Non-genotoxic conditioning regimens for stem cell transplantation

Abstract

To provide a composition for use in a clinically applicable method of stem cell transplantation that facilitates engraftment and reconstitutes immunocompetence of the recipient without requiring radiotherapy or chemotherapy and without development of GVHD or graft rejection.SOLUTION: A pharmaceutical composition is for use in a method of providing stem cell engraftment. The method has a conditioning regimen comprising: bringing targeted endogenous stem cells in a subject concomitantly into contact, at the ame time, with (i) an agent that specifically binds to endogenous stem cells in a targeted tissue and (ii) an agent that blocks interaction between CD47 and SIRPα; bringing the subject into contact with (iii) an agent that induces transient immunosuppression; and introducing a cellular composition comprising exogenous stem cells to the subject following a wash-out period of time sufficient to reduce the serum level of (i) and (ii) to non-toxic levels, the exogenous stem cells engrafting in the absence of myeloablative conditioning.SELECTED DRAWING: Figure 1-1

Description

[Background technology]

[0001] Stem cells, through their ability to self-renew and generate differentiated cells, provide an organism with a means of maintaining and repairing certain tissues. Clinically, bone marrow transplantation and hematopoietic stem cell transplantation are widely used as a means of providing blood cell-generating capacity to patients, typically those whose endogenous stem cells have been depleted by high-dose chemotherapy or radiation therapy.

[0002] Hematopoietic cell transplantation (HCT) generally involves the intravenous infusion of autologous or allogeneic hematopoietic cells, the active subset of which is hematopoietic stem cells (HSCs). These are harvested from bone marrow, peripheral blood, or umbilical cord blood and transplanted to reconstitute hematopoietic function in patients whose bone marrow or immune system is damaged or deficient. This procedure is often performed as part of treatment to eliminate bone marrow-infiltrating processes such as leukemia or to correct congenital immunodeficiencies.

[0003] Additionally, HCT allows cancer patients to receive higher doses of chemotherapy than their bone marrow can normally tolerate. Bone marrow function is then restored by replacing the marrow with previously harvested stem cells. It is also possible to transplant enriched or purified HSC populations that are free of other cells, many of which are harmful to the host.

[0004] Preparative or conditioning regimens are a critical component of hematopoietic cell transplantation (HCT). For successful transplantation, clearance of the bone marrow niche must be achieved for donor hematopoietic stem cells (HSCs) to engraft. Preparative regimens may also provide sufficient immunosuppression to prevent rejection of the transplanted graft and eradicate the disease for which the transplant is being performed. Current methods for clearing the niche space rely on radiation and / or chemotherapy, which can have toxic adverse effects that significantly limit the potential clinical utility of BMT. Traditionally, myeloablative conditioning regimens are used.

[0005] Myeloablative regimens can be classified as radiation-containing or radiation-free regimens, treatments developed by gradually increasing the dose of radiation or specific drugs to the maximum tolerated dose. Total body irradiation and cyclophosphamide or busulfan and cyclophosphamide are commonly used myeloablative therapies. These regimens are particularly used in advanced malignant tumors such as leukemia. However, such treatments have many disadvantages in terms of toxicity to patients.

[0006] Improved methods for the engraftment of stem cells, including hematopoietic stem cells, are of great clinical importance. The present invention addresses this need. Summary of the Invention

[0007] Methods and compositions are provided for long-term, multilineage engraftment of stem cells, including but not limited to hematopoietic stem cells, in a recipient by treating the recipient with a pre-transplant non-myeloablative, non-genotoxic conditioning regimen and administering an effective amount of a cell population containing exogenous stem cells. The conditioning regimen includes the administration of agents that act on endogenous cell populations for various purposes. The methods enable engraftment to treat hematologic disorders and can also be used to tolerize the recipient to the donor's HLA type for future organ transplants.

[0008] Endogenous stem cells are depleted by a pre-transplantation treatment regimen. Agents that deplete endogenous stem cells include, but are not limited to, antibodies specific to c-kit and agents that block CD47 activity. These agents can also deplete exogenous stem cells after administration, and therefore require a "washout" period between the time of administration of the agent and the time of administration of exogenous stem cells. The washout period is sufficient to reduce the serum level of the agent that depletes endogenous stem cells to a non-toxic level that does not cause stem cell depletion.

[0009] In some embodiments, the transplant conditioning regimen includes at least one agent that provides transient immunosuppression of cytotoxic lymphocytes. A variety of biologic and non-myeloablative medications are available for this purpose, including, but not limited to, agents that inhibit CD40 / CD40L activity, mycophenolic acid, cyclosporin A, rapamycin, FK506, corticosteroids, and the like. In some embodiments, the agent inhibits CD40L and is an antibody specific for CD40L. The transient immunosuppressant can be administered before or simultaneously with the exogenous stem cells, so long as the transient immunosuppressant is active at the time the exogenous stem cells are administered.

[0010] In some embodiments, the transplant conditioning regimen includes at least one agent that depletes one or both of T lymphocytes and natural killer (NK) cells. Agents that deplete T cells specifically include, but are not limited to, agents comprising antibodies specific for CD3, CD4, CD8, and the like. Agents that deplete T cells and NK cells specifically include, but are not limited to, agents comprising antibodies specific for CD2, CD52, CD45, antithymocyte globulin (ATG), and the like. Agents that deplete NK cells specifically include, but are not limited to, agents comprising antibodies specific for CD122, CD56, and the like. The depleting agent(s) can be administered prior to infusion of the exogenous stem cells, and optionally are active after infusion, so long as the target cells are depleted when the exogenous stem cells are administered.

[0011] In one embodiment, a method is provided for selecting and administering an appropriate set of agents for non-genotoxic conditioning prior to transplantation. It is shown herein that the requirements for a pre-transplant conditioning regimen for successful stem cell engraftment vary according to certain parameters, including the number of donor cells administered to the recipient, the purity of the donor cells, the degree of major histocompatibility incompatibility between the donor and recipient, and the immune status of the recipient. Selecting the appropriate set of agents for an individual and the timing of administration of the agents can optimize the therapeutic outcome of the transplant.

[0012] In some embodiments, the methods described herein involve HLA typing of donors and recipients to determine HLA-matched or HLA-mismatched pairs, CD34 + Obtain hematopoietic cells from a donor containing hematopoietic stem and progenitor cells, which may be referred to as HSPCs, optionally HSPCs of a desired phenotype, e.g., CD34 + The method may include isolating the cells and formulating an effective amount of HSPCs, selecting a set of agents for a non-genotoxic conditioning regimen for the recipient prior to infusion of the hematopoietic cells based on the number of donor cells to be administered to the recipient, the purity of the donor cells, the degree of major histocompatibility incompatibility between the donor and recipient, and the recipient's immune status, administering the set of agents for the non-genotoxic conditioning regimen, infusing the hematopoietic cells, and monitoring the recipient for hematopoietic stem cell engraftment. The methods described herein are applicable to both HLA-matched and HLA-mismatched transplant conditions, such as HLA-mismatched and haplo-mismatched transplants, haplo-identical transplants, etc.

[0013] In some embodiments, HSPCs are obtained from a donor hematopoietic cell sample. In some embodiments, the hematopoietic cell sample is bone marrow. In some embodiments, HSPCs are obtained from umbilical cord blood. In some embodiments, the hematopoietic cell sample is obtained by apheresis from donor mobilized peripheral blood. In some embodiments, HSPCs are generated in vitro. The HSPC donor can be allogeneic or autologous, for example, the HSPCs are genetically engineered by the introduction or deletion of genetic material, for example, during ex vivo culture, prior to reinfusion. The allogeneic donor can be MHC-matched to the recipient. The donor can be haploidentical or haplomismatched to the recipient. The donor can be mismatched at one or more MHC loci, for example, mismatched at 1, 2, 3, 4, 5, or 6 major loci for MHC matching.

[0014] HSPCs are optionally isolated from the hematopoietic cell sample for expression of CD34. Isolation may further include selection for expression of CD90. Purified HSPCs are characterized by the presence of CD34 in the population. + The effective amount of CD34 may be at least about 45% pure, at least about 50% pure, at least about 60% pure, at least about 70% pure, at least about 80% pure, or at least about 90% pure, as defined by the percentage of cells that are + The cells were administered at a dose of approximately 10 per kg of recipient body weight. 5 ~about 10 7 CD34 + cells per kg of recipient body weight, at least about 5 x 10 5 CD34 + cells, at least about 10 per kg of recipient body weight 6 CD34 + cells, at least about 3 x 10 per kg of recipient body weight 6 CD34 + cells, at least about 5 x 10 per kg of recipient body weight 6 CD34 + cells per kg of recipient body weight, 7 CD34+ CD34 in the infusion may be 100 or more cells. + The cell dose, cell purity, and total number of cells delivered, i.e., both CD34 + Cells and CD34 - The total cell dose is an important parameter for the selection of a non-genotoxic transplant conditioning agent.

