Human B7-H3 Research Articles

Development of a Specifically Labeled 89Zr Antibody for the Noninvasive Imaging of Tumors Overexpressing B7-H3.

B7-H3 has emerged as a promising target and potential biomarker for diagnosing tumors, evaluating treatment efficacy, and determining patient prognosis. Hu4G4 is a recombinant humanized antibody that selectively targets the extracellular domain of human B7-H3. In this study, we describe the radiolabeling of hu4G4 with the positron emission tomography (PET) emitter radionuclide zirconium 89 (89Zr) and evaluate its potency as an immuno-PET tracer for B7-H3-targeted imaging by comparing it in vitro and in vivo to [89Zr]Zr-DFO-DS-5573a using various models. The radiolabeled compound, [89Zr]Zr-desferrioxamine-hu4G4 ([89Zr]Zr-DFO-hu4G4), demonstrated a high radiochemical purity (RCP) of greater than 99% and a specific activity of 74 MBq/mg following purification. Additionally, it maintained stability in human serum albumin (HSA) and acetate buffer, preserving over 90% of its RCP after 7 days. Three cell lines targeting human B7-H3(U87/CT26-CD276/GL261-CD276) were used. Flow cytometry analysis indicated that the B7-H3-positive cells (U87/CT26-CD276/GL261-CD276) had a higher B7-H3 protein level with no expression in the B7-H3-negative cells (CT26-wt/GL261-wt) (P < 0.001). Moreover, the cellular uptake was 45.71 ± 3.78% for [89Zr]Zr-DFO-hu4G4 in CT26-CD276 cells versus only 0.93 ± 0.47% in CT26-wt cells and 30.26 ± 0.70% when [89Zr]Zr-DFO-hu4G4 in CT26-CD276 cells were blocked with 100× 8H9. The cellular uptake of [89Zr]Zr-DFO-hu4G4 was akin to that observed with [89Zr]Zr-DFO-DS-5573a with no significant differences (45.71 ± 3.78 % vs 47.07 ± 0.86 %) in CT26-CD276 cells. Similarly, the CT26-CD276 mouse model demonstrated markedly low organ uptake and elevated tumor uptake 48 h after [89Zr]Zr-DFO-hu4G4 injection. PET/CT analysis showed that the tumor-to-muscle (T/M) ratios were substantially higher compared to other imaging groups: 27.65 ± 3.17 in CT26-CD276 mice versus 11.68 ± 4.19 in CT26-wt mice (P < 0.001) and 16.40 ± 0.78 when 100× 8H9 was used to block [89Zr]Zr-DFO-hu4G4 in CT26-CD276 mice (P < 0.01) at 48 h post-injection. Additionally, the tracer showed markedly high accumulation in the tumor region (22.57 ± 3.03% ID/g), comparable to the uptake of [89Zr]Zr-DFO-DS-5573a (24.76 ± 5.36% ID/g). A dosimetry estimation study revealed that the effective dose for [89Zr]Zr-DFO-hu4G4 was 2.96 × 10-01 mSv/MBq, which falls within the acceptable range for further research in nuclear medicine. Collectively, these results indicated that [89Zr]Zr-DFO-hu4G4 was successfully fabricated and applied in B7-H3-targeted tumor PET/CT imaging, which showed excellent imaging quality and tumor detection efficacy in tumor-bearing mice. It is a promising imaging agent for identifying tumors that overexpress B7-H3 for future clinical applications.

A novel B7-H3 x CD3 bispecific T-cell engager for the treatment of B7-H3+ tumors.

e14509 Background: B7-H3 (CD276) is a transmembrane protein highly expressed in solid tumors but with limited expression in normal tissues. Its presence is strongly associated with the inhibition of T-cell activity, cancer progression, and invasion. Furthermore, B7-H3 is strongly associated with poor prognosis and reduced patient survival. Given its crucial role in tumor biology, B7-H3 has emerged as an attractive therapeutic target for drug development. We utilized ExMab heterodimer platform and common light chain technology to develop EX105, a next-generation B7-H3×CD3 bispecific antibody. This innovative T-cell engager exhibits excellent anti-tumor activity in mouse models and good safety profile in NHP tox study. Methods: EX105 was generated using ExMab heterodimer platform and common light chain technology to form a heterodimeric IgG1 structure. Preclinical pharmacology studies, efficacy, and safety were evaluated. Anti-tumor activity study of EX105 includes in vitro T-cell cytotoxicity assays and in vivo PBMC reconstituted xenograft mice models. Preclinical safety evaluation of EX105 was performed through GLP single-dose and repeat-dose tox studies in non-human primates (NHPs). Results: Based on ELISA and FACS analyses, EX105 can bind to both human and cynomolgus monkey B7-H3 and CD3 antigens. Furthermore, SPR data suggest that EX105 exhibits a high affinity to B7-H3 and a much lower affinity to CD3. In consistent with that, EX105 only induced relatively low cytokine release. Nevertheless, EX105 showed a potent in vitro T cell cytotoxicity against B7-H3+ tumor cell lines. The in vivo anti-tumor activity of EX105 was further determined in several tumor xenograft models. Treatment with EX105 resulted in remarkable tumor regression in both “hot” tumor model and “cold” tumor model, indicating its effectiveness across a range of tumor types. Tox study in NHPs showed minimal adverse effects and no liver impairment after EX105 administration, even at high doses. The cytokine release data observed in this study suggests a good safety profile of EX105, with much lower cytokine release compared to other T cell engagers. Conclusions: These findings collectively indicate that EX105 has the potential to become a promising therapeutic agent for the treatment of various B7-H3 positive cancers. The first-in-human clinical trial for patients with advanced B7-H3 positive cancers is due to start.

