Selection of functional human antibodies from retroviral display libraries

Abstract

Antibody library technology represents a powerful tool for the discovery and design of antibodies with high affinity and specificity for their targets. To extend the technique to the expression and selection of antibody libraries in an eukaryotic environment, we provide here a proof of concept that retroviruses can be engineered for the display and selection of variable single-chain fragment (scFv) libraries. A retroviral library displaying the repertoire obtained after a single round of selection of a human synthetic scFv phage display library on laminin was generated. For selection, antigen-bound virus was efficiently recovered by an overlay with cells permissive for infection. This approach allowed more than 10(3)-fold enrichment of antigen binders in a single selection cycle. After three selection cycles, several scFvs were recovered showing similar laminin-binding activities but improved expression levels in mammalian cells as compared with a laminin-specific scFv selected by the conventional phage display approach. Thus, translational problems that occur when phage-selected antibodies have to be transferred onto mammalian expression systems to exert their therapeutic potential can be avoided by the use of retroviral display libraries.

Figure 1

Schematic representations of the retrovirus constructs and the selection procedure. (A) All constructs contain the complete genome of the MoMLV being comprised of the gag, pol and env genes. A SfiI/NotI cassette is inserted in the env gene between the 3′ codon of the signal peptide (SP) and the 5′ codon of the SU domain. Polypeptides inserted into this cassette will be displayed on the viral Env protein via a factor Xa cleavage site (Xa). (B) Selection procedure for retroviral scFv libraries. The virus library is released from HEK-293T cells transfected with pscFv-XMo. The virus particles are then incubated with laminin-1 coated cell culture plates to allow binding. After binding and washing NIH-3T3 cells are added for infection. The infected cells are then cultivated to produce virus particles for the next round of selection.

Figure 2

Laminin-bound retroviruses can be recovered. (A) NIH-3T3 cells were added to immobilized laminin-1 incubated with 2.5 × 10⁶ particles of the 7A5-XMo (left panel) or the L36-XMo virus (right panel) after removal of unbound particles. Infected cells (appearing in red colour) were detected by immunostaining using specific antiserum for the viral capsid protein. (B) Recovery efficiency of the L36-XMo. Approximately 2.5 × 10⁷ 7A5-XMo virus particles were spiked with decreasing amounts (10-fold dilutions) of L36-XMo virus particles. The relative amounts of both viruses were determined upon ClaI digestion of RT–PCR fragments generated on viral genomic RNA before (Input, lanes 3–8) and after laminin binding and infection of NIH-3T3 cells (Output, lanes 9–14) to laminin-1. Each of the lanes 3–8 and 9–14 corresponds to 10-fold dilutions of L36-XMo. The ClaI-digested fragments derived from PCR using plasmids p7A5-XMo and pL36-XMo as template served as controls, respectively (lanes 1 and 2).

Figure 3

Fingerprint analysis of the selected scFvs. RT–PCR fragments derived from genomic viral RNA representing the scFv fragment coding sequences before (A) and after three rounds of selection (B) were cloned into bacteria. Colony-PCR fragments from 50 randomly picked bacterial clones were restricted with BstNI, respectively. Similar restriction patterns were grouped and the patterns of the most prominent clones L9, L28 and L6 are indicated.

Figure 4

Laminin-1-binding activity of virus pools and selected scFv viruses. (A) An aliquot of 130 mU RT activity of the unselected (input) and the selected (output) virus populations were incubated with laminin-1 or milk powder coated ELISA plates. After washing, RT activities of the retained virus particles were quantified, respectively. (B) Different amounts of RT activity (input) of the unselected (orange line) and the selected (red line) virus populations as well as of the reconstituted L6-XMo (closed triangles, blue line), L9-XMo (closed diamonds, blue line), L28-XMo (closed squares, blue line) and L36-XMo viruses (closed circles, grey line) were incubated with laminin-1 coated ELISA plates. After washing, RT activities of the retained virus particles were quantified, respectively (output). Average values of three independent experiments are given.

Figure 5

Characterization of the selected scFv-displaying viruses. (A) Infection rates of the selected viruses after binding to laminin-1. An aliquot of 1 mU of RT activity of the indicated viruses, respectively, was incubated with laminin-1 coated cell culture plates. After washing, NIH-3T3 cells were added and the number of infected cells was quantified 24 h post-infection by immunostaining using the MLV capsid-specific antiserum. The average ratio of the number of infected cells and the input RT activity, based on three independent experiments, is provided. (B) Env protein incorporation into viral particles. HEK-293T cells were transfected with plasmids, pL6-XMo (lane 5), pL28-XMo (lane 4) and pL36-XMo (lane 3), respectively, or left untransfected (lane 2). At day two after transfection, virus particles released from the cells were separated on a 10% SDS–polyacrylamide gel. MoMLV wild-type virus was loaded as a molecular weight marker control for the viral proteins. Immunoblotting was performed using SU and capsid protein-specific antisera.

Figure 6

Functional characterization of scFv antibody molecules produced by human cells. (A) Secretion of scFvs into the cell culture supernatant by HEK-293T cells transiently transfected with plasmid pCR3.1, pCR3.1-L36, pCR3.1-L6 or pCR3.1-L28. Antibody functionality was demonstrated by ELISA against plastic immobilized BSA and laminin-1, respectively. Bound scFv was detected with an anti-myc mAb. (B) Western-blot analysis of scFv secreted into the cell culture supernatant by pCR3.1 (lane 2), pCR3.1-L36 (lane 3), pCR3.1-L6 (lane 4) or pCR3.1-L28 (lane 5) transfected HEK-293T cells, respectively. An aliquot of 20 μl of supernatant of each transfectant was loaded in each lane. An aliquot of 1 μg and 500 ng of recombinant L36 scFv antibody purified from bacterial culture supernatant were loaded in lanes 1 and 6, respectively. Immunoblotting was performed using an anti-myc mAb. Intensity of individual bands was evaluated by PCBas software (Raytest, Germany). The values were normalized to the intensity of control and expressed underneath the respective blot lane.