scholarly journals A protease-resistant immunotoxin against CD22 with greatly increased activity against CLL and diminished animal toxicity

Blood ◽  
2009 ◽  
Vol 113 (16) ◽  
pp. 3792-3800 ◽  
Author(s):  
John E. Weldon ◽  
Laiman Xiang ◽  
Oleg Chertov ◽  
Inger Margulies ◽  
Robert J. Kreitman ◽  
...  
Keyword(s):  
Anticancer Agents ◽  
Proteolytic Processing ◽  
Lymphocytic Leukemia ◽  
Cleavage Sites ◽  
Pseudomonas Exotoxin A ◽  
Lysosomal Protease ◽  
Tumor Associated Antigen ◽  
Protease Digestion ◽  
Higher Doses ◽  
Increased Activity

Abstract Immunotoxins based on Pseudomonas exotoxin A (PE) are promising anticancer agents that combine a variable fragment (Fv) from an antibody to a tumor-associated antigen with a 38-kDa fragment of PE (PE38). The intoxication pathway of PE immunotoxins involves receptor-mediated internalization and trafficking through endosomes/lysosomes, during which the immunotoxin undergoes important proteolytic processing steps but must otherwise remain intact for eventual transport to the cytosol. We have investigated the proteolytic susceptibility of PE38 immunotoxins to lysosomal proteases and found that cleavage clusters within a limited segment of PE38. We subsequently generated mutants containing deletions in this region using HA22, an anti-CD22 Fv-PE38 immunotoxin currently undergoing clinical trials for B-cell malignancies. One mutant, HA22-LR, lacks all identified cleavage sites, is resistant to lysosomal degradation, and retains excellent biologic activity. HA22-LR killed chronic lymphocytic leukemia cells more potently and uniformly than HA22, suggesting that lysosomal protease digestion may limit immunotoxin efficacy unless the susceptible domain is eliminated. Remarkably, mice tolerated doses of HA22-LR at least 10-fold higher than lethal doses of HA22, and these higher doses exhibited markedly enhanced antitumor activity. We conclude that HA22-LR advances the therapeutic efficacy of HA22 by using an approach that may be applicable to other PE-based immunotoxins.

Primary care oncology model (PCOM): Implementation of a model integrating primary and oncology care for patients taking oral anticancer agents.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. TPS1587-TPS1587
Author(s):  
Emily R. Mackler ◽  
Karen B. Farris ◽  
Katie S. Gatwood ◽  
Amna Rizvi-Toner ◽  
Alex Wallace ◽  
...  
Keyword(s):  
Primary Care ◽  
Anticancer Agents ◽  
Chronic Conditions ◽  
Lymphocytic Leukemia ◽  
Chronic Obstructive ◽  
Multiple Chronic Conditions ◽  
Hiv Intervention ◽  
Patient Reported ◽  
Chronic Medications ◽  
Oral Anticancer Agents

