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Cancer is the second leading cause of death globally. To treat various types of cancers, several treatments including ‘chemotherapy’, to destroy rapidly growing cancer cells, ‘surgical procedure’, to remove the cancer tissues physically, ‘radiation therapy’, to deliver high doses of radiation on cancer cells, and ‘immunotherapy’, to invigorate our body’s anti-cancer immunity have been developed (Waldman
In recent years, ‘chimeric antigen receptor T (CAR-T) cell’ therapy has been one of the most actively studied treatments for a variety of cancers. In CAR-T cell therapy, T cells from a patient are harvested and genetically engineered to express a chimeric antigen receptor which can recognize specific tumor-associated antigens such as CD19 and B-cell maturation antigen (BCMA). Then these genetically engineered T cells are infused back into the patient (Teoh and Chng, 2021). In clinical studies involving CAR-T cell therapies, a 72-83% overall response rate against refractory or relapsed B cell acute lymphoblastic leukemia (Zheng
Programmed death-ligand 1 (PD-L1, a transmembrane protein also referred to as CD274 or B7 homolog 1) is an immune checkpoint molecule that causes the generation of inhibitory signals by binding with PD-1 on the counterpart cells (Qin
In search for novel and efficient anti-cancer immunotherapies, bispecific antibodies with anti-PD-L1 binding component and diverse binding partners are under development in several studies. They include PD-L1xCD3, PD-L1x4-1BB (CD137) and PD-L1xLAG-3, etc. (Horn
Therefore, in our current study, we generated αCD3xαPD-L1 bispecific antibody (BiTE) and tested the T cells and CAR-T cells tethered with BiTE for the cancer-targeting specificity and anti-cancer efficacy, respectively. We observed that such bispecific antibody-bound T cells (BiTE:T cells) showed a higher tumor killing activity
Murine colon adenocarcinoma cell line CT26 was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Ovalbumin (OVA)-transfected B16/F10 melanoma (MO5) was kindly provided by Dr. Kenneth Rock (University of Massachusetts, Worcester, MA, USA) and human HER-2/neu-expressing CT26 colon carcinoma (Her-2/CT26) was developed by transduction of CT26 using a retroviral vector system (Chung
The single chain fragment variable (scFv) sequence encoding ‘Her-2/neu (ErbB2)’-targeting ML39 was synthesized based on the cDNA sequence from the patent (PCT/US2007/024287). It was used to generate ErbB2-targeting CAR construct, which contains the mouse CD8α signal sequence followed by the ML39 scFv linked to the hinge domain of the CD8α molecule and intracellular signaling domains of the CD28 and CD3zeta molecules. Mock lentivirus vector was used to construct CAR-T for the negative control. The fragments were subcloned into the plasmid MSCV-IRES-Thy1.1 DEST (pMIT) vector which was purchased from Addgene (Watertown, MA, USA). High titer replication defective lentiviral vectors were produced and concentrated before use.
Negative selection using the CD8α+ T Cell Isolation Kit, mouse (MACS, Bergisch Gladbach, Germany) was applied to isolate the primary mouse T cells from spleen (or blood samples) from BALB/c or C57BL/6 mice. For the preparation of activated T cells, mouse T cells were incubated with 4 μg/mL concentration of anti-CD3 antibody (Biolegend, San Diego, CA, USA) and 2 μg/mL for anti-CD28 antibody (Biolegend). T cells were cultured in RPMI 1640 medium supplemented with GlutaMAXTM-I (Invitrogen) and 10% fetal bovine serum (Gibco, Carlsbad, CA, USA), 10 mM HEPES buffer, 100 μg/mL of Pen/Strep, 100 μg/mL of gentamycin, and 50 μM mercaptoethanol (all from Invitrogen). The end of stimulation was determined based on the downward shifts of the peaks from ‘CellTraceTM Violet’ (CTV) staining (Invitrogen), which usually took 2 days after stimulation.
All variable domains of the antibodies have been previously patented or published. Variable domains of anti-PD-L1 antibody were obtained by phage display as previously described (Choi
Expression construct was made with the DNA of αCD3xαPD-L1 BiTE followed by a His6 tag. DNA was transiently transfected into FreeStyleTM 293-F Cells (Gibco) by FectoPRO (Polyplus, Illkirch-Graffenstaden, France) following the manufacturer’s instructions. After 5 days of culture, supernatants from the transfected cells were purified by open-column chromatography using Ni-NTA agarose (Qiagen, MD, USA). Elution fractions were collected and dialyzed against PBS (pH 7.2). Protein concentration was determined using NanoDrop ND-2000 spectrophotometer (Thermo Scientific, MA, USA) based on absorption at 280 nm. Purity of protein was detected using SDS-PAGE and Coomassie brilliant blue staining.
Target cells and T cells were seeded in a 96-well plate (104 cells/well); the cells were treated with 0.1 μg/mL BiTE and incubated for 24 h at 37°C and 5% CO2. After the incubation, 10 μL of cell counting kit 8 solution (Cell Counting Kit-8, Dojindo Co., Kumamoto, Japan) was added to each well and incubated for an additional 2 h. The absorbance at 450 nm was measured by a SpectraMax i3 microplate reader (Molecular Devices, San Jose, CA, USA).