[0015] CD3 delivered with HSPC compositions + The maximum number of cells is approximately 10 per kg of recipient body weight. 6 CD3 or less + cells, approximately 5 × 10 per kg of recipient body weight 5 CD3 or less + cells, approximately 3 x 10 per kg of recipient body weight 5 CD3 or less + cells, approximately 10 per kg of recipient body weight 4 CD3 or less + The infusion may be CD3 + The number of cells can be a parameter for the selection of agents that inhibit cytotoxic lymphocytes, where CD3 + Increasing the number of cells may require administration immediately prior to or during the infusion of one or more agents that destroy T cells, including but not limited to antibodies specific for CD3, CD4, CD8, etc.

[0016] In some embodiments, the transplant is performed in the absence of myeloablative conditioning. In some embodiments, the recipient is immunocompetent. Administration of the pre-transplant conditioning regimen is repeated as necessary to achieve the desired level of destruction.

[0017] In some embodiments, CD47 blockade is achieved by administering a soluble SIRPα polypeptide, which may be a high-affinity SIRPα variant polypeptide, hi other embodiments, antibodies specific for one or both of SIRPα and CD47 are administered.

[0018] After transplantation with donor stem cells, the recipient may be chimeric or mixed chimeric with respect to the donor cells. The method of the present invention allows for effective stem cell engraftment in the absence of non-selective destructive methods, such as radiation or chemotherapy, which have the undesirable effect of destroying differentiated cells involved in the function of the target tissue, as well as undesirable side effects on other tissues (e.g., cells of the gastrointestinal system, hair growth), and increase the risk of secondary malignancies.

[0019] In one embodiment of the present invention, the stem cells are one or more of autologous hematopoietic stem cells, genetically modified hematopoietic stem cells, and allogeneic, typically allogeneic, hematopoietic stem cells, which are used in the treatment of various blood disorders, such as genetic disorders such as aplastic anemia, sickle cell disease, thalassemia, severe immunodeficiency, bone marrow failure states, immunodeficiencies, hemoglobinopathies, leukemia, lymphoma, immune tolerance induction, genetic disorders treatable by bone marrow transplantation, and other blood disorders.

[0020] The methods of the present invention are also useful for inducing tolerance in patients, such as tolerance to donor tissue in organ transplants, or tolerance to self-antigens in the treatment of autoimmune diseases. One embodiment of the present invention provides a method for inducing tolerance in a patient, comprising administering to the patient an agent that targets stem cells, including but not limited to, an antibody specific for c-kit and an agent that blocks CD47 activity, in combination with an effective amount of one or a set of agents that reduce the number or activity of cytotoxic lymphocytes, which may include, but are not limited to, T cells and natural killer (NK) cells. In some embodiments, at least one agent that provides transient immunosuppression of cytotoxic lymphocytes is included, including, but not limited to, an agent that inhibits CD40 / CD40L activity. In some embodiments, the agent is an antibody specific for CD40L. In some embodiments, the method is performed in the absence of genotoxic conditioning treatment. After the transplant conditioning regimen, the recipient is infused with an effective amount of hematopoietic stem and progenitor cells, thereby providing immune tolerance to the donor cells for future organ transplantation.

[0021] The invention will best be understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to scale. Conversely, the dimensions of the various features have been arbitrarily increased or reduced for the sake of clarity. The drawings include the following figures: [Brief explanation of the drawings]

[0022] [Figure 1-1]ACK2, Clone 3, MR-1, and CD122 demonstrate the ability of immunocompetent animals to effectively engraft haploidentical whole bone marrow. In Figure 1A, 30e6 AKR x Hz F1 whole bone marrow was harvested and transplanted retro-orbitally into individual Balb / c x C57BL / 6 recipients. Chimerism was determined by CD45 allelic differentiation. In Figure 1B, each antibody was administered on the designated days for conditioning. On day -8, 100 μg of Clone 3 was administered, followed by 500 μg of Clone 3 on all subsequent days. On day -6, 500 μg of ACK2 was administered. On day -1, 250 μg of Tm-b1 was administered. On day 0, 500 μg of MR1 was administered. [Figure 1-2] We demonstrate that ACK2, Clone 3, MR-1, and CD122 enable effective engraftment of haploidentical whole bone marrow into immunocompetent animals. Mice were conditioned with different combinations of each antibody. Total donor chimerism was measured over 13 weeks, in addition to T cell (Figure 1C), B cell (Figure 1D), granulocyte (Figure 1E), and granulocyte (Figure 1F) chimerism. Unchimeric mice in the ACK2+Clone 3+MR1 cohort were discontinued. [Figure 2] Figure 16 shows donor chimerism in antibody-transplant-conditioned mice at 16 weeks, showing the percentage of mice that are chimeric per cohort, as well as the mean levels of total donor, T cell, B cell, and granulocyte chimerism. Anchimeric mice in the ACK2+ clone 3+ MR1 cohort were censored. [Figure 3-1] This demonstrates that NK cell depletion is necessary for engraftment of low-cell dose bone marrow. In Figure 3A, various amounts of AKRxHz F1 whole bone marrow were harvested and transplanted retro-orbitally into individual Balb / cxC57BL / 6 recipients. Chimerism was determined by CD45 allelic differentiation. In Figure 3B, each antibody was given on the days marked for transplant conditioning. On day -8, 100 μg of clone 3 was given, followed by 500 μg of clone 3 on all subsequent days. On day -6, 500 μg of ACK2 was given. On day -2, 250 μg of Tm-b1 was given. On day 0, 500 μg of MR1 was given. [Figure 3-2]NK cell depletion is necessary for engraftment of low-cell-dose bone marrow. Each group was minimally conditioned with clone 3, ACK2, and MR1. CD122 was additionally added to two noted cohorts, thus receiving all four antibodies. Conditioned mice received either 30 x 10, 10, 3 x 10, or 10 whole bone marrow. Total donor chimerism was measured at week 3 in addition to T cell chimerism in Figure 3C, B cell chimerism in Figure 3D, granulocyte chimerism in Figure 3E, and chimerism in Figure 3F. [Figure 4-1] These results demonstrate that monoclonal antibody cocktails can induce long-term multilineage hematopoietic reconstitution. Figure 4A shows a haploidentical transplantation scheme using an AKRB6F1 donor and a CB6F1 recipient. Figure 4B shows flow cytometry analysis of MHC class I expression in donor and recipient strains. Figure 4C shows the dosing schedule for the transplant conditioning regimen. Figure 4D shows donor chimerism in the long-term HSC compartment (Lin-c-KIT+Sca1+CD150+Flk2-CD34-) after antibody transplant conditioning. [Figure 4-2] Monoclonal antibody cocktails can induce long-term multilineage hematopoietic reconstitution. Figures 4E-G show that antibody conditioning enables long-term multilineage chimerism after WBM transplantation. [Figure 4-3] These results demonstrate that monoclonal antibody cocktails can induce long-term multilineage hematopoietic reconstitution. Figure 4H shows CBCs after WBM antibody conditioning on day 0. Figure 4I shows the percentage of animals that are chimeric at various WBM doses with or without NK cell depletion. [Figure 5-1] We demonstrate that monoclonal antibody cocktails can induce long-term multilineage hematopoietic reconstitution of low-dose purified HSCs. Figure 5A shows the sorting scheme used to calculate and isolate LSK and c-KIT+ cells for transplantation. Figure 5B shows granulocyte chimerism after various hematopoietic cell transplants. Figure 5C shows the dosing regimen for LSK antibody conditioning treatment. [Figure 5-2]We demonstrate that monoclonal antibody cocktails can induce long-term multilineage hematopoietic reconstitution of low-dose purified HSCs. Figure 5D shows the abundance of mature immune cell populations in the bone marrow after LSK antibody transplant conditioning. Figure 5E shows total peripheral blood donor chimerism after LSK transplantation. Figure 5F shows the percentage of animals that are chimeric after excluding individual components of the LSK antibody cocktail. [Figure 6-1] Low-dose LSK transplantation via a non-genotoxic conditioning regimen demonstrates tolerance to donor tissue. Figures 6A and 6B show the abundance of donor-reactive host T cells in peripheral blood after WBM (A) and LSK (B) transplantation. Figure 6C shows a schematic diagram of ear-heart transplantation. Figure 6D shows donor heart survival. [Figure 6-2] Low-dose LSK transplantation via a non-genotoxic conditioning regimen enables tolerance to the donor tissue. Figure 6E shows gross examination, H&E, and IF of a representative ear-heart graft 34 days after tissue transplantation. [Figure 7-1] These results demonstrate that hematopoietic stem cells can engraft even in the presence of complete MHC mismatches. Figure 7A shows a transplantation schematic with DBA1 / J as the donor and CB6F1 as the host. Figure 7B shows the percentage of donor engraftment after WBM and LSK transplantation 8 weeks later. Figure 7C shows the overall survival of transplanted animals. [Figure 7-2] Hematopoietic stem cells can engraft even in the presence of complete MHC mismatches. Figure 7D outlines a pan-antibody conditioning regimen that can deplete endogenous HSCs and provide transient immunosuppression by targeting host T and NK cells. [Figure 8-1] FIG. 8A shows a classification scheme for determining peripheral blood chimerism by CD45 interallelic differences between host and donor. [Figure 8-2] Figures 8B-D show multilineage peripheral blood chimerism 16 weeks after WBM transplantation for T cells (B), B cells (C), and granulocytes (D). [Figure 9] 1 shows peripheral blood donor chimerism after monotherapy conditioning with a monoclonal antibody. [Figure 10]Figures 10A-C show complete blood counts (A), peripheral blood subpopulations (B), and spleen subpopulations (C) from animals 1 day after completion of conditioning without transplantation. [Figure 11] Shown are 16-weekend peripheral blood chimerism after various LSK antibody conditioning regimens. DETAILED DESCRIPTION OF THE INVENTION

