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BackgroundTriple-negative breast cancer (TNBC) represents a subtype of breast cancer with poorest prognosis due to limited effective targeted therapies. Chimeric antigen receptor T cell (CAR-T) therapy has shown remarkable efficacy in treating hematological cancers, but its application in TNBC requires further development. One major obstacle is the lack of suitable tumor-specific target in TNBC. Inspired by recent success of trophoblast cell-surface antigen 2 (TROP2) antibody-drug conjugate in TNBC, we developed a second-generation CAR that specifically targets TROP2 and formally evaluated its antitumor activity and safety profile using in vitro and in vivo models.MethodsA CAR molecule targeting TROP2 was constructed based on the clinically-validated humanized antibody Sacituzumab and expressed in primary human T cells using a retroviral vector. Tumor cytotoxicity, cytokine production and T-cell proliferation of TROP2 CAR-T cells were tested against multiple TNBC cell lines in vitro. Antitumor efficacy was evaluated using orthotopic and metastatic models of cell line-derived xenograft in NSG mice and in patient-derived xenograft (PDX) model. The safety profile of TROP2 CAR-T cells was assessed using TROP2-humanized immunocompetent mice and an “AND”-logic gated SynNotch CAR targeting B7-H3 and TROP2 was engineered to minimize off-tumor, on-target toxicity of TROP2 CAR-T cells.ResultsHuman TROP2 CAR-T cells demonstrated robust antitumor activity in vitro and in orthotopic/metastatic/PDX xenograft mouse models. TROP2 CAR-T cells caused lethal on-target, off-tumor toxicity in TROP2-humanized immunocompetent mice, causing severe tissue damage in lungs and systemic inflammation. The B7-H3/TROP2 “AND”-logic gated SynNotch CAR-T cells showed comparable antitumor efficacy without causing apparent adverse effects as in TROP2 CAR-T cells.ConclusionsThese data indicate that while CAR-T therapy targeting TROP2 possesses potent antitumor activity against TNBC cell lines and PDX, its potential side effects could be lethal due to TROP2 expression in vital organs such as the lung. Using an “AND”-logic gated CAR is a viable solution to overcome its in vivo toxicity. Our study lays the groundwork for future development of TROP2 CAR-T cell therapy for TNBC.

BackgroundPhosphatidylserine (PS) exposed on apoptotic cells promotes immune clearance of dead cells without inducing inflammation. Conversely, PS exposure on live tumor cells promotes an immunosuppressive tumor microenvironment that hinders antitumor immune responses. After confirming elevated PS levels in various tumor cell lines and cancer tissues, we aimed to investigate its potential as a target antigen for chimeric antigen receptor T cell (CAR-T) therapy.MethodsWe used two different approaches to target PS. First, we employed the adaptor proteins, EDAnnexin or BCMAnnexin comprising annexin V and EDA (extra domain A of fibronectin) or B-cell maturation antigen (BCMA) antigens, to redirect the lytic activity of EDA CAR-T or BCMA CAR-T cells toward PS-expressing tumor cells. In a second approach, we developed an annexin V-based CAR (Anxa CAR-T) to directly recognize PS-positive tumor cells.ResultsThe adaptors proteins EDAnnexin and BCMAnnexin successfully redirected EDA CAR-T or BCMA CAR-T cell activity, leading to an efficient recognition of PS+ tumor cells in vitro. However, the established immunological synapse differs significantly from that observed when CAR-T cells recognize the tumor cells directly. In vivo administration of the adaptor proteins, combined with the corresponding CAR-T cells, displayed antitumor activity in mice bearing PS+ tumors. Regarding the second approach, Anxa CAR-T cells effectively recognized and killed PS+ tumor cells in vitro. Nonetheless, PS exposure on T-cell membranes during T-cell activation impeded efficient Anxa CAR-T cell manufacturing due to fratricide. By optimizing retroviral dose to reduce Anxa CAR expression on the cell membrane, or by using the multikinase inhibitor dasatinib, the fratricide effect was mitigated, enabling successful Anxa CARLow-T cell production. Remarkably, Anxa CARLow-T cells demonstrated antitumor activity in in vivo murine models of PS+ hepatocarcinoma and teratocarcinoma. No signs of toxicity were observed after Anxa CAR-T cell administration.ConclusionsPS holds promise as a target antigen for CAR-T cell therapy, underscoring the need to address fratricide as a key challenge in the development of PS-targeting CAR-T cells.

