Control of acute myeloid leukemia and generation of immune memory in vivo using AMV564, a bivalent bispecific CD33 x CD3 T cell engager.

Off-the-shelf immunotherapeutics that suppress tumor growth and provide durable protection against relapse could enhance cancer treatment. We report preclinical studies on a CD33 x CD3 bivalent bispecific diabody, AMV564, that not only suppresses tumor growth, but also facilitates memory responses in a mouse model of acute myelogenous leukemia (AML). Mechanistically, a single 5-day treatment with AMV564 seems to reduce tumor burden by redirection of T cells, providing a time window for allogeneic or other T cells that innately recognize tumor antigens to become activated and proliferate. When the concentration of bispecific becomes negligible, the effector: target ratio has also shifted, and these activated T cells mediate long-term tumor control. To test the efficacy of AMV564 in vivo, we generated a CD33+ MOLM13CG bioluminescent human cell line and optimized conditions needed to control these cells for 62 days in vivo in NSG mice. Of note, not only did MOLM13CG become undetectable by bioluminescence imaging in response to infusion of human T cells plus AMV564, but also NSG mice that had cleared the tumor also resisted rechallenge with MOLM13CG in spite of no additional AMV564 treatment. In these mice, we identified effector and effector memory human CD4+ and CD8+ T cells in the peripheral blood immediately prior to rechallenge that expanded significantly during the subsequent 18 days. In addition to the anti-tumor effects of AMV564 on the clearance of MOLM13CG cells in vivo, similar effects were seen when primary CD33+ human AML cells were engrafted in NSG mice even when the human T cells made up only 2% of the peripheral blood cells and AML cells made up 98%. These studies suggest that AMV564 is a novel and effective bispecific diabody for the targeting of CD33+ AML that may provide long-term survival advantages in the clinic.

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.

Characterization of a novel T cell-engaging bispecific antibody for elimination of L1CAM-positive tumors.

Neural cell adhesion molecule L1 (L1CAM) is a cell-surface glycoprotein involved in cancer occurrence and migration. Up to today, L1CAM-targeted therapy appeared limited efficacy in clinical trials although quite a few attempts by monoclonal antibody (mAb) or chimeric antigen receptor T-cell therapy (CAR-T) have been reported. Therefore, the development of new effective therapies targeting L1CAM is highly desirable. It has been demonstrated that T cell-engaging bispecific antibody (TCE) plays an effective role in cancer immunotherapy by redirecting the cytotoxic activity of CD3+ T cells to tumor cells, resulting in tumor cell death. In this study, we designed and characterized a novel bispecific antibody (CE7-TCE) based on the IgG-(L)-ScFv format, which targets L1CAM and CD3 simultaneously. In vitro, CE7-TCE induced specific killing of L1CAM-positive tumor cells through T cells. In vivo, CE7-TCE inhibited tumor growth in human peripheral blood mononuclear cell/tumor cell co-grafting models. To overcome the adaptive immune resistance (AIR) that impairs the efficacy of TCEs, we conducted a combination therapy of CE7-TCE with Pembrolizumab (anti-PD1 mAb), which enhanced the anti-tumor activity of CE7-TCE. Our results confirmed the feasibility of using L1CAM as a TCE target for the treatment of solid tumors and revealed the therapeutic potential of CE7-TCE combined with immune checkpoint inhibitors.

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.

Pharmacokinetic models for first-in-human dose selection of immune-activating products in oncology.

Pharmacokinetic (PK) models are increasingly submitted to the FDA to support first-in-human (FIH) dose selection of immune-oncology products. To examine whether a simple PK modeling (SPM) using clearance for scaling was acceptable for dose estimation, FIH(SPM) doses were computed and compared to doses that were safely administered to patients. We concluded that the SPM approach is acceptable in FIH dose estimation, but the variables should be carefully selected for CD3 constructs. For CD3 constructs, use of 60 kg BWh, a clearance exponent of 0.75, and a targeted plasma concentration based on relevant and/or sensitive activity assays was an acceptable approach for FIH dose selection; use of 0.85 as the scaling factor is questionable at this time as it resulted in a FIH dose that was too close to the AHD for one product (7%). Immune activating mAbs were not sensitive to changes in the clearance exponent (0.75-0.85) or body weight (60-70 kg). For PD-1/PD-L1 mAbs, using products' in vitro EC50 in the model resulted in suboptimal FIH doses and clinical data of closely related products informed FIH dose selection. PK models submitted by sponsors were diverse in methods, assumptions, and variables, and the resulting FIH doses were not always optimal.

