4

Engineering of Allogeneic Donor Cells for Acceptance by the Host’s Immune System

The objective of the second session of the workshop was to explore recent advances in engineering allogeneic donor cells for acceptance by a host’s immune system, including gene editing approaches, universal donor cells, and immune evasion. Rachel Salzman, of the American Society of Gene and Cell Therapy, moderated the session.

MESENCHYMAL STROMAL CELLS IN IMMUNOMODULATORY THERAPIES

Katarina Le Blanc, professor of clinical stem cell research at the Karolinska Institute, discussed efforts to bring mesenchymal stromal cells (MSC) into the clinic with immunomodulatory therapies.

Function of Mesenchymal Stromal Cells in Multimodal Immunomodulation

Le Blanc provided an overview of the role of mesenchymal stromal cells in multimodal immunomodulation. MSCs interact in a number of ways with both innate and adaptive immune cells, with the end result being induction of regulatory T cells (Treg), she explained (Le Blanc and Mougiakakos, 2012). The mode of action of MSCs applied as a local injection is likely less complex than that of MSCs administered by intravenous infusion (Krampera and Le Blanc, 2021). This may explain why the majority of applications approved by regulatory agencies are delivered locally, she noted. Endothelial cells that are normally in contact with blood have anticoagulant properties, but when an injury ruptures the endothelial layers, MSCs, which are tissue-resident cells, can come into contact with blood. MSCs express surface markers that trigger platelet aggregation and coagulation cascade activation (Moll et al., 2014; Moll et al., 2011; Moll et al., 2012). In addition, MSCs activate the complement system, facilitating MSC

engulfment by phagocytes (Gavin et al., 2019b). This reaction is termed the instant blood-mediated inflammatory reaction, and it results in intravenously infused MSCs clotting in the lungs (de Witte et al., 2017; Goncalves et al., 2017). Cytotoxic cells further activate lysosomes and phagocytes that engulf membrane particles and secrete anti-inflammatory factors.

Mesenchymal Stromal Cell Therapies

The anti-inflammatory properties of MSCs through intravenous infusion and local injection have been evaluated in clinical trials for a number of diseases, Le Blanc said.¹ Although clinical experience confirms an exceptional safety profile, the efficacy of MSC therapy has been difficult to establish. This may be due in part to the tendency to generalize and combine results from different types of MSCs and patients with diverse disease characteristics. Although MSCs from different tissues may look similar under the microscope, they differ greatly in terms of functional characteristics. For instance, research has demonstrated that MSCs from one individual’s adipose tissue more closely resemble MSCs from another person’s adipose tissue than MSCs from the individual’s own bone marrow (Gregoire et al., 2019; Ho et al., 2018; Kehl et al., 2019; Menard et al., 2020; Menard et al., 2013). This result indicates tissue specificity in MSCs, Le Blanc emphasized. Furthermore, the pro-coagulant properties differ depending on the tissue of origin (Moll et al., 2014; Moll et al., 2012). For example, MSCs derived from placenta induced massive clotting in comparison to MSCs from bone marrow. The coagulation activation of MSCs from the umbilical cord and adipose tissue falls in between that of MSCs from placenta and bone marrow. Finally, primary fibroblasts induced massive clotting (Moll et al., 2014; Moll et al., 2012), she noted.

Role of the Recipient in Mesenchymal Stromal Cell Treatments

Projecting the effect of MSC treatment requires an understanding of the recipients, Le Blanc remarked. Most diseases are classified based on clinical features, without full understanding of the underlying disease biology, she added. For instance, Le Blanc and her colleagues examined the gut mucosa of patients who fulfilled clinical criteria for severe GVHD (Gavin et al., 2019a). Biopsies of gut mucosa were obtained prior to the MSC infusion, and gross histological staining and immunohistochemistry were

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¹ Intravenous infusion: graft-versus-host disease; multiple sclerosis; solid organ transplantation; myocardial infarction; heart failure; Crohn’s disease fistulas; asthma; aging frailty; acute respiratory distress syndrome. Local injection: vocal fold scarring; xerostomia; radiation injury; Beurger’s disease; osteoarthritis.

used to assess the immune cell profiles. From a biological point of view, samples from the patients who responded to MSC treatment differed in appearance from those of nonresponders, Le Blanc explained. In line with these findings, another study found that GVHD patients with high cytotoxic activity against the infused MSCs during in vitro testing were more likely to respond to MSC therapy (Galleu et al., 2017). Both studies found differences in the cells of responders compared to those of nonresponders, Le Blanc highlighted.