TPS1587 Background: Non-adherence to oral anticancer agents (OAA) has been reported among 30% of individuals. Often, individuals with cancer are not just managing their new OAA but also medications to treat multiple chronic conditions (MCC). Multiple factors contribute to the extent patients on OAAs and MCC medications adhere to therapy. The objective of this study is to improve medication, symptom, and disease management of patients with hematological malignancies and MCC through care coordination between pharmacists. Methods: Design. This is a multi-center prospective single arm pilot study at two academic medical centers in Michigan and Tennessee. Subjects. Ninety participants will be recruited, 60 from site 1 and 30 from site 2. Inclusion criteria are: adults > 18 years, diagnosed with and initiating oral treatment for chronic myeloid leukemia, chronic lymphocytic leukemia, or multiple myeloma, diagnoses of at least 2 chronic conditions, where one is type 2 diabetes, hypertension, congestive heart failure, depression/anxiety, gastroesophageal reflux disease, hyperlipidemia, or chronic obstructive pulmonary disease, taking at least two chronic medications, and able to provide electronic consent. Exclusion criteria are: inability to speak English, and diagnosis of type 1 diabetes or HIV. Intervention. Participants will complete two Patient Reported Outcome Measures (PROMs) for their OAA that will be reviewed by the oncology pharmacist, with follow-up to the care team if needed. Participants will be scheduled for a Comprehensive Medication Review with a primary care pharmacist for up to two visits for their chronic medications. The intervention over 2 months, and the oncology and primary care pharmacists communicate via electronic health record about medications, symptoms, and disease control. Outcomes. The primary endpoints are (a) dose-adjusted adherence by proportion days covered (PDC) for the OAA and (b) PDC for chronic condition medications, assessed using 6 months of prescription claims. Data will be collected from patients using REDCap surveys and abstracted data will be entered into REDCap. Implementation by pharmacists and patient acceptability will be examined. Analysis. The association of OAA and chronic medication adherence (PDC) will be examined via correlation. Participant demographics,clinical characteristics, and the symptom experience from the PROM will be described. Using CMR results, medication problems, recommendations, and changes will be provided. Program implementation will be assessed and patient perceptions obtained from post-CMR interviews. A joint display for the quantitative and qualitative data for feasibility, appropriateness, and acceptability from pharmacists will be completed. Results: Screening and recruitment has begun. Clinical trial information: NCT04595851 and NCT04663100.

scholarly journals Proteolytic maturation of α2δ represents a checkpoint for activation and neuronal trafficking of latent calcium channels

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Ivan Kadurin ◽  
Laurent Ferron ◽  
Simon W Rothwell ◽  
James O Meyer ◽  
Leon R Douglas ◽  
...  
Keyword(s):  
Calcium Channels ◽  
Proteolytic Cleavage ◽  
Proteolytic Processing ◽  
Calcium Currents ◽  
Cleavage Sites ◽  
Voltage Dependent ◽  
Neuronal Processes ◽  
Associated Proteins ◽  
Synaptic Boutons

The auxiliary α2δ subunits of voltage-gated calcium channels are extracellular membrane-associated proteins, which are post-translationally cleaved into disulfide-linked polypeptides α2 and δ. We now show, using α2δ constructs containing artificial cleavage sites, that this processing is an essential step permitting voltage-dependent activation of plasma membrane N-type (CaV2.2) calcium channels. Indeed, uncleaved α2δ inhibits native calcium currents in mammalian neurons. By inducing acute cell-surface proteolytic cleavage of α2δ, voltage-dependent activation of channels is promoted, independent from the trafficking role of α2δ. Uncleaved α2δ does not support trafficking of CaV2.2 channel complexes into neuronal processes, and inhibits Ca2+ entry into synaptic boutons, and we can reverse this by controlled intracellular proteolytic cleavage. We propose a model whereby uncleaved α2δ subunits maintain immature calcium channels in an inhibited state. Proteolytic processing of α2δ then permits voltage-dependent activation of the channels, acting as a checkpoint allowing trafficking only of mature calcium channel complexes into neuronal processes.

scholarly journals A Forward Genetic Strategy Reveals Destabilizing Mutations in the Ebolavirus Glycoprotein That Alter Its Protease Dependence during Cell Entry

2009 ◽  
Vol 84 (1) ◽  
pp. 163-175 ◽  
Author(s):  
Anthony C. Wong ◽  
Rohini G. Sandesara ◽  
Nirupama Mulherkar ◽  
Sean P. Whelan ◽  
Kartik Chandran
Keyword(s):  
Proteolytic Processing ◽  
Cell Entry ◽  
Cleavage Sites ◽  
Viral Glycoprotein ◽  
Enveloped Virus ◽  
Cysteine Cathepsins ◽  
Vesicular Stomatitis ◽  
Lysosomal Proteases ◽  
Conformational Rearrangements ◽  
Viral Membrane Fusion

