Construction of Lentiviruses Pseudotyped with Sindbis E2-Single Chain Antibody (SCA) Fusions or Membrane-Anchored SCAs for Targeted Therapies in Lymphoma.
Authors: Luong Quang Trong , Morizono Kouki , Chen Irvin SY , De Vos Sven 
Published: October 2019

Abstract
Abstract 3571
Poster Board III-508
Previously, we demonstrated specific targeting of CD20+ and CD30+ cells can be achieved in vitro using vectors of Morizono et al. [Cell Cycle 2005; Nature Medicine 2005]. These lentiviruses expressed modified Sindbis E2 proteins that contain the Fc receptor binding domain (ZZ) of protein A. The lentiviruses could be specifically and effectively directed to target cells by conjugation of an antibody to the modified ZZ Sindbis viral envelope (m168). CD30+ HL cell lines and CD20+ non-Hodgkin lymphoma (NHL) cell lines were used. Anti-CD30-labeled viruses (m168-αCD30) specifically transduced CD30+ HL cells while avoiding CD30− NHL cells; in addition, transduction of CD20+ NHL cells was achieved using m168-αCD20 viruses (unpublished data). However, these viruses were unsuitable for in vivo studies because of the unstable conjugation of antibody and envelope protein, and the ambiguity of the ZZ domains to bind Fc receptors. We developed two targeting approaches. For the first approach, we constructed vectors containing single chain antibodies (SCA) directly fused to the Sindbis E2 spike glycoprotein. Approximately 100 different construct-variants have been produced so far, that contain SCA-E2 fusions with a variety of flexible linkers to allow for adequate folding and expression of the fusion proteins. These constructs contain either anti-CD30-E2 (T405 or T215) or anti-CD20-E2 sequences. The second approach was to construct membrane-anchored SCA vectors that can be expressed on the envelope of the lentiviruses. We produced in excess of 30 variants containing anti-CD30 or anti-CD20 sequences with transmembrane domains of CD4 or VSV-G, or a Glycosylphosphoinositol (GPI)-anchor. Potentially, these constructs are more stable and might allow an in vivo targeting approach. We have tested these constructs for expression of SCA-E2 or membrane-anchored SCA by Western analyses. The expression of SCA-E2 fusion proteins was dependent on the length of the linker used with greater expression observed with longer linkers. The linker length should contain a minimum of 20 amino acids either side of the cloning/fusion site to permit adequate expression of SCA-E2 fusion proteins. SCA-GPI-anchor proteins, on the other hand, are not fused to the Sindbis E2 protein but contain similar length flexible linkers to permit proper folding of the SCA and GPI proteins. We tested whether the viruses produced with these modified envelope proteins or membrane-anchored proteins were able to infect CD30 or CD20 expressing target cells. We achieved up to 40% infection of 293-T CD20+ cells with anti-CD20-E2 constructs but only about 10% infection of 293-T CD30+ cells using T405-E2 (anti-CD30) constructs. In contrast, we found that T405-GPI (anti-CD30) constructs were able to infect 60% of target cells while anti-CD20-GPI infected less than 10% of target cells. Additionally, we found that T215-E2 (anti-CD30) pseudotyped viruses were not able to infect CD30+ target cells. We conclude from our data (1) that while single chain antibody sequences were the functional components that determine specificity of pseudotyped lentiviruses, not all single chain antibodies will be functional in these systems; and (2) the success of the different anchoring techniques seems to depend on the single chain antibody and the target antigen. These vectors will now be subjected to in vivo targeting.

Disclosures:
No relevant conflicts of interest to declare.