scholarly journals Novel pipeline of high-frequency neoantigens heathy donor-based validation in breast cancer

2019 ◽  
Author(s):  
Lili Qin ◽  
Ying Huang ◽  
Zhaoduan Liang ◽  
Geng Liu ◽  
Xiumei Lin ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Binding Affinity ◽  
High Frequency ◽  
Ex Vivo ◽  
Human Leukocyte ◽  
Genetic Mutation ◽  
Cross Reactivity ◽  
Target Cells ◽  
Cytotoxic Lymphocytes

SummaryNeoantigen, a peptide fragment formed by genetic mutation, gives immunologist a new target for cancer therapy. Development of biotechnology has opened a new era for discovering high-frequency neoantigens. The aim of our study was to identify breast cancer neoantigens for tumor immunotherapy using an efficient way. Here, we established a computational pipeline to identify neoantigens associated with breast cancer using data from database and evaluated the immunogenicity of neoantigens using the peripheral blood of healthy donators in vitro. We identified 39,401 missense mutation sites from 285,283 single nucleotide variations (SNVs) obtained from database, and confirmed candidate epitopes by analyzing the binding affinity of mutant epitopes and human leukocyte antigen (HLA) using 6 algorithms. Peptide-binding assay was used as a complement for affinity testing. The immunogenicity of candidate peptides with high affinity were assessed through enzyme-linked immunospot (ELISPOT) assay and Cytotoxicity assay. In our study, we identified 10 candidate peptides with high binding affinity of HLA-A*0201 alleles, and seven of ten peptides showed the ability of inducing specific cytotoxic lymphocytes(CTLs) ex vivo, in healthy HLA-A2+donors. We found that the peptide derived from TWISTNB have the highest immunogenicity and cytotoxicity among those candidate peptides. Furthermore, it can trigger the immune response of specific-CTLs to destroy target cells expressing this neoantigen in vitro, and without cross-reactivity with wild-type peptides. We conclude that the effective pipeline will provide potential possibilities to rapidly identify abundant high-frequency neoantigens and create neoantigen library for immunotherapy of breast cancer and even other tumors.

scholarly journals Selection of aptamers against triple negative breast cancer cells using high throughput sequencing

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Débora Ferreira ◽  
Joaquim Barbosa ◽  
Diana A. Sousa ◽  
Cátia Silva ◽  
Luís D. R. Melo ◽  
...  
Keyword(s):  
Breast Cancer ◽  
High Throughput ◽  
Triple Negative Breast Cancer ◽  
High Throughput Sequencing ◽  
Triple Negative ◽  
Metastatic Breast ◽  
Target Cells ◽  
Cancer Tissue ◽  
Surface Receptors

AbstractTriple-negative breast cancer is the most aggressive subtype of invasive breast cancer with a poor prognosis and no approved targeted therapy. Hence, the identification of new and specific ligands is essential to develop novel targeted therapies. In this study, we aimed to identify new aptamers that bind to highly metastatic breast cancer MDA-MB-231 cells using the cell-SELEX technology aided by high throughput sequencing. After 8 cycles of selection, the aptamer pool was sequenced and the 25 most frequent sequences were aligned for homology within their variable core region, plotted according to their free energy and the key nucleotides possibly involved in the target binding site were analyzed. Two aptamer candidates, Apt1 and Apt2, binding specifically to the target cells with $$K_{d}$$ K d values of 44.3 ± 13.3 nM and 17.7 ± 2.7 nM, respectively, were further validated. The binding analysis clearly showed their specificity to MDA-MB-231 cells and suggested the targeting of cell surface receptors. Additionally, Apt2 revealed no toxicity in vitro and showed potential translational application due to its affinity to breast cancer tissue sections. Overall, the results suggest that Apt2 is a promising candidate to be used in triple-negative breast cancer treatment and/or diagnosis.

scholarly journals Single-cell RNA sequencing reveals ex vivo signatures of SARS-CoV-2-reactive T cells through ‘reverse phenotyping’

