Soluble T-Cell Receptors Produced in Human Cells for Targeted Delivery

<div><p>Recently, technology has become available to generate soluble T-cell receptors (sTCRs) that contain the antigen recognition part. In contrast to antibodies, sTCRs recognize intracellular in addition to extracellular epitopes, potentially increasing the number of applications as reagents for target detection and immunotherapy. Moreover, recent data show that they can be used for identification of their natural peptide ligands in disease. Here we describe a new and simplified expression method for sTCRs in human cells and show that these sTCRs can be used for antigen-specific labeling and elimination of human target cells. Four different TCRs were solubilized by expression of constructs encoding the TCR alpha (α) and beta (β) chains lacking the transmembrane and intracellular domains, linked by a ribosomal skipping 2A sequence that facilitates equimolar production of the chains. Cell supernatants containing sTCRs labeled target cells directly in a peptide (p)-human leukocyte antigen (HLA)-specific manner. We demonstrated that a MART-1p/HLA-A*02:01-specific sTCR fused to a fluorescent protein, or multimerized onto magnetic nanoparticles, could be internalized. Moreover, we showed that this sTCR and two sTCRs recognizing CD20p/HLA-A*02:01 could mediate selective elimination of target cells expressing the relevant pHLA complex when tetramerized to streptavidin-conjugated toxin, demonstrating the potential for specific delivery of cargo. This simple and efficient method can be utilized to generate a wide range of minimally modified sTCRs from the naturally occurring TCR repertoire for antigen-specific detection and targeting.</p></div

List of primer sequences and their use and/or specificity.

a<p>I is desoxyinosine.</p>b<p>These primers are designed when clone sequence is known.</p>c<p>Sequence in italic is V-chain specific.</p

Electroporation of mRNA encoding MART-1 specific TcRs.

<p>(a) SupT1 were electroporated with or without 20 µg mRNA encoding the indicated TcR_2A. Twelve hours later, the cells were stained with HLA-A2/MART-1 multimer-PE and anti-CD3 Alexa Fluor 647. (b) Human PBMC were electroporated with 20 µg mRNA (same constructs as in (a)) and cultured for 4.5 hours. Following the addition of T2 cells pre-loaded or not (grey) with MART-1 peptide (10 µM final concentration), the cells were co-incubated for an additional 5 hours in the presence of anti-CD107a/b Alexa Fluor 647 antibodies, monensin and brefeldin A. Prior to analysis, cells were stained with anti-CD8 PE. The percentage of CD107a/b positive cells from the CD8 positive population is plotted. PHA was used to control for similar maximal degranulation levels regardless of the mRNA used for electroporation. Each bar represents the mean values of duplicates.</p

Multimerization of the sTCR increases sensitivity of antigen detection by flow cytometry.

<p>(a) SupT1 cells expressing SCT-CD20 were incubated with increasing concentrations of CD20 swap-sTCR monomer or tetramer, as indicated, and indirectly labeled with anti-FLAG AF647. Results shown are representative of two experiments, and error bars represent SD of duplicates (b) HLA-A2⁺ SupT1 cells were loaded with indicated concentrations of peptide and subsequently labeled with saturating amounts of either sTCR tetramer or nanobeads conjugated to sTCR monomers, followed by staining with anti-FLAG-AF647. Results shown are representative of two experiments. SupT1 cells expressing SCT-CD20 were used as a positive control. The scale shows the calculation of Arcsinh ratio of the Median. (c) SupT1 cells were loaded with increasing concentrations of CD20p (left panel), MART-1p or WT MART-1p (right panel), followed by labeling with saturating amounts of indicated sTCR tetramer, and indirectly stained with anti-FLAG AF647. Irrelevant peptide control (irr peptide) was used at 100 μM (disconnected symbols). Results shown are from one experiment representative of 3 (CD20p and MART-1p) or 2 (WT MART-1p) experiments. Error bars indicate SD of duplicates. (d) SupT1 cells expressing SCT-M1 were incubated with increasing concentrations of PE-conjugated sTCR tetramer.</p

Design of the sTCR and its use as a labeling reagent.

