Synthesis and biological evaluation of a peptide-paclitaxel conjugate which targets the integrin αvβ₆

Abstract

The integrin α(v)β(6) is an emergent biomarker for non-small cell lung cancer (NSCLC) as well as other carcinomas. We previously developed a tetrameric peptide, referred to as H2009.1, which binds α(v)β(6) and displays minimal affinity for other RGD-binding integrins. Here we report the use of this peptide to actively deliver paclitaxel to α(v)β(6)-positive cells. We synthesized a water soluble paclitaxel-H2009.1 peptide conjugate in which the 2'-position of paclitaxel is attached to the tetrameric peptide via an ester linkage. The conjugate maintains its specificity for α(v)β(6)-expressing NSCLC cells, resulting in selective cytotoxicity. Treatment of α(v)β(6)-positive cells with the conjugate results in cell cycle arrest followed by induction of apoptosis in the same manner as free paclitaxel. However, initiation of apoptosis and the resultant cell death is delayed compared to free drug. The conjugate demonstrates anti-tumor activity in a H2009 xenograft model of NSCLC with efficacy comparable to treatment with free paclitaxel.

Figure 1. Structure of H2009.1 peptide-paclitaxel conjugate (compound 3) and FITC-labeled H2009.1 peptide-paclitaxel conjugate (compound 4)

The H2009.1 peptide displays the sequence RGDLATLRQL and the scrambled H2009.1 peptide is DALRLQGTLR.

Figure 2. The paclitaxel-H2009.1 conjugate binds specifically to α_vβ₆–expressing cells

H2009 cells (α_vβ₆-positive) and H460 cells (α_vβ₆- negative) were incubated for 1 hour with 1 M paclitaxel-H2009.1 conjugate labeled with FITC (compound 4) or scrambled peptide-paclitaxel-dye conjugate. Binding of the conjugate was measured by flow cytometry. A total of 10,000 events were evaluated for fluorescence in channel 1 (excitation at 488 nm, emission at 500-550 nm).

Figure 3. Paclitaxel is released from the conjugate in the presence of esterase and serum

Compound 3 was dissolved in phosphate-buffered solutions (PBS, 0.01M) at pH 7.4 (▴), or esterase (18u/mL) in PBS (∎), or rat serum (●), individually. The solutions were incubated at 37° C. At time points 3, 8, 24, 48, 72, 96 h during incubation, the release of paclitaxel was analyzed by HPLC.

Figure 4. The H2009.1-paclitaxel conjugate displays α_vβ₆–selective cytotoxicity that is time dependent

Left Panel: H2009 cells were exposed to 1 μM Taxol, H2009.1 peptide-paclitaxel (compound 3) and scH2009.1 peptide-paclitaxel for 10min, washed, and incubated further in fresh medium for different time point. The cell viability is normalized to untreated control cells. Right Panel: H2009 cells and H460 cells were exposed to 1 μM Taxol and compound 3 for 10min, washed, and incubated further in fresh medium for indicated times. The short exposure time to the conjugates is necessary to assure that the affects observed are due to the conjugate and not paclitaxel released from the peptide before internalization.

Figure 5. The affect of H2009.1-paclitaxel conjugate on cell viability is blocked by pretreatment with H2009.1 peptide

H2009 cells were treated with or without 10 μM H2009.1 10-mer tetrameric peptide for 1 hour, washed, and then treated with 1 μM H2009.1-paclitaxel conjugate (compound 3) or scH2009.1 peptide-paclitaxel. Viability was determined at 96 h.

Figure 6. H2009.1-pacliatxel activates caspases and initiates apoptosis

H2009 cells were exposed to 1 μM H2009.1 H2009.1-paclitaxel conjugate for 10 min, washed, and incubated in fresh media for indicated times. Floating and attached cells were collected and subjected to caspases/PI staining using Vybrant® FAM Poly Caspases Assay and analyzed by flow cytometry. 10,000 events were measured using channel 1 (excitation 488 nm, emission 500 nm) and channel 3 (excitation 488 nm, emission 650 nm). The data represents the average of two experiments.

Figure 7. Tumor growth rate is slowed by free paclitaxel and H2009.1-paclitaxel conjugate by similar amounts

Subcutaneous tumors were established on the flank of NOD/SCID mice (n=5). At day 18, saline, free paclitaxel and compound 3 were injected intravenously via the tail vein with 5mg/kg, based on paclitaxel weight. Animals were treated at days 18, 21, 24, 27, and 30. Measurements were made with calipers by an independent scientist. Tumor volumes are calculated as V = l × 2w/2. Saline (●), Paclitaxel (∎), H2009.1-paclitaxel (▴).

Scheme I

Synthesis of 2maleimido-paclitaxel was prepared via coupling paclitaxel with 3-maleimidopropionic acid (compound 1).

Scheme II. Synthesis of tetrameric H2009 peptide by a convergent coupling reaction (compound 2)

Michael addition of cysteine containing monomeric peptides with the maleimide trilysine core results in formation of the tetrameric peptide. A PEG11 linker is placed between the peptide and the core to improve solubility and prevent peptide aggregation. Removal of the acetamidomethyl protein group from the trilysine core by treatment with the Ag(OAc) reveals a unique thiol for coupling to compound 1.