The role of small molecules in cell and gene therapy

Abstract

Cell and gene therapies have achieved impressive results in the treatment of rare genetic diseases using gene corrected stem cells and haematological cancers using chimeric antigen receptor T cells. However, these two fields face significant challenges such as demonstrating long-term efficacy and safety, and achieving cost-effective, scalable manufacturing processes. The use of small molecules is a key approach to overcome these barriers and can benefit cell and gene therapies at multiple stages of their lifecycle. For example, small molecules can be used to optimise viral vector production during manufacturing or used in the clinic to enhance the resistance of T cell therapies to the immunosuppressive tumour microenvironment. Here, we review current uses of small molecules in cell and gene therapy and highlight opportunities for medicinal chemists to further consolidate the success of cell and gene therapies.

Current and future uses of small molecules to improve the safety, efficacy and manufacturing of cell and gene therapies.

Introduction

The overlapping fields of cell and gene therapy (CGT) offer the potential for curative treatments for a wide range of diseases. Gene therapy is the delivery of genetic material into patient cells in vivo with the use of vectors that are typically viral-based. Gene therapy can enable the permanent correction of genetic-based disorders, for example by the delivery of a fully functioning gene to correct for the effects of a disease causing mutation. Cell therapy is defined as the administration of live cells, which can be derived from the patient receiving the therapy (autologous cell therapy) or from a difference source (allogeneic cell therapy). A type of cell therapy that overlaps with gene therapy is the treatment of patients with gene engineered cells. Examples of gene engineered cell therapies include stem cells corrected for genetic mutations and immune cells engineered with synthetic receptors to enable their recognition of antigens expressed on tumour cells. This review will focus on gene therapy and gene engineered cell therapy where there have been significant advancements in recent years.

In 2016, Strimvelis® became the first CGT product on the EU market and used autologous hematopoietic stem cells (HSCs) engineered with an adenosine deaminase gene to treat patients with adenosine deaminase deficiency severe combined immunodeficiency (ADA-SCID).¹ Since 2016, more than ten CGT products that have received market approval and groundbreaking achievements have been documented in other diseases, for example the reversal of blindness in patients with retinal dystrophies.² Significantly, CGTs are becoming available for indications beyond rare genetic diseases and two chimeric antigen receptor T cell (CAR-T) products, Yescarta® and Kymriah®, are demonstrating remarkable clinical success in the treatment of non-Hodgkin lymphoma (NHL) and acute lymphoblastic leukaemia (ALL), respectively. Investment in the field is soaring, with $2.3 billion invested into private CGT companies in the last decade.³ Furthermore, as of November 2017 there were over 2500 ongoing CGT clinical trials in a wide range of indications.⁴ With the advancement of genetic engineering tools and a deeper understanding of the immune system, there is belief in the field that treating patients with CGTs will become commonplace alongside small molecules and biopharmaceuticals.

However, the potential of CGTs is offset by the significant challenges facing their safety, efficacy and manufacturing. Gene insertions into the genome carry the risk of insertional mutagenesis.⁵ Cell therapies used in the treatment of cancer often result in acute toxicities such as cytokine release syndrome (CRS) or neurotoxicity, the latter of which was responsible for the death of 5 patients during a CD19 CAR-T clinical trial.⁶ With regards to efficacy, delivering genes to a sufficient number of target cells in patients and maintaining long-term expression of the therapeutic protein are major issues for gene therapies.⁷ For cell therapies, successful engraftment and persistence in patients is critical for durable responses.⁸ Transferred cells must also overcome significant barriers to clear diseases, such as the immunosuppressive tumour microenvironment (TME) which has been a major contributor to the lack of cell therapy success in solid tumour malignancies.⁹ Even with the potential development of safe and efficacious CGTs, there remain significant obstacles to bring these novel medicines to large patient populations at affordable prices. The manufacturing of CGTs is a complex multi-step process that involves various biological entities, and there is an urgent need to improve the scale and speed of CGT production.¹⁰

Small molecules have an established role in CGT via their use in conditioning chemotherapy and are increasingly used to address other challenges in the CGT lifecycle (Fig. 1 and Table 1). Researchers can select from a wide range of compounds to optimise yields in manufacturing processes or enhance the efficacy of cell therapies in clinical combinations. In addition to targeting endogenous pathways, small molecules can be used to control the activity of synthetic pathways introduced into CGTs via synthetic chemical control systems. When improving an existing CGT product, the use of small molecules may be preferred over genetic engineering, which can be time consuming and undesirable due to the need to register a new product. Compared with biopharmaceuticals, small molecules have the advantage of rapid kinetics, oral bioavailability and enhanced penetration across cellular and tissue membranes (e.g. blood brain barrier). It should be noted that these advantages mostly benefit the in vivo use of small molecules over biopharmaceuticals and may not be relevant for all applications in the CGT lifecycle. Additionally, small molecules are typically more affordable reagents than biopharmaceuticals. However, the decision to use a small molecule depends on whether the resulting improvements outweigh the additional complexity and how the cost–benefit compares with other options.

Fig. 1. Possible applications of small molecules in the lifecycle of T cell therapies. (1.) For the manufacturing of an autologous CAR-T cell therapy, peripheral blood mononuclear cells (PBMCs) are isolated from the patient by apheresis, then (2.) CD4+/CD8+ T cells are selected and activated. DNA plasmids encoding for components of a viral vector with a CAR transgene are (3.) transfected into mammalian cells (e.g. HEK293), from which viral vectors are (4.) produced and harvested. Small molecules can be used in synthetic chemical control systems to limit the expression of the CAR in mammalian cells in order to enhance viral vector titres. (5.) Viral vectors are used to transduce T cells with the CAR transgene and enhancers can be used to boost gene transfer efficiency. (6.) Engineered T cells are then expanded ex vivo and can be cultured with small molecules to regulate their phenotype. (7.) Cells are then formulated and cryopreserved with the potential addition of small molecules to prevent reduction in product quality. (8.) Patients undergo lymphodepleting conditioning chemotherapy prior to (9.) infusion of the T cell therapy product to enhance engraftment and efficacy. (10.) Small molecules can be used to manage toxicities such as graft-versus-host disease (GvHD) or boost efficacy of the therapy in the immunosuppressive TME. For allogeneic CAR-T therapies, CAR-T cells are generated from a source that is not from the patient and are commonly referred to as ‘off-the-shelf’. Many of the process steps are the same for autologous and allogeneic therapies, however there are significant differences that beneficially impact the costs of manufacturing allogeneic therapies.¹¹.

Selection of small molecules used in cell and gene therapy.

