CD3, CD8, IFN-γ, tumor and stroma inflammatory cells as prognostic indicators for surgically resected SCLC: evidences from a 10-year retrospective study and immunohistochemical analysis.

Current clinical guidelines limit surgical intervention to patients with cT1-2N0M0 small cell lung cancer (SCLC). Our objective was to reassess the role of surgery in SCLC management, and explore novel prognostic indicators for surgically resected SCLC. We reviewed all patients diagnosed with SCLC from January 2011 to April 2021 in our institution. Survival analysis was conducted using the Kaplan-Meier method, and independent prognostic factors were assessed through the Cox proportional hazard model. In addition, immunohistochemistry (IHC) staining was performed to evaluate the predictive value of selected indicators in the prognosis of surgically resected SCLC patients. In the study, 177 SCLC patients undergoing surgical resection were ultimately included. Both univariate and multivariate Cox analysis revealed that incomplete postoperative adjuvant therapy emerged as an independent risk factor for adverse prognosis (p < 0.001, HR 2.96). Survival analysis revealed significantly superior survival among pN0-1 patients compared to pN2 patients (p < 0.0001). No significant difference in postoperative survival was observed between pN1 and pN0 patients (p = 0.062). Patients with postoperative stable disease (SD) exhibited lower levels of tumor inflammatory cells (TIC) (p = 0.0047) and IFN-γ expression in both area and intensity (p < 0.0001 and 0.0091, respectively) compared to those with postoperative progressive disease (PD). Conversely, patients with postoperative SD showed elevated levels of stromal inflammatory cells (SIC) (p = 0.0453) and increased counts of CD3+ and CD8+ cells (p = 0.0262 and 0.0330, respectively). Survival analysis indicated that high levels of SIC, along with low levels of IFN-γ+ cell area within tumor tissue, may correlate positively with improved prognosis in surgically resected SCLC (p = 0.017 and 0.012, respectively). In conclusion, the present study revealed that the patients with pT1-2N1M0 staging were a potential subgroup of SCLC patients who may benefit from surgery. Complete postoperative adjuvant therapy remains an independent factor promoting a better prognosis for SCLC patients undergoing surgical resection. Moreover, CD3, CD8, IFN-γ, TIC, and SIC may serve as potential indicators for predicting the prognosis of surgically resected SCLC.

Expression of HLA-DR and KLRG1 enhances the cytotoxic potential and cytokine secretion capacity of CD3+ T cells in tuberculosis patients.

BACKGROUND: Human T cells play an important role in immunity against tuberculosis (TB) infection. Activating receptor HLA-DR and inhibitory receptor KLRG1 are critical regulators of T cell function during viral infection and tumorigenesis, but they have been less studied in TB infection. METHODS: In this study, we explored the relationship between CD3+ T cell expression of HLA-DR and KLRG1 receptors and function against TB infection. Flow cytometry was conducted to assess the immunomodulatory effects of HLA-DR and KLRG1 receptors on CD3+ T cells in patients with different TB infection status. RESULTS: We found activating receptors HLA-DR, NKG2C, CD57 and NKP46, and inhibitory receptors KLRG1 and KIR on CD3+ T cells in different TB infection status showed different distribution patterns; the cytotoxic potential and cytokine secretion capacity of CD3+ T cells after Mtb-specific antigen stimulation were significantly enhanced in TB infection groups. Further studies revealed HLA-DR+ T and KLRG1+ T cells expressed higher activating and inhibitory receptors than the negative population. In addition, the expression of cytotoxic potential and cytokine secretion capacity of HLA-DR+ T and KLRG1+ T cells was significantly higher than that of HLA-DR- T and KLRG1- T cells. CONCLUSIONS: Expression of HLA-DR and KLRG1 enhances the cytotoxic potential and cytokine secretion capacity of CD3+ T cells in TB patients, suggesting CD3+ T cells expressing HLA-DR and KLRG1 are important effector cell phenotypes involved in the host anti-TB infection. HLA-DR and KLRG1 expressed by CD3+ T cells may be potential predictive markers of TB disease progression and clinical immune assessment.