Targeting both GD2 and B7-H3 using bispecific antibody improves tumor selectivity for GD2-positive tumors.

Disialoganglioside 2 (GD2), overexpressed by cancers such as melanoma and neuroblastoma, is a tumor antigen for targeted therapy. The delivery of conventional IgG antibody technologies targeting GD2 is limited clinically by its co-expression on nerves that contributes to toxicity presenting as severe neuropathic pain. To improve the tumor selectivity of current GD2-targeting approaches, a next-generation bispecific antibody targeting GD2 and B7-H3 (CD276) was generated. Differential expression of human B7-H3 (hB7-H3) was transduced into GD2+ B78 murine melanoma cells and confirmed by flow cytometry. We assessed the avidity and selectivity of our GD2-B7-H3 targeting bispecific antibodies (INV34-6, INV33-2, and INV36-6) towards GD2+/hB7-H3- B78 cells relative to GD2+/hB7-H3+ B78 cells using flow cytometry and competition binding assays, comparing results an anti-GD2 antibody (dinutuximab, DINU). The bispecific antibodies, DINU, and a non-targeted bispecific control (bsAb CTRL) were conjugated with deferoxamine for radiolabeling with Zr-89 (t1/2 = 78.4 h). Using positron emission tomography (PET) studies, we evaluated the in vivo avidity and selectivity of the GD2-B7-H3 targeting bispecific compared to bsAb CTRL and DINU using GD2+/hB7-H3+ and GD2+/hB7-H3- B78 tumor models. Flow cytometry and competition binding assays showed that INV34-6 bound with high avidity to GD2+/hB7-H3+ B78 cells with high avidity but not GD2+/hB7-H3+ B78 cells. In comparison, no selectivity between cell types was observed for DINU. PET in mice bearing the GD2+/hB7-H3- and GD2+/hB7-H3+ B78 murine tumor showed similar biodistribution in normal tissues for [89Zr]Zr-Df-INV34-6, [89Zr]Zr-Df-bsAb CTRL, and [89Zr]Zr-Df-DINU. Importantly, [89Zr]Zr-Df-INV34-6 tumor uptake was selective to GD2+/hB7-H3+ B78 over GD2+/hB7-H3- B78 tumors, and substantially higher to GD2+/hB7-H3+ B78 than the non-targeted [89Zr]Zr-Df-bsAb CTRL control. [89Zr]Zr-Df-DINU displayed similar uptake in both GD2+ tumor models, with uptake comparable to [89Zr]Zr-Df-INV34-6 in the GD2+/hB7-H3+ B78 model. The GD2-B7-H3 targeting bispecific antibodies successfully improved selectivity to cells expressing both antigens. This approach should address the severe toxicities associated with GD2-targeting therapies by reducing off-tumor GD2 binding in nerves. Continued improvements in bispecific antibody technologies will continue to transform the therapeutic biologics landscape.

Development of a Human B7-H3-Specific Antibody with Activity against Colorectal Cancer Cells through a Synthetic Nanobody Library.

Nanobodies have emerged as promising tools in biomedicine due to their single-chain structure and inherent stability. They generally have convex paratopes, which potentially prefer different epitope sites in an antigen compared to traditional antibodies. In this study, a synthetic phage display nanobody library was constructed and used to identify nanobodies targeting a tumor-associated antigen, the human B7-H3 protein. Combining next-generation sequencing and single-clone validation, two nanobodies were identified to specifically bind B7-H3 with medium nanomolar affinities. Further characterization revealed that these two clones targeted a different epitope compared to known B7-H3-specific antibodies, which have been explored in clinical trials. Furthermore, one of the clones, dubbed as A6, exhibited potent antibody-dependent cell-mediated cytotoxicity (ADCC) against a colorectal cancer cell line with an EC50 of 0.67 nM, upon conversion to an Fc-enhanced IgG format. These findings underscore a cost-effective strategy that bypasses the lengthy immunization process, offering potential rapid access to nanobodies targeting unexplored antigenic sites.

Abstract 5087: ILB-3101, a novel B7H3-targeting ADC with Eribulin as payload, demonstrates strong tumor killing activities and favorable PK/TOX profile in preclinical evaluation

Abstract B7H3, a B7 family member of immune checkpoint proteins, is overexpressed in a broad spectrum of malignant tumors. It has become an attractive target for cancer therapies in recent years. ILB-3101, a potential best-in-class ADC, was constructed by conjugating a potent mitotic inhibitor Eribulin to a proprietary anti-B7H3 mAb through a proprietary linker. This ADC molecule has been developed and preclinically evaluated as both the 1st line ADC treatment with B7H3 expressed patients in Eribulin sensitive cancer types and the later line treatment for patients whose cancer are B7H3 positive but TopoI inhibitor resistant. The humanized antibody of ILB-3101 is IgG1 type and shows high affinity to both human and monkey B7H3 proteins. It also shows better internalization than benchmark antibodies such as that of DS-7300 analog. The DAR value of ILB-3101 is 3.5. ILB-3101 has potent and dose-dependent cytotoxicity to B7H3-positive tumor cells and shows better activities than DS-7300 analog as positive control. In vivo studies based on xenograft tumor models reveal that ILB-3101 has potent anti-tumor activities in various tumor types including TNBC, sarcoma, ovarian cancer and AML etc., which are superior or comparable to DS-7300 analog. Furthermore, ILB-3101 shows strong anti-tumor activity on Dxd based ADC resistant tumors in xenograft tumor models. The PK study of ILB-3101 in cynomolgus monkeys shows dose-dependent exposure and desirable PK profile. ILB-3101 was well tolerated in cynomolgus monkeys with HNSTD at 6 mg/kg. No serious adverse events were observed, and the main safety findings were limited and reversible hematological and immunological effects, which are consistent with the safety profile of Eribulin as an approved chemotherapy. The in vitro and in vivo preclinical results above demonstrate that ILB-3101 has good efficacy and safety profile and it is a promising and potentially best-in-class B7H3 targeting ADC drug candidate. ILB-3101 is worthy of further translational research and clinical trials. Its IND application has been submitted in China, followed by US FDA filing soon. Citation Format: Yushuan Shang, Junzhuan Qiu, Yanfei Xin, Min Zhao, Qiang Liu, Zhengsheng Wang, Xiaojing Gong, Yan Xu, Yan Li, Mingde Xia, Rulei Chen. ILB-3101, a novel B7H3-targeting ADC with Eribulin as payload, demonstrates strong tumor killing activities and favorable PK/TOX profile in preclinical evaluation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5087.