Small molecule	Structure	Molecular target used in CGT application/ Other targets	CGT application	Drug development stage/ CGT application development stage
H89		Protein kinase A inhibitor	Ex vivo inhibition of cryopreservation-induced cell death	Marketed/ Pre-clinical
Poloxamer synperonic F108		Decreases membrane microviscosity	Ex vivo lentiviral vector transduction enhancement	Pre-clinical/ Clinical
Phorbol 12-myristate 13-acetate		Protein kinase C activator	Ex vivo lentiviral vector transduction enhancement	Clinical/ Pre-clinical
Bortezomib		Proteasome inhibitor	Ex vivo AAV vector transduction enhancement	Marketed/ Pre-clinical
PTA		Farnesyl diphosphate synthase inhibitor	Ex vivo expansion of γδ T cells	Pre-clinical/ Pre-clinical
JQ1		BET inhibitor	Ex vivo inhibition of T cell differentiation during T cell expansion	Clinical/ Pre-clinical
TWS119		GSK-3β inhibitor	Ex vivo inhibition of T cell differentiation during T cell expansion	Pre-clinical/ Clinical
Idelalisib		PI3Kδ inhibitor	Ex vivo inhibition of T cell differentiation during T cell expansion	Marketed/ Pre-clinical
AKT inhibitor VIII		AKT inhibitor	Ex vivo inhibition of T cell differentiation during T cell expansion	Pre-clinical/ Pre-clinical
M1		Unknown	Ex vivo modulation of T cell metabolism during T cell expansion	Pre-clinical/ Pre-clinical
Mdivi-1		Inhibitor of GTPase dynamin-related protein-1	Ex vivo modulation of T cell metabolism during T cell expansion	Pre-clinical/ Pre-clinical
Dasatinib		Inhibitor of Bcr–Abl and Src family tyrosine kinases	Ex vivo inhibition of T cell differentiation during T cell expansion. In vivo inhibition of T cell therapies	Marketed/ Pre-clinical
Guanine		Synthetic aptamer/ Guanine deaminase, hypoxanthine–guanine phosphoribosyltransferase	Ex vivo or in vivo transgene expression control via synthetic riboswitch system	Pre-clinical/ Pre-clinical
Tetracycline		(a) Synthetic aptamer or (b) synthetic TetR/ 30S and 50S subunits of microbial ribosomes, TetR	Ex vivo or in vivo transgene expression control via synthetic (a) riboswitch system or (b) transcriptional system	Marketed/ Pre-clinical
Theophylline		Synthetic aptamer/ Phosphodiesterase inhibitor, adenosine receptor inhibitor and histone deacetylase activator	Ex vivo or in vivo transgene expression control via synthetic riboswitch system	Marketed/ Pre-clinical
RG-102240		Synthetic EcR/ EcR	Ex vivo or in vivo transgene expression control via synthetic transcriptional system	Pre-clinical/ Pre-clinical
Veledimex		Synthetic EcR/ EcR	Ex vivo or in vivo transgene expression control via synthetic transcriptional system (RheoSwitch Therapeutic System®)	Clinical/ Clinical
Shield-1		Stabiliser of synthetic destabilised FKBP12	Ex vivo or in vivo control of protein degradation via synthetic system	Pre-clinical/ Pre-clinical
IAA		Dimeriser of synthetic IAA17 and exogenous TIR1/ IAA17 and TIR1	Ex vivo or in vivo induction of protein degradation via synthetic system	Pre-clinical/ Pre-clinical
BI224436		Synthetic HIV integrase/ HIV integrase	In vivo CAR-T cell activation control via synthetic dimerisation system	Clinical/ Pre-clinical
AP21967		Dimeriser of synthetic FKBP12 and synthetic FRB/ FKBP12	In vivo CAR-T cell activation control via synthetic dimerisation system	Pre-clinical/ Pre-clinical
AP1903		Dimeriser of synthetic FKBP12	In vivo induction of caspase-9 apoptosis via synthetic system	Clinical/ Clinical
Ganciclovir		Exogenous HSV-TK/ HSV-TK	In vivo inducible cell death by GDEPT	Marketed/ Clinical
Plerixafor		CXCR4 inhibitor	In vivo HSC mobilisation	Marketed/ Clinical
Bendamustine		DNA alkylator	In vivo lymphodepleting conditioning	Marketed/ Clinical
IT-603		c-Rel inhibitor	In vivo inhibition of GvHD	Pre-clinical/ Pre-clinical
Decitabine		Inhibitor of DNA methyltransferases	In vivo enhancement and maintenance of antigen expression in tumour cells	Marketed/ Pre-clinical
CA-170		PD-L1 & VISTA inhibitor	In vivo inhibition of T cell suppression	Clinical/ Clinical
BMS-1166		PD-L1 inhibitor	In vivo inhibition of T cell suppression	Pre-clinical/ Pre-clinical
Birinapant		SMAC mimetic and IAP inhibitor	In vivo sensitisation of tumour cells to killing by CAR-T cells	Clinical/ Pre-clinical

The ideal properties for small molecules used in vivo for CGT applications are broadly similar to small molecules used in other areas of medicine: high specificity, high potency, low toxicity and suitable absorption, distribution, metabolism and excretion (ADME) properties. Small molecules used in vivo with synthetic chemical control systems should possess specificity for the engineered system with limited interaction with endogenous biology, and typically have short half-lives to enable rapid control. Small molecules used for ex vivo applications (e.g. in manufacturing) will not enter the patient, thus there are fewer requirements on their pharmacokinetic and ADME properties. Additionally, both clinically validated and pre-clinical compounds can be used as long as no residual compound is present in the formulated product. Small molecules used in manufacturing must, however, be cell penetrating, non-toxic to cells, physically stable and good manufacturing practices (GMP) grade.

This review will outline current and potential uses of small molecules to improve the safety, efficacy and manufacturing of CGTs. Limitations in currently used small molecules are discussed and opportunities to develop new chemical entities (NCEs) are highlighted. Cell therapies are diverse in the cell type that can be used and the disease that can be targeted, thus this article will focus predominantly on gene engineered T cells for immuno-oncology. There will be some coverage on gene engineered HSCs, however a comprehensive review on the use small molecules for stem cell therapy can be found elsewhere.^12–14 Unless otherwise specified, T cells will describe conventional CD8 or CD4 expressing effector T cells. For this review, we have adhered to the conventionally accepted definition of small molecules as <900 Daltons molecular weight compounds, along with the NCE descriptor. Therefore, potential applications of molecules such as siRNAs, miRNAs or anti-sense oligonucleotides in CGT will not be discussed.

Synthetic chemical control systems in cell and gene therapies

Effective control over the timing and strength of CGTs is required to limit toxicities and achieve the desired therapeutic response. This can be achieved via the use of small molecules that modulate the activation or persistence of CGTs. For example, dasatinib is a Bcr–Abl and Src family tyrosine kinase inhibitor that rapidly and reversibly ablates signalling in T cells and can be used as a pharmacological off-switch for engineered CAR-T and TCR-T cells.¹⁵ This approach is straightforward, however it is not specific for the engineered cells and only certain aspects of CGTs can be controlled in this way. Thus, methods that offer greater control of CGT responses with small molecules typically involve a synthetic chemical control system that is first genetically introduced into a CGT. Synthetic chemical control systems are diverse in their structure and function, but all consist of biological effectors under the control of small molecule responsive components. Here, we will describe several RNA- and protein-based synthetic chemical control systems and will highlight the level of chemical and biological engineering required to achieve effective control of CGTs using this approach.

RNA-based control systems

One method to control the expression of delivered genes is to incorporate small molecule responsive RNA motifs, called riboswitches, into the transcripts of transgenes (Fig. 2A). Riboswitches control the activity of transcripts by binding small molecules at their aptamer domains, which induces conformational changes in the RNA molecule. Researchers have exploited the modular architecture of riboswitches to design molecules which couple multiple small molecule inputs and gene expression outputs.^16,17 Riboswitches are attractive for CGTs because they are small in size and can be easily incorporated into gene delivery vectors, which are typically limited in space.¹⁸ Furthermore, as riboswitches are cis-regulatory elements they frequently exhibit low off-target effects and fast response rates.

Fig. 2. Synthetic chemical control systems in cell and gene therapies. Synthetic chemical control systems can be incorporated into CGTs in order to control their activity using small molecules. Aptazymes incorporated into the untranslated regions (UTRs) of RNA molecules encoding for transgenes enable users to control the expression of transgenes. (A) For example, ligand binding at the aptamer domain of a tetracycline-regulatable aptazyme results in a conformational change that leads to the autocatalytic cleavage of the RNA molecule and subsequent attenuation of transgene expression.¹⁹ (B) Incorporation of chemically induced dimerisation domains (e.g. FKBP12 and T2089L mutant of FRB) into a split-CAR enables small molecule control over CAR-T cell activation.²⁰ (C) Synthetic transcriptional control systems with small molecule responsive components can be used to regulate the transcription of delivered transgenes. In the EcR system, transcription is activated when EcR heterodimerises with RxR upon ligand binding, which results in the recruitment of a transcriptional activator (e.g. VP16) to the transgene.²¹ (D) Destabilised domains (e.g. L106P mutant of FKBP12) can be fused to a therapeutic protein in order to control its stability and activity.²² Ligand binding stabilises the fusion protein, whilst absence of the ligand results in the degradation of the entire fusion protein.

