Reduced Effector Function Research Articles

Intratumoral CXCL12 Gradients Contextualize Tumor Cell Invasion, Migration and Immune Suppression in Breast Cancer.

Although the CXCL12/CXCR4 pathway has been prior investigated for its prometastatic and immuno- suppressive roles in the tumor microenvironment, evidence on the spatiotemporal regulation of these hallmarks has been lacking. Here, we demonstrate that CXCL12 forms a gradient specifically around cancer cell intravasation doorways, also known as Tumor Microenvironment of Metastasis (TMEM) doorways, thus facilitating the chemotactic translocation of prometastatic tumor cells expressing CXCR4 toward the perivascular TMEM doorways for subsequent entry into peripheral circulation. Fur- thermore, we demonstrate that the CXCL12-rich micro-environment around TMEM doorways may cre- ate immunosuppressive niches, whereby CD8 + T cells, despite being attracted to these regions, often exhibit reduced effector functions, limiting their efficacy. While the CXCL12/CXCR4 pathway can mini- mally influence the overall composition of immune cell populations, it biases the distribution of CD8 + T cells away from TMEM doorways, justifying its prior-established role as immunosuppressive factor for CD8 + T cells. Our research suggests that the complex interactions between CXCL12 and the various tumor and immune cell types contributes not only to the completion of the initial steps of the metastatic cascade, but also offers an immunological "sanctuary" to prometastatic tumor cells homed around TMEM doorways. Overall, our study enhances our current understanding on the mechanisms, via which CXCL12 orchestrates tumor cell behavior and immune dynamics, potentially guiding future thera- peutic strategies to combat breast cancer metastasis and improve anti-tumor immunity.

CD4+ T cell help during early acute hepacivirus infection is critical for viral clearance and the generation of a liver-homing CD103+CD49a+ effector CD8+ T cell subset.

In hepatitis C virus (HCV) infection, CD4+ and CD8+ T cells are crucial for viral control. However, a detailed understanding of the kinetic of CD4+ T cell help and its role in the generation of different CD8+ T cell subsets during acute infection is lacking. The absence of a small HCV animal model has impeded mechanistic studies of hepatic antiviral T cell immunity and HCV vaccine development. In this study, we used a recently developed HCV-related rodent hepacivirus infection mouse model to investigate the impact of CD4+ T cell help on the hepatic CD8+ T cell response and viral clearance during hepacivirus infection in vivo. Our results revealed a specific kinetic of CD4+ T cell dependency during acute infection. Early CD4+ T cell help was essential for CD8+ T cell priming and viral clearance, while CD4+ T cells became dispensable during later stages of acute infection. Effector CD8+ T cells directly mediated timely hepacivirus clearance. An analysis of hepatic CD8+ T cells specific for two different viral epitopes revealed the induction of subsets of liver-homing CD103+CD49a+ and CD103-CD49a+ effector CD8+ T cells with elevated IFN-γ and TNF-α production. CD103+CD49a+ T cells further persisted as tissue-resident memory subsets. A lack of CD4+ T cell help and CD40L-CD40 interactions resulted in reduced effector functions and phenotypical changes in effector CD8+ T cells and a specific loss of the CD103+CD49a+ subset. In summary, our study shows that early CD4+ T cell help through CD40L signaling is essential for priming functional effector CD8+ T cell subsets, including unique liver-homing subsets, and hepacivirus clearance.

Targeting Tumor-Associated Sialic Acids Using Chimeric Switch Receptors Based on Siglec-9 Enhances the Antitumor Efficacy of Engineered T Cells.

Cancer exploits different mechanisms to escape T-cell immunosurveillance, including overexpression of checkpoint ligands, secretion of immunosuppressive molecules, and aberrant glycosylation. Herein, we report that IFNγ, a potent immunomodulator secreted in the tumor microenvironment, can induce α2,6 hypersialylation in cancer cell lines derived from various histologies. We focused on Siglec-9, a receptor for sialic acid moieties, and demonstrated that the Siglec-9+ T-cell population displayed reduced effector function. We speculated that Siglec-9 in primary human T cells can act as a checkpoint molecule and demonstrated that knocking out Siglec-9 using a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system enhanced the functionality of primary human T cells. Finally, we aimed to augment cancer-specific T-cell activity by taking advantage of tumor hypersialylation. Thus, we designed several Siglec-9-based chimeric switch receptors (CSR), which included an intracellular moiety derived from costimulatory molecules (CD28/41BB) and different hinge regions. In an antigen-specific context, T cells transduced with Siglec-9 CSRs demonstrated increased cytokine secretions and upregulation of activation markers. Moreover, T cells equipped with specific Siglec-9 CSRs mediated robust antitumor activity in a xenograft model of human tumors. Overall, this work sheds light on tumor evasion mechanisms mediated by sialylated residues and exemplifies an approach to improve engineered T cell-based cancer treatment. See related Spotlight by Abken, p. 1310.

