Development of an artificial antibody specific for HLA/peptide complex derived from cancer stem-like cell/cancer-initiating cell antigen DNAJB8

Background Peptide-vaccination therapy targeting tumour-associated antigens can elicit immune responses, but cannot be used to eliminate large tumour burden. In this study, we developed a therapeutic single-chain variable-fragment (scFv) antibody that recognises the cancer stem-like cell/cancer-initiating cell (CSC/CIC) antigen, DNAJB8. Methods We screened scFv clones reacting with HLA-A24:20/DNAJB8-derived peptide (DNAJB8_143) complex using naive scFv phage-display libraries. Reactivity and affinity of scFv clones against the cognate antigen were quantified using FACS and surface plasmon resonance. Candidate scFv clones were engineered to human IgG1 (hIgG1) and T-cell-engaging bispecific antibody (CD3xJB8). Complement-dependent cytotoxicity (CDC) and bispecific antibody-dependent cellular cytotoxicity (BADCC) were assessed. Results scFv clones A10 and B10 were isolated after bio-panning. Both A10-hIgG1 and B10-hIgG1 reacted with DNAJB8-143 peptide-pulsed antigen-presenting cells and HLA-A24(+)/DNAJB8(+) renal cell carcinoma and osteosarcoma cell lines. A10-hIgG1 and B10-hIgG1 showed strong affinity with the cognate HLA/peptide complex (KD = 2.96 × 10−9 M and 5.04 × 10−9 M, respectively). A10-hIgG1 and B10-hIgG1 showed CDC against HLA-A24(+)/DNAJB8(+) cell lines. B10-(CD3xJB8) showed superior BADCC to A10-(CD3xJB8). Conclusion We isolated artificial scFv antibodies reactive to CSC/CIC antigen DNAJB8-derived peptide naturally present on renal cell carcinoma and sarcoma. Immunotherapy using these engineered antibodies could be promising.


BACKGROUND
The first immunotherapy against cancer was performed using Coley's Toxins developed by William B. Coley in the nineteenth century. 1 He vaccinated sarcoma patients with live or inactivated bacteria. Various vaccination therapies using tumour-associated antigen (TAA)-derived peptides, autologous tumour lysates and tumour lysate-loaded dendritic cells have been developed to date. 2 We conducted peptide-vaccination trials using TAAderived peptides of SYT-SSX junctional peptides derived from specific chromosomal translocation for synovial sarcoma, papillomavirus-binding factor (PBF) for osteosarcoma and survivin for various carcinomas. 3,4 Although immune responses against the vaccinated peptides were frequently observed, the objective clinical response rates were low.
In contrast, cell-based immunotherapy using T cells exogenously expressing T-cell receptor (TCR) directed to TAA (TCR-T), especially for NY-ESO-I, showed clinical responses against melanoma and synovial sarcoma. 5 Anti-CD19 chimeric antigen receptor-expressing T cells (CAR-T) also showed dramatic responses in B-cell haematological malignancies. 6 However, CD19 CAR-T cells can destroy not only neoplastic B cells but also normal B cells. Target antigens are still restricted, and new antigens might be required for the development of immunotherapy for various cancers.
Considering the current status, we focused on (i) the identification of new TAAs that were expressed in cancer stem-like cells/ cancer-initiating cells (CSCs/CICs) but not in normal organs that also regulate tumour-initiating ability, and (ii) the development of antibodies directed to CSC/CIC antigen using antibody engineering. 3,7 Recently, various therapeutic antibodies targeting tumour antigens expressed on tumour cell surfaces have been developed. In particular, anti-CD20 monoclonal antibody rituximab and anti-CD19 T-cell-engaging bispecific antibody blinatumomab showed objective responses in B-cell haematological neoplasia. 8,9 In this study, we developed a therapeutic single-chain variablefragment (scFv) antibody recognising the HLA-A24/peptide derived from DNAJB8 complex as a prototype of CSC/CIC antigen. 10,11 Then, we engineered the scFv to bivalent and bispecific formats to assess its in vitro cytotoxic capacity in renal cell carcinoma and sarcoma.

METHODS
This study was performed in accordance with the guidelines established by the Declaration of Helsinki, and was approved by the Ethics Committee of Sapporo Medical University. The healthy donors provided informed consent for the use of blood samples in our research.
