Model-Informed Drug Development of Autologous Car-T Cell Therapy: Strategies to Optimize Car-T Cell Exposure Leveraging Cell Kinetic/Dynamic Modeling

Autologous Chimeric antigen receptor (CAR-T) cell therapy has been highly successful in the treatment of aggressive hematological malignancies and is also being evaluated for the treatment of solid tumors as well as other therapeutic areas. A challenge, however, is that up to 60% of patients do not sustain a long-term response. Low CAR-T cell exposure has been suggested as an underlying factor for a poor prognosis. CAR-T cell therapy is a novel therapeutic modality with unique kinetic and dynamic properties. Importantly, clear dose-exposure relationships do not seem to exist for any of the currently approved CAR-T cell products. In other words, dose increases have not led to a commensurate increase in the measurable in vivo frequency of transferred CAR-T cells. Therefore, alternative approaches beyond dose titration are needed to optimize CAR-T cell exposure. In this paper, we provide examples of actionable variables - design elements in CAR-T cell discovery, development, and clinical practice, which can be modified to optimize autologous CAR-T cell exposure. Most of these actionable variables can be assessed throughout the various stages of discovery and development as part of a well-informed research and development program. Model-informed drug development approaches can enable such study and program design choices from discovery through to clinical practice and can be an important contributor to cell therapy effectiveness and efficiency

T cell receptor engineering targeting FOXM1 for the treatment of lung cancer

https://openworks.mdanderson.org/sumexp23/1069/thumbnail.jp

Urinary T Cells Are Detected in Patients With Immune Checkpoint Inhibitor-Associated Immune Nephritis That Are Clonotypically Identical to Kidney T Cell Infiltrates

Acute kidney injury (AKI) occurs in ~20% of patients receiving immune checkpoint inhibitor (ICI) therapy; however, only 2-5% will develop ICI-mediated immune nephritis. Conventional tests are nonspecific in diagnosing disease pathology and invasive procedures (i.e. kidney biopsy) may not be feasible. In other autoimmune renal diseases, urinary immune cells correlated with the pathology or were predictive of disease activity. Corresponding evidence and analysis are absent for ICI-mediated immune nephritis. We report the first investigation analyzing immune cell profiles of matched kidney biopsies and urine of patients with ICI-AKI. We demonstrated the presence of urinary T cells in patients with immune nephritis by flow cytometry analysis. Clonotype analysis of T cell receptor (TCR) sequences confirmed enrichment of kidney TCRs in urine. As ICI therapies become standard of care for more cancers, noninvasively assessing urinary immune cells of ICI therapy recipients can facilitate clinical management and an opportunity to tailor ICI-nephritis treatment

Monocyte-derived dendritic cells loaded with a mixture of apoptotic/necrotic melanoma cells efficiently cross-present gp100 and MART-1 antigens to specific CD8(+ )T lymphocytes

BACKGROUND: In the present study, we demonstrate, in rigorous fashion, that human monocyte-derived immature dendritic cells (DCs) can efficiently cross-present tumor-associated antigens when co-cultured with a mixture of human melanoma cells rendered apoptotic/necrotic by γ irradiation (Apo-Nec cells). METHODS: We evaluated the phagocytosis of Apo-Nec cells by FACS after PKH26 and PKH67 staining of DCs and Apo-Nec cells at different times of coculture. The kinetics of the process was also followed by electron microscopy. DCs maturation was also studied monitoring the expression of specific markers, migration towards specific chemokines and the ability to cross-present in vitro the native melanoma-associated Ags MelanA/MART-1 and gp100. RESULTS: Apo-Nec cells were efficiently phagocytosed by immature DCs (iDC) (55 ± 10.5%) at 12 hs of coculture. By 12–24 hs we observed digested Apo-Nec cells inside DCs and large empty vacuoles as part of the cellular processing. Loading with Apo-Nec cells induced DCs maturation to levels achieved using LPS treatment, as measured by: i) the decrease in FITC – Dextran uptake (iDC: 81 ± 5%; DC/Apo-Nec 33 ± 12%); ii) the cell surface up-regulation of CD80, CD86, CD83, CCR7, CD40, HLA-I and HLA-II and iii) an increased in vitro migration towards MIP-3β. DC/Apo-Nec isolated from HLA-A*0201 donors were able to induce >600 pg/ml IFN-γ secretion of CTL clones specific for MelanA/MART-1 and gp100 Ags after 6 hs and up to 48 hs of coculture, demonstrating efficient cross-presentation of the native Ags. Intracellular IL-12 was detected in DC/Apo-Nec 24 hs post-coculture while IL-10 did not change. CONCLUSION: We conclude that the use of a mixture of four apoptotic/necrotic melanoma cell lines is a suitable source of native melanoma Ags that provides maturation signals for DCs, increases migration to MIP-3β and allows Ag cross-presentation. This strategy could be exploited for vaccination of melanoma patients