To develop combination immunotherapies for gastric cancers, immunologically well-characterized preclinical models are crucial. Here, we leveraged two transplantable murine gastric cancer cell lines, YTN2 and YTN16, derived from the same parental line but differing in their susceptibility to immune rejection. We established their differential sensitivity to immune checkpoint inhibitors (ICI) and identified neoantigens. Although anti-CTLA-4 mAbs eradicated YTN16 tumors in 4 of 5 mice, anti-PD-1 and anti-PD-L1 mAbs failed to eradicate YTN16 tumors. Using whole-exome and RNA sequencing, we identified two and three neoantigens in YTN2 and YTN16, respectively. MHC class I ligandome analysis detected the expression of only one of these neoantigens, mutated Cdt1, but the exact length of MHC binding peptide was determined. Dendritic cell vaccine loaded with neoepitope peptides and adoptive transfer of neoantigen-specific CD8+ T cells successfully inhibited the YTN16 tumor growth. Targeting mutated Cdt1 had better efficacy for controlling the tumor. Therefore, mutated Cdt1 was the dominant neoantigen in these tumor cells. More mCdt1 peptides were bound to MHC class I and presented on YTN2 surface than YTN16. This might be one of the reasons why YTN2 was rejected while YTN16 grew in immune-competent mice.

Understanding regional distribution of HLA frequencies is crucial for optimizing enrollment in HLA-restricted clinical trials and to promote trial diversity per the Food and Drug Administration’s 2020 mandate. Using US HLA frequency data and census demographics we developed a method to create high-resolution HLA class 1 genotypic frequency maps. Analyzing HLA-A*11:01 and HLA-B*58:01 as alleles of interest, we found significant US regional variations. HLA-A*11:01, which presents KRAS neoantigen mutations targeted by TCR T-cell therapies, showed 10–15% genotypic frequency (national average 11.2%), with western US states 1.5 times higher than average and local variations within California (10–19%). These insights can be used to guide clinical trial site selection, for example, in National Cancer Institute (NCI) cancer center catchment areas. For HLA-B*58:01, which reacts pharmacogenetically with allopurinol and results in severe cutaneous adverse reactions, Mississippi had a high frequency among US states, which could be used to guide potential public safety campaigns. This method can identify regions with high HLA type representation, aiding efficient patient identification and enrollment for HLA-specific clinical trials and health-awareness efforts.

BackgroundTumor-derived proprotein convertase subtilisin/kexin type 9 (PCSK9) facilitates tumor progression, but the role of immune cell-intrinsic PCSK9 in tumor control remains unclear.MethodsOrthotopic models of pancreatic cancer and melanoma in Pcsk9-deficient mice were established and tumor-infiltrating immune cells were analyzed using single-cell RNA sequencing and flow cytometry. The effect of genetic disruptions of PCSK9 on murine CD8+ T cells and human chimeric antigen receptor (CAR)-T cells was evaluated both in vitro and in vivo.ResultsAblation of host Pcsk9 remarkably suppressed tumor growth and prolonged the survival of tumor-bearing mice, while tumor cells still express PCSK9. The enhanced tumor suppression in Pcsk9-deficient mice depended on CD8+ T cells. Notably, PCSK9 expression was induced in CD8+ tumor-infiltrating lymphocytes (TILs). Consequently, Pcsk9 ablation potentiated the antitumor capacity of CD8+ T cells, showing increased intratumoral infiltration and improved cytotoxic function, along with higher proportions of both effector-memory precursor exhausted (TPEX) and terminally exhausted (TTEX) CD8+ TILs. Additionally, disruption of PCSK9 in both murine CD8+ T cells and human CAR-T cells, synergistic with PD-1 blockade, promoted tumor suppression.ConclusionThese findings indicate that PCSK9 inhibits the antitumor function of CD8+ T cells, suggesting it may be a promising target for enhancing T-cell-based cancer immunotherapy.

Although chimeric antigen receptor (CAR)-engineered T cell therapy has achieved encouraging clinical trial results for treating hematological cancers, further optimization can likely expand this therapeutic success to more patients and other cancer types. Most CAR constructs used in clinical trials incorporate single chain variable fragment (scFv) as the extracellular antigen recognition domain. The immunogenicity of nonhuman scFv could cause host rejection against CAR T cells and compromise their persistence and efficacy. The limited availability of scFvs and slow discovery of new monoclonal antibodies also limit the development of novel CAR constructs. Adnectin, a class of affinity molecules derived from the tenth type III domain of human fibronectin, can be an alternative to scFv as an antigen-binding moiety in the design of CAR molecules. We constructed adnectin-based CARs targeting epithelial growth factor receptor (EGFR) and found that compared to scFv-based CAR, T cells engineered with adnectin-based CARs exhibited equivalent cell-killing activity against target H292 lung cancer cells in vitro and had comparable antitumor efficacy in xenograft tumor-bearing mice in vivo. In addition, with optimal affinity tuning, adnectin-based CAR showed higher selectivity on target cells with high EGFR expression than on those with low expression. This new design of adnectin CARs can potentially facilitate the development of T cell immunotherapy for cancer and other diseases. Wang and colleagues show that a novel design of CAR based on adnectin, a class of scaffold molecules derived from the tenth type III domain of human fibronectin, can target EGFR-positive tumor cells and exhibit a similar functional profile as compared with the conventional scFv-derived CAR.

