Indiana University-Purdue University Indianapolis (IUPUI) / Triple-negative breast cancer (TNBC), which accounts for 15-20% of all breast cancers, is highly aggressive and metastatic with the poorest overall rates. While surgery, radiation, and chemotherapy remain the main treatment options, TNBC represents an unmet medical need for better treatment strategies. Tremendous efforts have been made to develop effective therapies over the past years. However, TNBC treatment options are still very limited due to the lack of good drug targets and the low response rate of current therapies. In this study, we developed two different strategies to treat TNBC based on its cancer genomic features: 1) heterozygous loss of chromosome 17p (17p loss) and 2) high mutation load. 17p loss is one of the most frequent genomic events in breast cancer including TNBC, rendering cancer cells vulnerable to the inhibition of POLR2A via α-amanitin (POLR2A-specific inhibitor). Here, we developed a new drug T-Ama (α-amanitin-conjugated trastuzumab) targeting HER2-low TNBC with 17p loss by combining the effects of α-amanitin and trastuzumab (HER2+ breast cancer therapy). Our results showed that T-Ama exhibited superior efficacy in treating HER2-low TNBC with 17p loss in vitro and in vivo, and surprisingly induced immunogenic cell death (ICD) which further enhanced T cell infiltration and cytotoxicity levels and delivered greater efficacy in combination with immune checkpoint blockade therapy. Collectively, the therapeutic window created by 17p loss and HER2 expression will make HER2-low TNBC clinically feasible targets of T-Ama. As another genetic feature of TNBC, the higher genomic instability and mutational burden results in more neoantigens presented on MHC-I, along with the higher level of tumor-infiltrating T cells, making TNBC a perfect model for immunotherapy compared to the other breast cancer subtypes. Here, we designed a deconvolution-algorithm-derived library screening to find new therapeutic targets and identified PIK3C2α as a key player that determines MHC-I turnover and reduces the MHC-I-restricted antigen presentation on tumor cells. In preclinical models, inhibition of PIK3C2α profoundly suppressed breast tumor growth, increased tumor-infiltrating CD8+ T cells, and showed high potential enhancing the efficacy of anti-PD-1 therapy, suggesting that PIK3C2α is a potential therapeutic target for TNBC immunotherapy. / 2025-05-22