Bio-panning with biotinylated antigen Bio-panning was performed according to our previous report with some modifications. 18 Biotinylated HLA--A24:02/DNAJB8_143 peptide and HLA-A24:02/HIV peptide complexes were used as antigens. The scFv phage-display libraries constructed by our laboratory (Library #1) and provided by Daiichi-Sankyo Co., Ltd. (Library #2) were used for Experiment #1 and Experiment #2, respectively. The phage library (0.25 mL) was mixed with PBS (0.25 mL) containing 4% (w/v) milk in a 1.5-mL tube and incubated with 100 μL of magnetic beads (Dynabeads M-280 Streptavidin, Life Technologies, Carlsbad, CA), which were prewashed with PBS with 0.1% Tween 20 (PBST). The magnetic beads (100 μL) were prewashed and blocked with 2% PBS-M for 1-2 h at room temperature. The phage supernatant was mixed with 0.5 mL of biotinylated HLA-A24:02/HIV peptide complex (1000 nM) and incubated for 60 min (negative panning). After incubation, the phage-antigen mixture was mixed with the magnetic beads for 15 min. The resultant phage supernatant (500 μL) was mixed with 0.5 mL of biotinylated HLA-A24:02/DNAJB8_143 peptide complex (500 nM in the first round and 100 nM in the second and subsequent rounds) and mixed for 60 min (positive panning). After incubation, the phage-antigen mixture was mixed with the magnetic beads, followed by additional incubation for 15 min. Specific phage binders were eluted from the magnetic beads by incubation with 1 mL of 100 mM triethylamine for 7 min. The eluted phage aliquot was immediately neutralised with 100 μL of 1 M Tris-HCl, pH 7.4. The resultant phage aliquot was used for phage rescue.
Phage rescue was performed as follows. Half of the phage aliquot after bio-panning was added to 10 mL of log-phage TG1 or XL1 blue and incubated at 37°C for 1 h with slow shaking. After incubation, ampicillin (final concentration 100 µg/mL), glucose and helper phage (M13K07 or VCM13) were added and incubated for 60 min. Then, 15 mL of fresh 2xYT containing ampicillin (100 μg/mL) and kanamycin (25 μg/mL) and 1% glucose was added to cultured E. coli infected with the phage and helper phage, followed by incubation at 26°C overnight. Overnight culture of bacteria, including the proliferated phage, was isolated using polyethylene glycol precipitation and used for the next round of bio-panning.
After bio-panning, soluble scFv expression of E. coli infected with the phage was induced in a microplate. The phage aliquot after bio-panning (400 μL) was added to 10 mL of log-phage E. coli and incubated at 37°C for 1 h with slow shaking. Following incubation, the E. coli aliquot was seeded on a 2xYTAG agar plate and incubated at 37°C overnight. The next day, 94 clones were picked up and inoculated independently into wells containing 100 μL of 2xYTAG in a 96-well microculture plate. The plate was incubated at 37°C with shaking for 5 h. After incubation, 20 μL of 2xYTAG with 3 mM isopropyl-β-D(−)-thiogalactopyranoside (IPTG) was added and incubated at 28°C overnight. Then, 50 μL of the supernatant was harvested and immediately used for ELISA screening according to the previous report. 18 Generation of scFv-hIgG and bispecific antibody scFv cDNA in phagemid vector was subcloned into pFX-hIgG1 for scFv-hIgG1 expression previously constructed by our laboratory. 18 For soluble expression of scFv-hIgG1, 4 μg of the plasmid was transfected using Lipofectamine 2000 (Life Technologies) into 293 T cells precultured on a 10-cm culture dish in DMEM supplemented with 10% FBS. After 4-5 h, the culture medium was replaced with fresh AIM-V (Life Technologies) without serum. The supernatant was harvested and replaced with fresh AIM-V at 24, 48 and 72 h after transfection. The collected supernatant was passed through a chromatography column with Protein G. The column was washed with 20 mM sodium phosphate (pH 7.0) and eluted by fraction (1 mL per fraction) with a total of 5 mL of 0.1 M glycine (pH 2.7), followed by immediate neutralisation with 1/10 volume of Tris-HCl (pH 9.0). Fractions containing antibodies were assessed by SDS-PAGE with or without DDT to confirm that oxidised scFv-hIgG formed a dimer protein.
Surface plasmon resonance analysis Surface plasmon resonance analysis was performed using a ProteOn XPR36 (Bio-Rad Laboratories, Inc., Tokyo, Japan) according to the protocol described by Nahshol et al. 20 Briefly, 1 μg/mL    Statistical analysis For comparisons, we used the unpaired t test in JMP software (SAS Institute Inc., Tokyo, Japan). Where relevant, figures indicate statistical parameters, including the value of n, means ± SD and statistical significance.
Next, the specificity of A10 scFv-hIgG1 and B10 scFv-hIgG1 was assessed using amino acid substitution peptides. The reactivities of the antibodies to amino acid substitution peptide-pulsed T2-A24 cells were determined using flow cytometry. The results showed that position 3 (P3) amino acid, P6 and P7 were critical for recognition (Fig. S2). P2 and P9 might be critical for binding HLA-A24 molecules as anchor amino acids. Considering the importance of the middle part of DNAJB8_143 peptide for specific recognition, A10 scFv and B10 scFv might react specifically with the cognate HLA/peptide complex similar to TCR.