ABSTRACT Ebolavirus (EBOV) entry into cells requires proteolytic disassembly of the viral glycoprotein, GP. This proteolytic processing, unusually extensive for an enveloped virus entry protein, is mediated by cysteine cathepsins, a family of endosomal/lysosomal proteases. Previous work has shown that cleavage of GP by cathepsin B (CatB) is specifically required to generate a critical entry intermediate. The functions of this intermediate are not well understood. We used a forward genetic strategy to investigate this CatB-dependent step. Specifically, we generated a replication-competent recombinant vesicular stomatitis virus bearing EBOV GP as its sole entry glycoprotein and used it to select viral mutants resistant to a CatB inhibitor. We obtained mutations at six amino acid positions in GP that independently confer complete resistance. All of the mutations reside at or near the GP1-GP2 intersubunit interface in the membrane-proximal base of the prefusion GP trimer. This region forms a part of the “clamp” that holds the fusion subunit GP2 in its metastable prefusion conformation. Biochemical studies suggest that most of the mutations confer CatB independence not by altering specific cleavage sites in GP but rather by inducing conformational rearrangements in the prefusion GP trimer that dramatically enhance its susceptibility to proteolysis. The remaining mutants did not show the preceding behavior, indicating the existence of multiple mechanisms for acquiring CatB independence during entry. Altogether, our findings suggest that CatB cleavage is required to facilitate the triggering of viral membrane fusion by destabilizing the prefusion conformation of EBOV GP.

scholarly journals Cleavage Map and Proteolytic Processing of the Murine Norovirus Nonstructural Polyprotein in Infected Cells

2006 ◽  
Vol 80 (16) ◽  
pp. 7816-7831 ◽  
Author(s):  
Stanislav V. Sosnovtsev ◽  
Gaël Belliot ◽  
Kyeong-OK Chang ◽  
Victor G. Prikhodko ◽  
Larissa B. Thackray ◽  
...  
Keyword(s):  
Cell Culture ◽  
Caspase 3 ◽  
Proteolytic Processing ◽  
Site Directed Mutagenesis ◽  
Cleavage Sites ◽  
Murine Norovirus ◽  
Permissive Cell ◽  
Additional Processing ◽  
Infected Cells

ABSTRACT Murine norovirus (MNV) is presently the only member of the genus Norovirus in the Caliciviridae that can be propagated in cell culture. The goal of this study was to elucidate the proteolytic processing strategy of MNV during an authentic replication cycle in cells. A proteolytic cleavage map of the ORF1 polyprotein was generated, and the virus-encoded 3C-like (3CL) proteinase (Pro) mediated cleavage at five dipeptide cleavage sites, 341E/G342, Q705/N706, 870E/G871, 994E/A995, and 1177Q/G1178, that defined the borders of six proteins with the gene order p38.3 (Nterm)-p39.6 (NTPase)-p18.6-p14.3 (VPg)-p19.2 (Pro)-p57.5 (Pol). Bacterially expressed MNV 3CL Pro was sufficient to mediate trans cleavage of the ORF1 polyprotein containing the mutagenized Pro sequence into products identical to those observed during cotranslational processing of the authentic ORF1 polyprotein in vitro and to those observed in MNV-infected cells. Immunoprecipitation and Western blot analysis of proteins produced in virus-infected cells demonstrated efficient cleavage of the proteinase-polymerase precursor. Evidence for additional processing of the Nterm protein in MNV-infected cells by caspase 3 was obtained, and Nterm sequences 118DRPD121 and 128DAMD131 were mapped as caspase 3 cleavage sites by site-directed mutagenesis. The availability of the MNV nonstructural polyprotein cleavage map in concert with a permissive cell culture system should facilitate studies of norovirus replication.