The immunologic abnormalities in patients with paroxysmal nocturnal hemoglobinuria are associated with disease progression.

OBJECTIVES: To suggest the presence of a hyperimmune state in patients, and indicate that immune system attack on glycosylphosphatidylinositol (+) (GPI+) cells while escaping GPI- cell immunity. METHODS: We retrospective the immune cell subtypes in peripheral blood from 25 patients visiting Tianjin Medical University General Hospital, Tianjin, China, with classical paroxysmal nocturnal hemoglobinuria (PNH) and 50 healthy controls. RESULTS: The total CD3+ and CD3+CD8+ cell levels were higher in patients with PNH. The CD3+ cells are positively, correlated with lactate dehydrogenase (LDH; r=0.5453, p=0.0040), indirect bilirubin (r=0.4260, p=0.0379) and Flear- cells in monocytes (r=0.4099, p=0.0303). However, a negative correlation was observed between CD3+ cells and hemoglobin (r= -0.4530, p=0.0105). The total CD19+ cells decreased in patients, and CD19+ cells were negatively correlated with LDH (r= -0.5640, p=0.0077) and Flear- cells in monocytes (r= -0.4432, p=0.0341). Patients showed an increased proportion of total dendritic cells (DCs), with a higher proportion of myeloid DCs (mDCs) within the DC population. Moreover, the proportion of mDC/DC was positively correlated with CD59- cells (II + III types) in red cells (r=0.7941, p=0.0004), Flear- cells in granulocytes (r=0.5357, p=0.0396), and monocytes (r=0.6445, p=0.0095). CONCLUSION: Our results demonstrated that immune abnormalities are associated with PNH development.

Zinc Ionophore Pyrithione Mimics CD28 Costimulatory Signal in CD3 Activated T Cells.

Zinc is an essential trace element that plays a crucial role in T cell immunity. During T cell activation, zinc is not only structurally important, but zinc signals can also act as a second messenger. This research investigates zinc signals in T cell activation and their function in T helper cell 1 differentiation. For this purpose, peripheral blood mononuclear cells were activated via the T cell receptor-CD3 complex, and via CD28 as a costimulatory signal. Fast and long-term changes in intracellular zinc and calcium were monitored by flow cytometry. Further, interferon (IFN)-γ was analyzed to investigate the differentiation into T helper 1 cells. We show that fast zinc fluxes are induced via CD3. Also, the intracellular zinc concentration dramatically increases 72 h after anti-CD3 and anti-CD28 stimulation, which goes along with the high release of IFN-γ. Interestingly, we found that zinc signals can function as a costimulatory signal for T helper cell 1 differentiation when T cells are activated only via CD3. These results demonstrate the importance of zinc signaling alongside calcium signaling in T cell differentiation.

ITPRIPL1 binds CD3ε to impede T cell activation and enable tumor immune evasion.

Cancer immunotherapy has transformed treatment possibilities, but its effectiveness differs significantly among patients, indicating the presence of alternative pathways for immune evasion. Here, we show that ITPRIPL1 functions as an inhibitory ligand of CD3ε, and its expression inhibits T cells in the tumor microenvironment. The binding of ITPRIPL1 extracellular domain to CD3ε on T cells significantly decreased calcium influx and ZAP70 phosphorylation, impeding initial T cell activation. Treatment with a neutralizing antibody against ITPRIPL1 restrained tumor growth and promoted T cell infiltration in mouse models across various solid tumor types. The antibody targeting canine ITPRIPL1 exhibited notable therapeutic efficacy against naturally occurring tumors in pet clinics. These findings highlight the role of ITPRIPL1 (or CD3L1, CD3ε ligand 1) in impeding T cell activation during the critical "signal one" phase. This discovery positions ITPRIPL1 as a promising therapeutic target against multiple tumor types.

Mesothelin CAR T Cells Secreting Anti-FAP/Anti-CD3 Molecules Efficiently Target Pancreatic Adenocarcinoma and its Stroma.

