34 research outputs found
Hexa-arginine enhanced uptake and residualization of selective high affinity ligands by Raji lymphoma cells
<p>Abstract</p> <p>Background</p> <p>A variety of arginine-rich peptide sequences similar to those found in viral proteins have been conjugated to other molecules to facilitate their transport into the cytoplasm and nucleus of targeted cells. The selective high affinity ligand (SHAL) (DvLPBaPPP)<sub>2</sub>LLDo, which was developed to bind only to cells expressing HLA-DR10, has been conjugated to one of these peptide transduction domains, hexa-arginine, to assess the impact of the peptide on SHAL uptake and internalization by Raji cells, a B-cell lymphoma.</p> <p>Results</p> <p>An analog of the SHAL (DvLPBaPPP)<sub>2</sub>LLDo containing a hexa-arginine peptide was created by adding six D-arginine residues sequentially to a lysine inserted in the SHAL's linker. SHAL binding, internalization and residualization by Raji cells expressing HLA-DR10 were examined using whole cell binding assays and confocal microscopy. Raji cells were observed to bind two fold more <sup>111</sup>In-labeled hexa-arginine SHAL analog than Raji cells treated with the parent SHAL. Three fold more hexa-arginine SHAL remained associated with the Raji cells after washing, suggesting that the peptide also enhanced residualization of the <sup>111</sup>In transported into cells. Confocal microscopy showed both SHALs localized in the cytoplasm of Raji cells, whereas a fraction of the hexa-arginine SHAL localized in the nucleus.</p> <p>Conclusion</p> <p>The incorporation of a hexa-D-arginine peptide into the linker of the SHAL (DvLPBaPPP)<sub>2</sub>LLDo enhanced both the uptake and residualization of the SHAL analog by Raji cells. In contrast to the abundant cell surface binding observed with Lym-1 antibody, the majority of (DvLPBaPPP)<sub>2</sub>LArg6AcLLDo and the parent SHAL were internalized. Some of the internalized hexa-arginine SHAL analog was also associated with the nucleus. These results demonstrate that several important SHAL properties, including uptake, internalization, retention and possibly intracellular distribution, can be enhanced or modified by conjugating the SHALs to a short polypeptide.</p
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Evaluation of integrin αvβ6 cystine knot PET tracers to detect cancer and idiopathic pulmonary fibrosis.
Advances in precision molecular imaging promise to transform our ability to detect, diagnose and treat disease. Here, we describe the engineering and validation of a new cystine knot peptide (knottin) that selectively recognizes human integrin αvβ6 with single-digit nanomolar affinity. We solve its 3D structure by NMR and x-ray crystallography and validate leads with 3 different radiolabels in pre-clinical models of cancer. We evaluate the lead tracer's safety, biodistribution and pharmacokinetics in healthy human volunteers, and show its ability to detect multiple cancers (pancreatic, cervical and lung) in patients at two study locations. Additionally, we demonstrate that the knottin PET tracers can also detect fibrotic lung disease in idiopathic pulmonary fibrosis patients. Our results indicate that these cystine knot PET tracers may have potential utility in multiple disease states that are associated with upregulation of integrin αvβ6
On Demand Biosensors for Early Diagnosis of Cancer and Immune Checkpoints Blockade Therapy Monitoring from Liquid Biopsy