Fibromodulin as a Novel Tumor-Associated Antigen (TAA) in Chronic Lymphocytic Leukemia (CLL) Which Allows Expansion of Specific CD8+ Autologous T Lymphocytes.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 175-175 ◽  
Author(s):  
Christine Mayr ◽  
Dagmar Bund ◽  
Martin Schlee ◽  
Andreas Moosmann ◽  
Michael Hallek ◽  
...  
Keyword(s):  
T Cells ◽  
In Vitro Culture ◽  
B Lymphocytes ◽  
Cd40 Ligand ◽  
Immune Monitoring ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Autologous Tumor ◽  
Tumor Associated Antigen

Abstract BACKGROUND: Fibromodulin (FMOD), a collagen binding protein, was shown to be highly overexpressed in CLL cells compared to normal B lymphocytes by gene expression profiling. Therefore FMOD might serve as potential tumor associated antigen (TAA) in CLL, enabling expansion of FMOD-specific T cells. FMOD is physiologically expressed in articular cartilage, tendon and ligament. Furthermore, interactions of FMOD with the growth factor TGF-b were described and it may be a biologically relevant modulator of TGF-b activity. Methods: Unpulsed native CLL cells and CD40 ligand (CD40L)-stimulated CLL cells as antigen presenting cells (APC) were used to expand autologous T cells from 13 patients. RESULTS: In CLL samples derived from 16 patients, high expression of FMOD by real-time RT-PCR was detectable in contrast to normal B lymphocytes. The number of T cells during four weeks of in vitro culture increased 2-fold with native CLL cells as APC and 3.5-fold with CD40L-stimulated CLL cells as APC. The amount of T cells recognizing HLA-A0201 binding FMOD-derived peptides detected by HLA-A2-dimer/peptide staining increased 10-fold during in vitro culture. The T cells expanded were also able to secrete IFN-g upon recognition of the antigen demonstrated by IFN-g-ELISPOT assays. T cells not only recognized HLA-A0201 binding FMOD peptides presented by TAP-deficient T2 cells, but also FMOD overexpressing autologous CLL cells in an HLA-A0201 restricted manner. Neither HLA-A0201 negative CLL cells nor non-malignant cells, i.e. PBMC from healthy donors or tonsilar B cells, were specifically recognized by T cells. When CD40L-stimulated CLL cells were used as APC, which were pulsed with FMOD peptides prior to coincubation with the T cells, significant higher amounts of T cells specifically recognized autologous CLL cells in IFN-g-ELISPOT assays P< 0.018). CONCLUSION: FMOD was shown for the first time to be naturally processed and ( presented as TAA in primary CLL cells. This enables the expansion of autologous tumor-specific T cells which might be applicable in clinical vaccination trials or as a tool for a CLL-specific immune monitoring in the context of vaccination approaches including CD40L-gene modified autologous leukemic cells.

Formin-Like Gene Expression in Chronic Lymphocytic Leukemia Is Associated with Unfavorable Prognostic Factors and Clinical Outcome

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4181-4181
Author(s):  
Eva Calpe ◽  
Antonio Martinez ◽  
Patricia Favaro ◽  
Marta Crespo ◽  
Carles Codony ◽  
...  
Keyword(s):  
B Cell ◽  
Recombinant Expression ◽  
Human Leukocyte ◽  
High Serum ◽  
Lymphocytic Leukemia ◽  
Expression Cloning ◽  
Cd38 Expression ◽  
Tumor Associated Antigen ◽  
Mantle Cell ◽  
B Cell Subsets

