Enzyme Catalyzed Decomposition of 4-Hydroxycyclophosphamide

According to general doctrine [1] canceroselectivity of Cyclophosphamide is based on different activities of the 4-hydroxycyclophosphamide (OHCP) detoxifying cellular enzyme aldehyde dehydrogenase in tumor and normal cells. Aldehyde dehydrogenase converts the OHCP tautomere aldophosphamide (ALDO) to the non-cytotoxic carboxyphosphamide. Due to different activities of the detoxifying enzyme more cytotoxic phosporamide mustard (PAM) is spontaneously released from OHCP/ALDO in tumor cells. PAM unfolds its cytotoxic activity by forming intrastrand and interstrand DNA crosslinks. This hypothesis is supported by in vitro experiments which show inverse correlations of aldehyde dehydrogenase activity and sensitivity of tumor cells against activated congeners of cyclophosphamide like mafosfamide which hydrolyses within a few minutes to OHCP. In protein free rat serum ultrafiltrate however free OHCP and its coexisting tautomer ALDO are stable compounds. Its half-life in protein free rat serum ultrafiltrate (pH7, 37oC) is more than 20 h. Contrary to protein free ultrafiltrate in whole serum ALDO is enzymatically decomposed to PAM and 3-hydroxypropionaldehyde (HPA) within minutes. The decomposing enzyme was identified as 3´-5´ phosphodiesterase, the Michaelis constant was determined to be 10-3M in human serum.The experiments presented clearly demonstrate that ALDO is not only cleaved base catalyzed yielding acrolein and PAM [2, 3] but also cleaved enzymatically by serum phosphodiesterases yielding HPA and PAM. It is discussed that the reason of the high canceroselectivity of cyclophosphamide is not only due to enrichment of OHCP/ALDO in tumor cells due to less detoxification of ALDO in tumor cells than in normal cells. It is discussed that there is a good reason for an additional mechanism namely the amplification of apoptosis of PAM damaged cells by HPA.A two step mechanism for the mechanism of action of OHCP/ALDO is discussed. During the first step, the DNA is damaged by alkylation by PAM. During the second step the cell containing damaged DNA is eliminated by apoptosis, supported by HPA.

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Hepatocyte-tumor cell interaction in vitro. I. Conditions for rosette formation and inhibition by anti-H-2 antibody.

Journal of Experimental Medicine ◽

10.1084/jem.151.4.984 ◽

1980 ◽

Vol 151 (4) ◽

pp. 984-989 ◽

Cited By ~ 64

Author(s):

V Schirrmacher ◽

R Cheingsong-Popov ◽

H Arnheiter

Keyword(s):

Tumor Cell ◽

Tumor Cells ◽

Metastatic Tumor ◽

Cell Interaction ◽

Rosette Formation ◽

Normal Cells ◽

Neuraminidase Treatment

Murine hepatocytes, isolated by an in situ collagenase-perfusion technique and cultured in Petri dishes, were shown to form rosettes with liver-metastasizing syngeneic tumor cells. Pretreatment of the tumor cells with neuraminidase generally increased the binding, whereas pretreatment of the liver cells with neuraminidase abolished the binding completely. The tumor-cell binding may be mediated by the previously described lectin-like receptor of hepatocytes that also was sensitive to neuraminidase treatment and that bound desialylated cells better than normal cells. Anti-H-2 sera could efficiently inhibit the rosette formation of metastatic tumor cells with the hepatocytes, which points to a possible role of H-2 molecules in this interaction of neoplastic and normal cells.

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122 A powerful, precise targeting system controlled by tumor deletions transforms CEA and MSLN CAR-T cells into tumor-selective agents

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.122 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A131-A131

Author(s):

Agnes Hamburger ◽

Han Xu ◽

Yuta Ando ◽

Grace Asuelime ◽

Kristian Bolanos-Ibarra ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Cells ◽

