Over the last two decades, advancements in genetic engineering have led to new cancer immunotherapies. Genetically modified T cells express customizable chimeric antigen receptors (CAR) that efficiently hunt down specific proteins on cancer cells and initiate T cell attack (1). The now six U.S. Food and Drug Administration (FDA)-approved CAR T cell therapies are a game changer for treating certain lymphomas and leukemias. However, their efficacy in treating solid tumors is limited. While researchers are looking into modifying CAR T cells for solid tumors, others are focusing their efforts on another big player in the immune system: macrophages.
A new CAR on the lot
Solid tumors work hard to limit T cell trafficking and create unwelcoming, immunosuppressive conditions. Unlike B cell malignancies, which share common available targets for CAR T cell therapies, it is very difficult to find a universal antigen expressed on all solid tumor cells. Therapies that target a single antigen ignore antigen negative cells within the tumor, causing selective pressure and eventual resistance to those therapies.
This is where macrophages come into play. As the most abundant immune cell in the tumor microenvironment, macrophages readily traffic into solid tumors. In fact, solid tumors welcome macrophages because they are plastic. Tumor cells can easily convert macrophages from an anti-inflammatory phenotype to an immunosuppressive phenotype to help tumors grow. Outside of the tumor environment, macrophages are central regulators of the innate immune system and direct an antitumor immune response. These qualities led to early attempts by scientists to harvest monocytes from patients, grow them into macrophages in the lab, and reintroduce them into patients in high volumes to attack tumor cells (2). While these studies failed to show antitumor effects, they provided valuable information on the safety and feasibility of using macrophages in the clinic.
“The natural armamentarium of the nonengineered macrophage was too weak to combat the cancer,” said Michael Klichinsky, a pharmacologist at Carisma Therapeutics. This led Klichinksy and a team of researchers at the University of Pennsylvania, which has a rich history of CAR T cell therapy innovations, to search for new ways to equip macrophages with CARs. They published their findings in Nature Biotechnology in 2020 (3).
The team of researchers quickly learned that engineering macrophages was more difficult than engineering T cells. Traditionally, researchers use retroviruses or lentiviruses to introduce CARs into cells, but these methods were ineffective, leading Klichinsky and his team to turn to adenoviruses. Macrophages express cluster of differentiation 46 (CD46), a protein that allows adenovirus 35 (Ad35) to attach to the cell and release its genetic cargo. Klichinsky and his team modified Ad35 to transport CARs, creating a new Ad5f35 vector that exhibited excellent efficiency in delivering engineered CARs to macrophages. Adenoviral infections activate an immune response, so the researchers hypothesized that the vector would induce a proinflammatory macrophage state regardless of the cargo inside the Ad5f35 vector. However, further investigation into Ad5f35 revealed a secondary effect. To their surprise, the vector also locked macrophages into a permanent proinflammatory state, thus preventing tumors from turning macrophages to their advantage.
“When CAR macrophages get to the tumor, not only do they resist immunosuppression, but they drive inflammation. They help warm up the otherwise cold tumor microenvironment,” said Klichinsky. Just like CAR T cells, CAR macrophages kill cells expressing the targeted antigen. However, because macrophages are professional antigen presenting cells, they also gobble up tumor cells, process other tumor-derived antigens, and use these to prime secondary T cell adaptive immune responses. “You are essentially therapeutically vaccinating the patient against their own tumor antigens,” said Klichinsky. Ultimately, this leads to long term immune memory that protects from antigen negative relapse.
CAR macrophages in the fast lane
These promising results from Klichinsky and colleagues prompted the U.S. FDA to grant Fast Track designation to CT-0508, a CAR macrophage designed to target HER2-positive solid tumors, in September 2021. The research team is now enrolling patients with HER2 overexpressing solid tumors for which treatments are either unavailable or have failed in a Phase 1 clinical trial for CT-0508 (4).
Currently, both CAR T cell and CAR macrophage therapies are autologous cell therapies, meaning they use a patient’s existing cells. For this clinical trial, patients receive a bone marrow stimulator to trigger the release of monocytes. Once extracted, researchers differentiate the monocytes into macrophages in the lab, transduce them with the Ad5f35 vector carrying the antiHER2 CAR, and cryopreserve the engineered cells for reinfusion.
To assess the safety and tolerability of CT-0508, the clinical team administered genetically modified macrophages across three separate infusions into the first seven patients enrolled in the trial. The researchers also investigated a number of secondary measures, including clinical efficacy, cell kinetics, and T cell characteristics.
