Bertozzi’s TwoStep Therapeutics to Target Solid Tumors With Engineered Peptide Drug Delivery Vehicle

Caitlyn Miller’s interest in cancer started when her stepfather was diagnosed with oral cancer when she was 10 years old. His diagnosis kicked off a 14-year battle that included different treatments including surgery, radiation therapy, and chemotherapy. Seeing the devastating side effects of treatment, she knew that she wanted to someday work on treatment approaches that were both effective and specific. As she told GEN, “What really drew me into the field of tumor-targeted therapies was seeing that we could design better drugs that would only kill the tumor tissue, but not the healthy tissue.”

Now Miller, PhD, is making those dreams a reality. She is one of the co-founders and CEO of TwoStep Therapeutics, a newly-minted company launched to commercialize a targeted drug delivery platform for solid tumors. Other founders on the list include Carolyn Bertozzi, PhD, professor of chemistry, investigator at Howard Hughes Medical Institute and 2022 chemistry Nobel Laureate, Jennifer Cochran, PhD, senior associate vice provost for research and professor of bioengineering, and Ronald Levy, MD, professor of medicine in Stanford’s division of oncology.

TwoStep officially launched this week announcing a $6.5 million seed round led by NFX with participation from other investors including 2048 Ventures, Alexandria Venture Investments, Cooley’s affiliated fund GC&H Investments, and the family office of the founder of Arcadia Investment Partners. The company is also the first to emerge from the Stanford Innovative Medicines Accelerator’s entrepreneurship-in-residence program. 

Proceeds from the seed round will go toward developing a pipeline of solid tumor-targeting therapies delivered using a unique peptide vehicle, called PIP, developed in the Cochran laboratory in Stanford’s bioengineering department. “In its compact form, PIP is a synthetic, ultra-stable peptide that has been engineered to localize and penetrate tumors—ideal properties for broad targeted therapy applications,” Cochran explained in a statement. 

The company’s initial focus will be on targeted cytotoxic drug delivery and immunotherapy. They’ll be focused specifically on solid tumors that lack well-defined targets and as such have few targeted treatment options. 

A first-of-its-kind targeted delivery vehicle

Miller was drawn to the Cochran lab during her graduate program because of their work on tumor-targeting agents. They developed what ultimately became the PIP molecule with an eye toward using it to image solid tumors. The imaging results were strong so the scientists began thinking about leveraging it for therapeutic delivery. 

Polyspecific integrin-binding peptides or PIPs are engineered peptides that belong to a broader class of peptides called Knottins. These are disulfide-constrained peptides that fold into an intramolecular pseudoknot, a conformation that makes them highly stable and resistant to degradation by proteases. This stability makes it an attractive scaffold for protein engineering in the context of drug delivery. 

In searching for a peptide that worked for targeted tumor delivery, the Cochran lab engineered and tested multiple PIP scaffolds relying on the combined expertise of its founding team in chemical biology, antibody-drug conjugates, and immuno-oncology. They also established in vivo proof of concept of its multi-targeted agent using various therapeutic payloads and fusion proteins.

The PIP that will underlie TwoStep’s therapeutic pipeline stood out from the other ones tested because of its ability to bind selectively to five tumor-associated integrins, Miller explained. And it does so in a conformation-specific manner. Specifically, it binds preferentially to open, active conformation of the integrins, which limits its binding to healthy tissues. 

Other characteristics that make the PIP agent well suited for targeted therapy delivery are its enhanced tumor penetration capabilities as well as the ability to fuse the peptide sequence with biologics such as antibodies. And the agent can be manufactured relatively cheaply, Miller said. 

There is some flexibility regarding the exact mechanism used to deliver treatments using PIPs. Tumor cells internalize the integrins quite rapidly. So a PIP molecule could be attached to a drug conjugate with a cleavable linker and sent into the cells where the drug needs to be released. PIPs could also be used to deliver treatments that stay on the surface of the cancer cells, Miller said. 

They could also be used to deliver different kinds of payloads. There are no particular constraints in terms of the size of the payload but there are important considerations such as the hydrophilicity of the therapeutic—hydrophobicity can affect the biodistribution of a drug in the body.

In the study published in Cell Chemical Biology, the scientists describe delivering a PIP conjugated to a synthetic immunostimulant called CpG, a toll-like receptor 9 agonist, to mouse models of breast and pancreatic cancer. They showed that targeted delivery of a therapeutic using their approach “induces tumor regression and enhances therapeutic efficacy” compared with nontargeted delivery of CpG. In the Journal of Immunology study, they showed that administering a PIP conjugated to CpG both “promoted the regression of existing mammary tumors” and delayed the “growth of independent newly arising tumors” in genetically engineered mice.

In terms of which tumors it will target with PIP, the TwoStep team is considering a few cancer types as well as different payloads. For its drug conjugate pipeline, Miller said that TwoStep will target patients with tumors that don’t already have an approved antibody-drug conjugate. In the context of immunotherapies, she said the company will likely run a Phase I clinical trial that tests PIP in five different tumor types. That list includes melanoma based on evidence from an earlier trial that tested intratumoral delivery of the CpG payload to melanoma patients. The data from that trial indicated that the payload worked well for this cancer type so TwoStep intends to investigate the feasibility of doing targeted delivery of the immunostimulant using PIP. 

Miller hopes to have a clear lead program by the end of this year ahead of a planned Series A fundraising round slated for next year.  

While TwoStep is the company targeting tumor-associated integrins using PIP, other companies are interested in the potential of targeting tumor-associated integrins. Novartis for example, has a Phase I clinical trial testing a radioligand therapy for patients with advanced or metastatic tumors that express two integrins. Pfizer also has a drug for solid tumors in testing that also targets an integrin—one of the same ones that PIP targets.

This article has been updated to correct a previously reported detail about the earlier trial for melanoma involving the immunostimulant CpG. That initial trial focused on intratumoral delivery of the immunostimulant not delivery using PIP as stated earlier.