PURPOSE: Targeting solid tumors with chimeric antigen receptor (CAR) T cells remains challenging due to heterogenous target antigen expression, antigen escape, and the immunosuppressive tumor microenvironment (TME). Pancreatic cancer is characterized by a thick stroma generated by cancer-associated fibroblasts (CAF), which may contribute to the limited efficacy of mesothelin-directed CAR T cells in early-phase clinical trials. To provide a more favorable TME for CAR T cells to target pancreatic ductal adenocarcinoma (PDAC), we generated T cells with an antimesothelin CAR and a secreted T-cell-engaging molecule (TEAM) that targets CAF through fibroblast activation protein (FAP) and engages T cells through CD3 (termed mesoFAP CAR-TEAM cells). EXPERIMENTAL DESIGN: Using a suite of in vitro, in vivo, and ex vivo patient-derived models containing cancer cells and CAF, we examined the ability of mesoFAP CAR-TEAM cells to target PDAC cells and CAF within the TME. We developed and used patient-derived ex vivo models, including patient-derived organoids with patient-matched CAF and patient-derived organotypic tumor spheroids. RESULTS: We demonstrated specific and significant binding of the TEAM to its respective antigens (CD3 and FAP) when released from mesothelin-targeting CAR T cells, leading to T-cell activation and cytotoxicity of the target cell. MesoFAP CAR-TEAM cells were superior in eliminating PDAC and CAF compared with T cells engineered to target either antigen alone in our ex vivo patient-derived models and in mouse models of PDAC with primary or metastatic liver tumors. CONCLUSIONS: CAR-TEAM cells enable modification of tumor stroma, leading to increased elimination of PDAC tumors. This approach represents a promising treatment option for pancreatic cancer.

CD3 downregulation identifies high-avidity human CD8 T cells.

CD8 T cells recognize infected and cancerous cells via their T-cell receptor (TCR), which binds peptide-MHC complexes on the target cell. The affinity of the interaction between the TCR and peptide-MHC contributes to the antigen sensitivity, or functional avidity, of the CD8 T cell. In response to peptide-MHC stimulation, the TCR-CD3 complex and CD8 co-receptor are downmodulated. We quantified CD3 and CD8 downmodulation following stimulation of human CD8 T cells with CMV, EBV, and HIV peptides spanning eight MHC restrictions, observing a strong correlation between the levels of CD3 and CD8 downmodulation and functional avidity, regardless of peptide viral origin. In TCR-transduced T cells targeting a tumor-associated antigen, changes in TCR-peptide affinity were sufficient to modify CD3 and CD8 downmodulation. Correlation analysis and generalized linear modeling indicated that CD3 downmodulation was the stronger correlate of avidity. CD3 downmodulation, simply measured using flow cytometry, can be used to identify high-avidity CD8 T cells in a clinical context.

CD6 triggers actomyosin cytoskeleton remodeling after binding to its receptor complex.

The T cell marker CD6 regulates both T cells and target cells during inflammatory responses by interacting with its receptors. However, only a few receptors binding to the extracellular domains of CD6 have been identified, and cellular events induced by CD6 engagement with its receptors in target cells remain poorly understood. In this study, we identified CD44 as a novel CD6 receptor by proximity labeling and confirmed the new CD6-CD44 interaction by biochemical and biophysical approaches. CD44 and the other 2 known CD6 receptors, CD166 and CDCP1, were distributed diffusely on resting retinal pigment epithelium (RPE) cells but clustered together to form a receptor complex upon CD6 binding. CD6 stimulation induced dramatic remodeling of the actomyosin cytoskeleton in RPE cells mediated by activation of RhoA, and Rho-associated kinase signaling, resulting in increased myosin II phosphorylation. Such actomyosin activation triggered the disassembly of tight junctions responsible for RPE barrier integrity in a process that required all components of the tripartite CD6 receptor complex. These data provided new insights into the mechanisms by which CD6 mediates T cell-driven disruption of tissue barriers during inflammation.

