Next-Generation Conditioning for Bone Marrow Transplantation: Paving the Way for CAR-T Cell-Based Conditioning
Authors: Kevin G. Haworth, Hans-Peter Kiem
Journal: Molecular Therapy (2018)

Conditioning regimens that can more specifically target bone marrow-repopulating cells while reducing or eliminating off-target effects in other tissues and organs would significantly advance the field of bone marrow transplantation (BMT) and hematopoietic stem cell gene therapy. These challenges have prompted researchers to investigate alternative, nonmyeloablative, and nongenotoxic methods of marrow conditioning to facilitate engraftment of blood or marrow stem cells without major off-target side effects. In this issue of Molecular Therapy, Arai et al.1Arai Y. Choi U. Corsino C.I. Koontz S.M. Tajima M. Sweeney C.L. Black M.A. Feldman S.A. Dinauer M.C. Malech H.L. Myeloid conditioning with c-kit-targeted CAR-T cells enables donor stem cell engraftment.Mol. Ther. 2018; 26 (this issue): 1181-1197Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar document the convergence of two types of emerging immunotherapy treatments to begin addressing these challenges. By combining chimeric antigen receptor (CAR)-modified T cells with antibody-mediated nongenotoxic conditioning against the hematopoietic stem cell marker CD117 (c-kit), they demonstrate successful depletion of recipient bone marrow prior to transplantation. To increase the efficiency of this cell-based conditioning, they included two additional factors to facilitate both the engraftment and trafficking of infused CAR-modified cells. By preconditioning recipient mice with low-dose cyclophosphamide (Cy), they achieved both persistence and expansion of infused CAR-modified cells and could specifically target these cells for migration to the bone marrow by co-expressing the CXCR4 receptor along with the CAR molecule. Since the development of BMT in the middle of the 20th century, over one million individuals have undergone this procedure to cure a range of disease from cancer to inherited genetic conditions. A common component of essentially all transplants is the use of a conditioning regimen designed to facilitate the engraftment of the donor cells and, in case of malignant diseases, reduce or eliminate any remaining cancer cells. Typically, conditioning regimens for myeloabaltive or reduced-intensity transplants involve either irradiation or chemotherapy with alkylating reagents or both. While effective, both approaches can be genotoxic and cause damage to residual hematopoiesis and other actively dividing cells throughout the body. Thus, while acceptable for malignant diseases, for nonmalignant diseases, nongenotoxic conditioning regimens would be preferable. Diseases such as Fanconi anemia and other DNA repair disorders are particularly susceptible to alkylating chemotherapy and, therefore, alternative strategies would be extremely beneficial in these settings. The first component for nongenotoxic conditioning presented by Arai et al.1Arai Y. Choi U. Corsino C.I. Koontz S.M. Tajima M. Sweeney C.L. Black M.A. Feldman S.A. Dinauer M.C. Malech H.L. Myeloid conditioning with c-kit-targeted CAR-T cells enables donor stem cell engraftment.Mol. Ther. 2018; 26 (this issue): 1181-1197Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar focuses on the stem cell marker CD117. This receptor is expressed on primitive hematopoietic stem cells and functions through binding of the cytokine stem cell factor (SCF), which is an essential component for maintaining hematopoietic potential. Early studies using antibodies to target and deplete this population through antibody-dependent cell-mediated cytotoxicity (ADCC) demonstrated the ability for donor stem cells to engraft and contribute to multi-lineage hematopoiesis.2Czechowicz A. Kraft D. Weissman I.L. Bhattacharya D. Efficient transplantation via antibody-based clearance of hematopoietic stem cell niches.Science. 2007; 318: 1296-1299Crossref PubMed Scopus (312) Google Scholar, 3Xue X. Pech N.K. Shelley W.C. Srour E.F. Yoder M.C. Dinauer M.C. Antibody targeting KIT as pretransplantation conditioning in immunocompetent mice.Blood. 2010; 116: 5419-5422Crossref PubMed Scopus (60) Google Scholar Recently, immunotoxin molecules such as saporin have been conjugated to antibodies to utilize non-ADCC pathways and directly eliminate target cells.4Palchaudhuri R. Saez B. Hoggatt J. Schajnovitz A. Sykes D.B. Tate T.A. Czechowicz A. Kfoury Y. Ruchika F. Rossi D.J. et al.Non-genotoxic conditioning for hematopoietic stem cell transplantation using a hematopoietic-cell-specific internalizing immunotoxin.Nat. Biotechnol. 2016; 34: 738-745Crossref PubMed Scopus (137) Google Scholar These methods have demonstrated robust donor engraftment in immunocompetent mouse models and, after identification of antibodies that specifically recognize epitopes of human CD117, these methods are currently under investigation for clinical translation. The second component utilizes advancements in the application of CAR-modified T cells. CAR molecules were first described in the 1990s and, over the past decade, have seen a substantial increase in both their functionality and clinical application.5Srivastava S. Riddell S.R. Chimeric antigen receptor T cell therapy: Challenges to bench-to-bedside Efficacy.J. Immunol. 