Background

Chimeric antigen receptor (CAR) T cells are a type of adoptive cellular therapy that redirects T cells’ specific functions toward target cells using a synthetic receptor.1 2 This therapy has provided an impressive outcome in hematological malignancies and has been approved by the Food and Drug Administration, including CD19-targeted and BCMA-targeted CARs.3 4 However, the clinical outcome of CAR T cell therapy in solid tumors remains limited due to the suppressive tumor microenvironment and the heterogeneity of target surface antigens.1 5 6

B7 homolog 3 (B7-H3) or CD276 is an immunoregulatory ligand in the B7 family. In humans, it comprises two isoforms that consist of either one or two identical pairs of immunoglobulin variable (IgV)-like and immunoglobulin constant (IgC)-like domains (2Ig-B7-H3 and 4Ig-B7-H3, respectively).7 8 Initially, B7-H3 was reported as a costimulatory molecule for T cell activation in development of allograft rejection.9 As evidence accumulates, however, this molecule has been shown to exhibit a positive correlation with tumor progression and metastasis; it was found to be exclusively expressed at a high level across various types of cancers.10–13 It is also identified as a detection marker for micro-metastatic tumor cells.14 Upregulation of B7-H3 is associated with a decrease in immune cell infiltration13 and downregulation of lymphocyte responses, especially NK and CD8+ T cells.15 16 This makes B7-H3 a potential target for immunotherapy, including CAR T cell therapy.

Preclinical evaluation of CAR targeting B7-H3 T cells has been validated in a range of solid tumors,17–21 which has led to the translation of B7-H3 CAR T cells in clinical trials, including brain (NCT04185038, NCT05241392 and NCT05474378), ovarian (NCT04670068) and liver cancers (NCT05323201), emphasizing the potential of B7-H3 as a target molecule for immunotherapy,

In this study, we have identified four specific clones of anti-B7-H3 monoclonal antibodies (mAbs) with defined binding kinetics. Using these novel antibodies, we have developed second generation B7-H3 CAR T cells. Our preclinical study demonstrates that these B7-H3-targeted CAR T cells effectively eliminated primary tumors and controlled rechallenge tumor xenografts. Additionally, we showed that the novel B7-H3 CAR T cells accumulate at the tumor site and expand following tumor rechallenge. These findings provide compelling evidence for potential clinical translation of our novel B7-H3 CAR T cells for the treatment of solid malignancies.

MethodsAnalysis of CD276 gene expression

The expression of B7-H3/CD276 across primary cancer samples of 695 patients and their adjacent normal tissues from TCGC, TARGET, and GTEx was downloaded from cBioPortal22 and UCSC Xena databases.23 An RNA-Seq data were transformed to the log2 of the transcript count per million (TPM); log2(TPM). A significant upregulation was defined at p value cut-off 0.05 by paired t-test.

Cell lines

NALM-6 (RRID:CVCL_0092), CaOV-3 (RRID:CVCL_0201), MDA-MB-231 (RRID:CVCL_0062), Capan-2 (RRID:CVCL_0026), HuCCT-1 (RRID:CVCL_0324), U87 (RRID:CVCL_0022), H4 (RRID:CVCL_1239) and LN229 (RRID:CVCL_0393) were purchased from American Type Culture Collection. Cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)/High glucose (Cytiva), Roswell Park Memorial Institute (RPMI)-1640 Medium (Cytiva) or McCoy’s medium (Cytiva) supplemented with 10% Fetal Bovine Serum (FBS) (Gibco), 1% GlutaMAX-I (Gibco) and 1% HyClone Penicillin-Streptomycin (Cytiva). Cells were cultured in a 37°C incubator with a humidified atmosphere of 5% CO2. Cell lines were routinely tested with MycoAlert Mycoplasma Detection kit (Lonza) and performed in all experiments with mycoplasma-free.

mAb generation

Anti-B7-H3 mAbs were generated as previously described.24 Briefly, BALB/c and ICR mice (Nomura Siam International) were immunized with human B7-H3 Fc (cat: Z03426; Genscript) that emulsified in AdjuLite Freund’s complete adjuvant (Pacific Immunology) every 4-week intervals for 16 weeks and a booster dose for an additional 3 months. Blood was collected from the facial vein, and mouse anti-human B7-H3-specific IgG titers were determined by ELISA. The mouse that provided the highest B7-H3-specific IgG titer was selected for final immunization. Four days later, the mouse was sacrificed, and the spleen was aseptically harvested for hybridoma generation. Splenocytes were isolated and fused with an SP2/o myeloma partner. Clonal selection was performed and screened for hybridoma clones producing B7-H3-specific IgG by ELISA. Selected hybridoma monoclones were cultured in serum-free medium for antibody production. Affinity-purified B7-H3 mAbs were derived from protein A column chromatography using the AKTA purification system. Surface plasmon resonance (SPR) was used to determine the binding kinetics of each mAb using Biacore T200, as previously described,25 in which B7-H3-His tagged protein (cat: NP_079516, R&D Systems) was used as an analyte. The equilibrium dissociation rate constant (K D) was calculated using the following formula:

