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Background: Thailanstatins are naturally occurring anti-proliferative compounds that target spliceosomes and modulate pre-mRNA splicing. Alterations in splicing machinery and mRNA splicing is common in cancer and represents a potential susceptibility that can be exploited by targeted delivery of a splicing modulator to tumors with antibody drug conjugates (ADCs). Results: PH1 payload is a novel derivative of Thailanstatin optimized for metabolic stability and anti-tumor activity. PH1 is an efficient modulator of splicing in vitro and in vivo and a poor substrate for the MDR1 transporter. Transcriptome analysis of PH1- treated NCI-N87 cells revealed multiple classes of mis-splicing events including elevated expression of transcripts with skipped exons (3364 genes/4x change) and retained introns (332 genes/2.5x change). Translation of transcripts with retained introns and novel exon-exon junctions results in generation of neoepitopes. Upon conjugation to Trastuzumab, Tras PH1 ADC exhibited nanomolar potency specific to HER2-expressing cancer cells, with durable anti-tumor response in vivo and favorable pharmacokinetic exposure in mice. Due to the potential for generation of neoepitopes, combination studies of Tras PH1 with a checkpoint inhibitor antibody was performed in a syngeneic MC38-HuHer2 model. Single agent and combination treatments induced tumor recruitment of CD11b+ positive myeloid cell subsets. While the combination treatment eradicated tumors, the treated animals also developed tumor-specific immunity. A toxicology study performed in cynomolgus monkeys confirmed favorable PK profile and a wide safety margin for Tras PH1 ADC. Conclusions: We present the development of a splicing modulator-based payload class with the ability to target tumor mRNA splicing, induce tumor neoepitopes, recruit myeloid cells and generate anti-tumor immunity. These findings support the development of ADCs using this novel class of immunostimulatory payload. Citation Format: Satyajit K. Mitra, Vasu Jammalamadaka, Jeffrey Kang, Teodora Losic, Greg Tuffy, Tony W. Liang, Kim Tipton, Adriana Lopez, Scott Savage, William Monteith, William E. Haskins, Melissa S. Jurica, Sanjeev Satyal, Mary Do. Development of a splicing modulator-based ADC payload class with immune stimulatory properties for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1832.

A hallmark of the clinical course of patients with Inflammatory Bowel Disease (IBD) is poorly healing erosions and ulcers in the intestinal mucosa. Despite the adverse clinical consequences of non-healing mucosal wounds in IBD, current front-line therapies that selectively target mucosal wound healing are not available. Recent studies revealed an association between colonic inflammation and aberrant glycosylation of epithelial CD44v6. Under conditions of inflammation, epithelial CD44v6 was shown to be decorated with the terminal glycan sialyl Lewis A. Importantly, targeting sialylated Lewis glycans on CD44v6 with a murine antibody GM35 was shown to promote mucosal wound healing in cell lines and in biopsy based wounding assays as well as dextran sodium sulfate (DSS) induced murine colitis models. We sequenced CDRs from GM35 and produced a humanized antibody. Eight candidate human IgG1 clones were produced and screened. hPTM-001.4772 was selected from the eight candidates based on glycan affinity and selectivity compared to GM35. In vitro wound healing studies revealed that PTM-001.4772 was as effective as GM35 in promoting repair of scratch wounds with human intestinal epithelial cells. Furthermore, intraperitoneal injection of mice with hPTM-001.4772 during induction of DSS colitis resulted in reduced weight loss compared to control IgG. These results suggest that hPTM-001.4772 is well-positioned as a unique potential candidate therapeutic for IBD that can be used to selectively promote healing of mucosal wounds and ulcers.

RAV12 is a chimeric antibody that recognizes an N-linked carbohydrate antigen (RAAG12) strongly expressed on multiple solid organ cancers. More than 90% of tumors of colorectal, gastric, and pancreatic origin express RAAG12, and a majority of these tumors exhibit uniform RAAG12 expression. RAV12 exhibits potent cytotoxic activity in vitro against COLO 205 colon tumor cells via an oncotic cell death mechanism. RAV12-treated COLO 205 cells undergo morphologic changes consistent with oncosis, including cytoskeletal rearrangement, rapid plasma membrane swelling, and cell lysis. RAV12 inhibited the growth of RAAG12-expressing gastrointestinal tumor xenografts in athymic mice. In the case of SNU-16 tumor cells, twice weekly treatment of established s.c. tumors with 10 mg/kg RAV12 caused a ∼70% suppression of tumor growth at the end of the study. This preclinical data has led to the initiation of a phase I/IIA clinical study of RAV12 in patients with metastatic or recurrent adenocarcinoma. [Mol Cancer Ther 2007;6(3):856–65]

