This review proposes a new targeted cancer therapeutic called an intracellular caspase-modulating chimeric antigen receptor (iCCAR) that functions inside malignant cells. The iCCAR would contain an antibody-based single-chain variable fragment (scFv) domain targeting a specific epitope in a mutant protein expressed by cancer cells. Upon binding this epitope, the iCCAR would directly induce apoptosis. Two possible configurations for achieving this controlled apoptosis are described. The review focuses on an approach using an scFv to control a constitutively active apoptosis effector embedded within via an intein. The goal is to develop a therapeutic that can target cancer cells more specifically than current CAR-T cell therapies, reducing the risk of serious adverse events
Assessing the clinical utility of cancer genomic and proteomic data across tu...Gul Muneer
Molecular profiling of tumors promises to advance the clinical
management of cancer, but the benefits of integrating
molecular data with traditional clinical variables have not been
systematically studied. Here we retrospectively predict patient
survival using diverse molecular data (somatic copy-number
alteration, DNA methylation and mRNA, microRNA and protein
expression) from 953 samples of four cancer types from The
Cancer Genome Atlas project. We find that incorporating
molecular data with clinical variables yields statistically
significantly improved predictions (FDR < 0.05) for three
cancers but those quantitative gains were limited (2.2–23.9%).
Additional analyses revealed little predictive power across
tumor types except for one case. In clinically relevant genes,
we identified 10,281 somatic alterations across 12 cancer types
in 2,928 of 3,277 patients (89.4%), many of which would
not be revealed in single-tumor analyses. Our study provides
a starting point and resources, including an open-access
model evaluation platform, for building reliable prognostic and
therapeutic strategies that incorporate molecular data
Identifying novel and druggable targets in a triple negative breast cancer ce...Thermo Fisher Scientific
In this study, we developed a CRISPR/Cas9-based high throughput loss-of-function screen for identifying target genes responsible for the tumor proliferation and growth in TNBC. Our initial focus was to identify essential kinases in MDA-MB-231 cell line using the Invitrogen™ LentiArray™ Human Kinase CRISPR Library, which targets 840 kinases with up to 4 different gRNAs per protein kinase for complete gene knockout. This functional screen identified over 90 protein kinases that are essential for cell viability and cell proliferation. Ten of these hits (CDK1, CDK2, CDK8, CDK10, CDK11A, CDK19, CDK19, CDC7, EPHA2 and WEE1) are well-known targets validated in the literature. Currently, we are in the process validating the novel hits through target gene sequencing, western blotting and target specific small molecule kinase inhibitors.
Proteogenomic analysis of human colon cancer reveals new therapeutic opportun...Gul Muneer
We performed the first proteogenomic study on a prospectively collected colon cancer cohort. Comparative proteomic and phosphoproteomic analysis of paired tumor and normal adjacent tissues produced a catalog of colon cancer-associated proteins and phosphosites, including known and putative new biomarkers, drug targets, and cancer/testis antigens. Proteogenomic integration not only prioritized genomically inferred targets, such as copy-number drivers and mutation-derived neoantigens, but also yielded novel findings. Phosphoproteomics data associated Rb phosphorylation with increased proliferation and decreased apoptosis in colon cancer, which explains why this classical tumor suppressor is amplified in colon tumors and suggests a rationale for targeting Rb phosphorylation in colon cancer. Proteomics identified an association between decreased CD8 T cell infiltration and increased glycolysis in microsatellite instability-high (MSI-H) tumors, suggesting glycolysis as a potential target to overcome the resistance of MSI-H tumors to immune checkpoint blockade. Proteogenomics presents new avenues for biological discoveries and therapeutic development.
Assessing the clinical utility of cancer genomic and proteomic data across tu...Gul Muneer
Molecular profiling of tumors promises to advance the clinical
management of cancer, but the benefits of integrating
molecular data with traditional clinical variables have not been
systematically studied. Here we retrospectively predict patient
survival using diverse molecular data (somatic copy-number
alteration, DNA methylation and mRNA, microRNA and protein
expression) from 953 samples of four cancer types from The
Cancer Genome Atlas project. We find that incorporating
molecular data with clinical variables yields statistically
significantly improved predictions (FDR < 0.05) for three
cancers but those quantitative gains were limited (2.2–23.9%).
