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Small Molecule Agents

F5446: A small molecule SUV39H1-selective inhibitor and the next generation epigenetic agent for human colorectal cancer immunotherapy

A. Intellectual Property
Patent: US 10,577,371 B2: Small Molecule Histone Methyltransferase SUV39H1 Inhibitor and Uses Thereof (approved on March 3, 2020)

Licensing Contact: Mr. Carl Clark, Director Innovation Commercialization, Augusta University, 1120 15th Street, Augusta. GA 30912. USA.
Tel: 1-706-721-4055

B. Colorectal Cancer Immunotherapy Background and Market Value

Human CRC does not respond to immune checkpoint inhibitor immunotherapy. Recent breakthroughs in immune checkpoint inhibitor (ICI) cancer immunotherapy, particularly anti-PD-1 immunotherapy, have resulted in durable efficacy in many types of human cancers. Although compelling data from human CRC patients and mouse tumor models demonstrate that CRC is a highly immunogenic tumor type(1-8), CRC, except for the small subset of microsatellite instable (MSI) CRC which accounts for approximately 4% of human CRC cases, does not respond to anti-PD-1 immunotherapy. Therefore, lack of response to ICI immunotherapy is another significant challenge in human CRC management. The underlying mechanism of CRC non-response to anti-PD-1 immune therapy is currently unknown. It has been generally believed that the high tumor mutation burdens (TMB) serve as neoantigens to generate tumor-reactive cytotoxic T lymphocytes (CTLs) in MSI CRC(9, 10). Our recent published preliminary studies determined that CTLs heavily infiltrates both MSI and microsatellite stable (MSS) human colon carcinoma(11), which thus challenges this above notion and indicates that CTL functional deficiency, not CTL infiltration level, is at least in part responsible for human CRC non-response to anti-PD-1 immunotherapy. Logically, targeting CTL function directly in the tumor microenvironment is potentially an effective immunotherapeutic approach to suppress CRC. Our agent F5446 serves as such a drug.

C. Scientific Data that support F5446 as a promising immunotherapeutic agent

C1. Our published data of F5446:

  1. Chunwan Lu, John D. Klement, Dafeng Yang, Thomas Albers, Iryna O. Lebedyeva, Jennifer L. Waller, and Kebin Liu. 2020. SUV39H1 regulates human colon carcinoma apoptosis and cell cycle to promote tumor growth. Cancer Lett. 28;476: 87-96.
  2. Chunwan Lu, Dafeng Yang, John D. Klement, II Kyu Oh, Natasha M. Savage, Jennifer L. Waller, Aaron H. Colby, Mark W. Grinstaff, Nicholas H. Oberlies, Cedric J. Pearce, Zhiliang Xie, Samuel K. Kulp, Christopher C. Coss, Mitch A. Phelps, Thomas Albers, Iryna O. Lebedyeva, and Kebin Liu. 2019. SUV39H1 represses the expression of cytotoxic T lymphocyte effectors to promote colon tumor immune evasion. Cancer Immunol. Res. 7:414-427. PMCID:PMC6397681.

C2. Determination of the SUV39H1-H3K9me3 axis as Gzmb repressor in tumor-infiltrating CTLs.
C2.1. CTL tumor-infiltration levels in MSS and MSI human colon carcinoma: Human MSS and MSI

Figure 1. Tumor-infiltration CTLs in MSS and MSI human colon carcinoma. A. Representative images of MSS (n=9) and MSI (n=8) human colon carcinoma showing CD8+ T cell infiltration. B. Quantification of CD8+ CTLs in the MSS and MSI colon carcinoma. Each dot represents number of CD8+ CTLs in one view area of a tumor specimen.

colon carcinoma specimens were analyzed by immunohistochemical staining of CD8 T cells. As expected, all eight MSI colon carcinoma specimens exhibit medium to high levels of tumor-infiltrating CTLs. However, eight of the nine MSS colon carcinoma specimens also exhibit medium levels of tumor-infiltration CTLs (Fig. 1A). No statistically significant difference in CTL tumor infiltration levels was observed between MSI and MSS colon carcinoma specimens (Fig. 1B).

C2.2. SUV39H1 is highly expressed in tumor cells and tumor-infiltrating CTLs in the tumor microenvironment:M The above observation that no significant difference in CTL levels exists between MSS and MSI colon carcinomas suggest that a lack of CTL infiltration might not be the sole mechanism underlying colon cancer immune escape. It is therefore possible that the functional status of the tumor-infiltrating CTLs is also responsible for colon cancer immune escape. We screened the TCGA database for altered gene expression between human colon carcinoma tissues and normal colon tissues to identify genes with known function in regulating CTL functions. We found that SUV39H1, which is an H3K9me3-specific histone methyltransferase recently shown to regulate CTL effector expression (22), is significantly elevated in the tumor tissue (Fig. 2A). Next, we sought to determine the expression levels of SUV39H1 in tumor-infiltrating CTLs in the MC38 colon carcinoma mouse model. CD45- tumor cells and CD8+ CTLs were isolated from fresh MC38 tumor tissues and analyzed for SUV39H1 mRNA levels. As in the human colon carcinoma tissues, SUV39H1 is highly expressed in the tumor-infiltrating CTLs and in the tumor cells in the MC38 tumor tissue (Fig. 2B).