NK cells with adhesion defects and reduced cytotoxic functions are associated with a poor prognosis in multiple myeloma

AbstractThe promising results obtained with immunotherapeutic approaches for multiple myeloma (MM) call for a better stratification of patients based on immune components. The most pressing being cytotoxic lymphocytes such as natural killer (NK) cells that are mandatory for MM surveillance and therapy. Here, we performed a single-cell RNA sequencing analysis of NK cells from 10 patients with MM and 10 age/sex–matched healthy donors that revealed important transcriptomic changes in the NK cell landscape affecting both the bone marrow (BM) and peripheral blood compartment. The frequency of mature cytotoxic CD56dim NK cell subsets was reduced in patients with MM at the advantage of late-stage NK cell subsets expressing NF-κB and interferon-I inflammatory signatures. These NK cell subsets accumulating in patients with MM were characterized by low CD16 and CD226 expression and poor cytotoxic functions. MM CD16/CD226Lo NK cells also had adhesion defects with reduced lymphocyte function-associated antigen 1 (LFA-1) integrin activation and actin polymerization that may account for their limited effector functions in vitro. Finally, analysis of BM-infiltrating NK cells in a retrospective cohort of 177 patients with MM from the Intergroupe Francophone du Myélome (IFM) 2009 trial demonstrated that a high frequency of NK cells and their low CD16 and CD226 expression were associated with a shorter overall survival. Thus, CD16/CD226Lo NK cells with reduced effector functions accumulate along MM development and negatively affect patients’ clinical outcomes. Given the growing interest in harnessing NK cells to treat myeloma, this improved knowledge around MM–associated NK cell dysfunction will stimulate the development of more efficient immunotherapeutic drugs against MM.

Expression of CD39 is associated with T cell exhaustion in ovarian cancer and its blockade reverts T cell dysfunction

ABSTRACT Immune exhaustion is a hallmark of ovarian cancer. Using multiparametric flow cytometry, the study aimed to analyze protein expression of novel immunological targets on CD3+ T cells isolated from the peripheral blood (n = 20), malignant ascites (n = 16), and tumor tissue (n = 6) of patients with ovarian cancer (OVCA). The study revealed an increased proportion of effector memory CD8+ T cells in OVCA tissue and malignant ascites. An OVCA-characteristic PD-1high CD8+ T cell population was detected, which differed from PD-1lowCD8+ T cells by increased co-expression of TIGIT, CD39, and HLA-DR. In addition, these OVCA-characteristic CD8+ T cells showed reduced expression of the transcription factor TCF-1, which may also indicate reduced effector function and memory formation. On the contrary, the transcription factor TOX, which significantly regulates terminal T cell-exhaustion, was found more frequently in these cells. Further protein and gene analysis showed that CD39 and CD73 were also expressed on OVCA tumor cells isolated from solid tumors (n = 14) and malignant ascites (n = 9). In the latter compartment, CD39 and CD73 were also associated with the expression of the “don’t eat me” molecule CD24 on tumor cells. Additionally, ascites-derived CD24+EpCAM+ tumor cells showed a higher frequency of CD39+ or CD73+ cells. Furthermore, CD39 expression was associated with unfavorable clinical parameters. Expression of CD39 on T cells was upregulated through CD3/CD28 stimulation and its blockade by a newly developed nanobody construct resulted in increased proliferation (eFluor), activation (CD25 and CD134), and production of cytotoxic cytokines (IFN-γ, TNF-α, and granzyme-B) of CD8+ T cells.

Iron controls the development of airway hyperreactivity by regulating ILC2 metabolism and effector function.

Group 2 innate lymphoid cells (ILC2s) rapidly induce a type 2 inflammation in the lungs in response to allergens. Here, we focused on the role of iron, a critical nutritional trace element, on ILC2 function and asthma pathogenesis. We found that transferrin receptor 1 (TfR1) is rapidly up-regulated and functional during ILC2 activation in the lungs, and blocking transferrin uptake reduces ILC2 expansion and activation. Iron deprivation reprogrammed ILC2 metabolism, inducing a HIF-1α-driven up-regulation of glycolysis and inhibition of oxidative mitochondrial activity. Consequently, we observed that in vivo iron chelation or induction of hypoferremia reduced the development of airway hyperreactivity in experimental models of ILC2-driven allergic asthma. Human circulating ILC2s rapidly induced TfR1 during activation, whereas inhibition of iron uptake or iron deprivation reduced effector functions. Last, we found a negative relationship between circulating ILC2 TfR1 expression and airway function in cohorts of patients with asthma. Collectively, our studies define cellular iron as a critical regulator of ILC2 function.

Dysfunctional natural killer cells can be reprogrammed to regain anti-tumor activity

Natural killer (NK) cells are critical to the innate immune system, as they recognize antigens without prior sensitization, and contribute to the control and clearance of viral infections and cancer. However, a significant proportion of NK cells in mice and humans do not express classical inhibitory receptors during their education process and are rendered naturally “anergic”, i.e., exhibiting reduced effector functions. The molecular events leading to NK cell anergy as well as their relation to those underlying NK cell exhaustion that arises from overstimulation in chronic conditions, remain unknown. Here, we characterize the “anergic” phenotype and demonstrate functional, transcriptional, and phenotypic similarities to the “exhausted” state in tumor-infiltrating NK cells. Furthermore, we identify zinc finger transcription factor Egr2 and diacylglycerol kinase DGKα as common negative regulators controlling NK cell dysfunction. Finally, experiments in a 3D organotypic spheroid culture model and an in vivo tumor model suggest that a nanoparticle-based delivery platform can reprogram these dysfunctional natural killer cell populations in their native microenvironment. This approach may become clinically relevant for the development of novel anti-tumor immunotherapeutic strategies.