Alternatively Spliced Variants of Murine CD247 Influence T Cell Development and Activation, Revealing the Importance of the CD3ζ C-Terminal Region.

CD247, also known as CD3ζ, is a crucial signaling molecule that transduces signals delivered by TCR through its three ITAMs. CD3ζ is required for successful thymocyte development. Three additional alternatively spliced variants of murine CD247 have been described, that is, CD3ι, CD3θ, and CD3η, that differ from CD3ζ in the C terminus such that the third ITAM is lost. Previous studies demonstrated defects in T cell development in mice expressing CD3η, but the TCR signaling pathways affected by CD3η and the impacts of the CD3ι and CD3θ on T cell development were not explored. In this study, we used a retrovirus-mediated gene transfer technique to express these three isoforms individually and examined the roles of them on T cell development and activation. Rag1-/- mice reconstituted with CD3θ-expressing bone marrow failed to develop mature T cells. CD3ι-expressing T cells exhibited similar development and activation as cells expressing CD3ζ. In contrast, thymic development was severely impaired in CD3η-reconstituted mice. Single-positive but not double-positive CD3η-expressing thymocytes had reduced TCR expression, and CD5 expression was decreased at the double-positive stage, suggesting a defect in positive selection. Peripheral CD3η-expressing T cells had expanded CD44hi populations and upregulation of exhaustion markers seen by flow cytometry and RNA sequencing analysis. Analysis of early signaling events demonstrated significantly reduced activation of both the PLCγ1 and Akt/mTOR signaling pathways. There was also a reduction in the frequency of activation of CD3η-expressing T cells. These studies reveal the importance of the CD3ζ C-terminal region in T cell development and activation.

CD38 marks the exhausted CD8+ tissue-resident memory T cells in hepatocellular carcinoma.

Introduction: Despite recent advances in immunotherapy for hepatocellular carcinoma (HCC), the overall modest response rate underscores the need for a better understanding of the tumor microenvironment (TME) of HCC. We have previously shown that CD38 is widely expressed on tumor-infiltrating leukocytes (TILs), predominantly on CD3+ T cells and monocytes. However, its specific role in the HCC TME remains unclear. Methods: In this current study, we used cytometry time-of-flight (CyTOF), bulk RNA sequencing on sorted T cells, and single-cell RNA (scRNA) sequencing to interrogate expression of CD38 and its correlation with T cell exhaustion in HCC samples. We also employed multiplex immunohistochemistry (mIHC) for validating our findings. Results: From CyTOF analysis, we compared the immune composition of CD38-expressing leukocytes in TILs, non-tumor tissue-infiltrating leukocytes (NIL), and peripheral blood mononuclear cells (PBMC). We identified CD8+ T cells as the dominant CD38-expressing TILs and found that CD38 expression was significantly higher in CD8+ TRM in TILs than in NILs. Furthermore, through transcriptomic analysis on sorted CD8+ TRM from HCC tumors, we observed a higher expression of CD38 along with T cell exhaustion genes, including PDCD1 and CTLA4, compared to the circulating memory CD8 T cells from PBMC. This was validated by scRNA sequencing that revealed co-expression of CD38 with PDCD1, CTLA4, and ITGAE (CD103) in T cells from HCC tumors. The protein co-expression of CD38 and PD-1 on CD8+ T cells was further demonstrated by mIHC on HCC FFPE tissues, marking CD38 as a T cell co-exhaustion marker in HCC. Lastly, the higher proportions of CD38+PD-1+ CD8+ T cells and CD38+PD-1+ TRM were significantly associated with the higher histopathological grades of HCC, indicating its role in the aggressiveness of the disease. Conclusion: Taken together, the concurrent expression of CD38 with exhaustion markers on CD8+ TRM underpins its role as a key marker of T cell exhaustion and a potential therapeutic target for restoring cytotoxic T cell function in HCC.

CAR T-Cell Therapy for Cancer: Latest Updates and Challenges, with a Focus on B-Lymphoid Malignancies and Selected Solid Tumours.

