Cost-effectiveness of treating advanced melanoma with tumor-infiltrating lymphocytes based on an international randomized phase 3 clinical trial.

INTRODUCTION: In a multicenter, open-label randomized phase 3 clinical trial conducted in the Netherlands and Denmark, treatment with ex vivo-expanded tumor-infiltrating lymphocytes (TIL-NKI/CCIT) from autologous melanoma tumor compared with ipilimumab improved progression-free survival in patients with unresectable stage IIIC-IV melanoma after failure of first-line or second-line treatment. Based on this trial, we conducted a cost-utility analysis. METHODS: A Markov decision model was constructed to estimate expected costs (expressed in 2021€) and outcomes (quality-adjusted life years (QALYs)) of TIL-NKI/CCIT versus ipilimumab in the Netherlands. The Danish setting was assessed in a scenario analysis. A modified societal perspective was applied over a lifetime horizon. TIL-NKI/CCIT production costs were estimated via activity-based costing. Through sensitivity analyses, uncertainties and their impact on the incremental cost-effectiveness ratio (ICER) were assessed. RESULTS: Mean total undiscounted lifetime benefits were 4.47 life years (LYs) and 3.52 QALYs for TIL-NKI/CCIT and 3.33 LYs and 2.46 QALYs for ipilimumab. Total lifetime undiscounted costs in the Netherlands were €347,168 for TIL-NKI/CCIT (including €67,547 for production costs) compared with €433,634 for ipilimumab. Undiscounted lifetime cost in the Danish scenario were €337,309 and €436,135, respectively. This resulted in a dominant situation for TIL-NKI/CCIT compared with ipilimumab in both countries, meaning incremental QALYs were gained at lower costs. Survival probabilities, and utility in progressive disease affected the ICER most. CONCLUSION: Based on the data of a randomized phase 3 trial, treatment with TIL-NKI/CCIT in patients with unresectable stage IIIC-IV melanoma is cost-effective and cost-saving, both in the current Dutch and Danish setting. These findings led to inclusion of TIL-NKI/CCIT as insured care and treatment guidelines. Publicly funded development of the TIL-NKI/CCIT cell therapy shows realistic promise to further explore development of effective personalized treatment while warranting economic sustainability of healthcare systems.

Competitive Repopulation and Allo-Immunologic Pressure Determine Chimerism Kinetics after T Cell-Depleted Allogeneic Stem Cell Transplantation and Donor Lymphocyte Infusion.

After allogeneic stem cell transplantation (alloSCT), patient-derived stem cells that survived the pretransplantation conditioning compete with engrafting donor stem cells for bone marrow (BM) repopulation. In addition, donor-derived alloreactive T cells present in the stem cell product may favor establishment of complete donor-derived hematopoiesis by eliminating patient-derived lymphohematopoietic cells. T cell-depleted alloSCT with sequential transfer of potentially alloreactive T cells by donor lymphocyte infusion (DLI) provides a unique opportunity to selectively study how competitive repopulation and allo-immunologic pressure influence lymphohematopoietic recovery. This study aimed to determine the relative contribution of competitive repopulation and donor-derived anti-recipient alloimmunologic pressure on the establishment of lymphohematopoietic chimerism after alloSCT. In this retrospective cohort study of 281 acute leukemia patients treated according to a protocol combining alemtuzumab-based T cell-depleted alloSCT with prophylactic DLI, we investigated engraftment and quantitative donor chimerism in the BM and immune cell subsets. DLI-induced increase of chimerism and development of graft-versus-host disease (GVHD) were analyzed as complementary indicators for donor-derived anti-recipient alloimmunologic pressure. Profound suppression of patient immune cells by conditioning sufficed for sustained engraftment without necessity for myeloablative conditioning or development of clinically significant GVHD. Although 61% of the patients without any DLI or GVHD showed full donor chimerism (FDC) in the BM at 6 months after alloSCT, only 24% showed FDC in the CD4+ T cell compartment. In contrast, 75% of the patients who had received DLI and 83% of the patients with clinically significant GVHD had FDC in this compartment. In addition, 72% of the patients with mixed hematopoiesis receiving DLI converted to complete donor-derived hematopoiesis, of whom only 34% developed clinically significant GVHD. Our data show that competitive repopulation can be sufficient to reach complete donor-derived hematopoiesis, but that some alloimmunologic pressure is needed for the establishment of a completely donor-derived T cell compartment, either by the development of GVHD or by administration of DLI. We illustrate that it is possible to separate the graft-versus-leukemia effect from GVHD, as conversion to durable complete donor-derived hematopoiesis following DLI did not require induction of clinically significant GVHD.