Recently, considerable interest has emerged in the development of biosensors to detect biomarkers and immune checkpoints to identify and measure cancer through liquid biopsies. The detection of cancer biomarkers from a small volume of blood is relatively fast compared to the gold standard of tissue biopsies. Traditional immuno-histochemistry (IHC) requires tissue samples obtained using invasive procedures and specific expertise as well as sophisticated instruments. Furthermore, the turnaround for IHC assays is usually several days. To overcome these challenges, on-demand biosensor-based assays were developed to provide more immediate prognostic information for clinicians. Novel rapid, highly precise, and sensitive approaches have been under investigation using physical and biochemical methods to sense biomarkers. Additionally, interest in understanding immune checkpoints has facilitated the rapid detection of cancer prognosis from liquid biopsies. Typically, these devices combine various classes of detectors with digital outputs for the measurement of soluble cancer or immune checkpoint (IC) markers from liquid biopsy samples. These sensor devices have two key advantages: (a) a small volume of blood drawn from the patient is sufficient for analysis, and (b) it could aid physicians in quickly selecting and deciding the appropriate therapy regime for the patients (e.g., immune checkpoint blockade (ICB) therapy). In this review, we will provide updates on potential cancer markers, various biosensors in cancer diagnosis, and the corresponding limits of detection, while focusing on biosensor development for IC marker detection
Development of a Novel Long-Lived ImmunoPET Tracer for Monitoring Lymphoma Therapy in a Humanized Transgenic Mouse Model
Positron emission tomography (PET) is an attractive imaging
tool
to localize and quantify tracer biodistribution. ImmunoPET with an
intact mAb typically requires two to four days to achieve optimized
tumor-to-normal ratios. Thus, a positron emitter with a half-life
of two to four days such as zirconium-89 [<sup>89</sup>Zr] (<i>t</i><sub>1/2</sub>: 78.4 h) is ideal. We have developed an
antibody-based, long-lived immunoPET tracer <sup>89</sup>Zr-Desferrioxamine-p-SCN
(Df-Bz-NCS)-rituximab (Zr-iPET) to image tumor for longer durations
in a humanized CD20-expressing transgenic mouse model. To optimize
the radiolabeling efficiency of <sup>89</sup>Zr with Df-Bz-rituximab,
multiple radiolabelings were performed. Radiochemical yield, purity,
immunoreactivity, and stability assays were carried out to characterize
the Zr-iPET for chemical and biological integrity. This tracer was
used to image transgenic mice that express the human CD20 on their
B cells (huCD20TM). Each huCD20TM mouse received a 7.4 MBq/dose. One
group (<i>n</i> = 3) received a 2 mg/kg predose (blocking)
of cold rituximab 2 h prior to <sup>89</sup>Zr-iPET; the other group
(<i>n</i> = 3) had no predose (nonblocking). Small animal
PET/CT was used to image mice at 1, 4, 24, 48, 72, and 120 h. Quality
assurance of the <sup>89</sup>Zr-iPET demonstrated NCS-Bz-Df: antibody
ratio (c/a: 1.5 ± 0.31), specific activity (0.44–1.64
TBq/mol), radiochemical yield (>70%), and purity (>98%). The
Zr-iPET
immunoreactivity was >80%. At 120 h, Zr-iPET uptake (% ID/g) as
mean
± STD for blocking and nonblocking groups in spleen was 3.2 ±
0.1% and 83.3 ± 2.0% (<i>p</i> value <0.0013.).
Liver uptake was 1.32 ± 0.05% and 0.61 ± 0.001% (<i>p</i> value <0.0128) for blocking and nonblocking, respectively.
