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Nucleic acid Aptamers as Potential Therapeutic and Diagnostic agents for

Lymphoma

Presented by

Naila SajjadPhD Biochemistry

10-arid-1766

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List of Abbreviation used

• ALCL Anaplastic large cell lymphoma• PEI Polyethyleneimine• BCL2 B cell lymphoma 2• AML Acute myeloid lymphoma• ALL Acute lymphoid lymphoma

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Aptamers

• Oligonucleotide or peptide molecules that bind to specific target molecules

• Created by selecting them from large random sequences

• Used for research and clinical purposes as macromolecular drug

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Conti…..

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Classification of Aptamers

DNA/RNA aptamers

• Consist of short strand of oligonucleotide

Peptide aptamers

• Consist of short variable peptide domain attached at both ends to a protein scaffold

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Nucleic acid Aptamers

• Nucleic acid species engineered through repeated rounds of in vitro selection or SELEX (Systematic evolution of ligand by exponential enrichment)

• Bind to molecular targets such as small molecule, protein, nucleic acid

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SELEX

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LYMPHOMA

• Blood cancers develops in the lymphatic system arise from affecting lymphocyte

• There are at least 35 types of lymphoma which are grouped based on cell histology, genetic mutations and expression of certain genes

• Hodgkin’s and non-Hodgekin’s lymphoma are two major categories

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Aptamers

• As a potential “next generation” of diagnostic tools

• Therapeutic applications of aptamers in lymphoma

• Aptamer “small bomb” mediates delivery of drugs to cancer cells

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Aptamers as a potential “next- generation” of diagnostic tools

• Lymphoma is fatal if left untreated or diagnosed at a late stage

• Burkitt lymphoma (BL)

Methods to diagnose• Immunophenotyping by flow cytometry (IFC)• Karyotyping analysis of peripheral blood & bone

marrow• Detection of malignant cell mutations through PCR

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BL

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Conti…

Limitations

• IFC requires the use of monoclonal antibodies. Complicating diagnose and increasing cost

• PCR can sensitively examine RNA expression of certain genes to detect cancer cells have very low signal levels

Advantages of nucleic acid aptamers over antibodies

• tolerant of chemical and temp changes• Longer shelf lives• Reduce manufacturing cost• Better tissue penetration & cellular uptake

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Anti-CD30 Aptamers

CD30 Antigen expressed in activated T & B

(Transmembrane protein cells but over expressed in ALCL

Receptors of TNF) Hodgkin’s lymphoma

Marker for diagnosing

these lymphoma

Anti-CD30 antibodies used in CD30 expression

Anti-CD30RNA Aptamers binds to CD30 Proteins

(identified using extracellular

domain of receptor activator of

nuclear factor KB)

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ALCL

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Use of anti-CD30 Aptamers as probe

• Explored using flow cytometry and immunostaining

Fluorescently labeled tested with cultured ALCL & Hodgkin’s

anti-CD30 aptamers lymphoma

flow ctometry & fluoroscene

microscopy shows

binding to lymphoma cells

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Immunostaining

Anti-CD30 Aptamers binds to CD30 Proteins serves as replace

& antibodies ment of

CD30 antibodies

Biotinylated anti-CD30 Aptamers

Used to study expression of CD30 on lymphoma cells subsequently visualized by horseradish peroxidase

Specifically stain the lymphoma cells and required a shorter incubation time and lower temp

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TD05 Aptamers

Burkitt’s lymphoma (BL)• Acute blood cancer

• Diagnosed through morphological inspection of blood cells & biopsied tissue sample

• TD05 aptamers have been designed to diagnosed BL

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Conti…….. TD05 Aptamers initially panel of DNA aptamers

(Evolved by applying cell-SELEX were selected for Ramos cells

Against BL cell lines i.e. Ramos cells

Not bind only recognize shows strong binding

To normal CD19 B Ramos cells affinity

cells or other bone marrow cells

Subsequent experiments immunoglobulin heavy mu chain (B-cell activated receptor)

expressed on the surface of BL cells

binding target for TD05 Aptamers

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Aptamer nanoparticle strip biosensors

• Flow based detection of BL• Development of simple, sensitive dipstick test • Litmus test type of assay

Gold nanoparticles aggregate immobiized on thiolated TD05

Responsive to Ramos cells aptamers

Was designed

Test zone of strip immobilized TE02 aptamer

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Principle of assay

sample solution or capillary action move along strip

If in the presence of Ramos cells TD05 aptamers immobilized on Au

nanoparticles interact with Ramos cells Au- nanoparticles

aptamers cell complex migrate along strip complex was captured by

TE02 immobilized on strip complex accumulation at test zone producing red line migration control zone