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
David S. Fischer ◽  
Meshal Ansari ◽  
Karolin I. Wagner ◽  
Sebastian Jarosch ◽  
Yiqi Huang ◽  
...  
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Ex Vivo ◽  
Severe Disease ◽  
Human Leukocyte ◽  
Cell Receptor ◽  
Single Cell Rna Sequencing

AbstractThe in vivo phenotypic profile of T cells reactive to severe acute respiratory syndrome (SARS)-CoV-2 antigens remains poorly understood. Conventional methods to detect antigen-reactive T cells require in vitro antigenic re-stimulation or highly individualized peptide-human leukocyte antigen (pHLA) multimers. Here, we use single-cell RNA sequencing to identify and profile SARS-CoV-2-reactive T cells from Coronavirus Disease 2019 (COVID-19) patients. To do so, we induce transcriptional shifts by antigenic stimulation in vitro and take advantage of natural T cell receptor (TCR) sequences of clonally expanded T cells as barcodes for ‘reverse phenotyping’. This allows identification of SARS-CoV-2-reactive TCRs and reveals phenotypic effects introduced by antigen-specific stimulation. We characterize transcriptional signatures of currently and previously activated SARS-CoV-2-reactive T cells, and show correspondence with phenotypes of T cells from the respiratory tract of patients with severe disease in the presence or absence of virus in independent cohorts. Reverse phenotyping is a powerful tool to provide an integrated insight into cellular states of SARS-CoV-2-reactive T cells across tissues and activation states.

scholarly journals HIV-1 subverts the complement system in semen to enhance viral transmission

2021 ◽  
Author(s):  
Bernadien M. Nijmeijer ◽  
Marta Bermejo-Jambrina ◽  
Tanja M. Kaptein ◽  
Carla M. S. Ribeiro ◽  
Doris Wilflingseder ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Virus Transmission ◽  
Sexual Contact ◽  
Target Cells ◽  
People Living With Hiv ◽  
Lectin Receptor ◽  
Pre Treatment ◽  
Living With Hiv ◽  
Hiv 1

AbstractSemen is important in determining HIV-1 susceptibility but it is unclear how it affects virus transmission during sexual contact. Mucosal Langerhans cells (LCs) are the first immune cells to encounter HIV-1 during sexual contact and have a barrier function as LCs are restrictive to HIV-1. As semen from people living with HIV-1 contains complement-opsonized HIV-1, we investigated the effect of complement on HIV-1 dissemination by human LCs in vitro and ex vivo. Notably, pre-treatment of HIV-1 with semen enhanced LC infection compared to untreated HIV-1 in the ex vivo explant model. Infection of LCs and transmission to target cells by opsonized HIV-1 was efficiently inhibited by blocking complement receptors CR3 and CR4. Complement opsonization of HIV-1 enhanced uptake, fusion, and integration by LCs leading to an increased transmission of HIV-1 to target cells. However, in the absence of both CR3 and CR4, C-type lectin receptor langerin was able to restrict infection of complement-opsonized HIV-1. These data suggest that complement enhances HIV-1 infection of LCs by binding CR3 and CR4, thereby bypassing langerin and changing the restrictive nature of LCs into virus-disseminating cells. Targeting complement factors might be effective in preventing HIV-1 transmission.

scholarly journals Effect of Chitosan Coating on PLGA Nanoparticles for Oral Delivery of Thymoquinone: In Vitro, Ex Vivo, and Cancer Cell Line Assessments

Coatings ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 6
Author(s):  
Sultan Alshehri ◽  
Syed Sarim Imam ◽  
Md Rizwanullah ◽  
Khalid Umar Fakhri ◽  
Mohd Moshahid Alam Rizvi ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Particle Size ◽  
Oral Delivery ◽  
Ex Vivo ◽  
Cancer Cell Line ◽  
Entrapment Efficiency ◽  
Plga Nanoparticles ◽  
Cancer Management ◽  
Mcf 7