Single chain variable fragment secretion and purification

Single chain variable fragments (scFvs) with a C-terminus 6 histidine tag (6 × His) were cloned into the pTwist CMV-WPRE(Neo) for scFv secretion using ExpiCHO-S cells according to the manufacturer’s instructions (Gibco). Filtered scFv was purified with HisTrap column. Briefly, 1 mL HisTrap Excel column (Cytiva) was equilibrated with binding buffer (20 mM sodium phosphate, 500 mM NaCl). 30 mL of culture supernatant was loaded onto the column at a flow rate of 1 mL/min. The column was washed with 20 mL of washing buffer (20 mM sodium phosphate, 500 mM NaCl, 30 mM Imidazole) to remove non-specifically bound proteins. Then, scFv-his was eluted using a linear gradient from 0% to 100% of washing buffer to eluting buffer (20 mM sodium phosphate, 500 mM NaCl, 500 mM Imidazole). An additional 5 mL of eluting buffer was applied to the column to ensure complete elution. Collected 15 fractions of 1 mL each during the elution process. The pooled fractions (4–15) were subjected to buffer exchange using a 10 kDa Amicon Ultra Centrifugal Filter (Merck) device to replace the elution buffer with PBS.

Isolation and activation of primary human T lymphocytes

Human peripheral blood mononuclear cells were isolated by gradient centrifugation using Ficoll-Paque Premium (Cytiva) and activated using T-Cell TransAct (Miltenyi Biotec) in 24-well plate. Activated T cells were cultured in complete TexMACS medium (Miltenyi Biotec) supplemented with 5% FBS and 50 U/mL human IL-2 (Miltenyi Biotec) for 3 days before use.

Generation of B7H3 CAR T cells

Gamma (γ)-retroviruses were generated by transfecting 293 T cells with the plasmid encoding the B7-H3-CAR constructs, the plasmid containing the MomLV gag-pol sequence, and the RD114 envelope plasmid using GeneJuice Transfection Reagent (Sigma-Aldrich) as previously described.26

For T cell transduction, non-tissue culture-treated 24-well plate were coated with 7 µL of RetroNectin (Takara Bio) in 1 mL PBS at 4°C overnight. After removal of the coating mixture, the retroviral supernatant of each CAR construct was added and centrifuged at 2000 × g for 90 min. The virus was gently discarded before seeding the well with 2 105 activated T cells in complete TexMAC medium supplemented with 10 ng/mL human IL-7 (Miltenyi Biotec) and 5 ng/mL human IL-15 (Miltenyi Biotec). The plates were centrifuged at 400 × g for 5 min, and the cells were left to expand in culture for experiments on days 7–12. The complete medium supplemented with 10 ng/mL human IL-7 (Miltenyi Biotec) and 5 ng/mL human IL-15 (Miltenyi Biotec) was changed every 2–3 days.

Flow cytometric staining

The following antibodies were used in this study; PE anti-mouse Ig (polyclonal, cat: 550589; BD Biosciences), 7-AAD (cat: 559 925BD; Biosciences), PerCP anti-human CD3 (clone UCHT1, cat: 560835; BD Biosciences), PE anti-human CD8 (clone SK1, cat: 340046; BD Biosciences), FITC mouse IgM, κ Isotype Control (clone G155228, cat: 564680; BD Biosciences), FITC anti-human CD45RA (clone HI100, cat: 304106; BioLegend), FITC anti-human CD4 (clone RPA-T4, cat: 564419; BD Biosciences), FITC anti-human TIM-3 (CD366) (clone 7D3, cat: 565568; BD Biosciences), VioBlue mouse IgG2b, κ Isotype Control (clone 27-35, cat: 562748; BD Biosciences), VioBlue anti-human CCR7 (clone 150503, cat: 560863; BD Biosciences), VioBlue anti-human LAG-3 (CD223) (clone T47-530, cat: 565720; BD Biosciences), VioGreen IgG2a, κ Isotype Control (clone MOPC-173, cat: 563483; BD Biosciences), VioGreen anti-human TIGIT (clone 741182, cat: 747482; BD Biosciences), PE-Cy7 Mouse IgG1 κ Isotype Control (clone MOPC-21, cat: 565573; BD Biosciences), PE-Cy7 anti-human CD279 (PD-1) (clone EH12.1, cat: 561272; BD Biosciences). APC anti-human CD19 (clone HIB19, cat: 555415; BD Biosciences) was used to analyze expression of tCD19. Human B7-H3 protein-Fc (cat: B73-H5253; Acro) and anti-human IgG, Fcγ fragment specific (polyclonal mouse IgG, cat: 109-136-170; Jackson ImmunoResearch) as secondary antibody were used for CAR detection. Flow cytometry was acquired using a MACSQuant Analyzer 10 Flow Cytometer (Miltenyi Biotec) and analyzed with FlowJo V.10.5.3.