Role of Teplizumab, a Humanized Anti-CD3 Monoclonal Antibody, in Managing Newly Diagnosed Type 1 Diabetes: An Updated Systematic Review and Meta-Analysis.

OBJECTIVE: Teplizumab has emerged as a potential disease-modifying drug in type 1 diabetes (T1D). This meta-analysis sought to summarize the therapeutic effect of teplizumab in newly diagnosed patients with T1D. METHODS: Randomized controlled trials involving patients with T1D receiving teplizumab in the intervention arm and placebo (or no active intervention) in the control arm were searched throughout the electronic databases. The primary outcome was the change in area under the curve of C-peptide levels from baseline. RESULTS: Seven reports from 6 studies involving 834 subjects met the inclusion criteria. Compared to teplizumab, greater reductions in area under the curve of C-peptide from the baseline values were observed in the control group after 6 months (mean difference [MD] 0.07 nmol/L [0.01, 0.13], P = .02), after 12 months (MD 0.07 nmol/L [0.04, 0.11], P = .0001), after 18 months (MD 0.10 nmol/L [0.06, 0.14], P < .00001), and after 24 months (MD 0.07 nmol/L [0.01, 0.14], P = .03) of interventions. Moreover, fewer patients treated with teplizumab had a decreased C-peptide response after 6 months (odds ratio [OR] 0.21), after 12 months (OR 0.17), after 18 months (OR 0.30), and after 24 months (OR 0.12) of treatment. The preservation of endogenous insulin production was supported by reduced use of exogenous insulin with maintenance of comparable glycemic control for up to 18 months post-treatment. Teplizumab imparted higher risks of grade 3 or higher adverse events, adverse events leading to study medication discontinuation, nausea, rash, and lymphopenia. CONCLUSION: The results of the meta-analysis support teplizumab as a promising disease-modifying therapy for newly diagnosed T1D.

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.

Teplizumab and ß-Cell Function in Newly Diagnosed Type 1 Diabetes.

BACKGROUND: Teplizumab, a humanized monoclonal antibody to CD3 on T cells, is approved by the Food and Drug Administration to delay the onset of clinical type 1 diabetes (stage 3) in patients 8 years of age or older with preclinical (stage 2) disease. Whether treatment with intravenous teplizumab in patients with newly diagnosed type 1 diabetes can prevent disease progression is unknown. METHODS: In this phase 3, randomized, placebo-controlled trial, we assessed ß-cell preservation, clinical end points, and safety in children and adolescents who were assigned to receive teplizumab or placebo for two 12-day courses. The primary end point was the change from baseline in ß-cell function, as measured by stimulated C-peptide levels at week 78. The key secondary end points were the insulin doses that were required to meet glycemic goals, glycated hemoglobin levels, time in the target glucose range, and clinically important hypoglycemic events. RESULTS: Patients treated with teplizumab (217 patients) had significantly higher stimulated C-peptide levels than patients receiving placebo (111 patients) at week 78 (least-squares mean difference, 0.13 pmol per milliliter; 95% confidence interval [CI], 0.09 to 0.17; P<0.001), and 94.9% (95% CI, 89.5 to 97.6) of patients treated with teplizumab maintained a clinically meaningful peak C-peptide level of 0.2 pmol per milliliter or greater, as compared with 79.2% (95% CI, 67.7 to 87.4) of those receiving placebo. The groups did not differ significantly with regard to the key secondary end points. Adverse events occurred primarily in association with administration of teplizumab or placebo and included headache, gastrointestinal symptoms, rash, lymphopenia, and mild cytokine release syndrome. CONCLUSIONS: Two 12-day courses of teplizumab in children and adolescents with newly diagnosed type 1 diabetes showed benefit with respect to the primary end point of preservation of ß-cell function, but no significant differences between the groups were observed with respect to the secondary end points. (Funded by Provention Bio and Sanofi; PROTECT ClinicalTrials.gov number, NCT03875729.).