Although exponential progress in treating advanced malignancy has been made in the modern era with immune checkpoint blockade, survival outcomes remain suboptimal. Cellular immunotherapy, such as chimeric antigen receptor T cells, has the potential to improve this. CAR T cells combine the antigen specificity of a monoclonal antibody with the cytotoxic 'power' of T-lymphocytes through expression of a transgene encoding the scFv domain, CD3 activation molecule, and co-stimulatory domains. Although, very rarely, fatal cytokine-release syndrome may occur, CAR T-cell therapy gives patients with refractory CD19-positive B-lymphoid malignancies an important further therapeutic option. However, low-level expression of epithelial tumour-associated-antigens on non-malignant cells makes the application of CAR T-cell technology to common solid cancers challenging, as does the potentially limited ability of CAR T cells to traffic outside the blood/lymphoid microenvironment into metastatic lesions. Despite this, in advanced neuroblastoma refractory to standard therapy, 60% long-term overall survival and an objective response in 63% was achieved with anti GD2-specific CAR T cells.

A dynamic biomimetic model of the membrane-bound CD4-CD3-TCR complex during pMHC disengagement.

The coordinated (dis)engagement of the membrane-bound T cell receptor (TCR)-CD3-CD4 complex from the peptide-major histocompatibility complex (pMHC) is fundamental to TCR signal transduction and T cell effector function. As such, an atomic-scale understanding would not only enhance our basic understanding of the adaptive immune response but would also accelerate the rational design of TCRs for immunotherapy. In this study, we explore the impact of the CD4 coreceptor on the TCR-pMHC (dis)engagement by constructing a molecular-level biomimetic model of the CD3-TCR-pMHC and CD4-CD3-TCR-pMHC complexes within a lipid bilayer. After allowing the system complexes to equilibrate (engage), we use steered molecular dynamics to dissociate (disengage) the pMHC. We find that 1) the CD4 confines the pMHC closer to the T cell by 1.8 nm at equilibrium; 2) CD4 confinement shifts the TCR along the MHC binding groove engaging a different set of amino acids and enhancing the TCR-pMHC bond lifetime; 3) the CD4 translocates under load increasing the interaction strength between the CD4-pMHC, CD4-TCR, and CD4-CD3; and 4) upon dissociation, the CD3-TCR complex undergoes structural oscillation and increased energetic fluctuation between the CD3-TCR and CD3-lipids. These atomic-level simulations provide mechanistic insight on how the CD4 coreceptor impacts TCR-pMHC (dis)engagement. More specifically, our results provide further support (enhanced bond lifetime) for a force-dependent kinetic proofreading model and identify an alternate set of amino acids in the TCR that dominate the TCR-pMHC interaction and could thus impact the design of TCRs for immunotherapy.

A simplified function-first method for the discovery and optimization of bispecific immune engaging antibodies.

Bi-specific T-cell engager antibodies (BiTEs) are synthetic fusion molecules that combine multiple antibody-binding domains to induce active contact between T-cells and antigen expressing cells in the body. Blinatumomab, a CD19-CD3 BiTE is now a widely used therapy for relapsed B-cell malignancies, and similar BiTE therapeutics have shown promise for treating various other forms of cancer. The current process for new BiTE development is time consuming and costly, requiring characterization of the individual antigen binding domains, followed by bi-specific design, protein production, purification, and eventually functional screening. Here, we sought to establish a more cost-efficient approach for generating novel BiTE sequences and assessing bioactivity through a function first approach without purification. We generate a plasmid with a bi-modular structure to allow high-throughput exchange of either binding arm, enabling rapid screening of novel tumour-targeting single chain variable (scFv) domains in combination with the well-characterized OKT3 scFv CD3-targeting domain. We also demonstrate two systems for high throughput functional screening of BiTE proteins based on Jurkat T cells (referred to as BiTE-J). Using BiTE-J we evaluate four EGFRvIII-scFv sequenced in BiTE format, identifying two constructs with superior activity for redirecting T-cells against the EGFRvIII-tumour specific antigen. We also confirm activity in primary T cells, where novel EGFRvIII-BiTEs induced T cell activation and antigen selective tumor killing. We finally demonstrate similar exchange the CD3-interacting element of our bi-modular plasmid. By testing several novel CD3-targeting scFv elements for activity in EGFRvIII-targeted BiTEs, we were able to identify highly active BiTE molecules with desirable functional activity for downstream development. In summary, BiTE-J presents a low cost, high-throughput method for the rapid assessment of novel BiTE molecules without the need for purification and quantification.