Additional analyses revealed little predictive power across
tumor types except for one case. In clinically relevant genes,
we identified 10,281 somatic alterations across 12 cancer types
in 2,928 of 3,277 patients (89.4%), many of which would
not be revealed in single-tumor analyses. Our study provides
a starting point and resources, including an open-access
model evaluation platform, for building reliable prognostic and
therapeutic strategies that incorporate molecular data
Identifying novel and druggable targets in a triple negative breast cancer ce...Thermo Fisher Scientific
In this study, we developed a CRISPR/Cas9-based high throughput loss-of-function screen for identifying target genes responsible for the tumor proliferation and growth in TNBC. Our initial focus was to identify essential kinases in MDA-MB-231 cell line using the Invitrogen™ LentiArray™ Human Kinase CRISPR Library, which targets 840 kinases with up to 4 different gRNAs per protein kinase for complete gene knockout. This functional screen identified over 90 protein kinases that are essential for cell viability and cell proliferation. Ten of these hits (CDK1, CDK2, CDK8, CDK10, CDK11A, CDK19, CDK19, CDC7, EPHA2 and WEE1) are well-known targets validated in the literature. Currently, we are in the process validating the novel hits through target gene sequencing, western blotting and target specific small molecule kinase inhibitors.
Proteogenomic analysis of human colon cancer reveals new therapeutic opportun...Gul Muneer
We performed the first proteogenomic study on a prospectively collected colon cancer cohort. Comparative proteomic and phosphoproteomic analysis of paired tumor and normal adjacent tissues produced a catalog of colon cancer-associated proteins and phosphosites, including known and putative new biomarkers, drug targets, and cancer/testis antigens. Proteogenomic integration not only prioritized genomically inferred targets, such as copy-number drivers and mutation-derived neoantigens, but also yielded novel findings. Phosphoproteomics data associated Rb phosphorylation with increased proliferation and decreased apoptosis in colon cancer, which explains why this classical tumor suppressor is amplified in colon tumors and suggests a rationale for targeting Rb phosphorylation in colon cancer. Proteomics identified an association between decreased CD8 T cell infiltration and increased glycolysis in microsatellite instability-high (MSI-H) tumors, suggesting glycolysis as a potential target to overcome the resistance of MSI-H tumors to immune checkpoint blockade. Proteogenomics presents new avenues for biological discoveries and therapeutic development.
A new generation of cancer immunotherapy called isac can achieve complete tum...DoriaFang
In a new study published in Nature Cancer on December 7, researchers from Bolt Biotherapeutics and Stanford University School of Medicine have developed an immune-stimulating antibody conjugates (ISAC). It combines the accuracy of antibody targeting tumors with the killing potential of the innate and adaptive immune system into a single drug, achieving complete tumor regression and durable anti-tumor immunity in multiple tumor models.
Osteoblasts remotely supply lung tumors with cancer-promoting SiglecFhigh neu...Gul Muneer
Systemic cross-talk between lung tumors and bones
Bone marrow–derived myeloid cells can accumulate within tumors and foster
cancer outgrowth. Local immune-neoplastic interactions have been intensively
investigated, but the contribution of the systemic host environment to tumor growth
remains poorly understood. Here, we show in mice and cancer patients (n = 70) that
lung adenocarcinomas increase bone stromal activity in the absence of bone
metastasis. Animal studies reveal that the cancer-induced bone phenotype involves
bone-resident osteocalcin-expressing (Ocn+) osteoblastic cells. These cells promote
cancer by remotely supplying a distinct subset of tumor-infiltrating SiglecFhigh
neutrophils, which exhibit cancer-promoting properties. Experimentally reducing
Ocn+ cell numbers suppresses the neutrophil response and lung tumor outgrowth.
