CAR-NKs, CAR-Ms, Next-Gen CARs, Off-the-Shelf Therapeutics

From CAR-T cells to CAR-innate immune cells

Chimeric antigen receptor (CAR; genetically engineered receptors that provide specific properties to an immune effector cell) cell therapy is facing a new revolution.

Since the FDA approval of CD19-CAR-T cells (for introduction into CAR-T cells read 2019 immuno-oncology post and historical overview provided by Styczyński, 2020), the field has shifted into a different dimension. While 4th generation CARs (see below for ON-switch-, Universal-, OR-gate-, AND-gate-, TRUCK- and Inhibitory- CARs) present further enhancement in antitumoral potency of CAR(-T) cells, and smart CARs (Sacheva et al., 2019) introduced further enhancement in the safety of CAR-T cell therapy, another innovative approach to driving CAR cell translation forward present CAR innate immune cells.

Figure 1. The full potential of CAR T cells will require parallel scientific progress to overcome primary and secondary resistance and addressing practical challenges relating to affordability and scalability (Schultz and Mackall, 2019).

What is wrong (main liabilities and limitations) with CAR-T cells ?

The main problems with CAR-T cell therapy are cytokine release syndrome, neurotoxicity, and on-target/off-tumor effects. Continued progress has demanded solutions to these SAFETY CHALLENGES as well as BIOLOGICAL CHALLENGES, including barriers that have limited the efficacy of CAR T cells in non–B cell malignancies (in particularly solid malignancies). Further, the field has been facing the MANUFACTURING CHALLENGES (related to requirement for personalized product) limiting accessibility of these therapies for patients. Namely, CAR-T cells are usually prepared from autologous peripheral blood (ie personalized), which is costly and time consuming.

The ability to use cells from healthy donors, referred to as ‘off-the-shelf ’ allogeneic CAR T cells (see side bar), could potentially address these issues (for comprehensive review discussing the main challenges associated with this approach and the different ways to improve the current versions of allogeneic CAR T cells read Depil et al., 2020). However, allogeneic approaches are associated with two major issues. First, the administered allogeneic T cells may cause life-threatening graft-versus-host disease (GVHD). Second, these allogeneic T cells may be rapidly eliminated by the host immune system, limiting their antitumour activity.

FIgure 2. Manufacturing of allogeneic CAR T cells. Allogeneic chimeric antigen receptor (CAR) T cells from a single manufacturing batch have the potential to benefit multiple patients. The manufacturing process for allogeneic CAR-T cell products starts with a source of third- party healthy T lymphocytes collected by leukapheresis. Technologies such as viral vector- mediated transgenesis or gene knock- in mediated by gene editing enable the permanent insertion of recombinant DNA coding for a CAR and possibly additional genes, such as a suicide gene or a costimulation receptor in said lymphocytes. Technologies can also eliminate expression of αβ T cell receptor (TCR) on said T cells (for example, gene editing- mediated TCRα [TRAC] knockdown) and CD52. T cells are then expanded using anti- CD3/anti- CD28 beads and cytokines. The remaining αβ TCR- positive cells are magnetically removed using anti- αβ TCR antibodies. The vials are then filled with the allogeneic CAR T cells. The product is then stored, frozen and shipped to hospitals when needed. (Depil et al., 2020)

Recent research and studies highlight the role of the innate immune system when considering immuno-therapeutic approaches and add another platform to the increasing number of cell therapy modalities, in particularly to treat solid tumors.

Advantage(s) of Natural Killer (NK)-CAR cells

CARs can be introduced into any effector cell which can elicit killing of the target cell. Mature natural killer (NK) cells provide attractive candidate effector cells to express CARs (CAR-NK) being potentially safer and more effective than CAR-T cells in that:

• Infusion of CAR-NK cells seldom results in cytokine release syndrome because the NK cells secrete mainly restricted levels of granulocyte-macrophage colony-stimulating factor and IFN-γ, while the pro-inflammatory cytokines IL-1 and IL-6 are seldom secreted.

• The low likelihood of triggering GVHD upon allogeneic infusion means that CAR-NK cells may be prepared as an “off-the-shelf” product and are not restricted to autologous cells.

• CAR-NK cells are able to induce tumor cell lysis in CAR-dependent and CAR-independent manners. Natural recognition receptors that recognize stress-induced ligands independent of CARs are retained on CAR-NK cells, and NK cells express FcγRIII (CD16) that can mediate antibody-dependent cellular cytotoxicity (ADCC). 