A phase I clinical study of vaccination of melanoma patients with dendritic cells loaded with allogeneic apoptotic/necrotic melanoma cells. Analysis of toxicity and immune response to the vaccine and of IL-10 -1082 promoter genotype as predictor of disease progression

<p>Abstract</p> <p>Background</p> <p>Sixteen melanoma patients (1 stage IIC, 8 stage III, and 7 stage IV) were treated in a Phase I study with a vaccine (DC/Apo-Nec) composed of autologous dendritic cells (DCs) loaded with a mixture of apoptotic/necrotic allogeneic melanoma cell lines (Apo-Nec), to evaluate toxicity and immune responses. Also, IL-10 1082 genotype was analyzed in an effort to predict disease progression.</p> <p>Methods</p> <p>PBMC were obtained after leukapheresis and DCs were generated from monocytes cultured in the presence of GM-CSF and IL-4 in serum-free medium. Immature DCs were loaded with gamma-irradiated Apo-Nec cells and injected id without adjuvant. Cohorts of four patients were given four vaccines each with 5, 10, 15, or 20 × 10⁶DC/Apo-Nec cell per vaccine, two weeks apart. Immune responses were measured by ELISpot and tetramer analysis. Il-10 genotype was measured by PCR and corroborated by IL-10 production by stimulated PBMC.</p> <p>Results</p> <p>Immature DCs efficiently phagocytosed melanoma Apo-Nec cells and matured after phagocytosis as evidenced by increased expression of CD83, CD80, CD86, HLA class I and II, and 75.2 ± 16% reduction in Dextran-FITC endocytosis. CCR7 was also up-regulated upon Apo-Nec uptake in DCs from all patients, and accordingly DC/Apo-Nec cells were able to migrate <it>in vitro </it>toward MIP-3 beta. The vaccine was well tolerated in all patients. The DTH score increased significantly in all patients after the first vaccination (Mann-Whitney Test, p < 0.05). The presence of CD8⁺T lymphocytes specific to gp100 and Melan A/MART-1 Ags was determined by ELISpot and tetramer analysis in five HLA-A*0201 patients before and after vaccination; one patient had stable elevated levels before and after vaccination; two increased their CD8 + levels, one had stable moderate and one had negligible levels. The analysis of IL-10 promoter -1082 polymorphism in the sixteen patients showed a positive correlation between AA genotype, accompanied by lower <it>in vitro </it>IL-10 production by stimulated PBMC, and faster melanoma progression after lymph nodes surgery (p = 0.04). With a mean follow-up of 49.5 months post-surgery, one stage IIC patient and 7/8 stage III patients remain NED but 7/7 stage IV patients have progressed.</p> <p>Conclusion</p> <p>We conclude that DC/Apo-Nec vaccine is safe, well tolerated and it may induce specific immunity against melanoma Ags. Patients with a low-producing IL-10 polymorphism appear to have a worst prognosis.</p> <p>Trial registration</p> <p>Clinicaltrials.gov (NHI) NCT00515983</p

Single-Cell CRISPR Immune Screens Reveal Immunological Roles of Tumor Intrinsic Factors

Genetic screens are widely exploited to develop novel therapeutic approaches for cancer treatment. With recent advances in single-cell technology, single-cell CRISPR screen (scCRISPR) platforms provide opportunities for target validation and mechanistic studies in a high-throughput manner. Here, we aim to establish scCRISPR platforms which are suitable for immune-related screens involving multiple cell types. We integrated two scCRISPR platforms, namely Perturb-seq and CROP-seq, with both in vitro and in vivo immune screens. By leveraging previously generated resources, we optimized experimental conditions and data analysis pipelines to achieve better consistency between results from high-throughput and individual validations. Furthermore, we evaluated the performance of scCRISPR immune screens in determining underlying mechanisms of tumor intrinsic immune regulation. Our results showed that scCRISPR platforms can simultaneously characterize gene expression profiles and perturbation effects present in individual cells in different immune screen conditions. Results from scCRISPR immune screens also predict transcriptional phenotype associated with clinical responses to cancer immunotherapy. More importantly, scCRISPR screen platforms reveal the interactive relationship between targeting tumor intrinsic factors and T cell-mediated antitumor immune response which cannot be easily assessed by bulk RNA-seq. Collectively, scCRISPR immune screens provide scalable and reliable platforms to elucidate molecular determinants of tumor immune resistance