These observations posit osteoblasts as remote regulators of lung cancer and
identify SiglecFhigh neutrophils as myeloid cell effectors of the osteoblast-driven
protumoral response
Nanodroplet processing platform for deep and quantitative proteome profiling ...Gul Muneer
Nanoscale or single-cell technologies are critical for biomedical applications. However, current mass spectrometry (MS)-based proteomic approaches require samples comprising a minimum of thousands of cells to provide in-depth profiling. Here, we report the development of a nanoPOTS (nanodroplet processing in one pot for trace samples) platform for small cell population proteomics analysis. NanoPOTS enhances the efficiency and recovery of sample processing by downscaling processing volumes to <200 nL to minimize surface losses. When combined with ultrasensitive liquid chromatography-MS, nanoPOTS allows identification of ~1500 to ~3000 proteins from ~10 to ~140 cells, respectively. By incorporating the Match Between Runs algorithm of MaxQuant, >3000 proteins are consistently identified from as few as 10 cells. Furthermore, we demonstrate quantification of ~2400 proteins from single human pancreatic islet thin sections from type 1 diabetic and control donors, illustrating the application of nanoPOTS for spatially resolved proteome measurements from clinical tissues.
Final presentation for BIOL405, NSC, Spring 2014. Presented by Kevin Hugins and Duy-Khiem Chanh Pham. This presentation addressed the use of Chimeric Antigen Receptors for gene therapy for cancer. Gene therapy was first conceptualized to alter debilitating fates of genetic diseases. Gene therapy technology can help introduce new functional DNA to replace mutated genes. The idea first arose in 1972 when Friedmann and Roblin authored a paper, “Gene therapy for human genetic disease?”, demonstrating that exogenous DNA can be taken up by mammalian cells (1). They proposed that the same procedure could be done on humans to correct genetic defects by introducing therapeutic DNA. Currently, genetic modification of T lymphocytes has been the major area of research for treating malignant tumors. This technique seeks to create chimeric antigen receptor (CAR) in T cells by genetically modifying them in vitro and reintroduce them back into blood circulation. The T cells are unique to every patient and the chimeric antigen receptors are unique to the tumor that it is targeting.
The IMPACT of INDEL realignment: Detecting insertions and deletions longer th...Ronak Shah
Cancer is a disease of the genome –most of its forms result from a buildup of genetic alterations that, directly or indirectly, allow the patient’s cells to proliferate without restraint. For decades, identifying and targeting cancer mutations for treatment was impractical due to the limitations of sequencing technology. However, the rise of high-throughput next-generation sequencing (NGS) tools has allowed researchers to rapidly and cheaply sequence large, targeted regions of DNA. MSK-IMPACT(Memorial Sloan Kettering-IntegratedMutation Profiling of Actionable Cancer Targets), a sequencing platform with an associated computational pipeline, takes advantage of improvements in sequencing technology to analyzetumor specimensfor clinically actionable variants in341 cancer-associatedgenes.Criticalto IMPACT’s efficacy is the detection of somatic DNAalterationslike INDELs, which are insertions or deletions of nucleotides. Current sequence aligners have difficulty accuratelymapping reads (short, overlapping DNA sequences) containing morethan a single base change, let alone reads containing INDELs. This flaw necessitates the use of INDEL realigners, whichrearrange reads inregions where INDELs might exist in order to identify them more easily. Currently, the INDEL realignment software associated withMSK-IMPACT’scomputational pipeline, the Genome Analysis Toolkit’s IndelRealigner (GATK), canonly efficiently resolveINDELsshorter than 30 base pairs, which limits theplatform’sreliability forINDELdetection. Thus, wetested and compared the performance of a new INDEL realigner called ABRA (Assembly BasedRe-Aligner) to that of GATK’s IndelRealigner.
Stem Cells in A New Era of Cell based Therapies - Creative BiolabsCreative-Biolabs
A stem cell can replicate itself or differentiate into cells that carry out the specific functions of the body. The application of stem cells in regenerative medicine and disease therapeutics is one of the most exciting advances in medical science today. In cell-based therapies, stem cells may play two roles. The first role is as drug-delivery vehicles. The second role is as therapeutic agents themselves. Stem cells also offer opportunities for scientific advances that go far beyond cell-based therapies. Creative Biolabs is dedicated to facilitate the research of stem cells in both basic science and therapeutics development. Please contact us if you are interested in our services or products.