2018; 200: 459-468Crossref PubMed Scopus (121) Google Scholar These artificially engineered cellular receptors are created by combining all necessary recognition and signaling domains normally present on several proteins in multiple complexes into one transmembrane protein. The resulting CAR molecule is independently capable of identifying specific antigen expression on target cell populations and eliciting cytotoxic immune activation. The antigen recognition domain of CARs is often comprised of the variable fragment from an antibody, meaning that any epitope that is specifically bound by an antibody can be targeted by CAR-modified cells. This circumvents the requirement for T cell receptor (TCR)-mediated antigen recognition and enables a method for directing the immune system to target specific cell populations, including self-antigens. While similar methods have been utilized before with limited success, Arai et al.1Arai Y. Choi U. Corsino C.I. Koontz S.M. Tajima M. Sweeney C.L. Black M.A. Feldman S.A. Dinauer M.C. Malech H.L. Myeloid conditioning with c-kit-targeted CAR-T cells enables donor stem cell engraftment.Mol. Ther. 2018; 26 (this issue): 1181-1197Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar combined this approach with another component to increase CAR-modified T cells' ability to deplete the bone marrow compartment. By including a secondary cell surface receptor, CXCR4, they increased the frequency of CAR cells in the bone marrow over 10-fold, which enhanced depletion of resident CD117-expressing cells and facilitated multi-lineage donor stem cell engraftment. The CXCR4 receptor is involved in homing and retention of hematopoietic cells to the marrow niche through interactions with its ligand, stromal cell-derived factor-1 (SDF-1). High expression of SDF-1 in bone marrow endothelial cells and osteoblasts directs CXCR4-expressing hematopoietic cells to this compartment and increases their retention.6Brenner S. Whiting-Theobald N. Kawai T. Linton G.F. Rudikoff A.G. Choi U. Ryser M.F. Murphy P.M. Sechler J.M. Malech H.L. CXCR4-transgene expression significantly improves marrow engraftment of cultured hematopoietic stem cells.Stem Cells. 2004; 22: 1128-1133Crossref PubMed Scopus (103) Google Scholar Such a method is not only relevant for bone marrow conditioning, but could also be utilized in other CAR-mediated therapies where directing migration of effector cells to specific tissues through receptor-ligand interactions could increase efficacy. By combining these factors for nongenotoxic immunotherapy conditioning, the authors demonstrate successful engraftment of donor bone marrow in a mouse model of chronic granulomatous disease. These studies provide the proof-of-concept that CAR-mediated bone marrow conditioning can be used in a therapeutic setting. However, a major limitation of the method will be the complete elimination of all CAR-modified infused T cells prior to donor cell infusion. If even small numbers of residual CAR-T cells are present in either peripheral circulation or in tissues, these could elicit killing of cells before they have had time to migrate to the marrow compartment or prevent efficient engraftment and hematopoietic development. Arai et al.1Arai Y. Choi U. Corsino C.I. Koontz S.M. Tajima M. Sweeney C.L. Black M.A. Feldman S.A. Dinauer M.C. Malech H.L. Myeloid conditioning with c-kit-targeted CAR-T cells enables donor stem cell engraftment.Mol. Ther. 2018; 26 (this issue): 1181-1197Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar utilize differential expression of mouse Thy1.1 and Thy1.2 for clearance of residual CAR-modified cells after marrow depletion but prior to donor infusion through a simple anti-Thy1.1 antibody injection. While effective in the mouse model, such a method will not be possible in patients, and alternative methods will be needed. This could be accomplished with the inclusion of a suicide gene alongside CAR expression7Jones B.S. Lamb L.S. Goldman F. Di Stasi A. Improving the safety of cell therapy products by suicide gene transfer.Front. Pharmacol. 2014; 5: 254Crossref PubMed Google Scholar or perhaps the development of alternative methods for the clearance of CAR-expressing cells, such as antibodies targeted against the CAR molecule itself. Aside from the benefit of reduced or eliminated systemic cytotoxicity by using cellular mediated conditioning, the patient's own T cells are engineered for this function, which will eliminate any potential rejection of modified cells or graft-versus-host disease, and thus increase the safety of this approach. By adding this type of conditioning regimen to the clinical toolbox, it allows for the selection of whichever nongenotoxic conditioning approach makes the most sense for each individual patient on a case-by-case basis. These studies are paving the way for the next generation of BMT by eliminating the requirement for genotoxic systemic conditioning regimens, which should increase both the safety and efficacy of this life-saving treatment, especially for patients with nonmalignant diseases. Myeloid Conditioning with c-kit-Targeted CAR-T Cells Enables Donor Stem Cell EngraftmentArai et al.Molecular TherapyMarch 9, 2018In BriefArai and colleagues demonstrate in a congenic murine transplant model that c-kit-targeted CAR-T cells forced to overexpress CXCR4 showed enhanced trafficking to bone marrow to achieve substantial depletion of endogenous hematopoietic stem cells (HSCs), allowing significant multilineage engraftment of donor HSCs and cure of a chronic granulomatous disease mouse model. Full-Text PDF Open Archive