For CGT applications, the most effective type of riboswitches combine ligand binding aptamers with self-cleaving RNA motifs known as ribozymes.^19,23,24 When incorporated into a transcript, ribozymes enable potent inhibition of gene expression through spontaneous self-cleavage of the mRNA. A ribozyme-based riboswitch, also known as an aptazyme, can be created through the precise positioning of a ligand binding aptamer domain into a transcript such that small molecule binding inhibits or activates self-cleavage of the transcript. Aptazymes are particularly attractive for therapies with viral vectors as robust gene regulation is maintained at high vector copy numbers following viral replication.^25–27 Aptazymes have been used in a number of CGT applications, such as a safety switch to control the replication of oncolytic adenoviruses.²⁸ In this example, a theophylline-dependent aptazyme was inserted into the adenovirus immediate early gene E1A of an oncolytic adenovirus.²⁹ E1A expression could then be conditionally inhibited upon addition of theophylline, which in turn enabled functional inhibition of viral replication, particle production and oncolysis. In addition to regulation of delivered transgenes, aptazymes have a useful application in the user controlled regulation of endogenous gene expression. To achieve this, aptazymes have been modified for the conditional induction of miRNA in mammalian cells and provide an attractive mechanism for the spatiotemporal control of RNA interference CGT approaches.^30,31 For example, theophylline-dependent aptazyme regulation of endogenous MAP4K expression in HepG2 cells demonstrates a potential strategy to silence metastatic genes in cancer cells.³² Aptazymes can also be used to control transgene expression in engineered T cells.³³ Chen et al. 2010, demonstrated tight regulation of T cell growth in vivo, and this was achieved using an aptazyme that was adaptable to different small molecule inputs and to different regulatory outputs, such as combinatorial expression of IL-2, IL-15 or the purification marker CD19.³⁴ One of the best performing aptazymes to date exhibited a 33-fold tetracycline-dependent change in HeLa cell transgene expression when delivered by adeno-associated virus (AAV) vectors.¹⁹ However, when the same aptazyme-containing AAV vector was injected into the gastrocnemius muscle of mice, the average fold change from the studies was dramatically less at 6.9-fold. Poor translation to the in vivo setting was believed to be a result of tetracycline's poor pharmacokinetics, specifically its rapid metabolism in mice and poor bioavailability to the muscle cells.³⁵ This is a broad issue: riboswitches with the highest performance (e.g. fast response rate, low basal activity, high level of gene induction) in vitro are responsive to theophylline, guanine or tetracycline, which all have poor bioavailability and are required at high concentrations for in vivo genetic control.³⁶

For RNA-based control systems to become clinically applicable, there is a need for riboswitches that are responsive to small molecules with excellent properties for clinical application. Aptamers can be generated de novo to bind to ligands using systematic evolution of ligands by exponential enrichment (SELEX), however this process has significant difficulties and limitations.^37–39 Consequently, there remains a lack of aptamers responsive to clinical small molecules, and aptamers responsive to (6R)-folinic acid are one of the few examples that exists. (6R)-Folinic acid was selected for aptamer development due to its marketed status, low toxicity and acceptable ADME properties.^40,41 Additionally, (6R)-folinic acid can be conjugated to resin and used with column chromatography purification, which significantly aids the identification of ligand binding aptamers during SELEX.⁴² (6R)-Folinic acid aptamer domains have been used in riboswitches to enable user control of T cell proliferation, which was achieved via the conditional miRNA targeting of endogenous cytokine receptor subunits IL-2Rβ and IL-2Rγc.³³ Advances in medicinal chemistry (e.g. biotinylation) have made resin conjugation accessible to more small molecules, but there are significant drawbacks to small molecule immobilisation for aptamer discovery such as the binding of aptamers to linking groups and the need to use modified ligands.⁴³ A method known as capture-SELEX now exists that circumvents the need for small molecule immobilisation and has been a major step forward in the identification of small molecule binding aptamers.^44,45 A significant challenge also exists in engineering suitable connections between novel aptamers and regulatory domains to generate riboswitches with sensitive and dynamic gene control. Here, in-format screening methods have been developed to rapidly assess libraries of riboswitch designs and have proven to be effective in selecting riboswitches with specific functions in cells.^46,47 Methods enabling the screening of libraries in their final configuration is something we believe will be transformative for the rapid discovery of all types of biological components with robust and effective activity in CGTs. Additionally, an area of chemistry that is likely to impact the success of riboswitches in CGT is the discovery of small molecule drugs that target RNA.^48,49 Work in this area is leading to a better understanding of chemical moieties that enable RNA binding and may lead to the development of small molecules with excellent properties for clinical application that can be used as part of high performance riboswitches.

Chemically controlled dimerisation

Use of small molecules to manipulate protein–protein interactions is another strategy to control the activity of CGTs. Chemically induced dimerisation (CID) is the most widely used method and involves the complex formation of proteins following the addition of a small molecule.^50–53 Conversely, small molecules can also be used to disrupt the dimerisation of proteins. Examples of chemically controlled dimerisation systems include those regulated by gibberellin, coumermycin or the HIV-1 integrase inhibitor BI224436, which have all been used to control the activity of CAR-T cells via the reversible dimerisation of split-CARs (Fig. 2B).^20,54,55 The most successful systems to date are based on FK506 binding protein 12 (FKBP12), which can be homodimerised upon addition of bivalent binders or heterodimerized with FKBP12–rapamycin binding protein (FRB) upon addition of rapamycin analogues (rapalogs). These CID systems were originally developed as chemical tools and their potential clinical application was limited due to the binding of dimerisers to endogenous FKBP12 or FRB.⁵⁶ Furthermore, binders of FKBP12 and FRB have known toxicities and are immunosuppressants, which make them undesirable for use in cellular immunotherapies.^57,58 In a landmark paper almost 3 decades ago, an orthogonal FKBP12 homodimerising system was generated by remodelling the ligand interface of FKBP12 with a de novo binding pocket.⁵⁹ A small molecule, AP1903, was then designed from FKBP12 binders that was sterically blocked from binding the endogenous FKBP12 but could bind the mutant FKBP12. This was achieved via a carbonyl to ethyl substitution in AP1903, which enabled a 1000-fold selectivity for the mutant FKBP12. This orthogonal CID system has been used to induce apoptosis in a number of cell therapies by controlling the dimerisation of caspase-9.^60–62 The inducible caspase-9 system controlled by AP1903 has been tested in the clinic on multiple occasions and can enable the elimination of up to 95% of transplanted T cells within 30 minutes to stop graft versus host disease (GvHD).^63,64 Importantly, no dose-limiting toxicities have been observed in humans treated with AP1903, providing strong evidence that AP1903 is orthogonal and has no significant impact on endogenous FKBP12 function.⁶⁵ The inducible caspase-9 system is currently incorporated into several CAR-T cell therapies undergoing clinical trials (NCT03696784 and NCT02414269). Results from these trials will inform the field whether the speed and efficiency of this technology are sufficient to control adverse effects of CAR-T therapy. Interestingly, researchers have been exploring the use of the hypomethylating agent 5-azacytidine to prevent methylation and silencing of the inducible caspase-9 gene in order to enhance the effectiveness of AP1903 induced apoptosis.⁶⁶ AP1903 is an excellent example of how developing NCEs rather than repurposing available compounds can have a significant impact on the clinical success of CGTs.

