and transduced with 41BBζ or 41BBζ-sIL15 GPC3-CARs prior to cell expansion, αβ T cell depletion and cryopreservation. In vitro characterization included: 1) co-culture assays with GPC3-expressing HCC targets HepG2 and PLC/PRF/5, 2) phenotypic analysis by flow cytometry, and 3) cytokine production by multiplexed immunoassay. For in vivo assessment of tumor control, immunodeficient NSG mice were subcutaneously injected with HepG2 cells and treated with a single dose of 41BBζ or 41BBζ-sIL15 GPC3-CAR Vδ1 T cells. Additionally, tissues were harvested 7 days post transfer and analyzed by flow cytometry for Vδ1 T cell tissue homing and proliferation, or at end of study and analyzed for GvHD by immunohistochemistry. Results: Vδ1 T cells expanded over 10,000-fold and routinely reached >80% purity. Expanded Vδ1 T cells showed a primarily naïve-like phenotype (CD45RA+CD27+) with minimal exhaustion receptor expression and displayed robust proliferation, cytokine production, and cytotoxic activity against HCC cell lines expressing low and high GPC3 levels in vitro. In a HepG2 mouse model, GPC3-CAR Vδ1 T cells primarily accumulated and proliferated in the tumor, and a single dose was able to efficiently control tumor burden without causing GvHD. Importantly, 41BBζ-sIL15 GPC3-CAR Vδ1 cells displayed enhanced tumor-specific proliferation that resulted in better tumor control without any toxicity. Conclusions: Our results show that expanded Vδ1 T cells engineered with GPC3-CAR and sIL-15 represent a promising platform for safe and effective off-the-shelf treatment of HCC.

Fully human antibody VH domains to generate mono and bispecific CAR to target solid tumors

BackgroundChimeric antigen receptor (CAR) T cells are effective in B-cell malignancies. However, heterogeneous antigen expression and antigen loss remain important limitations of targeted immunotherapy in solid tumors. Therefore, targeting multiple...

A Panel of Tumor-associated Autoantibodies for the Detection of Early-stage Breast Cancer.

We previously found a panel of autoantibodies against multiple tumor-associated antigens (BMI-1, HSP70, MMP-7, NY-ESO-1, p53 and PRDX6) that might facilitate early detection of esophagogastric junction adenocarcinoma and esophageal squamous cell carcinoma. Here we aimed at assessing the diagnostic performance of these autoantibodies in breast cancer patients. Enzyme-linked immunosorbent assay was applied to detect sera autoantibodies in 123 breast cancer patients and 123 age-matched normal controls. We adopted logistic regression analysis to identify optimized autoantibody biomarkers for diagnosis and receiver-operating characteristics to analyze diagnostic efficiency. Five of six autoantibodies, BMI-1, HSP70, NY-ESO-1, p53 and PRDX6 demonstrated significantly elevated serum levels in breast cancer compared to normal controls. An optimized panel composed of autoantibodies to BMI-1, HSP70, NY-ESO-1 and p53 showed an area under the curve (AUC) of 0.819 (95% CI 0.766-0.873), 63.4% sensitivity and 90.2% specificity for diagnosing breast cancer. Moreover, this autoantibody panel could differentiate patients with early stage breast cancer from normal controls, with AUC of 0.805 (95% CI 0.743-0.886), 59.6% sensitivity and 90.2% specificity. Our findings indicated that the panel of autoantibodies to BMI-1, HSP70, NY-ESO-1 and p53 as serum biomarkers have the potential to help detect early stage breast cancer.

Mechanism of EBV inducing anti-tumour immunity and its therapeutic use.