Loss Of Heterozygosity ◽

Tumor Antigens ◽

Allelic Loss ◽

List Type ◽

Mixed Cell ◽

Normal Cells

BackgroundMesothelin (MSLN) and carcinoembryonic antigen (CEA) are classic tumor-associated antigens that are expressed in many solid tumors including the majority of lung, colorectal and pancreatic cancers. However, both MSLN and CEA are also expressed in vital normal organs. This normal expression creates risk of serious inflammation for CEA- or MSLN-directed therapeutics. To date all active CEA- or MSLN-targeted investigational therapeutics have been toxic when administered systemically.MethodsWe have developed a safety mechanism to protect normal tissues without abrogating sensitivity of cytotoxic T cells directed at MLSN(+) or CEA(+) tumors in a subset of patients with defined loss of heterozygosity (LOH) in their tumors (figure 1). This dual-receptor (Tmod< sup >TM</sup >) system exploits common LOH at the HLA locus in cancer cells, allowing T cells to recognize the difference between tumor and normal tissue.1 2 T cells engineered with specific Tmod constructs contain: (i) a MSLN- or CEA-activated CAR; and, (ii) an inhibitory receptor gated by HLA-A*02. HLA-A*02 binding blocks T cell cytotoxicity, even in the presence of MSLN or CEA. The Tmod system is designed to treat heterozygous HLA class I patients, selected for HLA LOH. When HLA-A*02 is absent from tumors selected for LOH, the CARs are predicted to mediate potent killing of the A*02(-) malignant cells.ResultsThe Tmod system robustly protects surrogate normal cells even in mixed-cell populations in vitro while mediating robust cytotoxicity of tumor cells in xenograft models (see example in figure 2). The MSLN CAR can also be paired with other blockers, supporting scalability of the approach to patients beyond HLA-A*02 heterozygotes.Abstract 122 Figure 1Illustration of the Tmod T cell engaging with tumor cells with somatic loss of HLA-A*02 and with normal cells.Abstract 122 Figure 2Bioluminescence measurements show the average difference between the size of the MSLN(+)A*02(+) ‘normal’ graft compared to the MSLN(+)A*02(-) tumor graft on the two flanks of mice after T cell infusion. Both tumor and normal grafts are destroyed by CAR-Ts (CAR-3 and M5 benchmark) while the MSLN Tmod cells kill the tumor but not the normal graft.ConclusionsThe Tmod mechanism may provide an alternative route to leverage solid-tumor antigens such as MSLN and CEA in safer, more effective ways than previously possible.ReferencesHamburger AE, DiAndreth B, Cui J, et al. Engineered T cells directed at tumors with defined allelic loss. Mol Immunol 2020;128:298–310.Hwang MS, Mog BJ, Douglass J, et al. Targeting loss of heterozygosity for cancer-specific immunotherapy. Proc Natl Acad Sci U S A 2021;118(12):e2022410118.

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Circulating Lymphangioleiomyomatosis Tumor Cells With Loss of Heterozygosity in the TSC2 Gene Show Increased Aldehyde Dehydrogenase Activity

CHEST Journal ◽

10.1016/j.chest.2019.03.040 ◽

2019 ◽

Vol 156 (2) ◽

pp. 298-307 ◽

Cited By ~ 1

Author(s):

Gustavo Pacheco-Rodríguez ◽

Wendy K. Steagall ◽

Leigh Samsel ◽

Pradeep K. Dagur ◽

J. Philip McCoy ◽

...

Keyword(s):

Tumor Cells ◽

Dehydrogenase Activity ◽

Loss Of Heterozygosity ◽

Aldehyde Dehydrogenase ◽

Tsc2 Gene ◽

Aldehyde Dehydrogenase Activity

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Tumor-Specific Proteolytic Processing of Cyclin E Generates Hyperactive Lower-Molecular-Weight Forms

Molecular and Cellular Biology ◽

10.1128/mcb.21.18.6254-6269.2001 ◽

2001 ◽

Vol 21 (18) ◽

pp. 6254-6269 ◽

Cited By ~ 120

Author(s):

Donald C. Porter ◽

Ning Zhang ◽

Christopher Danes ◽

Mollianne J. McGahren ◽

Richard M. Harwell ◽

...

Keyword(s):

Molecular Weight ◽

Tumor Cells ◽

Serine Proteases ◽

Cyclin E ◽

S Phase ◽

Proteolytic Processing ◽

Full Length ◽

Lower Molecular Weight ◽

Normal Cells

ABSTRACT Cyclin E is a G1 cyclin essential for S-phase entry and has a profound role in oncogenesis. Previously this laboratory found that cyclin E is overexpressed and present in lower-molecular-weight (LMW) isoforms in breast cancer cells and tumor tissues compared to normal cells and tissues. Such alteration of cyclin E is linked to poor patient outcome. Here we report that the LMW forms of cyclin E are hyperactive biochemically and they can more readily induce G1-to-S progression in transfected normal cells than the full-length form of the protein can. Through biochemical and mutational analyses we have identified two proteolytically sensitive sites in the amino terminus of human cyclin E that are cleaved to generate the LMW isoforms found in tumor cells. Not only are the LMW forms of cyclin E functional, as they phosphorylate substrates such as histone H1 and GST-Rb, but also their activities are higher than the full-length cyclin E. These nuclear localized LMW forms of cyclin E are also biologically functional, as their overexpression in normal cells increases the ability of these cells to enter S and G2/M. Lastly, we show that cyclin E is selectively cleaved in vitro by the elastase class of serine proteases to generate LMW forms similar to those observed in tumor cells. These studies suggest that the defective entry into and exit from S phase by tumor cells is in part due to the proteolytic processing of cyclin E, which generates hyperactive LMW isoforms whose activities have been modified from that of the full-length protein.