Gene-directed enzyme prodrug therapy (GDEPT)

Inducible cell death of engineered cell therapies can also be achieved by GDEPT, which involves the introduction of an enzyme into cells that can convert an administered non-toxic prodrug into a toxic metabolite. Examples include cytosine deaminase that converts 5-fluorocytosine into the cytotoxic 5-fluorouracil and the viral target protein herpes simplex virus-thymidine kinase (HSV-TK) that converts the antiviral drug ganciclovir into toxic phosphorylated metabolites.^67,68 Both the HSV-TK and the cytosine deaminase GDEPT systems have the advantage that their primary prodrugs are approved clinical agents. The HSV-TK system has been used successfully in the clinic to aborogate GvHD following T cell infusion, however the activated metabolite of ganciclovir can only kill actively dividing cells and is thus less preferred than the inducible caspase-9 CID system for mitigating toxicities encountered with TCR-T or CAR-T cell therapy.⁶⁹

Synthetic transcriptional control systems

Synthetic transcriptional control systems are well established in CGTs and enable the use of small molecules to switch the transcription of delivered transgenes on or off. Several antibiotic-controlled transcriptional control systems exist, but the tetracycline-regulatable system is most extensively used. The tetracycline-regulatable system is based around the Tet repressor protein, which binds Tet-inducible promoters in the absence of tetracycline and can be used to block transcription or recruit transcriptional activators. The tetracycline-regulatable system has been effectively used in conjunction with lentivirus, AAV, retrovirus and high-capacity helper-dependent adenovirus (HC-Ad) vectors.⁷⁰ Recently, the system was used to achieve inducible expression of a CD33 CAR and a CD19 CAR in T cells in order to control T cell activation and killing.^71,72 Ligands for the tetracycline-regulatable system have already been tested in the clinic and exhibit good safety profiles.^70,73,74 Doxycycline and minocycline are preferred over tetracycline due to their higher tissue distribution, reduced toxicity and higher gene induction levels. There is a risk of immunogenicity with the tetracycline-regulatable system due to its non-human components and the system is yet to be tested in humans.

Synthetic transcriptional control systems based on steroid hormone receptors are also commonly used in CGTs, and the insect ecdysone receptor (EcR) is an exciting example of a chemical control system that has recently been tested in humans. Insect EcR forms a heterodimer with human or mouse retinoid x receptor (RxR), and the system is typically formatted in a two-hybrid system in which the EcR ligand binding domain is fused to a DNA binding domain and RxR is fused to a transcriptional activator (Fig. 2C).^75,76 The EcR system is another example of CID, however it is unusual in that the ligand only binds to the EcR domain and ligand binding induces a conformational change in EcR to enable binding to RxR. The EcR system is activated by natural ecdysteroids, which have suboptimal ADME profiles and are poorly suited for use in the clinic. Thus, efforts have been made to modify their physiochemical properties and one group took the approach of systematically alkylating hydroxyl groups on ecdysteroids in order to increase hydrophobicity and remove hydroxyl groups that may contribute to drug metabolism.⁷⁷ This work successfully yielded semi-synthetic ecdysteroids derivatives with better ADME profiles, and one derivative, ponasterone A alkylated at the 22-position, also had enhanced gene induction activity. However, despite enhancements to ecdysteroids, the EcR system is typically used with synthetic diacylhydrazines, which are better suited for the clinic and have higher affinity to EcR.⁷⁵ The most advanced iteration of the EcR system is the RheoSwitch Therapeutic System (RTS)®, which is controlled by the synthetic diacylhydrazine veledimex.⁷⁸ Veledimex was developed specifically for synthetic transcription control and thus has excellent in vivo gene regulation activity and physiochemical properties.⁷⁹ Veledimex enables rapid control of gene expression due to its short half-life (2.5–5 h) and has a high level of BBB permeability, with 40% of plasma concentrations detected in human glioblastomas.^79,80 RTS® was recently used to control an IL-12 transgene delivered to glioblastoma tumours via a replication incompetent adenoviral vector (Ad-RTS-hIL-12). IL-12 is a master regulator of the immune system and modifies the immunosuppressive tumour microenvironment to become more inflammatory. However, IL-12 is highly potent and has toxicity if administered systemically, thus the Ad-RTS-hIL-12 gene therapy was developed to control the local delivery of IL-12 in tumours.^81,82 Phase I clinical trials demonstrated that Ad-RTS-hIL-12 plus veledimex had potent anti-tumour activity in recurrent glioblastoma patients and increased median overall survival compared with historical controls (NCT02026271 and NCT03679754).^80,83 Importantly, adverse events including CRS correlated with veledimex dose and were effectively reversed with dose discontinuation. Additionally, there have been no reports of immunogenicity towards the RTS® components and neither veledimex nor RTS® components have a significant effect on endogenous gene expression or cell processes.⁸⁴ Ad-RTS-hIL-12 plus veledimex has been granted an FDA fast track designation for the treatment of recurrent glioblastoma and initial data on a combination clinical trial with PD-1 blocker nivolumab also look promising (NCT03636477).⁸³

Inducible protein degradation

Small molecule induced degradation of synthetic proteins is another emerging stratgey to achieve spatiotemporal control over CGTs.⁸⁵ This can be achieved by the formation of ternary complexes composed of a protein tag known as a degron, a small molecule and a ubiquitin ligase. For example, target proteins can be fused to a degron from the plant protein IAA17, which is recruited to the E3 ubiquitin ligase complex SKP1-CUL1-F-box protein (SCF) by the addition of auxins such as indole-3-acetic acid (IAA).^86,87 Unfortunately, this system also requires the exogenous expression of the plant-based F-box transport inhibitor response 1 (TIR1) protein to enable degradation in human cells and no auxins have been tested in the clinic. Recently, induced protein degradation systems using human-derived degrons controlled by clinically validated compounds were seperately reported by the Maus group, the Kaelin group and ourselves.^88–90 All three groups isolated degrons from the human protein Ikaros3, which is naturally targeted for degradation by IMiD compounds such as thalidomide or lenalidomide. This system has been used to control the degradation of CARs and may be another strategy to control CAR-T activity. Proteolysis targeting chimaeras (PROTACs) can also be used to target synthetic proteins for degradation.⁹¹ In a recent example, bromodomain targeting PROTACs, ARV-771 and ARV-825, were used to degrade CARs fused to bromodomains and effectively suppress activation in primary T cells.⁹² In addition to their in vivo use, PROTAC degradation of CARs could be exploited during the ex vivo expansion of CAR-T cells, for example to reduce CAR tonic signalling, which is associated with accelerated T cell exhaustion.⁹³ Furthermore, ex vivo degradation of CARs could enable the infusion of cells with reduced CAR expression in order to mitigate potential off-target toxicities.

Conditional induction of protein stability is another method to control protein degradation using small molecules (Fig. 2D). Using this strategy, proteins are engineered to be partially unfolded and constitutively targeted for proteasomal degration. When a small molecule is added, it sufficiently stablises the protein to enable expression and function of the protein. This technology was originally developed by the Wandless laboratory who demonstrated that mutations to FKBP12 ablated its expression and this process could be reversed by the binding of the ligand Shield-1.²² Shield-1 was generated from the FKBP12 binder SLF* by mutation of a carboxylic acid group to a morpholine group in order to achieve greater intracellular availability and potency. The capacity to control molecular processes using this strategy has been demonstrated in vitro and in vivo using mouse models.⁹⁴ Multiple versions of this system have since been developed using destablised domains derived from human proteins and using FDA approved small molecules.⁹⁵ This system is currently being used by Obsidian Therapuetics to control the expression of IL-12 and CD40L in CAR-T therapies.

Small molecules in cell and gene therapy manufacturing

An exciting application of small molecules in CGT is their use in manufacturing processes to enhance yields or the efficacy of products. Due to the lower restrictions on small molecules used ex vivo, the development of NCEs for CGT manufacturing is an exciting area of research for medicinal chemists.

Hematopoietic stem cell (HSC) mobilisation and harvesting

Engineered stem cell therapies such as Strimvelis® use HSCs that are harvested from patient bone marrow, genetically engineered and then engrafted back into the patient. An important step in this process is HSC mobilisation in which HSCs are stimulated out of the bone marrow space into the bloodstream so that they can be harvested. Typical clinical protocols for HSC mobilisation utilise cyclophosphamide and granulocyte colony stimulating factor (G-CSF), which have a variety of side effects including flu-like symptoms, bone pain and are frequently ineffective or require multiple cycles of mobilisation to provide sufficient HSCs.⁹⁶ There is thus a demand for improved mobilisation protocols and advancements have been made with repurposed clinical small molecules.^97,98 Plerixafor is a CXCR4 antagonist originally developed for the treatment of HIV, but can be used with G-CSF to improve mobilisation, particularly in patients previously failing G-CSF mobilisation.^99–101 Interestingly, modifying the mobilisation protocol also alters the different type of HSC populations obtained, which highlight further opportunities in this space to control HSC harvesting.

