Peptide-drug-conjugates for Personalized, Targeted Therapy of Chronic Lymphocytic Leukemia

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.