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Using a hybrid radioenhancer to realize tumor cell-targeted treatment for osteosarcoma: an in vitro study

Current Medicinal Chemistry ◽

10.2174/0929867327666201118155216 ◽

2020 ◽

Vol 27 ◽

Author(s):

Fu-I Tung ◽

Li-Chin Chen ◽

Yu-Chi Wang ◽

Ming-Hong Chen ◽

Pei-Wei Shueng ◽

...

Keyword(s):

Western Blot ◽

Tumor Cell ◽

Cell Viability ◽

Tumor Cells ◽

Clonogenic Assay ◽

Normal Cells ◽

Targeted Radiotherapy ◽

In Vitro Investigation ◽

Dose Radiation

: Osteosarcoma is insensitive to radiation. High-dose radiation is often used as a treatment, but causes side effects in patients. Hence, it is important to develop tumor cell-targeted radiotherapy that could improve radiotherapy efficiency on tumor cells and reduce the toxic effect on normal cells during radiation treatment. In this study, we developed an innovative method for treating osteosarcoma by using a novel radiation-enhancer (i.e., carboxymethyl-hexanoyl chitosan-coated selfassembled Au@Fe3O4 nanoparticles; CSAF NPs). CSAF NPs were employed together with 5-aminolevulinic acid (5-ALA) to achieve tumor cell-targeted radiotherapy. In this study, osteosarcoma cells (MG63) and normal cells (MC3T3-E1) were used for an in vitro investigation, in which a reactive oxygen species (ROS) assay, cell viability assay, clonogenic assay, and western blot were used to confirm the treatment efficiency. The ROS assay showed that the combination of CSAF NPs and 5-ALA enhanced radiation-induced ROS production in tumor cells (MG63); however, this was not observed in normal cells (MC3T3-E1). The cell viability ratio of normal cells to tumor cells after treatment with CSAF NPs and 5-ALA reached 2.79. Moreover, the clonogenic assay showed that the radiosensitivity of MG63 cells was increased by the combination use of CSAF NPs and 5-ALA. This was supported by performing a western blot that confirmed expression of cytochrome c (a marker of cell mitochondria damage) and caspase-3 (a marker of cell apoptosis). The results provide an essential basis for developing tumor-cell targeted radiotherapy by means of low-dose radiation.

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Replicative Senescence in Good-Risk Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v108.11.1922.1922 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1922-1922

Author(s):

Mark Zijlmans ◽

Susan Swiggers ◽

Maria Rife Soler ◽

Berna Beverloo

Keyword(s):

Cell Growth ◽

Tumor Cells ◽

Replicative Senescence ◽

Proliferative Capacity ◽

Telomere Shortening ◽

Normal Cells ◽

Beta Galactosidase ◽

Good Risk

Abstract Immortal cell growth is considered the hallmark of tumor cells. In contrast, normal cells have a limited proliferative capacity of 40–60 cell divisions, also known as the Hayflick limit. The limited proliferative capacity of normal cells relates to gradual telomere shortening as a consequence of the end-replication problem. Upon critical telomere shortening, cells enter a non-replicative but viable state referred to as replicative senescence. These replicative senescent cells stain blue in a beta-Galactosidase assay and activate DNA double-strand break repair pathways at telomeres (e.g. gamma-H2AX foci). In human fibroblast models, escape from senescence results from loss of p53 and Rb function. Escape is associated with reactivation of telomerase. High levels of telomerase, as observed in germ cells and most tumor cells, allow for immortal cell growth. Recently, we demonstrated low levels of telomerase in AML patients with t(8;21) or inv(16) (Swiggers et al, G.C.C. 2006). Interestingly, levels of telomerase in these AML samples were similar to levels of telomerase in normal bone marrow progenitor cells. We hypothesized that AML without re-activated telomerase may still have intact senescence pathways that limit the proliferative capacity of normal cells. This hypothesis was addressed by studying AML patient samples without telomerase re-activation, i.e., t(8;21), t(15;17) or inv(16) (n=10), and a control group of AML with telomerase re-activation (multiple gains/losses of genetic material, n=8). AML samples werelong-time cultured in vitro in the presence of hematopoietic growth factors (range 3–6 weeks),analyzed in vivo following transplantation in NOD-SCID mice andin patients at time of relapse. Cells with all characteristics of replicative senescence, i.e. enlarged, viable, non-proliferating, blue-coloring in beta-Galactosidase assay, critical short telomeres and gamma-H2AX foci at telomeres, were clearly observed in all AML samples with t(8;21), t(15;17) or inv(16). Gradual telomere shortening was observed in these AML cells in vitro upon long-term culture, in vivo after transplantation in NOD-SCID mice and in vivo in patients at relapse compared to time of diagnosis, indicating that these AML cells do not have an adequate telomere maintenance mechanism. We conclude that AML cells with t(8;21), t(15;17) or inv(16) are characterized by intact pathways that induce replicative senescence. Intact pathways that limit proliferative lifespan may be critical to the high cure rates following chemotherapy treatment of patients with good-risk AML.

