Enabling allogeneic therapies: CIRM‐funded strategies for immune tolerance and immune evasion

Abstract A major goal for the field of regenerative medicine is to enable the safe and durable engraftment of allogeneic tissues and organs. In contrast to autologous therapies, allogeneic therapies can be produced for many patients, thus reducing costs and increasing availability. However, the need to overcome strong immune system barriers to engraftment poses a significant biological challenge to widespread adoption of allogeneic therapies. While the use of powerful immunosuppressant drugs has enabled the engraftment of lifesaving organ transplants, these drugs have serious side effects and often the organ is eventually rejected by the recipient immune system. Two conceptually different strategies have emerged to enable durable engraftment of allogeneic therapies in the absence of immune suppression. One strategy is to induce immune tolerance of the transplant, either by creating “mixed chimerism” in the hematopoietic system, or by retraining the immune system using modified thymic epithelial cells. The second strategy is to evade the immune system altogether, either by engineering the donor tissue to be “invisible” to the immune system, or by sequestering the donor tissue in an immune impermeable barrier. We give examples of research funded by the California Institute for Regenerative Medicine (CIRM) in each of these areas, ranging from early discovery‐stage work through clinical trials. The advancements that are being made in this area hold promise that many more patients will be able to benefit from regenerative medicine therapies in the future.

enabled the engraftment of lifesaving organ transplants, these drugs have serious side effects and often the organ is eventually rejected by the recipient immune system. Two conceptually different strategies have emerged to enable durable engraftment of allogeneic therapies in the absence of immune suppression. One strategy is to induce immune tolerance of the transplant, either by creating "mixed chimerism" in the hematopoietic system, or by retraining the immune system using modified thymic epithelial cells. The second strategy is to evade the immune system altogether, either by engineering the donor tissue to be "invisible" to the immune system, or by sequestering the donor tissue in an immune impermeable barrier. We give examples of research funded by the California Institute for Regenerative Medicine (CIRM) in each of these areas, ranging from early discovery-stage work through clinical trials.
The advancements that are being made in this area hold promise that many more patients will be able to benefit from regenerative medicine therapies in the future. inhibitor, and steroids. These drugs are associated with cumulative side effects that increase the risk of cardiovascular disease, diabetes, hypertension, infection, cancer, and nephrotoxicity. 1 Other immunosuppressive agents that are used clinically are monoclonal antibodies to target and eliminate reactive host lymphocytes (eg, Alemtuzumab) 2,3 or to block costimulation of T lymphocytes (eg, Abatacept). 4 Despite this armamentarium of tools for suppressing the patient immune response, the risks of graft rejection remain high. Furthermore, when the donor tissue in question is hematopoietic stem cells (HSCs), patients are also at risk for suffering from graft vs host disease (GVHD), with side effects ranging from mild to life-threatening. With increasing numbers of allogeneic cell-based therapies being developed for testing in the clinic, the challenge of achieving long-term engraftment while avoiding potentially lifelong immune suppression is being actively approached by many different groups. These strategies can be divided into two conceptually different approaches; one approach is to induce immune tolerance of the donor tissue, while the second approach is to evade the immune response altogether. The California Institute for Regenerative Medicine (CIRM) funds the development of many types of cell therapies and thus it is critical also to fund the development of methods to ensure those therapies can be safely and durably transplanted to patients. In this perspective, we briefly describe some of the strategies being taken by our awardees to address the problem of immune rejection. Table 1 summarizes multiple CIRM-funded projects that are testing methods to induce immune tolerance, a situation in which the host immune system does not reject allogeneic transplanted tissue, and, in the case of HSC transplantation (HSCT), the donor immune cells do not attack the host tissue. Several of the clinical stage projects are using the induction of hematopoietic "mixed chimerism" to create immunological tolerance, while, in parallel, preclinical work is being done to develop ways to reprogram the immune system at the level of the thymus.

| Mixed chimerism
One approach to induce tolerance of an allogeneic donor graft is through generation of "mixed chimerism," characterized by coexistence of donor and recipient blood and immune cells. Persistent mixed chimerism has been demonstrated to be required for immune tolerance, minimizing, or preventing both graft rejection by the host and GVHD. 5,6 CIRM has funded several groups using this approach for a variety of different disease indications. In one example, Dr Sam Strober and his colleagues at Stanford have enrolled and treated kidney disease patients with either human leukocyte antigen (HLA) fully matched or haploidentical (match at 3/6 HLA antigen loci) related donor combined kidney and blood stem cell transplants (Table 1).
After receiving a kidney transplant, these patients were conditioned with lymphoid irradiation and antithymocyte globin and then infused with hematopoietic progenitor cell (CD34+) and T-cell (CD3+) populations from the same donor. This study showed that HLA fully The goal of CIRM "Discovery" stage awards is either to identify a candidate therapeutic that demonstrates reproducible disease modifying activity in a preclinical model relevant to the target indication, or to identify a medical device that demonstrates technical feasibility in meeting product design requirements and initial performance criteria.