Table 1. Current clinical trials of CAR-NK cells. Clinical trials featuring CAR-NK cells against various target antigens and diseases which are currently actively recruiting or are scheduled to begin recruiting in the near future. Data was obtained from clinicaltrials.gov. Abbreviations: iPSC, induced pluripotent stem cell. (Pfefferle and Huntington, 2020)

Crash course in NK cell biology - Licenced to kill cancer cells

NK cells are innate cytotoxic lymphocytes involved in the surveillance and elimination of transformed (eg. tumor) cells. They exert effector functions without the need for prior exposure to the antigen. NK cells possess an extensive repertoire of receptors (activating and inhibitory) that recognize altered protein expression on target cells, thereby controlling the cytolytic function. Uniquely, these receptors can distinguish normal from transformed cells via “missing-self” or “induced self” recognition models. Inhibitory receptors recognize MHC-I molecules, whose absence may result in NK activation, the so-called “missing-self recognition” . However lack of MHC expression is not sufficient or necessary to induce NK activation; rather, signaling from activating receptors is required. Activating receptors provide activating signals upon binding to stress-induced ligands on target cells, which is referred to as “induced-self recognition”. Ultimately, NK activation depends on the balance between activating and inhibitory signals triggered by these receptors. When activating signals prevail (recognition of transformed cell), NK cells respond, whereas when inhibitory signaling is stronger (encounter with healthy cell), NK cells do not respond. 

Despite their potent antitumor activity, NK cells face substantial challenges that hinder their efficacy. In particularly solid tumors present considerable challenges to the application of NK-cell-based therapies as it is difficult for NK cells to traffic and infiltrate into the tumor sites. Similar to T cells, NK cell function, activation, and phenotype are impaired by the tumor microenvironment, even rendering NK cells dysfunctional or exhausted. Thus, strategies to improve the cytolytic activity, durable persistence within tumor microenvironment, and activation of NK cells need to be employed similarly as with anti-tumor cytolytic T cells (for challenges of NK cell-based cancer immuno-therapies read Hodgins et al. 2019).  

NK cells introduce allogeneic therapy (“off-the-shelf” product)

The early studies of adoptive NK cell therapy focused on enhancing the antitumor activity of endogenous NK cells, selection of NK cells from a leukapheresis product and subsequently infusing the autologous NK cells into patients, followed by administration of systemic cytokines (most commonly IL-2) to provide additional in vivo stimulation and support their expansion. The lack of antitumor effect this strategy met related to the inhibition of autologous NK cells by self-HLA molecules.

An alternative strategy is to use allogeneic instead of autologous NK cells, thus taking advantage of the inherent alloreactivity afforded by the “missing self ” concept. A number of studies have shown that infusion of haploidentical NK cells (to exploit HLA alloreactivity) is safe and can mediate impressive clinical activity in some patients with AML. Miller et al. (2005) were among the first to show that adoptive transfer of ex vivo-expanded haploidentical NK cells (and subsequent subcutaneous IL-2 administration) after lymphodepleting chemotherapy is safe, and can result in expansion of NK cells in vivo without inducing graft-vs.-host disease (GVHD). In fact, algorithms have been developed to ensure selection of hematopoetic stem cell (HSC) donors with the greatest potential for NK cell alloreactivity for allogeneic HSCT. Since, allogeneic NK cells have demonstrated a proven track record of safety after infusion for adoptive immunotherapy in patients with cancer.

Figure 3. Manufacturing timeline of CAR-T cell and CAR-natural killer (NK) cell products. Comparison of the manufacturing time for CAR-T and CAR-NK cell products, from harnessing the cells for product processing to patient monitoring after treatment. Abbreviations: CRS, cytokine release syndrome; ICANS, immune e ector cell-associated neurologic syndrome; iPSC, induced pluripotent stem cell.(Pfefferle and Huntington, 2020)

So rather than drawing millions of a patient’s cells, treating them with a receptor over weeks or months and then reinjecting them into a patient, off-the-shelf CAR NK cells don’t cause graft-versus-host disease and avoid the need to extract and modify a cancer patient's own cells.

The patient's immune system will eventually reject any foreign NK cells. But before that happens, the donor NK cells will have a window of time during which they can combat cancer cells and most critical question is how to make them persist long enough to benefit patients.

iPSCs are superior source of NK-CARs

Whereas allogeneic CAR-T cells are currently not an option due to the risk of GVHD, as discussed above allogeneic NK cells are safe in this regard. To date, most of NK cell-based adoptive immunotherapy clinical trials have used primary NK cells isolated from donor’s peripheral blood (PB-NK cells) or umbilical cord blood (UCB-NK cells).