xCELLigence Real-Time Cell Analysis

Cytotoxic activity was determined using xCELLigence Real-Time Cell Analysis (RTCA). Briefly, 50 µL of complete medium was prepared in a 96-well electronic microtiter plate (E-plate) for blanking the plates. The E-plate was placed into xCELLigence (ACEA BioSciences, SP model), and the background of the wells was measured before adding 100 µL of target cells. Then, additional cells were settled at room temperature for 30 min before re-engagement onto the xCELLigence analyzer and allowed to attach to the wells overnight. Complete medium from each well (50 µL) was discarded before 100 µL of effector cells was added to each condition. Co-culture cells were incubated at room temperature for 30 min before re-engagement onto the xCELLigence analyzer, and an impedance value was continuously recorded as the cell index every 30 min. The time point close to 48 hours of co-culture was used for data analysis using RTCA Software Pro. Each condition was duplicated in every donor. Using NALM-6 as a target, E-plate was precoated with IMT assay (anti-CD40) Tethering Kit (Agilent) according to the manufacturer’s instructions for 2 hours.

CFSE Cell proliferation assay

T cells were stained with CellTrace CFSE Cell Proliferation Kit (ThermoScientific) according to the manufacturer’s instructions. The labeled cells were cultured with 25 gy γ-irradiated LN229 cells at effector-to-target (E:T) ratio of 2:1 in complete medium supplemented with or without 50 U/mL human IL-2 (Miltenyi Biotec) for 6 days. Cells were then harvested and stained with 7AAD and APC anti-human CD3 (clone OKT3, cat: 317318; BioLegend), and analyzed by flow cytometry.

ELISA

To test the specificity of secreted scFv, MaxiSorp ELISA plates (ThermoScientific) were coated with human B7-H3-Fc protein (50 ng/well, 100 µL) in 1 × PBS and then incubated at 4°C overnight. Plates were washed three times with PBS containing Tween 0.05% (PBST). Purified scFv 500 ng/mL was added to the first well (200 µL/well), and then 2-fold serial dilution (10 points) was performed. The plate was incubated at 37°C for 1 hour. After the washing step, mouse anti-His-HRP (cat: 4603-05; SouthernBiotech) at 1:8000 in PBST was added to the plate (100 µL/well) and then incubated at 37°C for 1 hour, followed by washing. The SIGMAFAST OPD substrate solution (Sigma-Aldrich) was added to the plate at 100 µL/well and incubated in the dark at room temperature for 15 min. The reaction was stopped by adding 50 µL/well of 2 n H2SO4. The absorbance was read at 492 nm.

For cytokine measurement, effector cells were cocultured with tumor cells at a 1:1 ratio in a 24-well plate without exogenous cytokines. After 24 hours, the supernatant was collected, and IL-2, IFN-γ, and TNF-α levels were quantified in duplicate using specific ELISA kits (Invitrogen) following the manufacturer’s instructions.

Spheroid co-culture assay

LN229 GFP cells were seeded at a density 1 × 104 cells/well in 96-well ultralow plates and centrifuged 300 × g for 5 min before placed into an IncuCyte S3 (Sartorius). Two days later, effector cells were tagged with Incucyte Cytolight Rapid Red Dye (Sartorius) following the manufacturer’s instructions and 5 × 103 cells were added for co-culture. Total Green Object Integrated Intensity of spheroid was recorded every hour at 10 × magnification using the IncuCyte ZOOM software (Sartorius). Cytotoxicity was calculated using the following formula:

