*4.3. Enzyme-Linked Immunosorbent Assay (ELISA)*

Binding activity and specificity of Abs to the purified CMVpp65495-503/HLA-A\*02:01 SCT protein were determined by ELISA, as described previously [17].

#### *4.4. Cell Cultures*

HLA-A\*02:01-expressing cell lines Malme-3M, MDA-MB-231, and HCT116 and an HLA-A\*02:01-negative LoVo cell line were purchased from the Korean Cell Line Bank and maintained and cultured in an RPMI-1640 medium (HyClone, Busan, Korea) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (HyClone, Busan, Korea), penicillin (100 U/mL), streptomycin (100 μg/mL), and amphotericin B (0.25 μg/mL; Hy-Clone) [35,36]. All cell lines were maintained at 37 ◦C in a humidified 5% CO2 incubator and routinely screened for *Mycoplasma* contamination (CellSafe, Yongin-si, Korea).

#### *4.5. Flow Cytometry*

To determine the expression levels of HLA-A\*02:01, cells (2.0 × <sup>10</sup><sup>5</sup> cells/mL) were incubated for 30 min with a PE-conjugated mouse anti-HLA-A2 monoclonal Ab (cat. # sc-32236 PE, Santa Cruz Biotechnology, diluted 1:100). After washing with 1 mL ice-cold PBS, cells were analyzed on a FACSCalibur flow cytometer (Becton-Dickinson, Franklin lakes, New Jersey, USA). All staining procedures were performed at 4◦C.

To detect pMHC on cell surfaces, cells (3.0 × <sup>10</sup><sup>5</sup> cells/mL) were pulsed with the vehicle, CMVpp65495-503, or HPVE711-19 peptide at the indicated concentration for 3 h at 37 ◦C, washed with fluorescence-activated cell sorting (FACS) buffer (1% FBS in PBS, pH 7.4), and resuspended at 1.5 × <sup>10</sup><sup>5</sup> cells/sample. All staining procedures were performed at 4 ◦C. Cells were incubated for 1 h with the TCR-like Ab at the indicated concentration, washed with 1 mL FACS buffer, and incubated with an Alexa Fluor 647-conjugated goat anti-mouse IgG-specific F(ab')2 polyclonal Ab (cat. # 115-606-008, Jackson ImmunoResearch, diluted 1:600) for 30 min. After washing with 1 mL ice-cold PBS, cells were analyzed on the FACSCalibur flow cytometer. Data were analyzed using FlowJo V10 software (Tree Star).

#### *4.6. Affinity Maturation of Abs*

The yeast strains and media compositions have been previously described in detail [34,35]. Library generation of Abs by complementarity-determining region (CDR) mutagenesis was performed in the scFab format involving a G4S-based 63-amino-acid linker between VL and VH, using YSD technology as described previously [17]. The yeast library was screened using magnetically activated cell sorting (MACS) and an FACS Aria III instrument (BD Biosciences) against biotinylated CMVpp65495-503/HLA-A\*02:01 SCT protein (with a gradual decrease in concentration from 2 μM to 0.4 nM) in the presence of a 10-fold higher concentration of non-biotinylated HPVE711-19/HLA-A\*02:01 SCT protein as a competitor, as specified in the text. In FACS, cell surface expression and antigen binding levels of the scFab library were monitored by indirect double immunofluorescence labeling of the CH1 C-terminal c-myc tag (anti-c-myc mouse Ab [9E10], diluted 1:100) with an Alexa 488-labeled goat anti-mouse IgG Ab (Invitrogen, diluted 1:600) and streptavidin-conjugated R-phycoerythrin (Invitrogen, diluted 1:600). Typically, the top 0.1–0.2% of target-binding cells were sorted. The final sorted yeast cells were plated on a selective medium, and individual clones were isolated and further analyzed. DNA from the screened yeast cells was recovered using a Zymoprep kit (Zymo Research, CA, USA) as previously described [34,35].

#### *4.7. Biolayer Interferometry*

Kinetic binding interactions of TCR-like Abs with CMVpp65495-503/HLA-A\*02:01 SCT protein were monitored at pH 7.4 using an Octet QKe System (ForteBio, California, USA), as described previously [17,35]. All data were globally fitted via the 1:1 Langmuir binding model, and association and dissociation rate constants were calculated using Octet Data Analysis Software, version 11.0 (ForteBio, Fremont, CA, USA).

#### **5. Patents**

Patents resulting from the work reported in this manuscript have been filed in the Republic of Korea (Application number: KR 10-2020-0138273) and PCT (application number: PCT/KR2020/017067).

**Supplementary Materials:** The following are available online at https://www.mdpi.com/1422-006 7/22/5/2349/s1.

**Author Contributions:** Conceptualization, Y.-S.K. and S.-Y.L.; methodology, S.-Y.L. and J.-A.K.; validation, D.-H.K. and M.-J.S.; investigation, S.-Y.L., D.-H.K., M.-J.S., J.-A.K., and K.J.; writing—original draft preparation, S.-Y.L. and Y.-S.K.; writing—review and editing, K.J. and Y.-S.K.; supervision, Y.-S.K.; project administration, Y.-S.K.; funding acquisition, Y.-S.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Samsung Future Technology Center (grant number SRFC-MA1802-09).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All data in this study are available within the article or from the authors on request.

**Conflicts of Interest:** Y.S.K. and S.Y.L. are listed as inventors on the patent application (KR 10-2020- 0138273; PCT/KR2020/017067) related to the technology described in this work. The other authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**