Novel strategies to improve efficacy of treatment with tumor-infiltrating lymphocytes (TILs) for patients with solid cancers.

PURPOSE OF REVIEW: Treatment with tumor-infiltrating lymphocytes (TILs) has shown remarkable clinical responses in patients with advanced solid tumors. Although the TIL production process is very robust, the original protocol stems from the early nineties and lacks effective selection for tumor-reactivity and functional activity. In this review we highlight the limitations of the current production process and give an overview of improvements that can be made to increase TIL efficacy. RECENT FINDINGS: With the recent advances in single cell sequencing technologies, our understanding of the composition and phenotype of TILs in the tumor micro environment has majorly increased, which forms the basis for the development of new strategies to improve the TIL production process. Strategies involve selection for neoantigen-reactive TILs by cell sorting or selective expansion strategies. Furthermore, gene editing strategies like Clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas9) can be used to increase TIL functionality. SUMMARY: Although combining all the possible improvements into a next generation TIL product might be challenging, it is highly likely that those techniques will increase the clinical value of TIL therapy in the coming years.

Tumor-Infiltrating Lymphocyte Therapy or Ipilimumab in Advanced Melanoma.

BACKGROUND: Immune checkpoint inhibitors and targeted therapies have dramatically improved outcomes in patients with advanced melanoma, but approximately half these patients will not have a durable benefit. Phase 1-2 trials of adoptive cell therapy with tumor-infiltrating lymphocytes (TILs) have shown promising responses, but data from phase 3 trials are lacking to determine the role of TILs in treating advanced melanoma. METHODS: In this phase 3, multicenter, open-label trial, we randomly assigned patients with unresectable stage IIIC or IV melanoma in a 1:1 ratio to receive TIL or anti-cytotoxic T-lymphocyte antigen 4 therapy (ipilimumab at 3 mg per kilogram of body weight). Infusion of at least 5×109 TILs was preceded by nonmyeloablative, lymphodepleting chemotherapy (cyclophosphamide plus fludarabine) and followed by high-dose interleukin-2. The primary end point was progression-free survival. RESULTS: A total of 168 patients (86% with disease refractory to anti-programmed death 1 treatment) were assigned to receive TILs (84 patients) or ipilimumab (84 patients). In the intention-to-treat population, median progression-free survival was 7.2 months (95% confidence interval [CI], 4.2 to 13.1) in the TIL group and 3.1 months (95% CI, 3.0 to 4.3) in the ipilimumab group (hazard ratio for progression or death, 0.50; 95% CI, 0.35 to 0.72; P<0.001); 49% (95% CI, 38 to 60) and 21% (95% CI, 13 to 32) of the patients, respectively, had an objective response. Median overall survival was 25.8 months (95% CI, 18.2 to not reached) in the TIL group and 18.9 months (95% CI, 13.8 to 32.6) in the ipilimumab group. Treatment-related adverse events of grade 3 or higher occurred in all patients who received TILs and in 57% of those who received ipilimumab; in the TIL group, these events were mainly chemotherapy-related myelosuppression. CONCLUSIONS: In patients with advanced melanoma, progression-free survival was significantly longer among those who received TIL therapy than among those who received ipilimumab. (Funded by the Dutch Cancer Society and others; ClinicalTrials.gov number, NCT02278887.).

Human T cells recognize HLA-DP-bound peptides in two orientations.

