Chimeric antigen receptor (CAR) T cell therapy has transformed cancer treatment, but it is not the only way scientists can harness the immune system. Other immune cells offer unique advantages for immunotherapy.
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BREAKING THROUGH IMMUNE SUPPRESSION WITH CAR T CELLS
Screening 12,000 genes helped researchers develop new generation CAR T cells for cancer therapy.
BY YUNING WANG, PHD
Since 2017, the United States Food and Drug Administration (FDA) has approved six chimeric antigen receptor (CAR) T cell therapies for treating blood cancers (1). Behind the excitement around these therapies is the concern about patients’ short-lived responses to CAR T cell infusions, limiting their chances of long-term survival. CAR T cells have even lower efficacy for solid tumors because immunosuppressive factors in the tumor microenvironment (TME) inhibit T cell activity, thereby promoting tumor development and evading therapies.
Scientists have made efforts to identify immunosuppressive genes that contribute to T cell dysfunction in the TME, such as using CRISPR to knock out genes for loss-of-function screening. In a recent study published in Nature, researchers at the New York Genome Center and New York University took a different approach by targeting positive regulators that boost T cell function, presenting the possibility of armoring CAR T cells with enhanced tumor killing abilities (2).
To discover genes that can improve a T cell’s antitumor activity, Neville Sanjana, a geneticist at the New York Genome Center, and his team screened nearly 12,000 genes. The researchers constructed a lentiviral library containing a collection of protein coding genes in the human genome and transduced the library into T cells. They monitored the proliferation of these T cells and captured numerous highly expressed genes involved in diverse immune processes, such as lymphocyte proliferation and interferon production. The top ranked gene, to the team’s surprise, was lymphotoxin-β receptor (LTBR).
LTBR is a tumor necrosis factor receptor that mediates cytokine release and cell apoptosis, which helps regulate lymphoid organogenesis and inflammation. Although T and B cells often express the ligands for LTBR, the receptor itself is typically absent in these cells (3).
To understand how exogenous LTBR impacts T cell functions, Sanjana and his team used single cell sequencing to profile T cell transcriptomes. They found that LTBR triggered the upregulation of various genes, enhancing multiple immune responses, including boosting cytokine secretion, promoting T cell proliferation, and decreasing cell apoptosis.
Based on their sequencing data, the authors pinpointed nuclear factor (NF)-κB, a key immune response regulator, as the most significant upregulated gene in LTBR-transduced T cells. NF-κB typically coordinates various transcription factors in different pathways to trigger inflammatory responses and promote immune cell development (4). In LTBR-transduced T cells, the researchers confirmed that NF-κB also upregulated several key inflammatory pathways and transcriptions factors, such NF-κB p65 and p52. These data helped Sanjana and his team identify which signaling pathways LTBR acts on to stimulate T cell immune activities.
The team then coexpressed LTBR with FDA-approved CARs and tested their antigen specific responses and cytotoxicity in patients’ T cells. They discovered that compared to the original CAR T cells, LTBR CAR T cells demonstrated increased cytokine secretion and cytotoxicity against tumor cells. These results highlight the potential of engineering T cells using immune enhancing genes as an approach to improving CAR T immunotherapy, especially for solid cancers.
References
1. CAR T Cells: engineering immune cells to treat cancer. National Cancer Institute. https://www.cancer.gov/about-cancer/treatment/research/car-t-cells
2. Legut, M. et al. A genome-scale screen for synthetic drivers of T cell proliferation. Nature 603, 728–735 (2022).
3. Norris, P. S. & Ware, C. F. The LTβR signaling pathway. Madame Curie Bioscience Database [Internet]. https://www.ncbi.nlm.nih.gov/books/NBK6515/
4. Sun, S.-C. The non-canonical NF-κB pathway in immunity and inflammation. Nat Rev Immunol 17, 545–558 (2017).
Engineering Guided by Biology: CAR NK Cells
BY SUNITHA CHARI, PHD | DESIGNED BY KRISTYN REID
Scientists use chimeric antigen receptors (CARs)- engineered extracellular fusion proteins - to enhance the natural ability of T cells to target and eliminate tumor cells. Now, scientists are expanding the CAR portfolio to natural killer (NK) cells, leveraging the inherent ability of NK cells to target and eliminate tumor cells with minimal toxicity (1).