[0023] A method is provided for stem cell engraftment in a subject by treatment with a non-genotoxic, non-myeloablative conditioning regimen prior to infusion of a cell composition comprising stem and progenitor cells.

[0024] The present invention aims to provide a clinically applicable method of stem cell transplantation that facilitates engraftment and reconstitutes the recipient's immune competence without the need for radiation therapy or chemotherapy, or without the development of GVHD or graft rejection. Guidelines are also provided for selecting an appropriate pre-transplantation regimen based on the characteristics and dose of the donor stem cell population and the HLA compatibility between the donor and the recipient.

[0025] Aspects of the present invention are based on the discovery that the use of agents that target endogenous stem cells, e.g., anti-c-kit antibodies, combined with agents that enhance killing of endogenous stem cells by blocking the interaction of CD47 and SIRPα, optionally in combination with transient immunosuppression, achieves depletion of the endogenous stem cell niche that facilitates effective engraftment of hematopoietic stem cells (HSCs), optionally in combination with agents that deplete T and / or NK cells, allowing safe engraftment of donor cells. Specifically, the present invention combines this improved selective destruction of endogenous stem cells in conjunction with the administration of exogenous stem cells to the recipient to result in effective, long-term engraftment and tolerance.

[0026] It is to be understood that the invention is not limited to the particular methodology, products, devices, and agents described, as methods, devices, and formulations may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention, which may be limited only by the appended claims.

[0027] It should be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a drug candidate" refers to one or a mixture of such candidates, reference to "the method" includes reference to equivalent steps and methods known to those skilled in the art, and so forth.

[0028] All technical and scientific terms used herein, unless clearly defined otherwise, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing the devices, formulations, and methods described in the publications and that can be used in connection with the presently described invention.

[0029] Where a range of values ​​is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of that stated range, and any other stated or intervening value, is encompassed within the invention, unless the context clearly dictates otherwise. Independently, the upper and lower limits of these smaller ranges that may be included in smaller ranges are encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of both limits, ranges excluding either or both of those inclusive limits are also included in the invention.

[0030] In order to ensure a thorough understanding of the present invention, the following description sets forth numerous specific details. However, it will be apparent to one skilled in the art that the present invention may be practiced without one or more of these specific details. Additionally, well-known features and procedures known to those skilled in the art have not been described in order to avoid obscuring the present invention.

[0031] Generally, conventional methods of protein synthesis, recombinant cell culture, and protein isolation, as well as recombinant DNA technology, within the skill of the art, are used in the present invention. Such techniques are fully explained in the literature, see, for example, Maniatis, Fritsch & Sambrook, Molecular Cloning: A Laboratory Manual (1982); Sambrook, Russell and Sambrook, Molecular Cloning: A Laboratory Manual (2001); Harlow, Lane and Harlow, Using Antibodies: A Laboratory Manual: Portable Protocol No. 1, Cold Spring Harbor Laboratory (1998); and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory; (1988).

[0032] Conditioning regimens. Patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) are prepared using so-called conditioning regimens that can suppress the recipient's immune system and deplete endogenous stem cells to allow engraftment of donor stem cells.

[0033] The intensity of conventional transplant conditioning regimens can vary significantly.The description of regimen can refer to genotoxic or non-genotoxic regimen, and can overlap with myeloablative or non-myeloablative regimen.For example, see Bacigalupo et al.(2009)Biol Blood Marrow Transplant.15(12):1628-1633, which is specifically incorporated herein by reference.

[0034] Genotoxic regimens involve, at least in part, the administration of agents that have direct or indirect effects on DNA, inducing mutations, activating events at the wrong time, and directly damaging DNA, resulting in mutations. Examples of genotoxic agents include radiation and certain chemotherapeutic agents, such as alkylating agents, intercalating agents, and inhibitors of enzymes involved in DNA replication. The methods of the present invention are non-genotoxic and therefore exclude the use of such agents.

[0035] Myeloablative conditioning regimens are combinations of agents expected to produce severe hypopancytosis and bone marrow destruction within 1 to 3 weeks of administration. The hypopancytosis is prolonged, usually irreversible, and in most cases fatal unless hematopoiesis is restored by hematopoietic stem cell infusion. Examples include total body irradiation and / or administration of alkylating agents, fludarabine, dimethylbusulfan, and etoposide (VP16). There is significant overlap between genotoxic and myeloablative agents.

[0036] Nonmyeloablative conditioning regimens typically cause minimal cytopenias and little initial toxicity, but are immunosuppressive to the extent that they result in engraftment of donor lymphohematopoietic stem cells when effective doses of HSPCs are subsequently administered.

[0037] The transplant conditioning regimens provided herein are non-genotoxic and non-myeloablative, and primarily utilize agents that target endogenous stem cell depletion to prevent engraftment without causing log-lasting pancytopenia. The methods do not utilize genotoxic chemotherapeutic agents or radiation, although in some cases, non-genotoxic, targeted immunosuppressants, such as cyclosporine A, corticosteroids, etc., can be used for transient immunosuppression.

[0038] "Concomitant administration" of an active agent in the methods of the invention refers to administration with an agent at a time when the agent has a therapeutic effect at the same time. Such concomitant administration may necessarily involve simultaneous (concurrent) (i.e., at the same time), prior, or subsequent administration of the agents. Those of skill in the art will have no difficulty determining the appropriate timing, sequence, and dosages of administration for particular drugs and compositions of the invention.

[0039] Stem cell markers. Exemplary markers for antibody-mediated destruction of human hematopoietic stem cells include CD34, CD90 (thy-1), CD59, CD110 (c-mpl), c-kit (CD-117), and the like. Markers useful for the destruction of mesodermal stem cells include FcγRII, FcγRIII, Thy-1, CD44, VLA-4α, LFA-1β, HSA, ICAM-1, CD45, Aa4.1, Sca-1, and the like. Neural crest stem cells can be positively selected using antibodies specific for the low-affinity nerve growth factor receptor (LNGFR). Neural stem / progenitor cells have been described in the art, and their use in various therapeutic protocols has been widely discussed. For example, see Uchida et al. (2000) Proc Natl Acad Sci US A. 97(26):14720-5, among others. U.S. Patent No. 6,638,501, Bjornson et al.; U.S. Patent No. 6,541,255, Snyder et al.; U.S. Patent No. 6,498,018, Carpenter; U.S. Patent Application No. 2002 / 0012903, Goldman et al.; Palmer et al. (2001) Nature 411(6833):42-3; Palmer et al. (1997) Mol Cell Neurosci. 8(6):389-404; Svendsen et al. (1997) Exp. Neurol. 148(1):135-46 and Shihabuddin (1999) Mol Med Today. 5(11):474-80, each of which is specifically incorporated herein by reference. Human mesenchymal stem cells can be disrupted using markers such as SH2 (CD105), SH3 and SH4, and Stro-1.