Applications in vector production and gene delivery

During the production of viral vectors, the expression of payload proteins in mammalian producer cells may negatively impact vector titres by causing protein toxicity or resource limitations. Thus, a useful strategy is to use small molecules in synthetic chemical control systems to attenuate expression of the payload during vector production. For example, in the production of an AAV vector encoding for TGFβ1, a guanine-activated aptazyme was incorporated into the transgene in order to attenuate TGFβ1 expression during vector production and this enhanced vector titres by 23-fold.¹⁰² A tetracycline-regulatable transcriptional system has also been used to control the expression of lentiviral vector components in mammalian producer cells.¹⁰³ This method enables the generation of vector producing stable cell lines and improves vector titres by inducing vector production only after producer cell expansion.

Whether viral vectors are used to genetically modify cells ex vivo or in vivo, maximising the efficiency of transduction reduces vector use and may also improve the efficacy of the therapy. A broad range of small molecules have been used as transduction enhancers and typically function by enhancing vector entry, targeting intracellular pathways involved in transduction or blocking cellular antiviral pathways.^104,105 For retroviral and lentiviral vectors, cationic polymers such as polybrene and protamine sulfate neutralise the electrostatic repulsion between the viral envelope and cell membrane, and can enhance transduction in cochlear hair cells, mesenchymal stem cells and T cells.^106–109 Non-ionic amphiphilic poloxamers, such as poloxamer P338 and poloxamer synperonic F108, enhance lentivector particle entry by decreasing membrane microviscosity and increasing lipid exchange. Poloxamers have been shown to have lower toxicity and higher lentiviral transduction efficiency than cationic polymers in a range of cells including murine T cells, human CD30+ lymphoma cell lines and human T cells.^110–112 Transduction requires multiple steps, some of which are poorly understood, to take place in order to successfully establish permanent gene expression. Thus, the use of cell-based phenotypic screens that cover all the steps of transduction is critical to the discovery of effective transduction enhancers. Phenotypic screening with cells is not straightforward but is powerful for identifying synergies or novel modulators of transduction, which may also reveal insights into the mechanics of viral vector gene transfer. Some successful outputs of phenotypic cell-based screening include the combination of poloxamer synperonic F108 with protamine sulfate, which results in enhanced transduction efficiency and vector copy number (VCN) when transducing CD34+ HSCs with lentiviral vectors.¹⁰⁴ Additionally, the protein kinase C agonist phorbol 12-myristate 13-acetate (PMA) was found in a HTS to enhance the lentiviral transduction of human hematopoietic cell lines and CD34+ HSCs.¹¹³ Mechanistic deconvolution is an important step following phenotypic screening and further work is required to understand exactly how PMA enhances lentiviral transduction.

Proteasome inhibitors, such as bortezomib and carfilzomib, have been shown to enhance AAV vector transduction rates although the exact mechanisms of enhancement are unclear.^114,115 Use of proteasomal inhibitors also results in reduced presentation of AAV capsid-derived peptides on MHC class I molecules, potentially lowering the host immune response against AAV vector transduced cells.¹¹⁶ AAV vector transduction can additionally be enhanced by blocking undesired repair pathways like non-homologous end joining (NHEJ) in order to promote the use of homologous recombination. The natural product vanillin acts as a potent inhibitor of NHEJ and can increase hepatic gene targeting using AAV vectors.¹¹⁷ The delivery of genes using CRISPR/Cas9 is also dependent on NHEJ and small molecule inhibition of NHEJ is also effective in boosting CRISPR/Cas9 editing efficiency.¹³ As with lentiviral vectors, cell-based phenotypic screening approaches have also been useful in identifying a large diversity of small molecules types that can enhance AAV transduction in the malignant liver cell line HepG2, mouse retinal cells and mouse liver cells in vivo.^118,119

Ex vivo expansion of γδ T cells

γδ T cells are an atypical subset of immune cells which combine features of the innate and adaptive immune systems. γδ T cells can be engineered with synthetic immune receptors (e.g. CARs) to complement their innate tumour killing activity and are potentially suitable for ‘off-the-shelf’ allogenic oncology cell therapies as they lack HLA specificity.¹²⁰ The Vγ9Vδ2 subset of γδ T cells recognise phosphoantigen by-products, such as isopentenyl pyrophosphate (IPP), of the non-mevalonate pathway of cholesterol biosynthesis in certain cancer cells.¹²¹ For the ex vivo expansion of Vγ9Vδ2 cells, IPP or the IPP synthetic analogue bromohydrin pyrophosphate (BrHPP) can be used.¹²² Alternatively, γδ T cells can be expanded with APCs treated with bisphosphonates such as pamidronate or zoledronic acid, which inhibit farnesyl diphosphate synthase and lead to the accumulation of IPP in APCs.^123–125 In order to generate a bisphosphonate with superior cellular uptake and thus activity, the prodrug tetrakis-pivaloyloxymethyl 2-(thiazole-2-ylamino) ethylidene-1,1-bisphosphonate (PTA) was synthesised with negatively charged phosphonate groups masked with pivoxil esters for enhanced hydrophobicity.¹²⁶ The purity of Vγ9Vδ2 cells expanded ex vivo with PTA was significantly higher than that observed with zoledronic acid, with up to 99% purity achieved.¹²⁷ Due to the enhanced purity, Vγ9Vδ2 cells expanded with PTA had enhanced engraftment in immunodeficient NOG mice and a lower level of contaminating conventional T cell outgrowth was observed.

Phenotype of ex vivo expanded T cells

A burgeoning area of research surrounds the use of small molecules to modulate the phenotype of conventional effector T cell therapies during their ex vivo expansion. The expansion and long-term persistence of T cells re-infused in cancer patients is highly correlated with objective clinical response in CAR-T, TCR-T and TIL therapies, both in liquid and solid tumours.^128–131 Preclinical in vivo data and emerging correlative clinical data point to the less differentiated subtypes, in particular stem cell memory T cells (T_SCM), for driving remission of tumours thanks to their enhanced metabolic fitness and ability to expand, persist and continuously generate cytotoxic T cells.^132–137 To date most manufacturing processes have prioritised total cell counts, which have encouraged the use of extensive T cell activation and long expansion times (10–14 days), and have consequently favoured more differentiated subsets.^138–140 Thus, the development of processes that enrich for less differentiated subtypes or promote stem/memory-like phenotypes in T cell products is emerging as a potential strategy to maintain the immune surveillance of T cell therapies over long periods. As part of this strategy, researchers have been using small molecules to target signalling, metabolic and epigenetic pathways in order to modulate the differentiation and ultimately phenotype of ex vivo expanded T cells.

Following T cell activation via the TCR complex and CD28 costimulation, the PI3K/AKT/mTOR signalling pathway is central to inducing changes in T cell activity. Inhibitors of PI3K, such as idelalisib, and of AKT, such as AKT Inhibitor VIII, have been used to limit T cell differentiation during ex vivo expansion and promote more memory-like T cells without significantly impairing cell growth.^141–146 T cells expanded with PI3K or AKT inhibitors demonstrate enhanced persistence and tumour clearance in vivo, and there are on-going clinical trials assessing the performance of CAR-T cells cultured with either PI3K inhibitors (NCT03274219) or AKT inhibitors (NCT03139370). Wnt–β-catenin signalling can be induced by glycogen synthase-3β (GSK-3β) inhibitor TWS119, and TWS119 is currently being used alongside IL-7 and IL-21 to generate large numbers of T_SCM CD19-CAR-T cells from naïve T cells for a clinical investigation (NCT01087294).¹⁴⁷ The CAR-T product from this method exhibited long-term persistence and enhanced anti-tumour responses in mouse models. However, these T cells had lower vector transduction efficiency and had impaired ex vivo fold expansion compared with T cells cultured using the standard method. Inhibition of mTOR also promotes less differentiated subtypes at the expense of T cell growth and both results are unsurprising given that many pathways regulating differentiation also drive proliferation in T cells.¹⁴³ Therapeutic cell doses could be achieved with the TWS119 treated process, which is critical given that the benefit of a small molecule would always be outweighed by any potential increase in manufacturing failure.