IL-22-producing CD3 + CD8- T cells increase in immune clearance stage of chronic HBV infection and correlate with the response of Peg-interferon treatment.

Interleukin (IL)-22 regulates host defense. This study investigated the predominant IL-22-producing cell subsets under HBV associated immune stages. We found circulating IL-22-producing CD3 + CD8- T cells were significantly increased in immune active (IA) stage than those in immunotolerant stage, inactive carrier and healthy controls (HCs). The plasma IL-22 level was higher in IA and HBeAg-negative CHB compared to HCs. Importantly, CD3 + CD8- T cells were identified as the predominant source of plasma IL-22 production. Up-regulated IL-22-producing CD3 + CD8- T cells obviously correlated with the grade of intrahepatic inflammation. The proportions of IL-22-producing CD3 + CD8- T cells were significantly down-regulated after 48 weeks of Peg-interferon treatment, and the differences were of great significance in patients with normalize ALT levels at 48 weeks, rather than those with elevated ALT levels. In conclusion, IL-22 might play a proinflammatory function in. chronic HBV infected patients with active inflammation and Peg-interferon treatment could attenuate the degree of liver inflammation through down-regulating IL-22-producing CD3 + CD8- T cells.

The immune-metabolic crosstalk between CD3+C1q+TAM and CD8+T cells associated with relapse-free survival in HCC.

Introduction: Although multiple targeted treatments have appeared, hepatocellular carcinoma (HCC) is still one of the most common causes of cancer-related deaths. The immunosuppressive tumor microenvironment (TME) is a critical factor in the oncogenesis and progression of HCC. The emerging scRNA-seq makes it possible to explore the TME at a high resolution. This study was designed to reveal the immune-metabolic crosstalk between immune cells in HCC and provide novel strategies to regulate immunosuppressive TME. Method: In this study, we performed scRNA-seq on paired tumor and peri-tumor tissues of HCC. The composition and differentiation trajectory of the immune populations in TME were portrayed. Cellphone DB was utilized to calculate interactions between the identified clusters. Besides, flow cytometry, RT-PCR and seahorse experiments were implemented to explore potential metabolic and epigenetic mechanisms of the inter-cellular interaction. Result: A total of 19 immune cell clusters were identified and 7 were found closely related to HCC prognosis. Besides, differentiation trajectories of T cells were also presented. Moreover, a new population, CD3+C1q+ tumor-associated macrophages (TAM) were identified and found significantly interacted with CD8+ CCL4+T cells. Compared to the peri-tumor tissue, their interaction was attenuated in tumor. Additionally, the dynamic presence of this newly found cluster was also verified in the peripheral blood of patients with sepsis. Furthermore, we found that CD3+C1q+TAM affected T cell immunity through C1q signaling-induced metabolic and epigenetic reprogramming, thereby potentially affecting tumor prognosis. Conclusion: Our study revealed the interaction between CD3+C1q+TAM and CD8+ CCL4+T cells and may provide implications for tackling the immunosuppressive TME in HCC.

Changing roles of CD3 + CD8 low T cells in combating HIV-1 infection.