SummaryTumour-associated antigens (TAAs) comprise a large collection of non-mutated cellular antigens recognized by T cells in human and murine cancers. Their potential as immunotherapy targets has been explored for over two decades1, yet the genesis of TAA-specific T cells remains elusive. While tumour cells may be an important source of TAAs for T cell priming2, several recent studies suggest that infection with some viruses including Epstein-Barr virus (EBV) and influenza virus can elicit T cell responses against abnormally expressed cellular antigens that function as TAAs3,4. However, the cellular and molecular basis of such responses remains undefined. Here, we show that expression of the EBV signaling protein LMP1 in B cells provokes T cell responses to multiple TAAs. LMP1 signaling leads to overexpression of many cellular antigens previously shown to be TAAs, their presentation on MHC-I and -II (mainly through the endogenous pathway), and the upregulation of costimulatory ligands CD70 and OX40L, thereby inducing potent cytotoxic CD4+ and CD8+ T cell responses. These findings delineate a novel mechanism of infection-induced anti-tumour immunity. Furthermore, by ectopically expressing LMP1 in patient tumour B cells and thereby empowering them to prime T cells, we develop a general approach for rapid production of autologous cytotoxic CD4+ T cells against a broad array of endogenous tumour antigens, such as TAAs and neoantigens, for treating B-cell malignancies. This work stresses the need to revisit classical concepts concerning viral and tumour immunity, which will be critical to fully understand the impact of common infections on human health and to improve the rational design of immune approaches for cancers.

FT576: Multi-Specific Off-the-Shelf CAR-NK Cell Therapy Engineered for Enhanced Persistence, Avoidance of Self-Fratricide and Optimized Mab Combination Therapy to Prevent Antigenic Escape and Elicit a Deep and Durable Response in Multiple Myeloma

Multiple redundancy within the spectrum of an immune response is required to prevent antigen escape or adaptation of the targeted population to host defenses. As adoptive cell therapies continue to evolve, multi-modal engineering of effector cells offers the prospect of tackling increasingly complex disease settings such as multiple myeloma (MM), where targeting of a single tumor associated antigen is frequently confounded by antigen shedding and escape variation resulting in the inability to develop a curative therapy. There are multiple advantages in expanding treatment options beyond autologous primary T and NK cells, including the use of induced pluripotent stem cells (iPSC) to derive effector cells that can be uniformly manufactured at scale from renewable starting cellular material and where precision genetic engineering can be achieved at the clonal level which can be applied sequentially in order to build multiple specificities and functional modalities. To create a platform targeted toward MM, a multiplexed edited base iPSC-derived NK (iNK) cell configuration consisting of a CD38 KO iPSC modified to overexpress a recombinant IL-15 signaling complex (IL15RF) for autonomous persistence and a functionally enhanced high-affinity, non-cleavable CD16 (hnCD16) was developed. Introduction of IL15RF enabled expansion of iNK cells without additional exogenous cytokine support during the manufacturing process and greatly improved functional persistence of iNK cells both in vitro and in various xenograft mouse models (Figure 1). To target MM in a broad and comprehensive manner, we tested our novel BCMA-CAR in combination with different myeloma targeted antibodies. In combination with hnCD16, co-expression of BCMA-CAR and IL15RF culminates in an iNK cell therapeutic, termed FT576, capable of multiantigen-specificity through combinatorial use of CAR and hnCD16 with monoclonal antibodies to tackle antigen escape. Chimerization of an anti-BCMA scFv shown to elicit higher affinity onto the CAR platform produced specific in vitro recognition of BCMA+ myeloma cells in short-term and long-term NK cell cytotoxicity assays. Specificity of the BCMA-CAR was demonstrated using NALM6 overexpressing BCMA using a short range 4H caspase assay (NALM6_BCMA EC50 14.4, NALM6wt EC50 39.1, p*<0.0001). Utilizing a long range clearance assay, serial restimulation by repeated rounds of exposure to fresh MM1S MM target cells was tested, showing remarkable persistence and antigen-mediated expansion of CAR function in isolation or combined with antibody through 3 rounds of stimulation in the absence of exogenous cytokine support (Figure 2). Continuous long-range clearance assays demonstrated levels of BCMA targeting activity of FT576 alone was equivalent to primary BCMA-targeted CAR-T cells against a panel of BCMA+ target cells. Utilizing hnCD16, BCMA-CAR was tested in combination with anti-CD38 (daratumumab), anti-SLAMF7 (elotuzumab), or anti-CD19, showing synergistic increase in tumor targeting through various tumor associated antigens (TAAs). Polyfunctionality of FT576 stimulated either through CAR or ADCC was similarly measured by both Isoplexis and single cell RNA sequencing. Specificity for plasma cells was confirmed using primary bone marrow samples from either healthy donors or patients. In animal models, as a monotherapy, FT576 achieved sustained tumor control against disseminated MM1s with persistence profile suggestive of antigen mediated expansion (Figure 3). In combination with daratumumab, FT576 was able to achieve complete clearance of MM1S. Combination with other monoclonal antibodies displayed a similar response demonstrating the unique ability of FT576 to be directed to target multiple TAAs. Together, these studies demonstrate the versatility of FT576 as a highly effective multi-antigen targeting and cost-effective off-the-shelf BCMA-CAR iNK cell product and supports the rational for a first-of-kind Phase I Study as a monotherapy or in combination with therapeutic mAbs targeted to MM-associated surface antigens, driving a path towards a curative therapeutic in MM. Disclosures Goodridge: Fate Therapeutics, Inc: Current Employment. Bjordahl:Fate Therapeutics: Current Employment. Mahmood:Fate Therapeutics, Inc: Current Employment. Reiser:FATE THERAPEUTICS: Current Employment. Gaidarova:Fate Therapeutics, Inc: Current Employment. Blum:Fate Therapeutics: Current Employment. Cichocki:Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding. Chu:Fate Therapeutics, Inc: Current Employment. Bonello:Fate Therapeutics, Inc: Current Employment. Lee:Fate Therapeutics, Inc.: Current Employment. Groff:Fate Therapeutics, Inc: Current Employment. Meza:Fate Therapeutics, Inc: Current Employment. Chu:Roche Holding AG: Current equity holder in publicly-traded company; Fate Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Walcheck:Fate Therapeutics: Consultancy, Research Funding. Malmberg:Vycellix: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Consultancy, Patents & Royalties. Miller:Vycellix: Consultancy; Onkimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees; Nektar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding; GT Biopharma: Consultancy, Patents & Royalties, Research Funding. Valamehr:Fate Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company.