Chimeric Antigen Receptors (paper with corresponding power point)Kevin B Hugins
Gene therapy was first conceptualized to alter debilitating fates of genetic diseases. Gene therapy technology can help introduce new functional DNA to replace mutated genes. The idea first arose in 1972 when Friedmann and Roblin authored a paper, “Gene therapy for human genetic disease?”, demonstrating that exogenous DNA can be taken up by mammalian cells (1). They proposed that the same procedure could be done on humans to correct genetic defects by introducing therapeutic DNA. Currently, genetic modification of T lymphocytes has been the major area of research for treating malignant tumors. This technique seeks to create chimeric antigen receptor (CAR) in T cells by genetically modifying them in vitro and reintroduce them back into blood circulation. The T cells are unique to every patient and the chimeric antigen receptors are unique to the tumor that it is targeting.
Developing a framework for for detection of low frequency somatic genetic alt...Ronak Shah
Cancer is a complex, heterogeneous disease of the genome. Most cancers result
from an accumulation of multiple genetic alterations that lead to dysfunction of cancer-associated
genes and pathways. Recent advances in sequencing technology have enabled comprehensive
profiling of genetic alterations in cancer. We have established a targeted sequencing platform
(IMPACT: Integrated Mutation Profiling of Actionable Cancer Targets) using hybridization capture and
next-generation sequencing (NGS) technology, which can reveal mutations, indels and copy number
alterations involving 340 cancer related genes.
This ppt file represents a simple overview on what is antibody validation & how to validate an antibody before performing any research.
Used references are also included.
A new generation of cancer immunotherapy called isac can achieve complete tum...DoriaFang
In a new study published in Nature Cancer on December 7, researchers from Bolt Biotherapeutics and Stanford University School of Medicine have developed an immune-stimulating antibody conjugates (ISAC). It combines the accuracy of antibody targeting tumors with the killing potential of the innate and adaptive immune system into a single drug, achieving complete tumor regression and durable anti-tumor immunity in multiple tumor models.
Osteoblasts remotely supply lung tumors with cancer-promoting SiglecFhigh neu...Gul Muneer
Systemic cross-talk between lung tumors and bones
Bone marrow–derived myeloid cells can accumulate within tumors and foster
cancer outgrowth. Local immune-neoplastic interactions have been intensively
investigated, but the contribution of the systemic host environment to tumor growth
remains poorly understood. Here, we show in mice and cancer patients (n = 70) that
lung adenocarcinomas increase bone stromal activity in the absence of bone
metastasis. Animal studies reveal that the cancer-induced bone phenotype involves
bone-resident osteocalcin-expressing (Ocn+) osteoblastic cells. These cells promote
cancer by remotely supplying a distinct subset of tumor-infiltrating SiglecFhigh
neutrophils, which exhibit cancer-promoting properties. Experimentally reducing
Ocn+ cell numbers suppresses the neutrophil response and lung tumor outgrowth.
These observations posit osteoblasts as remote regulators of lung cancer and
identify SiglecFhigh neutrophils as myeloid cell effectors of the osteoblast-driven
protumoral response
Nanodroplet processing platform for deep and quantitative proteome profiling ...Gul Muneer
Nanoscale or single-cell technologies are critical for biomedical applications. However, current mass spectrometry (MS)-based proteomic approaches require samples comprising a minimum of thousands of cells to provide in-depth profiling. Here, we report the development of a nanoPOTS (nanodroplet processing in one pot for trace samples) platform for small cell population proteomics analysis. NanoPOTS enhances the efficiency and recovery of sample processing by downscaling processing volumes to <200 nL to minimize surface losses. When combined with ultrasensitive liquid chromatography-MS, nanoPOTS allows identification of ~1500 to ~3000 proteins from ~10 to ~140 cells, respectively. By incorporating the Match Between Runs algorithm of MaxQuant, >3000 proteins are consistently identified from as few as 10 cells. Furthermore, we demonstrate quantification of ~2400 proteins from single human pancreatic islet thin sections from type 1 diabetic and control donors, illustrating the application of nanoPOTS for spatially resolved proteome measurements from clinical tissues.