[0040] In one embodiment of the present invention, the marker for depletion is c-kit (CD117). CD117 is a type III receptor tyrosine kinase that binds to stem cell factor (a substance that induces the proliferation of certain types of cells), also known as "steal factor" or "c-kit ligand." When this receptor binds to stem cell factor (SCF), it forms a dimer that activates its intrinsic tyrosine kinase activity, which in turn phosphorylates and activates signaling molecules that propagate signals within the cell. See, for example, human refseq entries Genbank NM_000222 and NP_000213. CD117 is an important cell surface marker used to identify certain types of hematopoietic (blood) progenitor cells within the bone marrow. Hematopoietic stem cells (HSCs), multipotent progenitor cells (MPPs), and common myeloid progenitor cells (CMPs) express high levels of CD117. Many antibodies that specifically bind to human CD117 are known in the art and commercially available, including, but not limited to, SR1, 2B8, ACK2, YB5-B8, 57A5, 104D2, etc. Of interest is the humanized form of SR1, AMG191, which is described in U.S. Patent Nos. 8,436,150 and 7,915,391, and is an aglycosylated IgG1 humanized antibody.

[0041] An effective amount of anti-CD117 antibody may be administered in one or more doses, including a single dose, which may be administered at least about one week, at least about five days, or at least about three days prior to transplantation. The period between administration and transplantation is sufficient to substantially eliminate the anti-CD117 antibody from the recipient's circulation. For example, the reduction in peak serum levels after administration is typically sufficient time for the levels to decrease by at least about 10-fold, typically at least about 100-fold, 1000-fold, 10,000-fold, or less from the peak level. After the washout period, donor stem cells are preferably introduced within the empty niche "window," typically within about 3 days, about 2 days, about 1 day, or upon clearance.

[0042] In some embodiments, an effective amount of an anti-CD117 antibody is up to about 10 mg / kg, up to about 5 mg / kg, up to about 1 mg / kg, up to about 0.5 mg / kg, up to about 0.1 mg / kg, or up to about 0.05 mg / kg, and dosages may vary depending on the particular antibody and recipient.

[0043] Anti-CD47 Agents. As used herein, the terms "anti-CD47 agent" or "agent that provides CD47 blockade" refer to any agent that reduces binding of CD47 (e.g., on target cells) to SIRPα (e.g., on phagocytes). Non-limiting examples of suitable anti-CD47 reagents include high-affinity SIRPα polypeptides, SIRPα reagents including, but not limited to, anti-SIRPα antibodies, soluble CD47 polypeptides, and anti-CD47 antibodies or antibody fragments. In some embodiments, a suitable anti-CD47 agent (e.g., an anti-CD47 antibody, SIRPα reagent, etc.) specifically binds to CD47 and reduces binding of CD47 to SIRPα.

[0044] An effective amount of an anti-CD47 agent can vary depending on the agent, but generally can be in the range of up to about 50 mg / kg, up to about 40 mg / kg, up to about 30 mg / kg, up to about 20 mg / kg, up to about 10 mg / kg, up to about 5 mg / kg, up to about 1 mg / kg, up to about 0.5 mg / kg, up to about 0.1 mg / kg, or up to about 0.05 mg / kg, and the dosage can vary depending on the particular antibody and the recipient. Agents that bind to CD47, such as soluble SIRPα polypeptides and anti-CD47 antibodies, can be administered in higher doses where more cells express CD47 in the body.

[0045] The anti-CD47 agent may be administered one or more days prior to transplantation, and in some embodiments, is administered daily for about 1, about 2, about 3, about 4, about 5, about 6, about 7, or more days, i.e., about 1 to 7 days, about 1 to 5 days, about 1 to 3 days, etc. As with anti-c-kit agents, targeting CD47 may affect donor stem cells after infusion, so a washout period is required before infusion of hematopoietic cells. The washout period may be shorter than with c-kit antibodies, but is typically at least about 24 hours, at least 36 hours, or at least 48 hours, and may be up to about 1 week, up to about 5 days, up to about 3 days, etc.

[0046] In some embodiments, a suitable anti-CD47 agent (e.g., an anti-SIRPα antibody, a soluble CD47 polypeptide, etc.) specifically binds to SIRPα and reduces binding of CD47 to SIRPα. Suitable anti-CD47 agents that bind to SIRPα do not activate SIRPα (e.g., in phagocytes that express SIRPα). The effectiveness of a suitable anti-CD47 agent can be evaluated by assaying the agent. In an exemplary assay, target cells are incubated in the presence or absence of a candidate agent. Agents used in the methods of the invention upregulate phagocytosis by at least 5% (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 120%, at least 140%, at least 160%, at least 180%, at least 200%, at least 500%, at least 1000%) compared to phagocytosis in the absence of the agent. Similarly, in vitro assays for the level of tyrosine phosphorylation of SIRPα show a decrease in phosphorylation by at least 5% (e.g., at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%) compared to phosphorylation observed in the absence of the candidate agent.

[0047] In some embodiments, the anti-CD47 agent does not activate CD47 upon binding. Activation of CD47 can result in a process similar to apoptosis (i.e., programmed cell death) (Manna and Frazier, Cancer Research, 64, 1026-1036, February 1, 2004). Thus, in some embodiments, the anti-CD47 agent does not directly induce cell death of CD47-expressing cells.

[0048] SIRPα Reagents. SIRPα reagents comprise a portion of SIRPα typically located between the signal sequence and the transmembrane domain sufficient to bind CD47 with appreciable affinity, or a fragment thereof that retains binding activity. Suitable SIRPα reagents reduce (e.g., block, prevent, etc.) the interaction between the native protein SIRPα and CD47. SIRPα reagents typically comprise at least the d1 domain of SIRPα.

[0049] In some embodiments, the subject anti-CD47 agents are "high-affinity SIRPα reagents" that include SIRPα-derived polypeptides and analogs thereof (e.g., CV1-hlgG4 and CV1 monomers). High-affinity SIRPα reagents are described in International Application PCT / US13 / 21937, specifically incorporated herein by reference. High-affinity SIRPα reagents are variants of native SIRPα proteins. The amino acid changes that result in increased affinity are localized in the d1 domain; thus, high-affinity SIRPα reagents include the d1 domain of human SIRPα with at least one amino acid change relative to the wild-type sequence within the d1 domain. Such high-affinity SIRPα reagents optionally include additional amino acid sequences, such as antibody Fc sequences, native proteins or fragments thereof, portions of the wild-type human SIRPα protein other than the d1 domain, including, but not limited to, residues 150-374, typically contiguous with the d1 domain. High affinity SIRPα reagents can be monomeric or multimeric, i.e., dimeric, trimeric, tetrameric, etc. In some embodiments, the high affinity SIRPα reagents are soluble, wherein the polypeptide lacks the SIRPα transmembrane domain and contains at least one amino acid change relative to the wild-type SIRPα sequence, which amino acid change enhances the affinity of the SIRPα polypeptide binding to CD47, e.g., by reducing the off-rate by at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 500-fold, or more.

[0050] Optionally, the SIRPα reagent is a fusion protein, e.g., fused in frame with a second polypeptide. In some embodiments, the second polypeptide can increase the size of the fusion protein, e.g., so that the fusion protein is not rapidly cleared from the circulation. In some embodiments, the second polypeptide is part or all of an immunoglobulin Fc region. The Fc region assists phagocytosis by providing an "eat me" signal, which enhances the blocking of the "don't eat me" signal provided by the high-affinity SIRPα reagent. In other embodiments, the second polypeptide is any suitable polypeptide substantially similar to an Fc, providing increased size, a multimerization domain, and / or additional binding or interaction with Ig molecules.

[0051] Anti-CD47 Antibodies. In some embodiments, a subject anti-CD47 agent is an antibody that specifically binds to CD47 (i.e., an anti-CD47 antibody) and reduces the interaction between CD47 on one cell (e.g., an infected cell) and SIRPα on another cell (e.g., a phagocyte). In some embodiments, a suitable anti-CD47 antibody does not activate CD47 upon binding. Some anti-CD47 antibodies do not reduce the binding of CD47 to SIRPα (and therefore are not considered "anti-CD47 agents" herein); such antibodies can be referred to as "non-blocking anti-CD47 antibodies." Suitable anti-CD47 antibodies that are "anti-CD47 agents" can be referred to as "CD47-blocking antibodies." Non-limiting examples of suitable antibodies include clones B6H12, 5F9, 8B6, and C3 (e.g., as described in International Patent Publication WO 2011 / 143624, specifically incorporated herein by reference). Suitable anti-CD47 antibodies include fully human, humanized, or chimeric versions of such antibodies. Humanized antibodies (e.g., hu5F9-G4) are particularly useful for in vivo applications in humans due to their low antigenicity. Similarly, caninized, feline, and other antibodies are particularly useful for applications in dogs, cats, and other species, respectively. Antibodies of interest include humanized antibodies, or caninized, feline, equine, bovine, porcine, and other antibodies, and variants thereof.