BACKGROUND: Cluster of differentiation 8 (CD8 T) cells play critical roles in eradicating human immunodeficiency virus (HIV)-1 infection, but little is known about the effects of T cells expressing CD8 at low levels (CD8 low ) or high levels (CD8 high ) on HIV-1 replication inhibition after HIV-1 invasion into individual. METHODS: Nineteen patients who had been acutely infected with HIV-1 (AHI) and 20 patients with chronic infection (CHI) for ≥2 years were enrolled in this study to investigate the dynamics of the quantity, activation, and immune responses of CD3 + CD8 low T cells and their counterpart CD3 + CD8 high T cells at different stages of HIV-1 infection. RESULTS: Compared with healthy donors, CD3 + CD8 low T cells expanded in HIV-1-infected individuals at different stages of infection. As HIV-1 infection progressed, CD3 + CD8 low T cells gradually decreased. Simultaneously, CD3 + CD8 high T cells was significantly reduced in the first month of AHI and then increased gradually as HIV-1 infection progressed. The classical activation of CD3 + CD8 low T cells was highest in the first month of AHI and then reduced as HIV-1 infection progressed and entered the chronic stage. Meanwhile, activated CD38 - HLA-DR + CD8 low T cells did not increase in the first month of AHI, and the number of these cells was inversely associated with viral load ( r = -0.664, P = 0.004) but positively associated with the CD4 T-cell count ( r = 0.586, P = 0.014). Increased programmed cell death protein 1 (PD-1) abundance on CD3 + CD8 low T cells was observed from the 1st month of AHI but did not continue to be enhanced, while a significant T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibition motif (ITIM) domains (TIGIT) abundance increase was observed in the 12th month of infection. Furthermore, increased PD-1 and TIGIT abundance on CD3 + CD8 low T cells was associated with a low CD4 T-cell count (PD-1: r = -0.456, P = 0.043; TIGIT: r = -0.488, P = 0.029) in CHI. Nonetheless, the nonincrease in PD-1 expression on classically activated CD3 + CD8 low T cells was inversely associated with HIV-1 viremia in the first month of AHI ( r = -0.578, P = 0.015). Notably, in the first month of AHI, few CD3 + CD8 low T cells, but comparable amounts of CD3 + CD8 high T cells, responded to Gag peptides. Then, weaker HIV-1-specific T-cell responses were induced in CD3 + CD8 low T cells than CD3 + CD8 high T cells at the 3rd and 12th months of AHI and in CHI. CONCLUSIONS: Our findings suggest that CD3 + CD8 low T cells play an anti-HIV role in the first month of infection due to their abundance but induce a weak HIV-1-specific immune response. Subsequently, CD3 + CD8 low T-cell number decreased gradually as infection persisted, and their anti-HIV functions were inferior to those of CD3 + CD8 high T cells.

Talquetamab, a T-Cell-Redirecting GPRC5D Bispecific Antibody for Multiple Myeloma.

BACKGROUND: G protein-coupled receptor, family C, group 5, member D (GPRC5D) is an orphan receptor expressed in malignant plasma cells. Talquetamab, a bispecific antibody against CD3 and GPRC5D, redirects T cells to mediate killing of GPRC5D-expressing myeloma cells. METHODS: In a phase 1 study, we evaluated talquetamab administered intravenously weekly or every other week (in doses from 0.5 to 180 µg per kilogram of body weight) or subcutaneously weekly, every other week, or monthly (5 to 1600 µg per kilogram) in patients who had heavily pretreated relapsed or refractory multiple myeloma that had progressed with established therapies (a median of six previous lines of therapy) or who could not receive these therapies without unacceptable side effects. The primary end points - the frequency and type of dose-limiting toxic effects (study part 1 only), adverse events, and laboratory abnormalities - were assessed in order to select the recommended doses for a phase 2 study. RESULTS: At the data-cutoff date, 232 patients had received talquetamab (102 intravenously and 130 subcutaneously). At the two subcutaneous doses recommended for a phase 2 study (405 µg per kilogram weekly [30 patients] and 800 µg per kilogram every other week [44 patients]), common adverse events were cytokine release syndrome (in 77% and 80% of the patients, respectively), skin-related events (in 67% and 70%), and dysgeusia (in 63% and 57%); all but one cytokine release syndrome event were of grade 1 or 2. One dose-limiting toxic effect of grade 3 rash was reported in a patient who had received talquetamab at the 800-µg dose level. At median follow-ups of 11.7 months (in patients who had received talquetamab at the 405-µg dose level) and 4.2 months (in those who had received it at the 800-µg dose level), the percentages of patients with a response were 70% (95% confidence interval [CI], 51 to 85) and 64% (95% CI, 48 to 78), respectively. The median duration of response was 10.2 months and 7.8 months, respectively. CONCLUSIONS: Cytokine release syndrome, skin-related events, and dysgeusia were common with talquetamab treatment but were primarily low-grade. Talquetamab induced a substantial response among patients with heavily pretreated relapsed or refractory multiple myeloma. (Funded by Janssen Research and Development; MonumenTAL-1 ClinicalTrials.gov number, NCT03399799.).