Human leukocyte antigen (HLA) molecules present small peptide antigens to T cells, thereby allowing them to recognize pathogen-infected and cancer cells. A central dogma over the last 50+ y is that peptide binding to HLA molecules is mediated by the docking of side chains of particular amino acids in the peptide into pockets in the HLA molecules in a conserved N- to C-terminal orientation. Whether peptides can be presented in a reversed C- to N-terminal orientation remains unclear. Here, we performed large-scale identification of peptides bound to HLA-DP molecules and observed that in addition to peptide binding in an N- to C-terminal orientation, in 9 out of 14 HLA-DP allotypes, reverse motifs are found, compatible with C- to N-terminal peptide binding. Moreover, we isolated high-avidity human cytomegalovirus (CMV)-specific HLA-DP-restricted CD4+ T cells from the memory repertoire of healthy donors and demonstrate that such T cells recognized CMV-derived peptides bound to HLA-DPB1*01:01 or *05:01 in a reverse C- to N-terminal manner. Finally, we obtained a high-resolution HLA-DPB1*01:01-CMVpp65(142-158) peptide crystal structure, which is the molecular basis for C- to N-terminal peptide binding to HLA-DP. Our results point to unique features of HLA-DP molecules that substantially broaden the HLA class II bound peptide repertoire to combat pathogens and eliminate cancer cells.

Amino acids at position 5 in the peptide/MHC binding region of a public virus-specific TCR are completely inter-changeable without loss of function.

Anti-viral T-cell responses are usually directed against a limited set of antigens, but often contain many T cells expressing different T-cell receptors (TCRs). Identical TCRs found within virus-specific T-cell populations in different individuals are known as public TCRs, but also TCRs highly-similar to these public TCRs, with only minor variations in amino acids on specific positions in the Complementary Determining Regions (CDRs), are frequently found. However, the degree of freedom at these positions was not clear. In this study, we used the HLA-A*02:01-restricted EBV-LMP2FLY -specific public TCR as model and modified the highly-variable position 5 of the CDR3ß sequence with all 20 amino acids. Our results demonstrate that amino acids at this particular position in the CDR3ß region of this TCR are completely inter-changeable, without loss of TCR function. We show that the inability to find certain variants in individuals is explained by their lower recombination probability rather than by steric hindrance.

TIL classified to memory state are correlated with response to immune checkpoint blockade.

Tumor reactivity mediated by tumor infiltrating lymphocytes (TILs) is one of the hallmarks of the clinical effect of immune checkpoint blockade (ICB). Jaiswal et al. used a functional genomics approach to better characterize TIL phenotypes predictive for response to ICB.1.

Public T-Cell Receptors (TCRs) Revisited by Analysis of the Magnitude of Identical and Highly-Similar TCRs in Virus-Specific T-Cell Repertoires of Healthy Individuals.

Since multiple different T-cell receptor (TCR) sequences can bind to the same peptide-MHC combination and the number of TCR-sequences that can theoretically be generated even exceeds the number of T cells in a human body, the likelihood that many public identical (PUB-I) TCR-sequences frequently contribute to immune responses has been estimated to be low. Here, we quantitatively analyzed the TCR-repertoires of 190 purified virus-specific memory T-cell populations, directed against 21 epitopes of Cytomegalovirus, Epstein-Barr virus and Adenovirus isolated from 29 healthy individuals, and determined the magnitude, defined as prevalence within the population and frequencies within individuals, of PUB-I TCR and of TCR-sequences that are highly-similar (PUB-HS) to these PUB-I TCR-sequences. We found that almost one third of all TCR nucleotide-sequences represented PUB-I TCR amino-acid (AA) sequences and found an additional 12% of PUB-HS TCRs differing by maximally 3 AAs. We illustrate that these PUB-I and PUB-HS TCRs were structurally related and contained shared core-sequences in their TCR-sequences. We found a prevalence of PUB-I and PUB-HS TCRs of up to 50% among individuals and showed frequencies of virus-specific PUB-I and PUB-HS TCRs making up more than 10% of each virus-specific T-cell population. These findings were confirmed by using an independent TCR-database of virus-specific TCRs. We therefore conclude that the magnitude of the contribution of PUB-I and PUB-HS TCRs to these virus-specific T-cell responses is high. Because the T cells from these virus-specific memory TCR-repertoires were the result of successful control of the virus in these healthy individuals, these PUB-HS TCRs and PUB-I TCRs may be attractive candidates for immunotherapy in immunocompromised patients that lack virus-specific T cells to control viral reactivation.