[0052] Anti-SIRPα antibodies. In some embodiments, a subject anti-CD47 agent is an antibody that specifically binds to SIRPα (i.e., an anti-SIRPα antibody) and reduces the interaction between CD47 on one cell (e.g., an infected cell) and SIRPα on another cell (e.g., a phagocyte). Suitable anti-SIRPα antibodies can bind to SIRPα without activating or stimulating signaling through SIRPα, since activation of SIRPα inhibits phagocytosis. Instead, suitable anti-SIRPα antibodies promote preferential phagocytosis of infected cells over normal cells. Cells (e.g., infected cells) that express high levels of CD47 relative to other cells (non-infected cells) are preferentially phagocytosed. Thus, suitable anti-SIRPα antibodies specifically bind to SIRPα (without activating / stimulating a signaling response sufficient to inhibit phagocytosis) and block the interaction between SIRPα and CD47. Suitable anti-SIRPα antibodies include fully human, humanized, or chimeric versions of such antibodies. Humanized antibodies are particularly useful for in vivo applications in humans due to their low antigenicity. Similarly, caninized, feline, and other antibodies are particularly useful for applications in dogs, cats, and other species, respectively. Antibodies of interest include humanized antibodies, or caninized, feline, equine, bovine, porcine, and other antibodies, and variants thereof.

[0053] Soluble CD47 Polypeptides. In some embodiments, a subject anti-CD47 agent is a soluble CD47 polypeptide that specifically binds to SIRPα and reduces the interaction between CD47 on one cell (e.g., an infected cell) and SIRPα on another cell (e.g., a phagocyte). A suitable soluble CD47 polypeptide can bind to SIRPα without activating or stimulating signaling through SIRPα, since activation of SIRPα inhibits phagocytosis. Instead, a suitable soluble CD47 polypeptide promotes preferential phagocytosis of infected cells over non-infected cells. Cells (e.g., infected cells) that express high levels of CD47 relative to normal non-target cells (normal cells) will be preferentially phagocytosed. Thus, a suitable soluble CD47 polypeptide specifically binds to SIRPα without activating / stimulating a signaling response sufficient to inhibit phagocytosis.

[0054] In some cases, a suitable soluble CD47 polypeptide can be a fusion protein (e.g., as structurally described in U.S. Patent Publication US20100239579, specifically incorporated herein by reference). However, only fusion proteins that do not activate / stimulate SIRPα are suitable for the methods provided herein. Suitable soluble CD47 polypeptides also include any peptide or peptide fragment, including variants, or naturally occurring CD47 sequences (e.g., extracellular domain sequences or extracellular domain variants) that can specifically bind to SIRPα and inhibit the interaction between CD47 and SIRPα without stimulating sufficient SIRPα activity to inhibit phagocytosis.

[0055] In certain embodiments, a soluble CD47 polypeptide comprises the extracellular domain of CD47, including the signal peptide, such that the extracellular portion of CD47 is typically 142 amino acids in length. Soluble CD47 polypeptides described herein also include CD47 extracellular domain variants comprising at least 65%-75%, 75%-80%, 80%-85%, 85%-90%, or 95%-99% (or any percent identity between 65%-100% not specifically recited) amino acid sequence, which variants retain the ability to bind to SIRPα without stimulating SIRPα signaling.

[0056] In certain embodiments, the signal peptide amino acid sequence may be replaced with a signal peptide amino acid sequence derived from another polypeptide (e.g., an immunoglobulin or CTLA4). For example, unlike full-length CD47, which is a cell surface polypeptide that crosses the outer cell membrane, soluble CD47 polypeptides are secreted. Thus, a polynucleotide encoding a soluble CD47 polypeptide may include a nucleotide sequence encoding a signal peptide associated with a polypeptide that is normally secreted from a cell.

[0057] In other embodiments, the soluble CD47 polypeptide comprises an extracellular domain of CD47 lacking the signal peptide. As described herein, the signal peptide is not exposed on the cell surface of secreted or transmembrane proteins because the signal peptide is cleaved during protein translocation or because the signal peptide remains anchored to the outer cell membrane (such peptides are also referred to as signal anchors). The signal peptide sequence of CD47 is believed to be cleaved from CD47 polypeptide precursor cells in vivo.

[0058] In other embodiments, the soluble CD47 polypeptide comprises a CD47 extracellular domain variant. Such a soluble CD47 polypeptide retains the ability to bind to SIRPα without stimulating SIRPα signaling. The CD47 extracellular domain variant can have an amino acid sequence that is at least 65%-75%, 75%-80%, 80-85%, 85%-90%, or 95%-99% identical to a native CD47 sequence (including any percent identity between any one of the recited ranges).

[0059] Transient immunosuppressants. Transient immunosuppressants block the activity of immune cells, particularly T lymphocytes, for a short period of time, usually during or shortly before administration of donor cells. Transient immunosuppression, i.e., effective serum levels of immunosuppressant(s), can be maintained for at least about 3 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, and can be maintained for up to 1 month, up to 2 months, up to 3 months, up to 4 months, up to 5 months, up to 6 months, or longer. In some embodiments, a single dose of the agent is administered prior to or simultaneously with the donor cells. Such agents are typically suppressive without destroying immune cell populations. The initial administration of the agent is given within about 3 days, within about 2 days, within about 1 day, or at the time of administration of the donor cells.

[0060] Transient immunosuppression can be achieved by administering pharmacological immunosuppressants, including, but not limited to, calcineurin inhibitors, which inhibit calcineurin activity in combination with binding proteins. These include, for example, tacrolimus and cyclosporine A. Both cyclosporine and tacrolimus levels must be carefully monitored. Initially, levels can be maintained in the 10-20 ng / mL range, but after three months, levels can be kept low (5-10 ng / mL) to reduce the risk of nephrotoxicity. Other pharmacological agents for this purpose include steroids, azathioprine, mycophenolate mofetil, and sirolimus.

[0061] In some embodiments, the transient immunosuppressant blocks the interaction of CD40 with CD40 ligand. CD40 is a costimulatory protein found on antigen-presenting cells (APCs) and is required for their activation. These APCs include phagocytes (macrophages and dendritic cells) and B cells. CD40 is part of the TNF receptor family. The primary activating signaling molecules for CD40 are IFNγ and CD40 ligand (CD40L).

[0062] "CD40 ligand" (also referred to as "CD40L" and "CD154") is a type II transmembrane protein. CD40L was initially thought to be restricted to activated T lymphocytes, where it functions as a mediator of T cell-dependent B cell activation, proliferation, and differentiation. Expression of CD40L plays a functional role as a central mediator of immunity and inflammation in the tumor necrosis factor (TNF) gene superfamily. CD40 / CD40L interaction is essential for the development of thymus-dependent humoral immune responses. CD40L regulates physiological processes such as T cell-mediated effector functions and general immune responses required for adequate host defense, but also triggers the expression of proinflammatory mediators, such as cytokines, adhesion molecules, and matrix-degrading activity, all of which are associated with the pathogenesis of chronic inflammatory diseases, such as autoimmune disorders, arthritis, atherosclerosis, and cancer.

[0063] Given its important role in mediating many aspects of the immune response, the CD40 / CD40L pathway provides a therapeutic target for the prevention of transplant rejection. Interfering with the CD40 / CD40L signaling pathway using anti-CD40L antibodies can be effective in preventing acute allograft rejection and alloantibody responses in animal models and clinical use. Subsequent studies have demonstrated the beneficial effects of anti-CD40L on the prolongation of graft survival in several rodent models (pancreatic islet, limb, cornea, and bone marrow).