Metabolic reprogramming is an important component of T cell differentiation and the superior anti-tumour activity of less differentiated subtypes is driven by their metabolic fitness.^148,149 As T cells differentiate, their metabolism changes from a state dependent on oxidative phosphorylation and fatty acid oxidation to a state driven by aerobic glycolysis. Consequently, glycolysis inhibitors (2-deoxyglucose), LDH inhibitors (NCI-737) and compounds that modulate the mitochondrial dynamics to mimic those seen in memory T cells (M1 and Mdivi-1) have all been effective in enhancing the proportion of less differentiated subtypes, in vivo persistence and anti-tumour activity of ex vivo expanded T cells.^150–152 Signalling pathways downstream of TCR and CD28 initiate most of the metabolic changes during differentiation, thus small molecules modulating PI3K, AKT, mTOR and GSK-3β also alter the metabolism of T cells either directly and/or indirectly.^{145,147,153–155} Additionally, a functional starvation state can be induced in T cells by using 2-hydroxycitrate (2-HC), an inhibitor of enzyme ATP citrate lyase, to reduce the abundance of nucleocytosolic acetyl-coenzyme A (AcCoA).¹⁵⁶ Reduction of nucleocytosolic AcCoA metabolically reprograms T cells towards a mitochondrially oriented state and leads to the maintenance of less differentiated phenotypes. As expected, these 2-HC treated T cells demonstrated enhanced persistence and tumour control in mouse models. Interestingly, functional starvation by 2-HC can be phenocopied by culturing T cells in a high extracellular concentration of potassium, as this also leads to the reduction of nucleocytosolic AcCoA. Manipulation of ex vivo culture conditions (e.g. oxygen levels) and media components (e.g. nutrients) may be an effective approach to promote the expansion of less differentiated T cells, particularly if synergies with small molecule treatments can be identified.^157–159

As T cells differentiate they undergo significant epigenetic modifications on their DNA and histones.^160,161 These epigenetic changes are responsible for initiating and maintaining gene expression changes that give rise to the phenotypes of each differentiation state. Epigenetic pathways can be targeted with small molecules to inhibit differentiation during ex vivo expansion, but effective manipulation relies on balancing the extensive interactions between regulatory transcription factors and epigenetic readers and writers. Bromodomain and extraterminal domain (BET) proteins recognise acetylated lysines on histone tails and regulate transcription at genes important in many T cell processes including differentiation. T cells treated with the BET inhibitor JQ1 have an increased population of T_SCM cells ex vivo and these cells exhibit greater persistence and tumour clearance in vivo.¹⁶² This is believed to be a result of reduced expression of transcription factor BATF, which increases the repressive activity of the histone deacetylase sirtuin 1 (SIRT1).¹⁶³ However, BET inhibition induces many gene expression changes in T cells including the repression of the proliferative enhancer c-Myc, which is likely responsible for the reduction of T cell proliferation in ex vivo cultures treated with BET inhibitors.¹⁶⁴ DNA and histone modifying enzymes are highly dependent on metabolic products for their function, thus pharmacological targeting of metabolic pathways may also be used to manipulate epigenetic mechanisms driving differentiation.¹⁶⁵ As previously mentioned, treatment with 2-HC reduces nucleocytosolic AcCoA levels, and one of the mechanism in which this prevents effector differentiation is by limiting histone acetylation.¹⁵⁶

Given the progressive nature of T cell differentiation, the timing of pharmacological intervention in an ex vivo process is an important variable to optimise. Dasatinib is an inhibitor of the proximal TCR signalling kinase Lck and treatment with dasatinib potently inhibits T cell signalling and significantly impairs T cell expansion.¹⁶⁶ However, if T cells are temporarily treated with dasatinib then the compound can induce transient periods of rest in T cells.¹⁶⁷ These rested T cells have increased expression of T_SCM-associated markers and exhibit enhanced persistence and tumour clearance. Recently, a single cell proliferative tracing and mass cytometry approach was used to dissect the contribution of time, cell division and regulatory protein expression on T cell differentiation during ex vivo expansion.¹⁶⁸ The results led to the use of ibrutinib, a BTK and ITK inhibitor, in an ex vivo expansion process, and the administration of ibrutinib prior to T cell activation most effectively directed cultures towards T_SCM-like phenotypes. Additional high-dimensional datasets, particularly those incorporating metabolism and epigenetics, will be powerful in enabling the selection and optimal application of small molecules in ex vivo expansion processes. It should be noted that work is still ongoing to define the ideal T cell for optimal clinical efficacy and it may not be a classical T cell subtype such as T_SCM that is defined by surface markers.^9,134 Thus, functional screening approaches that assay multiple desirable T cell characteristics are likely to lead to the identification of superior chemically induced T cell phenotypes. An example of this was recently demonstrated by Gurusamy et al., who screened CRISPR–Cas9 edited primary T cells on 4 phenotypes of therapeutically effective anti-tumour T cells: cell expansion, stemness, metabolic fitness and limited genomic stress.¹⁶⁹ Their screen identified the TCR kinase p38 as a target for superior T cell characteristics, and CD19 CAR-T cells expanded ex vivo with a p38 inhibitor, doramapimod, had enhanced tumour clearance and persistence in mouse models.

Formulation and cryopreservation

Cryopreservation is a critical step in the manufacturing process of CGTs. Cryopreservation is required for the transportation of CGT from manufacturing sites to bedsides, and suboptimal cryopreservation can lead to significant loss in viability and functionality of the medicine. Small molecules can be added as cryopreservation agents or formulation additives in order to help minimise the impacts of freezing or thawing on product quality.¹⁷⁰ For example, proline, lactose, and mannitol can be included in viral vector formulations to help reduce the level of vector loss due to container absorption and freeze–thaw cycling.¹⁷¹ Interestingly, optimal additives may differ depending on the viral vector type. For example, enveloped viral vectors appear to be more stable in trehalose whilst non-enveloped vectors appear more stable in mannitol.¹⁷² Formulations or post-thaw culture media that include small molecule excipients can also minimise the onset of cell death due to cryopreservation.¹⁷³ Examples include protein kinase A inhibitors, such as H89, and reactive oxygen species scavengers, such as glutathione, both of which have a beneficial effect on the cryopreservation of stem cells.^174,175

Clinical applications of small molecules with cell and gene therapies

The successful application of CGTs in patients requires a careful management of factors related to the therapy, host and disease. The versatile, controllable and fast acting nature of small molecules have made them valuable tools in the clinic to mitigate the toxicities and enhance the efficacy of CGTs. Here, we will cover several clinical applications of small molecules that have been or have the potential to be impactful to the success of CGTs. Although small molecules used in synthetic chemical control systems are also administered directly to the patient, these examples will not be covered as they have already been covered in a previous section (see section ‘Synthetic chemical control systems in cell and gene therapies’).