Given the difficulties of sourcing abundant numbers of cytotoxic NK cells from peripheral blood, additional strategies have been investigated to provide readily available banks of NK cells for patients. More readily accessible NK sources to engineer using CARs present cell lines (NK92 [NK92 cells are aneuploid and need to be irradiated before use which limits the in vivo survival and expansion of these cells - known to be a key determinant of anti-tumor activity], KyHG1), or allogeneic NKs derived from iPSCs.

The human cell line NK92, widely used for preclinical applications, has been clinically investigated as an allogeneic NK therapeutic. Tang et al. (2018) reported the results of the first-in-man phase 1 clinical trial of off-the-shelf CD33-CAR NK-92 cells for three relapsed and refractory AML patients showing no significant adverse effects at doses of CD33-CAR NK92 cells up to 5 billion with suppression of minimal residual disease (MRD) biomarkers.

Liu et al. (2018) transduced cord blood-derived NK cells with a retroviral vector incorporating the gene for CAR-CD19, IL-15 and inducible caspase-9 as a suicide gene (iC9). The team has shown they can manufacture hundreds of doses of CAR NK cells from a single unit of cord blood and their ultimate plan is to freeze and store these CAR NK cells in a cell bank to be immediately availeble to treat the patients. In a recently reported Phase I and II clinical trial they administered HLA-mismatched anti-CD19 CAR-NK cells derived from cord blood to 11 patients with relapsed or refractory CD19-positive cancers (non-Hodgkin’s lymphoma or chronic lymphocytic leukemia [CLL]) who had already been through a median of 4 lines of therapy in a single infusion after lymphodepleting chemotherapy. None of the patients experienced GVHD. After 13.8 months, seven were still disease-free [8 (73%) had a response; 7 (4 with lymphoma and 3 with CLL) had a complete remission, and 1 had remission of the Richter’s transformation component but had persistent CLL], although the researchers said the actual duration of response couldn’t be measured because patients went on other therapies after receiving the CAR-NK (Li et al. 2020).

“With an exclusive license and research agreement Takeda received access to MD Anderson’s CAR NK platform and the exclusive rights to develop and commercialize up to four programs, including a CD19-targeted CAR NK-cell therapy and a B-cell maturation antigen (BCMA)-targeted CAR NK-cell therapy. Takeda and MD Anderson also will conduct a research collaboration to further develop these CAR NK programs.”

— MD Anderson News Release November 05, 2019

In contrast, human embryonic stem cell (hESC)-NK cells and human induced pluripotent stem cell (hiPSC-)NK cells can generate essentially unlimited cells sources suitable for clinical use. Importantly, hESC/hiPSC-NK cells exhibit similar phenotype, transcriptome and functions as primary NK cells. Moreover, hESC/iPSC-NK cells can be routinely genetically modified on the undifferentiated pluripotent stem cells to produce a uniform population of NK cells with the desired effect by any of several methods including transposon and viral vectors (eg the CRISPR/Cas9 or TALEN). Induced pluripotent stem cell (iPSC) technology has the potential to enable the next frontier in the development of NK-CARs. The advent of iPSCs, with their capacity to be genetically engineered and indefinitely expanded in culture (ie unlimited self-renewing capacity), has served to create a potentially unlimited cell source for differentiation into specialized cell types and development of “off-the-shelf” NK-CARs. 

COMPANIES IN THE iPSC CELL THERAPY SPACE

Fate Therapeutics: Fate Therapeutics led the next generation cell therapy pioneering effort with the first-ever IND cleared for an iPSC derived therapy (FT500) as well as the first ever IND cleared for a genetically engineered iPSC-derived therapy (FT516).

Evotec: Evotec's iPSC platform has been developed over the last six years with the goal to industrialise iPSC-based drug screening in terms of throughput, reproducibility and robustness to reach highest industrial standards. This effort was initially enabled by a research collaboration with Harvard University involving world-leading scientists at the Harvard Stem Cell Institute. More recently, Evotec established collaborations with Celgene, Sanofi, Center for Regenerative Therapies Dresden, Censo Biotechnologies and Fraunhofer IME-SP to support Evotec's growing iPSC activities.

BlueRock: Acquired in August 2019 by Bayer focusing on differentiated non-oncologic iPSC platform for neurology, cardiology and immunology.

Allogene Therapeutics, which signed an agreement with Notch Therapeutics to develop induced pluripotent stem cell (iPSC) allo-CAR therapy products for initial application in non-Hodgkin lymphoma, leukemia and multiple myeloma.

Mesoblast: Mesoblast Limited announced that it has entered into a partnership with Cartherics Pty Ltd to develop allogeneic ‘off-the-shelf’ CAR-T cells armed with multiple targeting receptors for use in solid cancers.