[0064] As used herein, the terms "anti-CD40L agent" or "agent that provides CD40L blockade" refer to any agent that reduces binding of CD40L to CD40 (e.g., on target cells). Non-limiting examples of suitable anti-CD40L reagents include anti-CD40 antibodies, and anti-CD40L antibodies or antibody fragments. Agents of interest include, but are not limited to, antibody fragments and small molecules. For example, CDP7657 is a high-affinity PEGylated monovalent Fab' anti-CD40L antibody fragment. An effective amount of an antibody can be up to about 50 mg / kg, up to about 25 mg / kg, up to about 10 mg / kg, up to about 5 mg / kg, up to about 1 mg / kg, up to about 0.5 mg / kg, or less, where dosages can vary with the particular antibody and recipient. As an alternative to antibodies, small molecule inhibitors are described, for example, in Chen et al. (2017) J. Med. Chem. 60, 8906-8922, which is specifically incorporated herein by reference.

[0065] T Cell Depletion. For some transplant situations, it is desirable to also deplete endogenous T cells, as outlined in Table 1. In some embodiments, the depleting agent is specific for T cells, while in others, it also acts on NK cells. Antibodies that target T cells include, for example, antibodies specific for CD2, CD3, CD4, CD8, CD52 (campath), CD45, and ATG. With regard to timing, it is desirable that the T cell depleting agent be active at or shortly before administration of the donor cells. Therapeutic levels of the depleting agent can be maintained for at least about 3 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, and for up to 1 month, up to 2 months, up to 3 months, up to 4 months, up to 5 months, up to 6 months, or more after administration of the donor cells. In some embodiments, a dose of the agent is administered within about 3 days, about 2 days, about 1 day, or at the same time as administration of the donor cells, and, depending on the antibody, may be administered daily for several days, e.g., 2, 3, 4 days, etc., prior to infusion. An effective amount of antibody may be up to about 50 mg / kg, up to about 25 mg / kg, up to about 10 mg / kg, up to about 5 mg / kg, up to about 1 mg / kg, up to about 0.5 mg / kg, or less, e.g., up to about 100 μg / kg, up to about 50 μg / kg, up to about 10 μg / kg, or up to about 1 μg / kg, where dosage may vary depending on the particular antibody and recipient. Antibody-based therapies may use monoclonal antibodies (e.g., muromonab CD3) or polyclonal antibodies, anti-CD25 antibodies (e.g., basiliximab, daclizumab), etc. Antibodies include, for example, ATG preparations, KT3, BTI-322® (US Pat. No. 5,730,979, the disclosure of which is incorporated herein by reference).

[0066] Several anti-human CD3 mAbs are in clinical development, including teplizumab. MGA031 is a humanized IgG1 antibody developed by grafting the complementarity-determining regions of OKT3 onto a human IgG1 backbone. Otelixizumab (ChAglyCD3, TRX4, GSK2136525) is a humanized IgG1 derived from the rat antibody YTH12.5 with a single mutation in the gamma 1 Fc region to avoid glycosylation and thus inhibit FcR binding. Visilizumab (Nuvion, HuM291) is a humanized IgG2 antibody that has been rendered non-mitogenic by two point mutations in its Fc region. Foralaumab (28F11-AE; NI-0401) is a fully human anti-CD3 mAb.

[0067] Useful agents for depletion of T cells and NK cells include anti-CD52 antibodies, such as the clinically approved antibody Campath (alemtuzumab), a recombinant DNA-derived humanized monoclonal antibody against CD52, a 21-28 kD cell surface glycoprotein. Campath-1H is an IgG1 kappa antibody with human variable framework and constant regions and complementarity-determining regions derived from a mouse (rat) monoclonal antibody (Campath-1G). Campath can be administered, for example, at currently approved clinical doses, e.g., titrated up to a maximum single dose of 30 mg over a period of about 3 to about 7 days.

[0068] NK Cell Depletion. For some transplant situations, it is desirable to also deplete endogenous NK cells, as outlined in Table 1. As noted above, some agents act on both T cells and NK cells, such as antibodies against CD2, CD52, etc. Other agents are specific for NK cells and can be administered in combination with T cell targeting agents. Antibodies that selectively target NK cells include, for example, antibodies specific for CD122 and CD56.

[0069] With regard to timing, it is desirable that the NK cell depleting agent be active at or shortly before administration of the donor cells. Therapeutic levels of the depleting agent can be maintained for at least about 3 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, and for up to 1 month, up to 2 months, up to 3 months, up to 4 months, up to 5 months, up to 6 months, or more after administration of the donor cells. In some embodiments, a dose of the agent is administered within about 3 days, within about 2 days, within about 1 day, or at the same time as administration of the donor cells, and, depending on the antibody, can be administered daily for several days, e.g., 2, 3, 4 days, etc., prior to infusion. An effective amount of antibody can be up to about 50 mg / kg, up to about 25 mg / kg, up to about 10 mg / kg, up to about 5 mg / kg, up to about 1 mg / kg, up to about 0.5 mg / kg, or less, e.g., up to about 100 μg / kg, up to about 50 μg / kg, up to about 10 μg / kg, up to about 1 μg / kg, where dosage can vary with the particular antibody and recipient.

[0070] "CD122" (also called "interleukin-2 receptor subunit beta," IL2RB) is a type I membrane protein. CD122 is a subunit of the interleukin-2 receptor (IL2R), which is involved in T cell-mediated immune responses and exists in three forms depending on its ability to bind interleukin-2. The low-affinity form of IL2R is a monomer of the alpha subunit and is not involved in signal transduction. The intermediate-affinity form consists of an alpha / beta subunit heterodimer, while the high-affinity form consists of an alpha / beta / gamma subunit heterotrimer. Both the intermediate-affinity and high-affinity forms of the receptor are involved in receptor-mediated endocytosis and the transmission of mitogenic signals from interleukin-2. The use of alternative promoters results in multiple transcript variants encoding the same protein.

[0071] As used herein, the term "anti-CD122 agent" or "agent that provides CD122 blockade" refers to any agent that depletes CD122-positive cells, including natural killer (NK) cells. Non-limiting examples of suitable anti-CD122 reagents include anti-IL-2 antibodies, and anti-CD122 antibodies or antibody fragments.

[0072] Antibodies targeting CD56 are in clinical development and are used for NK cell depletion. For example, IMGN901 is a CD56-targeting antibody-drug conjugate designed for the selective delivery of the cytotoxic maytansinoid DM1, with a maximum tolerated dose (MTD) of approximately 75 mg / m. 2 This is, for example, about 1 to about 60 mg / m 2 may be administered at a dose of

[0073] "Major histocompatibility complex antigens" (also called "MHC" or "human leukocyte antigens," or HLA) are protein molecules expressed on the cell surface that confer a unique antigenic identity to these cells. MHC / HLA antigens are target molecules recognized by T cells and natural killer (NK) cells as derived from hematopoietic stem cells of the same origin as the immune effector cells ("self") or from hematopoietic reconstituting cells of a different origin ("non-self"). Two major classes of HLA antigens are recognized: HLA class I and HLA class II. HLA class I antigens (A, B, and C in humans) enable cells to recognize themselves as "self," while HLA class II antigens (DR, DP, and DQ in humans) are involved in reactions between lymphocytes and antigen-presenting cells. Both have been implicated in the rejection of transplanted organs.

[0074] An important aspect of the HLA gene system is its polymorphism. Each gene, MHC class I (A, B, and C) and MHC class II (DP, DQ, and DR), exists in different alleles. HLA alleles are represented by numbers and subscripts. For example, two unrelated individuals may have class I HLA-B, genes B5 and Bw41, respectively. The allelic gene products differ by one or more amino acids in the α and / or β domain(s). Large panels of specific antibodies or nucleic acid reagents are used to type an individual's HLA haplotype using white blood cells expressing class I and class II molecules. The most important genes for HLA typing are the six MHC class I and class II proteins, with two alleles for each of HLA-A, HLA-B, and HLA-DR.

[0075] HLA genes are clustered in a "superlocus" located at chromosome 6p21, which encodes six classical HLA genes and at least 132 protein-encoding genes that play important roles in regulating the immune system and several other fundamental molecular and cellular processes. The complete locus is approximately 3.6 Mb long and contains at least 224 genes. One effect of this clustering is that "haplotypes," or sets of alleles inherited from one parent on a single chromosome, tend to be inherited as a group. Sets of alleles inherited from each parent form haplotypes when some alleles tend to associate with each other. Identifying a patient's haplotype can predict the probability of finding a compatible donor and aid in the development of search strategies, because some alleles and haplotypes are more common than others and are distributed at different frequencies in different racial and ethnic groups.

