Clinical Trial Details
— Status: Not yet recruiting
Administrative data
| NCT number |
NCT02828774 |
| Other study ID # |
2015050 |
| Secondary ID |
|
| Status |
Not yet recruiting |
| Phase |
N/A
|
| First received |
June 13, 2016 |
| Last updated |
July 7, 2016 |
| Start date |
August 2016 |
| Est. completion date |
June 2020 |
Study information
| Verified date |
June 2016 |
| Source |
Assuta Medical Center |
| Contact |
Ofer Shpilberg, MD |
| Phone |
+972-37644364 |
| Email |
ofers[@]assuta.co.il |
| Is FDA regulated |
No |
| Health authority |
Israel: Ministry of Health |
| Study type |
Observational
|
Clinical Trial Summary
Using phage libraries extensively pre-absorbed on a series of normal cell types, we will
isolate phage specifically internalized by B-CLL cells from newly diagnosed and untreated
CLL patients. Peptide sequences are then derived by Next Generation Sequencing (NGS).
NGS-based studies are contributing to an improved understanding of cancer heterogeneity in
order to tailor treatment to patients based on the individual makeup of their tumor. However
the use of NGS to derive phage displayed peptide sequences is so far rare (22).
Traditionally, after exposure to a target and recovery by elution, the phage clones are
isolated by titration on bacterial lawns. It is technically demanding and labour intensive
to select and analyze more than about 15 of the sometimes thousands clones recovered.
Therefore information on other potentially important sequences is missed. NGS allows
sequencing of the entire recovered phage pool and provides far more detailed bioinformatic
analyses of peptide sequences or motifs. RNA from the CLL cells is used for RNA-seq
expression sequencing. The wide application of NGS in combination with bioinformatics tools
has begun to revolutionize cancer research, diagnosis and therapy. The peptide and RNA
sequencing data will afford bioinformatic testing of correlations of exome expression and
clinical parameters with the pattern of peptide sequences internalized by CLL cells of
different patients. This information is crucial to answering questions 1, 2 and 3 discussed
on page 1 above. The results of this analysis will probably not allow identification of
specific receptors targeted by the peptides. The aim at this stage of the research is to
identify candidate targeting peptides. Once identified, further research will be needed to
identify the receptors to which they bind. Regarding question 4, there is currently very
little published information on the therapeutic potential of PDCs in leukemia. Using two
peptides we have isolated that target murine A20 leukemic cells, we will prepare multi-drug
PDCs (using technology we have developed) and in an animal model, test their ability to
enhance the survival and quality of life of CLL bearing animals.
Description:
Methodology A: CLL specific peptides: (Specific aim 1) Whole blood samples are received from
healthy volunteers and newly diagnosed and untreated CLL patients according to our Helsinki
permit no. 0432-13-RMC. (Rabin Medical Center) Acquisition of samples at follow up and from
an additional 80 patients will be covered by additional Helsinki submissions now underway.
Lymphocytes are separated using Ficoll and the B cells isolated using a B-cell isolation
kit. The B-cells from the healthy volunteers and an aliquot of those isolated cells from
each patient are frozen for later use (see D: below). The remaining patient cells are
divided into two fractions. One is used for RNA extraction. The other is exposed to an
"Absorbed" phage library pre-absorbed on normal cells. After incubation, cells are washed
and phage bound to the cell membrane are eluted and recovered. Cells are then lysed and the
lysate retained. Phage in the eluate and lysate (membrane-bound and internalized
respectively) are amplified separately in E.coli. The amplified phage are isolated and DNA
prepared for NGS. Sequencing is carried out in an external core facility (The Technion
Genome Center).
. B: Bioinformatic analyses of peptide sequences (Specific aim 1) I) Sequencing of peptides:
Sequence reads are obtained from phage DNA libraries generated and analyzed with
high-throughput sequencing (Illumina HiSeq system). Quality control checks are performed
using FastQC tool. The reads are processed to trim adaptors and remove the consensus
sequences from either side using the Cutadapt software (23). The remaining fragment tags
contain the peptides' DNA sequence and are expected to be 21 nucleotides in length. Next,
the BioString package from the Bioconductor framework are used to translate these nucleotide
sequences, and the number of occurrences of each peptide sequence is calculated to find the
most frequent peptides. Following normalization to library size, peptide sequence counts
from different patient libraries are compared to find common sequences. Sequences from the
membrane-bound pool fraction are subtracted from the internalized pool fraction so as to
remove membrane-bound clones that may not have been successfully eluted off the CLL cell.