Abstract 4012: SOT302: Dual expression of exogenous glutamine oxaloacetate transaminase (GOT2) and TP53-induced glycolysis and apoptosis regulator (TIGAR) enhance CAR T cell activity in preclinical solid tumor models

Abstract Background: Despite success in hematological malignancies, CAR T therapy has had limited efficacy against solid tumors. The suppressive tumor microenvironment limits CAR T activity through various mechanisms including competition for available nutrients and chronic stimulation resulting in reduced effector functions or T cell exhaustion. CAR T cells with enhanced metabolic fitness or more durable memory phenotype could potentially improve the clinical outcome in solid tumors. GOT2 plays an important role in mitochondrial function and maintenance of redox homeostasis. We have previously demonstrated enhancement of CAR T cells overexpressing GOT2. TIGAR is an enzyme known to promote antioxidative activites and reduce reactive oxygen species and has a role in protecting against apoptosis. Here we tested SOT302 CAR T cells expressing both GOT2 and TIGAR in preclinical solid tumor models. Results: Expression of GOT2 and TIGAR transgenes were confirmed by qRT-PCR and western blot. Activity of GOT2 and TIGAR were confirmed by measuring aminotransferase activity (AST) and glutathione production, respectively. Compared to control CAR T cells, SOT302 is enriched for CD8+ Tscm cells and has a higher percentage functional, non-senescent (CD28+CD57-) cells. SOT302 has similar cytokine expression and cytotoxicity in standard co-culture assays compared to controls. SOT302 cells were chronically stimulated with plate-bound antigen every 3-4 days for 4 rounds. SOT302 had greater expansion of cells and were less exhausted and had better cytolytic capacity compared to controls. SOT302 cells were then evaluated in tumor xenograft mouse models. Consistent with our previous findings, CAR T cells expressing the single GOT2 transgene were more efficacious compared to CAR T cells alone. Expression of the single TIGAR transgene had no apparent benefit over CAR alone, however SOT302 cells expressing both GOT2 and TIGAR had superior anti-tumor activity compared to CAR alone and CAR+GOT2. No overt toxicity or significant body weight loss was observed. Ex vivo analyses to measure peripheral expansion, tumor infiltration and exhaustion were also performed. Preliminary findings indicate that SOT302 has better peripheral expansion compared to CAR alone and similar expansion compared to CAR+GOT2 controls. SOT302 cells had better tumor infiltration compared to both the CAR alone and CAR+GOT2 cells. Tumor infiltrating SOT302 cells also exhibited lower levels of exhaustion compared to CAR alone or CAR+GOT2 controls. Summary: SOT302 cells express exogenous GOT2 and TIGAR transgenes and have superior anti-tumor activity in preclinical solid tumor models. These data suggest that SOT302 may be a promising candidate for patients with solid tumor cancers. Citation Format: Emily Kuiper, Samyabrata Bhaduri, Daniel Garafola, Kshitij Sharma, Jennifer Coccia, Kathleen Whiteman, Amy Jensen-Smith. SOT302: Dual expression of exogenous glutamine oxaloacetate transaminase (GOT2) and TP53-induced glycolysis and apoptosis regulator (TIGAR) enhance CAR T cell activity in preclinical solid tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4012.

CD8+ T Cells Mediate Lethal Lung Pathology in the Absence of PD-L1 and Type I Interferon Signalling following LCMV Infection.

CD8+ T cells are critical to the adaptive immune response against viral pathogens. However, overwhelming antigen exposure can result in their exhaustion, characterised by reduced effector function, failure to clear virus, and the upregulation of inhibitory receptors, including programmed cell death 1 (PD-1). However, exhausted T cell responses can be "re-invigorated" by inhibiting PD-1 or the primary ligand of PD-1: PD-L1. Further, the absence of the type I interferon receptor IFNAR1 also results in T cell exhaustion and virus persistence in lymphocytic choriomeningitis virus Armstrong (LCMV-Arm)-infected mice. In this study, utilizing single- and double-knockout mice, we aimed to determine whether ablation of PD-1 could restore T cell functionality in the absence of IFNAR1 signalling in LCMV-Arm-infected mice. Surprisingly, this did not re-invigorate the T cell response and instead, it converted chronic LCMV-Arm infection into a lethal disease characterized by severe lung inflammation with an infiltration of neutrophils and T cells. Depletion of CD8+ T cells, but not neutrophils, rescued mice from lethal disease, demonstrating that IFNAR1 is required to prevent T cell exhaustion and virus persistence in LCMV-Arm infection, and in the absence of IFNAR1, PD-L1 is required for survival. This reveals an important interplay between IFNAR1 and PD-L1 with implications for therapeutics targeting these pathways.

TCell Exhaustion.

Tcell responses must be balanced to ensure adequate protection against malignant transformation and an array of pathogens while also limiting damage to healthy cells and preventing autoimmunity. Tcell exhaustion serves as a regulatory mechanism to limit the activity and effector function of Tcells undergoing chronic antigen stimulation. Exhausted Tcells exhibit poor proliferative potential; high inhibitory receptor expression; altered transcriptome, epigenome, and metabolism; and, most importantly, reduced effector function. While exhaustion helps to restrain damage caused by aberrant Tcells in settings of autoimmune disease, it also limits the ability of cells to respond against persistent infection and cancer, leading to disease progression. Here we review the process of Tcell exhaustion, detailing the key characteristics and drivers as well as highlighting our current understanding of the underlying transcriptional and epigenetic programming. We also discuss how exhaustion can be targeted to enhance Tcell functionality in cancer. Expected final online publication date for the Annual Review of Immunology, Volume 42 is April 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Chronic chromosome instability induced by Plk1 results in immune suppression in breast cancer

