Introduction
You look up, watching the grim faces around the room. The doctor just confirmed your worst fears, your mother has liver cancer. Holding the report, you are trembling, knowing what is coming. Months upon months of torturous treatments, going in and out of the hospital watching your mother grow weaker and weaker by the day, ravaged by the aggressive and cytotoxic therapeutics. Traveling for hours to the hospital every day, alternating between caring for your mother’s ailing body and earning money wherever you can while you watch your bank account drain away, all for the slim chance of surviving a few more years while the nightmare of a recurrence shadows you everywhere.
This is the reality that faces 20 million families in 2022 alone as their loved ones are newly diagnosed with cancer (Global cancer burden growing, 2024). The future looks even grimmer, with a projected 35 million new cancer cases in 2050, a 77% increase from 2022 (Global cancer burden growing, 2024). With cancer being such a prolific killer, causing one in six deaths globally in 2020 even during the midst of COVID-19, it would not be a stretch to argue that cancer is the greatest enemy human healthcare faces (Cancer, 2022).
Yet existing cancer therapeutics all have their fatal flaws. Surgery often fails to completely eliminate cancer cells, chemotherapy and radiotherapy are highly damaging to patients and can lead to resistance, and many immunotherapies still pose a major safety risk. More importantly, advanced cancer therapies are simply not accessible to most of the global population. Every existing cancer therapeutics requires large amounts of highly trained and highly specialized doctors, researchers, technicians, each of them taking decades to hone their skill. Every existing cancer therapeutic requires a huge amount of capital, from both the patients and their public health system. Yet it's exactly transitioning countries like India, who have the highest increase in relative cancer incidence, who lack the infrastructure, skilled workforce and resources to tackle this explosion in cancer incidence (Bray et al., 2024). As we stare down this titan of public health challenge, existing cancer therapeutics are simply not enough. Not only do we need an effective solution, but also a practical one. A therapeutic that is cheap to produce, easy to transport, requires minimal expertise to administer, has little side effect, and universal to various kinds of cancer. As cancer poses an unprecedented threat to human welfare, now more than ever do we need a solution that is simple, safe, effective, and accessible.
Hence, CAR_Ma was created. Chimeric Antigen Receptor (CAR) has emerged as a spearhead in modern immunotherapy, equipping our powerful immune system to detect and destroy cancer. Current CAR-T therapies fuse immune T-cells with immunoglobulin-derived regions combined with T-cell receptor (TCR)-derived regions together to target cancer cells with unprecedented specificity, achieving remarkable success and recently obtaining FDA approval (Car T cells, 2022; Kuwana et al., 1987). A truly beautiful system, carrying a beautifully fatal flaw. Solid tumors, which constitute up to 90% of cancer (NCI, 2015), fail to respond against CAR-T. Solid tumors are encompassed within their own Tumour Microenvironment (TME), a powerful physical barrier preventing the penetration into cancer by CAR-T cells (Gajewski et al., 2013). The TME halts penetration, and produces chemokines that suppress T-cell functionality. An achilles’ heel to our most powerful therapeutic yet. Compounding this, M2-like phenotype tumor-associated macrophages (TAMs) and chemokines within the TME would often promote tumor growth, leading to immunosuppression of CAR-T cells. Such an adverse environment pathetically renders our most advanced therapies inert.
Facing down such a monumental problem, an equally monumental solution is required. A new chapter of cancer immunotherapeutic we call CAR_Ma, CAR Multi-Modal Advanced. The beginning of a karmic end, to a long interminable foe.
The next generation CAR_Ma
CAR_Ma is based on CAR-M, where the chimeric antigen receptor is expressed on macrophage instead of T-cells. Despite being conceptualized in 1987 (Kuwana et al., 1987), it wasn’t until 2021 that the first clinical trial of CAR-M was held (Sloas et al., 2021), where the anti-HER2 CAR-M designed by Klichinsky et al. in 2020 were put to test.
Building upon the successes of our initial proof of concept. The next generation of CAR_Ma builds upon the platform developed in 2023 (HKU iGEM, 2023), but is reworked completely from the ground up to be the future of cancer immunotherapy based on the powerful technologies developed.
CAR_Ma is built upon a new idea, one first created by HKU iGEM. Vector multi-modality, the ability to use a single unified vector through different modes of operation and function. The multi-modal revolution is spearheaded by our first generation multi-modal RNA vector; this vector leverages a new, next-generation, high-fidelity RNA replication circuit to enable three modes of function. Transient expression, for genes that only need to be expressed for a short period of time. Sustained and controlled expression, powered by new high-fidelity replication machinery powering genes that need to remain expressed for sustained periods of time. And an RNA-release mechanism, to release functional non-coding RNA sequences such as guide RNAs for Cas-related operations, and shRNA for RNAi. This combination creates a robust expression system that can be used from CAR expression to gene therapies in the future.
Solid tumors are known to express immune evasion checkpoints such as SIRPα/CD47 (Chao et al., 2012) and PD-1/PD-L1 (Cui et al., 2024), which aids in tumor evasion, by circumventing detection by immune cells and phagocytosis. Building on CRISPR-Cas9g gene editing, a multiplex Cas12a system knocks out these systems simultaneously with other proteins that weaken macrophage response and T-cell activation to dramatically enhance phagocytosis and improve immune cell coordination.