In the present study, thymoquinone (TQ)-encapsulated chitosan- (CS)-coated poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles (NPs) were formulated using the emulsion evaporation method. NPs were optimized by using 33-QbD approach for improved efficacy against breast cancer. The optimized thymoquinone loaded chitosan coated Poly (d,l-lactide-co-glycolide) nanoparticles (TQ-CS-PLGA-NPs) were successfully characterized by different in vitro and ex vivo experiments as well as evaluated for cytotoxicity in MDA-MB-231 and MCF-7 cell lines. The surface coating of PLGA-NPs was completed by CS coating and there were no significant changes in particle size and entrapment efficiency (EE) observed. The developed TQ-CS-PLGA-NPs showed particle size, polydispersibility index (PDI), and %EE in the range between 126.03–196.71 nm, 0.118–0.205, and 62.75%–92.17%. The high and prolonged TQ release rate was achieved from TQ-PLGA-NPs and TQ-CS-PLGA-NPs. The optimized TQ-CS-PLGA-NPs showed significantly higher mucoadhesion and intestinal permeation compared to uncoated TQ-PLGA-NPs and TQ suspension. Furthermore, TQ-CS-PLGA-NPs showed statistically enhanced antioxidant potential and cytotoxicity against MDA-MB-231 and MCF-7 cells compared to uncoated TQ-PLGA-NPs and pure TQ. On the basis of the above findings, it may be stated that chitosan-coated TQ-PLGA-NPs represent a great potential for breast cancer management.

Cord blood comprises antigen-experienced T cells specific for maternal minor histocompatibility antigen HA-1

Blood ◽  
2005 ◽  
Vol 105 (4) ◽  
pp. 1823-1827 ◽  
Author(s):  
Bregje Mommaas ◽  
Janine A. Stegehuis-Kamp ◽  
Astrid G. van Halteren ◽  
Michel Kester ◽  
Jürgen Enczmann ◽  
...  
Keyword(s):  
T Cells ◽  
Cord Blood ◽  
Ex Vivo ◽  
Cytotoxic T Cells ◽  
Cord Blood Transplantation ◽  
Human Leukocyte ◽  
Leukemic Cells ◽  
Target Cells ◽  
Graft Versus Leukemia

AbstractUmbilical cord blood transplantation is applied as treatment for mainly pediatric patients with hematologic malignancies. The clinical results show a relatively low incidence of graft-versus-host disease and leukemia relapse. Since maternal cells traffic into the fetus during pregnancy, we questioned whether cord blood has the potential to generate cytotoxic T cells specific for the hematopoietic minor histocompatibility (H) antigen HA-1 that would support the graft-versus-leukemia effect. Here, we demonstrate the feasibility of ex vivo generation of minor H antigen HA-1-specific T cells from cord blood cells. Moreover, we observed pre-existing HA-1-specific T cells in cord blood samples. Both the circulating and the ex vivo-generated HA-1-specific T cells show specific and hematopoietic restricted lysis of human leukocyte antigen-A2pos/HA-1pos (HLA-A2pos/HA-1pos) target cells, including leukemic cells. The cord blood-derived HA-1-specific cytotoxic T cells are from child origin. Thus, the so-called naive cord blood can comprise cytotoxic T cells directed at the maternal minor H antigen HA-1. The apparent immunization status of cord blood may well contribute to the in vivo graft-versus-leukemia activity after transplantation. Moreover, since the fetus cannot be primed against Y chromosome-encoded minor H antigens, cord blood is an attractive stem cell source for male patients. (Blood. 2005;105:1823-1827)

scholarly journals Allergenic Characterization of New Mutant Forms of Pru p 3 as New Immunotherapy Vaccines