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Phenobarbital Enhances the Aldehyde Dehydrogenase Activity of Rat Hepatocytes in Vitro and in Vivo

Acta Pharmacologica et Toxicologica ◽

10.1111/j.1600-0773.1986.tb00191.x ◽

2009 ◽

Vol 59 (5) ◽

pp. 403-409 ◽

Cited By ~ 11

Author(s):

Marios Marselos ◽

George Michalopoulos

Keyword(s):

Dehydrogenase Activity ◽

Aldehyde Dehydrogenase ◽

Rat Hepatocytes ◽

Aldehyde Dehydrogenase Activity

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120 P-MUC1C-ALLO1: An allogeneic car-t for multiple solid tumor indications

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0120 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A131-A131

Author(s):

Anna Kozlowska ◽

Yan Zhang ◽

Jacqueline Fritz ◽

Steven Wang ◽

Rebecca Codde ◽

...

Keyword(s):

T Cells ◽

Tumor Cells ◽

Triple Negative ◽

Xenograft Model ◽

Cancer Model ◽

Normal Cells ◽

Car T Cells ◽

Car T

BackgroundMUC1 is a highly glycosylated protein that is expressed at the apical border of mucosal epithelium where it plays a protective role. MUC1 is comprised of an N-terminal subunit (MUC1N) tethered to a C-terminal subunit (MUC1C), forming a stable complex on the cell surface. A proteolytic ‘stump’ of MUC1C that may be aberrantly glycosylated is over-represented in cancer, making it an attractive therapeutic target. Here we report generation of allogeneic MUC1C-specific CAR T cells, P-MUC1C-ALLO1, that are designed to leverage the learnings of our P-BCMA-ALLO1 program. P-MUC1C-ALLO1 targets a MUC1C epitope and has the potential for efficacy against a wide range of solid tumors, without targeting normal epithelial cells.Methods mRNA-generated MUC1C CAR-T cells were evaluated for specificity and function by degranulation assay against various solid tumor and normal cells and cell lines. Autologous and allogeneic MUC1C CAR-T cells were produced using the piggyBac® DNA Modification System, a nonviral CAR-T manufacturing method that produces CAR-T products with an exceptionally high percentage of T stem cell memory (TSCM) cells. To produce allogeneic cells, multiplex editing of both TRBC and B2M was performed with the Cas-CLOVER™ Site-Specific Gene Editing System to reduce or eliminate GvHD and host versus graft alloreactivity, respectively. To determine in vivo antitumor efficacy of MUC1C CAR-T cells, we employed the MDA.MB.468 triple negative breast cancer model and the OVCAR3 disseminated ovarian cancer model.ResultsSpecific degranulation of transiently-expressing CAR+ T cells was observed against multiple tumor cells, with no observable activity against normal human primary cells. In assay of stable P-MUC1C-101 CAR-T cells, more than 95% expressed CAR, and were comprised of an exceptionally high-percentage of TSCM cells (CD45RA+CD62L+CD45RO-). In vitro, P-MUC1C-ALLO1 cells specifically proliferated, lysed, and secreted IFN-γ against MUC1C+ breast and ovarian tumor cell lines. In breast cancer in vivo xenograft model, both unedited (MUC1C CAR-T) and edited (P-MUC1C-ALLO1) MUC1C CAR-T eliminated established, triple negative MDA.MB.468 tumor cells to undetectable levels, demonstrating the efficacy of the MUC1C CAR-T and the robustness of the allogeneic platform. In the OVCAR3 xenograft model, intraperitoneally administered MUC1C CAR-T eliminated established tumor cells to levels below the limit of detection.ConclusionsP-MUC1C-ALLO1 is Poseida’s allogeneic CAR TSCM product that has a potential to treat multiple MUC1-expressing indications. P-MUC1C-ALLO1 displayed in vitro specificity for tumor vs normal cells, and in vivo efficacy against xenograft models of breast and ovarian cancer. We anticipate an IND filing and initiation of a Phase 1 clinical trial in 2021.

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