A10 scFv-hIgG1 and B10 scFv-hIgG1 induced complementdependent cytotoxicity Next, we investigated the capacity of the scFv-hIgG1 clones to induce complement-dependent cytotoxicity (CDC). CDC-induced cells were detected by complement C3b positivity on cell membranes and DAPI positivity in nuclei, which was the result of membrane permeability induced by the classical pathway of     CDC. We observed that each of the scFv-hIgG1 clones induced CDC in HLA-A24(+)/DNAJB8(+) renal cell carcinoma cell line CAKI-1 and osteosarcoma cell line HOS-A24 (Fig. 3a, b). The proportion of CDC-induced cells was determined using flow cytometry. Similar to the observation by fluorescence microscopy, we also noted that each scFv-hIgG1 mediated CDC activity on CAKI-1 (5.72% and 29.12%, respectively) and HOS-A24 (20.89% and 18.11%, respectively) by A10 scFv-hIgG1 and B10 scFv-hIgG1 (Fig. 3c).

DISCUSSION
In this study, we (i) isolated specific scFv clones reacting to the HLA-A24-restricted CSC/CIC antigen DNAJB8-derived peptide, and found that (ii) A10 scFv and B10 scFv showed high specificity and strong affinity for the cognate antigen, (iii) bivalent A10 scFv-hIgG1 and B10 scFv-hIgG1 could induce CDC to HLA-24 (+)/DNAJB8(+) carcinoma and sarcoma cell lines and (iv) T-cellengaging bispecific antibody B10-(CD3xJB8) induced T-cellmediated cellular cytotoxicity against HLA-A24(+)/DNAKB8(+) carcinoma and sarcoma cell lines. These results suggested that such artificial antibodies might be of benefit for antibody-based therapy in patients with carcinoma and sarcoma.
To date, many monoclonal antibodies have been approved and yielded clinical response in haematological malignancies and some solid tumours. However, acquired resistance of tumour cells can occur in monoclonal antibody therapy. 22 In addition, loss of target antigens has also occurred. 23 Therefore, new targets and formats of antibodies might be promising for clinical applications. DNAJB8 was previously reported as a CSC/CIC antigen expressed in RCC. Transduction of DNAJB8 increased tumorigenicity, while knockdown of DNAJB8 decreased tumorigenicity in ACHN in vitro and in vivo. 10 Therefore, the antibody targeting DNAJB8-positive tumour cells might be an attractive candidate.
Recently, T-cell-engaging bispecific antibodies have been used particularly in haematological B-cell malignancies. 24 Blinatumomab (CD3xCD19) showed 43% complete response (CR) and CR with partial haematological recovery (CRh) in patients with Philadelphia-chromosome-negative, primary refractory or relapsed leukaemia. 25 T cells were activated by CD3 scFv only when they were engaged with CD19-expressing cells via blinatumomab. 24 Clinical trials of various T-cell-engaging bispecific antibodies, including carcinoembryonic antigen (CEA) × CD3 for gastric cancer, epithelial cell adhesion molecule (EpCAM) × CD3 for epithelial cancer, B-cell maturation antigen (BCMA) × CD3 for multiple myeloma and prostate-specific membrane antigen (PSMA) × CD3 for prostate cancer, are currently being conducted. Most bispecific antibodies target cell surface proteins overexpressed on tumour cells; however, the challenge to develop bispecific antibodies directed to intracellular antigen-derived peptides presented by HLA class I remains. Dao et al. reported the potential efficacy of bispecific antibody directed to WT1derived peptide in the context of HLA-A2. 26 Ahmed et al. reported the TCR-like bispecific antibody targeting EBV LMP2A-derived peptide in the context of HLA-A2. 27 Such bispecific antibodies with TCR specificity might be promising for targeting various tumour-specific intracellular proteins with oncogenic functions. DNAJB8 is a cancer stem-cell/cancer-initiating antigen and not expressed in normal organs, except for testis lacking HLA expression like cancer-testis antigen. Hence, the bispecific antibody targeting DNAJB8, B10-(CD3xJB8), might have therapeutic potential without adverse effects on normal organs.
B10-(CD3xJB8) showed cytotoxicity against HLA-A24 (+)/DNAJB8(+) tumour cells; however, the frequencies of IL-2 and IFNγ-secreting cells were lower than those of TNFα. We previously observed that the mRNA expression of TNFα was detectable but not that of IL-2 and IFNγ in naive and stem-cell memory T cells. 28 Therefore, these observations suggested that B10-(CD3xJB8) might activate T cells close to naive subsets. Further studies using in vivo experiments might be required to prove the efficacy of B10-(CD3xJB8) in the preclinical setting.
In conclusion, we developed scFv clones specific for HLA-A24/ cancer stem-like antigen DNAJB8-derived peptide with high specificity and strong affinity. The T-cell-engaging bispecific antibody targeting DNAJB8, B10-(CD3xJB8), might hold promise for antibody-based therapy in patients with carcinoma and bone sarcoma.