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
C. Gómez-Casado ◽  
M. Garrido-Arandia ◽  
P. Gamboa ◽  
N. Blanca-López ◽  
G. Canto ◽  
...  
Keyword(s):  
Food Allergy ◽  
Ex Vivo ◽  
Binding Capacity ◽  
Cross Reactivity ◽  
T Cell Epitopes ◽  
Native Form ◽  
Ige Binding ◽  
Pru P 3

Nowadays, treatment of food allergy only considered the avoidance of the specific food. However, the possibility of cross-reactivity makes this practice not very effective. Immunotherapy may exhibit as a good alternative to food allergy treatment. The use of hypoallergenic molecules with reduced IgE binding capacity but with ability to stimulate the immune system is a promising tool which could be developed for immunotherapy. In this study, three mutants of Pru p 3, the principal allergen of peach, were produced based on the described mimotope and T cell epitopes, by changing the specific residues to alanine, named asPru p 3.01, Pru p 3.02, andPru p 3.03.Pru p 3.01showed very similar allergenic activity as the wild type byin vitroassays. However,Pru p 3.02andPru p 3.03presented reduced IgE binding with respect to the native form, byin vitro,ex vivo,and in vivo assays. In addition,Pru p 3.03had affected the IgG4 binding capacity and presented a random circular dichroism, which was reflected in the nonrecognition by specific antibodies anti-Pru p 3. Nevertheless, bothPru p 3.02andPru p 3.03maintained the binding to IgG1 and their ability to activate T lymphocytes. Thus,Pru p 3.02andPru p 3.03could be good candidates for potential immunotherapy in peach-allergic patients.

O-191 Assessing the use of tumour-specific DARPin-toxin fusion proteins for ex vivo purging of cancer metastases from human ovarian cortex tissue fragments before autotransplantation

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
L Eijkenboom ◽  
V Palacio-Castañeda ◽  
F A Groenman ◽  
D D M Braat ◽  
C C M Beerendonk ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Fusion Proteins ◽  
Breast Cancer Cells ◽  
Ex Vivo ◽  
Ovarian Tissue ◽  
In Vitro Growth ◽  
Ovarian Cortex ◽  
Positive Breast Cancer

Abstract Study question Is it possible to eradicate cancer cells from ovarian cortex by using tumour-specific designed ankyrin repeat protein (DARPin)-toxin fusion proteins, without compromising the ovarian tissue? Summary answer Purging ovarian cortex ex vivo from experimentally induced breast cancer tumour foci is possible by tumour-targeted DARPin-toxin fusion proteins trough inhibition of protein synthesis. What is known already Ovarian tissue cryopreservation and autotransplantation is a successful technique for fertility restoration in cancer patients. The procedure is not without risk since malignant cells may still be present in the graft. Procedures to detect cancer cells render the tissue fragment useless for autotransplantation. Strategies to circumvent this problem such as in vitro maturation of follicles or the construction of artificial ovaries are pursued but are still experimental. Alternatively, we have shown ex vivo purging of ovarian cortex is possible by elimination of rhabdomyosarcoma after treatment with verteporfin. This allows treatment of cortex fragments before autotransplantation without compromising ovarian tissue integrity. Study design, size, duration Human ovarian cortex fragments harbouring breast cancer tumour foci were exposed for 24 h to DARPins fused to the translocation and catalytic domain of Pseudomonas aeruginosa exotoxin A (DARPin-toxin fusion proteins) targeting EpCAM or HER2. After treatment with the DARPin-toxin fusion proteins the tissue was cultured for an additional 6 days to allow any remaining tumour cells to form foci. In addition, the functional integrity of the ovarian tissue was analysed after purging. Participants/materials, setting, methods Breast cancer cell lines expressing different levels of EpCAM and HER2 were introduced in human ovarian tissue to form tumour foci. After purging with DARPin-toxin fusion proteins, the presence of any remaining cancer cells in the tissue was analysed with (immuno)histochemistry and RT-qPCR. Possible detrimental effects on the viability of ovarian cortex and follicles were determined by (immuno)histology, a follicular viability assay and an assay to determine the in vitro growth capacity of small follicles. Main results and the role of chance Ovarian cortex harbouring EpCAM-positive breast cancer cells showed a significant decrease in the number of tumour foci after treatment with the EpCAM-targeted DARPin-toxin fusion proteins. Although exposure to the EpCAM-specific DARPin had no effect on morphology or viability of follicles, a decrease in oocyte viability after in vitro growth experiments was observed, presumably due to low level expression of EpCAM on oocytes. In contrast to the EpCAM-specific DARPin-toxin fusion protein, the DARPin-toxin fusion protein targeting HER2 had no detrimental effects on morphology, viability or in vitro growth of follicles while foci of HER2-positive breast cancer cells were severely affected as indicated by the presence of apoptotic bodies, tumour cell remnants and the absence of viable tumour cells. The histological results after purging with the HER2-specific DARPin-toxin fusions proteins were confirmed by RT-qPCR, showing a decrease to basal levels of HER2 mRNA in the ovarian cortex tissue. Limitations, reasons for caution The effect of DARPin-toxin fusion proteins depends heavily on the expression of their target on the cancer cell. The target protein should not be expressed by ovarian cortex as this may lead to tissue damage. The functional integrity of ovarian cortex after the treatment requires further investigation in vivo. Wider implications of the findings Purging metastases from ovarian cortex without harming ovarian tissue is possible by targeting tumour specific surface expressed antigens with DARPin-toxin fusion proteins. Purging ovarian cortex tissue with DARPin-toxin fusion proteins provides a feasible therapeutic strategy to prevent reintroduction of cancer by autotransplantation in case of malignancies expressing tumour-specific surface markers. Trial registration number not applicable