Final presentation for BIOL405, NSC, Spring 2014. Presented by Kevin Hugins and Duy-Khiem Chanh Pham. This presentation addressed the use of Chimeric Antigen Receptors for gene therapy for cancer. Gene therapy was first conceptualized to alter debilitating fates of genetic diseases. Gene therapy technology can help introduce new functional DNA to replace mutated genes. The idea first arose in 1972 when Friedmann and Roblin authored a paper, “Gene therapy for human genetic disease?”, demonstrating that exogenous DNA can be taken up by mammalian cells (1). They proposed that the same procedure could be done on humans to correct genetic defects by introducing therapeutic DNA. Currently, genetic modification of T lymphocytes has been the major area of research for treating malignant tumors. This technique seeks to create chimeric antigen receptor (CAR) in T cells by genetically modifying them in vitro and reintroduce them back into blood circulation. The T cells are unique to every patient and the chimeric antigen receptors are unique to the tumor that it is targeting.
The IMPACT of INDEL realignment: Detecting insertions and deletions longer th...Ronak Shah
Cancer is a disease of the genome –most of its forms result from a buildup of genetic alterations that, directly or indirectly, allow the patient’s cells to proliferate without restraint. For decades, identifying and targeting cancer mutations for treatment was impractical due to the limitations of sequencing technology. However, the rise of high-throughput next-generation sequencing (NGS) tools has allowed researchers to rapidly and cheaply sequence large, targeted regions of DNA. MSK-IMPACT(Memorial Sloan Kettering-IntegratedMutation Profiling of Actionable Cancer Targets), a sequencing platform with an associated computational pipeline, takes advantage of improvements in sequencing technology to analyzetumor specimensfor clinically actionable variants in341 cancer-associatedgenes.Criticalto IMPACT’s efficacy is the detection of somatic DNAalterationslike INDELs, which are insertions or deletions of nucleotides. Current sequence aligners have difficulty accuratelymapping reads (short, overlapping DNA sequences) containing morethan a single base change, let alone reads containing INDELs. This flaw necessitates the use of INDEL realigners, whichrearrange reads inregions where INDELs might exist in order to identify them more easily. Currently, the INDEL realignment software associated withMSK-IMPACT’scomputational pipeline, the Genome Analysis Toolkit’s IndelRealigner (GATK), canonly efficiently resolveINDELsshorter than 30 base pairs, which limits theplatform’sreliability forINDELdetection. Thus, wetested and compared the performance of a new INDEL realigner called ABRA (Assembly BasedRe-Aligner) to that of GATK’s IndelRealigner.
Stem Cells in A New Era of Cell based Therapies - Creative BiolabsCreative-Biolabs
A stem cell can replicate itself or differentiate into cells that carry out the specific functions of the body. The application of stem cells in regenerative medicine and disease therapeutics is one of the most exciting advances in medical science today. In cell-based therapies, stem cells may play two roles. The first role is as drug-delivery vehicles. The second role is as therapeutic agents themselves. Stem cells also offer opportunities for scientific advances that go far beyond cell-based therapies. Creative Biolabs is dedicated to facilitate the research of stem cells in both basic science and therapeutics development. Please contact us if you are interested in our services or products.
Chimeric Antigen Receptors (paper with corresponding power point)Kevin B Hugins
Gene therapy was first conceptualized to alter debilitating fates of genetic diseases. Gene therapy technology can help introduce new functional DNA to replace mutated genes. The idea first arose in 1972 when Friedmann and Roblin authored a paper, “Gene therapy for human genetic disease?”, demonstrating that exogenous DNA can be taken up by mammalian cells (1). They proposed that the same procedure could be done on humans to correct genetic defects by introducing therapeutic DNA. Currently, genetic modification of T lymphocytes has been the major area of research for treating malignant tumors. This technique seeks to create chimeric antigen receptor (CAR) in T cells by genetically modifying them in vitro and reintroduce them back into blood circulation. The T cells are unique to every patient and the chimeric antigen receptors are unique to the tumor that it is targeting.