Animal study

All animal procedures were reviewed and approved by the Chulalongkorn University Animal Care and Use Committee, protocol No. 2 273 033. At 8 weeks old, NOD.Cg-Prkdcscid Il2rgtm1Wjl /SzJ (NSG) mice were s.c. grafted on right flank with 1×106 LN229-Luc cells in Matrigel Basement Membrane Matrix (Corning). Seven days later, the mice were treated intravenous with 5×106 T cells in PBS. Tumor growth was weekly monitoring by bioluminescent imaging using IVIS Spectrum In Vivo Imagine System (PerkinElmer) and expressed as total flux (photons/s). Body weight, tumor size, and the onset of Graft Versus Host Disease (GVHD) were monitored weekly. All efforts were made to minimize animal suffering. Mice were euthanized when they visibly showed signs of discomfort and/or tumor total flux reached 1×1010 photons/s and/or tumor volume reached 12.5 mm in diameter. Tumor volume was calculated using the following formula:

Tissue pathological examination

The formalin-fixed tissues were processed for histological sectioning by the Exclusive Veterinary Professional Lab Center (Bangkok, Thailand). A section (5 µm thickness) of the tissue obtained was mounted on glass slides to stain with H&E and CD3 (polyclonal, cat: 103A-76; Cell Marque). Thereafter, the histopathology slides were observed under an optical microscope by a veterinary pathologist to investigate CD3 positive in the tissues.

Statistical analysis

All statistical tests were performed using Prism V.8 (GraphPad). Comparisons were analyzed using one-way or two-way analysis of variance, followed by Tukey’s multiple comparison test to assess differences between groups or between each group and the indicated control. Survival determined from the time of tumor cell injection was analyzed by the Kaplan-Meier method, and differences in survival between groups were compared by the Gehan-Breslow-Wilcoxon test. Data are shown as the mean±SD. *p≤0.05; **p<0.01; ***p<0.001 as significant.

ResultsExpression of B7-H3 across multiple types of solid tumors

First, we conducted an RNA-seq analysis of B7-H3/CD276 transcription from TCGC, TARGET, and GTEx using paired tumor and adjacent normal tissues across 695 patients. We observed a significant upregulation of CD276 to normal tissues in 17 of 22 types of solid cancers including bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), cholangiocarcinoma (CHOL), colon adenocarcinoma (COAD), esophageal carcinoma (ESCA), head and neck squamous cell carcinoma, kidney cancer (KICH, KIRC and KIRP), liver hepatocellular carcinoma (LIHC), lung cancer (LUAD and LUSC), prostate adenocarcinoma (PRAD), rectum adenocarcinoma (READ), stomach adenocarcinoma (STAD), thyroid carcinoma (THCA) and thymoma (THYM) (figure 1A). Our RNA seq data align with previous analyses that also showed upregulation of B7-H3 expression across multiple tumors.27 Expression of B7-H3 as a tumor surface antigen was also screened on various cancer cell lines by flow cytometry to confirm the result of RNA-seq analysis. Using a leukemia cell line (NALM-6) as a negative control, our flow cytometry results showed high expression of B7-H3 on ovarian (CaOV-3), breast (MDA-MB-231), pancreatic adenocarcinoma (PAAD) (Capan-2), CHOL (HuCCT-1) and glioblastoma (GBM) (U87, H4 and LN229) cell lines (figure 1B). These findings supported the potential use of B7-H3 as a target surface antigen for common solid cancer treatments.

Figure 1

B7-H3 is overexpressed in various solid cancers. (A) B7-H3/CD276 transcription analysis of TCGC, TARGET, and GTEx RNA-Seq data derived from human primary cancers compared with normal tissue was investigated. The distributions of log2 transcript count per million (TPM) for each CD276 transcript across primary tumor and adjacent-normal sections of same patients were represented with individual paired dot plots. Paired t-test, *p<0.05, **p<0.01, ***p<0.001. (B) Tumor surface B7-H3 was analyzed throughflow cytometry. Positive detection of tumor surface B7-H3 is displayed on each histogram. The number represents the ratio of median fluorescence intensity (MFI) of anti-B7-H3 to that of isotype control. BRCA, breast invasive carcinoma, n=113; BLCA, bladder urothelial carcinoma, n19; CESC, cervical squamous cell carcinoma, n=3; CHOL, cholangiocarcinoma, n=9; COAD, colon adenocarcinoma n=26; ESCA, esophageal carcinoma, n=13; HNSC, head and neck squamous carcinoma, n=13; KICH, kidney chromophobe, n=25; KIRC, kidney renal clear cell carcinoma n=72; KIRP, kidney renal papillary cell carcinoma, n=32; LIHC, liver hepatocellular carcinoma, n=50; LUAD, lung adenocarcinoma, n=58; LUSC, lung squamous cell carcinomas, n=50; PAAD, pancreatic adenocarcinoma, n=4; PCPG, pheochromocytoma and paraganglioma, n=3; PRAD, prostate adenocarcinoma, n=52; READ, rectum adenocarcinoma, n=9; SARC, sarcoma, n=2; STAD, stomach adenocarcinoma, n=35; THCA, thyroid carcinoma n=59; THYM, thymoma, n=11; UCEC, uterine corpus endometrial carcinoma, n=7.

