Targeting of phage particles towards endothelial cells by antibodies selected through a multi-parameter selection strategy

Abstract

One of the hallmarks of cancer is sustained angiogenesis. Here, normal endothelial cells are activated, and their formation of new blood vessels leads to continued tumour growth. An improved patient condition is often observed when angiogenesis is prevented or normalized through targeting of these genomically stable endothelial cells. However, intracellular targets constitute a challenge in therapy, as the agents modulating these targets have to be delivered and internalized specifically to the endothelial cells. Selection of antibodies binding specifically to certain cell types is well established. It is nonetheless a challenge to ensure that the binding of antibodies to the target cell will mediate internalization. Previously selection of such antibodies has been performed targeting cancer cell lines; most often using either monovalent display or polyvalent display. In this article, we describe selections that isolate internalizing antibodies by sequential combining monovalent and polyvalent display using two types of helper phages, one which increases display valence and one which reduces background. One of the selected antibodies was found to mediate internalization into human endothelial cells, although our results confirms that the single stranded nature of the DNA packaged into phage particles may limit applications aimed at targeting nucleic acids in mammalian cells.

Figure 1. Schematic selection for internalization.

In a basic selection for internalization the phage library is incubated with the live cells at 37 °C in order to allow internalization to happen. Washing steps are performed to remove the library clones not internalized. The cells are then lysed to release the internalized phage and the lysate is mixed with E. coli for infection. The bacteria surviving (due to phage encoded antibiotic resistance) on selective agar plates containing antibiotics can be used for production of new phage particles for additional rounds of selection or for screening.

Figure 2. Comparing helperphages with different properties.

Functionalized helper phages like the KM13 and the Hyperphage have been developed for rescuing phagemids into phage particles. Normal phagemid rescue results in only 1–10% of phage particles displaying a single antibody fragment. When rescuing phagmids with KM13 helperphage the trypsin cleavage site between domain 2 and 3 of pIII results in the non-displaying pIII from the helper phage being rendered non-infective. PIII fused with antibody encoded by the phagemid retains infectivity. Hyperphage is deleted in the gene encoding pIII so that no pIII can be derived from the helper phage, which in theory leads to 100% antibody display. The background can, however, not be removed when using Hyperphage.

Figure 3. Selection scheme and initial effect on selection output.

(A) Three different phagemid based parent libraries i.e. Tomlinson I, Tomlinson J and Garvan were each packaged into phage particles using either the gIII deficient Hyperphage or the protease sensitive KM13. The resulting six libraries were used in selections on HMEC-1 cells for surface binders and the resulting phage output from the selections were again packaged using either KM13 or Hyperphage as helper phage resulting in a total of twelve different sub-libraries. Selections using phage packaged with different combinations of helper phage were used for evaluating the effect of the helper phage derived characteristics on internalization. (B) The effect of using either Hyperphage or KM13 for rescue of phage particles was evaluated. The results showed a clear effect on the output of phage particles after selection for internalization, with Hyperphage packaging generally resulting in more CFU than KM13. (C) Selections for internalization were made on HMEC-1 cells using the Tom I parent library or one of four Tom I sub-libraries enriched for antibodies binding HMEC-1 surface antigens. HMEC-1 cells were fixed and permeabilized for ELISA. All enriched sub-libraries give a signal above the unselected parent library. Again a clear difference in signal between the sub-libraries with higher signal from the sub-libraries packaged with Hyperphage after the selection for enrichment is evident. Only results for the Tomlinson I libraries are shown.

Figure 4. ELISA validation of cellular specificity.

ELISAs were made to test the cell type specificity of the cherry picked clones. Each clone was tested for binding to HMEC-1 cells, ASF-2 fibroblasts and hMSC bone marrow stromal cells in parallel. The results have been split into clones originated from the Tomlinson I & J libraries (left) and clones from the Garvan library (right). The ELISAs were made with phage particles rescued with Hyperphage.

Figure 5. Comparing helperphage effects on ELISA signals.

ELISA was made on HMEC-1 cells using 15 cherry picked clones. The clones were rescued with KM13 and Hyperphage to compare the effect of the helperphage on the ELISA signal. The results were normalized based on cell number measured by Janus green staining.

Figure 6. Staining HMEC-1 cells to detect internalization.

(A,B) HMEC-1 cells incubated with either soluble H8 antibody or secondary antibody only. Incubations were made under internalizing conditions and followed by fixation, permeabilization and staining of internalized antibody (red). Cell nuclei are stained by dapi (blue). (C–E) HMEC-1 cells incubated with transferrin (red) and H8 antibody (green). Incubations were made under internalizing conditions and followed by fixation and permeabilization to stain H8. Scale bars = 10 μm.

Figure 7. Tracking antibody-phage internalization by ICC.

HMEC-1 cells incubated with either non-displaying KM13 helper phage (green) or phage displaying the H8 rescued with KM13 (green). Incubations were made under internalizing conditions and followed by permeabilization and staining of phage particles. (A) Cells incubated with 10¹¹ H8 displaying phages pr. well. (B) Cells incubated with 10¹¹ non-displaying KM13 helper phage. (C) Cells fixed in PFA and incubated with 10¹¹ H8 displaying phages pr. well. (D) HMEC-1cells incubated 48 hours without phage or with 5 × 10¹¹ H8-pFROG rescued with Hyperphage (E) or KM13 (F). The cells were washed, fixed and permeabilized before staining of phage particles (green). Cell nuclei are stained in blue. Scale bars = 10 μm.

Figure 8. Expression efficiency from ss-DNA versus ds-DNA in various cell lines.

Cell lines were transfected using either single or double stranded form of the GFP encoding 8H-pFROG plasmid (green) and imaged with phase contrast to visualize non-fluorescent cells. HMEC-1 cells transfected with ssDNA (A) and ds-DNA (B). MCF-7 breast cancer cells transfected with ssDNA (C) and with ds-DNA (D). HEK293 cells transfected with ssDNA (E) and with ds-DNA (F). ASF-2 cells transfected with ss-DNA (G) and dsDNA (H). HUVEC cells transfected with ss-DNA (I) and ds-DNA (J). Scale bars = 1 μm.