NK Cell
Unlike T cell activation, NK cell activation does not depend on antigen presentation. Instead, NK cells express natural cell surface receptors that activate or inhibit their cytotoxic activity (2).
Enhancing NK cytotoxicity with different CARs
CARs derived from activating receptors such as natural killer group 2D (NKG2D) receptors and cluster of differentiation 16 (CD16) receptors act synergistically with their naturally occurring counterparts to amplify NK cell cytotoxicity (2).
NKG2D CARs bind stress ligands on cancer cells and release the perforin protein, which punctures holes in cancer cell membranes, allowing CAR NK cells to deliver enzymes that initiate the programmed cell death cascade (2).
Bispecific CARs such as NKG2D CARs and human epidermal growth factor receptor 2 (HER2) CARs target two different antigens on breast cancer cells for precision targeting (3).
CD16 CARs recognize immunoglobulin G antibodies bound to cancer cells and enhance NK cell-mediated antibody-dependent cellular cytotoxicity (4).
Immune checkpoint inhibitors are also popular targets for developing engineered CARs. NK cells expressing immune checkpoint CARs recognize and eliminate cancer cells expressing antigens such as cytotoxic T lymphocyte antigen 4 or programmed death-ligand 1 (4, 5).
REFERENCES
1. Biederstädt, A., Rezvani, K. Engineering the next generation of CAR-NK immunotherapies. Int J Hematol 114(5), 554-571 (2021).
2. Khawar, M. B., Sun, H. CAR-NK cells: from natural basis to design for kill. Front Immunol 12, 707542 (2021).
3. Zhang, C., et al. Bispecific antibody-mediated redirection of NKG2D-CAR natural killer cells facilitates dual targeting and enhances antitumor activity. J Immunother Cancer 10, e002980 (2021).
4. Xie, G., et al. CAR-NK cells: a promising cellular immunotherapy for cancer. EBioMedicine 59, 102975 (2020).
5. Bajor, M., et al. PD-L1 CAR effector cells induce self-amplifying cytotoxic effects against target cells. J Immunother Cancer 1, e002500 (2022).
MAKING CAR T CELL THERAPY MORE ACCESSIBLE
Allogeneic T cells may serve as universal, ready-to-use therapeutic agents for large scale clinical applications.
BY YUNING WANG, PHD
Chimeric antigen receptor (CAR) T cell therapy is a remarkable achievement for fighting diseases using the body’s immune system, but there is a pressing need to make CAR T cells available for large patient groups. Most CAR T cell therapies, including all United States Food and Drug Administration-approved products, adopt an autologous approach. This means that clinicians take a patient’s own T cells, engineer them to express CARs, and infuse them back into the same patient. These autologous CAR T cells require a costly, lengthy, and individualized manufacturing process, often delaying and preventing patient access to treatments (1).
To mitigate these issues, scientists are exploring the possibility of utilizing allogeneic T cells from healthy donors for CAR T cell generation. Unlike patient-derived T cells, which may have become dysfunctional due to exposure to malignant tumors or infections, T cells from healthy blood demonstrate greater proliferative capacity and antitumor effects (2).
Using allogeneic T cells ensures CAR T cell product quality and facilitates the standardization of CAR T cell manufacturing. Researchers can select donors based on specific immune characteristics and produce batches of homogenous CAR T cells ahead of time, making treatments immediately available for patients. Since a single T cell collection can generate multiple therapeutic doses, the treatment may also cost less.
However, like many tissue and organ transplants, allogeneic CAR T cell infusions can cause undesirable alloimmune responses, including graft-versus-host disease (GvHD), a life-threatening condition where donor-derived CAR T cells recognize the recipient’s cells as non-self via T cell αβ receptors (αβ TCRs) and attack the recipient’s cells. Researchers have developed strategies to reduce GvHD by using T cell subsets that comprise lower αβ TCR diversities, such as T cells differentiated from hematopoietic stem cells, virus specific memory T cells, and non-αβ T cells (2).