Safety and efficacy of post-haematopoietic cell transplantation maintenance therapy with blinatumomab for relapsed/refractory CD19-positive B-cell acute lymphoblastic leukaemia: protocol for a phase I-II, multicentre, non-blinded, non-controlled trial (JPLSG SCT-ALL-BLIN21).

INTRODUCTION: Relapsed and refractory B-cell acute lymphoblastic leukaemia (R/R-B-ALL) is linked to a significant relapse rate after allogeneic haematopoietic cell transplantation (allo-HCT) in children, adolescents and young adults (CAYA). No standard treatment has been established to prevent relapse after allo-HCT for R/R-B-ALL, which is an unmet medical need. The administration of blinatumomab after allo-HCT is expected to enhance the antileukaemic effect on residual CD19-positive blasts by donor-derived CD3-positive T-cells. METHODS AND ANALYSIS: The goal of this multicentre, open-label, uncontrolled, phase I-II clinical trial is to assess the safety and effectiveness of post-transplant maintenance therapy with blinatumomab for CAYA patients (25 years old or younger) with CD19-positive R/R-B-ALL who have received allo-HCT beyond first complete remission (CR) and have CR with haematological recovery between 30 and 100 days after allo-HCT. Eighty-five paediatric institutions in Japan are participating in this study. Forty-one patients will enrol within 2.25-year enrolment period and follow-up period is 1 year. The primary endpoints are the treatment completion rate for phase I study and the 1-year graft-versus-host disease-free/relapse-free survival rate for phase II study, respectively. ETHICS AND DISSEMINATION: This research was approved by the Central Review Board at National Hospital Organization Nagoya Medical Center (Nagoya, Japan) on 21 January 2022 and was registered at the Japan Registry of Clinical Trials (jRCT) on 3 March 2022. Written informed consent is obtained from all patients and/or their guardians. The results of this study will be disseminated through peer-reviewed publications and conference presentations. TRIAL REGISTRATION NUMBER: jRCTs041210154.

Aberrant expression of T cell receptors in monocyte/macrophage RAW 264.7 cells: FCγRII/III compensates the need for CD3.

Conventionally T-cell receptors (TCRs) have so far been considered as a T-lymphocyte privilege. However, recent findings also place TCR expression in non-lymphoid cells, namely neutrophils, eosinophils and macrophages. In order to examine the ectopic expression of TCR, this study focused on RAW 264.7 cells, which have been broadly used for their macrophage properties. Immunofluorescence staining detected 70% and 40% of the cells to express TCRαß and TCRγδ respectively, which was also verified by RT-PCR experiments and confocal microscopy analysis. Interestingly, except from the predicted 292 and 288 bp gene products for the α- and γ-chain, additional products at 220 and 550 bp could be detected, respectively. RAW 264.7 cells also expressed the co-stimulatory CD4 and CD8 markers at a percentage of 61% and 14% respectively, which supported the expression of TCRs. However, only low numbers of cells expressed CD3ε and CD3ζ (9% and 7% respectively). Such observations contradicted the existing knowledge, and indicated that TCRs would be supported by other molecules for reaching the membrane and transducing their signal. Such candidate molecules could be the Fcγ receptors (FcγRs). Indeed, the FcγRII/III receptor was found to be expressed in 75% of the cells, which also expressed at a percentage of 25% major histocompatibility complex (MHC) class II molecules. Engagement of the FcγRII/III receptor by a recombinant IgG2aCH2 fragment, except from stimulating the macrophage-dependent properties of the cells, was shown to reduce expression of TCRαß and γδ indicating that FcγRII/III was indeed used by TCRs for their transport to the cell membrane. In order to examine the ability of RAW 264.7 cells to simultaneously display antigen presenting- and T-cell properties, functional experiments as to antigen-specific antibody and IL-2 production were performed. In in vitro immunization assays in the presence of naïve B cells, RAW264.7 failed to promote antibody production. However, RAW 264.7 cells could compete with antigen-stimulated macrophages but not T cells when applied to a system of in vivo antigen-sensitized cells followed by an in vitro immunization protocol. Interestingly, simultaneous addition of antigen and the IgG2aCH2 fragment to RAW 264.7 cells could promote IL-2 production from the cells, indicating that FcγRII/III activation could also support TCR stimulation. Extrapolating these findings to cells of the myeloid origin, the above results dictate novel regulatory mechanisms towards the alteration of the immune response.