Chromosome instability (CIN) contributes to resistance to therapies and tumor evolution. Although natural killer (NK) cells can eliminate cells with complex karyotypes, high-CIN human tumors have an immunosuppressive phenotype. To understand which CIN-associated molecular features alter immune recognition during tumor evolution, we overexpress Polo-like kinase 1 (Plk1) in a Her2+ breast cancer model. These high-CIN tumors activate a senescence-associated secretory phenotype (SASP), upregulate PD-L1 and CD206, and induce non-cell-autonomous nuclear factor κB (NF-κβ) signaling, facilitating immune evasion. Single-cell RNA sequencing from pre-neoplastic mammary glands unveiled the presence of Arg1+ macrophages, NK cells with reduced effector functions, and increased resting regulatory Tcell infiltration. We further show that high PLK1-expressing human breast tumors display gene expression patterns associated with SASP, NF-κβ signaling, and immune suppression. These findings underscore the need to understand the immune landscape in CIN tumors to identify more effective therapies, potentially combining immune checkpoint or NF-κβ inhibitors with current treatments.

T-Bet Overexpression Increases 4-1BB-Costimulated CAR T Cell Potency

Data from our lab and others has demonstrated that CAR T cells form a mixed Th1/Th2 population which, in contrast to canonical T cell differentiation, produce both interferon gamma (IFN-γ) and IL-4. Whereas a growing body of evidence has highlighted the importance of IFN-γ in the CAR T cell response, IL-4 can have immune-suppressive functions. IFN-γ from CD4+ CAR T cells (CAR4s), has been shown to promote cytotoxic effector functions of CD8+ CAR T cells (CAR8s), as well as bolster activation of endogenous immune cells including CD8 T cells and NK cells (Boulch et al Sci Immunol. 2021). Given the importance of IFN-γ to effective anti-tumor immune responses, we generated murine CAR T cells that target CD19 with a CD28 costimulatory domain (1928z) and compared them to CAR T cells that overexpress T-bet (T-bet-1928z) in an effort to skew CAR T cells away from IL-4 production and towards IFN-γ production. Upon stimulation of CAR T cells with the murine B-cell acute lymphoblastic leukemia (B-ALL) cell line E2A-PBX, fewer T-bet-1928z CAR4s produced IL-4 compared to 1928z CAR4s. This suggests T-bet overexpression is a viable strategy to reduce Th2-like programming of effector functions in CAR4s. To determine the impact of T-bet overexpression on early CAR T cell efficacy in vivo, we adoptively transferred 1928z or T-bet-1928z CAR T cells into leukemia-bearing C57Bl6/J recipients and analyzed CAR T cells from the bone marrow at peak expansion of CAR T cells as well as post-contraction. Both CARs eliminated CD19+ cells from the bone marrow and mediated long-term survival. T-bet-1928z CAR4s demonstrated increased expansion at Day 4 compared to 1928z CAR4s (p=0.0412), although there was no significant difference in CAR8 numbers at this timepoint. At Day 11 post-CAR, we observed an increase in the number of T-bet-1928z CAR T cells present in the bone marrow (p<0.0001), observed in both CAR4s (p<0.0001) and CAR8s (p=0.0074). While levels of memory-associated transcription factors TCF1 and FOXO1 were the same between CARs, fewer T-bet-1928z CAR T cells expressed IL7Rα (p=0.0002) and eomesodermin (p<0.0001), which are associated with the ability of T cells to self-renew and persist in the bone marrow. To determine the impact of T-bet on the persistence and renewal capacity of 1928z CAR T cells, we adoptively transferred 1928z or T-bet-1928z CAR T cells into leukemia-bearing RAG1-knockout recipients to eliminate endogenous T cell responses to E2A-PBX. After initial leukemia clearance, we rechallenged these mice with E2A-PBX and analyzed the bone marrow after 13 days. Both CARs were able to clear the leukemia rechallenge. While CAR4s were present in the bone marrow at similar numbers, the number of CAR8s was significantly higher among mice treated with T-bet-1928z CAR T cells (p=0.0317). Our results suggest that while T-bet may enhance in vivo expansion of CAR T cells, it provides no long-term benefit or deficit to the CAR T cell response. 4-1BB co-stimulated CAR T cells are less potent than CD28-costimulated CAR T cells and have reduced effector functions. We therefore hypothesized that T-bet overexpression would improve the efficacy of 4-1BB-costimulated CAR T cells by boosting effector functions and potency. To test this, we transduced T cells with a CAR targeting CD19 that contains a 4-1BB costimulatory domain (19BBz), or a 19BBz CAR and T-bet (19BBz-T-bet). We adoptively transferred 19BBz or 19BBz-T-bet CAR T cells into leukemia-bearing C57Bl6/J recipients. Despite no difference in CAR T cell numbers in the bone marrow, we observed fewer leukemia cells in mice treated with 19BBz-T-bet CAR T cells (p=0.0318) at Day 4 post-CAR. Treatment with 19BBz-T-bet CAR T cells also significantly improved survival of leukemia-bearing mice compared to 19BBz CAR T cells (p=0.0494). Our data demonstrate T-bet overexpression improves CD28-costimulated CAR T cell expansion but does little to augment long-term responses and diminishes features associated with capacity for self-renewal. In contrast, T-bet overexpression boosts the potency of 4-1BB-costimulated CAR T cells without impairing persistence, resulting in improved overall efficacy of 4-1BB-costimulated CAR T cell therapies.