[0076] As used herein, the term "HLA-matched" refers to a donor-recipient pair in which all HLA antigens are not mismatched between the donor and recipient. HLA-matched (i.e., all six alleles are matched) donor / recipient pairs have a reduced risk of graft-versus-host disease (GVHD) compared to mismatched pairs (i.e., at least one of the six alleles is mismatched). HLA haploidentical refers to a match in which one chromosome is matched at least for HLA-A, HLA-B, and HLA-DR, and may be matched at minor histocompatibility loci on the chromosome but not necessarily on the second chromosome. Such donors often occur in families; for example, parents may be haploidentical to their children, and siblings may be haploidentical.

[0077] As used herein, the term "HLA-mismatched" refers to a donor-recipient pair in which at least one HLA antigen is mismatched between the donor and recipient, particularly with respect to HLA-A, HLA-B, and HLA-DR. In some cases, one haplotype is matched and the other is mismatched. This situation is frequently observed with organs from living or deceased donors. HLA-mismatched donor / recipient pairs have an increased risk of GVHD compared to fully matched pairs (i.e., all six alleles are matched).

[0078] HLA alleles are typically described at various levels of detail. Most nomenclature begins with the HLA and locus name, followed by an * and several (even-numbered) numbers designating the allele. The first two numbers designate the allele group. Older typing methods often could not completely distinguish alleles and remained at this level. Numbers three and four designate synonymous alleles. Numbers five and six indicate any synonymous mutations within the coding frame of the gene. Numbers seven and eight identify mutations outside the coding region. Letters such as L, N, Q, or S may follow the allele nomenclature to designate expression levels or other non-genomic data known about it. Thus, a fully described allele can be up to nine numbers long, not including the HLA prefix and locus designation.

[0079] As used herein, a "recipient" is an individual receiving an organ, tissue, or cells from another individual (donor), generally of the same species. For purposes of this disclosure, the recipient and donor are either HLA-matched or HLA-mismatched.

[0080] As used herein, "antibody" includes reference to an immunoglobulin molecule that immunologically reacts with a specific antigen, and includes both polyclonal and monoclonal antibodies. The term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies. The term "antibody" also includes antigen-binding forms of antibodies, including fragments that retain antigen-binding ability (e.g., Fab', F(ab')2, Fab, Fv, and rIgG). The term also refers to recombinant single-chain Fv fragments (scFv). The term antibody also includes bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies.

[0081] The selection of antibodies for endogenous stem cell destruction and transient immunosuppression may be based on a variety of criteria, including selectivity, affinity, cytotoxicity, etc. The phrases "specifically (or selectively) bind" or "specifically (or selectively) immunoreact with" an antibody, when referring to a protein or peptide, refer to a binding reaction that determines the presence of that protein in a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, a designated antibody binds to a particular protein sequence at least twice background, more typically 10-100 times background. Generally, antibodies of the invention bind to antigens on the surface of target cells in the presence of effector cells (such as natural killer cells or macrophages). Fc receptors on the effector cells recognize the bound antibody. Cross-linking of the Fc receptors signals the effector cells to kill the target cell by cytolysis or apoptosis. In one embodiment, this induction is achieved via antibody-dependent cellular cytotoxicity (ADCC). In an alternative embodiment, the antibody is active in inhibiting the proliferation of targeted cells, destruction being achieved by interfering with growth factor signaling, e.g., an antibody specific for a growth factor receptor such as c-kit.

[0082] Antibodies that immunologically react with a specific antigen can be produced by recombinant methods, such as selecting libraries of recombinant antibodies in phage or similar vectors, or by immunizing animals with the antigen or DNA encoding the antigen. Methods for preparing polyclonal antibodies are known to those skilled in the art. Alternatively, the antibody may be a monoclonal antibody. Monoclonal antibodies can be prepared using the hybridoma method. In the hybridoma method, a suitable host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells.

[0083] Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. Similarly, human antibodies can be generated by introducing human immunoglobulin loci into transgenic animals, such as mice, in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire.

[0084] Antibodies also exist as several well-characterized fragments produced by digestion with various peptidases. Thus, pepsin digests antibodies that are disulfide-linked in the hinge region, itself split into V fragments by disulfide bonds. H -C H1 The resulting product is a dimer of Fab, a light chain joined to a light chain of Fab, F(ab'). The F(ab)' can be reduced under mild conditions to cleave the disulfide linkage in the hinge region, thereby converting the F(ab')2 dimer to a Fab' monomer. The Fab' monomer is essentially a Fab with a portion of the hinge region. While various antibody fragments are defined in terms of the digestion of an intact antibody, those skilled in the art will understand that such fragments can be synthesized de novo, either chemically or using recombinant DNA methodology. Thus, the term "antibody," as used herein, also includes antibody fragments produced by the modification of whole antibodies, or antibody fragments synthesized de novo using recombinant DNA methodology (e.g., single-chain Fv) or identified using a phage display library.

[0085] A "humanized antibody" is an immunoglobulin molecule that contains minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, humanized antibodies comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions are those of a human immunoglobulin consensus sequence. Optimally, humanized antibodies also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0086] Antibodies of interest for destruction may be tested for their ability to induce ADCC (antibody-dependent cellular cytotoxicity). Antibody-associated ADCC activity can be monitored and quantified by detecting either the release of label or lactate dehydrogenase from lysed cells, or by detecting a decrease in target cell survival (e.g., annexin assay). Assays for apoptosis may be performed by terminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end labeling (TUNEL) assay (Lazebnik et al., Nature: 371, 346 (1994)). Cytotoxicity may also be detected directly by detection kits known in the art, such as the cytotoxicity detection kit from Roche Applied Science (Indianapolis, Ind.). Preferably, the antibodies of the present invention induce at least 10%, 20%, 30%, 40%, 50%, 60%, or 80% cytotoxicity of target cells.

[0087] In some embodiments, the antibody is conjugated to an effector moiety. The effector moiety can be any number of molecules, including a labeling moiety such as a radioactive or fluorescent label, or can be a cytotoxic moiety. Cytotoxic agents are numerous and varied, including, but not limited to, cytotoxic drugs or toxins or active fragments of such toxins. Suitable toxins and their corresponding fragments include diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin, saporin, auristatin E, and the like. Cytotoxic agents also include radiochemicals created by conjugating radioisotopes to antibodies. Targeting cytotoxic moieties to transmembrane proteins helps increase the localized concentration of the cytotoxic moiety in the target area.

[0088] The term stem cell is used herein to refer to mammalian cells that have both the ability to self-renew and to generate differentiated progeny (see Morrison et al. (1997) Cell 88:287-298). Generally, stem cells possess one or more of the following characteristics: the ability to undergo asynchronous or symmetric replication, i.e., extensive self-renewal capacity, whereby the two daughter cells after division can have different phenotypes; the ability to exist in a mitotically quiescent form; and the ability of hematopoietic stem cells to reconstitute all hematopoietic lineages, all tissues in which they reside.

[0089] For engraftment purposes, a composition containing hematopoietic stem cells is administered to a patient. Such methods are well known in the art. The stem cells are optionally, but not necessarily, purified. Numerous reports have explored various methods for stem cell purification and subsequent engraftment, including flow cytometry; the Isolex system (Klein et al. (2001) Bone Marrow Transplant. 28(11):1023-9; Prince et al. (2002) Cytotherapy 4(2):137-45); immunomagnetic separation (Prince et al. (2002) Cytotherapy 4(2):147-55; Handgretinger et al. (2002) Bone Marrow Transplant. 29(9):731-6; Chou et al. (2005) Breast Cancer. 12(3):178-88); and the like. Each of these references is specifically incorporated herein by reference, particularly with respect to procedures, cell compositions and dosages for hematopoietic stem cell transplantation.

[0090] Hematopoietic stem cells can be obtained from bone marrow or by harvesting from peripheral blood. Bone marrow is typically aspirated from the posterior iliac crest while the donor is under local or general anesthesia. Additional bone marrow can be obtained from the anterior iliac crest. 1 x 10 per kilogram 8 and 2 x 10 8 A single bone marrow mononuclear cell population is typically considered desirable for establishing engraftment in autologous and allogeneic bone marrow transplants, respectively. Bone marrow can be primed with granulocyte colony-stimulating factor (G-CSF; filgrastim [Neupogen]) to expand the number of stem cells. Reference to "whole bone marrow" for purposes herein generally refers to a composition of mononuclear cells derived from bone marrow that has not been selected for a particular immune cell subset. "Fractionated bone marrow" refers to a composition of mononuclear cells derived from bone marrow that has not been selected for a particular immune cell subset, e.g., T cells, e.g., CD8 + cells, CD52 + cells, CD3 + Cells etc. are depleted, CD34 + It may be enriched for cells, etc.

