Design, manufacturing, purification, and characterization of Immuno-STAT and Neo-STAT proteins
Immuno-STAT proteins bearing H-2Db were genetically fused to an effector-attenuated murine IgG2a28. Immuno-STAT and Neo-STAT proteins bearing human HLAs were fused to an effector-attenuated human IgG1 and linked to CMV pp65495-503 or MART126-35, immunodominant epitopes in the context of human cytomegalovirus infection or malignant melanoma29,30,31. Domains within the Immuno-STAT and Neo-STAT frameworks are linked via G4S linkers. Cysteine substitutions R12C of β2m and A236C of HLA-A*0201 result in a stabilizing disulfide bond between these polypeptides. Neo-STAT also contains a disulfide bond comprising cysteine substitutions at Y84C and A139C of HLA-A*0201, which stabilizes the “empty” (i.e. epitope-less) Neo-STAT precursor prior to conjugation with the peptide epitope of interest32. An engineered cysteine, E44C, within β2m serves as the attachment site for maleimide conjugated peptides.
Immuno-STAT and Neo-STAT proteins were expressed by transient transfection in Expi-CHO cells (ThermoFisher). Proteins were purified from the conditioned media using a two-step method of ProteinA capture with MabSelect SuRe (GE) followed by size exclusion chromatography. For SDS-PAGE analysis, proteins were boiled in SDS sample buffer with or without reducing agent for 5 min before loading 2 µg per gel lane.
Empty Neo-STAT precursor proteins were linked to maleimide-conjugaged peptides using standard maleimide chemistry. Briefly, empty Neo-STAT precursors were exchanged into and partially reduced with a TCEP-based reducing buffer before two rounds of conjugation with a 20 fold molar excess of peptide-maleimide in the absence of TCEP. Conjugated Neo-STAT proteins were washed at low pH to remove excess unconjugated peptides in solution before purification by size exclusion and mass confirmation by electrospray ionization time of flight mass spectrometry (ESI-TOF MS).
Animals studies
Spleens were collected from C57BL/6J and P14 T cell receptor (specific for LCMV gp33-41/H-2Db) transgenic mice (Jackson Labs) at least six weeks of age following euthanasia by CO2 inhalation and confirmation of euthanasia by cervical dislocation. All studies requiring animal tissues were approved by the Institutional Animal Care and Use Committee for SmartLabs (Cambridge, MA) and were performed in compliance with federal guidelines and in accordance with the ARRIVE guidelines 2.0.
pSTAT5 assay and phosphoflow analysis
Spleens were harvested from C57BL/6J and P14 T cell receptor (specific for LCMV gp33-41/H-2Db) transgenic mice (Jackson Labs). Splenocytes from 10 to 15 mice (depending on the number of conditions tested) per strain were pooled and CD8 T cells were isolated using Dynabeads Untouched Mouse CD8 kit (ThermoFisher). CD8 splenocytes were resuspended in RPMI culture media (ATCC) supplemented with 10% fetal bovine serum (Hyclone). 1 × 105 C57BL/6J CD8 splenocytes or P14 TCR Tg CD8 splenocytes were seeded in 96-well U-bottom plates and stimulated with Immuno-STAT at a given concentration per well for 20 min at 37 °C (initial Immuno-STAT-IL2 framework variant screen) or 5 min at 37 °C (follow up studies) in a final volume of 100 µl. For the initial Immuno-STAT framework variant screen, mouse splenic CD8 T cells were incubated with Immuno-STAT proteins at 0.01, 0.1, 1, 10, 100, 250, 500 and 1000 nM. For confirmatory studies of LCMV-IST-IL2.FH4, LCMV-IST-IL2.F4, LCMV-IST-IL24, LCMV-IST-IL22, IL2.FH4-Fc and rhIL-2 (Peprotech), mouse splenic CD8 T cells were incubated with test articles at 0.00119, 0.00477, 0.0191, 0.0763, 0.305, 1.22, 4.88, 19.5, 78.1, 313, 1250, and 5000 nM. Cells were immediately fixed with IC fixation buffer (ThermoFisher), permeabilized with 100% methanol, and stained with a 1/10 dilution of anti-pY694 STAT5 antibody (BD Biosciences) for 30 min at room temperature, followed by flow cytometry on an iQue Screener (Intellicyt) for initial IST variant screen or an Attune NxT cytometer (Invitrogen) for follow up studies. The percent of positively stained cells was determined using FlowJo software (TreeStar). Non-linear curve fits and EC50 determinations from pSTAT5 dose response data were performed using Prism analysis software (Graphpad).