Clinical Research of Dendritic Cell-Mediated Tumor-Associated Antigen-Specific Cytotoxic T Lymphocytes in the Treatment of Multiple Myeloma and Non-Hodgkin Lymphoma

To investigate the safety and efficacy of tumor-associated antigen-specific cytotoxic T lympho- cytes (TAA-CTL) in the treatment of multiple myeloma (MM) and non-Hodgkin lymphoma (NHL). Peripheral blood mononuclear cells (PBMNC)of patients were collected. Dendritic cells (DC) were loaded with multiple tumor-associated antigens (TAA) (NY-ESO-1, MAGE-A3, MAGE-A4, WT1, Survivin, PRAME, LMP1 and LMP2A), then co-cultured with PBMNC to induce cytotoxic T lymphocytes (CTL). The phenotypes of cell products were detected, and the disease statuse was evaluated in 7 patients during or after infusion. The changes of TAA-CTL amount in the PBMNC of patients were measured by using IFN-γ ELISpot assay. TAA-CTL products were generated comprising CD3+ T cells (mean 82.98%) with a mixture of CD4+ (mean 42.09%) and CD8+ (mean 25.32%) T cells. Among them, 70% expressed effectors memory markers (CD45RO+CD62L-CCR7-). Each patient received TAA-CTL infusions for 1-4 times, and none of them showed obvious adverse reactions. The clinical symptoms and laboratory or imaging examination of 5 patients achieved positive effects. After cell therapy, the spot-forming cells (SFC) levels of most patients gradually increased and the peak often appeared about 2-3 weeks after the infusion. TAA-CTLs preliminarily show its safety and efficacy in MM and NHL patients, however, a larger population sample is needed to explore its clinical application value.