Another study found that patients with high levels of established markers of poor prognosis were more likely to benefit from MSC infusion, Le Blanc noted (Kasikis et al., 2021). Patients with poor prognosis were given the MSC treatment remestemcel-L or the best available therapy. The risk of death six months post-transplant was compared for both groups of patients, and those who received MSC treatment in the high-risk group were more likely to survive, she said. In a trial for chronic GVHD, an immunological analysis indicated that patients who responded to MSC therapy had higher levels of naïve T and B cells than nonresponder patients did. Unlike their counterparts, responder patients mobilized Tregs rapidly after each infusion (Boberg et al., 2020). Within hours or days after each infusion, specific cytokine responses to infused MSCs were evident in responder patients and were maintained for subsequent treatments. The results suggest that there are clear differences between those who respond to MSC treatment and those who do not, Le Blanc remarked.

Potential Markers of Responsiveness to Mesenchymal Stromal Cell Therapies

Le Blanc commented that she and Mauro Krampera, a collaborator of hers from the University of Verona, were asked to speculate on the markers that could predict responsiveness to MSCs or indicate patient responses after infusions. They separated biomarkers of disease activity from markers of MSC effect (Krampera and Le Blanc, 2021). Understanding the biological markers of disease activity may be crucial in future patient selection efforts, she noted. A diagnosis made solely on clinical symptoms will never allow optimal patient selection, she reiterated. The algorithms involved are quite complex, and much remains to be learned about MSC clinical use. This may be another reason why the MSC products approved to-date largely employ local injection. Accumulating clinical data will aid in selecting patients most likely to benefit from MSC treatment, she added.

PROTECTING TRANSPLANTED CELLS FROM IMMUNE REJECTION

Sonja Schrepfer, head of the hypoimmune platform at Sana Biotechnology and professor of surgery at University of California, San Francisco, described approaches to protecting transplanted cells from immune rejection, which she called “one of the keys to unlock the potential for the regenerative medicine field.”

Regenerative Stem Cell Therapy

Even recent advances in stem cell biology can be associated with immune recognition and rejection. One might assume that a stem cell therapy approach that regenerates a patient’s own cells and transplants them back into that same patient would avoid immune recognition or rejection of the graft, said Schrepfer (see Figure 4-1). However, the immune system can recognize autologous cell products that are generated from the same patient’s cells (Deuse et al., 2019; Deuse et al., 2015). Another approach produces induced pluripotent stem cells (iPSCs) from a group of healthy human leukocyte antigen (HLA) donors with different HLA types (Kawamura et al., 2016). The iPSCs are then banked in off-the-shelf formulas to use in allogeneic transplantation into HLA-matched patients. This approach is based on the theory that HLA molecules serve as a fingerprint of the cell that is recognized as foreign when transplanting cells from one person to another and that rejection will not take place when the fingerprints match, she said. Both approaches have resulted in cases of antigen recognition of the alloantigen, Schrepfer noted. In addition to the contribution of HLA in immune recognition and rejection, molecules such as minor histocompatibility antigens (miHA) can also be recognized. Autologous cell products carry the risk that miHA neoantigens will form and allogeneic HLA-matched cell products from HLA banks can also present miHA, leading to immune rejection.

Autologous Regenerative Stem Cell Therapy

In the autologous cell products approach, somatic cells from a patient hospitalized with organ failure, for example, are isolated and reprogrammed into iPSCs, Schrepfer explained. This reprogramming generates an autologous cell product that is then transplanted back into the same patient. Because the cells are harvested from the patient who will receive the cell product, it is expected that mismatch will be avoided and that the immune system will recognize the transplant as autologous and not become activated. However, this does not always prove to be the case. For example,

a cell type such as a cardiomyocyte has mitochondria that carry mitochondrial DNA (mtDNA), which is not as well protected as nuclear DNA, meaning that mtDNA mutations can occur, said Schrepfer. The mtDNA codes for 13 proteins within the respiratory chain; any mutations to these proteins will cause the immune system to recognize the cells as foreign, which could trigger immunoactivation (Deuse et al., 2019).