Conditioning chemotherapy

Conditioning chemotherapy is the administration of cytotoxic small molecules to patients prior to them receiving adoptive cell therapies. Many tissues maintain a homeostasis on the number of cells, which may limit or impact the ability to introduce new cells into a patient. Thus, conditioning chemotherapy is often used to deplete endogenous cells in order to achieve efficient engraftment of engineered cells. Combinations involving the DNA alkylating agent busulfan are widely used during HSC transplantation and present clinicians with an alternative to total body irradiation.¹⁷⁶ For Strimvelis®, busulfan-based conditioning proved to be critical for the successful engraftment of ADA-edited CD34+ marrow cells which allowed patients to stop taking immunoglobulin replacement and enzyme replacement therapy.^177,178

Conditioning chemotherapy is also an important component of the successful clinical application of adoptive T cell therapies. CAR-T cells rarely achieve substantial engraftment in patients that have not received lymphodepleting conditioning chemotherapy.^179–182 Lymphodepleting conditioning results in the higher expansion and longer persistence of adoptively transferred T cells and this is via a multitude of mechanisms beside the actual depletion of lymphocytes.^183,184 Lymphodepleting conditioning eliminates immunosuppressive cells (e.g. regulatory T cells), removes sinks for homeostatic cytokines (e.g. IL-2, IL-7 and IL-15), induces expression of costimulatory molecules and promotes the downregulation of tumor indoleamine 2,3-dioxygenase.^185–188 Cyclophosphamide and fludarabine are most commonly used in lymphodepleting conditioning regimens for CAR-T therapies.^189,190 Recent evidence demonstrates that the addition of fludarabine drives higher IL-15 levels and results in greater CAR-T expansion and persistence compared with conditioning with cyclophosphamide alone.¹⁹¹ Most patients receiving ACTs will have relapsed and refractory (r/r) diseases, thus lymphodepleting conditioning typically has a negligible effect on the tumour load. However, the identification of lymphodepleting drugs that effectively reduce the tumour burden may have a significant impact on patient outcome. A high tumour volume is associated with a shorter long-term survival and the development of acute toxicities such as CRS during CAR-T therapy.^192,193 With the aim of reducing tumour burden, researchers are trialing the use of bendamustine for lymphodepleting conditioning of r/r chronic lymphocytic leukemia and r/r B-cell lymphoma patients receiving CD19 CAR-T therapy.^194,195 The diseases of these patients are often cyclophosphamide-resistant, and current results suggest a beneficial improvement in efficacy and safety when using bendamustine conditioning compared with cyclophosphamide conditioning.

Immune responses to cell and gene therapies and graft versus host disease (GvHD)

B cell and T cell mediated immune responses to viral delivery vectors and to expressed transgene proteins have been extensively reported both pre-clinically and clinically.^196–198 Pre-existing or acquired immunity to viral vector subtypes can limit the durability, efficacy or prevent re-dosing of therapies.^199,200 Many of the same immunosuppressive approaches used in organ transplantation can be used to control immune responses to CGTs. For example, administration of prednisolone or other corticosteroids was used to downregulate the T cell response to AAV capsids in a clinical trial in which factor IX was expressed in hemophilia B patients.²⁰¹ More sophisticated formulation approaches have also been adopted to control host immune responses, such as synthetic vaccine particles encapsulating rapamycin.²⁰² When co-administered with AAV vectors these particles prevented the induction of anti-capsid humoral and cell mediated responses, enabling vector re-administration in mice and non-human primates.

As more allogeneic immune cell therapies enter the clinic, the risk of GvHD (a condition where immune cells from the donor attack the recipient patient's tissues) will need to be carefully managed.²⁰³ Broad immunosuppressive compounds are typically used to treat GvHD, but are undesirable for use with engineered immune cell therapies due to their negative effect on the functional activity of therapies and their generally high toxicities.^204,205 Thus, identification of targeted small molecules could be important for the successful clinical application of allogenic cell therapies. One exciting development in this space is the pharmacological inhibition of the NF-κB family member c-Rel.²⁰⁶ Loss of c-Rel activity reduces allo-activation but does not affect TCR activation or antigen-specific cytotoxicity of human T cells. Following HTS of a library of 15 000 compounds, structure–activity relationship (SAR) studies were carried out to identify the hydrophobic compound pyrimidinetrione and its derivatives, which have a 20- to 200-fold higher selectivity for c-Rel and NF-κB than other transcription factors such as OCT1 and AP1. Further chemical development yielded the compound IT-603, which can be successfully used to control GvHD without comprising anti-tumor activity of T cell therapies in mouse models.

Small molecule combinations with T cell therapies

Despite significant advances in the treatment of cancers with T cell therapies, most therapies fail to completely eradicate tumours from patients.¹⁸⁴ Clinical efficacy is particularly low for solid tumours where the immunosuppressive microenvironment poses significant barriers to the survival and activity of cell therapies.⁹ More advanced cell therapies are being developed with additional built-in functions in order to overcome these barriers. For example, CAR-T cells can be genetically engineered to secrete cytokines into the local environment or engineered with chemokine receptors to enhance tumour-directed trafficking.^207,208 Cell therapies can also be used in combination with clinically validated small molecules that enhance or complement their activity. Factors intrinsic to the therapy, host immune system and disease may all contribute to therapy failure, thus there is an enormous design space to be explored for T cell therapy combinations with small molecules.^209,210 Here, we will provide an overview of some combination approaches that are currently in clinical trials or have demonstrated promising pre-clinical results.

An increasingly popular strategy is the use of small molecules to remodel of the immunosuppressive TME to a more pro-inflammatory state. This can be achieved through a number of approaches including the use of inducers of immunological cell death of tumour cells or drugs that selectively deplete immunosuppressive myeloid-derived suppressor cells (MDSCs), tumour associated macrophages (TAMs) or regulatory T cells.^211–214 Antigen expression levels on tumour cells correlate with CAR-T efficacy and antigen downregulation is major mechanism of CAR-T resistance.^215,216 Thus, an attractive approach involves the use of small molecules to enhance or preserve antigen expression on tumour cells during T cell therapy. Examples of this include the use of the gamma-secretase inhibitors to increase BCMA expression in myeloma cells or hypomethylating agents, such as decitabine, that can promote the demethylation of antigen genes and boost expression of CD19, CD33 or cancer testes antigens.^217–220

Promising improvements in T cell therapy efficacy have also been observed with combinations of immunomodulatory small molecules targeting T cell pathways. IMiD compounds such as lenalidomide induce the degradation of Ikaros transcription factors in T cells, resulting in enhanced CAR-T proliferation and cytokine production.^221,222 Interestingly, evidence suggests that lenalidomide also enhances the quality of immune synapse formed by CAR-T cells, which is believed to drive the enhanced cytotoxic activity and tumour clearance of lenalidomide treated CAR-T cells specific for epidermal growth factor receptor variant III (EGFRvIII).²²³ Lenalidomide has also been shown to enhance NK cell responses against tumour cells by lowering the threshold for NK cell activation and augmenting actin cytoskeletal remodeling at immune synapses.²²⁴ In addition to limiting differentiation during the ex vivo expansion of T cell therapies, the BTK and ITK inhibitor ibrutinib can also enhance CAR-T efficacy if administered in vivo.²²⁵ Ibrutinib significantly increases the numbers of circulating T cells in CLL patients and reduces the expression of immune checkpoint receptors PD-1 and CTLA4 on T cells.²²⁶ Ibrutinib also disrupts the migration of TAMs into the TME and decreases the expression of the inhibitory receptor CD200 on tumour cells.^225,227 The synergistic activity of ibrutinib on T cells and the tumour have made it an ideal candidate for T cell therapy combinations, and early readouts from clinical trials in combination with CD19 CAR-T for CLL patients look promising.^228,229