Complex captured by DNA sequence complimentary to TD05 aptamer

2nd red line

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Conti…

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In vivo studies by TD05 Aptamers as molecular probe

• TD05 aptamers were labeled with fluorescent dyes such as cy 5 & injected into mice engrafted with Ramos cells

• Cy 5 labeled TD05 Aptamer accumulate in tumor tissues

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PEI Aptamer probe

• Used in vivo cancer imaging

• Degradation ratio of aptamer is reduced when TD05 aptamer are immobilized on PEI Polymer

• Fluorescence signal remained elevated for five hours post injection while signal when naked TD05 only stayed for 45 minutes

• Feasible method to image tumor in vivo with high specificity and sensitivity

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Sgc 8 Aptamers

• DNA aptamers generated for specific recognition of leukemia cells

• Two tumor cell lines were selected as ligands

• An ALL cell line, CCRF-CEM

• Ramos cell line was used as counter selection

• Protein tyrosine kinase 7 (PTK 7) is a molecular target of sgc 8 aptamers

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Conti..

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In vivo visualization of tumor

• Reporter bearing probes are used

• Cell recognizing ligend are labeled with reporter mol such as fluorescent dye & then bind to target cancer cells

• Signals are generated

Acivable aptamer reporter probe

• Tumor targeting

• A poly T linker

• Short DNA sequence complementary to part of aptamer that has a fluorophore & a quencher linked at either terminus

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Conti….

• In absence of target protein probe is in active form

• It keeps fluorophore & quencher in close proximity leading to quenched fluorescence

• In presence of cancer cells conformation of aptamer probe is recognized on binding

• Fluorophore get separated from quencher

• Strong signals are generated in response to interaction of probe with target cells

• Thus sgc 8 aptamers diagnosed cancer cells shorten the diagnostic time

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Therapeutic Applications of Aptamers in Lymphoma

• In past radiotherapy & chemotherapy were used

• Aptamers based therapeutic have emerged as an alternative

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AS1411 Aptamer

• A 26-nt G quadruplex DNA aptamer identified as a first anticancer nucleic acid agent for treatment of renal cell carcinoma

AS1411+nucleolin+nuclear activate transcription factor

Factor KB essential NK-KB

modulator

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Conti…..

Nucleolin over expressed in cancer cells

(multifunctional protein

Roles in cell growth & death)

Binds Au-rich element modulate interleukin 9

In 3 UTR of expression in T- cell lymphoma

Bcl2 mRNA

Protect Anti-apoptic Bcl2 from

degradation & stabilized Bcl2 mRNA

overproduction of Bcl2 in

cancer cells

AS1411 inhibit proliferation of tumor cell lines & suppressed tumor growth

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Conti….

• Phase I clinical trials (involving 30 patients) were completed in 2006 and indicated that AS1411 was safe and well-tolerated

• Phase II trials for AML are showing positive interim results (ClinicalTrials.gov Identifier: NCT00512083 and NCT01034410)

• Recent research has explored the use of AS1411 as a carrier for the delivery of cancer drugs specifically into cancer cells by nucleolin mediated internalization

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NOX-A12 Spiegelmers

• 45-nucleotide long (L Steroisomer) RNA aptamer

• High binding affinity, selectivity to the target, passive immunogenicity

• Evolved from natural D-RNA library by using mirror image target D-amino acid

• Synthesized as L-RNA to bind natural target (L amino acid)

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Conti….

NOXA12 Antagonize CXCL12/SDF-1

CXCR5 & CXCR7

metastasis

pathophysiology of chronic

lymphocytic leukemia

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Conti…

• A Phase 1 clinical trial involving 48 healthy subjects was launched in 2009 and showed that NOX-A12 spiegelmers were safe and well- tolerated

• Recently, two Phase IIa clinical studies of NOX-A12 have begun for hematological oncology indications, including non-Hodgkin’s lymphoma, chronic lymphocytic leukemia and multiple myeloma (Clinical- Trials.gov Identifier: NCT01486797 and NCT-01486797)

• This study will involve 33 relapsed CLL patients who will receive NOX-A12 spiegelmers in combination with a background therapy of chemotherapeutic agents. In-terim results are expected to be available in early 2013.

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Aptamer “smart bomb” mediates delivery of drugs to cancer cells

• Antibody drug conjugates have a cytotoxic potential of chemotherapy

• Two antibody –drug conjugates have been approved by FDA for treatment of hematological cancer

• Gemutuzumab ozogamium was 1st FDA approved designed for patients with replased AML

• It target CD33 antigen expressed on leukemia blast cells

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Conti….