Fibroblasts from six mice were isolated, reprogrammed into iPSCs, and then cultured in a study that illustrated some of the immunological hurdles related to autologous cell products in regenerative stem cell therapy (Deuse et al., 2019; Deuse et al., 2015). Over time, the iPSCs were passaged, or split, numerous times, Schrepfer described. Researchers found that iPSCs that were passaged 37 (P37) times had a higher frequency of mutations in mtDNA than did iPSCs passaged 7 (P7) times. In a heat map of mtDNA sequencing data, P37 mitochondrially encoded cytochrome c oxidase I—also called Co1—was found to have 92 percent heteroplasmy, meaning that 92 percent of the mitochondria in the cells carried that mutation. After transplanting these cells into the same mouse they were originally harvested from—an autologous or syngeneic transplant setting—researchers found that all the P7 cells survived. In contrast, when P37 cells were transplanted, only 40 percent of the grafted cells survived and the grafts in three of the mice were rejected. This indicates that the immune system can recognize even a single nucleotide polymorphism, demonstrating that autologous HLA can present neoantigens leading to rejection of autologous iPSCs, Schrepfer explained.

Human Leukocyte Antigen Banking for Pluripotent Stem Cells

The HLA banking concept aims to achieve matched transplantation that avoids autoimmune recognition, which has been tested by Teruhisa Kawamura and colleagues, said Schrepfer (Kawamura et al., 2016). Nonhuman primates (NHPs) matched for major histocompatibility complex (MHC) were used to imitate an HLA bank for humans. Researchers transplanted iPSC-generated cardiomyocyte patches under the skin of recipient MHC-matched monkeys and then grouped them by immunosuppression treatment administration. One group received no immunosuppression, and despite using MHC-matched grafts, all grafts in this group were rejected. Another group received tacrolimus with trough levels above 10 nanograms per milliliter, which is equivalent to what would be administered to a transplant patient for heart transplantation; the grafts in this group did not survive. A third group received a heavier immunosuppression regimen comprised of tacrolimus, prednisolone, and mycophenolate mofetil. The survival of grafts two months post-transplant for this group was 100 percent. These findings are a clear indication that HLA banking does not avoid

the risk of rejection and antigen recognition, which subsequently leads to rejection and loss of the grafts, Schrepfer stated.

Hypoimmune Cell Products: Protecting Allogenic Cells from Immune Destruction

The beauty of stem cell–based therapy is the accessibility of cells and the longer time frame during which they can be modified, Schrepfer remarked. Whereas solid organs have a short window of approximately six hours in which to be transplanted, researchers have time to modify stem cells and iPSCs. If researchers can utilize gene editing tools on iPSCs from healthy donors to create hypoimmune cell products, these could be available off the shelf for any patient. Advantages of such technology over other approaches are considerable, she said (see Box 4-1). Cells that are truly hypoimmune eliminate the need for immunosuppression treatment to avoid immune rejection. A well-characterized master cell line could be developed from one healthy donor, avoiding the need for cumbersome banking of huge numbers of cell lines and enabling easier manufacturing and quality control, Schrepfer explained.

Adaptive and Innate Immune Responses to Human Leukocyte Antigen in Allogeneic Cells

The field of hypoimmunity started years ago when researchers began studying a naturally occurring allogeneic graft—the fetus in a pregnant

mother, said Schrepfer. Half of fetal proteins are from the father, and the mother’s immune system recognizes the proteins as allogeneic (i.e., foreign) yet does not reject the fetus. Schrepfer and colleagues in the field studied feto–maternal immune tolerance, including molecules that are upregulated and downregulated, in an effort to understand the combination of molecules that could create a hypoimmune cell product. This concept of hypoimmune is an alternative approach to overcoming the immune barrier; hypoimmunity differs from tolerance induction in that it is based on the idea of immune evasion, whereby the immune system does not recognize the cell product after transplantation, Schrepfer noted. Adaptive immunity presents a major challenge in cellular transplantation (see Figure 4-2). When T cells recognize the HLA molecules of allogeneic cells, they kill and reject those cells. Researchers have come to understand that HLA removal overcomes the T-cell response. However, HLAs also inhibit other cell types, such as natural killer (NK) cells, that are part of the innate immune system. Thus, removing the HLAs introduces a new issue to allogeneic transplantation—that is, killing by innate immune cells.

