Biochemie, Chemie und Pharmazie
In vivo selection of retroviral display libraries for tumor homing
Lydia Jasmin Dürner
- The display of foreign polypeptides and proteins on the surface of viruses or cells provides an important tool for the engineering of biomolecules and the analysis of their interactions with binding partners. The most extensively used display platform is the coat protein of the filamentous bacteriophage (Smith, 1985). Phage display libraries have often been selected for polypeptides, e.g. single chain (sc) antibodies that bind to a protein of interest, but in vivo selection could only be demonstrated for peptides so far. An alternative display platform is the retrovirus murine leukemia virus (MLV). Here, polypeptides are displayed at the N-terminus of the viral envelope glycoprotein. Proof of principle for this platform was demonstrated for protease substrate libraries, which can be selected through coupling proteolytic activation with viral infectivity (Buchholz et al., 1998). Selection of the library CX4A on living cells resulted in viruses with more than three orders of magnitude improved spreading efficiency through tumor cells (Hartl et al., 2005). Also scAb libraries have recently been displayed and selected using retroviruses (Urban et al., 2005). The library scFvlibxMo displays the repertoire of phage display preselected sc antibodies for laminin-1 binding. The retrovirus based selection process resulted in laminin-specific sc antibodies with improved expression levels in mammalian cells.
This thesis describes the in vivo (i.e. in mouse tumor models) selection of the C-X4-A and scFvlibxMo for tumor homing upon systemic delivery.
For selection of the protease substrate library C-X4-A a subcutaneous tumor was induced in SCID mice followed by three systemic injections of the library. The selection process was monitored over a period of 34 days. After the incubation period mice were sacrificed and virus load in organs and tumor determined. PCR analysis after 34 days showed that virus from the library had preferentially infected the tumor. Sequence analysis showed the selection of protease substrates with the most prominent one with a frequency of over 65%. The four most prominent protease substrate variants where reconstituted into the original viral backbone for further investigation (C-SK-A, C-HI-A, C-HM-A and C-HS-A). Interestingly, these viruses exhibited a reduced spreading capacity in vitro on HT1080 cells as compared to the C-AK-A virus, which had previously been selected on HT1080 cells. When assayed for tumor homing, however, viruses C-HI-A and C-HS-A had clearly improved in comparison to C-AK-A. Tumor tissue had been infected at rates of over 55% while virus load of extratumoral organs was very low (infection rates <0.7 for C-HS-A and <0.02 for C-HI-A). Tumor targeting capacity had thus been improved over 10-fold by the in vivo selection of the C-X4-A library.
The experimental set up for the in vivo selection of the scFvlibxMo library was performed according to that of the C-X4-A library. Fingerprint analysis of the selected viruses that infected tumor tissue resulted in the identification of seven antibody variants showing unique CDR3 sequences. Two prominent clones (M49T-A and M49T-B) were cloned back into the MoMLV genome for further analysis of the reconstituted viruses. While variant B bound laminin-1 efficiently, variant A was unable to do so, although it was selected at highest frequency (76%). Both reconstituted viruses were equally well infectious and spread through HT1080rec1 cells at a similar efficiency as MoMLV. In an in vivo competition experiment the selected viruses clearly out-competed a laminin-1 binding reference virus L36xMo for tumor homing. To understand the molecular driving forces behind the in vivo selection process the epitope of the selected scFv M49T-A was identified using a phage peptide library approach. In silico analysis led to the identification of a small group of possible antigens, including tenascin, fibronectin and collagen.
The data described in this thesis demonstrate that the retrovirus display platform is capable of allowing the in vivo selection of protease substrates and scFvs. Notably, the replication competence of the system introduced an additional level of complexity to the library. The performed in vivo selections significantly enhanced tumor tropism. Selective infection of tumor cells combined with transfer of anti-tumoral genes is an attractive strategy for cancer therapy being in focus of current research. The viruses selected in this thesis build prime candidates for targeted retrovirus based tumor therapy.