The sequences remaining in the internalized pool serve as candidates for peptide-conjugate
therapy. A univariate logistic regression approach will then be used to test possible
associations of peptide counts with clinical and genetic findings. When clinical parameters
are treated as continuous variables, linear regression will be implemented. Candidate
peptides will be queried against the PepBank database (24) in order to check if they are
known to be related to cancer or other diseases and conditions such as apoptosis or
angiogenesis. Finally, a bioinformatic pipeline script will be developed automating the
above bioinformatics analysis steps.
II) RNA-seq to detect gene expression patterns and fusion genes:
The main aim of the RNA-seq analysis is to detect specific gene expression patterns which
can be correlated with peptide sequences and clinical parameters. These expression patterns
will give clues to cell surface receptor expression and intracellular pathway activity.
Patients will be divided based on the peptide sequences internalized by B-CLL cells, and a
statistical test will be performed in order to detect differentially expressed genes.
Pathway analysis will be performed in order to infer functional changes which may shade a
light on the possible role of the peptide receptors in the cell signaling cascade. Trim
galore software will be used for adapter trimming, and for removing low quality bases from
the ends of reads. Trimmed reads will be mapped to the human genome (hg38) using TopHat2
software. The number of reads overlaping each of the annotated genes will be counted using
the HT-seq python package. DESeq within the Bioconductor framework will be used for
normalization and differential expression analysis using variants of Fisher's exact test.
The Generally Applicable Gene-set Enrichment (GAGE) method will be applied to detect up and
down-regulated pathways. RNA-seq is also used to detect fusion gene events in the patient's
cancer cells and can be used as an independent validation method to test the sensitivity of
the peptide-dye conjugate used as for disease monitoring. For this aim, three different
tools TopHat-fusion, defuse and ChimeraScan will be used to identify fusion transcripts.
Several filtration steps will be applied in order to remove low-quality candidates as
described before, and only those candidates detected by all the three tools will be chosen
for RT-PCR validation.
C: Syntheses of Peptides and conjugates (Specific aims 1,2,3) Peptides will be synthesized
using solid-phase chemistry. In the first stage of the project, the two peptides specific
for A20 mouse leukemic cells already identified will be synthesized, namely HIS SER THR PRO
SER SER PRO (Peptide 1) and ASP SER SER LEU PHE ALA LEU (Peptide 2). In later stages (see
below), selected human CLL specific peptides will be synthesized as their sequences become
available. Only peptides will multiple repeat reads (>5) (see B above) will be selected
(maximum of 3 per patient) as these represent phage clones with greater propensity to induce
uptake into cells. Peptides will be synthesized both as fluorescent dye- and drug-conjugates
based on our previous work with targeted multi-drug conjugates (21). We will employ drugs
with differing mechanisms of action, such as the nitrogen mustard Chlorambucil used to treat
elderly CLL patients, and the microtubule inhibitor Combretastatin 4A, known to induce
apoptosis in CLL cells (25). Purity and composition of the products will be verified by HPLC
and LC/MS. Syntheses will be carried out by an external supplier.
D: In vitro validation of peptide specificity and PDC activity: (Specific aims 1, 2, 3)
Specificity of peptides: The specificity of targeting peptides will be demonstrated by
incubating peptide-dye conjugates with target and controls cells. For the mouse CLL system,
A20 cells will be used as target cells and normal mouse splenic lymphocytes and primary
cultures of mouse skin and epithelial cells will be used as controls cells. For the human
CLL system, patient CLL cells and normal human lymphocytes and HUVEC cells will be used as
target and controls cells respectively. Cells will be incubated with FITC-labeled peptides
at both 4oC and 37oC, washed and analyzed for binding and internalization respectively.
Binding will be evaluated by flow cytometry and internalization by confocal microscopy.
Cytotoxicity of PDCs: Target and control cells will be incubated for 24 and 48 hrs with
increasing concentrations (0-50uM drug equivalents) of free drug or PDCs. Percent Growth
Inhibition will be assessed by the XTT assay. Cytotoxic A20 specific PDCs will then be
evaluated in vivo (see E: below).