The small animal PET/CT image shows the spleen specific uptake of
Zr-iPET in mice at 120 h after tracer injection. Compared to the liver,
the spleen specific uptake of Zr-iPET is very high due to the expression
of huCD20. We optimized the radiolabeling efficiency of <sup>89</sup>Zr with Df-Bz-rituximab. These radioimmunoconjugate lots were stable
up to 5 days in serum <i>in vitro</i>. The present study
showed that <sup>89</sup>Zr is well-suited for mAbs to image cancer
over an extended period of time (up to 5 days)
Development of a Novel Long-Lived ImmunoPET Tracer for Monitoring Lymphoma Therapy in a Humanized Transgenic Mouse Model
Positron emission tomography (PET) is an attractive imaging
tool
to localize and quantify tracer biodistribution. ImmunoPET with an
intact mAb typically requires two to four days to achieve optimized
tumor-to-normal ratios. Thus, a positron emitter with a half-life
of two to four days such as zirconium-89 [<sup>89</sup>Zr] (<i>t</i><sub>1/2</sub>: 78.4 h) is ideal. We have developed an
antibody-based, long-lived immunoPET tracer <sup>89</sup>Zr-Desferrioxamine-p-SCN
(Df-Bz-NCS)-rituximab (Zr-iPET) to image tumor for longer durations
in a humanized CD20-expressing transgenic mouse model. To optimize
the radiolabeling efficiency of <sup>89</sup>Zr with Df-Bz-rituximab,
multiple radiolabelings were performed. Radiochemical yield, purity,
immunoreactivity, and stability assays were carried out to characterize
the Zr-iPET for chemical and biological integrity. This tracer was
used to image transgenic mice that express the human CD20 on their
B cells (huCD20TM). Each huCD20TM mouse received a 7.4 MBq/dose. One
group (<i>n</i> = 3) received a 2 mg/kg predose (blocking)
of cold rituximab 2 h prior to <sup>89</sup>Zr-iPET; the other group
(<i>n</i> = 3) had no predose (nonblocking). Small animal
PET/CT was used to image mice at 1, 4, 24, 48, 72, and 120 h. Quality
assurance of the <sup>89</sup>Zr-iPET demonstrated NCS-Bz-Df: antibody
ratio (c/a: 1.5 ± 0.31), specific activity (0.44–1.64
TBq/mol), radiochemical yield (>70%), and purity (>98%). The
Zr-iPET
immunoreactivity was >80%. At 120 h, Zr-iPET uptake (% ID/g) as
mean
± STD for blocking and nonblocking groups in spleen was 3.2 ±
0.1% and 83.3 ± 2.0% (<i>p</i> value <0.0013.).
Liver uptake was 1.32 ± 0.05% and 0.61 ± 0.001% (<i>p</i> value <0.0128) for blocking and nonblocking, respectively.
The small animal PET/CT image shows the spleen specific uptake of
Zr-iPET in mice at 120 h after tracer injection. Compared to the liver,
the spleen specific uptake of Zr-iPET is very high due to the expression
of huCD20. We optimized the radiolabeling efficiency of <sup>89</sup>Zr with Df-Bz-rituximab. These radioimmunoconjugate lots were stable
up to 5 days in serum <i>in vitro</i>. The present study
showed that <sup>89</sup>Zr is well-suited for mAbs to image cancer
over an extended period of time (up to 5 days)
Development of a Novel Long-Lived ImmunoPET Tracer for Monitoring Lymphoma Therapy in a Humanized Transgenic Mouse Model
Positron emission tomography (PET) is an attractive imaging
tool
to localize and quantify tracer biodistribution. ImmunoPET with an
intact mAb typically requires two to four days to achieve optimized
tumor-to-normal ratios. Thus, a positron emitter with a half-life
of two to four days such as zirconium-89 [<sup>89</sup>Zr] (<i>t</i><sub>1/2</sub>: 78.4 h) is ideal. We have developed an
antibody-based, long-lived immunoPET tracer <sup>89</sup>Zr-Desferrioxamine-p-SCN
(Df-Bz-NCS)-rituximab (Zr-iPET) to image tumor for longer durations
in a humanized CD20-expressing transgenic mouse model. To optimize
the radiolabeling efficiency of <sup>89</sup>Zr with Df-Bz-rituximab,
multiple radiolabelings were performed. Radiochemical yield, purity,
immunoreactivity, and stability assays were carried out to characterize
the Zr-iPET for chemical and biological integrity. This tracer was
used to image transgenic mice that express the human CD20 on their
B cells (huCD20TM). Each huCD20TM mouse received a 7.4 MBq/dose. One
group (<i>n</i> = 3) received a 2 mg/kg predose (blocking)
of cold rituximab 2 h prior to <sup>89</sup>Zr-iPET; the other group
(<i>n</i> = 3) had no predose (nonblocking). Small animal
PET/CT was used to image mice at 1, 4, 24, 48, 72, and 120 h. Quality
assurance of the <sup>89</sup>Zr-iPET demonstrated NCS-Bz-Df: antibody
ratio (c/a: 1.5 ± 0.31), specific activity (0.44–1.64
TBq/mol), radiochemical yield (>70%), and purity (>98%). The
Zr-iPET
immunoreactivity was >80%. At 120 h, Zr-iPET uptake (% ID/g) as
mean
± STD for blocking and nonblocking groups in spleen was 3.2 ±
0.1% and 83.3 ± 2.0% (<i>p</i> value <0.0013.).