Characterization of novel mAbs and their derivative scFvs targeting hB7-H3

In order to target B7-H3, we generated novel anti-human B7-H3 mAbs by immunizing BALB/c and ICR mice with recombinant hB7-H3-Fc protein. Spleens of those that generated high hB7-H3-specific IgG titers were harvested for hybridoma generation, which were then screened to confirm their specificity to hB7-H3 protein by ELISA. Their binding kinetics were measured by SPR and equilibrium dissociation rate constants (K D) were derived. We identified 4 mAb clones against hB7-H3 with various affinities ranging from nanomolar to picomolar designated as G7A5, 11G5, 12G5 and A2H4 (figure 2A). To assess their specificity for cell surface B7-H3, we stained various B7-H3 positive cancer cells with these new mAbs. Non-specific binding to the B7-H3 negative NALM-6 cell line was observed with the clone G7A5. In contrast, clones 11G5, 12G5 and A2H4 specifically bound to surface B7-H3 on tumor cells, with the clone 12G5 showing the lowest binding (figure 2B). We next generated single-chain variable fragments (scFvs) from these three mAb clones, along with the previously published anti-human B7-H3 mAb clone 376.9617 28 (figure 2C) and tested for their specific binding to hB7-H3 protein using ELISA. As shown in figure 2D, the scFv derived from clone A2H4 demonstrated the highest affinity among all clones including the previously reported 376.96.

Figure 2

Characterization of high-affinity mAbs and scFvs targeting B7-H3. (A) Sensorgram of hB7-H3-His analyte with concentrations of 6.30, 12.60, 25.20, 50.40 and 100.80 nM over 2 µg/mL of immobilized anti-B7-H3 mAbs clone G7A5, 12G5, 11G5 and A2H4. ka, association rate; kd, dissociation rate; K D, equilibrium dissociation constant. (B) Expression of endogenous B7-H3 on various tumor cell lines detected by new anti-B7-H3 mAbs and PE goat anti-mouse IgG (H+L). (C) Representation of scFvs constructs in expression vector. Each scFv was fused to a human Ig kappa (IgK) leader sequence for secretion and 6 Histidine (6His) tag to purification and detection by ELISA. (D) Binding of purified scFvs-His derived from anti-B7-H3 mAbs 12G5 (scFv.12G5), 11G5 (scFv.11G5), A2H4 (scFv.A2H4) and a published clone (scFv.376.96) using 2-fold serial dilution to plate-coated human B7-H3-Fc. Data are shown as mean±SD, n, three independent experiments.

Development and characteristics of B7-H3 targeting CAR T cells derived from novel mAbs

Next, we generated second-generation CAR constructs derived from these scFvs. Each CAR construct comprised human IgG2-CH3-derived spacer linked to the 4-1BB and CD3ζ intracellular signaling domains. Truncated-CD19 (tCD19) was added as a detection marker for transduction efficiency (figure 3A). On day 4 post-transduction, the transduction efficiency of all CAR constructs was similar, as indicated by expression of tCD19 (figure 3B). We also detected the CAR expression by staining with hB7-H3 protein. All CAR constructs were efficiently detected by B7-H3 protein, although the clone 12G5 showed lower CAR expression compared with the other clones (figure 3C). The immunophenotypes of B7-H3 CAR T cells were analyzed 8 days after transduction. There were no differences in CD4+ to CD8+ T cell ratio between B7H3.CARs and non-transduced (NT) T cells. However, a higher percentage of CD8+T cell population was observed across all groups (figure 3D). All B7H3.CAR T cells exhibited phenotypic differentiation, showing an increase in central memory T cells (TCM) and a decrease in naïve (TN) T cells in both CD4+and CD8+ subsets when compared with NT cells. There were no significant differences in memory phenotype between each CAR construct (figure 3E,F). Background exhaustion markers were also examined. Compared with NT, B7H3.CARs T cells showed an upregulation of T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), but not programmed cell death protein 1 (PD-1), lymphocyte activation gene-3 (LAG-3), or T cell immunoreceptor with Ig and ITIM domains (TIGIT) (figure 3G). Moreover, B7H3.CARs T cells proliferated robustly without a significant difference in fold expansion compared with NT or between CAR constructs (figure 3H).