Abstract Formin proteins are large proteins evolutionarily conserved that govern cell shape, adhesion, cytokinesis, and morphogenesis by remodeling the actin and microtubule cytoskeletons. The human leukocyte formin, named as FMNL1, corresponds to an extended cDNA of the 5′-truncated KW-13 which was reported as a tumor-associated antigen in CLL, using a serologic identification by recombinant expression cloning (SEREX). In addition, FMNL1 co-immunoprecipitates with P-Akt in CLL cells, suggests that this protein may contribute to regulate cell survival. The aim of this study was to analyze the expression of FMNL1 in normal B-cell subsets and in a series of 73 patients (median age, 59 years; male/female 40/33; Binet A: 90.2%) with CLL. FMNL1 expression was analyzed by Western Blot, Immunohistochemistry and by Real-Time RT-PCR using expression in Jurkat as baseline. In normal lymphocytes subsets, FMNL1 was only expressed in memory B-cells, and in T-cells, whereas germinal center lymphocytes were negative. Among lymphoid B-cell malignancies, FMNL1 was expressed in mantle-cell lymphomas, Burkitt’s lymphomas and in DLBCL of GCB-type. In CLL cases mean of FMNL1 expression by QRT-PCR was 2.18 AU (SD, 1.01 AU). Using an arbitrary cut-off of 3.2 AU, cases with increased expression of FMNL1 were associated with a younger age at diagnosis (&lt; 50 yrs), elevated lymphocyte count, high serum β2-microglobulin (β2-m) levels, increased ZAP-70 and CD38 expression, shorter time to progression, and shorter survival as compared to cases with low FMNL1 expression. No relationship was observed with genetic abnormalities. In summary, increased expression of FMNL1 gene couples with adverse clinical and biological parameters in patients with CLL. Finally, the interaction between FMNL1 and AKT protein in lymhoproliferative disorders is under investigation.

scholarly journals Targeted Deletion of MIC5 Enhances Trimming Proteolysis of Toxoplasma Invasion Proteins

2006 ◽  
Vol 5 (12) ◽  
pp. 2174-2183 ◽  
Author(s):  
Susannah D. Brydges ◽  
Xing Wang Zhou ◽  
My-Hang Huynh ◽  
Jill M. Harper ◽  
Jeffrey Mital ◽  
...  
Keyword(s):  
Cell Invasion ◽  
Limited Proteolysis ◽  
Opportunistic Pathogen ◽  
Proteolytic Processing ◽  
Secretory Proteins ◽  
Membrane Association ◽  
Cleavage Sites ◽  
Targeted Deletion ◽  
Genetic Ablation ◽  
Substrate Cleavage

ABSTRACT Limited proteolysis of proteins transiently expressed on the surface of the opportunistic pathogen Toxoplasma gondii accompanies cell invasion and facilitates parasite migration across cell barriers during infection. However, little is known about what factors influence this specialized proteolysis or how these proteolytic events are regulated. Here we show that genetic ablation of the micronemal protein MIC5 enhances the normal proteolytic processing of several micronemal proteins secreted by Toxoplasma tachyzoites. Restoring MIC5 expression by genetic complementation reversed this phenotype, as did treatment with the protease inhibitor ALLN, which was previously shown to block the activity of a hypothetical parasite surface protease called MPP2. We show that, despite its lack of obvious membrane association signals, MIC5 occupies the parasite surface during invasion in the vicinity of the proteins affected by enhanced processing. Proteolysis of other secretory proteins, including GRA1, was also enhanced in MIC5 knockout parasites, indicating that the phenotype is not strictly limited to proteins derived from micronemes. Together, our findings suggest that MIC5 either directly regulates MPP2 activity or it influences MPP2's ability to access substrate cleavage sites on the parasite surface.

scholarly journals Proteolytic Processing of the Open Reading Frame 1b-Encoded Part of Arterivirus Replicase Is Mediated by nsp4 Serine Protease and Is Essential for Virus Replication

1999 ◽  
Vol 73 (3) ◽  
pp. 2027-2037 ◽  
Author(s):  
Leonie C. van Dinten ◽  
Sietske Rensen ◽  
Alexander E. Gorbalenya ◽  
Eric J. Snijder
Keyword(s):  
Serine Protease ◽  
Proteolytic Processing ◽  
Equine Arteritis Virus ◽  
Open Reading Frame ◽  
Cleavage Sites ◽  
Nonstructural Proteins ◽  
Site Mutation ◽  
Reading Frame ◽  
Infected Cells ◽  
Unusual Phenotype