Developing a framework for for detection of low frequency somatic genetic alt...Ronak Shah
Cancer is a complex, heterogeneous disease of the genome. Most cancers result
from an accumulation of multiple genetic alterations that lead to dysfunction of cancer-associated
genes and pathways. Recent advances in sequencing technology have enabled comprehensive
profiling of genetic alterations in cancer. We have established a targeted sequencing platform
(IMPACT: Integrated Mutation Profiling of Actionable Cancer Targets) using hybridization capture and
next-generation sequencing (NGS) technology, which can reveal mutations, indels and copy number
alterations involving 340 cancer related genes.
This ppt file represents a simple overview on what is antibody validation & how to validate an antibody before performing any research.
Used references are also included.
For many years scientists yearned for the possibility of performing flow cytometry to analyse small bio-nanoparticles that are too small to be measured by conventional and high sensitivity instruments. These entities, extracellular vesicles, gene therapy vectors, viruses and drug delivery particles, are promised to become the next generation of therapeutics, but they have been hard to handle and characterise due to their small size and biological or chemical heterogeneity. There is therefore a strong case for bringing flow cytometry capability to the sub-200nm scale.
NanoFCM has developed the NanoAnalyzer platform that now enables true flow-cytometry measurement at the sub-micron scale, and down to particle sizes unreachable by any other flow cytometers (10-40nm depending on the nature of the substrate). Nano-flow cytometry, the technology that underpins the NanoAnalyzer, removes bias and uncertainty stemming from the use of fluorescence signal triggering by using its highly sensitive side-scatter channel to trigger particle events. The single-particle nature of the measurement prevents uncontrolled swarming events, reinforcing data integrity. High resolution of both scatter and fluorescence channels allows the assessment of subpopulations, based on size or on bio-chemical properties.
Nano-flow cytometry’s ability to measure simultaneously a (bio)-nanoparticle population for size, size distribution and bio-chemical properties on a single instrument dramatically improves data quality and throughput compared to the traditional, multiple-techniques approach involving particle characterisation and counting (DLS, NTA, RPS), combined with chemical and biological function assessment (ELISA, Western Blot, Flow Cytometry, PCR). Quantitative measurement of the active and contaminant particles in a single preparation opens up the possibility of characterisation-based nanomedicine regulatory approval, and allows the conduct of large-scale clinical studies. From the research laboratory to the quality control department, NanoFCM delivers comprehensive bio-nano analysis.
Crimson Publishers: Improved Version of Cancer Evo-Dev, a Novel Scientific Hy...CrimsonGastroenterology
Chronic but active inflammation, which is activated and maintained by stimulants such as infection and the interactions of stimulants with genetic predisposition, facilitates the occurrence and recurrence of cancers of various histotypes. Chronic inflammation, apparent or unapparent, is indispensible for the development of most malignancies, which has been clarified in hepatitis B virus (HBV)-induced hepatocellular carcinoma (HCC). Based on our previous work and the advances of researches on HBV-induced HCC and other inflammation-associated cancers, we presented the framework of a novel cancer theory termed Cancer Evolution and Development (Cancer Evo-Dev) [1-3]. Actually, Cancer Evo-Dev can be applied in cancers of many histotypes.
CHI's Targeting Stromal Cells in Cancer and Inflammatory Diseases Conference ...James Prudhomme
This virtual meeting will highlight cutting-edge science and provide insight into recent developments towards therapeutic stromal cell targeting in cancer and chronic inflammatory diseases. View full details and register: https://www.healthtech.com/stroma-conference
1. Please cite this article in press as: Tarone, S. Intracellular caspase-modulating chimeric antigen receptor, Drug Discov Today (2015), http://dx.doi.org/10.1016/j.drudis.2015.02.001
Drug Discovery Today Volume 00, Number 00 February 2015 REVIEWS
Intracellular caspase-modulating
chimeric antigen receptor
Scott Tarone
Tinton Falls, NJ, USA
In this review, a targeted cancer therapeutic is proposed providing
direct targeting of tumor-specific or -associated antigens from
within malignant cells: (i) with an antibody-based biological
agent; (ii) utilizing a constitutively active apoptosis effector that
is inhibited or nonfunctional until the antibody binds its agonist.