Liver uptake was 1.32 ± 0.05% and 0.61 ± 0.001% (<i>p</i> value <0.0128) for blocking and nonblocking, respectively.
The small animal PET/CT image shows the spleen specific uptake of
Zr-iPET in mice at 120 h after tracer injection. Compared to the liver,
the spleen specific uptake of Zr-iPET is very high due to the expression
of huCD20. We optimized the radiolabeling efficiency of <sup>89</sup>Zr with Df-Bz-rituximab. These radioimmunoconjugate lots were stable
up to 5 days in serum <i>in vitro</i>. The present study
showed that <sup>89</sup>Zr is well-suited for mAbs to image cancer
over an extended period of time (up to 5 days)
Recent Trends and Opportunities for the Targeted Immuno-Nanomaterials for Cancer Theranostics Applications
The targeted delivery of cancer immunotherapies has increased noticeably in recent years. Recent advancements in immunotherapy, particularly in blocking the immune checkpoints (ICs) axis, have shown favorable treatment outcomes for multiple types of cancer including melanoma and non-small-cell lung cancer (NSLC). Engineered micromachines, including microparticles, and nanoplatforms (organic and inorganic), functionalized with immune agonists can effectively deliver immune-targeting molecules to solid tumors. This review focuses on the nanomaterial-based strategies that have shown promise in identifying and targeting various immunological markers in the tumor microenvironment (TME) for cancer diagnosis and therapy. Nanomaterials-based cancer immunotherapy has improved treatment outcomes by triggering an immune response in the TME. Evaluating the expression levels of ICs in the TME also could potentially aid in diagnosing patients who would respond to IC blockade therapy. Detecting immunological checkpoints in the TME using noninvasive imaging systems via tailored nanosensors improves the identification of patient outcomes in immuno-oncology (IO). To enhance patient-specific analysis, lab-on-chip (LOC) technology is a rapid, cost-effective, and accurate way of recapitulating the TME. Such novel nanomaterial-based technologies have been of great interest for testing immunotherapies and assessing biomarkers. Finally, we provide a perspective on the developments in artificial intelligence tools to facilitate ICs-based nano theranostics toward cancer immunotherapy
Multiscale Framework for Imaging Radiolabeled Therapeutics
The
resistance of a tumor to a drug is the result of bulk properties
of the tumor tissue as well as phenotypic variations displayed by
single cells. Here, we show that radioisotopic detection methods,
commonly used for tracking the tissue distribution of drug compounds,
can be extended to the single-cell level to image the same molecule
over a range of physical scales. The anticancer drug rituximab was
labeled with short-lived radionuclides (<sup>89</sup>Zr/<sup>64</sup>Cu) and its accumulation at the organ level was imaged using PET
in a humanized transgenic mouse model of non-Hodgkin’s lymphoma.
To capture the distribution of the drug at a finer scale, tissue sections
and single living cells were imaged using radioluminescence microscopy
(RLM), a novel method that can detect radionuclides with single-cell
resolution. In vivo PET images (24 h postinjection) showed that [<sup>89</sup>Zr]Ârituximab targeted the intended site of human CD20 expression,
the spleen. Within this organ, RLM was used to resolve radiotracer
accumulation in the splenic red pulp. In a separate study, RLM highlighted
marked differences between single cells, with binding of the radiolabeled
antibody ranging from background levels to 1200 radionuclides per
cell. Overall, RLM images demonstrated significantly higher spatial
resolution and sensitivity than conventional storage-phosphor autoradiography.
In conclusion, this combination of PET and RLM provides a unique opportunity
for exploring the molecular mechanism of drugs by tracking the same
molecule over multiple physical scales, ranging from single living
cells to organs substructures and entire living subjects