New Cell Contenders in the CAR Field (Part 2)

By Michael A. Evans, BSci MSci 

We began our discussion of alternative CAR-cell types, with CAR-NK cells being the most popular alternative CAR-based therapy. Why do we need new cell contenders in the CAR field? Well, different CAR immune cells have distinct advantages and sometimes unique challenges or limitations.

The expansion of CARs to other cell types has already begun and shows promising results. We continue our exploration with macrophages.

CAR-Endowed Macrophages

The utility of CAR-endowed macrophages (CAR-MF) has recently been demonstrated, and their amazing potential realized. The novelty of CAR-MFs means much of the experimental data remains proprietary. However, the available information, in combination with what is known about macrophage biology, allows us to develop solid insights into their potential.

Macrophages are prominent members of the innate immune system and have many diverse bodily roles. Scientists typically characterize macrophages based on surface markers and secreted factors closely associated with

·       inflammation pathogen defense and tumor defense (M1)

·       anti-inflammation and wound healing (M2a)

·       immunoregulation (M2b)

·       immunosuppression and tissue remodeling (M2c)

·       angiogenesis and tumor promotion (M2d) [1]

Notably, the remarkable fluidity of macrophage polarization means these cells can exist as combinations of these subsets, making this classification system a guideline [2].

Overview of CAR macrophages and activation of the immune response (figure from https://carismatx.com/. © CARISMA Therapeutics Inc., 2019.)

Macrophage Functionality

While triggered by different receptors, the effector functions of T-cells and NK-cells are centered around activating apoptotic pathways in target cells [3]. The functions macrophages can exert towards tumors are quite different and focus on tumor cell ingestion (phagocytosis and trogocytosis), antigen presentation, and cytokine and effector molecule secretion [4-6].

CARs for macrophages include cytoplasmic signaling domains from phagocytotic receptors  (CD64 and Megf10), Toll-like receptors, as well as those more similar to CARs for CAR-T (CD3) [6-8].

The extracellular domain of these CARs instigates the identification of target molecules on cancer cells by macrophages, which can trigger phagocytosis (full cancer cell engulfment) or trogocytosis (biting pieces off), both of which can induce cancer cell death directly [6,7,9]. In addition, CAR-MF can incite other therapeutic effects.

How CAR-T Therapies Work

Clinically approved CAR-T therapies rely on a single target antigen to mount their anti-tumor response [10]. However, cancers often mutate, leading to antigen escape [11]. Targets phagocytosed or trogocytosed by macrophages can be processed for antigen presentation to T-cells [9].

Previous studies have demonstrated the importance of macrophage antigen presentation for tumor immune responses [12,13]. Many clinical trials are studying the benefit of anti-CD47 for treating cancer. CD47 is a cell surface marker commonly over-expressed in many cancers, which inhibits phagocytosis by macrophages.

By blocking this receptor, the macrophage phagocytosis rate increases, allowing macrophages to more efficiently present the antigens to CD8+ T-cells (primarily through an MHC I pathway) [13]. CAR-MF can present antigens from tumor cells to T-cells [6,7]. T-cell recognition of neoantigens could act as a second line of defense in cases where cancer cells adapt to evade CAR recognition.

Tumor Microenvironment

The makeup of the tumor microenvironment often mimics a chronic wound [14]. Because of this, the immune system responds appropriately to wound healing leading to immunosuppression, tumor metastasis, apoptosis resistance, and angiogenesis [14].

In addition to phagocytosis, target identification by CAR-MF promotes the release of inflammatory cytokines such as TNF-a, IL-6, IFN-g, and IL-127 [8,15]. These factors shift the tumor microenvironment and immune response towards tumor rejection by increasing Teff cell recruitment and function, tumor growth and angiogenesis suppression, and increased sensitivity to apoptosis [15].  

Because of the high prevalence of the target antigen in a tumor, CAR-MF are likely under constant stimulation to maintain this anti-tumor environment [7,8].

CAR-MF vs. CAR-T and CAR-NK

CAR-T and CAR-NK cells have struggled to effectively treat solid tumors due to the immunosuppressive nature of the tumor microenvironment, which actively inhibits their migration [16,17]. Research has demonstrated that macrophages are normally abundant in solid tumors and can sometimes make up 50% of their mass [18].

This high prevalence indicates that CAR-MF could target solid tumors better than other cell types [7].

Possible Complications

One challenge CAR-MF cells could face is inefficient gene incorporation [19]. Using viral vectors, CAR-T manufacturing produces a bottleneck and is a major cost driver [20,21]. Unfortunately, macrophages are notoriously resistant to viral transduction due to their ability to identify and destroy the viruses via phagocytosis and their low rates of cell division [19].

These issues result in low average macrophage transfection efficiencies for g-retroviruses (25%) and lentiviruses (37.5%), which are the vectors currently used in clinically approved CAR-T therapies and some preclinical CAR-MF therapies [8,22,23]. Some reports suggest adenoviral transduction CAR-MF cells have promise with CAR-expression rates up to 70% [7,8].

However, adenoviruses can be highly immunogenic and induce an inflammatory phenotype in edited cells, which adds significant risk. An early clinical trial involving adenovirus-based transduction reported a patient death due to a systemic inflammatory reaction caused by the viral vector. Since then, these vectors have been refined to minimize hazards [19]. These risks still exist in therapeutic environments.

Overcoming These Issues

Attempts to overcome these issues with non-viral methods have been challenging. Electroporation with macrophages is inefficient and induces widespread cell death [19]. We believe Hydropore™, a microfluidic vortex shedding (µVS) hardware developed at Indee labs, could solve macrophage gene delivery challenges.

Unlike viruses and particle-based methods, µVS allows for efficient gene delivery by creating vortices-induced holes in the cell membrane, which should help it avoid phagocytosis. In addition, results with other cell types indicate that µVS can provide high transfection efficiency and recovery without the cytotoxicity seen from electroporation [24].

About the Author

Michael A. Evans, BSci MSci is a bioengineering Ph.D. candidate at Harvard University with 7 years of experience in immunology, cell therapies, drug delivery, and organic chemistry. His current research focuses on using macrophages as carriers for nanoparticles to improve their targeting towards inflamed tissues. He is an author of 4 peer reviewed publications with three more in press. He attended Furman University and the University of California, Santa Barbara (UCSB), where he received a B.S. and M.S. in chemistry, respectively. He received the 2014 John Sampey Award for the Chemical Sciences and was a 2017 UCSB New Venture Competition Finalist and the People’s Choice Award recipient. Michael will be finishing his PhD in late 2019 and is excited to begin his career in gene-modified cell therapy.

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