Promiscuity of Peptides Presented in HLA-DP Molecules from Different Immunogenicity Groups Is Associated With T-Cell Cross-Reactivity.

In the context of HLA-DP-mismatched allogeneic stem cell transplantation, mismatched HLA-DP alleles can provoke profound allo-HLA-DP-specific immune responses from the donor T-cell repertoire leading to graft-versus-leukemia effect and/or graft-versus-host disease in the patient. The magnitude of allo-HLA-DP-specific immune responses has been shown to depend on the specific HLA-DP disparity between donor and patient and the immunogenicity of the mismatched HLA-DP allele(s). HLA-DP peptidome clustering (DPC) was developed to classify the HLA-DP molecules based on similarities and differences in their peptide-binding motifs. To investigate a possible categorization of HLA-DP molecules based on overlap of presented peptides, we identified and compared the peptidomes of the thirteen most frequently expressed HLA-DP molecules. Our categorization based on shared peptides was in line with the DPC classification. We found that the HLA-DP molecules within the previously defined groups DPC-1 or DPC-3 shared the largest numbers of presented peptides. However, the HLA-DP molecules in DPC-2 segregated into two subgroups based on the overlap in presented peptides. Besides overlap in presented peptides within the DPC groups, a substantial number of peptides was also found to be shared between HLA-DP molecules from different DPC groups, especially for groups DPC-1 and -2. The functional relevance of these findings was illustrated by demonstration of cross-reactivity of allo-HLA-DP-reactive T-cell clones not only against HLA-DP molecules within one DPC group, but also across different DPC groups. The promiscuity of peptides presented in various HLA-DP molecules and the cross-reactivity against different HLA-DP molecules demonstrate that these molecules cannot be strictly categorized in immunogenicity groups.

Identification of Functional HLA-A*01:01-Restricted Epstein-Barr Latent Membrane Protein 2-Specific T-Cell Receptors.

BACKGROUND: Adoptive transfer of genetically engineered T cells expressing antigen-specific T-cell receptors (TCRs) is an appealing therapeutic approach for Epstein-Barr virus (EBV)-associated malignancies of latency type II/III that express EBV antigens (LMP1/2). Patients who are HLA-A*01:01 positive could benefit from such products, since no T cells recognizing any EBV-derived peptide in this common HLA allele have been found thus far. METHODS: HLA-A*01:01-restricted EBV-LMP2-specific T cells were isolated using peptide major histocompatibility complex (pMHC) tetramers. Functionality was assessed by production of interferon gamma (IFN-γ) and cytotoxicity when stimulated with EBV-LMP2-expressing cell lines. Functionality of primary T cells transduced with HLA-A*01:01-restricted EBV-LMP2-specific TCRs was optimized by knocking out the endogenous TCRs of primary T cells (∆TCR) using CRISPR-Cas9 technology. RESULTS: EBV-LMP2-specific T cells were successfully isolated and their TCRs were characterized. TCR gene transfer in primary T cells resulted in specific pMHC tetramer binding and reactivity against EBV-LMP2-expressing cell lines. The mean fluorescence intensity of pMHC-tetramer binding was increased 1.5-2 fold when the endogenous TCRs of CD8+ T cells was knocked out. CD8+/∆TCR T cells modified to express EBV-LMP2-specific TCRs showed IFN-γ secretion and cytotoxicity toward EBV-LMP2-expressing malignant cell lines. CONCLUSIONS: We isolated the first functional HLA-A*01:01-restricted EBV-LMP2-specific T-cell populations and TCRs, which can potentially be used in future TCR gene therapy to treat EBV-associated latency type II/III malignancies.