Innate Cell Engager (ICE®) AFM13 Combined with Preactivated and Expanded (P+E) Cord Blood (CB)-Derived Natural Killer (NK) Cells for Patients with Refractory CD30-Positive Lymphomas: Final Results

CONCLUSIONS Pts with refractory Hodgkin (HL) and other CD30+ lymphomas have few effective therapies available and targeted NK cell immunotherapy is an active area of research. Transfer of non-targeted NK cells has shown limited clinical benefit as target recognition of cancer cells by NK cells constitutes a barrier. The innate cell engager AFM13 is a first-in-class CD30/CD16A bispecific antibody construct that induces selective killing of CD30+ tumor cells by engaging and activating NK cells. As a single agent, AFM13 has limited clinical activity against HL, likely due to the reduced effector function of autologous NK cells in these pts, which provides the rationale for combining AFM13 with allogeneic NK cells. Our prior preclinical work identified a promising combination of CB-derived NK cells that were first IL-12/IL-15/IL-18-preactivated followed by ex vivo expansion (P+E) with engineered K562 feeder cells expressing membrane-bound IL-21, 4-1BBL and CD48 and exogenous IL-2, and subsequently complexed with AFM13 just before infusion; these cytokine-induced memory-like NK cells presented chimeric antigen receptor (CAR)-NK-like features and increased in vitro and in vivo antitumor activity compared to either non-AFM13-precomplexed P+E NK cells or to AFM13 alone (Kerbauy et al, Clin Cancer Res 2021). Building on this work we wished to study this CB NK-cell therapy in pts with refractory CD30+ lymphomas. This investigator-initiated single-center phase I-II trial (NCT04074746) evaluated the safety and activity of P+E AFM13-precomplexed CB NK cells followed by systemic AFM13. Pts ages 15-75 with CD30+ lymphomas refractory to brentuximab vedotin were enrolled. The goals were to identify the recommended phase 2 dose (RP2D) of NK cells, estimate the overall (ORR) and complete response (CR), event-free (EFS) and overall survival (OS) rates, and study NK cell activation and persistence. Each treatment cycle consisted of fludarabine/cyclophosphamide (days −5 to −3) followed by infusion (day 0) of the AFM13-precomplexed CB NK cells, cultured for 14 days as described above, and three weekly IV infusions of AFM13 (200 mg, days 7, 14 and 21). Two cycles were planned for pts 1-19 and up to 4 cycles from patient 20 on. Response was evaluated on day 28 of each cycle. Enrollment proceeded at 3 dose levels (DL) of 10 6, 10 7 and 10 8 NK/Kg, respectively. Each patient received the same NK dose in all cycles. Between 09/20-12/22 we enrolled 42 pts (37 HL, 5 NHL), pretreated with a median 7 lines, at DL1 (N=3), DL2 (N=3) and DL3 (N=36), with 1 (N=2), 2 (N=21), 3 (N=3) or 4 cycles (N=16). All had active progressive disease at enrollment and no bridging therapy was given. The cords used for each of the 117 cycles were selected without consideration for HLA match, which was 0/6 (N=53), 1/6 (N=46), 2/6 (N=14), 3/6 (N=3) and 4/6 (N=1). The product infused consisted of >94% NK cells (expanded >2,700 fold), with only 0.02% T cells. The NK cells were 96% viable, 91% bound to AFM13, and 97.6% CD16+. The products were infused fresh. There was 1 grade 2 infusion-related reaction (IRR) in 115 infusions of AFM13-NK (0.8%) and 27 IRR infusion related reactions in 350 infusions of AFM13 (7.7%) (1 G3, 26 G2). We saw no cases of CRS, ICANS or GVHD of any grade. DL3 (10 8 NK/Kg) was established as the RP2D. The ORR and CR were 92.8% and 66.7%, respectively (94.4% and 72.2%, respectively, in 36 pts treated at the RP2D). Nine pts had a response consolidated with SCT (5 allo, 4 auto). At median follow-up of 14 (6-34) months, the EFS/OS rates are 31%/76%; median EFS/OS are 8 months/not reached. No relapses were associated with antigen loss. In the subset of pts planned for 4 cycles at RP2D, 15 of 23 pts remained event free at 6 mo (4 pts with and 11 without SCT consolidation), for 6-month EFS/OS rates of 65%/83%. Overall, 8 of the 9 pts (89%) receiving a consolidation SCT are event free at 10 to 34 months. AFM-bound CB NK cells were detected in blood from day 1 post infusion for up to 3 weeks. Donor NK levels followed similar patterns after each cycle, which argues against a sensitization after the first NK infusion. CyTOF studies showed increased expression of AFM13 and activation markers in donor NK cells. We confirmed trafficking of AFM13-bound donor NK cells to tumor sites shortly after infusion. CB-derived cytokine-induced memory-like NK cells precomplexed with AFM13 have excellent tolerability and activity for pts with heavily pretreated and refractory CD30+ lymphoma.

High-throughput Western Blot for in vitro and in vivo assessment of human Ig specific cleaving enzymes

Abstract Antibodies play a vital role in the body’s immune defense mechanism. However, pathogenic Ig antibodies are thought to contribute to several autoimmune conditions and transplant rejection. Proteases derived from human pathogens can specifically cleave IgG into F(ab’)2 and Fc fragments, thus allowing for rapid clearance of IgG. This unique trait suggests a novel opportunity to use these molecules to treat autoantibody mediated diseases. Using our IMPACT platform leveraging machine learning, we have engineered Ig proteases that specifically cleave IgG, and treatment with these proteases causes cleavage below the hinge region, which can result in the generation of the following degradation products: 1) Fab’2 and Fc fragments lacking effector function; or 2) single chain cleaved fragments with reduced effector functions. To detect these species, we developed a method using JESS, an automated high throughput western blot instrument. Using this system, we can detect intact, single cleaved and fully cleaved IgG in a time and dose dependent manner in both in vitro and in vivo assays. In conclusion, we successfully optimized a high throughput assay format that is highly specific and sensitive in evaluating the cleavage activity of different Ig proteases and could be a powerful tool to assess efficacy of IgG cleaving enzymes in the clinic.