Abstract 5569: Discovery of tumor-specific T cell receptors (TCRs) by reverse genetic engineering platform

Abstract TCR-T therapy is an ideal cell therapeutic strategy against solid tumors. However, the traditional method of TCR cloning has two major problems. First, we could only develop TCRs against previously discovered tumor antigen epitopes. Second, the affinity maturation of TCR sometimes leads to mortal toxicities due to the unexpected cross-reactivities. Therefore we hereby present a reverse genetic engineering platform to clone tumor specific TCRs from immunotherapies benefit cancer patients. Multi-antigen stimulating cell therapy (MASCT) is a cell-based immunotherapy combining therapeutic DC vaccine and adoptive T cell transfer, the IND application of which was approved in China. We observed dynamic T cell responses against multiple tumor-associated antigens used in MASCT-treated HCC patients, which showed a significant correlation with clinical outcome. Based on this observation, we propose that expanded tumor-specific T cells in MASCT-benefit cancer patients could be good sources to isolate clinical relevant TCRs. A HPV positive metastatic cervical cancer patient has shown partially response after MASCT treatment alone, and survived over 5 years. Moreover, a metastatic lung cancer patient has also shown partially response and survived for longer than 2 years after combination treatment of MASCT and anti-PD1 antibody. Enhanced and persist T cell responses against several TAAs as well as HPV antigens were detected in PBMCs of both patients. We successfully expended and isolated tumor antigen-specific T cells by IFNγ-secreting cell enrichment, and obtained alpha/beta chains paired TCR sequences using single T cell NGS technology. We selected 14 pairs of TCRs isolated from cervical cancer patient for further validation. TCRs from lung cancer patient were currently under synthesis or sequencing. Among 14 TCRs, a TAA-specific TCR and a HPV18E7-specific TCR showed superior antigen specificity and optimum avidity, and would be applied for TCR-T development. Of notes, our reverse genetic engineering of TCR-T (ReGET) platform.can not only identify both HLA class I and II-restricted TCRs, but also identify novel epitopes of tumor antigens, which may provide new targets for TCR-T therapy against solid tumors. Citation Format: Yanyan Han, Xiaoling Liang, Shudan Ou, Xihe Chen, Minjun Ma, Jun Li, Yifan Ma, Xiangjun Zhou. Discovery of tumor-specific T cell receptors (TCRs) by reverse genetic engineering platform [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 5569.

Abstract 601: Chimeric antigen receptors incorporating novel binding domains targeting CD123 direct potent antitumor activity of T cells: Correlation between affinity and activity

Abstract Unlike the tumor targets associated with initial CAR T cell successes, the vast majority of tumor associated antigens are not unique, but rather expressed at elevated levels compared to normal tissue. For example, while CD123 is overexpressed on many AML tumor cells, concerns regarding on-target, off-tumor toxicities - often associated low-level CD123 expression on normal endothelial cells - has hindered clinical success. Therefore, future generations of CAR T therapies will require novel strategies that have the ability to discriminate between normal and elevated antigen levels - potentially through mechanisms that involve 1) the simultaneous or sequential targeting of multiple tumor associated antigens and 2) agents that contain affinity-tuned targeting domains. Due to the large inventory of validated antibodies against a variety of therapeutic targets, scFv naturally emerged as the obvious and justifiable targeting domain for chimeric antigen receptors. However, many of the characteristics that have made antibodies versatile and effective recombinant therapeutics (e.g., high affinity, bivalency, antibody-dependent cytotoxicity, complement-dependent cytotoxicity, FcRn recycling, and low renal filtration rates) are not advantageous for membrane associated chimeric receptors. Furthermore, scFv are not native protein structures and their development, particularly as it pertains to solubility and aggregation, can be challenging. Therefore, we sought to develop a simple, highly selective targeting domain that could be engineered into complex, potentially multispecific therapeutics. We describe the design and development of non-scFv-derived binding domains. Using phage display and targeted mutagenesis, we identify a series of binding domains that target CD123 with high specificity - as characterized by functional and tissue binding studies. These domains exhibit affinities that range over 2 logs and demonstrate in vitro potencies that correlate with their affinities. CARs comprised of higher affinity binding domains mediate potent T-cell activation and cytolysis of CD123-expressing target cells and induce complete durable remission in two AML xenograft models. We also describe a strategy of engineering less immunogenic binding domains through the identification and removal of putative T cell epitopes and demonstrate that the resultant variants retain biological activity. Finally, we demonstrate further potential of our binding domains by generating functional, bi-specific CARs comprised of a CD123-specific binding domain and a CD19-specific scFv. The ability to incorporate our target-specific binding domains with a range of affinities into complex chimeric receptors, affords a viable alternative to scFv as targeting domains in CAR T cell therapeutics. Citation Format: David W. LaFleur, Haiying Qin, Justin P. Edwards, Liubov Zaritskaya, Ankit Gupta, C. Jenny Mu, Laura K. Richman, Terry J. Fry, David M. Hilbert. Chimeric antigen receptors incorporating novel binding domains targeting CD123 direct potent antitumor activity of T cells: Correlation between affinity and activity [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 601.