Paving the way to a bolstered immune landscape
In June 2022, the researchers presented data at the American Society of Clinical Oncology conference (5). They demonstrated a favorable safety profile with no major toxicities for CT-0508. Importantly, none of the patients exhibited neurotoxicity or major cytokine release syndrome, both of which are potential serious side effects for approved CAR T cell therapies. The authors found an initial cytokine surge in the bloodstream that quickly dissipated and corresponded with increased levels of CT-0508 in the tumor microenvironment. This aligned with the fact that mature macrophages do not linger in the bloodstream. “They’re there for a minute, and then they go park in the tissue,” said Kim Reiss, a medical oncologist at the University of Pennsylvania and principal investigator on the trial.
With respect to the clinical profile, at eight weeks post-infusion, four of the seven patients had stable disease, meaning minimal tumor shrinkage or growth, while the other three patients exhibited progressive disease, defined as at least a 20 percent increase in tumor growth.
The researchers ran T cell receptor sequencing on a subset of the patients to monitor changes in T cell repertoire following treatment. Reflective of an active immune response, they observed T cell expansion in the tumor periphery and microenvironment. These findings suggest that CT-0508 initiates an immune response and may also drive antitumor immunity.
To dig deeper, the authors used single cell RNA sequencing to assess remodeling of the tumor microenvironment following CAR macrophage treatment. After four weeks, the tumor microenvironment shifted towards an inflammatory state, evidenced by elevated proinflammatory macrophages as well as activated CD8 and CD4 T cells. “These findings suggest that these new T cells were not just randomly coming in, they were in fact, tumor-reactive,” said Klichinsky.
It is important to note the small sample size of this study. However, the authors are optimistic and are currently enrolling patients for group two of the Phase 1 trial. Group two patients will receive a single infusion instead of three spaced infusions. “We're looking to see if [fewer infusions] change the safety profile, but it is not expected to,” said Reiss. Additionally, Carisma Therapeutics is opening a combination study using CT-0508 alongside pembrolizumab, an anti programed cell death protein 1 (anti-PD1) antibody and T cell checkpoint inhibitor. The researchers hope this will combat T cell exhaustion, a common problem in late stage cancer, and thus work synergistically with the CAR macrophage to bolster immune system attack.
“Nowadays, if you can quickly retarget, repurpose, or reprogram an immune cell, that will hopefully be beneficial to patients with a range of cancers and other diseases,” said Michel Sadelain, an immunologist at Memorial Sloan Kettering and pioneer of CAR T cell therapy who was not involved in the recent studies. The preliminary results from the CAR macrophage trial are promising for the treatment of not only solid tumors but autoimmune disorders and other immune-related disorders as well. “There's a big exploration now; let's see what comes out of it all. It’s research, not everything will work,” said Sadelain. “But there are so many opportunities and possibilities that certainly many of us believe and hope that there will be many more cell therapies in the years to come.”
Learn more about advances in immuno-oncology in this ebook.
References
- June, C.H., & Sadelain, M. Chimeric antigen receptor therapy. NEJM 379, 64-73 (2018). https://www.nejm.org/doi/full/10.1056/NEJMra1706169
- Andreesen, R., Hennemann, B., & Krause, S.W. Adoptive immunotherapy of cancer using monocyte- derived macrophages: rationale, current status, and perspectives. J Leukoc Biol 64, 419–426. (1998). https://academic.oup.com/jleukbio/article-abstract/64/4/419/6976852?redirectedFrom=fulltext
- Klichinsky, M. et al., Human chimeric antigen receptor macrophages for cancer immunotherapy. Nature Biotechnology 38, 947-953 (2020). https://www.nature.com/articles/s41587-020-0462-y
- National Library of Medicine (U.S.). (2020, December - ). CAR-macrophages for the treatment of HER2 overexpressing solid tumors. Identifier NCT04660929. https:// clinicaltrials.gov/ct2/show/NCT04660929
- Reiss, K.A., et al., A phase 1, first-in-human (FIH) study of the anti-HER2 CAR macrophage CT-0508 in subjects with HER2 overexpressing solid tumors [abstract]. In: ASCO Annual Meeting; 2022 June 3-7; Chicago, IL. Abstract nr 2533. https://meetings.asco.org/abstracts-presentations/210453