Precision-activated T-cell engagers targeting HER2 or EGFR and CD3 mitigate on-target, off-tumor toxicity for immunotherapy in solid tumors.

To enhance the therapeutic index of T-cell engagers (TCEs), we engineered masked, precision-activated TCEs (XPAT proteins), targeting a tumor antigen (human epidermal growth factor receptor 2 (HER2) or epidermal growth factor receptor (EGFR)) and CD3. Unstructured XTEN polypeptide masks flank the N and C termini of the TCE and are designed to be released by proteases in the tumor microenvironment. In vitro, unmasked HER2-XPAT (uTCE) demonstrates potent cytotoxicity, with XTEN polypeptide masking providing up to 4-log-fold protection. In vivo, HER2-XPAT protein induces protease-dependent antitumor activity and is proteolytically stable in healthy tissues. In non-human primates, HER2-XPAT protein demonstrates a strong safety margin (>400-fold increase in tolerated maximum concentration versus uTCE). HER2-XPAT protein cleavage is low and similar in plasma samples from healthy and diseased humans and non-human primates, supporting translatability of stability to patients. EGFR-XPAT protein confirmed the utility of XPAT technology for tumor targets more widely expressed in healthy tissues.

Construction of a novel TROP2/CD3 bispecific antibody with potent antitumor activity and reduced induction of Th1 cytokines.

Many cancers, including triple-negative breast cancer, overexpress TROP2 on the surface of tumor cells. TROP2 has become a promising tumor associated antigen for the development of novel antibody-based targeted therapy. Herein, we constructed a novel bispecific antibody with the ability to simultaneously target TROP2 on the tumor surface and bind to CD3 to activate T cells. Given that the excessive production of Th1 cytokines induced by CD3-mediated T-cell overactivation may lead to toxicity in the clinic, we devised a strategy to modify this CD3-induced T cell activation by a two-step reduction in the bispecific antibody binding affinity for CD3 to a level that retained the ability of the bispecific antibody to effectively inhibit tumor growth while greatly reducing the amount of Th1 cytokines secreted by T cells. Thus, we provide insight into the design of T cell engagers that exhibit a promising toxicity profile while retaining inhibitory effects on tumor growth.

Microenvironment-responsive anti-PD-L1 × CD3 bispecific T-cell engager for solid tumor immunotherapy.

Bispecific T-cell Engager (BiTE) antibodies can redirect T-cells to tumor cells, and turn on the targeted lysis of tumor cells. However, BiTE has been challenging in solid tumors due to short plasma half-life, "off-target" effect, and immunosuppression via PD-1/PD-L1 axis. This study designed a safe, long-acting, and highly effective Protease-Activated PSTAGylated BiTE, named PAPB, which includes a shielding polypeptide domain (PSTAG), a protease-activated linker, and a BiTE core. The BiTE core consists of two scFvs targeting PD-L1 and CD3. BiTE core bound PD-L1 and CD3 in a dose-dependent manner, and PAPB can release BiTE core in response to MMP2 in the tumor microenvironment to exert antitumor activity. The plasma half-life of PAPB in mice was significantly prolonged from 2.46 h to 6.34 h of the BiTE core. In mice bearing melanoma (A375) xenografts, PAPB significantly increased infiltration of T lymphocytes in tumor tissue, and inhibited tumor proliferation without activating T-cells in the peripheral blood. Overall, the engineering protein PAPB could be a promising drug candidate for solid tumor immunotherapy.