A heat-shocked melanoma cell lysate vaccine enhances tumor infiltration by prototypic effector T cells inhibiting tumor growth

BackgroundImmune checkpoint blocker (ICB) therapy has shown survival benefits for some patients with cancer. Nevertheless, many individuals remain refractory or acquire resistance to treatment, motivating the exploration of complementary immunotherapies....

The Role of Human γδ T Cells in Anti-Tumor Immunity and Their Potential for Cancer Immunotherapy.

γδ T cells are a distinct subset of T cells whose T cell receptors consist of γ chains and δ chains, different from conventional αβ T cells. γδ T cells are considered as a member of the innate immunity because of their non-MHC restricted antigen recognition, rapid response to invading pathogens and sense early changes of malignant cells. Upon activation, they can further promote the activation of adaptive immune cells, such as T cells and B cells, by secreting various cytokines. Thus, γδ T cells are regarded as a bridge between innate immunity and acquired immunity. γδ T cells are involved in a variety of immune response processes, including immune defense and immune surveillance against infection and tumorigenesis. γδ T cells recognize multiple tumor-associated antigens or molecules in T cell receptors (TCRs)-dependent and natural killer cell receptors (NKRs)-dependent ways. γδ T cells not only display a direct killing capacity on a variety of tumors, but also exert anti-tumor immune responses indirectly by facilitating the function of other immune cells, such as dendritic cells (DCs), B cells and CD8+ T cells. In this review, we summarize the major subpopulations, the tumor recognition mechanisms, and the anti-tumor effects of human γδ T cells, particularly the potential of γδ T cells for cancer immunotherapy.

Alarmin-painted exosomes elicit persistent antitumor immunity in large established tumors in mice

Treating large established tumors is challenging for dendritic cell (DC)-based immunotherapy. DC activation with tumor cell-derived exosomes (TEXs) carrying multiple tumor-associated antigen can enhance tumor recognition. Adding a potent adjuvant, high mobility group nucleosome-binding protein 1 (HMGN1), boosts DCs’ ability to activate T cells and improves vaccine efficiency. Here, we demonstrate that TEXs painted with the functional domain of HMGN1 (TEX-N1ND) via an exosomal anchor peptide potentiates DC immunogenicity. TEX-N1ND pulsed DCs (DCTEX-N1ND) elicit long-lasting antitumor immunity and tumor suppression in different syngeneic mouse models with large tumor burdens, most notably large, poorly immunogenic orthotopic hepatocellular carcinoma (HCC). DCTEX-N1ND show increased homing to lymphoid tissues and contribute to augmented memory T cells. Importantly, N1ND-painted serum exosomes from cancer patients also promote DC activation. Our study demonstrates the potency of TEX-N1ND to strengthen DC immunogenicity and to suppress large established tumors, and thus provides an avenue to improve DC-based immunotherapy.

Adjuvant Screen Identifies Synthetic DNA-Encoding Flt3L and CD80 Immunotherapeutics as Candidates for Enhancing Anti-tumor T Cell Responses.

