Towards Physiologically and Tightly Regulated Vectored Antibody Therapies

Abstract

Cancers represent highly significant health issues and the options for their treatment are often not efficient to cure the disease. Immunotherapy strategies have been developed to modulate the patient's immune system in order to eradicate cancerous cells. For instance, passive immunization consists in the administration at high doses of exogenously produced monoclonal antibodies directed either against tumor antigen or against immune checkpoint inhibitors. Its main advantage is that it provides immediate immunity, though during a relatively short period, which consequently requires frequent injections. To circumvent this limitation, several approaches, reviewed here, have emerged to induce in vivo antibody secretion at physiological doses. Gene delivery vectors, such as adenoviral vectors or adeno-associated vectors, have been designed to induce antibody secretion in vivo after in situ cell modification, and have driven significant improvements in several cancer models. However, anti-idiotypic antibodies and escape mutants have been detected, probably because of both the continuous expression of antibodies and their expression by unspecialized cell types. To overcome these hurdles, adoptive transfer of genetically modified B cells that secrete antibodies either constitutively or in a regulated manner have been developed by ex vivo transgene insertion with viral vectors. Recently, with the emergence of gene editing technologies, the endogenous B cell receptor loci of B cells have been modified with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease (Cas-9) system to change their specificity in order to target a given antigen. The expression of the modified BCR gene hence follows the endogenous regulation mechanisms, which may prevent or at least reduce side effects. Although these approaches seem promising for cancer treatments, major questions, such as the persistence and the re-activation potential of these engineered cells, remain to be addressed in clinically relevant animal models before translation to humans.

Keywords: adoptive transfer; antibody; cell engineering; checkpoint inhibitors; gene editing; neutralization; reprogramming; viral vectors.

Figure 1

Reprogramming approach for the ectopic expression of membrane anchored and/or secreted antibodies. (a) Clinical approach. B cells from patients would be collected, modified, and then re-infused. Ex vivo B cell receptor insertion would be achieved by transduction with a lentiviral vector carrying the antigen-specific immunoglobulin sequence. (b) The molecular rheostat approach. A LV construct encoding the membrane-anchored and/or secreted form of an antigen-specific-IgG1/M driven by Igκ light chain promoter (FEEK) was generated. The constant IgG1 heavy chain (CH) and κ light chain (CL) genes were fused to the variable regions (VL and VH) of a monoclonal antibody directed against a specific antigen. Co-expression of CHs and VHs were obtained by introduction of the F2A peptide sequence. A 2A peptide was included between the secreted γ1 exon and between the μM domain. By mutating the 2A peptide, modulation of the secreted IgG and surface chimeric IgG/M BCR ratio can be achieved. (c) The FAM2 approach. A LV construct encoding the membrane-anchored and/or secreted form of an antigen-specific-IgG1 driven by Igκ light chain promoter (FEEK) was generated. The constant IgG1 heavy chain (CH) and κ light chain (CL) genes were fused to the variable regions (VL and VH) of a monoclonal antibody directed against a specific antigen. Co-expression of CHs and VHs were obtained by introduction of the F2A peptide sequence. Two short intronic sequences were included between the CH3 and M1 exons and between the M1 and M2 exons as well as two polyadenylation signals respectively before and after the transmembrane domains. The production of secreted or membrane-anchored immunoglobulins closely mimics the natural expression of these two distinct immunoglobulin forms, which is tightly controlled by alternative splicing and polyadenylation mechanisms during B-cell lymphopoiesis.

Figure 2

Reprogramming approach to edit of the endogenous BCR. (a) Cutting sites and DNA templates for BCR editing. The endogenous loci coding antibodies are displayed. The heavy chain is located on the chromosome 14 and comprises variable, joining, diversity and constant regions. The two types of light chains, κ and λ, are respectively located on chromosome 2 and 22 and comprises variable, joining and constant regions. The cutting sites targeted by different groups to edit genetically antibody loci are indicated by arrows (red [88], green [87], yellow [86], blue [85]). (b) Antibodies after editing. The resulting antibodies after gene editing are displayed bellow. The modified parts (different from the endogenous ones, in black) are indicated in blue.