Molecules to Prevent Killing of HLA-knockout Target Cells by Innate Immune Response

Researchers are investigating potential molecules that, when overexpressed, are hypothesized to inhibit innate immune cell killing. Schrepfer and colleagues tested the separate effects of overexpression of four molecules—HLA-E, HLA-G, programmed death-ligand 1, and CD47—using a cell line that does not express HLAs and was cultured with NK cells. When the assay was conducted with primary NK cells, such as those found in the human body, most of the molecules were not able to prevent the NK cell population from activating and killing the allogeneic cells. The CD47 molecule, however, can inhibit both subpopulations of NK cells and the entire NK cell population in a human body. With the creation of hypoimmune cells as a genetic endpoint, the engineering approach to off-the-shelf cell therapies involves taking iPSCs from healthy donors, removing HLA class I and II molecules, and then overexpressing CD47 (Deuse et al., 2021a; Deuse et al., 2019; Deuse et al., 2021b; Hu et al., 2021). The goal of creating off-the-shelf therapies is to eliminate the need for immunosuppression and to have these therapies available for anyone, at anytime, anywhere, Schrepfer highlighted.

Recent research has demonstrated that this method not only overcomes the allogeneic barrier but also enables the transplantation of fully functional cells. Researchers differentiated iPSCs from mice into endothelial cells and found that in T-cell activation assays of unmodified endothelial cells, allogeneic activation of T cells was much higher than in syngeneic transplantation, a process in which immune rejection is not expected (Deuse et al., 2019). Similarly, in subsequent antibody binding, immunoglobulin M (IgM) antibodies were higher in allogeneic activated samples than in syngeneic transplants. However, for hypoimmune endothelial cells, T-cell activation and IgM antibodies were similar for allogeneic activation and syngeneic transplantation (Deuse et al., 2019). Furthermore, studies in a disease model for hind-limb ischemia in mice showed that the unmodified, wild-type endothelial cells died and the hypoimmune cells survived (Deuse et al., 2021b). The animals injected with wild-type iPSC-derived endothelial cells experienced limb loss. In contrast, the hypoimmune iPSC-derived endothelial cells not only survived but also restored vascularization in mice that received them without immunosuppression (Deuse et al., 2021b). The goal of regenerative therapy extends beyond overcoming the immune barrier to enable cells to function as intended in vivo, Schrepfer emphasized.

Immunosuppression-Free Future of Regenerative Therapies

Nonhuman-primate (NHP) studies set a high bar for immune studies, because the NHP immune system is so active, Schrepfer noted. Researchers found that when unmodified iPSCs were injected into monkeys, T-cell activation was high. However, transplanted hypoimmune iPSCs avoided immune system detection and antibody production in allogeneic NHP recipients. She noted that immune evasion was achieved even after prior sensitization in monkeys, an important finding given that regenerative medicine should consider the needs of sensitized patients who may require redosing, or repetitive treatment. Researchers also found in NHPs that the CD47 molecule prevents the killing of hypoimmune iPSCs; the cells will survive when CD47 is present but are killed when CD47 is blocked. Finally, the unmodified iPSCs were rejected in rhesus monkeys after three weeks, whereas the hypoimmune iPSCs survived. When all immune system components are considered—NK cells as well as T cells—immune evasion with hypoimmune cells appears to be an achievable goal, Schrepfer said.

Schrepfer was optimistic that the future of regenerative therapies could be immunosuppression free, given that cellular transplantation without immunosuppression appears to be achievable using hypoimmune cells. Together, the findings she presented show that hypoimmune cells (1) evade allogeneic immune rejection; (2) do not activate the “missing self” response from NK cells and macrophages; (3) can be transplanted into sensitized recipients, offering the possibility of redosing; and (4) do not alter the recipient’s immune system (Box 4-1).