Century Therapeutcs: Created by Versant Ventures and partnered with Fujifilm Cellular Dynamics Inc. (FCDI) to develop iPSC-derived adaptive and innate immune effector cell therapies, emerged from stealth mode in 2019 with Bayer led investment and commitment to develop next-generation immune oncology treatments.

“In April 2020, the Fate Therapeutics entered into a global collaboration and option agreement with Janssen Biotech, Inc. (Janssen), one of the Janssen Pharmaceutical Companies of Johnson & Johnson, to develop iPSC-derived chimeric antigen receptor (CAR) NK and CAR T-cell product candidates targeting up to four tumor-associated antigens for which Janssen is contributing proprietary antigen binding domains.”

— Fate Therapeutics, Ltd

Fate Therapeutics has established itself as a leader in the space of iPSC-derived cell therapies, and over the last decade has refined its footprint-free iPSC manufacturing process, which can now robustly and reproducibly convert donor-derived somatic cells (eg fibroblasts or blood cells) into a nearly unlimited supply of fully differentiated NK-cell products.

Their platform has addressed key challenges in iPSC culture [nonintegrative methods have proven to be inefficient and labor intensive, often requiring additional reprogramming factors, propensity of iPSCs for spontaneous differentiation and the inability to single-cell culture hiPSCs in a feeder-free (FF) environment] and supports efficient and expedited episomal reprogramming using just OCT4/SOX2/SV40LT  combination (0.5%–4.0%, between days 12 and 16) in a completely feeder-free environment. The resulting hiPSCs are transgene-free, readily cultured, and expanded as single cells while maintaining a homogeneous and genomically stable pluripotent population. In principle, Fate’s approach could allow a homogeneous cell therapy to be manufactured in sufficient quantity to meet the needs of an entire product lifecycle.

Engineered Next-generation CAR-NKs may be the next great cancer immuno-therapy

The field continues to explore strategies to enhance CAR-NK efficacy, such as changing the CAR intracellular domains. Historically, the CD3ζ chain has been used alone or in combination with CD28, 4-1BB, or OX40 signaling domains - designed to promote T cell responses (Jackson et al., 2016), but also activating NK cells (Mehta and Rezvani, 2018). More recently, the signaling domains of adaptor molecules associated with activating NK receptors are being used to mimic physiological NK signaling. While most of these studies explored a few closely related CAR-NK constructs, Li et al. (2018) conducted a comprehensive screen and found that a 2B4 costimulatory plus CD3ζ intracellular signaling domain mediated better specific cytotoxicity than other combinations of CD3ζ, DAP10, DAP12, CD28, 2B4, and CD137 domains. As the field continues to expand, a better understanding of what dictates efficacy of different CAR constructs in various situations will likely follow.

Figure 4. Examples of 4th generation CAR construct organized by subgroups. Abbreviations: scFv, single-chain variable fragment; TM, transmembrane; TRUCK, T cells redirected for universal cytokine killing. (Pfefferle and Huntington, 2020)

The next-generation CAR designs can be classified into subgroups based on their rational.

• ON-switch CARs rely on a small molecule to assemble the fragmented CAR construct, allowing for controlled CAR activation through the administration of a drug.

• Universal CARs are another type of fragmented CAR design, whereby the antigen-specific portion can be exchanged to facilitate the targeting of numerous different cancer types through the same TM and intracellular signaling construct.

• OR-gate CARs aim to prevent tumor escape by providing two scFv domains against different targets, which are either bound to a single TM and intracellular domain (Tandem CAR) or are simply two complete CAR constructs expressed on the same cell (Dual CAR). Signaling through either scFv will activate the T cell.

• AND-gate CARs, as the name implies, also feature two scFvs, but require the presence of both antigens on the same cell before signal propagation. This approach allows for the targeting of non-tumor specific antigens, as tumor specificity is achieved by the dual expression of both antigens (Combinatorial CAR and synNotch Receptor) [44–46].

• T cells redirected for universal cytokine killing (TRUCK)—CAR constructs carrying a transgenic ‘payload’are a novel design for targeting solid tumors. These constructs feature the CAR-inducible expression of a transgene product, such as cytokine(s), co-stimulatory ligands and enzymes that can degrade the extracellular matrix in solid tumors, facilitating release of the ‘payload’ at the tumor site to modulate the tumor microenvironment (TME).

• Inhibitory CARs are focused on turning an immunosuppressive signal from a tumor cell into an activating signal by fusing the extracellular inhibitory domain, for example, PD-1, to an activating intracellular CAR domain.