Figure 3

Immune characteristics of B7-H3 targeting CAR T cells derived from novel mAbs (A) Schematic structure of the retroviral vector encoding the B7H3.CARs and tCD19 fragment. Variable light chain (VL) and variable heavy chain (VH) were from mAb clones 12G5 (12G5 CAR), 11G5 (11G5 CAR), A2H4 (A2H4 CAR) or 376.96 (376.96 CAR). (B, C) Representative histograms and summary data of CAR expression as measured by tCD19 (CD3+tCD19+), n=5 donors (B) and B7-H3-Fc protein (CD3+CAR+), n=10 donors (C) 4 days after transduction. Data are shown as mean±SD. One-way ANOVA with Tukey’s multiple comparison test, *p<0.05, **p<0.01, ***p<0.001. (D) Stacking bar graph represents percentage of CD4+ and CD8+ population in the CD3+T cells. Data are shown as mean±SD, n=4 donors. Two-way ANOVA with Tukey’s multiple comparison test. (E, F) Memory phenotypes of CD4+ (E) or CD8+T cells (F) of NT and B7H3.CAR T cells are represented by contour plots. Naïve T cell (TN), CCR7+CD45RA+; Central memory T cell (TCM), CCR7+CD45RA-; Effector memory T cell (TEM), CCR7-CD45RA-; Effector T cell (TEMRA), CCR7-CD45RA+. The proportions are shown as mean±SD, n=4 donors. Two-way ANOVA with Tukey’s multiple comparison test. ***p<0.001 for TN of NT compared with B7H3.CARs, ##p<0.01 for TCM of NT compared with B7H3.CARs, ###p<0.001 for TCM of NT compared with B7H3.CARs and $p≤0.05 for TEM of NT compared with B7H3.CARs. (G) Relative PD-1, TIM-3, LAG-3 and TIGIT expressions are shown as relative MFIs to isotype control. Data are shown as mean±SD, n=5 donors. (H) Fold expansion of live total T cells on days 0, 4, 8 and 12 post-transduction. Data are shown as mean±SD, n=4 donors. One-way ANOVA with Tukey’s multiple comparison test. *p<0.05, **p<0.01, ***p<0.001. ANOVA, analysis of variance; LAG-3, lymphocyte activation gene 3; MFI, median fluorescence intensity; PD-1, programmed cell death protein 1; TIGIT, T cell immunoreceptor with Ig and ITIM domains; TIM-3, T cell immunoglobulin and mucin domain-containing protein 3.

B7H3.CARs T cells exhibited potent cytotoxic function against B7-H3+ solid cancer cells in vitro

To demonstrate the specific antitumor activity of B7-H3 CAR T cells, we cultured them with NALM-6 and two different B7-H3-positive cell lines, MDA-MB-231 and LN229, at fixed numbers of target cells while varying the effector cells to achieve E:T ratios of 1:2, 1:1, and 2:1. The Cell Index of tumor cells was monitored in real time using an impedance-based assay, and the percentage of cytotoxicity was calculated after 48 hours of co-culture. None of the B7H3.CAR T cells showed anti-tumor activity against NALM-6, indicating that their response is antigen-specific (figure 4A). All B7-H3 CAR constructs exhibited comparable cytolytic function against B7-H3-positive cell lines MDA-MB-231 and LN229 in a dose-dependent manner (figure 4B,C). Notably, we observed that the cytotoxic function of B7H3.CAR T cells against LN229 was higher than that against MDA-MB-231, which correlates with the higher expression of B7H3 in LN229 compared with MDA-MB-231, as detected by flow cytometry in figure 1B. To further investigate, we evaluated the proliferative capacity of B7H3.CARs T cells on antigen stimulation. CAR T cells and NT cells were labeled with CellTrace CFSE and stimulated with irradiated LN229 cells. After 6 days in culture with or without IL-2 supplementation, we determined CD3+T cell proliferation based on CFSE dilution. As shown in figure 3D, B7H3.CARs T cell, but not NT cells, proliferated in response to antigen stimulation in the absence of exogenous IL-2. Interestingly, B7H3.CAR T cells derived from the 376.96 clone underwent significantly fewer cell divisions compared with those derived from newly generated clones. In the presence of IL-2, all T cells including NT cells exhibited proliferative capacity with no significant differences between groups (figure 3E). In addition, cytokine analysis revealed significantly increased levels of IL-2, IFN-γ, and TNF-α by B7H3 CAR T cells on exposure to B7-H3-positive targets. Notably, B7-H3.CAR T cells derived from clones 12G5 and A2H4 produced significantly higher levels of TNF-α compared with the 376.96 on antigen stimulation (online supplemental figure S1A–C).