Magnitude of Off-Target Allo-HLA Reactivity by Third-Party Donor-Derived Virus-Specific T Cells Is Dictated by HLA-Restriction.

T-cell products derived from third-party donors are clinically applied, but harbor the risk of off-target toxicity via induction of allo-HLA cross-reactivity directed against mismatched alleles. We used third-party donor-derived virus-specific T cells as model to investigate whether virus-specificity, HLA restriction and/or HLA background can predict the risk of allo-HLA cross-reactivity. Virus-specific CD8pos T cells were isolated from HLA-A*01:01/B*08:01 or HLA-A*02:01/B*07:02 positive donors. Allo-HLA cross-reactivity was tested using an EBV-LCL panel covering 116 allogeneic HLA molecules and confirmed using K562 cells retrovirally transduced with single HLA-class-I alleles of interest. HLA-B*08:01-restricted T cells showed the highest frequency and diversity of allo-HLA cross-reactivity, regardless of virus-specificity, which was skewed toward multiple recurrent allogeneic HLA-B molecules. Thymic selection for other HLA-B alleles significantly influenced the level of allo-HLA cross-reactivity mediated by HLA-B*08:01-restricted T cells. These results suggest that the degree and specificity of allo-HLA cross-reactivity by T cells follow rules. The risk of off-target toxicity after infusion of incompletely matched third-party donor-derived virus-specific T cells may be reduced by selection of T cells with a specific HLA restriction and background.

Comprehensive diagnostics of acute myeloid leukemia by whole transcriptome RNA sequencing.

Acute myeloid leukemia (AML) is caused by genetic aberrations that also govern the prognosis of patients and guide risk-adapted and targeted therapy. Genetic aberrations in AML are structurally diverse and currently detected by different diagnostic assays. This study sought to establish whole transcriptome RNA sequencing as single, comprehensive, and flexible platform for AML diagnostics. We developed HAMLET (Human AML Expedited Transcriptomics) as bioinformatics pipeline for simultaneous detection of fusion genes, small variants, tandem duplications, and gene expression with all information assembled in an annotated, user-friendly output file. Whole transcriptome RNA sequencing was performed on 100 AML cases and HAMLET results were validated by reference assays and targeted resequencing. The data showed that HAMLET accurately detected all fusion genes and overexpression of EVI1 irrespective of 3q26 aberrations. In addition, small variants in 13 genes that are often mutated in AML were called with 99.2% sensitivity and 100% specificity, and tandem duplications in FLT3 and KMT2A were detected by a novel algorithm based on soft-clipped reads with 100% sensitivity and 97.1% specificity. In conclusion, HAMLET has the potential to provide accurate comprehensive diagnostic information relevant for AML classification, risk assessment and targeted therapy on a single technology platform.

Effect of alemtuzumab-based T-cell depletion on graft compositional change in vitro and immune reconstitution early after allogeneic stem cell transplantation.

BACKGROUND AIMS: To reduce the risk of graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (alloSCT), T-cell depletion (TCD) of grafts can be performed by the addition of alemtuzumab (ALT) "to the bag" (in vitro) before transplantation. In this prospective study, the authors analyzed the effect of in vitro incubation with 20 mg ALT on the composition of grafts prior to graft infusion. Furthermore, the authors assessed whether graft composition at the moment of infusion was predictive for T-cell reconstitution and development of GVHD early after TCD alloSCT. METHODS: Sixty granulocyte colony-stimulating factor-mobilized stem cell grafts were obtained from ≥9/10 HLA-matched related and unrelated donors. The composition of the grafts was analyzed by flow cytometry before and after in vitro incubation with ALT. T-cell reconstitution and incidence of severe GVHD were monitored until 12 weeks after transplantation. RESULTS: In vitro incubation of grafts with 20 mg ALT resulted in an initial median depletion efficiency of T-cell receptor (TCR) α/ß T cells of 96.7% (range, 63.5-99.8%), followed by subsequent depletion in vivo. Graft volumes and absolute leukocyte counts of grafts before the addition of ALT were not predictive for the efficiency of TCR α/ß T-cell depletion. CD4pos T cells were depleted more efficiently than CD8pos T cells, and naive and regulatory T cells were depleted more efficiently than memory and effector T cells. This differential depletion of T-cell subsets was in line with their reported differential CD52 expression. In vitro depletion efficiencies and absolute numbers of (naive) TCR α/ß T cells in the grafts after ALT incubation were not predictive for T-cell reconstitution or development of GVHD post- alloSCT. CONCLUSIONS: The addition of ALT to the bag is an easy, fast and generally applicable strategy to prevent GVHD in patients receiving alloSCT after myeloablative or non-myeloablative conditioning because of the efficient differential depletion of donor-derived lymphocytes and T cells.