Endogenous glutamine is rate-limiting for anti-CD3 and anti-CD28 induced CD4+ T-cell proliferation and glycolytic activity under hypoxia and normoxia.

To meet the demand for energy and biomass, T lymphocytes (T cells) activated to proliferation and clonal expansion, require uptake and metabolism of glucose (Gluc) and the amino acid (AA) glutamine (Gln). Whereas exogenous Gln is converted to glutamate (Glu) by glutaminase (GLS), Gln is also synthesized from the endogenous pool of AA through Glu and activity of glutamine synthase (GS). Most of this knowledge comes from studies on cell cultures under ambient oxygen conditions (normoxia, 21% O2). However, in vivo, antigen induced T-cell activation often occurs under moderately hypoxic (1-4% O2) conditions and at various levels of exogenous nutrients. Here, CD4+ T cells were stimulated for 72 h with antibodies targeting the CD3 and CD28 markers at normoxia and hypoxia (1% O2). This was done in the presence and absence of the GLS and GS inhibitors, Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide (BPTES) and methionine sulfoximine (MSO) and at various combinations of exogenous Gluc, Gln and pyruvate (Pyr) for the last 12 h of stimulation. We found that T-cell proliferation, viability and levels of endogenous AA were significantly influenced by the availability of exogenous Gln, Gluc and Pyr as well as inhibition of GLS and GS. Moreover, inhibition of GLS and GS and levels of oxygen differentially influenced oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Finally, BPTES-dependent down-regulation of ECAR was associated with reduced hexokinase (HK) activity at both normoxia and hypoxia. Our results demonstrate that Gln availability and metabolism is rate-limiting for CD4+ T-cell activity.

LAG3 associates with TCR-CD3 complexes and suppresses signaling by driving co-receptor-Lck dissociation.

LAG3 is an inhibitory receptor that is highly expressed on exhausted T cells. Although LAG3-targeting immunotherapeutics are currently in clinical trials, how LAG3 inhibits T cell function remains unclear. Here, we show that LAG3 moved to the immunological synapse and associated with the T cell receptor (TCR)-CD3 complex in CD4+ and CD8+ T cells, in the absence of binding to major histocompatibility complex class II-its canonical ligand. Mechanistically, a phylogenetically conserved, acidic, tandem glutamic acid-proline repeat in the LAG3 cytoplasmic tail lowered the pH at the immune synapse and caused dissociation of the tyrosine kinase Lck from the CD4 or CD8 co-receptor, which resulted in a loss of co-receptor-TCR signaling and limited T cell activation. These observations indicated that LAG3 functioned as a signal disruptor in a major histocompatibility complex class II-independent manner, and provide insight into the mechanism of action of LAG3-targeting immunotherapies.

IBI379, a novel B cell maturation antigen/CD3 bispecific T-cell engager, displays high antitumor efficacy in preclinical models of multiple myeloma.

Multiple myeloma (MM) is a hematological malignancy that results from the malignant proliferation of plasma cells in the bone marrow. B cell maturation antigen (BCMA) is highly selectively expressed in malignant plasma cells and is a novel therapeutic target for MM. Here, we developed a bispecific T cell engager, IBI379, that targets BCMA and CD3, and investigated its antitumor efficacy against MM. IBI379 showed strong binding affinity with both BCMA and CD3, which triggered T cell activation, proliferation, and cytokine release. An in vitro study demonstrated that IBI379 induced the lysis of MM cells expressing differing levels of BCMA on the cell surface. Administration of IBI379 in H929 or Daudi-BCMA cell xenograft mouse models significantly inhibited tumor growth without inducing body weight loss. The mechanism of action study revealed the accumulation of CD4+CD8+ T cells and granzyme B-positive T cells in tumors that were treated with IBI379. Moreover, administration of low dose of IBI379 in cynomolgus monkeys was well-tolerated and induced the depletion of BCMA+ B cells and a mild transient increase of cytokines. Collectively, these results demonstrate that IBI379 is a highly potent therapeutic strategy for depleting BCMA-positive B cells and is a promising approach for the treatment of MM.