Abstract 2453: Soluble B7H3 has a potential of novel serum biomaker for colorectal cancer

Abstract B7H3 (also known as CD276, an immune checkpoint molecule) is aberrantly over expressed in many types of cancer. Recently, several reports indicate a crucial role for B7H3 in promoting carcinogenesis and metastasis. We investigated whether concentration of the soluble B7H3 could be used as a new prognostic predictor of colorectal cancer by measuring serum level of B7H3. Recently we established a new mAb to B7H3 and developed sandwich ELISA-system detecting human serum B7H3 with high sensitivity and specificity. Measuring the levels of B7H3 in the serum specimens of colorectal cancer donors, we examined the usefulness of B7H3 as a novel biomarker for colorectal cancers. We tested the blood from the 82 donors of colorectal cancers and 9 healthy donors who agreed in writing to this research, just before the operation. The mean value + 2SD in 9 healthy donors was set as the cutoff value. We classified cancer patients into 2 groups with high value of B7H3 and low value of B7H3. We also analyzed the relationships between serum B7H3 levels and disease-free survival (DFS) and overall survival (OS). Serum B7H3 levels were also compared with diagnostic biomarkers such as CEA and CA19-9. B7H3 immunostaining was also performed on tissue samples. The serum levels of B7H3 in patients with colorectal cancer were significantly higher than those of healthy donors. However, there were no correlation between B7H3 and other diagnostic markers CEA and CA19-9. Regarding DFS, the higher value group of serum B7H3 showed a tendency for DFS to decrease in Stage II and III. Regarding OS, there was no significant difference of serum levels of B7H3 in Stage I and IV cases, however, in Stage II and III, OS decreased significantly in the high B7H3 group. In B7H3 immunostaining, the tissue was not sufficiently stained. These data indicate that serum B7-H3 should be useful for novel biomarker for a rationale of disease monitoring and prognosis of colorectal cancer. Citation Format: Akihiro Koizumi, Tomoaki Ito, Kazunori Kato, Takumi Iwasawa, Tomoyuki Kushida, Koichi Sato, Chikako Okamoto. Soluble B7H3 has a potential of novel serum biomaker for colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2453.

Antitumor activity of the investigational B7-H3 antibody-drug conjugate, vobramitamab duocarmazine, in preclinical models of neuroblastoma

IntroductionB7-H3 is a potential target for pediatric cancers, including neuroblastoma (NB). Vobramitamab duocarmazine (also referred to as MGC018 and herein referred to as vobra duo) is an investigational duocarmycin-based antibody-drug...

SOLUBLE FORMS OF THE NKG2D RECEPTOR AND LIGAND NKG2DL1 IN PATIENTS WITH COLORECTAL CANCER

Relevance. Natural killer (NK) cells are considered to be effective cytotoxic cells of the tumor microenvironment, whose function is to effectively eliminate tumor cells. Their activation can occur when the NKG2D receptor interacts with the NKG2DL ligand expressed, for example, on tumor cells, which leads to the death of the latter. The aim of the study was a comparative study of the content of soluble forms of the sNKG2D receptor and its ligand sNKG2DL1 in the blood serum of healthy donors and patients with colorectal cancer (CRC), taking into account the clinical and morphological characteristics of the disease and prog-nosis. Material and methods. We examined 65 patients with CRC (32 women and 33 men), who were treated at the “National Medical Research Center of Oncology named after N.N. Blokhin” of the Ministry of Health of Russia aged (median 60 years) and 30 healthy donors (20 women and 10 men) aged (median 47 years). The clinical diagnosis in all patients was confirmed by the data of the morphological study of the tumor according to the Interna-tional Histological Classification of Tumors of the Digestive System (WHO, 2019), all had adenocarcinoma. The concentration of NKG2D and NKG2DL proteins was determined by ELISA in blood serum before treatment with Human B7-H3 Quantikine ELISA Kit reagents firm R&amp;D (USA) in accordance with the manufacturer's instructions. The measurements were carried out on an automatic enzyme immunoassay analyzer BEP 2000 Advance (Siemens Healthcare Diagnostics, Germany). Statistical analysis of the obtained results was carried out using GraphPad Prizm v. 9. Overall survival analysis was performed by constructing survival curves using the Kaplan-Meier method. Results. In patients with colorectal cancer, a significant increase in the content of sNKG2DL in blood serum was found compared to the control group. sNKG2D concentrations did not differ between controls and CRC patients. No significant associations were found between the content of sNKG2D and sNKG2DL1 proteins in the blood serum of CRC patients and the main clinical and morphological characteristics of the disease. A decrease in the con-centration of sNKG2D was noted with the progression of the disease, and the highest content of sNKG2D was found in the localization of the tumor in the caecum. For the ligand sNKG2DL1, the opposite pattern was observed, namely, its lowest concentration in blood serum in tumors located in the caecum. For the ligand sNKG2DL1, the opposite pattern was observed, namely, its lowest concentration in blood serum in tumors located in the cae-cum. At the same time, the levels of sNKG2D and sNKG2DL1 in the control group directly and significantly correlated with each other (r=0.453; p=0.014), while no such pattern was found in the group of CRC patients (r=0.014; p=0.935). sNKG2DL1 concentrations are not a prognostic factor in CRC, while high levels of sNKG2D tended to lead to a favorable prognosis of the disease, which indirectly confirms the positive significance of tumor in-filtration by NK cells. Conclusions. The mechanisms of the relationship between CRC avoidance of immune surveillance and the expression of the sNKG2D receptor and the sNKG2DL ligand are discussed as one of the directions in the study of immune checkpoints. We believe that the effect of CRC immunotherapy can be achieved by increasing the expression of NKG2D on immune cells and inducing the expression of NKG2DL in tumor cells, which is the basis of the con-cept of this line of research.