CD3 Target Affinity Chromatography Mass Spectrometry as a New Tool for Function-Structure Characterization of T-Cell Engaging Bispecific Antibody Proteoforms and Product-Related Variants.

T-cell engaging bispecific antibodies (TCBs) targeting CD3 and tumor-specific antigens are very promising therapeutic modalities. Since CD3 binding is crucial for the potency of TCBs, understanding the functional impact of CD3 antigen-binding fragment modifications is of utmost importance for defining critical quality attributes (CQA). The current CQA assessment strategy requires the integration of structure-based physicochemical separation and functional cell-based potency assays. However, this strategy is tedious, and coexisting proteoforms with potentially different functionalities may not be individually assessed. This increases the degree of ambiguities for defining meaningful CQAs, particularly for complex bispecific antibody formats such as TCBs. Here, we report for the first time a proof-of-concept study to separate and identify critically modified proteoforms of TCBs using functional CD3 target affinity chromatography (AC) coupled with online mass spectrometry (MS). Our method enabled functional distinction of relevant deamidated and glycosylated proteoforms and the simultaneous assessment of product-related variants such as TCB mispairings. For example, CD3 AC-MS allowed us to separate TCB mispairings with increased CD3 binding (i.e., knob-knob homodimers) within the bound fraction. The functional separation of proteoforms was validated using an established workflow for CQA identification based on thoroughly characterized ion-exchange fractions of a 2+1 TCB. In addition, the new method facilitated the criticality assessment of post-translational modifications in stress studies and structural variants in early stage clone selection. CD3 AC-MS has high impact for streamlining the integration of functional and structural characterizations of the large landscape of therapeutic CD3 targeting TCBs from early stage research to late stage characterization.

CAR-tropic extracellular vesicles carry tumor-associated antigens and modulate CAR T cell functionality.

Tumor-derived extracellular vesicles (EVs) are active contributors in metastasis and immunosuppression in tumor microenvironment. At least some of the EVs carry tumor surface molecules such as tumor-associated antigens (TAAs) and/or checkpoint inhibitors, and potentially could interact with T cells or CAR T cells. Upon contact with T cells, EVs could alter their phenotype and functions by triggering signaling through TCR or CAR reprogramming them to escape immune response. We hypothesize that EVs that possess TAA on the surface will probably interact with CAR T cells which can recognize and bind corresponding TAA. This interaction between EVs and CAR T cells may change the outcome of CAR T-based cancer immunotherapy since it should affect CAR T cells. Also, EVs could serve as adjuvants and antigenic components of antitumor vaccines. Herein, we isolated EVs from B cell precursor leukemia cell line (pre-B ALL) Nalm-6 and demonstrated that recognition and binding of CD19+EVs with CD19-CAR T cells strongly depends on the presence of CD19 antigen. CD19+EVs induce secretion of pro-inflammatory cytokines (IL-2 and IFN-y) and upregulated transcription of activation-related genes (IFNG, IFNGR1, FASLG, IL2) in CD19-CAR T cells. Tumor necrosis factor receptor superfamily (TNFRSF4 and TNFRSF9) and T-cell exhaustion markers (CTLA4, LAG3, TIM3 and PDCD1LG2) were also upregulated in CD19-CAR T cells after incubation with CD19+EVs. Long-term cultivation of CD19+ or PD-L1+EVs with CD19-CAR T cells led to increased terminal differentiation and functional exhaustion according to elevated expression of PD-1, TIGIT, CD57. In summary, our results suggest that chronic exposure of CD19-CAR T cells to CD19+EVs mediates activation and systemic exhaustion in antigen-specific manner, and this negative effect is accompanied by the impaired cytotoxic activity in vitro.