HA-1H T-Cell Receptor Gene Transfer to Redirect Virus-Specific T Cells for Treatment of Hematological Malignancies After Allogeneic Stem Cell Transplantation: A Phase 1 Clinical Study.

Graft-vs.-leukemia (GVL) reactivity after HLA-matched allogeneic stem cell transplantation (alloSCT) is mainly mediated by donor T cells recognizing minor histocompatibility antigens (MiHA). If MiHA are targeted that are exclusively expressed on hematopoietic cells of recipient origin, selective GVL reactivity without severe graft-vs.-host-disease (GVHD) may occur. In this phase I study we explored HA-1H TCR gene transfer into T cells harvested from the HA-1H negative stem-cell donor to treat HA-1H positive HLA-A*02:01 positive patients with high-risk leukemia after alloSCT. HA-1H is a hematopoiesis-restricted MiHA presented in HLA-A*02:01. Since we previously demonstrated that donor-derived virus-specific T-cell infusions did not result in GVHD, we used donor-derived EBV and/or CMV-specific T-cells to be redirected by HA-1H TCR. EBV and/or CMV-specific T-cells were purified, retrovirally transduced with HA-1H TCR, and expanded. Validation experiments illustrated dual recognition of viral antigens and HA-1H by HA-1H TCR-engineered virus-specific T-cells. Release criteria included products containing more than 60% antigen-specific T-cells. Patients with high risk leukemia following T-cell depleted alloSCT in complete or partial remission were eligible. HA-1H TCR T-cells were infused 8 and 14 weeks after alloSCT without additional pre-conditioning chemotherapy. For 4/9 included patients no appropriate products could be made. Their donors were all CMV-negative, thereby restricting the production process to EBV-specific T-cells. For 5 patients a total of 10 products could be made meeting the release criteria containing 3-280 × 106 virus and/or HA-1H TCR T-cells. No infusion-related toxicity, delayed toxicity or GVHD occurred. One patient with relapsed AML at time of infusions died due to rapidly progressing disease. Four patients were in remission at time of infusion. Two patients died of infections during follow-up, not likely related to the infusion. Two patients are alive and well without GVHD. In 2 patients persistence of HA-1H TCR T-cells could be illustrated correlating with viral reactivation, but no overt in-vivo expansion of infused T-cells was observed. In conclusion, HA-1H TCR-redirected virus-specific T-cells could be made and safely infused in 5 patients with high-risk AML, but overall feasibility and efficacy was too low to warrant further clinical development using this strategy. New strategies will be explored using patient-derived donor T-cells isolated after transplantation transduced with HA-1H-specific TCR to be infused following immune conditioning.

Long-term in vitro persistence of magnetic properties after magnetic bead-based cell separation of T cells.

Magnetic-activated cell sorting (MACS) using magnetic nanoparticles coated with specific antibodies is commonly used in immunology research. For in vitro isolation purposes, it is important to know to what extent the magnetic properties remain present in the isolated cell populations and whether it has consequences for sequential isolations. We hypothesized that only upon cell division, cells will lose their magnetic properties via dilution of the particles in/on their daughter cells. We analysed residual magnetic properties of cells that divided vs cells that did not divide after magnetic bead-based cell separation. As a model, we isolated T cells using beads targeting the non-modulating surface molecule CD45RO. Cells were labelled with the cell division tracking dye PKH and cultured under different conditions to induce variable degrees of cell division. We demonstrate that T cells that underwent no, or only minimal, cell divisions after MACS retained magnetic properties for up to at least 2 weeks of in vitro culture. The presence of nanoparticles was detected on their cell surface and intracellularly using Labeling Check reagent. These results have important consequences for procedures requiring repetitive isolation rounds after in vitro culture.