Figure 2. SUV39H1 expression is elevated in both colon carcinoma cells and tumor-infiltrating CTLs. A. Data sets of SUV39H1 mRNA level of human colon carcinoma (n=383) and normal colon tissues (n=50) were extracted from TCGA database and compared. B. Tumors were excised from two MC38 tumor-bearing mice and digested with collagenase. Portion of the tumor cells were incubated with anti-CD45 mAb-conjugated magnetic beads to deplete leukocytes to isolate tumor cells. Another portion of the tumor cells was incubated with anti-CD8 mAb-conjugated magnetic beads to isolated CD8+ tumor-infiltrating CTLs. RNA was prepared and analyzed by qPCR using SUV39H1-specific primers. Upper panel: the two bars indicate relative Suv39h1 mRNA level of the indicated cells from two mice. Bottom panel: agarose gel showing SUV39H1 DNA fragment. Rpl13a was used as normalization control.

C2.3. The Gzmb promoter is enriched with H3K9me3 in T cells: Mining ChIP-Seq data sets in GEO database revealed that H3K9me3 is enriched in the promoter regions of several T cell effectors including Gzmb in human T cells (GEO accession# GSM1058783)(Fig. 3A), suggesting that Gzmb may be repressed by the Suv39h1-H3K9me3 axis in T cells. We then purified CD3+ T cells from C57BL/6 mouse spleens and performed ChIP analysis using an H3K9me3-specific antibody and PCR primers that cover the promoters approximately from -2000 to +1000 relative to the Gzmb transcription start sites (Fig. 3B), and determined that H3K9me3 is enriched in the Gzmb promoter in resting mouse T cells (Fig. 3C) and tumor-infiltration T cells (Fig. 3D)(11). These observations indicate that the Suv39h1-H3K9me3 axis suppresses Gzmb expression of in resting T cells.

Figure 3. The Gzmb promoter is enriched with H3K9me3 in T cells. A. A snapshot of the H3K9me3 peak at the GZMB promoter in human T cells. B. Mouse Gzmb promoter structure. The ChIP PCR primer locations are indicated. C. Mouse CD3+ T cells were analyzed by ChIP using H3K9me3-specific antibody and gene-specific PCR primers as shown in B. D. CD8+ CTLs were isolated from pooled tumor tissues of five CT26 tumor-bearing mice and analyzed by ChIP with anti-H3K9me3 antibody and gene-specific PCR primers as indicated in B.

D. Development of the SUV39H1-selective small molecule inhibitor F5446.
The above observations provided strong rationale to develop a SUV39H1-specific inhibitor to target H3K9me3 to up-regulate Gzmb expression. In collaboration with a chemist, we developed the SUV39H1-selective small molecule inhibitor F5446 through a three-year effort as outlined in Fig. 4A. We have also developed a large-scale chemical synthesis procedure of F5446 through a commercial contract service (Fig. 4B in ref (11)). F5446 has a molecular weight of 552.95 g/mol (Fig. 4B) and exhibits an EC50 of 0.496 µM in an in vitro enzymatic activity assay (Fig. 4C)(11).

E. F5446 Efficacy and Toxicity Test in Mouse Models

E1. F5446 increases T cell effector expression to suppress colon carcinoma growth in vivo:
The perforin-granzyme cytotoxic pathway is one of the two effector mechanisms by which CTLs induce apoptosis of tumor cells(12-14). We then hypothesized that F5446 inhibits Suv39h1 to decrease H3K9me3 deposition at the Gzmb promoter in tumor-infiltrating CTLs to suppress colon carcinoma development. To test this hypothesis,

Figure 5. F5446 targets the H3K9me3 to increase Gzmb expression to suppress colon carcinoma growth in vivo. A. CT26 (2x105 cells) were injected to the right flanks of thirty BLAB/c mice. Twenty mice with similar sized tumors were randomly assigned into four groups at day 11 after tumor cell injection. The four groups of tumor-bearing mice were treated with vehicle and IgG (control), F5446 (10 mg/kg), anti-PD-1 mAb (200 g/mouse), and F5446+anti-PD-1 mAb, respectively, every two days for 14 days. Shown are tumor images (left panel) and tumor size (right panel). ** p<0.01. B. Tumor weight at the end of the experiments. C. CD8+ CTLs were isolated from pooled tumor tissues of five CT26 tumor-bearing mice from the control and F5446 treatment groups as shown in A. The isolated CTLs were then analyzed by ChIP with anti-H3K9me3 antibody as show in Fig. 3C &D. D. The isolated CTLs as shown in C were also analyzed by qPCR for the expression of granzyme B. Differences between control and the treatment groups were determined by two-tail t test with p<0.05 as being statistically significant. Column: mean; Bar, SEM.