OFF-THE-SHELF ENGINEERED IPSC-DERIVED NATURAL KILLER AND T CELLS FOR THE TREATMENT OF CANCER

Bob Valamehr, chief research and development officer at Fate Therapeutics, provided an overview of his company’s cell therapy platform. Although the iPSC-derived off-the-shelf platform is novel, the Fate Therapeutics approach provides similar benefits to more conventional strategies and drug development. The process begins with a starting material that is uniform and consistent for manufacture; in this case, the starting material is a master cell bank. The key reagent is a stem cell that has been highly engineered, banked, and fully characterized, he explained. Consistent starting material enables mass production of the cell type of choice. Multiple cell types can be made from stem cells, including NK and T cells. Mass production now allows the creation of a uniform product that is frozen in bags and ready to be shipped to hospitals. The frozen material can be thawed and directly infused, representing a new treatment paradigm in cell

therapy, in which treatment is available on demand and provided in outpatient settings, he explained.

Off-the-Shelf Cell Therapy Platform

At the heart of the master cell bank are iPSCs, which can be made into any of the 200 cell types found in the body, said Valamehr. When these cells are properly maintained, they have unlimited self-renewal capacity. Changing the culture conditions enables the cells to differentiate. For instance, Fate Therapeutics has been working on hematopoietic cell lineage differentiation, and they have established control over the cells to the extent that they are now able to dissociate iPSCs into single cells. Those individual cells are engineered to expand a uniform population of multi-edited engineered products (see Figure 4-3). The uniform composition and fully characterized master cell bank enable a renewable clonal cell line that can be used to create homogenous cell products. Valamehr explained that iPSCs are first edited, individual cells are allowed to expand into clonal populations, and then researchers compare individual clones for desired attributes. Screening occurs at both the molecular and functional level to select for desired attributes and avoid undesired traits, such as off-target editing or genomic instability. Thousands of clones are screened to select an individual clone that is then used to create a master cell bank. This renewable starting material can be used to make high-quality NK and T cells that are frozen in bags, delivered, thawed, and directly infused in outpatient settings. This off-the-shelf platform allows for multiplex engineering of a homogenous product, mass production, and off-the-shelf cell therapy application, he said.

Developing Novel Multiplexed Engineered Cells with Multi-Antigen Specificity

Ongoing work at Fate Therapeutics aims to develop novel multiplexed engineered iNK and iT cells with multi-antigen specificity to combat tumor heterogeneity and treatment resistance. Multiple components can be edited into a cell to create highly effective effector cells, Valamehr explained, and Fate Therapeutics focuses on multiple attributes in producing high-quality NK and T cells. He remarked that cancer is smart, and therefore it must be attacked in a multipronged manner. This involves putting multiple targeting entities onto the cell that enable the cell to attack the target through chimeric antigen receptor (CAR) T-cell receptor, CD16 receptor, and other novel strategies. For instance, cells are targeted to attack antigens specific to cancer. Once targeting entities are added to the cell, the cell product is combined with other effective therapeutic agents, such as monoclonal antibodies, checkpoint blockade therapy, T and NK cell engagers, and radiation

therapy (Saetersmoen et al., 2019). The manufacturing of NK and T cells allows different effector cells to be combined to introduce both innate and adaptive immunity into a patient, Valamehr explained.

Chimeric Antigen Receptor T-Cell Therapy

Fate Therapeutics is focusing their pipeline on CAR-targeting strategies, Valamehr said. The pipeline consists of multifaceted, multi-targeted CAR T and NK cells that can be synergized with current therapeutic agents. A CAR-19 NK therapy—dubbed FT596—that targets CD19 for B-cell malignancies is currently in clinical trials. This novel dual-antigen targeting strategy of FT596 is designed to overcome tumor heterogeneity and antigen escape for a durable response in B-cell malignancies. FT596 is an NK cell containing three specific antitumor modalities (Woan et al., 2021); the first is a high-affinity noncleavable CD16 (hnCD16) that maximizes antibody-dependent cell-mediated cytotoxicity (ADCC) (Jing et al., 2015). The modality hnCD16 enables effective elimination by engaging with the antibody, which in turn engages with the cancer cell (Zhu et al., 2020). The second antitumor modality is a CAR that targets CD19 and facilitates targeting of multiple antigens on a given lymphoma (Li et al., 2018). For instance, the CAR that targets CD19 can be combined with a monoclonal antibody (mAb) targeting CD20, CD22, or CD123. Third, FT596 has an interleukin (IL)-15 receptor fusion that causes NK cells to be more resilient and persistent, enabling them to survive without endogenous cytokine support (Woan et al., 2021). Typically, NK cells are challenging to engineer because they respond to the input of a transgene by expelling it, said Valamehr. However, since it starts from a clonal population of engineered iPSCs, the Fate Therapeutics final product is a pure population of NK cells—as defined by the CD45/CD56 population—that carries uniform expression of CARs and CD16. A single vial of banked starting material can produce over 1 trillion iPSC-derived NK cells, and by expanding the volume from 100 liters to 10,000 liters, the number of produced NK cells can increase to 100 trillion. Preclinical studies indicate that a multidose application of FT596 controls the NALM6 cell line in mice, Valamehr said.