A minority of T cells recognizing tumor-associated antigens presented in self-HLA can provoke antitumor reactivity.

Tumor-associated antigens (TAAs) are monomorphic self-antigens that are proposed as targets for immunotherapeutic approaches to treat malignancies. We investigated whether T cells with sufficient avidity to recognize naturally overexpressed self-antigens in the context of self-HLA can be found in the T-cell repertoire of healthy donors. Minor histocompatibility antigen (MiHA)-specific T cells were used as a model, as the influence of thymic selection on the T-cell repertoire directed against MiHA can be studied in both self (MiHApos donors) and non-self (MiHAneg donors) backgrounds. T-cell clones directed against the HLA*02:01-restricted MiHA HA-1H were isolated from HA-1Hneg/HLA-A*02:01pos and HA-1Hpos/HLA-A*02:01pos donors. Of the 16 unique HA-1H-specific T-cell clones, five T-cell clones derived from HA-1Hneg/HLA-A*02:01pos donors and one T-cell clone derived from an HA-1Hpos/HLA-A*02:01pos donor showed reactivity against HA-1Hpos target cells. In addition, in total, 663 T-cell clones (containing at least 91 unique clones expressing different T-cell receptors) directed against HLA*02:01-restricted peptides of TAA WT1-RMF, RHAMM-ILS, proteinase-3-VLQ, PRAME-VLD, and NY-eso-1-SLL were isolated from HLA-A*02:01pos donors. Only 3 PRAME-VLD-specific and one NY-eso-1-SLL-specific T-cell clone provoked interferon-γ production and/or cytolysis upon stimulation with HLA-A*02:01pos malignant cell lines (but not primary malignant samples) naturally overexpressing the TAA. These results show that self-HLA-restricted T cells specific for self-antigens such as MiHA in MiHApos donors and TAAs are present in peripheral blood of healthy individuals. However, clinical efficacy would require highly effective in vivo priming by peptide vaccination in the presence of proper adjuvants or in vitro expansion of the low numbers of self-antigen-specific T cells of sufficient avidity to recognize endogenously processed antigen.

What does cell therapy manufacturing cost? A framework and methodology to facilitate academic and other small-scale cell therapy manufacturing costings.

BACKGROUND AIMS: Recent technical and clinical advances with cell-based therapies (CBTs) hold great promise in the treatment of patients with rare diseases and those with high unmet medical need. Currently the majority of CBTs are developed and manufactured in specialized academic facilities. Due to small scale, unique characteristics and specific supply chain, CBT manufacturing is considered costly compared to more conventional medicinal products. As a result, biomedical researchers and clinicians are increasingly faced with cost considerations in CBT development. The objective of this research was to develop a costing framework and methodology for academic and other small-scale facilities that manufacture cell-based therapies. METHODS: We conducted an international multi-center costing study in four facilities in Europe using eight CBTs as case studies. This study includes costs from cell or tissue procurement to release of final product for clinical use. First, via interviews with research scientists, clinicians, biomedical scientists, pharmacists and technicians, we designed a high-level costing framework. Next, we developed a more detailed uniform methodology to allocate cost items. Costs were divided into steps (tissue procurement, manufacturing and fill-finish). The steps were each subdivided into cost categories (materials, equipment, personnel and facility), and each category was broken down into facility running (fixed) costs and operational (variable) costs. The methodology was tested via the case studies and validated in developer interviews. Costs are expressed in 2018 euros (€). RESULTS: The framework and methodology were applicable across facilities and proved sensitive to differences in product and facility characteristics. Case study cost estimates ranged between €23 033 and €190 799 Euros per batch, with batch yield varying between 1 and 88 doses. The cost estimations revealed hidden costs to developers and provided insights into cost drivers to help design manufacturing best practices. CONCLUSIONS: This framework and methodology provide step-by-step guidance to estimate manufacturing costs specifically for cell-based therapies manufactured in academic and other small-scale enterprises. The framework and methodology can be used to inform and plan cost-conscious strategies for CBTs.