Overcoming tolerance to tumor-associated antigens remains a hurdle for cancer vaccine-based immunotherapy. A strategy to enhance the anti-tumor immune response is the inclusion of adjuvants to cancer vaccine protocols. In this report, we generated and systematically screened over twenty gene-based molecular adjuvants composed of cytokines, chemokines, and T cell co-stimulators for the ability to increase anti-tumor antigen T cell immunity. We identified several robust adjuvants whose addition to vaccine formulations resulted in enhanced T cell responses targeting the cancer antigens STEAP1 and TERT. We further characterized direct T cell stimulation through CD80-Fc and indirect T cell targeting via the dendritic cell activator Flt3L-Fc. Mechanistically, intramuscular delivery of Flt3L-Fc into mice was associated with a significant increase in infiltration of dendritic cells at the site of administration and trafficking of activated dendritic cells to the draining lymph node. Gene expression analysis of the muscle tissue confirmed a significant up-regulation in genes associated with dendritic cell signaling. Addition of CD80-Fc to STEAP1 vaccine formulation mimicked the engagement provided by DCs and increased T cell responses to STEAP1 by 8-fold, significantly increasing the frequency of antigen-specific cells expressing IFNγ, TNFα, and CD107a for both CD8+ and CD4+ T cells. CD80-Fc enhanced T cell responses to multiple tumor-associated antigens including Survivin and HPV, indicating its potential as a universal adjuvant for cancer vaccines. Together, the results of our study highlight the adjuvanting effect of T cell engagement either directly, CD80-Fc, or indirectly, Flt3L-Fc, for cancer vaccines.

Cooperative Immune-Mediated Mechanisms of the HDAC Inhibitor Entinostat, an IL15 Superagonist, and a Cancer Vaccine Effectively Synergize as a Novel Cancer Therapy.

Immunotherapy has demonstrated clinical efficacy in subsets of patients with solid carcinomas. Multimodal therapies using agents that can affect different arms of the immune system and/or tumor microenvironment (TME) might increase clinical responses. We demonstrate that entinostat, a class I histone deacetylase inhibitor, enhances the antitumor efficacy of the IL15 superagonist N-803 plus vaccine in 4T1 triple-negative breast and MC38-CEA colon murine carcinoma models. A comprehensive immune and gene-expression analysis was performed in the periphery and/or TME of MC38-CEA tumor-bearing mice. Although N-803 plus vaccine induced peripheral CD8+ T-cell activation and cytokine production, there was no reduction in tumor burden and poor tumor infiltration of CD8+ T cells with minimal levels of granzyme B. For the first time, we demonstrate that the addition of entinostat to N-803 plus vaccine promoted significant tumor control, correlating with increased expression of genes associated with tumor inflammation, enhanced infiltration of activated CD8+ T cells with maximal granzyme B, T-cell responses to multiple tumor-associated antigens, increased serum IFNγ, reduction of regulatory T cells in the TME, and decreased expression of the checkpoint V-domain Ig suppressor of T-cell activation (VISTA) on multiple immune subsets. Collectively, these data demonstrate that the synergistic combination of entinostat, N-803, and vaccine elicits potent antitumor activity by generating a more inflamed TME. These findings thus form the rationale for the use of this combination of agents for patients harboring poorly or noninflamed solid carcinomas.

Multi-Specific CAR Targeting to Prevent Antigen Escape.

Chimeric antigen receptor (CAR) T cell therapy has demonstrated remarkable remission induction rates for relapsed/refractory B cell malignancies. However, loss of the CAR-targeted antigen, known as antigen escape, accounts for a substantial percentage of relapses following CAR therapy and is a major barrier to durable remission. Here, we discuss mechanisms for antigen escape and strategies to prevent this pattern of relapse, including the use of multi-specific CARs, which recognize and target multiple tumor-associated antigens simultaneously. Preclinical and early clinical trial data indicates that multi-specific CAR therapy for B cell malignancies is both safe and effective. Optimal combinations of target antigens, as well as different multi-specific CAR formats, are currently being evaluated. Although still in early stages of development, multi-specific CAR therapy represents a promising approach to mitigate antigen loss-related relapses and improve durability of remission in patients with refractory B cell malignancies, and may be applicable to other types of cancer.