Figure 4

Cytotoxicity and proliferation analysis of B7H3.CARs T cells (A–C) Normalized cell indices of NALM-6 (A), MDA-MB-231 (B), or LN229 (C) that were co-cultured with NT or B7H3.CARs at E:T ratios of 1:2, 1:1 and 2:1 were monitored in real time using xCELLigence impedance-based assay. Cytotoxicity at 48 hours of co-culture was calculated and presented as mean±SD, n=3 donors. One-way ANOVA with Tukey’s multiple comparison test, *p<0.05, **p<0.01, ***p<0.001. (D, E) Cell proliferation assay using CellTrace CFSE. T cell labeled with CFSE were stimulated with γ-irradiated LN229 cells and cultured for 6 days in media either without IL-2 supplementation (D) or with IL-2 supplementation (E). Live T cells were identified by gating on the 7AAD-CD3+ population. CFSE fluorescence histogram peaks were analyzed to determine the number of cell divisions. Data are shown as mean±SD, n=3 donors. Two-way ANOVA with Tukey’s multiple comparison test. *p<0.05, **p<0.01 indicate significant differences in division 0 among B7H3.CARs T cells. ANOVA, analysis of variance; E:T, effector-to-target.

We further assessed the in vitro antitumor activity of B7H3.CARs T cells in a spheroid assay to mimic the 3D structure of solid tumors. As shown in figure 5A, 12G5-derived CAR T cells demonstrated superior cytotoxicity at early time points, with statistical significance on day 2 compared with other constructs. By day 7, all B7-H3 CAR T-cell constructs efficiently infiltrated and eliminated the spheroids. Following spheroid elimination, residual T cells collected from day 7 were analyzed for exhaustion markers. There was no statistically significant difference in PD-1 expression among the 11G5, 12G5, A2H4, and 376.96 constructs. However, PD-1 expression was significantly higher in the 376.96 clone compared with the NT control. While the other new clones showed a trend toward increased PD-1 expression relative to NT, these differences were not statistically significant (figure 5B). Finally, we evaluated the persistence of B7H3.CARs T cells in a tumor rechallenge assay. Interestingly, after three rounds of co-culture with tumor cells, the A2H4 clone demonstrated significantly higher effector cell numbers compared with the 11G5 and 376.96 clones (online supplemental figure S2).

Figure 5

B7H3 CAR T cells exhibited potent In vitro anti-tumor activity in a spheroid assay. (A) Representative images of residual target (green) and effector (red) cells after co-culturing at E:T of 1:1 for 7 days. The scale bars are 400 µm. Total Green Object Integrated Intensity of spheroid was recorded and used to calculate cytotoxicity. Cytotoxicity was plotted as mean±SD, n= 4 donors. *p<0.05, **p<0.01, ***p<0.001, (B) After day 7 of co-culture, residual T cells were harvested. The expression of T cell exhaustion markers including PD1, TIM3, LAG3 and TIGIT were analyzed based on CD3+ population. Data are shown as mean±SD of Relative MFI to isotype, n=3 donors. (A, B) One-way ANOVA with Tukey’s multiple comparison test. **p<0.01, ***p<0.001. ANOVA, analysis of variance; E:T, effector-to-target; MFI, median fluorescence intensity.

Robust antitumor efficacy of B7H3.CARs T cells in glioblastoma model

To investigate the antitumor activity of B7H3.CARs T cells in an animal model of glioblastoma, NSG mice were subcutaneously (s.c.) implanted with 1 × 106 LN229-Luc cells on the right flank. Seven days later, we administered 5×106 total effector cells via intravenous injection to the tumor-bearing mice. Tumor size was monitored using bioluminescent imaging (BLI) and T cell persistence was detected weekly in sampled blood (figure 6A). All B7-H3 CAR T cells except those derived from the 376.96 clone were able to eradicate the primary engrafted tumor, demonstrating superior antitumor activity (figure 6B,C). To determine the persistent antitumor activity of the B7-H3 CAR T cells, we subsequently rechallenged the mice with fresh 1×106 LN229 cells on the left flank and monitored tumor growth. As shown in figure 6B–D, CAR T cells derived from clone 376.96 could not eliminate the rechallenged tumor, resulting in tumor progression. In contrast, B7-H3 CAR T cells derived from clone 12G5, 11G5, and A2H4 were able to control the rechallenged tumor over time. Notably, CAR T cells derived from clones 11G5 and A2H4 demonstrated superior antitumor activity compared with those from clone 12G5, where the tumor eventually relapsed. Survival analysis revealed a significant prolongation of survival in mice that received B7H3 CAR T cells derived from novel antibodies compared with those treated with clone 376.96 (figure 6E). All mice that received B7-H3 CAR T cell clone A2H4 and 11G5 survived until the end of the experiment.

