Development of A humanized OPN blockade antibody for human colorectal and pancreatic cancer immunotherapy
A. Intellectual Property
B. There is an unmet need and hugh market for new immunotherapeutic drugs for human colorectal and pancreatic cancer patients
C. Chemedimmune is developing an Osteopontin (OPN) blockade antibody as a new class of immunotherapeutic drug for colorectal and pancreatic cancer treatment.
C1. Our scientific publications that support OPN blockade antibody as an new class and effective immunotherapy for human colorectal and pancreatic cancers.
- John D. Klement, Amy V. Paschall, Priscilla S. Redd, Mohammed L. Ibrahim, Chunwan Lu, Dafeng Yang, Esteban Celis, Scott I. Abrams, Keiko Ozato, and Kebin Liu. 2018. An osteopontin/CD44 immune checkpoint controls CD8+ T cell activation and tumor immune evasion. Journal of Clinical Investigation. 128:5549-5560. PMID: 30395540.
- Hannah R. Moorman, Dakota Poschel, John D. Klement, Chunwan Lu, Priscilla S. Redd, and Kebin Liu.2020. Osteopontin: A Key Regulator of Tumor Progression and Immunomodulation. Cancers 12:3379. PMID: 33203146.
- John D. Klement, Dakota B. Poschel, Chunwan Lu, Alyssa D. Merting, Dafeng Yang, Priscilla S. Redd, and Kebin Liu. 2021. Osteopontin Blockade Immunotherapy Increases Cytotoxic T Lymphocyte Lytic Activity and Suppresses Colon Tumor Progression. Cancers 13:1006. PMID: 33670921.
- Christopher Gromisch, Motaz Qadan, Mariana Albuquerque Machado, Kebin Liu, Yolonda Colson and Mark W. Grinstaff. 2020. Pancreatic Adenocarcinoma: Unconventional Approaches for an Unconventional Disease. Cancer Res. 80:3179-3192.
OPN datasets in colorectal cancer and pancreatic cancer patients were extracted from TCGA database and analyzed. OPN expression level is significantly higher in colorectal carcinoma as compared to the non-neoplastic colon (Fig. 1A) and significantly higher in pancreatic tumor as compared to normal pancreas (Fig. 1E). ELISA analysis of serum samples revealed that OPN protein level is significantly higher in both colorectal and pancreatic cancer patient than in healthy donors (Fig. 1B & F). IHC analysis indicates that OPN protein level is higher in colon tumor cells (Fig. 1C), pancreatic tumor cells (Fig. 1G) and tumor-infiltrating immune cells (Figs. 1C & G) than in normal epithelial cells. OPN mRNA expression level is inversely corrected with survival time of colorectal (Fig. 1D) and pancreatic cancer patients (Fig. 1H).
Figure 1. OPN promotes human colorectal (A-D) and pancreatic (E-H) cancer progression. A & E. OPN expression level in normal tissues and tumors. B & F. OPN protein level in serum from healty donors and colorectal (B) and pancreatic (F) cancer patients. C & G. Matched pairs of human colon carcinoma and non-neoplastic colon (C, n=5) and human pancreatic adenocarcinoma and pancreas (G, n=10) were stained with OPN-specific antibody. Shown are representative images. Scale bar=100 mM. Green arrow: normal colon or pancreas cells; red arrow: colon or pancreatic tumor cells; yellow arrow: immune cells. D and H. OPN expression and survival datasets were extracted from human colorecatal (D) and pancreatic (H) cancer datasets and plotted for survival.
To investigate the cellular source of OPN in cancer patients, we mined scRNA-Seq datasets deposited in the GEO database (GSE146771). Colorectal tumor-resident immune cells were annotated. Cellular subtype analysis demonstrated that principally myeloid cells, as well as innate lymphoid cells (ILCs) and malignant cells, drove increased OPN levels (Fig. 2). Expression of SPP1 was enriched in colorectal tumor tissues compared to matched PBMC and healthy colon.
Figure 2. OPN expression profiles in the single cell level in human colorectal cancer patients. A. UMAP projection of human colorectal carcinoma 10x scRNA-seq data. Cells are annotated by clustering. B. Expression of SPP1 in indicated cell types from the single cell mRNA expression quantification, subsetted by tissue of origin. N: Normal Colon; P: PBMC; T: colorectal tumor.
To validate the function of OPN in tumor development, we made use of the highly immunogenic methylcholanthrene (MCA)-induced tumor model 31. WT mice exhibited a dramatically higher (70.3% or 26/37) MCA-induced tumor incidence rate than OPN KO mice (18.8, or 6/32)(Fig. 3A). Analysis of tumor size indicates that OPN deficiency results in a significant decrease in the overall tumor growth rate in tumor-bearing mice (Fig. 3B). These findings validate that OPN promotes tumor initiation and growth in vivo.