Reducing α-Synuclein in Human CSF; An Evaluation of Safety, Tolerability, Pharmacokinetics and Pharmacodynamics of MEDI1341, an α-Synuclein-specific antibody, in Healthy Volunteers and Parkinson’s Disease Patients (P1-11.007)

<h3>Objective:</h3> To evaluate the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of MEDI1341 (TAK-341) in single (SAD; NCT03272165) and multiple ascending dose (MAD; NCT04449484) studies undertaken in healthy volunteers (HVs) and Parkinson’s disease patients, respectively. <h3>Background:</h3> MEDI1341 is a human IgG1λ monoclonal antibody, engineered for selective, high-affinity binding of α-synuclein (ASN) and for reduced effector function. MEDI1341 recognises both monomeric and aggregated forms of human ASN. In rats and cynomolgus monkeys, MEDI1341 decreases free ASN in brain interstitial fluid and cerebrospinal fluid (CSF). In a mouse model, MEDI1341 attenuates ASN axonal propagation and accumulation in the brain. <h3>Design/Methods:</h3> HVs aged 18–80 years were eligible for the SAD study. Subjects eligible for the MAD study were aged 40–85 years with a diagnosis of mild-moderate Parkinson’s disease according to the UK Parkinson’s Disease Society Brain Bank criteria and stage 1–3 on the Hoehn and Yahr scale. In the SAD study, N=49 subjects received placebo or MEDI1341 administered intravenously (IV) across six dose levels. In the MAD study, two dose levels were evaluated in Parkinson’s patients (N=25), administered IV every 4 weeks for 3 infusions. <h3>Results:</h3> Administration of MEDI1341 or placebo to HVs or Parkinson’s patients was not associated with any medically important changes in safety parameters across all doses tested. Dose-dependent changes in serum concentrations for MEDI1341 were observed across cohorts. CSF concentrations demonstrated central nervous system penetration of MEDI1341. PD data showed dose-dependent suppression of free ASN in CSF, consistent with the results seen in preclinical species. <h3>Conclusions:</h3> MEDI1341 was generally well tolerated following IV administration to HVs or Parkinson’s patients after single or repeat doses, respectively. The PK and PD data provide evidence of dose-dependent and selective ASN target engagement by MEDI1341 in the central compartment. Full study design and data from the Phase 1 studies will be presented. <b>Disclosure:</b> Dr. Shering has received personal compensation for serving as an employee of AstraZeneca. An immediate family member of Dr. Shering has received personal compensation for serving as an employee of eResearch Technology Inc. Dr. Shering has stock in AstraZeneca. Dr. Shering has received intellectual property interests from a discovery or technology relating to health care. Michael Pomfret has nothing to disclose. Dr. Kubiak has received personal compensation for serving as an employee of AstraZeneca. Dr. Kubiak has stock in AstraZeneca. Mrs. Pouliquen has received personal compensation for serving as an employee of AstraZeneca. Mrs. Pouliquen has received personal compensation for serving as an employee of GSK. Mrs. Pouliquen has stock in GSK. Mrs. Pouliquen has stock in AstraZeneca. Wei Yin has received personal compensation for serving as an employee of Takeda. Wei Yin has stock in Takeda. Arthur Simen has received personal compensation for serving as an employee of Takeda. Dr. Ratti has received personal compensation for serving as an employee of Takeda. Dr. Ratti has received personal compensation for serving as an employee of Biogen. Dr. Ratti has stock in Takeda. Dr. Ratti has stock in Biogen. Dr. Perkinton has stock in AstraZeneca. Dr. Tan has received personal compensation for serving as an employee of AstaZeneca. An immediate family member of Dr. Tan has received personal compensation in the range of $50,000-$99,999 for serving as a Consultant for SOBI. Dr. Tan has stock in AstraZeneca. Dr. Chessell has received personal compensation in the range of $100,000-$499,999 for serving as a Employee with AstraZeneca. Dr. Ostenfeld has received personal compensation for serving as an employee of AstraZeneca. Dr. Ostenfeld has stock in AstraZeneca. Dr. Ostenfeld has received intellectual property interests from a discovery or technology relating to health care.

TET2 guards against unchecked BATF3-induced CAR T cell expansion.

Further advances in cell engineering are needed to increase the efficacy of chimeric antigen receptor (CAR) and other T cell-based therapies1-5. As T cell differentiation and functional states are associated with distinct epigenetic profiles6,7, we hypothesized that epigenetic programming may provide a means to improve CAR T cell performance. Targeting the gene that encodes the epigenetic regulator ten-eleven translocation 2 (TET2)8 presents an interesting opportunity as its loss may enhance T cell memory9,10, albeit not cause malignancy9,11,12. Here we show that disruption of TET2 enhances T cell-mediated tumour rejection in leukaemia and prostate cancer models. However, loss of TET2 also enables antigen-independent CAR T cell clonal expansions that may eventually result in prominent systemic tissue infiltration. These clonal proliferations require biallelic TET2 disruption and sustained expression of the AP-1 factor BATF3 to drive a MYC-dependent proliferative program. This proliferative state is associated with reduced effector function that differs from both canonical T cell memory13,14 and exhaustion15,16 states, and is prone to the acquisition of secondary somatic mutations, establishing TET2 as a guardian against BATF3-induced CAR T cell proliferation and ensuing genomic instability. Our findings illustrate the potential of epigenetic programming to enhance T cell immunity but highlight the risk of unleashing unchecked proliferative responses.