The expression of immune checkpoints proteins such as PD-L1 by tumour cells enables the suppression of anti-tumour responses by T cells and is a major mechanism for immune evasion. Antibody-based immune checkpoint inhibitors (ICIs) that block the PD-1–PD-L1 interaction have been transformative in cancer immunotherapy by disrupting inhibitory T cell signalling and reinvigorating dysfunctional T cells.²³⁰ However, the effectiveness of ICIs is dependent on the pre-existence of tumour reactive T cells. Thus, the combination of T cells engineered with receptors recognising antigens on tumour cells and PD-1 blockade, either pharmacological and genetic, is an exciting approach.^231–233 Current clinical trials are only testing antibody-based ICIs with T cell therapies, however there is interest in the use of small molecule ICIs due to their potential benefits.²³⁴ For example, the more rapid PK of small molecule ICIs may allow the avoidance of immune related toxicities that are believed to be caused by the extended half-life of humanised antibody ICIs.^235,236 Despite their appeal there are few small molecule ICIs to date, which is mostly due to the difficulty in developing small molecule inhibitors capable of occupying the shallow hydrophobic surfaces at the interface of immune checkpoint protein interactions.²³⁷ Some promising examples include inhibitors based on a 2-methyl-3-biphenyl-methanol scaffold developed by Bristol-Myers Squibb (BMS-8, BMS-200, BMS-202, BMS-242, BMS-1001, BMS-1166). These small molecule inhibitors block the PD-L1/PD-1 interaction by inducing the dimerisation of PD-L1 molecules.^238–240 Additionally, Curis Inc. has developed a first-in-class small molecule inhibitor, CA-170, that simultaneously blocks immune checkpoints PD-L1 and VISTA. Results from phase I (NCT02812875) and phase II (CTRI/2017/12/011026) clinical trials have demonstrated that CA-170 has excellent safety as an oral drug and progression-free survival (PFS) that is comparable with antibody ICIs in 2nd and 3rd line non-small-cell lung carcinoma (NSCLC) patients.^241,242

Combinations with small molecules hold great potential to enhance the efficacy of T cell therapies, however extensive exploration of the possibilities and accurate selection of candidates to test in the clinic are significant challenges for the field. Functional genomics has emerged as a powerful tool for immunotherapy researchers and Dufva et al. recently used an integrated drug and genome-wide CRISPR knock-out screening approach to identify pathways and small molecules that enhance CAR-T cytotoxicity.^243–245 The authours found that birinapant, a mimetic of the protein second mitochondrial-derived activator of caspases (SMAC) and antagonist of inhibitor of apoptosis proteins (IAPs), sensitised malignant B cells to killing by CD19 CAR-T cells.²⁴⁶ CRISPR-based screens were then used to identify genes in malignant B cells that confer resistance or further sensitivity to birinapant. The death receptor genes RIPK1 and TNFRSF1A were found to be important for enhancement of CAR-T cytotoxicity by birinapant, however the dependence on each gene varied between B cell malignancies. Furthermore, the level of sensitisation by birinapant also varied between B cell malignancies and the data suggest that this may be driven by heterogeneity in the genetics of different B cell malignancies. For SMAC mimetics, as with all potential small molecule combinations with T cell therapies, achieving a robust understanding of the genetic drivers of responses will be critical for selecting the appropriate patient population for each small molecule combination. Additional studies have demonstrated the ability of birinapant to enhance CAR-T efficacy in a HER2 solid tumour murine model, and there is a strong rationale for testing SMAC mimetic combinations with CAR-T in clinical trials.^209,247 There are, however, safety concerns due to the potential for enhanced CAR-T cytotoxicity against healthy tissues and due to the fact that CRS has been reported for both SMAC mimetics and T cell therapies.^248–250 A reduced therapeutic window is a general issue for combination therapies as small molecules and cell therapies can have overlapping toxicities that may become synergistic. Effective pre-clinical safety research and clinical strategies to mitigate toxicities will be essential for the success of small molecule combinations with T cell therapies.

Conclusions and perspectives

Progress in the fields of immunology, stem cell biology and vectorology have made treating patients with CGTs a reality. However, significant roadblocks remain in order to achieve durable responses in large numbers of patients. The application of small molecules within the lifecycle of CGTs has already been significant in moving CGT towards the status of mainstream medicine and there are many more opportunities to be explored. Advancements in synthetic chemical control systems have provided the possibility of controlling CGTs using the more predictable pharmacokinetics of small molecule dosing. However, these technologies are limited by their use of repurposed clinical validated compounds. Use of repurposed small molecules simplifies the CGT clinical developmental process, but these compounds often exhibit poor ADME properties or undesirable side effects. Thus, development of NCEs specifically for CGT synthetic chemical control systems is most likely necessary for the successful application of these technologies in the clinic. There is also considerable opportunity for the development of NCEs for CGT manufacturing, as small molecules used ex vivo have fewer restrictions on their physiochemical properties and do not have to be validated in the clinic. With the demand for more affordable CGTs and the potential for manufacturing methods to influence therapy efficacy, small molecule development in this area is poised to have a significant impact on the clinical and commercial success of CGT. Due to the complexity of CGTs, identifying small molecules most likely to have the greatest effect on efficacy is a major challenge for researchers. Guidance from CRISPR screens and use of multi-phenotypic assays which measure the most clinically relevant parameters will be important in maximising success in these efforts. Additionally, we believe that both CGT and medicinal chemistry will benefit greatly from collaborative initiatives involving biologists and chemists at early stages of the discovery of small molecules for CGT applications.

Conflicts of interest

All authors are GSK shareholders. C. H. is an Adaptimmune Therapeutics shareholder. L. L. B. and C. M. F. are inventors on patent WO2020078925. L. L. B., C. M. F. and C. H. are inventors on patent WO2019007869. There are no other conflicts of interest to declare.

Biographies

Biography

Lewis Brayshaw.

Lewis Brayshaw received his PhD in biochemistry and cell biology from University College London in 2016. Following his PhD, he joined the Discovery Research Department for Cell and Gene Therapy at GSK as a Post-Doctoral Researcher. He currently holds the position of an Investigator at GSK, and his research focuses on developing novel approaches to improve engineered T cell therapy responses in oncology.

Biography

Carlos Martinez Fleites.

Carlos Martinez Fleites obtained his PhD degree in chemistry from the University of York in 2007 under the guidance of Prof. Gideon Davies, FRS. He then moved into industry working at Astex Pharmaceuticals, UCB Celltech and more recently at GSK. He is the author of more than 20 research publications and patent applications in the field of biochemistry and structural biology. Carlos currently has the role of Scientific Leader in the Protein Degradation Group at the Medicine Design Department at GSK.

Biography

Takis Athanasopoulos.

Takis Athanasopoulos is Director, Head of Vector & Transgene in the Discovery Research Department for Cell & Gene Therapy at GSK. Previously, he was a lecturer in molecular biotechnology at the University of Wolverhampton (2011–2016) and academic visitor and research consultant for the Gene Therapy Group at Royal Holloway University. He is additionally the founder & CEO of Gene & Cell Ltd and an advisor of Quethera and Genethon. He holds a BSc in biochemistry from University of Patras and a PhD in biomedical sciences from University of London.

Biography

Thomas Southgate.

Thomas Southgate has worked in the design and development of cell and gene therapeutics for over 25 years for the treatment of solid and haematological malignancies, degenerative neurological disease and childhood metabolic disorders. Joining GSK 10 years ago he leads chemical and gene therapy oncology medicines through their early clinical development.

Biography

Laurent Jespers.

Laurent Jespers heads the Discovery Research Department for Cell & Gene Therapy at GSK since 2015. After his Ph.D. in biochemistry at the University of Leuven, Belgium, he completed a postdoc in Cambridge, UK, under the leadership of 2018 Nobel laureate Greg Winter. His academic and applied research interests have mainly focussed on protein and antibody engineering for application in oncology and other diseases areas. He joined GSK in 2007 following the acquisition of Domantis and took responsibility for discovery platforms on innovative modalities.

Biography

Chris Herring.

Chris Herring was at the time of writing Director, Head of Cell Engineering in the Discovery Research Department for Cell & Gene Therapy at GSK. Prior to joining the Cell & Gene Therapy group, he worked within GSK and the biotech company Domantis on a range of novel biopharmaceuticals, including domain antibodies, bispecific antibodies, as well as the half-life modulation of cytokines and peptides. He holds a BSc in biochemistry from the University of Surrey and a PhD in DNA repair from the University of Manchester. Chris is currently Senior Director, Head of Discovery Research at Adaptimmune Therapeutics plc.