Our published data revealed that OPN expression level is positively correlated with impaired T cell activation in vivo, suggesting that OPN may suppress CD8+ T cell activation. To test this hypothesis, we cultured T cells in the presence of recombinant mouse OPN protein and analyzed T cell activation. Indeed, OPN protein reproducibly inhibited CD8+ T cell activation and proliferation in a dose-dependent manner. Consistent with T cell activation inhibition, OPN inhibited IFNg serection in T cells. To determine whether the above findings can be translated to humans, CD3+ T cells were purified from 5 healthy donors and stimulated with anti-human CD3/CD28 in the presence of human OPN protein. Analysis of T cells revealed that OPN inhibited human CD8+ T cell activation and proliferation in a dose-dependent manner (Fig. 4A & B), and IFNg production in T cells (Fig. 4C).
To determine the function of OPN in tumor-T cell interactions, we knocked out Spp1, the gene that encodes OPN protein, in CT26 cells (Fig. 5A). Analysis of CTL effector function in a tumor and tumor-specific CTL co-culture model determined that knocking out OPN in the target tumor cells results in a significant increase in efficacy of the tumor-specific CTL in killing the target tumor cells (Fig. 5B). To determine whether this finding can be translated to tumor suppression in vivo, CT26 scramble and CT26 OPN KO tumor cells were injected to mice. Significantly more tumor nodules were formed in the WT mice than in the OPN KO mice in the experimental metastasis mouse model (Fig. 5C).
OPN is a secreted protein that is elevated in human cancer patient peripheral blood. The literature and our above findings indicate that OPN is expressed in various resident cells in the tumor microenvironment, suggesting that targeting OPN protein, rather than targeting a particular cell type, is potentially a more effective approach in cancer immunotherapy. Our published data thus provide a strong rationale to develop an OPN neutralization monoclonal antibody. We have now developed four OPN monoclonal antibodies (Fig. 6A). All four mAbs have high OPN protein-binding affinity (Fig. 6B). Three of the four mAb clones significantly increased the lytic activity of a tumor-specific CTL s in lysing the target CT26 tumor cells in vitro (Fig. 6C). Furthermore, these three OPN mAbs are effective in blocking OPN-mediated suppression of T cell activation in vitro (Fig. 6D).
To determine whether the above findings can be translated to tumor growth suppression in vivo, we injected CT26 tumor cells to mice. The tumor-bearing mice were then treated with OPN mAbs alone or in combination with anti-PD-1 mAb. OPN mAb clones 100D3 and 103D6 significantly suppressed CT26 tumor growth in mouse lungs (Fig. 7). 100D3 and 103D6 also increased the efficacy of anti-PD-1 immunotherapy, albeit to a small degree (Fig. 7). In summary, OPN mAb clones 100D3 and 103D6 are effective in increasing CTL lytic activity and in enhancing colon tumor growth control in immune competent mice.
Figure 7. OPN blockade immunotherapy suppresses colon tumor growth in vivo. CT26 cells were injected i.v. into mouse tail vein. Three days later, IgG and the indicated OPN mAbs (200 mg/mouse) were injected to the tumor-bearing mice s.c. either alone or in combination with anti-PD-1 (n=5) every 3 days. Mice were sacrificed on day 14 and analyzed for lung tumor nodules. Shown are representative tumor-bearing lungs of each treatment group (left). The tumor nodule number was presented at the bottom.
These 4 OPN mAb were purified in large quantity with low endotoxin level. To determine whether these antibodies have affinity for human OPN protein, we performed ELISA using recombinant OPN protein. Human OPN protein was coated to the tissue culture plate. Purified OPN mAbs were added to the culture plates and analyzed by ELISA using an anti-mouse ELISA kit and the TMB Substrate Set. Among the 4 mAb clones, 100D3 showed high binding affinity to human OPN protein (Fig. 8).
The high affinity of 100D3 for human OPN protein and its excellent OPN-blocking activity (Fig. 6), and in vivo tumor suppression efficacy in immune competent mice (Fig. 7) made 100D3 an ideal candiate for humanization and translation to human cancer immunotherapy.
Figure 9. Development of humanized h103D3 antibodies. A. SDS-PAGE results of purified humanized antibodies under non-reducing and reducing (top) and reducing conditions (bottom). Shown are antibodies 1-11 of the 16 antibodies listed in C. B. Examples of sensor-grams of human OPN binding to chimeric antibody (top) and humanized OPN antibody (bottom, VH1+VL3). C. The affinity of human OPN protein to 16 humanized OPN antibodies.
We are preparing for IND-enabling studies of hu100D3 to move hu100D3 to a phase I clinical trial in human colorectal and pancreatic cancer patients.