Antibodies directed to specific disease-related antigens have proven to be very successful therapeutics for a variety of disease indications. In spite of their high affinity and specificity for target antigen, target-mediated toxicity constitutes a major limitation for the development of antibodies to certain targets. We have addressed this type of on-target toxicity by developing a new class of targeting antibodies (Probody™ therapeutics) that remain in an inert, masked form until proteolytically activated at the site of disease. As a proof-of-concept for the construction of a Probody, we used cetuximab as a starting point. Cetuximab is an EGFR-targeted antibody approved for the treatment of colorectal and head-and-neck cancers that produces an on-target toxicity in the form of a skin rash that afflicts 88% of patients treated with the antibody. We engineered an EGFR Probody by incorporating an inhibitory masking peptide fused to the antibody light chain. Masking of the Probody is achieved through a linker that also incorporates a substrate that is cleaved by one or more proteases up-regulated in cancer. In vitro, EGFR binding and cell-based activities of the masked Probody were diminished compared to those of cetuximab, but treatment with exogenous target proteases activated the Probody and restored activity comparable to cetuximab. Using tumor xenograft models in mice, we demonstrated that the Probody remained masked in systemic circulation but was activated and accumulated in the tumor microenvironment. Tumor activation of the Probody translated to efficacy similar to that seen with cetuximab. Consistent with our results in mice, the Probody remained efficiently masked in non-human primates and did not cause skin toxicity such as that observed in animals treated with cetuximab. Together, these results demonstrate that antibody activity can be specifically targeted to diseased tissue by utilizing locally overexpressed proteases as activating agents, suggesting that a variety of antigens not previously amenable to an antibody therapeutic approach may be successfully addressed with Probodies. Citation Format: Luc R. Desnoyers, Annie Yang, Tony W. Liang, Stephen Moore, Jason Sagert, Daniel R. Hostetter, Elizabeth Menendez, Fei Han, Michael Krimm, Ken Wong, Jennifer Richardson, Jim W. West, Shouchun Liu, Olga Vasiljeva, Henry B. Lowman. Development of a proteolytically activatable EGFR Probody for cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4570. doi:10.1158/1538-7445.AM2013-4570

Antigens that are tumor-specific or over-expressed on cancer cells represent opportunities for developing target-specific antibody-based therapeutics. Utilizing a non-target-biased intact cell-based immunization approach, we have generated greater than 1600 mAbs to cell-surface proteins that are expressed on cancer cells. A subset of the mAbs exhibit differential reactivity to tumor cells compared to normal cells. One of the differentially expressed proteins identified was B7-H3 (also referred to as CD276). B7-H3 is a member of the B7 family of immune regulatory proteins. Over-expression of B7-H3 has been correlated with disease severity and outcome in a growing number of cancer types, and several lines of evidence support a functional role for B7-H3 in cancer. In this report we describe the development of a humanized anti-B7-H3 mAb, designated MGA271, which bears an engineered Fc domain that imparts enhanced antibody-dependent cellular cytotoxicity (ADCC) through increased affinity for the human activating Fc-gamma receptor IIIA (CD16A) and decreased affinity for the human inhibitory Fc-gamma receptor IIB (CD32B). MGA271 displays strong reactivity to multiple tumors including kidney, prostate, pancreatic, breast, colon, gastric and ovarian cancer as well as melanoma, but limited reactivity toward normal human tissues. Independently of the donor's CD16A genotype, MGA271 mediates ADCC in vitro against human tumor cell lines representing these tumor types, as well as to human lung and gastrointestinal cell lines that exhibit properties of cancer stem cells. MGA271 exhibits potent anti-tumor activity toward B7-H3-expressing tumor xenografts in mice knocked-out for the murine CD16 gene and transgenic for the low affinity allele of human CD16A. Single- and repeat-dose toxicology studies were carried out in cynomolgus monkeys and no significant test article-related safety findings were observed. Taken together, these data support the clinical development of MGA271 for the treatment of patients who have B7-H3-expressing cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2725. doi:1538-7445.AM2012-2725