The clinical protocol for FT596 tested the treatment on B-cell lymphoma as a monotherapy and in combination with rituximab. The chemotherapy cyclophosphamide was used to make space for the FT596 by eliminating immune cells through lympho-conditioning and lymphodepletion. Cyclophosphamide also increases homeostatic cytokines in the body. Furthermore, this drug rids the environment of CD8, CD4 cells, or NK cells that could potentially eliminate FT596 in the allogeneic setting. After cyclophosphamide treatment, one dose of FT596 was given either as a monotherapy or in combination with rituximab. The treatment was

assessed 29 days later, and the study found that FT596 demonstrated dose-dependent efficacy. A dose of 30 million cells did not indicate clinical response. However, patients responded to single doses of 90 million and 300 million cells administered as fourth or fifth lines of therapy. FT596 is one of the first off-the-shelf iPSC-derived products in an allogeneic setting that shows antitumor activity and contribution to disease control, Valamehr remarked. Of the 14 patients tested, 10 achieved overall response and 7 achieved complete response.

Strategies for Evading the Immune System

The three approaches Fate Therapeutics uses to evade the host immune system include cloak, eliminate, and attack and proliferate, Valamehr outlined. The cloaking strategy involves knocking out genes associated with CD8, CD4, and NK cell engagement. This process cloaks the product from detection by dismissing engagement of T and NK cells with the cell product. The elimination strategy utilizes mAb CD38 to eliminate activated effector cells found in the body. When T and NK cells are activated, they express CD38; therefore, daratumumab, an anti-CD38 mAb, is administered to a patient with hnCD16 to eliminate any activated NK or T cells in the vicinity, Valamehr explained. The product avoids self-destruction because it is a CD38 knockout, while still supporting the killing of other activated cells that come near it. The final strategy of attack and proliferation utilizes an alloimmune defense strategy designed for patients with treatment regimens that do not include cyclophosphamide (Mo et al., 2021). A novel synthetic receptor eliminates nearby alloreactive immune cells and simultaneously provides a biological signal to promote positive cellular proliferation. This product not only survives in the host immune system, but it also becomes activated through engagement with the host immune system, Valamehr remarked. Fate Therapeutics is advancing both these novel immune evasion strategies and off-the-shelf T and NK cell products, which may be combined to combat cancer and its process of evolution, he said.

DISCUSSION

Cell Therapy Risk Mitigation

A member of the community asked Schrepfer and Valamehr how to mitigate the risk of engineered cells becoming malignant or out of control. The safety aspects of cellular transplants that evade immune recognition must be considered, Schrepfer replied. One concern is the possibility that viral load could lead to HLA-negative cells becoming infected. The risk of that occurring is yet to be determined, and it merits further research,

especially when considering that knockout mice have been able to clear viruses, she stated. Another concern is that, theoretically, iPSCs carry the risk of teratoma formation. Furthermore, the cells may not function as they are designed to function, and mitigating risk involves controlling for that. Control is not only needed for immune evasion strategies but also for tolerance induction approaches, she added. Should something go awry in a setting of tolerance induction, such as an insulin-producing beta cell not functioning as it should, a safety backup may be required. Beyond overcoming immune barriers and avoiding cell rejection, strategies to control the cells are needed as well as research on the benefits and risks for specific patient populations, said Schrepfer.

The beauty of off-the-shelf products is that they can be highly characterized, Valamehr remarked. Before a developed product is administered to a patient, it is studied for months to determine standard dosage, the maximal dosage that would ever be reached, and tumor toxicity. The product is fully characterized for genomic stability, in vitro oncogenicity, and safety perspectives in the tumor toxicity study. Whereas the critical mission of an autologous treatment is administration to the patient, the off-the-shelf setting allows for thorough testing to enable confidence that the product will not revert, has no residuals, and is effective, said Valamehr.