Differential expression of CD8 defines phenotypically distinct cytotoxic T cells in cancer and multiple sclerosis.

Cytotoxic T lymphocytes take on a leading role in many immune-related diseases. They function as key effector immune cells fighting cancer cells, but they are also considerably involved in autoimmune diseases. Common to both situations, CD8+ T cells need to adapt their metabolism and effector function to the harsh and nutrient-deprived conditions of the disease-associated microenvironment. We used an in vitro starvation as well as rapamycin treatment protocol mimicking nutrient deprivation to generate CD8Low versus CD8High T cells and performed FACS-Sorting followed by transcriptomic profiling of the cytotoxic T cell subsets. Prominent markers identified in the CD8Low versus the CD8High T cells were then used to investigate the presence of these cell subsets in immune-related human diseases. Employing cancer tissue microarrays and PhenOptics multispectral imaging as well as flow cytometry, we studied these CD8+ T cell subsets in cancer and relapsing-remitting multiple sclerosis patients. Starvation induced a decreased expression of CD8, yielding a CD8Low T cell subpopulation with an altered transcriptomic signature and reduced effector function. CD8Low T cell showed enhanced ST2L and IL6ST (CD130) expression compared to CD8High T cells which expressed elevated KLRD1 (CD94) and granzyme B levels within the tumour microenvironment (TME). Spatial analysis revealed the presence of CD8High T cells in close proximity to tumour cells, while the CD8Low T cells resided at the tumour boundaries. Importantly, the number of tumour-infiltrating CD8Low T lymphocytes correlated with a poor prognosis as well as with enhanced cancer progression in human mammary carcinoma. We also found a reduced frequency of CD8Low T lymphocytes in a cohort of relapse (disease active) multiple sclerosis patients compared to healthy subjects during immune cell starvation in vitro. In summary, our data show that functionally distinct cytotoxic T lymphocytes can be identified based on their expression of CD8. Indicating a more general role in CD8 T cell immunity, these cells may play opposing roles in the TME, and also in the pathophysiology of autoimmune diseases such as multiple sclerosis.

Innate Cell Engager AFM13 Combined with Preactivated and Expanded Cord Blood-Derived NK Cells for Patients with Double Refractory CD30+ Lymphoma

Patients with multiple relapsed or refractory (R/R) Hodgkin lymphoma (HL) and other CD30+ lymphoma have few effective therapies available and targeted adoptive natural killer (NK) cell immunotherapy is an active area of investigation. Transfer of non-targeted NK cells has shown limited clinical benefit as target recognition of cancers by NK cells remains a substantial barrier. The innate cell engager (ICE®) construct, AFM13, is a first-in-class CD30/CD16A bispecific antibody construct that induces specific and selective killing of CD30+ tumor cells by engaging and activating NK cells. As a single agent, AFM13 has limited clinical activity against HL, likely due to the reduced effector function of autologous NK cells in these patients, which provides the rationale for the combination of AFM13 with allogeneic NK cells. Our prior preclinical work identified a promising combination of cord blood (CB)-derived NK cells that were first IL-12/IL-15/IL-18-preactivated followed by expansion (P+E) with engineered K562 feeder cells expressing membrane-bound IL-21, 4-1BB ligand and CD48 ex vivo, and subsequently complexed with AFM13 just prior to infusion (Kerbauy et al, Clin Cancer Res 2021; 27:3744-56). These cytokine-induced memory-like CB-derived NK cells precomplexed with AFM13 (AFM13-NK) presented chimeric antigen receptor (CAR)-NK cell-like features and showed an increased in vitro and in vivo antitumor activity compared to either P+E NK or AFM13 alone. Building on this work we wished to clinically study adoptive AFM13-NK cell therapy in pts with R/R CD30+ lymphomas. METHODS: This single-center phase I-II trial (NCT04074746) evaluates the safety and activity of AFM13-NK, followed by AFM13 monotherapy. Patients aged 15-75 years with R/R CD30+ lymphomas refractory to brentuximab vedotin are enrolled. The trial goals are to identify the recommended phase 2 dose (RP2D) of AFM13-NK cells, estimate the overall (ORR) and complete response (CR) rates, event-free (EFS) and overall survival (OS) rates, and studies of NK cell activation and persistence. Each treatment cycle consisted of fludarabine/cyclophosphamide (days −5 to −3), followed by infusion of the AFM13-NK cells, cultured for 14 days as described above (day 0), and three weekly IV infusions of AFM13 (200 mg, days 7, 14 and 21). Two cycles were administered to patients 1-19 and 4 cycles were given starting with patient 20. Response was evaluated on day 28 of each cycle. Patients were enrolled at 3 dose levels: DL1 (106 NK/Kg), DL2 (107NK/Kg) and DL3 (108 NK/Kg). Each patient received the same NK dose in all cycles. RESULTS: As of 7/31/22, 30 pts have been enrolled and treated (Table) at DL1 (N=3), DL2 (N=3) and DL3 (N=24), with 1 (N=1), 2 (N=24), 3 (N=3) or 4 cycles (N=3). All patients had active progressive disease at enrollment and no bridging therapy was given. The cords used for each of the 69 cycles were selected with no consideration for HLA match, which was 0/6 (N=31), 1/6 (N=31), 2/6 (N=5), 3/6 (N=1) and 4/6 (N=1). The product infused consisted of >99% NK cells (expanded >2,700 fold), with only 0.02% T cells. The NK cells were >98% viable and 92% of them were bound to AFM13. The products were infused fresh. There were no infusion-related reactions (IRR) after infusing AFM13-NK and 11 IRR in 182 infusions of AFM13 alone (6%) (1 grade 3, 10 grade 2). We saw no cases of CRS, ICANS or GVHD of any grade. DL3 (108 NK/Kg) was established as the RP2D. The ORR in the entire study is 97% with 63% CRs. All 24 patients treated at the RP2D responded (100% ORR) with 70.8% (17/24) CRs. Five patients had a response consolidated with a SCT. At median follow-up of 8 (range 1-23) months, the EFS and OS rates of all 30 patients are 57% and 83%, respectively. AFM-NK cells were detected in serum from day 1 post infusion and persisted for 3-4 weeks. Donor NK levels followed similar patterns after each cycle, which argues against a sensitization effect in the patient after the first NK infusion. Donor NK cells showed by CyTOF increased binding to AFM13 and expression of activation markers. CONCLUSIONS: This is the first clinical trial to date using an ICE® construct precomplexed with cytokine-induced memory-like CB-NK cells to treat patients with CD30+ R/R HL and NHL. Our preliminary results indicate that this ICE® precomplexed CB-NK cell therapy has an excellent tolerability profile and is highly active in patients with heavily pretreated R/R CD30+ lymphomas and warrants further investigation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Tumor-Associated Lymphatics Upregulate MHC-II to Suppress Tumor-Infiltrating Lymphocytes.