Bispecific antibody targeting both B7-H3 and PD-L1 exhibits superior antitumor activities.

Clinical application of PD-1andPD-L1 monoclonal antibodies (mAbs) is hindered by their relatively low response rates and the occurrence of drug resistance. Co-expression of B7-H3 with PD-L1 has been found in various solid tumors, and combination therapies that target both PD-1/PD-L1 and B7-H3 pathways mayprovide additional therapeutic benefits. Up to today, however, no bispecific antibodies targeting both PD-1 and B7-H3 have reached the clinical development stage. In this study, we generated a stable B7-H3×PD-L1 bispecific antibody (BsAb) in IgG1-VHH format by coupling a humanized IgG1 mAb against PD-L1 with a humanized camelus variable domain of the heavy-chain of heavy-chain antibody (VHH) against human B7-H3. The BsAb exhibited favorable thermostability, efficient T cell activation, IFN-γ production, and antibody-dependent cell-mediated cytotoxicity (ADCC). In a PBMC humanized A375 xenogeneic tumor model, treatment with BsAb (10 mg/kg, i.p., twice a week for 6 weeks) showed enhanced antitumor activities compared to monotherapies and, to some degree, combination therapies. Our results suggest that targeting both PD-1 and B7-H3 with BsAbs increases their specificities to B7-H3 and PD-L1 double-positive tumors and induces a synergetic effect. We conclude that B7-H3×PD-L1 BsAb is favored over mAbs and possibly combination therapies in treating B7-H3 and PD-L1 double-positive tumors.

Synthesis and Evaluation of Clinically Translatable Targeted Microbubbles Using a Microfluidic Device for In Vivo Ultrasound Molecular Imaging.

The main aim of this study is to synthesize contrast microbubbles (MB) functionalized with engineered protein ligands using a microfluidic device to target breast cancer specific vascular B7-H3 receptor in vivo for diagnostic ultrasound imaging. We used a high-affinity affibody (ABY) selected against human/mouse B7-H3 receptor for engineering targeted MBs (TMBs). We introduced a C-terminal cysteine residue to this ABY ligand for facilitating site-specific conjugation to DSPE-PEG-2K-maleimide (M. Wt = 2.9416 kDa) phospholipid for MB formulation. We optimized the reaction conditions of bioconjugations and applied it for microfluidic based synthesis of TMBs using DSPE-PEG-ABY and DPPC liposomes (5:95 mole %). The binding affinity of TMBs to B7-H3 (MBB7-H3) was tested in vitro in MS1 endothelial cells expressing human B7-H3 (MS1B7-H3) by flow chamber assay, and by ex vivo in the mammary tumors of a transgenic mouse model (FVB/N-Tg (MMTV-PyMT)634Mul/J), expressing murine B7-H3 in the vascular endothelial cells by immunostaining analyses. We successfully optimized the conditions needed for generating TMBs using a microfluidic system. The synthesized MBs showed higher affinity to MS1 cells engineered to express higher level of hB7-H3, and in the endothelial cells of mouse tumor tissue upon injecting TMBs in a live animal. The average number (mean ± SD) of MBB7-H3 binding to MS1B7-H3 cells was estimated to be 354.4 ± 52.3 per field of view (FOV) compared to wild-type control cells (MS1WT; 36.2 ± 7.5/FOV). The non-targeted MBs did not show any selective binding affinity to both the cells (37.7 ± 7.8/FOV for MS1B7-H3 and 28.3 ± 6.7/FOV for MS1WT cells). The fluorescently labeled MBB7-H3 upon systemic injection in vivo co-localized to tumor vessels, expressing B7-H3 receptor, as validated by ex vivo immunofluorescence analyses. We have successfully synthesized a novel MBB7-H3 via microfluidic device, which allows us to produce on demand TMBs for clinical applications. This clinically translatable MBB7-H3 showed significant binding affinity to vascular endothelial cells expressing B7-H3 both in vitro and in vivo, which shows its potential for clinical translation as a molecular ultrasound contrast agent for human applications.