ABSTRACT The open reading frame (ORF) 1b-encoded part of the equine arteritis virus (EAV) replicase is expressed by ribosomal frameshifting during genome translation, which results in the production of an ORF1ab fusion protein (345 kDa). Four ORF1b-encoded processing products, nsp9 (p80), nsp10 (p50), nsp11 (p26), and nsp12 (p12), have previously been identified in EAV-infected cells (L. C. van Dinten, A. L. M. Wassenaar, A. E. Gorbalenya, W. J. M. Spaan, and E. J. Snijder, J. Virol. 70:6625–6633, 1996). In the present study, the generation of these four nonstructural proteins was shown to be mediated by the nsp4 serine protease, which is the main viral protease (E. J. Snijder, A. L. M. Wassenaar, L. C. van Dinten, W. J. M. Spaan, and A. E. Gorbalenya, J. Biol. Chem. 271:4864–4871, 1996). Mutagenesis of candidate cleavage sites revealed that Glu-2370/Ser, Gln-2837/Ser, and Glu-3056/Gly are the probable nsp9/10, nsp10/11, and nsp11/12 junctions, respectively. Mutations which abolished ORF1b protein processing were introduced into a recently developed infectious cDNA clone (L. C. van Dinten, J. A. den Boon, A. L. M. Wassenaar, W. J. M. Spaan, and E. J. Snijder, Proc. Natl. Acad. Sci. USA 94:991–997, 1997). An analysis of these mutants showed that the selective blockage of ORF1b processing affected different stages of EAV reproduction. In particular, the mutant with the nsp10/11 cleavage site mutation Gln-2837→Pro displayed an unusual phenotype, since it was still capable of RNA synthesis but was incapable of producing infectious virus.

scholarly journals Mode of cleavage of pig big endothelin-1 by chymotrypsin. Production and degradation of mature endothelin-1

1990 ◽  
Vol 270 (2) ◽  
pp. 541-544 ◽  
Author(s):  
M Takaoka ◽  
Y Miyata ◽  
Y Takenobu ◽  
R Ikegawa ◽  
Y Matsumura ◽  
...  
Keyword(s):  
Amino Acid ◽  
Major Product ◽  
Proteolytic Processing ◽  
Intermediate Form ◽  
Cleavage Sites ◽  
Amino Acid Residues ◽  
Disulphide Bridge ◽  
Endothelin 1 ◽  
Hydrolysis Of

Pig endothelin-1 [ET-1-(1-21)] seems to be produced via proteolytic processing between Trp-21 and Val-22 of an intermediate form consisting of 39 amino acid residues, termed big ET-1-(1-39), by a chymotrypsin-like proteinase. We examined the chymotryptic-cleavage sites of big ET-1-(1-39) by reverse-phase h.p.l.c. and sequence analysis, and found that chymotrypsin cleaved initially the Tyr-31-Gly-32 bond of big ET-1-(1-39), followed by cleavage between Trp-21 and Val-22. Furthermore, chymotrypsin hydrolysed the generated ET-1-(1-21), producing a single major product that had the same amino acid sequence as ET-1-(1-21) with a cleavage between Tyr-13 and Phe-14. The disulphide bridge between Cys-1 and Cys-15 remained intact. These results indicate that the conversion of big ET-1-(1-39) into ET-1-(1-21) catalysed by chymotrypsin requires hydrolysis of the Tyr-31-Gly-32 bond before that of the Trp-21-Val-22 bond, an event followed by cleavage between Tyr-13 and Phe-14 within the loop of ET-1-(1-21). Thus a chymotrypsin-like proteinase might be involved not only in the production but also in the degradation of ET-1-(1-21) in vivo.

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