Steady-state lymphatic endothelial cells (LECs) can induce peripheral tolerance by presenting endogenous antigens on MHC class I (MHC-I) molecules. Recent evidence suggests that lymph node LECs can cross-present tumor antigens on MHC-I to suppress tumor-specific CD8+ T cells. Whether LECs can act as immunosuppressive cells in an MHC-II dependent manner in the local tumor microenvironment (TME) is not well characterized. Using murine heterotopic and spontaneous tumor models, we show that LECs in the TME increase MHC-II expression in the context of increased co-inhibitory signals. We provide evidence that tumor lymphatics in human melanoma and breast cancer also upregulate MHC-II compared to normal tissue lymphatics. In transgenic mice that lack LEC-specific MHC-II expression, heterotopic tumor growth is attenuated, which is associated with increased numbers of tumor-specific CD8+ and effector CD4+ T cells, as well as decreased numbers of T regulatory CD4+ cells in the TME. Mechanistically, we show that murine and human dermal LECs can take up tumor antigens in vitro. Antigen-loaded LECs in vitro can induce antigen-specific proliferation of CD8+ T cells but not CD4+ T cells; however, these proliferated CD8+ T cells have reduced effector function in the presence of antigen-loaded LECs. Taken together, our study suggests LECs can act as immunosuppressive cells in the TME in an MHC-II dependent manner. Whether this is a result of direct tumor antigen presentation on MHC-II requires additional investigation.

Multimodal single-cell profiling of intrahepatic cholangiocarcinoma defines hyperactivated Tregs as a potential therapeutic target.

The landscape and function of the immune infiltrate of intrahepatic cholangiocarcinoma (iCCA), a rare, yet aggressive tumor of the biliary tract, remains poorly characterized, limiting development of successful immunotherapies. Herein, we aimed to define the molecular characteristics of tumor-infiltrating leukocytes with a special focus on CD4+ regulatory T cells (Tregs). We used high-dimensional single-cell technologies to characterize the T-cell and myeloid compartments of iCCA tissues, comparing these with their tumor-free peritumoral and circulating counterparts. We further used genomics and cellular assays to define the iCCA-specific role of a novel transcription factor, mesenchyme homeobox 1 (MEOX1), in Treg biology. We found poor infiltration of putative tumor-specific CD39+ CD8+ T cells accompanied by abundant infiltration of hyperactivated CD4+ Tregs. Single-cell RNA-sequencing identified an altered network of transcription factors in iCCA-infiltrating compared to peritumoral T cells, suggesting reduced effector functions by tumor-infiltrating CD8+ T cells and enhanced immunosuppression by CD4+ Tregs. Specifically, we found that expression of MEOX1 was highly enriched in tumor-infiltrating Tregs, and demonstrated that MEOX1 overexpression is sufficient to reprogram circulating Tregs to acquire the transcriptional and epigenetic landscape of tumor-infiltrating Tregs. Accordingly, enrichment of the MEOX1-dependent gene program in Tregs was strongly associated with poor prognosis in a large cohort of patients with iCCA. We observed abundant infiltration of hyperactivated CD4+ Tregs in iCCA tumors along with reduced CD8+ T-cell effector functions. Interfering with hyperactivated Tregs should be explored as an approach to enhance antitumor immunity in iCCA. Immune cells have the potential to slow or halt the progression of tumors. However, some tumors, such as intrahepatic cholangiocarcinoma, are associated with very limited immune responses (and infiltration of cancer-targeting immune cells). Herein, we show that a specific population of regulatory T cells (a type of immune cell that actually suppresses the immune response) are hyperactivated in intrahepatic cholangiocarcinoma. Targeting these cells could enable cancer-targeting immune cells to act more effectively and should be looked at as a potential therapeutic approach to this aggressive cancer type.