With Athletes and individuals developing osteoarthritis and chondral defects at younger ages, long term treatments are in high demand. Total knee replacements only last for 10-15 years, so younger individuals would need to have multiple knee replacements within their lifetime. Allograft transplantation has shown to last long term and have high success rates, but the lack of donors and the possibility of damaging other areas of the knee to obtain tissue grafts has become a large concern. Xenografts derived from porcine cartilage is cost effective and the supply is abundant. Two antigen removal processes were examined: a short term antigen removal process to maintain the mechanical stability of the tissue, and a long antigen removal process to minimize the risk of triggering an immune response. The antigen removal processes were compared, and the future precautions were determined to enhance the probability of creating a viable osteochondral xenograft preparation technique.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24495号 / 医博第4937号 / 新制||医||1063(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 杉田 昌彦, 教授 鈴木 実 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Die Identifizierung endogener Stammzellen mit kardiogenem Potenzial und die Möglichkeit, deren Differenzierung zu steuern, würde einen Meilenstein in der kardioregenerativen Therapie darstellen. Innerhalb der Gefäßwand konnten unterschiedliche Stamm- und Vorläuferzellen identifiziert werden, die sog. Gefäßwand-residenten Stammzellen (VW-SCs). Zuletzt konnten aus CD34(+) VW-SCs, ohne genetische Manipulation, Kardiomyozyten generiert werden. Zusätzlich fungiert die Gefäßwand als Quelle inflammatorischer Zellen, die essenziell für die kardiogene Differenzierung der VW-SCs zu sein scheinen. Ziel dieser Arbeit war es, das Verhalten von CD44(+) VW-SCs zu untersuchen, um herauszufinden, inwieweit dieser Stammzelltyp eine endogene Generierung von Kardiomyozyten unterstützen könnte. Dabei wurde mit infarzierten Mäuseherzen, dem Aortenringassay (ARA) und dem kardialen Angiogeneseassay (CAA) gearbeitet. Sowohl in vivo in ischämischen Arealen infarzierter Mäuseherzen als auch ex vivo im CAA kam es zu einem signifikanten Anstieg von CD44(+) Zellen. Mittels Färbungen auf CD44 und Ki-67 konnte die Teilungsfähigkeit dieser Zellen demonstriert werden. Ex vivo ließen sich aus CD44(+) Zellen F4/80(+) Makrophagen generieren. Die CD44(+) VW-SCs können sich dabei sowohl zu pro-inflammatorischen iNOS(+) M1- als auch zu anti-inflammatorischen IL-10(+) M2-Makrophagen differenzieren. Eine Modulation der kardialen Inflammation könnte einen entscheidenden Einfluss auf die Kardiomyogenese haben. Unter VEGF-A kam es im CAA zu einer deutlichen Zunahme von CD44(+) Zellen. Unter Lenvatinib blieb das kardiale Sprouting gänzlich aus, die Anzahl der CD44(+) Zellen stagnierte und die VW-SCs verblieben in ihren physiologischen Nischen innerhalb der Gefäßwand. Warum es nach einem MI kaum zu einer funktionellen Herzmuskelregeneration kommt, ist weiterhin unklar. Die therapeutische Beeinflussung koronaradventitieller CD44(+) VW-SCs und inflammatorischer Prozesse könnte dabei zukünftig eine wichtige therapeutische Option darstellen. / The identification of endogenous stem cells with cardiogenic potential and the possibility to control their differentiation would represent a milestone in cardioregenerative therapy. Within the vascular wall, different stem and progenitor cells could be identified, the so-called vascular wall-resident stem cells (VW-SCs). Most recently, cardiomyocytes could be generated from CD34(+) VW-SCs, without genetic manipulation. In addition, the vascular wall acts as a source of inflammatory cells which appear to be essential for cardiogenic differentiation of VW-SCs. The objective of this work was to investigate the behavior of CD44(+) VW-SCs to see to what extent this stem cell type could support endogenous generation of cardiomyocytes. This was done using infarcted mouse hearts, the aortic ring assay (ARA), and the cardiac angiogenesis assay (CAA). There was a significant increase in CD44(+) cells in vivo in ischemic areas of infarcted mouse hearts and ex vivo in the CAA. A double staining for CD44 and Ki-67 demonstrated the ability of these cells to proliferate. Ex vivo, F4/80(+) macrophages could be generated from CD44(+) cells. Thereby, the CD44(+) VW-SCs can differentiate into both pro-inflammatory iNOS(+) M1 and anti-inflammatory IL-10(+) M2 macrophages. Modulation of cardiac inflammation may have a critical impact on cardiomyogenesis. Under VEGF-A, there was a clear increase in CD44(+) cells in the CAA. Under lenvatinib, cardiac sprouting was completely absent, the number of CD44(+) cells stagnated, and VW-SCs remained in their physiological niches within the vessel wall. Why there is little functional myocardial regeneration after MI remains unclear. Therapeutic manipulation of coronary adventitial CD44(+) VW-SCs and inflammatory processes may represent an important therapeutic option in the future.

No description available.

No description available.