Figure 6

B7H3.CAR T cells derived from new mAbs exhibit superior tumor rejection in a xenograft mouse model of high grade GBM. (A) Schema for glioblastoma xenograft model in NSG mice. Blood sampling and tumor progression monitoring by bioluminescence imaging (BLI) were conducted in a weekly basis. (B) Representative tumor bioluminescence images of mice (C) Tumor growth curve measured by BLI. Data are shown as mean±SD or individual mouse, n=4. Two-way ANOVA with Tukey’s multiple comparison test, ***p<0.001. (D) Mean tumor volume measured by caliper are shown. Data are shown as the mean±SD, n=4. (E) Survival probability of mice grafted with LN229, n=4 mice per arm. Gehan-Breslow-Wilcoxon test. *p<0.05, **p<0.01. (F) Circulating T cells (CD45+CD3+) in mice after T cells infusion were examined by flow cytometry, n=4. Two-way ANOVA with Tukey’s multiple comparison test, ***p<0.001. ANOVA, analysis of variance.

Moreover, when monitoring the number of CD45+CD3+ T cells in the blood weekly postinfusion (figure 6F), we observed that B7-H3 CAR T cells derived from clone A2H4 showed the highest proliferation. These expanded cells started to decline on day 14, which correlated with the elimination of the primary tumor and the absence of the target antigen. Interestingly, B7-H3 CAR T cell clones A2H4 and 11G5 demonstrated the ability to expand after tumor rechallenge, resulting in long-term tumor control and prolonged survival.

A2H4-derived B7H3 CAR T cells demonstrated superior T cell persistence at tumor site

Next, we sought to examine the in vivo kinetics of B7-H3 CAR T cells, T cells were engineered to express GFP-firefly luciferase and infused into NSG mice implanted with LN229 on their right flank, and BLI was monitored every 3–4 days (figure 7A). On day 3 post-CAR T infusion, nearly all T cell signals were observed at the tumor site in the group of mice that received B7-H3 CAR T cells derived from clones 11G5 and A2H4. In contrast, mice treated with clones 12G5 and 376.96 displayed distribution of T cell signals in various tissues before accumulating at the tumor site by day 7, particularly the clone 376.96 which has been reported to have murine cross-reactivity.17 The T cell signals in all groups diminished by day 10. Notably, the highest signal intensity at the tumor site was observed with A2H4-B7H3 CAR T cells (figure 7B). To support these findings, tumor sections were obtained 7 days post-T cell infusion, followed by CD3 immunohistochemistry. Tumors isolated from mice treated with 11G5, 12G5, and A2H4 CAR T cells exhibited more than 15 CD3+cells per high-power field, whereas very few CD3+cells were detected in tumors from mice treated with 376.96-derived CAR T cells (figure 7C).

Figure 7

A2H4-derived B7H3 CAR T cells exhibited the highest T cell accumulation at tumor site. (A) Experimental scheme of in vivo T cell tracking monitored by BLI. (B) Representative T cell bioluminescence images of mice. Time course of total bioluminescence at tumor site are shown as mean±SD n=3 for NT and 376.96 CAR and n 4 for 12G5, 11G5 and A2H4 CAR. Two-way ANOVA with Tukey’s multiple comparisons test, *p<0.05, **p<0.01, ***p<0.001. The area under curve were analyzed and represented on bar graph with mean±SD. One-way ANOVA with Tukey’s multiple comparisons test, *p<0.05, **p<0.01, ***p<0.001 (C) CD3 immunohistochemistry staining of residual tumors isolated from mice treated with NT or B7H3.CAR T cells on the day 7. Scale bar represents 50 µm. ANOVA, analysis of variance.

Discussion

In this study, we identified four mAbs that bound to hB7-H3, among which three exhibited specific and high-affinity binding. These three clones were subsequently engineered into second-generation CAR T cells to evaluate their therapeutic potential. The preclinical efficacy of these newly developed B7-H3-targeted CAR T cells was assessed in comparison with the B7-H3 CAR T derived from previously reported mAb. Notably, novel B7-H3 CAR T cells derived from clone A2H4, eradicated tumors in glioblastoma xenograft mice. This particular clone provided superior T cell proliferation and accumulation at the tumor site. More importantly, A2H4-derived B7-H3 CAR T cells were able to repopulate and eliminate rechallenge tumors, resulting in significantly prolonged survival in mice.

B7-H3 is highly expressed in tumors compared with normal tissues. A restricted expression of this antigen was found to positively correlate with stage progression in many cancers.17 27 Here, mAbs specific to recombinant hB7-H3 protein, with an aff