Abstract 5199: B7-H3 &amp; PD-L1 double-humanized BALB/c mouse: a novel animal model for preclinical studies of human B7-H3 antibodies or bispecific antibodies

Abstract Immune checkpoint molecule B7-H3, also known as CD276, is a member of the B7-CD28 family of immunomodulatory proteins. It is a type I membrane protein with sequence similarities to the extracellular domain of programmed death-ligand 1 (PD-L1). B7-H3 is highly expressed in most human cancers, but has limited distribution in normal tissues, remaining elusive of its receptor. Due to its promising safety as a dominant tumor target, various strategies have been developed to modulate the effect of B7-H3 via monoclonal antibodies, bispecific antibodies, ADC, or CAR-T. To study the effect of these therapies in an immunocompetent mouse model, we established a double humanized B7-H3 and PD-L1 mouse model on BALB/c background (BALB/c-hB7-H3/hPD-L1). In this model, the extracellular domain of murine fragments was replaced by the human counterparts while the trans-membrane and cytoplasmic domain were kept intact. When engrafted with CT26 colon cancer cells, which stably overexpress human B7-H3 and PD-L1 while endogenous murine counterparts were knocked out, the tumor growth was inhibited certain degree by anti-B7-H3 antibody (8H9 Biosimilar, 20mpk, TGI=18.56%) treatment while inhibited significantly after the monotherapy of anti-PD-L1 (Tecentriq, 3 mpk, TGI=55.89%, p&amp;lt;0.001). The same dosage combination of anti-B7-H3 and anti-PD-L1 had a significant inhibition on tumor growth (TGI=76.85%, p&amp;lt;0.001), and had a synergistic effect (CDI&amp;lt;0.7) compared with the monotherapy. Analysis of tumor-infiltrating lymphocytes (TILs) at the end of the efficacy study showed that the proportion of CD45+ immune cells was significantly increased in all of the treated groups. The NK cells were significantly increased and the Treg cells were significantly decreased especially in the combined treatment group. Based on the above, the B7-H3 and PD-L1 double humanized mouse model is suitable for the pre-clinical evaluation of mono or combined immune checkpoint blockade with anti-human B7-H3 and PD-L1 therapy. Citation Format: Shiying Guo, Lingyu Song, Xin Qin, Jianming Xu, Hongyan Sun, Cunxiang Ju, Hongyu Wang, Santi Suryani Chen, Zhiying Li, Mark Wade Moore, Jing Zhao, Xiang Gao. B7-H3 &amp; PD-L1 double-humanized BALB/c mouse: a novel animal model for preclinical studies of human B7-H3 antibodies or bispecific antibodies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5199.

Abstract 1528: 6B5, an anti-human B7-H3 therapeutic antibody that enhances antibody-dependent cellular cytotoxicity and inhibits tumor growth in B7-H3-humanized mice

Abstract B7-H3 (CD276) is a type I transmembrane protein belonging to the B7 family of immune-regulatory ligands. Initially reported as a costimulatory signal of human T cells, increasing evidence suggests that it may also have coinhibitory function. TLT-2 has been identified as a potential cognate receptor for B7-H3, but others may exist. Overexpression of B7-H3 protein is found in a variety of human cancers, including lung adenocarcinomas, neuroblastomas, gliomas, and pancreatic tumors—and is associated with poor prognosis and survival. While B7-H3 can promote T cell activation and anti-tumor responses in the context of CD3 stimulation, its coinhibitory function inhibits T and NK cell activity in the tumor microenvironment. We generated 6B5, a fully human B7-H3 monoclonal antibody, from murine B7-H3 knock-out RenMab™ mice, which contain the full human immunoglobulin variable domain. 6B5 is cross-reactive with rodent and cynomolgus monkey B7-H3. It displays higher affinity for tumor cell lines than Enoblituzumab. 6B5 has no staining on normal human tissues. In vitro antibody-dependent cellular cytotoxicity (ADCC) against H520 squamous cell carcinoma cells was potentiated by 6B5 relative to Enoblituzumab and, critically, did not exhibit the hook effect at high dosage. In vivo efficacy of 6B5 in humanized B7-H3 mice bearing murine EL4 lymphomas was comparable to Enoblituzumab. ADCC enhancement in vivo was comparable to afucosylation or five-point mutations. Epitope mapping revealed non-overlapping B7-H3 binding epitopes of 6B5 and Enoblituzumab. Taken together, these data suggest that 6B5 is a promising therapeutic anti-human B7-H3 IgG monoclonal antibody that may find clinical application in a range of cancers, including current refractory types. Citation Format: Yunyun Chen, Jiangmei Li, Yanan Guo, Wenqi Hu, Ting Mao, Li Hui, W. Frank An, Yi Yang, Feng Li. 6B5, an anti-human B7-H3 therapeutic antibody that enhances antibody-dependent cellular cytotoxicity and inhibits tumor growth in B7-H3-humanized mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1528.

Radioimmunotherapy Targeting B7-H3 in situ glioma models enhanced antitumor efficacy by Reconstructing the tumor microenvironment.