scholarly journals In Vitro and In Vivo Comparison of Lymphocytes Transduced with a Human CD16 or with a Chimeric Antigen Receptor Reveals Potential Off-Target Interactions due to the IgG2 CH2-CH3 CAR-Spacer

2015 ◽  
Vol 2015 ◽  
pp. 1-13 ◽  
Author(s):  
Béatrice Clémenceau ◽  
Sandrine Valsesia-Wittmann ◽  
Anne-Catherine Jallas ◽  
Régine Vivien ◽  
Raphaël Rousseau ◽  
...  
Keyword(s):  
Chimeric Antigen Receptor ◽  
Tumor Regression ◽  
Critical Role ◽  
Antigen Receptor ◽  
Target Cells ◽  
Cytotoxic Lymphocytes ◽  
Her2 Positive ◽  
Positive Target

The present work was designed to compare two mechanisms of cellular recognition based on Ab specificity: firstly, when the anti-HER2 mAb trastuzumab bridges target cells and cytotoxic lymphocytes armed with a Fc receptor (ADCC) and, secondly, when HER2 positive target cells are directly recognized by cytotoxic lymphocytes armed with a chimeric antigen receptor (CAR). To compare these two mechanisms, we used the same cellular effector (NK-92) and the same signaling domain (FcεRIγ). The NK-92 cytotoxic cell line was transfected with either a FcγRIIIa-FcεRIγ(NK-92CD16) or a trastuzumab-based scFv-FcεRIγchimeric receptor (NK-92CAR). In vitro, the cytotoxic activity against HER2 positive target cells after indirect recognition byNK-92CD16was always inferior to that observed after direct recognition byNK-92CAR. In contrast, and somehow unexpectedly, in vivo, adoptive transfer ofNK-92CD16+ trastuzumab but not ofNK-92CARinduced tumor regression. Analysis of the in vivo xenogeneic system suggested that the human CH2-CH3 IgG2 used as a spacer in our construct was able to interact with the FcR present at the cell surface of the few NSG-FcR+ remaining immune cells. This interaction, leading to blockage of theNK-92CARin the periphery of the engrafted tumor cells, stresses the critical role of the composition of the spacer domain.

scholarly journals Participation of the H-2 antigens of tumor cells in their lysis by syngeneic T cells.