Salzman asked the speakers whether generation methods for making iPSCs can cause mutations in mitochondria and, if so, whether that potential problem can be mitigated. Mutations are dependent on how cells are handled; for instance, freezing and thawing increases the risk of mutation, Schrepfer responded. Processes can be optimized to build quality control by analyzing mutations and methods that increase risk, she added. At the center of Fate Therapeutics’ strategy is screening clones, Valamehr noted. By screening 1,000 clones, researchers can detect undesired effects and changes in the host mitochondrial genome. Over the course of multiple years, they follow the entire development of the clone and establish deep familiarity with it. Clone selection provides options of different attributes that can be selected or deselected to avoid undesirable mutations, he said. It is different with MSCs since they are natural cells and are limited by nature, Le Blanc added.

Mesenchymal Stromal Cell Therapy

Given that most MSC products are approved for local injection rather than infusion, Salzman asked Le Blanc about the risk–benefit profile of MSCs and how this applies on a translational level. There may be more safety data on infusion delivery than on local delivery, and these safety data are extraordinary, Le Blanc responded. It is rare for a treatment to have as few side effects as does intravenous injection of MSCs; however, efficacy has

not been achieved thus far, she added. Furthermore, greater understanding is needed regarding who should be targeted with MSCs. Drawing a parallel to a scenario in which all fever patients are treated with penicillin despite the fact that not all fevers are caused by penicillin-sensitive bacteria, she stated that the biology of disease should be better understood in order to target treatment appropriately. Salzman added that the opportunity for a perfect solution seems to lie in finding a treatment that provides the right amount of benefit—neither too little nor too much—while avoiding high risk.

Since MHC concerns do not apply to MSCs, Salzman asked Le Blanc whether MSCs are created alike or if variability is found across a population in terms of external profiling. Salzman asked how wide the spectrum of variability among donor MSCs is in contrast to hematopoietic stem cells or other cell sources. MSCs do not engraft and instead disappear in the blood stream within minutes, Le Blanc replied. She posited that rapid destruction is more likely at play than pure rejection or lack of engraftment. This is seen in local injection in the vocal fold system. The vocal fold is a fairly immune privileged site, yet MSCs are not found a day or two after injection. Immune responses have been detected, but these are minor, and true rejection is therefore unlikely to be the cause of the rapid disappearance of cells, she said.

Specifics of Hypoimmunity

Salzman asked Schrepfer about the stage of cell development at which the effects of engineered hypoimmunity become apparent in stem cells such as iPSCs, embryonic stem cells (ESCs), mesoderm, and hematopoietic stem cells. Schrepfer replied that when she entered the field in 2005, many researchers hoped that ESCs—and later iPSCs—held the potential to evade detection by the immune system due to their early stage of development, therefore these cells or their derivatives could be transplanted into anyone without immune recognition. The current understanding is that regardless of the state, the HLAs and the alloantigens they present will eventually be recognized by the immune system. Thus, no cell state protects against immune recognition and subsequent rejection, she said. Another concept focuses on the transplant site rather than on the cell. Some researchers believe that certain sites, such as the brain and eye, may be immunoprivileged. Cell transplantation typically breaks the blood–brain barrier, resulting in the immunoprivileged organ losing its immune privilege, she explained. Clinical practices must therefore consider the handling of tissue and the environment. Schrepfer stated that she does not think tissues, organs, or allogenic cells are immune privileged, although some cell types such as MSCs have immune-privileged capabilities. Despite this, the risk of MSCs being recognized and rejected in an allogenic setting persists.

Given that CD47 is known to emit a signal directing macrophages not to eat the cells, Schrepfer was asked whether overexpression of CD47 inhibits phagocytosis-mediated clearance of apoptotic donor cells by tissue-resident macrophages. While CD47 is known as the “don’t eat me” protein, Schrepfer calls it the “don’t kill me” protein because it also works on NK cells with a different mechanism than the one it utilizes on macrophages. Necrotic and apoptotic cells will still be cleared when CD47 is overexpressed because the molecules are no longer functional and are therefore unable to prevent cell clearance. Some cell death will occur during cell transplantation due to the stress of the process and the lack of oxygen, and dead cells need to be cleared. Apoptosis and necrosis render the molecules unable to inhibit clearance by macrophages, she explained.