Systemic sclerosis (SSc) is a chronic autoimmune disease of the connective tissue. Immune system dysregulation, excessive deposition of collagen and microvascular damage in the skin and multiple internal organs are the main pathologic characteristics of the disease. Little is known about the mechanisms that are responsible for the pathogenesis of SSc. However, evidence has been accumulated demonstrating that T cells play a key role in the initiation and propagation of the disease. Previous studies in our laboratory have identified the presence of high proportions of identical β–chains TCR transcripts, demonstrating the presence of clonal expansion of T cells in skin biopsies from patients with SSc of recent onset. These T cells have undergone proliferation and clonal expansion in response to as yet unidentified antigen(s). The hypothesis that has been tested in this study is whether clonally expanded T cells in skin biopsies of patients with SSc of recent onset recognize self or non–self (possibly viral) putative SSc antigens, including DNA topoisomerase I, cytomegalovirus (CMV) and parvovirus. With the objective to identify the antigens recognized by clonally expanded T cells in skin biopsies of patients with SSc, we examined the presence of α– and β–chain TCR transcripts. Amplification of α–chain TCR transcripts by the non–palindromic adaptor PCR (NPA–PCR)/Vα specific PCR followed by cloning and sequencing revealed the presence of several clonally expanded α–chain TCR transcripts in skin biopsies from four patients with SSc and peripheral blood from one of these patients. Additionally, several clonally expanded β–chain TCR transcripts were identified in skin biopsies from all three of these patients with SSc examined, after NPA–PCR/Vβ specific amplification followed by cloning and sequencing. To identify the antigens recognized by these in vivo clonally expanded α– and β–chain TCR clones, full length α– and β– chain TCR transcripts containing the identified CDR3 regions from the clonally expanded TCR clones from the patients SSc–21 and SSc–22 were constructed. Pairs of clonally expanded, full length α– and β–chain TCR transcripts and appropriate controls were expressed in mutant TCR negative cells of the Jurkat T cell line (J.RT3–T3.5) by using a retroviral gene transfer and expression system. Each clonally expanded α–chain TCR transcript was combined with each clonally expanded β–chain TCR transcript from the same patient, generating T cells lines containing all pairing combinations of the clonally expanded TCR transcripts for each SSc patient. A total of 52 T cell lines were generated, including 10 control T cell lines. The surface expression of the TCR complex on these T cell lines was verified by flow cytometric analysis using antibodies against the α/β TCR and CD3epsilon. We employed an intracellular calcium mobilization assay to examine whether the Jurkat T cell lines transduced with the clonally expanded TCR transcripts from skin biopsies from patients with SSc (SSc–21 and SSc–22) recognize putative SSc antigens or their peptides presented by autologous EBV–transformed B cell lines. The putative SSc antigens that were tested are the self–antigen, DNA topoisomerase I and the viral antigens, cytomegalovirus and parvovirus which have been previously suggested to be involved in the pathogenesis of SSc. Significant intracellular calcium mobilization was observed in response to 3 DNA topoisomerase I and 2 CMV peptides by 5 T cell lines transduced with clonally expanded α– and β–chain TCR transcripts from patients SSc–21 and SSc–22. / Microbiology and Immunology

EBV episomes replicate once per cellular S phase, during latent infection of host cells. Only one viral protein, Epstein-Barr Nuclear Antigen-1 (EBNA-1) is required for replication; the rest of the replication machinery is provided by the cell. EBNA-1 is an excellent model to study the molecular events required for DNA replication and its regulation because viral replication is limited to once per cell cycle. EBNA-1 is a member of a special class of DNA binding proteins called origin binding proteins (OBPs). These specialized proteins bind to distinct DNA sequences in the genome called origins of replication, where DNA replication is initiated. Origin binding proteins may serve to distort the DNA at the origin and may also attract the cellular replication machinery. Structural studies of the DNA binding and dimerization region of EBNA-1 using X-ray crystallography were undertaken in order to better understand how OBPs bind to origin DNA sequences and facilitate the assembly of the cellular replication apparatus. Six truncation mutants of EBNA-1, all containing the DNA binding and dimerization region of EBNA-1, were cloned, over-expressed in bacteria and purified to apparent homogeneity. Four of these clones were crystallized using the method of hanging-drop vapour-diffusion. Two fragments, EBNA₄₇₀₋₆₁₉ and EBNA₄₇₀₋₆₀₇, formed well-ordered crystals that diffracted beyond 2.5 Å resolution. In addition , this study also demonstrates the value of finding the most suitable piece of the protein for crystallization. This piece should fold into a compact domain for efficient packing into a crystal. Finding the optimal piece of the protein reduces the time spent searching for crystallization conditions. / Thesis / Master of Science (MS)