Radionuclide drug conjugates (RDCs) with antibodies serve as a novel approach for the treatment of malignant tumors including glioblastoma. However, RDCs require optimal antibodies to work efficiently. Hu4G4, a novel B7-H3-targeting humanized monoclonal IgG1 antibody, is highly specific for the human B7-H3 protein (a marker of tumor cells, including glioblastoma cells). Herein, we established 131I-labeled hu4G4 (131I-hu4G4) and showed that it specifically bound to B7-H3 with high affinity (Kd = 0.99 ± 0.07 nM) and inhibited the growth of U87 cells in vitro. 131I-hu4G4 displayed potent in situ antitumor activity in a mouse model of glioma based on GL261 Red-Fluc-B7-H3 cells. More importantly, 131I-hu4G4 remodeled the tumor microenvironment and promoted the transformation of glioma from "cold" to "hot" tumors by promoting CD4+ and CD8+ T cell infiltration and the polarization of M2 to M1. Therefore, the antitumor activity observed with 131I-hu4G4, together with its ability to enhance antitumor immune responses, makes it a novel candidate for radioimmunotherapy of glioblastoma.

Abstract 2761: FT573: Preclinical development of multiplexed-engineered iPSC-derived NK cells expressing a novel camelid nanobody chimeric antigen receptor (CAR) targeting pan-cancer antigen B7-H3

Abstract Introduction: B7-H3 (CD276) has gained significant clinical interest as a pan-tumor target antigen for development of various immuno-oncology agents. Due to its broad expression on a wide variety of solid tumors and minimal expression on normal tissues, B7-H3 is an ideal tumor antigen target. Additionally, high levels of B7-H3 are found on “immunologically cold” tumors such as glioblastoma multiforme, prostate cancer, head and neck cancer and soft tissue sarcomas, which typically have poor response to approved immune therapies. To effectively target B7-H3 with an off-the-shelf cellular therapy, we describe here the development of camB7-H3 CAR-NK cell utilizing our iPSC platform to engineer multiple modalities into a clonal iPSC line, which can serve as the starting cell source for mass production of off-the-shelf, iPSC-derived CAR-NK cells (CAR-iNK cells). Methods: A camelid nanobody specific for human B7-H3 (camB7-H3) was discovered using a phage display library and validated in functional assays. camB7-H3 CAR-iNK cells were designed to 1) express membrane-bound IL-15/IL-15 receptor fusion for enhanced persistence, 2) have a CD38 knockout to improve metabolic fitness, 3) express a high-affinity non-cleavable CD16 to maximize ADCC when combined with a therapeutic antibody, and 4) express an anti-camB7-H3 CAR optimized for NK cell signaling. As the initial preclinical study, camB7-H3 CAR-iNK cells were assessed using flow cytometry-based functional assays evaluating CD107a and IFNγ or xCelligence target killing assays against B7-H3 transgenic or naturally expressing tumor cell lines. Results: The camelid single domain B7-H3 initially tested in CAR-T exhibited B7-H3 specific binding and specific activity against several solid tumor cell lines (breast, ovarian, prostate, lung). We next produced camB7-H3 CAR-iNK cells and demonstrated superior B7-H3-specific target elimination compared to untransduced iNK cells. These results were seen across multiple tumor lines representing various solid tumor indications. Further enhancement of anti-tumor efficacy was seen when combined with therapeutic antibodies, including trastuzumab and cetuximab. Conclusions: We have successfully produced and validated the specificity and function of an engineered camB7-H3 CAR-iNK cell exhibiting robust target killing and on-target specificity. To our knowledge, this is the first camelid nanobody antigen recognition domain reported in a CAR-NK cell to be used as an off-the-shelf immunotherapy. The combining camB7-H3 CAR-NK with monoclonal antibodies targeting HER2 and EGFR as a dual targeting approach will add additional tumor specificity, further increase the efficacy of tumor cell elimination and prevent antigen escape. Additional camB7-H3 CAR-iNK cell preclinical studies are in process and will be discussed. Citation Format: Nicholas Zorko, Frank Cichocki, John Goulding, Bryan Hancock, Robert Blum, Mochtar Pribadi, Bjoern Gaertner, Tom Lee, Martin Felices, Ryan Bjordahl, Bahram Valamehr, Jeffrey S. Miller. FT573: Preclinical development of multiplexed-engineered iPSC-derived NK cells expressing a novel camelid nanobody chimeric antigen receptor (CAR) targeting pan-cancer antigen B7-H3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2761.

Abstract 1652: A humanized mouse model of the promising immune checkpoint molecule B7-H4

Abstract B7-H4, a member of the B7-family of immune-regulatory ligands, has emerged in recent years as a promising target for immune checkpoint therapy development. B7-family ligands bind via extracellular tandem immunoglobulin-like domains to CD28-family receptors on lymphocytes to communicate co-stimulatory or co-inhibitory signals. B7-H4 remains an orphan ligand, although evidence from knockout mice suggests a co-inhibitory role and its expression on cancer cells and tumor-associated macrophages is associated with inhibition of proliferation, cell cycle progression, and cytokine secretion in T cells. B7-H4 is highly expressed in a variety of tumors (e.g., renal, gastric, breast, and ovarian carcinomas) and plays less well-appreciated roles in autoimmune diseases, viral infections, and maternal-fetal immunity. Negative regulation of T cell responses by B7-H4 has also been shown to induce tolerance of islet allografts in mouse models of Type I diabetes. A range of B7-H4-targeted immunotherapy agents have been successfully developed, including monoclonal antibodies, antibody-drug conjugates, B7-H4/CD3-targeting bispecific antibodies, and B7-H4-specific CAR-T cells. To evaluate the efficacy of a B7-H4 antibody, Biocytogen generated humanized B-hB7-H4 mice. Our targeting strategy was to replace the murine B7-H4 extracellular domain sequences with their corresponding human sequences. We verified mRNA and protein expression of human B7-H4 in the humanized B-hB7-H4 mice by RT-PCR and Western Blot, respectively. An in vivo efficacy study demonstrated that B7-H4 antibodies significantly inhibited tumor growth in B-hB7-H4 mice bearing tumors derived from the B16-F10 murine melanoma line. These data confirm that the B-hB7-H4 humanized mouse model is a powerful tool for evaluating the preclinical potential of B7-H4-targeting immune-therapeutics. Keywords: Humanized mice, B7-H4, Immunotherapy Citation Format: Ruili Lyu, Chonghui Liu, John Charpentier, Suman Zhao, Luke (Zhaoxue) Yu. A humanized mouse model of the promising immune checkpoint molecule B7-H4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1652.