Six weeks old female mice including BALB/c and C57BL/6 were purchased from KOATECH (Pyeongtaek, Korea). The mice were maintained under specific pathogen-free conditions in the experimental facilities at Kangwon National University (Chuncheon, Korea). All the animal experiments were performed according to the approved guidelines of the Institutional Animal Care and Use committee of Kangwon National University (KW-201007-1). To establish a mouse tumor model, seven weeks old mice were challenged with 2×106 MO5 cells or Her-2/CT26 cells subcutaneously. Tumor length, height, and width were measured using calipers and the tumor volume was calculated as 1/6π×length (mm)×height (mm)×width (mm).
Tumor tissues were harvested and minced using sterile razor blades. The tumor pieces were digested using an enzyme mixture containing 400 U/mL collagenase type IV purchased from Worthington Biochemical Corporation (Lakewood, NJ, USA) and 0.02 mg/mL DNase I purchased from Sigma Aldrich (St. Louis, MO, USA) in RPMI 1640 and incubated at 37°C for 45 min and passed through 70 μm cell strainers (BD Bioscience). A Percoll gradient from GE Healthcare (Chicago, IL, USA) was then used to separate cancer cells and enrich lymphocytes.
Statistical analyses were performed using Graphpad Prism 9 (GraphPad Software, LLC, San Diego, CA, USA). Unpaired two-tailed Student’s t-tests were used when the data had a Gaussian distribution with similar variances. One-way analysis of variance (ANOVA) was used to compare more than two groups followed by post hoc tests (Bonferroni test). The threshold for statistical significance was
A bispecific T-cell engager molecule, a form of bispecific antibody, was generated with two tandemly linked single chain Fvs (scFvs) (Fig. 1A), with the scFvs originated from anti-CD3ε antibody (145-2C11) and anti-PD-L1 antibody (KL001-13). To test the functionality of this bispecific T-cell engager molecule on the activation of T cells, we attempted to determine the minimal concentration of BiTE molecule which can induce T cell proliferation. Incubation of BiTE molecules (0.01, 0.1 µg/mL concentration) with T cells for 1 h could not achieve proliferative activation of naïve CD8 T cells. However, after 48 h of incubation, CD8 T cells acquired sufficient proliferative activation, which was shown as substantial dye dilution profile for CTV-labeled CD8 T cells (Fig. 1B). This data suggests that T cells can be incorporated with BiTE molecules without strong proliferative activation during a short period of incubation (~1 h). However, T cells can still retain their functional activation after a relatively long period of incubation (~48 h) with a certain concentration of BiTE molecules.
The T cells are usually co-incubated with the beads coated with anti-CD3/CD28 antibodies or soluble anti-CD3 antibody with IL-2 to achieve the required activation during the manufacturing process of therapeutic CAR-T cells (Li and Kurlander, 2010; Zhang
To verify that BiTE molecule can exert cancer killing activities through bridging between cancer cells and T cells, mouse CD3 (mCD3)xPD-L1-targeting BiTE was loaded on to polyclonal CD8 T cells that were isolated from splenocytes of C57BL/6 mice. Briefly, isolated T cells were activated using anti-CD3/CD28 antibodies for 48 h. Then, those CD8 T cells were incubated with 0.1 µg/mL of mCD3xPD-L1-targeting BiTE for 2 h. After washing out non-bound BiTE, mCD3xPD-L1 BiTE:T cells were obtained and co-cultured with MO5 cells, which highly express PD-L1 on their surface, for 16 h. The effector cells were seeded with the represented ratio (Target cell : Effector cell). The cancer killing activity of BiTE:T cells was determined by CCK8 activities of MO5 cells (Fig. 1D). mCD3xPD-L1 BiTE:T cells significantly suppressed CCK8 activity more than when MO5 co-cultured with plain T cells (Fig. 1D). These results suggest that BiTE:T cells can effectively target and kill cancer cells expressing tumor antigen on their surface
To ascertain whether the BiTE-loaded CD8 T cells can exert anti-cancer function as CAR-T cell does
Through the experiment with B16.MO5 cells and OT-1 cells in C57BL/6 mouse model, we confirmed that mCD3/PD-L1 BiTE molecule can induce enhanced tumor-growth inhibition of OT-1 T cells compared to the matched tumor cells treated with plain OT-1 T cells. Further, we extended the utility of BiTE-bound T cells from antigen-specific OT-1 T cells to polyclonal CD8 T cells. To evaluate the anti-tumor activity of CD8 T cells bound with mCD3/PD-L1 BiTE, 2×105 of mCD3/PD-L1 BiTE:T cells were intravenously administered to MO5-bearing mice on the 10th day after initial tumor cell graft (Fig. 3A). Introduced mCD3xPD-L1 BiTE:T cells significantly suppressed tumor growth compared to both 2 μg/kg of BiTE intraperitoneal injection and plain T cell treatment (Fig. 3B). Additionally, analysis of T cell population in tumor showed that effector memory CD8+ T cells were increased compared to the control. However, central memory CD8+ T cells were decreased (Fig. 3C), suggesting an enhanced ratio of effector memory to central memory T cells. This result is consistent with the reports of previous studies suggesting that the increased effector memory to central memory T cells ratio in tumor serves as a predictive biomarker of enhanced anticancer immune response against several cancers (Liu