Abstract 2224: A panel of autoantibodies against multiple tumor-associated antigens in the early immunodiagnosis of lung cancer

Abstract Lung cancer is the most common cancers worldwide and with very low 5-year survival rate due to lack of effective early diagnostic methods. In our previous study, nine autoantibodies against tumor-associated antigens (TAAs) were identified to be potential biomarkers in detection of lung cancer and this study aimed to find the optimal panel of TAAs to detect lung cancer in the early stage. Methods A total of 1,130 serum samples were included in this study, including patients with lung cancer, normal control and benign lung diseases patients. Autoantibodies against PSIP1,TOP2A,ACTR3,RPS6KA5,HMGB3,MMP12,GREM1,ZWINT and NUSAP1 were detected by enzyme-linked immunosorbent assay. Logistic regression models were generated from the training set and further validated in another independent set. We also evaluated the ability of the model to detecting benign lung diseases and early-stage lung cancer. Results Two greater models were selected. One model including 5 autoantibodies panel (GREM1,HMGB3,ZWINT,TOP2A and PSIP1) and another model including 3 autoantibodies panel (GREM1,HMGB3 and PSIP1). Finally, the panel of 3 autoantibodies showed high diagnostic accuracy with areas under the curve (AUC) of 0.711(95 % CI 0.674-0.746) in training cohort and 0.891(95% CI 0.845-0.927) in validation cohort, respectively. The AUC of the model of 3 autoantibodies panel was 0.948(95% CI 0.912-0.972) in discriminate lung cancer and benign pulmonary disease. This model could discriminate early-stage lung cancer patients from normal controls, with AUC of 0.686(95 % CI 0.633-0.735) in training cohort and AUC of 0.911(95 % CI 0.849-0.954) in validation cohort, and the overall AUC for early-stage lung cancer was 0.779(95 % CI 0.738-0.816) when the two cohorts were combined. Conclusions In summary, it would be more cost-effective and more efficient to use the model with 3 autoantibodies panel which has a high diagnostic performance for lung cancer detection, especially for early-stage lung cancer. And the model may apply a new help for the discrimination for benign pulmonary disease and lung cancer. Note: This abstract was not presented at the meeting. Citation Format: Di Jiang, Tingting Wang, Lu Pei, Peng Wang, Hua Ye, Chunhua Song, Kaijuan Wang, Jianying Zhang, Liping Dai. A panel of autoantibodies against multiple tumor-associated antigens in the early immunodiagnosis of lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2224.

Abstract LB-076: Personal neoantigen immunotherapy in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is one of the most common cancers and the leading causes of mortality in China with limited therapeutic options. To investigate whether neoantigens could induce tumor specific immune responses in patients with HCC, neoantigen stimulating cellular therapy (neo-MASCT) was applied after radical resection. Neo-MASCT was a cell-based immunotherapy with injections of mature dendritic cells (DCs) loaded with a peptide pool of multiple shared tumor associated antigens (TAAs) and several personalized neoantigens followed by autologous T cells stimulated by these DCs. To obtain neoantigens, fresh tumor samples were acquired immediately after biopsy or surgery for subsequently WES and RNAseq. All mutations including SNV, non-frame shift insert/deletion (Indel), and neoORF were detected by comparing NGS data from tumors to normal tissues. All neo-epitope candidates were predicted by our own proprietary algorithm MASNEO according to the patient’s HLA class I genotypes. Long neoantigen peptides containing 25-31 amino acids with higher affinities were selected for further synthesis and treatment. Peptides containing multiple neo-epitopes were preferred. Until now, eight patients had received neo-MASCT, and seven of them were tested for specific immune responses against both TAAs and neoantigens using IFNγ Elispot assay. The results showed that Neo-MASCT induced TAA-specific immune responses in five patients (5/7, 71%), and induced neoantigen-specific immune responses in six patients (6/7, 86%), respectively. One patient demonstrated specific T cell responses against all the five neoantigen peptides (5/5, 100%) after neo-MASCT treatment. The average responsive rate of neoantigens was 65% (22/34 peptides) in the six responding patients. In conclusion, our results demonstrate that neo-MASCT is well-tolerated in HCC patients and elicits immune responses against multiple tumor antigens, especially personalized neoantigens. We will continuously monitor the immune responses against neoantigens, and use the results to train and optimize our predicting algorithm. We will also evaluate the clinical benefit of neo-MASCT in further clinical trials with more patients. Citation Format: Sui Peng, Zhenwei Peng, Lixia Xu, Jie Mei, Longqing Tang, Yanyan Han, Ming Kuang. Personal neoantigen immunotherapy in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-076.