Abstract 452: Development and implementation of image analysis-based Quantitative Continuous Score (QCS) for B7-H4 IHC to understand AZD8205 pharmacodynamics

Abstract Antibody-Drug-Conjugates (ADCs) are biopharmaceutical drugs designed for targeted tumor therapy, meant to improve therapeutic index by restricting drug delivery to tumor cells that express the target antigen. ADCs bind to the target molecule on the cell membrane, which triggers internalization, linker cleavage, and ultimately drug release inside the target cell. Prospective patient selection can be done by quantifying the level of target expression in the tumor using immuno-histochemistry (IHC). However, this process typically involves pathologists and is time consuming, expensive, and prone to human bias. We have developed a supervised deep learning algorithm that segments IHC images of invasive tumor epithelium into individual epithelial cells and their membrane, cytoplasm and nucleus with high accuracy. On unseen test data its performance for epithelial cell detection and segmentation is comparable to the inter-pathologist consensus. With our algorithm, we can describe the target molecule distribution of individual cells in a fully quantitative fashion after using standard IHC methods: we call our approach Quantitative Continuous Score (QCS). We applied QCS to interrogate the mechanism of action of AZD8205, a B7-H4 directed ADC incorporating a novel topoisomerase I linker-warhead. Pharmacodynamic effects were evaluated in vivo, using a human tumor xenograft mouse model and the cell line HT29-huB7-H4 Clone 26, engineered to express human B7-H4. After tumors grew in volume to approximately 250 to 300 mm3, animals were randomized and each mouse received an IV injection of either AZD8205 (1.25, 3.5, or 7 mg/kg) or control articles. Tumors were collected at designated timepoints, fixed in 10% neutral buffered formalin and subsequently embedded into paraffin blocks. IHC and QCS were then used to examine human IgG, γH2AX foci, cleaved caspase-3, and epithelial cell density in tumor samples over time. Using our novel approach we could quantitatively measure the level of AZD8205 bound to tumor cells, with the highest level of ADC on the cell membrane detected at 24-48 hrs. Increased dose levels accelerated the binding kinetics of the drug and led to to a 4- and 3-fold excess of γH2AX and CC-3 respectively, as well as more cells being killed, with up to 2/3 of all epithelial cells dead at the highest dose studied. In summary, we here set the basis for future mechanistic investigation of model systems using computational pathology to improve our understanding of ADC effects. Computational pathology has the potential to determine molecule abundance quantitatively, increase throughput and avoid human bias. Our data implies QCS has the potential to identify patients who may respond to AZD8205, which we will interrogate further and integrate into future clinical studies. Citation Format: Philipp Wortmann, Tze Heng Tan, Susanne Haneder, Andrea Ennio Storti, Ansh Kapil, Jon Chesebrough, Daniel Sutton, Michal Sulikowski, Arthur Lewis, Sofia Koch, Steve Sweet, Zifeng Song, David Chain, Yeoun Jin Kim, Nadia Luheshi, Krista Kinneer, Zachary A. Cooper, Marlon Rebelatto, Günter Schmidt, Hadassah Sade, J. Carl Barrett. Development and implementation of image analysis-based Quantitative Continuous Score (QCS) for B7-H4 IHC to understand AZD8205 pharmacodynamics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 452.

B7-H3 Specific CAR T Cells for the Naturally Occurring, Spontaneous Canine Sarcoma Model.

One obstacle for human solid tumor immunotherapy research is the lack of clinically relevant animal models. In this study, we sought to establish a chimeric antigen receptor (CAR) T-cell treatment model for naturally occurring canine sarcomas as a model for human CAR T-cell therapy. Canine CARs specific for B7-H3 were constructed using a single-chain variable fragment derived from the human B7-H3-specific antibody MGA271, which we confirmed to be cross-reactive with canine B7-H3. After refining activation, transduction, and expansion methods, we confirmed target killing in a tumor spheroid three-dimensional assay. We designed a B7-H3 canine CAR T-cell and achieved consistently high levels of transduction efficacy, expansion, and in vitro tumor killing. Safety of the CAR T cells were confirmed in two purposely bred healthy canine subjects following lymphodepletion by cyclophosphamide and fludarabine. Immune response, clinical parameters, and manifestation were closely monitored after treatments and were shown to resemble that of humans. No severe adverse events were observed. In summary, we demonstrated that similar to human cancers, B7-H3 can serve as a target for canine solid tumors. We successfully generated highly functional canine B7-H3-specific CAR T-cell products using a production protocol that closely models human CAR T-cell production procedure. The treatment regimen that we designed was confirmed to be safe in vivo. Our research provides a promising direction to establish in vitro and in vivo models for immunotherapy for canine and human solid tumor treatment.

AAMP is a binding partner of costimulatory human B7-H3.