To extend the application of BiTE-bound T cells to CAR-T cell system, we generated BiTE-bound CAR-T cells (BiTE:ML39 CAR-T) by incubation of ML39 CAR-T cells expressing anti-human Her2/neu (ErbB2) scFv with 0.1 µg/mL of BiTE for 1 h (Fig. 4A). To evaluate the activity of BiTE:ML39 CAR-T, we adopted human Her2/neu-expression CT26 colon cancer transplantation model (Ko
Bispecific antibody and CAR-T cell therapies have been successful therapeutic options for several hematological malignancies. These have been recent breakthroughs in the research of oncology and immune-checkpoint inhibitory agents (Edeline
To achieve highly efficacious anti-tumor activities and devise simpler, versatile T cell-based therapeutic agents, we have investigated bispecific antibody-bound T cells in the murine tumor models. When mCD3xPD-L1-targeting BiTE was bound to OT-1 T cells and applied to B16.MO5-bearing mice, it showed higher tumor growth inhibition than treatment with OT-1 cells alone. This shows that BiTE binding on T cells augments the tumor-killing activities of OT-1 T cells through BiTE-mediated T cell receptor (TCR) activation, additional to the intrinsic activation through direct OT-1 T cell and OVA:MHC-I interaction.
To extend the utility of BiTE-bound T cells, we also tested BiTE binding to polyclonal CD8 T cells, which are composed of T cells with diverse TCR repertoire. Introduced mCD3xPD-L1 BiTE:T cells significantly suppressed tumor growth compared to both 2 μg/mL of BiTE or plain T cell treatments. This further confirmed that BiTE-mediated TCR activation on diverse T cell population is enough to suppress the tumor growth. Enhanced effector memory T cells in tumor also indicated that mCD3xPD-L1 BiTE can direct polyclonal CD8 T cells to tumor and inhibit the tumor growth regardless of their TCR specificities towards tumor antigens.
When BiTE-bound CAR-T cells were applied to the human Her2/neu-expressing CT26 murine colon tumor model, similar to the BiTE-bound OT-1 cell activation, BiTE-bound CAR-T cells also showed an enhanced tumor killing activity compared to that from sole CAR construct. This result reiterated the advantage of using BiTE-bound CAR-T cells against tumor cells.
The BiTE molecule, which is composed of the tumor antigen binding scFvs and anti-CD3 binding scFv, has T cell recruiting activity towards tumor sites when it is administered intravenously to the patients. One such molecule, blinatumomab, which targets B cells in acute lymphoblastic leukemia, showed strong tumor killing activities and got US FDA approval in 2014 (Franquiz and Short, 2020). However, owing to its small size and the associated short half-life in blood circulation, it needs to be administered as continuous infusion for almost 4 weeks either in hospital setting or at home during the first treatment cycle, which has serious patient compliance issues. In comparison with blinatumomab and CAR-T cell therapy, BiTE-bound T cell or CAR-T cell therapy has several advantages. As BiTE molecules are incubated with T cells and these T cells are infused back to patient’s blood stream, BiTE-bound T cell strategy does not need continuous infusion similar to blinatumomab and shows better tumor killing activities. This could result in highly improved patient compliance and enhanced anti-tumor responses. Additionally, in contrast to the complicated process of CAR-T cell manufacture using lentiviral transduction of CAR construct into activated T cells from leukapheresis of patient’s blood, the procedure of BiTE binding on the peripheral blood mononuclear cells (PBMC) is much simpler. BiTE-bound T cells can be manufactured using a low-cost process; however, further molecular and process optimization are needed. Furthermore, the strategy of applying BiTE-bound T cells is highly versatile. Therefore, several BiTE molecules can be incorporated into the patient’s PBMC to cope with the heterogeneous nature of tumor cells. This strategy has the potential to apply combinatory BiTE molecules on patient’s autologous T cells simultaneously or in sequential application of different BiTE molecules during each treatment cycle.
Interestingly enough, our experiments on BiTE-bound CAR-T cells also showed enhanced tumor killing activities
Because pharmacokinetics is an important aspect in the development of any therapeutic agent, it will be of great interest to investigate the pharmacokinetic properties of BiTE-bound T cells. As shown
In summary, our study using bispecific antibody-bound T cells (BiTE:T cells) showed highly efficacious tumor killing activities
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No.2020R1A5A8019180).
This research was supported by the Basic Science Research Program of the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT, and Future Planning (NRF-2017M3A9C8060390).