CD160 serves as a negative regulator of NKT cells in acute hepatic injury

[EN] CD160 and BTLA both bind to herpes virus entry mediator. Although a negative regulatory function of BTLA in natural killer T (NKT) cell activation has been reported, whether CD160 is also involved is unclear. By analyzing CD160−/− mice and mixed bone marrow chimeras, we show that CD160 is not essential for NKT cell development. However, CD160−/− mice exhibit severe liver injury after in vivo challenge with α-galactosylceramide (α-GalCer). Moreover, CD160−/− mice are more susceptible to Concanavalin A challenge, and display elevated serum AST and ALT levels, hyperactivation of NKT cells, and enhanced IFN-γ, TNF, and IL-4 production. Lastly, inhibition of BTLA by anti-BTLA mAb aggravates α-GalCer-induced hepatic injury in CD160−/− mice, suggesting that both CD160 and BTLA serve as non-overlapping negative regulators of NKT cells. Our data thus implicate CD160 as a co-inhibitory receptor that delivers antigen-dependent signals in NKT cells to dampen cytokine production during early innate immune activationSIWe thank the NIH Tetramer Core Facility for providing PBS 57 ligand loaded CD1d Tetramers. Further, we thank the staffs of Gyerim Experimental Animal Resource Center for animal care and technical assistance. K.-M. Lee was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT, and Future planning (NRF-2016M3A9B6948342, NRF- 2017R1A2B3004828, and NRF-2018M3A9D3079288). S.-J. Kim was supported by the Korea Health Industry Development Institute (KHIDI-HI14C2640) grant funded by Korea Government. S.-J. Ha was supported by a grant from the NRF (NRF- 2018R1A2A1A05076997). T.-J. Kim was additionally supported by a grant from the NRF (NRF-2016R1A6A3A04009698

Fludarabine Modulates Immune Response and Extends In Vivo Survival of Adoptively Transferred CD8 T Cells in Patients with Metastatic Melanoma

Adoptive T cell therapy involving the use of ex vivo generated antigen-specific cytotoxic T lymphocytes provides a promising approach to immunotherapy. It has become increasingly apparent that anti-tumor efficacy using adoptively transferred T cells is linked to their duration of in vivo persistence and can only be achieved when combined with some form of pre-infusion patient conditioning regimen. An optimal conditioning regimen that provides a positive benefit without serious toxicities has yet to be defined. We have established a unique clinical model that allows for evaluation of a given conditioning regimen on adoptively transferred T cells in humans. In this first-in-human study (FHCRC #1796), we evaluate the use of fludarabine, an FDA-approved reagent with predictable lymphodepleting kinetics and duration of action, as a conditioning regimen that promotes homeostatic upregulation of cytokines and growth signals contributing to in vivo T cell persistence.We conducted a phase I study in patients with refractory metastatic melanoma. Patients received two infusions of a single tumor-reactive antigen-specific CTL clone expanded to 10(10)/m(2); the first infusion was given without fludarabine conditioning, and the second CTL infusion was given after a course of fludarabine (25 mg/m(2)/dayx5 days). This design permits intra-patient comparison of in vivo T cell persistence pre- and post-fludarabine. Nineteen CTL infusions were administered to ten patients. No serious toxicities were observed. Three of nine evaluable patients experienced minor response or stable disease for periods of 5.8-11.0 months with two additional patients demonstrating delayed disease stabilization. The median overall survival in this heavily pre-treated population was 9.7 months. Fludarabine led to a 2.9 fold improvement in the in vivo persistence of transferred CTL clones from a median of 4.5 days (range 0-38+) to 13.0 days (range 2-63+) (p<0.05). Fludarabine lymphodepletion increased plasma levels of the homeostatic cytokines IL-7 and IL-15. Surprisingly, fludarabine also increased the relative percentage of CD4+ T cells expressing the regulatory protein Foxp3.Lymphodepletion with fludarabine enhances transferred T cell persistence but suggest that additional improvements to optimize T cell survival and address regulatory T cells are critical in providing anti-tumor efficacy.ClinicalTrials.gov NCT00317759

Evolution and modulation of antigen-specific T cell responses in melanoma patients

Analyzing antigen-specific T cell responses at scale has been challenging. Here, we analyze three types of T cell receptor (TCR) repertoire data (antigen-specific TCRs, TCR-repertoire, and single-cell RNA + TCRαβ-sequencing data) from 515 patients with primary or metastatic melanoma and compare it to 783 healthy controls. Although melanoma-associated antigen (MAA) -specific TCRs are restricted to individuals, they share sequence similarities that allow us to build classifiers for predicting anti-MAA T cells. The frequency of anti-MAA T cells distinguishes melanoma patients from healthy and predicts metastatic recurrence from primary melanoma. Anti-MAA T cells have stem-like properties and frequent interactions with regulatory T cells and tumor cells via Galectin9-TIM3 and PVR-TIGIT -axes, respectively. In the responding patients, the number of expanded anti-MAA clones are higher after the anti-PD1(+anti-CTLA4) therapy and the exhaustion phenotype is rescued. Our systems immunology approach paves the way for understanding antigen-specific responses in human disorders.</p