Combined IL-2, agonistic CD3 and 4-1BB stimulation preserve clonotype hierarchy in propagated non-small cell lung cancer tumor-infiltrating lymphocytes.

BACKGROUND: Adoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TIL) yielded clinical benefit in patients with checkpoint blockade immunotherapy-refractory non-small cell lung cancer (NSCLC) prompting a renewed interest in TIL-ACT. This preclinical study explores the feasibility of producing a NSCLC TIL product with sufficient numbers and enhanced attributes using an improved culture method. METHODS: TIL from resected NSCLC tumors were initially cultured using (1) the traditional method using interleukin (IL)-2 alone in 24-well plates (TIL 1.0) or (2) IL-2 in combination with agonistic antibodies against CD3 and 4-1BB (Urelumab) in a G-Rex flask (TIL 3.0). TIL subsequently underwent a rapid expansion protocol (REP) with anti-CD3. Before and after the REP, expanded TIL were phenotyped and the complementarity-determining region 3 ß variable region of the T-cell receptor (TCR) was sequenced to assess the T-cell repertoire. RESULTS: TIL 3.0 robustly expanded NSCLC TIL while enriching for CD8+ TIL in a shorter manufacturing time when compared with the traditional TIL 1.0 method, achieving a higher success rate and producing 5.3-fold more TIL per successful expansion. The higher proliferative capacity and CD8 content of TIL 3.0 was also observed after the REP. Both steps of expansion did not terminally differentiate/exhaust the TIL but a lesser differentiated population was observed after the first step. TIL initially expanded with the 3.0 method exhibited higher breadth of clonotypes than TIL 1.0 corresponding to a higher repertoire homology with the original tumor, including a higher proportion of the top 10 most prevalent clones from the tumor. TIL 3.0 also retained a higher proportion of putative tumor-specific TCR when compared with TIL 1.0. Numerical expansion of TIL in a REP was found to perturb the clonal hierarchy and lessen the proportion of putative tumor-specific TIL from the TIL 3.0 process. CONCLUSIONS: We report the feasibility of robustly expanding a T-cell repertoire recapitulating the clonal hierarchy of the T cells in the NSCLC tumor, including a large number of putative tumor-specific TIL clones, using the TIL 3.0 methodology. If scaled up and employed as a sole expansion platform, the robustness and speed of TIL 3.0 may facilitate the testing of TIL-ACT approaches in NSCLC.

Three-dimensional colon cancer organoids model the response to CEA-CD3 T-cell engagers.

Rationale: The CEA-CD3 T cell bispecific antibody cibisatamab (CEA-TCB) is currently undergoing clinical trials. Here we study its performance against three-dimensional tumor organoids in cocultures with T cells as compared to a higher affinity CEACAM5-CD3 (CEACAM5-TCB) bispecific antibody using time-lapse confocal microscopy. Methods: Pre-labelled spheroids derived from colon cancer cell lines and primary organoids derived from four colorectal cancer surgical specimens, which expressed different graded levels of CEA, were exposed in cocultures to T lymphocytes. Cocultures were treated with CEA-CD3 T-cell engagers and were followed by live confocal microscopy. Caspase 3 activation detected in real-time was used as an indicator of tumor cell death. Co-cultures were also set up with autologous tumor-associated fibroblasts to test the co-stimulatory effect of a fibroblast activated protein (FAP)- targeted 4-1BBL bispecific antibody fusion protein currently undergoing clinical trials. Results: Tumor-cell killing of 3D colon carcinoma cultures was dependent on the levels of surface CEA expression, in such a way that the lower affinity agent (CEA-TCB) did not mediate killing by human preactivated T cells below a certain CEA expression threshold, while the high affinity construct (CEACAM5-TCB) remained active on the low CEA expressing organoids. Modelling heterogeneity in the levels of CEA expression by coculturing CEA high and low organoids showed measurable but weak bystander killing. Cocultures of tumor organoids, autologous fibroblasts and T cells allowed to observe a costimulatory effect of anti-FAP-4-1BBL both to release IFNγ and to attain more efficacious tumor cell killing. Conclusion: Three-dimensional tumor cocultures with T cells using live confocal microscopy provide suitable models to test the requirements for colon-cancer redirected killing as elicited by CEA-targeted T-cell engagers undergoing clinical trials and treatment allow combinations to be tested in a relevant preclinical system.