CT26 colon carcinoma mouse model was used. CT26 cell line is highly metastatic to the liver and lung(15-17). CT26 tumor-bearing mice were treated with IgG, F5446, anti-PD-1 mAb and F5446+anti-PD-l. Both F5446 and anti-PD-1 treatments significantly reduced tumor growth (Fig. 5A & B). F5446 and anti-PD-1 did not show additive or synergistic effects in this tumor model (Fig. 5A & B). CD8+ CTLs were then isolated from the tumor tissues and analyzed by ChIP for H3K9me3 level at the Gzmb promoter region. F5446 therapy significantly decreased H3K9me3 deposition at the Gzmb promoter (Fig. 5C) and increased granzyme B expression in tumor-infiltrating CTLs (Fig. 5D).

E2. In vivo tolerability of F5446: We then collaborated with the Mitch Phelps Lab at Ohio State University to perform F5446 in vivo toxicity studies. Mice were dosed every two days with vehicle, 10 or 20 mg/kg body weight F5446 for a total of 7 doses. Observations of body weights during the 2-weeks dosing period indicated that mice in the 20 mg/kg group lost body weight on average less than 10%. The 10 mg/kg body weight group maintained a stable body weight throughout the dosing period, whereas the vehicle-treated control group

increased average body weight by ~10% (Fig. 6 and supplementary table S2 in ref (11)). Complete blood counts indicated no significant differences between vehicle and the F5446 treatment groups (Supplementary Table S3 in ref(11)). Serum chemistry data suggested no difference between vehicle and drug-treated groups with all markers, except amylase and lipase, which were reduced, although still within normal levels in the F5446 treatment groups (Supplementary Table S4 in ref(11)). These preliminary data determined that F5446 is well-tolerated at a dose twice as the efficacious dose.

Figure 6. Mouse weight change kinetics. Mice were treated with vehicle (n=5), F5446 (n=5) at the indicated dose every 2 days for 7 times.

F. Summary of F5446 mechanism of action

ICI immune therapy (e.g., anti-PD-1 mAb Keytruda and Opediv) acts through blocking PD-1 and PD-L1 interactions to unleash PD-L1-suppressed tumor-infiltrating CTLs to kill tumor cells. Our published data determined that CTLs infiltrates both MSI and MSS human colorectal carcinoma. We also determined that PD-L1-independent immune suppressions, such as MDSC-mediated immune suppression, plays a key role in colorectal cancer immune evasion. Moreover, we determined that the SUV39H1-H3K9me3 epigenetic axis silences Gzmb expression in tumor-infiltrating CTLs to impair CTL effector function. Based on this mechanism, we developed a human SUV39H1-selective small molecule inhibitor F5446 that is effective in directly activates Gzmb expression in CTLs, which bypass all types of immune suppressions to activate CTL functions to suppress CRC development (Fig. 7). F5446 thus can be a monotherapeutic agent or can be used in combination with anti-PD-1 immunotherapy.

Figure 7. F5446 mechanism of actin

G. Market Need
According to Clarivate Analytics, the global market for ICI cancer immunotherapy in 2022 is projected at about $34.652 billion. In 2016, the sales of drugs for colorectal cancer in the United States is $6.1 billion (chemotherapies $1.0 billion, anti-VEGF therapies $3.5 billion, anti-EGFR therapies $1.4 billion, and other targeted agents $0.2 billion). The market projection for 2026 is about $7.6 billion (chemotherapies $0.7 billion, anti-VEGF therapies $3.2 billion, anti-EGFR therapies $1.4 billion, BRAF or MEK inhibitors $0.7 billion, other TKIs $0.2 billion, and Check point inhibitor $1.1 billion. It is very clear that the PD-1 and PD-L1 inhibitors are the biggest growth area in CRC market. There are no reliable projections of ICI immunotherapy for colorectal cancer since there is no current development and sale data for the projection, but once ICI becomes effective in colorectal cancer, the annual market value should be at least in the billions. In addition, human pancreatic cancer also does not respond to ICI immunotherapy. As shown in Figure 7. F5446 can directly activate granzyme B to activate CTLs and therefore F5446 is effectively for immunotherapy of human colorectal cancer, and pancreatic cancer, and theoretically for all human cancer. We estimate the annual value of F5446 is in the billions if successfully developed.

H. References

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