Two possible configurations to achieve this end are provided. One
embeds an scFv control into the apoptosis-inducing effector using
an intein and the second employs a zinc-finger-based targeting
and activating mechanism based on an approach known as se-
quence-enabled reassembly of proteins. Although the latter might
provide a broader range of targets, because zinc fingers bind
directly to DNA rather than transcribed protein, this review focus-
es on the former owing to the large body of clinical data available.
Targeting within the cell
Cancer immunotherapy with chimeric antigen receptors (CARs)
involves the transfection of autologous T cells with receptors
specific for a tumor antigen. The main requirement in the devel-
opment of a clinically successful CAR is that this antigen is
available on the malignant cell surface. CAR T cells have achieved
great success in trials with complete remission observed in patients
with chemorefractory chronic lymphocytic leukemia (CLL) and
acute lymphoblastic leukemia [1,2]. However, there have been
tragedies in other trials: one patient receiving erbB-2-targeted
CARs experienced respiratory distress within 15 min and died four
days later. In the case report investigators speculated that an on-
target/off-tissue event occurred with the T cells releasing cytokines
upon recognition of low levels of erbB-2 on lung epithelial cells [3].
The main requirement of CAR-based therapy has become its
greatest obstacle because the search for suitable cell surface tumor
targets has not yielded a group of tumor-specific antigens but
rather those shared with normal cells. The most recent release
on the growth of the T-cell-defined tumor antigen database reports
its 6th selection criteria: ‘a certain level of tumor- or tissue-speci-
ficity should be documented, as ubiquitous antigens do not qualify
as tumor antigens’ [4]. The potential for on-target/off-tissue seri-
ous adverse events (SAEs) remains as long as the targets are shared
even when preferential binding has been observed. Even the
highly successful CD-19-targeted CARs invariably result in chronic
hypogammaglobulinemia. To mitigate this risk, a biological agent
is proposed, hereafter it will be referred to as an intracellular
caspase-modulating chimeric antigen receptor (iCCAR), to be
delivered directly into the cytosol and subcellular compartments,
which is capable of detecting conserved signature sequences in
mutant proteins and, upon detection, directly inducing apoptosis
(Fig. 1).
The antigen-binding domain
In the iCCAR, the antigen-binding domain is an scFv derived from
a mAb engineered with specificity for a signature epitope within
the sequence of mutant protein expressed in malignant cells of a
given phenotype. In most cases, cells from malignant tissue from
an afflicted individual will be sequenced to identify the epitope,
thus requiring on-demand engineering of the applicable chimera.
However, this might not always be the case. Regarding onco-
genes, the research suggests it is not the function of the protein
product that is necessarily altered in oncogenic mutation but
rather a change in transcription regulation, a substantial decrease
in the ubiquitination and proteasome turnover rate or its active
state changing from induced to constitutive that is the actual
driver of oncogenesis [5,6]. From the latter two points, it seems
plausible that the mutations should involve only a few sequence
substitutions given that the protein itself is left functionally intact.
Whether these ‘hotspots’ arise from single nucleotide polymor-
phisms (SNPs) or consistently aligned translocations, it appears
that there could be epitopes that are common across cancer types.
This could leave only one major question: whether or not a
sufficient level of specificity is achievable to ensure off-target
toxicity is not a probable outcome. The fact that it has beenE-mail addresses: starone@synapticsynthase.com, starone@comcast.net.