A HSV 2 immediate early antigen was prepared and used as an immunogen in an attempt to produce monoclonal antibodies to this set to proteins. The results of three screening assays, ELISA, immunodiffusion and radioimmunoprecipitation with cell extracts and Protein A-Sepharose beads indicated that the hybrid cell lines are nonsecretors of antiHSV 2 antibodies. Other monoclonal cell lines from Dr. Bacchetti's laboratory were characterized by radioimmunoprecipitation with Protein A-Sepharose beads and cell extracts. / Thesis / Master of Science (MS)

BACKGROUND: Patients with relapsed or refractory large B-cell lymphoma (LBCL) who have relapsed after at least 2 lines of therapy had a poor prognosis before the introduction of chimeric antigen receptor (CAR) T-cell therapy. The FDA approved three CD19 CAR T-cell products, axicabtagene ciloleucel (axi-cel), tisagenlecleucel, and lisocabtagene maraleucel (liso-cel), based on the results of pivotal phase 2 clinical trials. High response rates and long-term remissions in these multiply relapsed patients led to randomized trials as a second-line therapy against the current standard of care for primary refractory and early relapsing LBCL. Axi-cel and liso-cel are now approved as second-line treatments for patients with relapsed or refractory large B-cell lymphoma based on these trials, while tisagenlecleucel failed to improve upon second-line standard of care. This has led to greater axi-cel and liso-cel usage as compared with tisagenlecleucel. Clinical trials and real-world trials show a higher toxicity profile for axi-cel as compared to liso-cel with similar efficacy outcomes, leading to selection of liso-cel for older patients with more medical comorbidities. However, axi-cel manufacturing is faster and more reliable making it a preferred choice for rapidly progressive lymphomas. No direct comparison has been made between the two in order to optimally inform product selection. OBJECTIVE: We aimed to compare the toxicity profile and efficacy outcomes between two cohorts, one treated with axi-cel and the other with liso-cel, ideally well matched, during the same period of time. METHODS: We retroactively gathered patient data for patients treated between June 2021 to September 2022 with both products. We compared the cohorts for patient characteristics that are proven to affect the toxicity and efficacy in order to identify significant differences that could influence our results and to increase the likelihood that the two cohorts were well matched. We then assessed associated toxicities and long-term efficacy outcomes. RESULTS: The two cohorts were comparable for all patient and disease variables other than age (median age of 62 years old in axi-cel compared to 71 years old liso-cel [p < 0.001]). There was no significant difference between high-grade cytokine release syndrome (CRS) (3% vs 5% for axi-cel vs. liso-cel cohorts, respectively; p = 0.58), high-grade immune effector cell-associated neurotoxicity syndrome (ICANS) (18% [ASTCT] or 19% [CTCAE], 14% [ASTCT] or 12% [CTCAE] for axi-cel vs. liso-cel cohorts, respectively, p = 0.055). There were higher rates of any grade CRS with axi-cel, and duration of hospitalization was longer for axi-cel vs. liso-cel (10 vs. 14 days, respectively). Best overall response rates (ORR) (93% vs. 84% axi-cel vs. liso-cel, respectively) and complete response (CR) rates (71% vs. 56% axi-cel vs liso-cel, respectively) did not statistically differ between the two groups. 12-month overall survival (OS) (76% vs. 81% axi-cel vs. liso-cel, respectively) and progression free survival (PFS) (61% vs. 45% of patients axi-cel vs. liso-cel, respectively) did not statistically differ between the two groups (p =0.94, p =0.51 for OS and PFS, respectively). CONCLUSIONS: Our study showed both products are similar in their high-grade toxicity profile as well as their efficacy. While axi-cel has more any grade CRS and ICANS, the lack of significantly higher high-grade toxicities likely reflects better and more aggressive toxicity mitigation strategies when patients present with low grade side effects. As a result, axi-cel in our study was found to be less toxic than previously seen in past clinical trials as well as real-world studies. Many factors go into selection of a CAR T-cell product, ranging from product performance attributes like safety and efficacy, to product manufacturing qualities like turnaround time and fidelity of manufacturing. With equivalency with regards to product performance, manufacturing qualities may then be most important in guiding product selection for LBCL patients.