1976 ◽  
Vol 143 (3) ◽  
pp. 601-614 ◽  
Author(s):  
J W Schrader ◽  
G M Edelman
Keyword(s):  
T Cells ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Target Cell ◽  
Cytotoxic T Cells ◽  
Hybrid Origin ◽  
Target Cells ◽  
Cytotoxic Lymphocytes ◽  
Effector Cells

Cytotoxic T lymphocytes were generated in vitro against H-2 compatible or syngeneic tumor cells. In vitro cytotoxic activity was inhibited by specific anti-H2 sera, suggesting that H-2 antigens are involved in cell lysis. Two observations directly demonstrated the participation of the H-2 antigens on the tumor cells in their lysis by H-2-compatible T cells. First, coating of the H-2 antigens on the target tumor cell reduced the number of cells lysed on subsequent exposure to cytotoxic T cells. Second, when cytotoxic T cells were activated against an H-2 compatible tumor and assayed against an H-2-incompatible tumor, anti-H-2 serum that could bind to the target cell, but not to the cytotoxic lymphocyte, inhibited lysis. H-2 antigens were also shown to be present on the cytotoxic lymphocytes. Specific antisera reacting with these H-2 antigens, but not those of the target cell, failed to inhibit lysis when small numbers of effector cells were assayed against H-2-incompatible target cells or when effector cells of F1-hybrid origin and bearing two H-2 haplotypes were assayed against a tumor cell of one of the parental strains. These findings suggest that it is the H-2 antigens on the tumor cell and not those on the cytotoxic lymphocytes that are important in cell-mediated lysis of H-2-compatible tumor cells.

scholarly journals ADAM22/LGI1 complex as a new actionable target for breast cancer brain metastasis

BMC Medicine ◽  
2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Sara Charmsaz ◽  
Ben Doherty ◽  
Sinéad Cocchiglia ◽  
Damir Varešlija ◽  
Attilio Marino ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Metastatic Breast Cancer ◽  
Brain Metastasis ◽  
In Silico ◽  
Ex Vivo ◽  
Metastatic Breast ◽  
Peptide Mimetic ◽  
Breast Cancer Brain Metastasis

Abstract Background Metastatic breast cancer is a major cause of cancer-related deaths in woman. Brain metastasis is a common and devastating site of relapse for several breast cancer molecular subtypes, including oestrogen receptor-positive disease, with life expectancy of less than a year. While efforts have been devoted to developing therapeutics for extra-cranial metastasis, drug penetration of blood–brain barrier (BBB) remains a major clinical challenge. Defining molecular alterations in breast cancer brain metastasis enables the identification of novel actionable targets. Methods Global transcriptomic analysis of matched primary and metastatic patient tumours (n = 35 patients, 70 tumour samples) identified a putative new actionable target for advanced breast cancer which was further validated in vivo and in breast cancer patient tumour tissue (n = 843 patients). A peptide mimetic of the target’s natural ligand was designed in silico and its efficacy assessed in in vitro, ex vivo and in vivo models of breast cancer metastasis. Results Bioinformatic analysis of over-represented pathways in metastatic breast cancer identified ADAM22 as a top ranked member of the ECM-related druggable genome specific to brain metastases. ADAM22 was validated as an actionable target in in vitro, ex vivo and in patient tumour tissue (n = 843 patients). A peptide mimetic of the ADAM22 ligand LGI1, LGI1MIM, was designed in silico. The efficacy of LGI1MIM and its ability to penetrate the BBB were assessed in vitro, ex vivo and in brain metastasis BBB 3D biometric biohybrid models, respectively. Treatment with LGI1MIM in vivo inhibited disease progression, in particular the development of brain metastasis. Conclusion ADAM22 expression in advanced breast cancer supports development of breast cancer brain metastasis. Targeting ADAM22 with a peptide mimetic LGI1MIM represents a new therapeutic option to treat metastatic brain disease.

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