Salzman also asked Schrepfer about the barriers involved in the placenta during pregnancy and how barriers may be used to generate hypoimmunity. Pregnancy is unique and fetal–maternal tolerance does not translate directly to hypoimmunity, Schrepfer replied. Pregnancy involves the inhibition of various immune populations at different times. For instance, high numbers of Tregs are needed during implantation of the fetus, but not later in pregnancy. Schrepfer and her colleagues have used the concept of pregnancy in studying the effects of molecules on immune populations. The barrier from fetus to mother is open, allowing for the trafficking of immune cells. Unfortunately, this process cannot be translated to cell transplantation, but the concept is used to start understanding the molecules involved, she said.

Strategies for Evading the Immune System

Valamehr was asked by a community member to speculate about which of the Fate Therapeutics’ strategies is best suited for transplants versus short-term use of iPSCs, such as immune cell therapy. The strategies of elimination and of attack and proliferation are only for cells that have the ability to kill, Valamehr replied. Eliciting an ADCC or CAR response is reserved for transient effector cells. The first strategy of cloaking breaks engagement and is used for regenerative medicine. He noted that other approaches exist beyond those that utilize T and NK cells, and he and his colleagues are working on a strategy that will disengage all interactions.

Expansion of Cellular Therapy Distribution

Salzman asked Valamehr about processes for dosing and repeat dosing off-the-shelf products and about the next frontier in off-the-shelf cell therapy. Distributing cell products into community hospitals is the next goal, he replied. Few people live near a cellular therapy research facility,

and therefore distributing products into as many locations as possible will increase the reach of therapeutic benefit to more people. Fate Therapeutics currently has two manufacturing sites and is planning to grow their manufacturing capacity. Ideally, patients will eventually be able to go to a pharmacy or local hospital and have the prescribed product thawed and administered on an outpatient basis in the community setting. He added that an overall goal is to enable multidosing. Just as aspirin can be taken every four hours until a headache goes away, cellular therapy could be administered until desired results are achieved. Currently, at low-scale production, each dose costs less than $5,000. Valamehr described a vision in which everyone has access to cellular therapy administered in a multidose manner until the cancer is gone.

Patient Profiling and Conditioning

Salzman asked Valemehr to elaborate on the benefits of conditioning patients before cell therapy and whether pathways in the future may eliminate the burden of conditioning on the patient. Conditioning in a cancer setting serves multiple purposes, Valamehr replied. Conditioning acts as an antitumor agent and creates space for the cell therapy. Competition within the body for cytokines limits available space in bone marrow. Conditioning allows for reducing endogenous immune cells, CD8s, CD4s, and NK cells that may reject the product while increasing homeostatic cytokines such as IL15. These effects hold value while simultaneously depleting the host immune system. FT596 has been administered in doses containing up to 900 million cells without being rejected, albeit in a population of cancer patients with exhausted immune systems, Valamehr noted. In a situation involving a solid tumor where the product is being used as frontline treatment, light conditioning is preferred to traditional conditioning. This involves creating a setting in which the product can work in concert with and take advantage of the endogenous immune system and induce a second wave of immunity. CAR therapy relies heavily on cyclophosphamide, but strategies involving allodefense receptors may overcome the need for conditioning in the future, said Valamehr.

Given that various patient profiles respond differently to treatment, Salzman asked the speakers what aspects of the target product profile should be considered and how the cell product could best match the profile of a patient experiencing an unmet health need. Schrepfer responded that researchers must consider that each disease is different, each patient has different viral infections, and that the immune system needs to be in a certain state in order to establish tolerance. In contrast, immune evasion does not require a specific immune system status because the goal of this concept

is to completely avoid cell recognition. Because evasion overcomes the need for the patient to have a certain immune state, evasion complements tolerance therapies, she added. Many clinical diagnoses of inflammatory disorders are made based on symptoms rather than on biology, Le Blanc remarked. Including biology in both staging and diagnosing disease will shed light on treatment requirements, she reiterated. Valamehr commented on the importance of universality and multi-antigen targeting. Targeting a cancer in multiple ways and targeting the immune system will enable cancer patients to be treated and to persist long enough to receive benefit from the treatments.