OPN Antibody

Development of A humanized OPN blockade antibody for human colorectal and pancreatic cancer immunotherapy

A. Intellectual Property

US patent 62/944,777: Osteopontin Monoclonal Antibodies for Cancer and Osteoporosis Immunotherapy.

B. There is an unmet need and hugh market for new immunotherapeutic drugs for human colorectal and pancreatic cancer patients

Despite the breakthrough of cancer immunotherapy with durable efficacy in many types of human cancers, colorectal and pancreatic cancers do not respond to current immuntherapies. There is therefore an unmet need and huge market for new immunotherapuetic agents for colorectal and pancreatic cancers.

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.

  1. 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.
  2. 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.
  3. 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.
  4. 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.
C2. Scientific data support the development of OPN blockade immunotherapy for human colorectal and pancreatic cancers
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.

C2.2. OPN is primarily expressed in tumor cells, myeloid cells and ILCs in human colorectal cancer
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.

C2.3. OPN promotes tumorigenesis and tumor development
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.
Figure 3. OPN promotes tumor development. A. WT and Spp1 KO mice were injected with MCA and monitored for tumor development. Shown are percentages of WT and OPN KO mice with tumor 102 days after MCA injection. B. The tumor sizes were measured in WT and OPN KO mice and plotted.
C2.4. OPN is an immune checkpoint that negatively regulates CD8+ T cell activation
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).
Figure 4. OPN inhibits human T cell activation. A. CD3+ T cells were purified from PBMC of healthy donors, labeled with CFSE, and cultured in plates coated with anti-CD3 and anti-CD28 mAbs and human OPN at the indicated concentrations for 3 days. CD8+ T cells were analyzed for CFSE intensity. Shown is representative data from one of five healthy donors. B. CFSE intensity as shown in A was quantified as division index. C. Culture supernatants in A were collected and measured for IFNg protein level by ELISA.
C2.5. OPN inhibits T cell activation to suppress CTL cytotoxicity to promoter tumor growth
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).
Figure 5. OPN inhibits tumor-specific CTL lytic activity. A. CT26 cells were transduced with lentiviral particles containing nontargeting sgRNA (scramble) or Spp1-targeting sgRNA (OPN.KO). Following puromycin selection to establish stable celll lines. Supernatants were collected from culture and measured for OPN protein by ELISA. B. CT26.Scramble and CT26.OPN.KO cells were co-cultured with the tumor-specific CTLs for 24h and analyzed by flow cytometry for cell death. C. CT26.Scramble and CT26.OPN.KO cells were injected to BALB/c mice intravenously. Lungs were examined 14 days later for tumor nodules (left panel) and tumor nodules were quantified (right panel).
C2.6. OPN neutralization increases CTL lytic activity
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).
Figure 6. OPN neutralization mAbs increase CTL tumor-lysing activity. A. Scheme of OPN neutralization monoclonal antibody generation. B. The four OPN mAb clones were tested for binding affinity to OPN protein by ELISA. C. CT26 cells were seeded in 96-well plates for 24h. IgG and the indicated OPN mAbs and CTLs were added to the tumor cell culture for 24h. Cell mixtures were collected, stained with CD8, Annexin V, and PI, and analyzed by flow cytometry. Tumor cell death was quantified. D. CSFE-labeled CD3+ T cells were cultured in 96-well plates coated with anti-CD3, anti-CD28, and OPN as indicated. Cells were analyzed for CSFE intensity 3 days later by flow cytometry. Shown are quantification of % divided cells as determined by CFSE intensity.
C2. 7. OPN blockade immunotherapy suppresses colon tumor growth in vivo
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.

C2.8. OPN clone 100D3 has high affinity for human OPN protein
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).
Figure 8. OPN clone 100D3 has high affinity for human OPN protein. Human OPN protein was coated to a 96-well plate. OPN mAb clones and IgG were added at the indicated concentrations and incubated for 1h, washed throughly, probed with anti-mouse IgG-HRP, and detected using Biolegend ELISA detection kit. The absorbance was plotted against the OPN mAb concentration.
C2. 9. Development of humanized OPN antibodies
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 contracted the 100D3 humanization project to a CRO. Complementarity-determining regions (CDR) grafting plus back mutation method was used to create humanized antibodies without sacrificing the binding affinity of the parental (chimeric) antibody. The CDR amino acids of OPN 100D3 was determined by protein sequencing. The DNA sequences encoding the chimeric antibody heavy and light chains were synthesized and inserted into the pcDNA3.4 vector to construct expression plasmids of full-length human IgG1. Expression of the chimeric antibody was conducted in Expi293F cells and the supernatants were purified with protein A affinity column. The chimeric antibody was validated by its binding adffinity to human OPN protein by a Surface Plasmon Resonance (SPR) biosensor, Biacore 8K (GE Healthcare) (Fig. 9) and ELISA (Data not shown). The structure of parental antibody was modeled by computer-aided homology modeling program. Humanized antibodies were designed using CDR grafting. Briefly, the CDRs of parental antibody were grafted into the human acceptors to obtain humanized light chains and humanized heavy chains for each parental antibody. Four heavy chains (VH1, VH2, VH3 and VH4) and 4 light chains (VL1, VL2, VL3 and VL4) were paired with each other for affinity ranking experiment. A total of 16 humanized antibody expression vectors and cell lines have been created. The purified antibodies have the correct protein sizes under native and denaturing conditions and were purified to 99% purity (Fig. 9A). These antibodies retained binding affinity to human OPN protein as determined by SPR assay (Fig. 9B & C) and ELISA. Antibody h100D3 (VH1+VL3, hu100D3) is currently our lead agent for clinical development.
C3. Current development status
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.