As one of the high-potential examples, FT596 is an investigational, universal, off-the-shelf natural killer (NK) cell cancer immunotherapy derived from a clonal master iPSC line engineered with three anti-tumor functional modalities:

1. a proprietary chimeric antigen receptor (CAR) optimized for NK cell biology, which contains a NKG2D transmembrane domain, a 2B4 co-stimulatory domain and a CD3-ζ signaling domain, that targets B-cell antigen CD19;

2. a novel high-affinity 158V, non-cleavable CD16 Fc receptor that has been modified to augment antibody-dependent cellular cytotoxicity by preventing CD16 down-regulation and enhancing CD16 binding to tumor-targeting antibodies;

3. an IL-15 receptor fusion (IL-15RF) that promotes enhanced NK cell activity.

The FDA has allowed investigation of FT596 in an open-label Phase 1 clinical trial as a monotherapy, in combination with rituximab for the treatment of advanced B-cell lymphoma, and in combination with obinutuzumab for the treatment of chronic lymphocytic leukemia. Fate Therapeutics plans to initiate enrollment in Early 2020.

In preclinical studies, FT596 has been shown to prevent tumor progression and to promote sustained long-term survival in a B-cell leukemia xenograft model. Moreover, as proof-of-concept for the mitigation of antigen escape, FT596 in combination with rituximab completely eliminates CD19+ and CD19- B-cell tumor cells in a co-culture cytotoxicity assay.

FT596, a next generation multi-MOA CAR-NK expresses: 1. CAR, tailor-made for NK cell anti-tumor activity, a novel high-affinity, non-cleavable variant of CD16 (hnCD16) that enhances its binding to therapeutic antibodies and prevents its down-regulation, which can significantly inhibit anti-tumor activity, IL15/R to enable NK cell persistence without the need for cytokine support.

Combination therapy of CD16-expressing CAR-NK cells together with monoclonal antibody therapy is one possibility for utilizing the full cytotoxic potential of NK cells through both target-specific lysis and ADCC.

Macrophages are driving CAR-Ms

The biotech company Carisma Therapeutics is ready to initiate a phase I trial to assess CAR macrophages (CAR-Ms) in patients with metastatic HER2-overexpressing tumours. 

As T cells and NK cells can’t easily find, penetrate and survive in the tumor microenvironment, other immune cell types have been explored as CAR platforms to circumvent some of the limitations of CAR-T and CAR-NK cells. Klichinsky et al. (2020) have engineered macrophages to express CARs that target their phagocytic activity towards tumour cells. As central effectors of the innate immune response, macrophages detect and eliminate abnormal and infected cells. Macrophages are also abundant within the TME and therefore could be particularly well suited to trafficking to and surviving within it. 

Early clinical trials in the 1990s and early 2000s of non-engineered macrophage infusions for patients with metastatic solid cancers, in which high numbers of autologous monocyte-derived macrophages were expanded and administered to patients, proved that the approach was safe. However, the trials did not show antitumour efficacy. Klichinsky et al. hypothesized that redirecting the phagocytic function of macrophages and stimulating the adaptive immune system with a CAR could change this outcome.

In their Nat Biotech 2020 publication the team led by Gill shows that HER2-targeted CAR-Ms convert bystander M2 macrophages to M1, induce activation and maturation of dendritic cells, and recruitment of resting and activated T cells in vitro. In different models of immunodeficient mice with diminished macrophage function, mice injected with SKOV3 — a HER2+ ovarian cancer cell line — and treated with a single dose of HER2-targeted CAR-Ms showed a substantial reduction in tumour burden and prolonged survival, although all mice eventually progressed. In xenograft models of five different types of solid tumor, CAR-M trafficked to all five types. CAR-Ms were further shown to induce a potentiation of T cell antitumour activity by shaping pro-inflammatory TME and boost anti-tumor T cell activity in humanized mouse models (CAR-Ms and donor-derived polyclonal (non-specific) T cells). This provides evidence for cross-presentation and for T cell co-stimulation, thereby raising the possibility that CAR-Ms could directly diminish tumor burden, sculpt the TME and generate a vaccinal effect. 

COVID-19 has had a huge and negative effect on cancer treatment and research.

In emerging Immuno-oncology field existing trials continue to be disrupted or suspended due to the COVID-19 pandemic (oncology field in general is getting the biggest hit, see report by Carlisle 2020), and there have been no new cell therapy trials.

As R&D community we need to secure the health care support for patients with cancer and ensure trial participants can receive the best possible care even in these exceptionally difficult times (for hidden costs and risks of I-O patients read previous I-O post, March 23rd 2020).