Building upon the advantages brought about by multi-modal RNA, sustained
transience is key. Alongside CRISPR based approaches facilitated by our novel
vector, we also developed a new alternative shRNA pipeline build for CAR_Ma.
Rather than relying on a permanent knockout affecting the host genome, a small
hairpin RNA (Taxman et al., 2010) facilitates the silencing of SIRPa to carry
the same effect as CRISPR-mediated knockout in transient nature. In conjunction
with the sustained transience of multi-modal RNA allows the silencing of SIRPa
to be sustained as long as the system is active, but also controlled
simultaneously. This also means, once the therapeutic is finished, the
multi-modal RNA can be deactivated allowing for the cell to return to a native
state.
Further development of our shRNA included ribozymes allow for specific release
of shRNA preserving structure and guaranteeing specificity. Next generation
designs allowing for multiplexed silencing have also been developed bringing the
next generation of CAR_Ma.
Our updated CAR receptor is the heart of CAR_Ma. Redesigned from the ground up, the powerhouse powering our CAR is our humanized nanobody targeting GPC3, an antigen highly expressed among cancers such as Hepatocellular carcinoma. These nanobodies represent a modular domain that can easily be switched or multiplexed to target one or more antigens allowing for the targeting of a vast collection of cancers. Enhanced with upgraded and redesigned co-stimulator domains to further enhance phagocytosis, trogocytosis, the engulfment of large targets and even the coordination of the rest of the immune system. The inclusion of IFN-y expression presents a powerful polarization factor to push transfected macrophages and even TAMs towards an anti-cancer, pro-inflammatory state. Culminating this is a powerful TMP small molecule based variable control system, allowing us to tightly and specifically control the RNA self-replication and expression of our CAR and SIRPa knockout systems, ensuring absolute safety and control over CAR_Ma expression.
The machinery behind many of CAR_Ma’s benefits is its sustained self-replication. RNA has had the potential to be an incredible vector to therapeutics, but it’s transient nature often limited their functionality. Self-replicating RNA (srRNA) solves nearly every issue of RNA therapeutics, while retaining all the benefits of a transient vector. Being an RNA vector, its semi-transient nature forbids integration into the genome, creating a safer and cheaper vector than plasmid and viral based vectors. The addition of self-replication is simply the cherry on top, matching or exceeding the long-term expression of certain viral systems. This is essential for CAR-Macrophages as receptor-mediated phagocytosis leads to the internalization and destruction of receptors (Mellman et al., 1983), the need to replace the receptors overtime becomes a great necessity. This makes it the ideal vector for use in both in vivo and in vitro applications. With our next generation of srRNA we have further evolved our system to include high-fidelity RNA-dependent RNA polymerase proofreading domains to minimize mutations further extending the lifespan of our vector by multiple fold. The addition of the control receptors synergises with our advanced CAR to allow its replication to be controlled as well. The replication of the system can be tightly controlled ensuring minimal strain on the cellular system whilst extending the lifetime of the srRNA.
CAR_Ma will only ever be as impactful as its delivery. Leveraging advanced microfluidics, our CAR_Ma system will be packaged into a state-of-the-art lipid or protein nanoparticle. This system will enable CAR_Ma to be deployed intravenously and generate CAR_Ma cells in vivo directly, lowering the barrier of entry to the most advanced immunotherapeutics, removing the need for CAR cells to be generated ex vivo. To ensure the safety and specificity, CAR_Ma’s nanoparticles will be functionalized with anti-CD14 nanobodies to ensure the specific and direct transfection of monocytes and macrophages. Further, by leveraging the robust manufacturing and logistics systems created and tested during the COVID-19 pandemic, CAR_Ma is poised to not only be simpler and cheaper to manufacture, but its simplified routes of administration may allow CAR_Ma to be used globally in places immunotherapies were never available to before.
Nanobodies are powerful heavy-chain-only recombinant antibodies developed from camelids or sharks (Jin et al., 2023). These powerful proteins are used throughout CAR_Ma and enable some of its incredible capabilities. Being smaller, simpler, cheaper and easier to produce than regular antibodies or single-chain variable fragments (scfvs), coupled with their inherently lowered immunogenic effect and greatly enhanced tolerance in the human body, nanobodies are almost the ideal candidate for use in therapeutics. The primary factor holding back their widespread adoption is the extremely steep cost of entry, as libraries can only be developed from the aforementioned camelids and sharks. Countering this, we have developed a powerful model to generate, test and evolve libraries of nanobodies in silico. Dubbed the Nanobody Initiative, target antigen sequences are uploaded where the model quickly generates a series of nanobody sequences to test against. These sequences can then be used in wet lab experiments to validate and be used for further development, cutting out the barrier to entry for the use of nanobodies. Our model combines with the CAR_Ma platform allowing for nanobodies in the CAR and the lipid nanoparticle to be replaced. This allows for the CAR_Ma to be easily adapted to target a multitude of cancer types, and our lipid nanoparticle to target a multitude of cells opening up new avenues of therapy for not only advanced immunotherapies, but future systems such as gene therapy.
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