BackgroundTargeted immunotherapies are of growing interest in the treatment of various cancers. B7 homolog 3 protein (B7-H3), a member of the co-stimulatory/-inhibitory B7-family, exerts immunosuppressive and pro-tumorigenic functions in various cancer types and is under evaluation in ongoing clinical trials. Unfortunately, interaction partner(s) remain unknown which restricts the druggability.MethodsAiming to identify potential binding partner(s) of B7-H3, a yeast two-hybrid and a mass spectrometry screen were performed. Potential candidates were evaluated by bimolecular fluorescence complementation (BiFC) assay, co-immunoprecipitation (co-IP), and functionally in a 3H-thymidine proliferation assay of Jurkat cells, a T-cell lineage cell line. Prognostic value of angio-associated migratory cell protein (AAMP) and B7-H3 expression was evaluated in isocitrate dehydrogenase 1 wildtype (IDH1wt) glioblastoma (GBM) patients from The Cancer Genome Atlas (TCGA)-GBM cohort.ResultsOf the screening candidates, CD164, AAMP, PTPRA, and SLAMF7 could be substantiated via BiFC. AAMP binding could be further confirmed via co-IP and on a functional level. AAMP was ubiquitously expressed in glioma cells, immune cells, and glioma tissue, but did not correlate with glioma grade. Finally, an interaction between AAMP and B7-H3 could be observed on expression level, hinting toward a combined synergistic effect.ConclusionsAAMP was identified as a novel interaction partner of B7-H3, opening new possibilities to create a targeted therapy against the pro-tumorigenic costimulatory protein B7-H3.

Co-immunizing with HMGB1 enhances anti-tumor immunity of B7H3 vaccine in renal carcinoma

Metastatic renal carcinoma is a kind of tumor with high degree of malignancy, but there are no effective treatment methods and strategies at present. In this study, we designed a folate-grafted PEI600-CyD (H1) nanoparticle-mediated DNA vaccine containing an adjuvant of high mobility group box 1 protein (HMGB1) and a tumor-specific antigen of B7H3 (CD276) for renal carcinoma therapy. Mice bearing subcutaneous human B7H3 (hB7H3)-Renca tumor were immunized with H1-pHMGB1/pB7H3, H1-pB7H3, H1-pHMGB1, or Mock vaccine. Compared to other control groups, the growth of the tumor was significantly inhibited in H1-pHMGB1/pB7H3 vaccine group. The increased proportion and mature of CD11c+ DCs were observed in the spleen of H1-pHMGB1/pB7H3 treated mice. Likewise, HMGB1 promoted B7H3 vaccine to induce tumor-specific CD8+ T cell proliferation and CTL responses. Beyond that, H1-pHMGB1/pB7H3 vaccine strengthened the induction of functional CD8+ T cells. With the depletion of CD8+ T cells, the anti-tumor effect of H1-pHMGB1/pB7H3 also disappeared, indicating that CD8+ T cells are the key factor of the anti-tumor activity of the vaccine. So, to sum up, H1-pHMGB1/pB7H3 vaccine could achieve the desired anti-tumor effect by enhancing the response of tumor-specific functional CD8+ T cell responses. H1 nanoparticle-based vaccines may have great potential and prospect in the treatment of primary solid tumors.

Development of a novel anti-B7-H4 antibody enhances anti-tumor immune response of human T cells

BackgroundB7-H4 is a member of the B7 superfamily that is expressed on the surface of tumors and exhibits limited expression on normal tissue. B7-H4 negatively regulates tumor immunity by interacting with the B7-H4 receptor, which is expressed by activated CD8 + T cells. Hence, we sought to generate an immunomodulatory antibody that targets B7-H4 and blocks the immunosuppressive activity of B7-H4. MethodsAnti-B7-H4 antibodies were generated using the hybridoma technique and screened by a binding assay based on B7-H4-expressing tumor cells. The B7-H4 antagonistic antibodies were further screened based on their checkpoint blockade activity using a SEB-stimulated peripheral blood mononuclear cell (PBMC) assay, which comprised B7-H4-expressing antigen presenting cells (APCs) and activated T cells. To assess the immunomodulatory activity of anti-B7-H4 antibodies, activated human CD8+ T cells were cultured in B7-H4 protein-coated plates, and the production of IL-2 and the proliferation rate of CD8+ T cells were measured. In addition, we evaluated the ADCC effect of anti-B7-H4 antibodies against tumor cell lines. The in vivo antitumor efficacy of the anti-B7-H4 antibody was also evaluated in human T cell-engrafted NOG mice. ResultsA panel of anti-B7-H4 antibodies was generated. The top 23 antibodies were screened to identify antibodies that disabled B7-H4-mediated inhibition. Antibody 17 exhibited the greatest induction of the production of IL-2 and IFN-gamma in SEB-stimulated PBMCs. Antibody 17 was constructed as a chimeric antibody (CH17) with a human IgG1 constant domain. CH17 showed high affinity for human B7-H4 and fully cross-reacted with cynomolgus B7-H4. Additionally, CH17 mediated potent antibody-dependent cell cytotoxicity (ADCC) against different B7-H4-positive tumor cell lines. More importantly, CH17 relieved B7-H4-mediated T cell suppression by enhancing IL2 production and promoting T cell proliferation. In an MDA-MB-468-bearing mouse model in which human pan-T cells were engrafted, CH17 delayed tumor growth by engaging T cells and exerted a synergistic effect in combination with an anti-human PD-1 antibody. ConclusionsWe successfully generated an immunomodulatory antibody targeting B7-H4 that possesses both T cell immune checkpoint inhibitory activity and ADCC activity in B7-H4-positive tumors. B7-H4-targeting antibodies might represent a promising immunotherapy for B7-H4-expressing tumors.