<p>(a) TCRα/β chains in full-length (upper part) and in soluble form (lower part). V: Variable domain, C: Constant domain, TM: transmembrane, CYTO: cytosolic domain. Interchain cystein bridges are indicated with S. (b) Design of the expression construct in which truncated TCRα and β chains are separated by a ribosome skipping sequence (2A) and tagged on their 3’-end. The tags used in this study are listed at their respective position. To increase interchain stability, a high affinity leucine zipper (LZ) was added to some sTCR constructs. (c) HLA-A2^pos T2 cells were loaded O/N with 10 μM MART-1p (red, blue) or 10 μM of an irrelevant peptide (CD20p_188–196) (filled grey). Ten μL of supernatant from HEK293 cells producing the DMF5 sTCR (red), or from mock transfected HEK 293 (blue), was added to the T2 cells and incubated for 15 minutes at RT followed by labeling with anti-His-647 antibodies and flow cytometric analysis (d) SupT1 cells expressing SCT-M1 (red) or SCT-CD20 (filled grey) were incubated with the DMF5 sTCR supernatant as described in (c) and stained using anti-His-647. The results in (c) and (d) are representative of at least two experiments and histograms are gated on viable cells, displayed as FSC^hi, SSC^hi events.</p

DMF5 sTCR is internalized upon specific ligand binding.

<p>(a) HeLa cells transfected with either SCT-M1-mCherry or SCT-CD20-mCherry (red) were incubated with His-tagged DMF5 sTCR labeled with an anti-His antibody and visualized using anti-mouse AF488 (green). Co-localization of the sTCR and SCT-M1 is shown in yellow (arrow). (b) Sup-T1 cells expressing SCT-M1 were incubated with DMF5 supernatants containing monomeric sTCR mCherry-His at 37°C for 30 minutes. The cells were subsequently put on ice to block endocytosis and stained with anti-His-AF647 (His). The nucleus was visualized by Hoechst stain (blue). (c) Sup-T1 cells expressing SCT-M1 were incubated with biotinylated DMF5 sTCR-mCherry bound to SA-Miltenyi nanobeads at 37°C.</p

TcR composition.

#<p>: frameshift.</p><p>nd: not determined.</p

Antigen-specific elimination of target cells by sTCR-Saporin as measured by ³H-thymidine incorporation.

<p>(a) SupT1 cells constitutively expressing SCT-M1 or SCT-CD20 were incubated with or without (NT) 10–40 nM DMF5 sTCR-Sap for 3 days. Cells were then labeled with ³H-thymidine and radioactive incorporation was measured (cpm). (b) SupT1 cells expressing SCT-M1 were incubated with increasing amounts of DMF5 sTCR-Sap for 3 days and ³H-thymidine incorporation was plotted as in (a). (c) HEK293 cells were pulsed with or without 10 μM MART-1p in the presence or absence of 20 nM DMF5 sTCR-Sap for 3 days, and ³H-thymidine incorporation was determined. The experiments shown in (a-c) are each representative of two performed and error bars represent SD from triplicates.</p

TcR structure and cloning strategy.

<p>(a) TcRα and β mRNA structures are similar: The 5′-situated Variable (V) domain encodes for the N-terminal part of the extracellular domain (EC) of the TcR. It starts with the signal sequence (L) upstream of the V-region. It ends at the recombination site with the D domain (only in TcRβ) and Joining domain (J), representing the hypervariable CDR3 domain. On the 3′-side of the messenger lies the Constant (C) region, which encodes for the carboxy-terminal part of the EC, the transmembrane region (TM) and the short intracellular domain (IC). (b) The 5′-RACE of the TcR is performed by reverse-transcription of mRNA into cDNA using an oligo dT primer. The cDNA is then polyC-tailed by TdT and this reaction is followed by two sequential amplifications using nested primers (pC1 and pC2) together with a polyC annealing primer (pGI). The final product is cloned into pTA and sequenced. The sequenced portion results in the full length V-domain and the CDR3 if the tailing has occurred on the 5′-UTR.</p

Retroviral delivery of MART-1 specific TcRs.

<p>(a) B44, B60 and DMF5 TcR_2A were expressed in the pMP71-G vector and transduced into SupT1 cells. After 3 days, cells were co-stained with anti-CD3 PB and HLA-A2/MART-1 multimer-PE. (b) Jurkat cells were transduced with the same constructs as in (a) or with a GFP pMP71-vector (Mock) and incubated for 24 hours with T2 cells loaded with or without MART-1 peptide (10 µM final concentration). IL-2 release was monitored by ELISA assay, and plotted as pg of IL-2 per mL of medium. Each bar represents the mean values of duplicates. Similar results were observed in two separate experiments.</p