• Brentuximab vedotin was 2nd FDA approved for treatment of Hodgkin’s lymphoma

• It target CD30 which is a establish marker for hodgkin’s lymphoma

• Aptamers have advantages over antibodies used in target delivery, particularly for cancer therapy

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TD05 Aptamer-mediated delivery of photosensitizer

• TD05 aptamer were engineered with photosensitizer for selective photodynamic therapy of targeted tumor cells

• It exploit the ability of non toxic light sensitive compound called photosensitizer to produce cytotoxic reactive oxygen species

• An aptamer photosensitizer conjugate (APC) was engineered

• Conjugate contain cytotoxic porphyrin-based photosensitizer reagent chlorin 6

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Conti….

• APC was selective to Ramos cells

• TD05 aptamers were designed to attach a lipid tail at the end of aptamers to form self-assembled aptamer-micelle nanostructure

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AS1411 Nucleolin Aptamer-mediated drug delivery

• Nuclear delivery

• It acts as a shuttle to transfer molecules between cell surface & nucleus

• Scientist have reported a novel strategy of using AS1411 aptamers as drug carrier to target cancer cells for photodynamic therapy

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Conti…

AS1411 aptamer+ multivalent AS1411apt-

6photodynamic molecules TMPy4 TMPy4 conjugate

nucleolin expressing cells

damage cells upon exposure

to light

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Lipid polymer hybrid nanoparticles (LPHN)

• These lipid-polymer hybrid nanoparticles were composed of 6 elements

• 1) a hydrophobic polymeric core made of poly D, L lactic-co-glycolic acid (PLGA)

• 2) a chemotherapeutic drug, paclitaxel• 3) a fluorescent dye• 4) a lipid layer made of lecithin,• 5) a PEGylated phospholipid, DSPE-PEG2000-COOH

and • 6) AS1411 aptamers on the surface

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Theranostic probe (TP)

• Scientist developed a cancer-targeting probe that can detect & inhibit cancer cells at the same time

• Probe used micro-RNA as a cancer biomarker & magnetic fluorescence nanoparticle conjugated with AS1411 aptamers and an miRNA-221 molecular beacon to simultaneously target cancer cells

• Collectively, resulting nanoparticles displayed superior selectivity in nucleolin expressing cancer cells

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Conti….

• When incubated with target cells, the nanoparticles were internalized and released the miR-221 molecular beacon in the cytoplasm

• In the presence of miR-221, the unloaded miR-221 molecular beacon hybridized with endogenous miR-221, resulting in a strong fluorescence signal & simultaneously miRNA-221 inhibition, thereby reducing expression of oncogenes

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Conti…

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Anti-CD Aptamer-mediated siRNA Delivery

• Used to deliver siRNA to Anaplastic lymphoma kinase (ALK) positive ALCL,which is an aggressive T-cell lymphoma that over expresses ALK oncogenes & CD30 surface proteins

• Nanoparticle of 140nm were formed

• ALK ,siRNA and CD30 aptamer were incorporated onto cationic PEI-Citrate carriers to form nanoparticles

• Inside the cell ALK siRNA were released from the nanoparticles to specifically silence ALK gene expression

• Result in growth arrest and apoptosis in CD30-expressing ALCL cells

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Conclusions

• Lymphoma are cancers that arise from white blood cells & usually present as solid tumors

• Aptamers are single stranded DNA or RNA oligonucleotides

• The immense diversity in function and structure of nucleic acids enable numerous aptamers to be generated through in vitro selection technique known as SELEX

• Aptamers may inhibit the function of target proteins, and can be used as potential therapeutic and diagnostic agents for lymphoma

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References

• Shum., K. T., J. Zhou and J. J. Rossi. 2013. Nucleic acid aptamers as potential therapeutic and diagnostic agents for lumphoma. J. Cancer therapy. 4: 872-890.

• Hu, S. Z., Y. Xu-Monette, A. Balasubramanyam, G. C. Manyam, C. Visco, A. Tzankov. 2013. “CD30 Expression Defines a Novel Subset of Diffuse Large B-Cell Lym- phoma with Favorable Prognosis and Distinct Gene Ex- pression Signature: A Report from the International DLBCL Rituximab-CHOP Consortium Program Study,” Blood. 4: 2715-2724.

• Mori., T. A. Oguro, T. Ohtsu and Y. Nakamura.2004. “RNA Aptamers Selected against the Receptor Activator of NF-kappaB Acquire General Affinity to Proteins of the Tumor Necrosis Factor Receptor Family,” Nucleic Acids Research. 32. 6120-6128.

• Shieh., Y. A., S. J. Yang, M. F. Wei and M. J. Shieh.2010. “Ap- tamer-Based Tumor-Targeted Drug Delivery for Photo- dynamic Therapy,” ACS Nano, Vol.3: 1433-1442.