Gene editing technologies have also emerged as effective tools for preventing GvHD. Scientists use engineered transcription activator-like effector nucleases (TALENs), restriction enzymes that recognize and cleave DNA at specific sites to eliminate αβ TCR expression. For example, researchers from the University College London used TALEN-edited allogeneic CAR T cells to treat two infants with leukemia. Using TALEN enzymes that target αβ TCR genes, they abolished αβ TCR expression on T cells. The two children achieved sustained remissions after the therapy and only showed mild skin GvHD, in contrast to typical multisystem and fulminant GvHD symptoms (3).
In addition to TALENs, scientists from the Vrije Universiteit Brussel recently established a CRISPR-Cas9-mediated approach for generating GvHD-free allogeneic CAR T cells (4). They knocked out αβ TCR genes in T cells derived from human peripheral blood using CRISPR-Cas9 and expressed a leukemia-specific CAR. The leukemia mice infused with modified T cells showed reduced tumor burden and prolonged survival without developing any GvHD symptoms.
By employing various T cell sources, gene editing techniques, and cell delivery systems, researchers around the world keep improving the efficacy and safety of CAR T cell therapy and developing new strategies for large scale CAR T cell manufacturing. With a number of allogeneic CAR T cell therapies for blood cancer and solid tumors under preclinical and clinical investigation, a universal off-the-shelf immunotherapy accessible for many more patients may soon be a reality.
REFERENCES
1. Köhl, U., Arsenieva, S., Holzinger, A. & Abken, H. CAR T Cells in Trials: Recent Achievements and Challenges that Remain in the Production of Modified T Cells for Clinical Applications. Hum Gene Ther 29, 559–568 (2018).
2. Depil, S., Duchateau, P., Grupp, S. A., Mufti, G. & Poirot, L. ‘Off-the-shelf’ allogeneic CAR T cells: development and challenges. Nat Rev Drug Discov 19, 185–199 (2020).
3. Qasim, W. et al. Molecular remission of infant B-ALL after infusion of universal TALEN gene-edited CAR T cells. Sci Transl Med 9, eaaj2013 (2017).
4. Tipanee, J., Samara-Kuko, E., Gevaert, T., Chuah, M. K. & VandenDriessche, T. Universal allogeneic CAR T cells engineered with Sleeping Beauty transposons and CRISPR-CAS9 for cancer immunotherapy. Mol Ther 30, 3155–3175 (2022).
MODIFYING MACROPHAGES TO ATTACK SOLID TUMORS
A team of industry and academic researchers developed a new way to engineer the immune system, leading to the first clinical trial using genetically modified macrophages.
BY DANIELLE GERHARD, PHD ver 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 efficiency 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, that 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 I 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, researcwdata 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% 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 I 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.”
The lifecycle of CAR macrophage therapy.
(1) After patients receive a bone marrow stimulator to encourage white blood cell release from the bone marrow, clinicians isolate the monocytes using apheresis. (2) The monocytes arrive at the lab, where scientists differentiate them into mature macrophages. (3) Next, they transduce the macrophage with the anti-HER2 CAR CT-0508. (4) Finally, scientists produce millions of these engineered cells, cryopreserve them, and ship them back to the clinic. (5) Clinicians reintroduce the patient’s genetically engineered macrophages, (6) which enter the solid tumor and kill HER2-expressing cells. In a secondary line of attack, macrophages identify other antigens expressed by the cancer cells and prime T cells with this information.
Michael Klichinsky (left) helped design the CAR macrophage used in the clinical trial (Carisma Therapeutics). Kim Reiss (middle) is a principal investigator on the clinical trial (University of Pennsylvania). Michel Sadelain (right) is a pioneer in CAR T cell therapies (Memorial Sloan Kettering).
REFERENCES
1. June, C.H., & Sadelain, M. Chimeric antigen receptor therapy. NEJM, 379, 64-73 (2018).
2. 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).
3. Klichinsky, M. et al., Human chimeric antigen receptor macrophages for cancer immunotherapy. Nature Biotechnology, 38, 947-953 (2020).
4. 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
5. 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.