Changes of CD3+CD56+ γδ T cell number and apoptosis during hospital admission are related to mortality in septic patients.

Immunoparalysis and apoptosis of T cells are serious problems for the evolution of septic patients. We aimed to relate changes in the number of αß and γδ T cells during hospital stay to the poor evolution of sepsis. In this prospective study, we recruited a total of 92 septic patients from the Emergency and Intensive Care Departments of two Hospitals, according to the latest criteria for the definition and management of sepsis. According to the severity of the septic process, there was a progressive decrease in T cells, being much more intense in γδ T cells. This decrease recovered in surviving patients, but CD3+CD56+ γδ T cells continued to decreased during hospital stay in non-surviving patients. Apoptosis increased in sepsis. Cell death of CD3+CD56+ γδ T cells progressively increased according to the severity of sepsis, especially in non-surviving patients.

CD81 costimulation skews CAR transduction toward naive T cells.

Adoptive cellular therapy using chimeric antigen receptors (CARs) has revolutionized our treatment of relapsed B cell malignancies and is currently being integrated into standard therapy. The impact of selecting specific T cell subsets for CAR transduction remains under investigation. Previous studies demonstrated that effector T cells derived from naive, rather than central memory T cells mediate more potent antitumor effects. Here, we investigate a method to skew CAR transduction toward naive T cells without physical cell sorting. Viral-mediated CAR transduction requires ex vivo T cell activation, traditionally achieved using antibody-mediated strategies. CD81 is a T cell costimulatory molecule that when combined with CD3 and CD28 enhances naive T cell activation. We interrogate the effect of CD81 costimulation on resultant CAR transduction. We identify that upon CD81-mediated activation, naive T cells lose their identifying surface phenotype and switch to a memory phenotype. By prelabeling naive T cells and tracking them through T cell activation and CAR transduction, we document that CD81 costimulation enhanced naive T cell activation and resultantly generated a CAR T cell product enriched with naive-derived CAR T cells.

Enriched CD45RA-CD62L+ central memory T and decreased CD3+CD56+ natural killer T lymphocyte subsets in the rectum of ulcerative colitis patients.

OBJECTIVES: To investigate the distinctive features of lymphocytes promoting inflammation in ulcerative colitis. METHODS: We performed flow cytometric analysis of peripheral blood mononuclear cells (PBMCs) and colorectal mucosa lymphocytes in ulcerative colitis patients (n = 13) and control patients (n = 5). RESULTS: CD62L+/CD3+CD4+ (35.7 ± 14.0% vs. 19.9 ± 6.4%) and CD62L+/CD3+CD4- cells (17.1 ± 17.4% vs. 2.4 ± 3.9%) were higher in the rectum of ulcerative colitis patients than in control patients. Subpopulation analysis revealed that CD45RA-CD62L+/CD3+CD4+, that is, central memory T cell fraction in CD4+ T cells, was significantly increased in the rectum of ulcerative colitis, compared to that in control patients (23.3 ± 10.5% vs. 8.2 ± 4.0%). Comparison of rectum and colon samples in ulcerative colitis patients indicated that CD56+/CD3+ was decreased in the rectum compared to that in the colon (11.3 ± 12.5% vs. 21.3 ± 16.5%). The ratio of CD56+/CD3+ was also decreased in the rectum of active ulcerative colitis patients compared to that in ulcerative colitis patients at the endoscopic remission stages (2.8 ± 1.7% vs. 18.5 ± 13.3%). CONCLUSION: We demonstrated that CD62L+ T lymphocytes, particularly the CD45RA-CD62L+ T cell subset that represents central memory T cells, were increased in the rectum of patients with ulcerative colitis. In addition, the CD56+/CD3+ subset (natural killer T cells) was decreased in the rectum compared to that of less inflamed colonic mucosa. These results suggest that the enrichment of central memory T lymphocytes and the reduction of natural killer T cells in the gut mucosa are involved in the pathogenesis of ulcerative colitis.