Human T cell expansion, tetramer staining and flow cytometry analysis
Human healthy donor peripheral blood mononuclear cells (PBMC) were obtained as frozen stocks (Astarte Biologics) or isolated from leukopaks (HemaCare); washed and resuspended in ImmunoCult-XF Cell Expansion Media (Stemcell Technologies). 1 × 107 PBMC were seeded in a 6 well plate with specific Immuno-STAT or Neo-STAT at a given concentration or media control in a total volume of 4 ml. Cells were incubated with 0, 0.1, 1, 3, 10, 30, or 100 nM CMV-IST-IL2.FH4 or MART-IST-IL2.FH4, with each concentration evaluated in 1–4 independent expansion trials. 0.3 nM Immuno-STAT was also evaluated for CMV-IST-IL2.FH4. For Neo-STAT expansion studies, PBMC were incubated with CMV-IST-FH4 or CMV-NST-FH4 at 0, 0.1, 0.3, 1, 3, 10 and 30 nM and PBMC were incubated with control MART-NST-IL2.FH4 at 0, 1, 3, 10 and 30 nM. For experiments examining the combined activity of CMV-IST-IL2.FH4 and CMV-IST-CD802, cells were incubated with CMV-IST-IL2.FH4 and CMV-IST-CD802 at 1 and 100 nM, respectively, or with media alone or with 5 µg/ml CMV pp65495-503 (NLVPMVATV) peptide plus 50 IU/ml IL-2. Immuno-STAT or peptide expansion cultures were maintained in a 37 °C CO2 incubator with replacement of half the culture media at day 5 and day 7. Cells were harvested on day 10, washed, resuspended in FACS buffer on ice and stained for viability using Fixable Viability Stain 780 (BD Biosciences) before staining with relevant tetramers (MBL International, MA) on ice for 30 min. Tetramer-stained cells were then washed and stained on ice with antibodies against CD3 (clone SK7, BioLegend), CD14 (clone M5E2, BioLegend), CD19 (clone HIB19, BioLegend), CD56 (clone HCD56, BioLegend), CD4 (clone SK3, BioLegend) and CD8 (clone SK1, BD Biosciences) for 30 min. Tetramer flow cytometric data was acquired using the Attune NxT cytometer (ThermoFisher) and analyzed using FlowJo software (Tree Star). Peak fold expansion per donor per trial was calculated as the maximum tetramer-positive frequency observed for PBMC expanded with specific IST-IL2.FH4 over tetramer-positive frequency for media-incubated PBMC. Peak fold expansion data was derived from between one and three expansion trials per donor, with mean peak fold expansion values used for donors with multiple expansion trials.
Intracellular cytokine and phenotypic staining of in vitro-expanded human CD8 T cells
A total of 2 to 4 × 106 human PBMCs expanded with specific Immuno-STAT or peptide were pretreated with brefeldin A (BFA) and monensin (ThermoFisher), plated in a 24-well plate, and stimulated at a 1:1 ratio with T2 cells (ATCC) that had been loaded with CMV pp65495–503 (NLVPMVATV) or Mart126–35 (ELAGIGILTV) or HIV-1 p17 Gag77–85 (SLYNTVATL; SL9) peptide for 2 h and washed twice. Cells were stimulated for 5 h, washed, stained with Fixed Viability Stain 780 (BD Biosciences), antibodies against CD3 (clone SK7, BioLegend), CD8 (clone SK1, BD Biosciences) and CD107a (clone H4A3, BD Biosciences), and fixed using IC fixation buffer (ThermoFisher). Cells were next washed in permeabilization buffer (eBioscience), stained with antibodies against TNF-α (clone MAb11, BD Biosciences), IFN-γ (clone 4S.B3, BioLegend), and granzyme B (clone GB11, BD Biosciences) for 30 min at room temperature, washed, and analyzed. For representative FACS plots and pairwise marker quantitation, PBMC were stimulated as described above using T2 cells loaded with 100 nM peptide. For peptide titration studies, PBMC were stimulated as described above using T2 cells loaded with 1 × 10–13, 1 × 10–12, 1 × 10–11, 1 × 10–10, 1 × 10–9, 1 × 10–8, 1 × 10–7, 1 × 10–6, or 1 × 10–5 g/ml peptide.
TCR sequencing
PBMCs from healthy donors were expanded in vitro with CMV pp65495-503 or Mart126-35 peptide plus IL-2, or with 100 nM specific Immuno-STAT in 10-day cultures. Expanded cells were harvested, tetramer stained, and CMV- or MART1-specific CD8+ T cells were single cell sorted (Sony SH800) and the α and β TCR chains were coamplified (iRepertoire). TCR library sequencing used an Illumina MiSeq v2 Nano Kit. Data for each individual well were demultiplexed, mapped, and analyzed using the iRmap VDJ pipeline and the iPair Analyzer.