Adoptive cell therapy (ACT) has seen a steep rise of new therapeutic approaches in its immune-oncology pipeline over the last years. This is in great part due to the recent approvals of chimeric antigen receptor (CAR)-T cell therapies and their remarkable efficacy in certain soluble tumors. A big focus of ACT lies on T cells and how to genetically modify them to target and kill tumor cells. Genetically modified T cells that are currently utilized are either equipped with an engineered CAR or a T cell receptor (TCR) for this purpose. Both strategies have their advantages and limitations. While CAR-T cell therapies are already used in the clinic, these therapies face challenges when it comes to the treatment of solid tumors. New designs of next-generation CAR-T cells might be able to overcome these hurdles. Moreover, CARs are restricted to surface antigens. Genetically engineered TCR-T cells targeting intracellular antigens might provide necessary qualities for the treatment of solid tumors. In this review, we will summarize the major advancements of the CAR-T and TCR-T cell technology. Moreover, we will cover ongoing clinical trials, discuss current challenges, and provide an assessment of future directions within the field.

Natural killer (NK) cells are lymphocytes with a key role in the defense against viral infections and tumor cells. Although NK cells are classified as innate lymphoid cells (ILCs), under certain circumstances they exhibit adaptive and memory-like features. The latter may be achieved, among others, by a brief stimulation with interleukin (IL)-12, IL-15 and IL-18. These cytokine-induced memory-like (CIML) NK cells resemble the trained immunity observed in myeloid cells. CIML NK cells undergo transcriptional, epigenetic and metabolic reprogramming that, along with changes in the expression of cell surface receptors and components of cytotoxic granules, are responsible for their enhanced effector functions after a resting period. In addition, these memory-like NK cells persist for a long time, which make them a good candidate for cancer immunotherapy. Currently, several clinical trials are testing CIML NK cells infusions to treat tumors, mostly hematological malignancies. In relapse/refractory acute myeloid leukemia (AML), the adoptive transfer of CIML NK cells is safe and complete clinical remissions have been observed. In our review, we sought to summarize the current knowledge about the generation and molecular basis of NK cell memory-like responses and the up-to-date results from clinical trials with CIML NK cells.

The rationale behind cancer immunotherapy is based on the unequivocal demonstration that the immune system plays an important role in limiting cancer initiation and progression. Adoptive cell therapy (ACT) is a form of cancer immunotherapy that utilizes a patient’s own immune cells to find and eliminate tumor cells, however, donor immune cells can also be employed in some cases. Here, we focus on T lymphocyte (T cell)-based cancer immunotherapies that have gained significant attention after initial discoveries that graft-versus-tumor responses were mediated by T cells. Accumulating knowledge of T cell development and function coupled with advancements in genetics and data science has enabled the use of a patient’s own (autologous) T cells for ACT (TIL ACTs). In TIL ACT, tumor-infiltrating lymphocytes (TILs) are collected from resected tumor material, enhanced and expanded ex-vivo , and delivered back to the patient as therapeutic agents. ACT with TILs has been shown to cause objective tumor regression in several types of cancers including melanoma, cervical squamous cell carcinoma, and cholangiocarcinoma. In this review, we provide a brief history of TIL ACT and discuss the current state of TIL ACT clinical development in solid tumors. We also discuss the niche of TIL ACT in the current cancer therapy landscape and potential strategies for patient selection.

Adoptive cell therapy (ACT) achieves substantial efficacy in the treatment of hematological malignancies and solid tumours, while enormous endeavors have been made to reduce relapse and extend the remission duration after ACT. For the genetically engineered T cells, their functionality and long-term anti-tumour potential depend on the specificity of the T cell receptor (TCR) or chimeric antigen receptor (CAR). In addition, the therapeutic benefit is directly to sufficient activation and proliferation of engineered T cells. Artificial antigen-presenting cells (aAPCs), as powerful boosters for ACT, have been applied to provide sustained stimulation of the cognate antigen and facilitate the expansion of sufficient T cells for infusion. In this review, we summarize the aAPCs used to generate effector cells for ACT and underline the mechanism by which aAPCs enhance the functionality of the effector cells. The manuscript includes investigations ranging from basic research to clinical trials, which we hope will highlight the importance of aAPCs and provide guidance for novel strategies to improve the effectiveness of ACT.