1359-6446/ß 2015 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.drudis.2015.02.001 www.drugdiscoverytoday.com 1
![Please cite this article in press as: Tarone, S. Intracellular caspase-modulating chimeric antigen receptor, Drug Discov Today (2015), http://dx.doi.org/10.1016/j.drudis.2015.02.001
Drug Discovery Today Volume 00, Number 00 February 2015 REVIEWS
Intracellular caspase-modulating
chimeric antigen receptor
Scott Tarone
Tinton Falls, NJ, USA
In this review, a targeted cancer therapeutic is proposed providing
direct targeting of tumor-specific or -associated antigens from
within malignant cells: (i) with an antibody-based biological
agent; (ii) utilizing a constitutively active apoptosis effector that
is inhibited or nonfunctional until the antibody binds its agonist.
Two possible configurations to achieve this end are provided. One
embeds an scFv control into the apoptosis-inducing effector using
an intein and the second employs a zinc-finger-based targeting
and activating mechanism based on an approach known as se-
quence-enabled reassembly of proteins. Although the latter might
provide a broader range of targets, because zinc fingers bind
directly to DNA rather than transcribed protein, this review focus-
es on the former owing to the large body of clinical data available.
Targeting within the cell
Cancer immunotherapy with chimeric antigen receptors (CARs)
involves the transfection of autologous T cells with receptors
specific for a tumor antigen. The main requirement in the devel-
opment of a clinically successful CAR is that this antigen is
available on the malignant cell surface. CAR T cells have achieved
great success in trials with complete remission observed in patients
with chemorefractory chronic lymphocytic leukemia (CLL) and
acute lymphoblastic leukemia [1,2]. However, there have been
tragedies in other trials: one patient receiving erbB-2-targeted
CARs experienced respiratory distress within 15 min and died four
days later. In the case report investigators speculated that an on-
target/off-tissue event occurred with the T cells releasing cytokines
upon recognition of low levels of erbB-2 on lung epithelial cells [3].
The main requirement of CAR-based therapy has become its
greatest obstacle because the search for suitable cell surface tumor
targets has not yielded a group of tumor-specific antigens but
rather those shared with normal cells. The most recent release
on the growth of the T-cell-defined tumor antigen database reports
its 6th selection criteria: ‘a certain level of tumor- or tissue-speci-
ficity should be documented, as ubiquitous antigens do not qualify
as tumor antigens’ [4]. The potential for on-target/off-tissue seri-
ous adverse events (SAEs) remains as long as the targets are shared
even when preferential binding has been observed. Even the
highly successful CD-19-targeted CARs invariably result in chronic
hypogammaglobulinemia. To mitigate this risk, a biological agent
is proposed, hereafter it will be referred to as an intracellular
caspase-modulating chimeric antigen receptor (iCCAR), to be
delivered directly into the cytosol and subcellular compartments,
which is capable of detecting conserved signature sequences in
mutant proteins and, upon detection, directly inducing apoptosis
(Fig. 1).
The antigen-binding domain
In the iCCAR, the antigen-binding domain is an scFv derived from
a mAb engineered with specificity for a signature epitope within
the sequence of mutant protein expressed in malignant cells of a
given phenotype. In most cases, cells from malignant tissue from
an afflicted individual will be sequenced to identify the epitope,
thus requiring on-demand engineering of the applicable chimera.
However, this might not always be the case. Regarding onco-
genes, the research suggests it is not the function of the protein
product that is necessarily altered in oncogenic mutation but
rather a change in transcription regulation, a substantial decrease
in the ubiquitination and proteasome turnover rate or its active
state changing from induced to constitutive that is the actual
driver of oncogenesis [5,6]. From the latter two points, it seems
plausible that the mutations should involve only a few sequence
substitutions given that the protein itself is left functionally intact.
Whether these ‘hotspots’ arise from single nucleotide polymor-
phisms (SNPs) or consistently aligned translocations, it appears
that there could be epitopes that are common across cancer types.
This could leave only one major question: whether or not a
sufficient level of specificity is achievable to ensure off-target
toxicity is not a probable outcome. The fact that it has beenE-mail addresses: starone@synapticsynthase.com, starone@comcast.net.
1359-6446/ß 2015 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.drudis.2015.02.001 www.drugdiscoverytoday.com 1](https://image.slidesharecdn.com/7d765ac3-4032-430a-aa2c-f5c906de5a52-160906013335/85/Published-PageOne-1-320.jpg)