Significance statement
For cell and tissue therapies to become widely accessible will ultimately require the success of off-the-shelf allogeneic products that can be administered to patients regardless of immune compatibility with the donor tissue. Since the long- A mixed chimerism approach is also being taken by Dr Joseph Rosenthal at City of Hope Medical Center, who is conducting a CIRMfunded phase 1 trial to treat sickle cell disease (SCD) patients with a haploidentical HSCT using nonmyeloablative conditioning ( Table 1).
The concept behind this approach was inspired by earlier observations that stable mixed chimerism sometimes occurs after standard myeloablative HSCT for SCD patients, and donor chimerism between 11% and 74% was found to be curative in the absence of GVHD. 9 The nonmyeloablative method that is used to generate mixed rather than complete chimerism is less toxic than that used for standard HSCT, and thus may be tolerated by patients with severe SCD, who would otherwise be ineligible for a transplant due to pre-existing organ damage and other comorbidities. The use of haploidentical donors may also increase patient access to this therapy by expanding the donor pool, as many family members may be suitable matches.
This trial aims to test the safety and feasibility as well as the efficacy of the approach.
Another pioneering and potentially transformative approach to developing tolerance by inducing mixed chimerism is being taken by  Table 2 summarizes multiple approaches to immune evasion being developed by CIRM grantees. These can be broadly divided into two main categories: (a) engineering cells to be immunologically "incognito" by manipulating the expression of immune system proteins, and (b) developing encapsulation devices that shield the cells from the immune system. We briefly describe some of the approaches below. Other disease areas are also being tackled with this approach; for example, Dr Tracy Grikscheit of Children's Hospital Los Angeles (CHLA) plans to engineer human iPSC to be nonimmunogenic before deriving hepatic progenitors to be tested in liver disease models, and Dr Lili Yang plans to engineer nonimmunogenic HSC to derive invariant natural killer T (iNKT) cells for use as a cancer therapeutic.

| Cell engineering to prevent immunogenicity
In a variation of the above approaches, Dr Sonja Schrepfer at UCSF was funded by CIRM to couple HLA class I and II disruption with CD47 overexpression in human iPSC, followed by derivation of cardiomyocytes, smooth muscle, and endothelial cells that were used to create a hypoimmunogenic cardiac patch. In preclinical testing, this patch evaded immune rejection after transplantation into fully major histocompatibility complex (MHC) mismatched allogeneic recipients. 23 The results indicate that CD47 expression may prevent NK cell responses, consistent with an association between CD47 expression and protection from NK cell-mediated cytotoxicity that was described previously in a study of squamous cell carcinoma lines. 24 As an added benefit, overexpression of CD47 prevents phagocytosis by macrophages. 25 Naturally occurring suppressors of the immune system have also suggested other ways to engineer cellular therapies to prevent recognition by the host immune system. The goal of CIRM "Discovery" stage awards is either to identify a candidate therapeutic that demonstrates reproducible disease modifying activity in a preclinical model relevant to the target indication, or to identify a medical device that demonstrates technical feasibility in meeting product design requirements and initial performance criteria.
We note that the above strategies, should they be successful, would prevent the immune system from recognizing donor tissue even if became infected or cancerous. Therefore, many researchers also include an inducible "kill switch" such as iCasp9 27 or herpes simplex virus thymidine kinase 28 so it would be possible to destroy the engrafted cells should the need arise.

| Encapsulation devices
Among the strategies to avoid the immune system, encapsulation is perhaps conceptually the simplest way to protect a cellular therapeutic while at the same time being the most limited in its application. Both microencapsulation devices, in which relatively small numbers of cells are embedded in matrices such as alginate, polyethylene glycol (PEG), or gelatin, and macroencapsulation devices (>1 mm), capable of containing larger amounts of cells, have arisen as viable approaches to evading the immune system. 29,30 The requirements for these encapsulation modalities are generally (a) that they are biostable within the given implantation site, (b) that they permit sufficient nutrient flow into the capsule as well as release of secreted molecules out of the capsule, and (c) that they do not induce a foreign body response and fibrotic encapsulation. Since encapsulation shields the therapeutic from any direct physical interaction with the immune system and other body tissues, this type of therapy is suited for enzyme or hormone replacement therapies, rather than regeneration of large amounts of tissues or organs.
One disease for which this strategy seems appropriate is T1D, where regulation of blood glucose by insulin production from pancreatic beta cells is impaired. Encapsulation of allogeneic insulin-producing pancreatic beta cells within semipermeable membranes would allow for the diffusion of insulin from the cell therapeutic into the blood system, while also allowing for the diffusion of oxygen and glucose into the capsule to support the viability of the cells within.
In this area, CIRM has supported the development of ViaCyte's Encaptra macroencapsulation device for the treatment of T1D with hESC-derived pancreatic precursor cells (PEC-01 cells). 31  insulin between the encapsulated cells and the blood system, the iBAP is implanted directly in-line with a blood vessel to provide enhanced kinetics of glucose sensing and insulin secretion while still isolating the allogeneic human stem cell derived pancreatic beta cells from the immune system using a silicon nanopore membrane. 33

| CONCLUSION
The ability to use allogeneic cells and tissues for regenerative medicine will be critical to enable large-scale, reproducible manufacturing that will both increase availability of the treatment and manage the cost of the products. Thus, as more regenerative cell therapies enter the clinic and begin to show efficacy in patients, it is critical, in parallel, to develop safe and easily implemented methods to overcome immunological barriers in order to facilitate durable engraftment of transplanted allogeneic tissues. Herein, we have described some of the approaches being taken by CIRM grantees to enable successful engraftment of allogeneic tissues, which are a small subset of the many efforts being made in this field. For example, other methods being used to induce immune tolerance include adoptive transfer of cell types such as Tregs, tolerogenic dendritic cells, and mesenchymal stromal cells. [34][35][36] In addition, there are many groups working on different ways to encapsulate or otherwise shield grafted cells from the immune response, 29 and to create immune evasive cells. 37 Although many of these approaches are still relatively new and have not yet been implemented clinically, there is great promise that improved methods for enabling engraftment of allogeneic cells, organs, or tissues will enable much broader use of regenerative therapies as they are developed.

ACKNOWLEDGMENT
Many thanks to Dr Lila Collins and Dr Kelly Shepard for helpful editorial suggestions.

CONFLICT OF INTEREST
The authors declared no potential conflicts of interest.