Immunopeptidome Analysis of HLA-DPB1 Allelic Variants Reveals New Functional Hierarchies.

HLA-DP alleles can be classified into functional T cell epitope (TCE) groups. TCE-1 and TCE-2 are clearly defined, but TCE-3 still represents an heterogeneous group. Because polymorphisms in HLA-DP influence the presented peptidome, we investigated whether the composition of peptides binding in HLA-DP may be used to refine the HLA-DP group classification. Peptidomes of human HLA-DP-typed B cell lines were analyzed with mass spectrometry after immunoaffinity chromatography and peptide elution. Gibbs clustering was performed to identify motifs of binding peptides. HLA-DP peptide-binding motifs showed a clear association with the HLA-DP allele-specific sequences of the binding groove. Hierarchical clustering of HLA-DP immunopeptidomes was performed to investigate the similarities and differences in peptidomes of different HLA-DP molecules, and this clustering resulted in the categorization of HLA-DP alleles into 3-DP peptidome clusters (DPC). The peptidomes of HLA-DPB1*09:01, -10:01, and -17:01 (TCE-1 alleles) and HLA-DPB1*04:01, -04:02, and -02:01 (TCE-3 alleles) were separated in two maximal distinct clusters, DPC-1 and DPC-3, respectively, reflecting their previous TCE classification. HLA-DP alleles categorized in DPC-2 shared certain similar peptide-binding motifs with DPC-1 or DPC-3 alleles, but significant differences were observed for other positions. Within DPC-2, divergence between the alleles was observed based on the preference for different peptide residues at position 9. In summary, immunopeptidome analysis was used to unravel functional hierarchies among HLA-DP alleles, providing new molecular insights into HLA-DP classification.

Priming of Allo-HLA-DP-Specific Reactivity from the Naïve T Cell Compartment Is Not Exclusively Mediated by Professional Antigen-Presenting Cells.

Allogeneic (allo) stem cell transplantation is applied to patients suffering from hematologic malignancies to replace the diseased hematopoietic system with cells derived from a donor stem cell graft. The majority of 10/10-matched unrelated donors are HLA-DP-mismatched, and this may result in varying degrees of the graft-versus-leukemia (GVL) effect with or without the occurrence of graft-versus-host disease (GVHD). Allo-HLA-reactive T cells are commonly present in the donor T cell repertoire, and thus a very profound alloreactive immune response can be provoked in the HLA-DP-mismatched setting. The magnitude and the diversity of the allo-HLA-DP-specific immune response likely dictates the balance between the occurrence of GVL and/or GVHD after transplantation. To understand the nature of the allo-HLA-DP-specific immune response provoked under different stimulatory conditions, immune responses were induced from both the naïve and memory T cell compartments using either HLA-DP-mismatched professional antigen-presenting cells (APCs) (monocyte-derived dendritic cells [allo-DCs]) or HLA-DP-mismatched nonprofessional APCs (skin-derived fibroblasts [allo-fibroblasts]) as stimulator cells. In this study, we observed that allo-HLA-DP-reactive T cells could be provoked from both the naïve and memory compartments by both types of APCs. However, the magnitude of the allo-HLA-DP-specific immune response was greater when stimulation was performed with allo-DCs. Moreover, we found that the frequency of allo-HLA-DP-reactive T cells was greater in the naïve T cell compartment compared with the memory T cell compartment, but we observed a comparable lineage specificity of these allo-HLA-DP-specific reactivities. Overall, the data from this study illustrate that the presence of professional APCs of recipient origin will mostly dictate the magnitude of the allo-HLA-DP-specific immune response derived from both the naïve and memory T cell compartments, but does not exclusively mediate the induction of these immune responses.