Cell culture systems: The A20 cell line is grown and maintained in FBS supplemented RPMI
culture medium. Normal primary cultures are maintained with selective media. On the other
hand, the survival of human CLL cells in culture is dependent on several unique
microenvironmental factors, including antigen stimulation of the B-cell BCR, T cell help
through the CD40-CD40L interaction, stimulation from the bone marrow stromal cell-derived
CXCL12 chemokine and stimulation of Toll-Like Receptor 9. These conditions will be mimicked
in vitro (26) by addition to the culture medium of anti-IgM (to crosslink the BCRs),
co-culture with fibroblasts expressing CD40L, addition of recombinant CXCL12 and CpG
dinucleotides respectively. These conditions will be used to test the specificity and
efficacy of human PDCs against cryopreserved samples of patient CLL cells and normal human
lymphocytes (controls).
E: Animal studies using A20 mouse model of CLL (Specific aim 3) While several antibody-drug
conjugates for the treatment of CLL have been used or are being tested (16), there is little
published information as to the efficacy of PDCs for this disease. Therefore we aim to study
the behavior of our A20 specific PDCs in an animal model of CLL, according to protocols
approved by the Ariel University Institutional Animal Ethics Committee, permit no.
IL-47-08-13. To validate the calibration of CLL development, A20 cells will be injected
intravenously into Balb/c mice and the development of CLL symptoms monitored. Usually this
takes between 40-50 days.
Study A): At this point, we know little about the most effective treatment schedule with
PDCs. For this reason, and based on published literature, we will first test the systemic
tolerance of the animals to the drugs in the PDCs by injecting groups of non-tumor bearing
mice intraperitoneally with 1, 7.5 or 15 mg/Kg of one Chlorambucil and Combretastatin 4A
containing PDC every third days for 3 weeks. Animals will be monitored for systemic
morbidity and survival. On the basis of these results, an initial treatment schedule will be
selected to compare two concentrations for each of the two PDCs. Animals showing initial
signs of CLL will be treated and followed for survival and development of CLL symptoms for
at least and additional 70 days.
Study B: Using the Study A results, the most effective PDC will be studied further. The PK
of the drug conjugate will be analyzed by administering it in 5 drug concentrations,
including that used for Study A. Blood samples will be drawn from the tail vein at 0.25,
0.5, 1, 2, 5, 10 and 24 hrs. PDC will be extracted and analyzed by LC/MS for content and
integrity. Drug concentrations will be analyzed by HPLC and used to calculate standard PK
parameters [Cmax; TCmax; T1/2]. Based on these experiments, an additional treatment protocol
will be devised aimed at maintaining a maximum blood PDC concentration over the treatment
period. The protocol will be tested on an additional group of animals. After the treatment
period, animals will be followed for survival and development of CLL symptoms for at least
and additional 70 days.
F: Detection of CLL cells in animal and patient whole blood. (Specific aims 1 and 2) The aim
of these experiments is to assess the potential of CLL-specific peptide-dye conjugates to
detect CLL cells in blood samples. Calibration experiments will be carried out on three
levels.
1. Cell doping: Cryopreserved samples of patient CLL cells will be added to either RPMI
culture medium or normal human blood (in three concentrations). B-cells isolated as
described in A above will be incubated with the appropriate Peptide-dye conjugate for
30mins at 37OC. Cells will be washed and analyzed for peptide binding by flow
cytometry. Similarly, A20 cells will be added to normal mouse blood and tested with A20
specific peptide-dye conjugates.
2. Animal model: Using the animal model described above, blood samples will be taken
once/week during the tumor induction phase. After RBC lysis, the remaining cells will
be incubated with peptide-dye conjugates, washed and analysed by flow cytometry.
Furthermore, samples will be taken once/fortnight from animals surviving Study B and
analysed for Minimal residual Disease and relapse.
3. Patient follow up: Aliquots of blood samples taken during routine follow up of the CLL
patients will be used. Cells will be incubated with the appropriate Peptide-dye
conjugate for 30mins at 37OC. Cells will be washed and analyzed for peptide binding by
flow cytometry.
G: Statistics Cytotoxicity of PDCs and free drug will be assessed by calculating % Growth
Inhibition of treated versus untreated cells. Experiments will be performed in triplicate
and repeated thrice. In animal studies, survival will be assessed using Kaplan-Meier curves.
Additional statistics related to the bioinformatic analyses are described in detail above in
section 1.7-B.