[0091] Hematopoietic stem cells can also be obtained from umbilical cord blood. Umbilical cord blood is a nearly unlimited source of hematopoietic stem cells for allogeneic hematopoietic stem cell transplantation. Umbilical cord blood banks (CBBs) have been established for related and unrelated UCBT, with over 400,000 units available, and over 20,000 cord blood transplants performed in children and adults. UCB hematopoietic progenitor cells are enriched for atomic stem / progenitor cells capable of generating long-term repopulating stem cells in vivo. However, the number of cells available from any single donor can be relatively small compared to other sources.

[0092] Mobilization of stem cells from bone marrow to peripheral blood using cytokines such as G-CSF or GM-CSF has led to the widespread application of peripheral blood progenitor cell collection by apheresis for hematopoietic stem cell transplantation. The dose of G-CSF used for mobilization is 10 μg / kg / day. However, in heavily conditioned autologous donors, doses up to 40 μg / kg / day can be given. Mozobil may be used in combination with G-CSF to mobilize hematopoietic stem cells into peripheral blood for collection.

[0093] The dose of stem cells administered may depend on the desired purity of the infused cell composition and the source of the cells. Current guidelines suggest that the minimum dose required for engraftment is 1-2 x 10 for autologous and allogeneic transplants. 6 CD34 + It has been shown that the dose is 3×10 cells / kg body weight. Higher doses, e.g., 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 10 7 or more. Often, the dose is limited by the number of cells available. Typically, regardless of source, the dose is determined based on the CD34 + Calculated based on the number of cells. CD34 +The percentage of cells may be low for unfractionated bone marrow or mobilized peripheral blood, in which case the total number of cells administered is much higher.

[0094] CD34 + Cells may be selected from a donor hematopoietic cell sample by affinity methods, including, but not limited to, magnetic bead selection, flow cytometry, etc. HSPC compositions may be selected by identifying CD34 + The HSPC composition may be at least about 50% pure, at least about 75% pure, at least about 85% pure, at least about 95% pure, or more, as defined by the percentage of cells that are + The maximum number of cells is approximately 10 per kg of recipient body weight. 6 CD3 or less + cells, approximately 10 per kg of recipient body weight 5 CD3 or less + cells, approximately 10 per kg of recipient body weight 4 CD3 or less + Alternatively, the cell population may be selected in tandem for expression of CD34 and CD90, and the cell population may be highly purified, e.g., at least about 85% CD34 + CD90 + Cells, at least about 90% CD34 + CD90 + Cells, at least about 95% CD34 + CD90 + cells, up to about 99% CD34 + CD90 + Alternatively, naive bone marrow or mobilized peripheral blood populations are used.

[0095] Hematopoietic stem cells can also be generated in vitro, e.g., from pluripotent embryonic stem cells, induced pluripotent cells, etc. See, e.g., Sugimura et al. (2017) Nature 545:432-438, which is specifically incorporated herein by reference, detailing protocols for the generation of hematopoietic progenitor cells.

[0096] The cells employed may be fresh, frozen, or precultured. They may be fetal, neonatal, adult, etc. Hematopoietic stem cells may be obtained from fetal liver, bone marrow, blood, particularly G-CSF- or GM-CSF-mobilized peripheral blood, or any other conventional source. Cells for engraftment are optionally isolated from other cells; the manner in which stem cells are separated from hematopoietic or other lineages is not critical to this specification. If desired, a substantially homogeneous population of stem or progenitor cells can be obtained by selectively isolating cells that exhibit epitope characteristics associated with stem cells but do not contain markers associated with differentiated cells.

[0097] Cells can be genetically modified to introduce genes useful in differentiated cells, such as repair of genetic defects in an individual, selectable markers, or genes useful in selection against undifferentiated ES cells. Cells can also be genetically modified to enhance survival, control proliferation, etc. Cells can be genetically modified by transfection or transduction with a suitable vector, homologous recombination, or other suitable techniques so that they express a gene of interest. In one embodiment, cells are transfected with a gene encoding the telomerase catalytic component (TERT) under a heterologous promoter, which typically increases telomerase expression above that occurring under the endogenous promoter (see International Patent Application WO 98 / 14592). In other embodiments, a selectable marker is introduced to provide a higher purity of the desired differentiated cells. Cells can be genetically modified using vector-containing supernatant for 8-16 hours, then switched to growth medium for 1-2 days. Genetically modified cells are selected using a drug selection agent such as puromycin, G418, or blasticidin, and then recultured.

[0098] The cells of the present invention can also be genetically modified to enhance their ability to participate in tissue regeneration or to deliver therapeutic genes to the administration site. Vectors are designed using a known coding sequence for a desired gene operably linked to a promoter that is constitutive, pan-specific, or particularly active in differentiated cell types. A suitable inducible promoter is activated in either the desired target cell type, the transfected cell, or its progeny. By transcription activation, it is intended that transcription be increased by at least about 100-fold, more usually at least about 1000-fold, above the basal level in the target cell. Various promoters that are induced in different cell types are known.

[0099] Many vectors useful for transferring exogenous genes into target mammalian cells are available. Vectors can be episomal, e.g., plasmids, virus-derived vectors such as cytomegalovirus and adenovirus, or can integrate into the target cell genome through homologous recombination or random integration, e.g., retrovirus-derived vectors such as MMLV, HIV-1, and ALV. For stem cell modification, lentiviral vectors are preferred. Lentiviral vectors, e.g., those based on HIV or FIV gag sequences, can be used to transfect non-dividing cells, e.g., quiescent human stem cells. A combination of a retrovirus and a suitable packaging cell line can also be used, in which case the capsid protein is useful for infecting target cells. Typically, the cells and virus are incubated in culture medium for at least about 24 hours. The cells are then allowed to grow naturally in culture medium for a short period of time, e.g., 24-73 hours, or at least two weeks, in some applications, and may be grown for up to five weeks or more before analysis. Commonly used retroviral vectors are "defective," i.e., unable to produce viral proteins required for productive infection. Replication of the vector requires propagation in a packaging cell line. Vectors may contain genes that must subsequently be removed, e.g., using a recombinase system such as Cre / Lox, or cells expressing them are destroyed by including genes that allow for selectable toxicity, e.g., herpesvirus TK, bcl-Xs, etc.

[0100] Chimerism, as used herein, generally refers to hematopoietic chimerism unless otherwise indicated. Whether an individual is a complete chimera, a mixed chimera, or a non-chimeric product is determined by analyzing hematopoietic cell samples, such as peripheral blood or bone marrow, from the transplant recipient, as known in the art. Analysis may be performed by any conventional typing method. In some embodiments, the degree of chimerism among total mononuclear cells, T cells, B cells, CD56+ NK cells, and CD15+ neutrophils is periodically monitored using PCR with microsatellite analysis probes. For example, commercially available kits are available that identify polymorphisms within the short terminal repeat lengths of donor and host origin. Automated readers provide the percentage of donor-type cells based on a standard curve from an artificial donor and host cell mixture.

[0101] Individuals who exhibit more than 95% donor cells in a given blood cell lineage at any time after transplantation by such analysis are said to have complete donor chimerism in this transplant patient group. Mixed chimerism is defined as more than 1% donor but less than 95% donor DNA in such analysis. Individuals exhibiting mixed chimerism may be further classified according to the evolution of chimerism, with improvement of mixed chimerism being defined as a continuous increase in the proportion of donor cells over a period of at least 6 months. Stable mixed chimerism is defined as a change in the percentage of recipient cells over time without a complete loss of donor cells.

[0102] A "patient" for purposes of the present invention includes both humans and other animals, particularly mammals, including pets and laboratory animals, e.g., mice, rats, rabbits, etc. Thus, the methods are applicable to both human therapy and veterinary applications. In one embodiment, the patient is a mammal, preferably a primate. In another embodiment, the patient is human.

[0103] Additional Terminology. The terms "treatment," "treating," "treat," and the like are used generally herein to refer to obtaining a desired pharmacological and / or physiological effect. The effect can be preventative, in terms of complete or partial prevention of a disease or its symptom(s), and / or therapeutic, in terms of partial or complete stabilization or cure of the disease and / or side effects caused by the disease. The term "treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes (a) preventing the disease and / or symptom(s) from occurring in a subject who may be affected by the disease or condition but has not yet been diagnosed as affected, (b) inhibiting the disease and / or symptom(s), i.e., halting their development, or (c) alleviating disease symptom(s), i.e., causing regression of the disease and / or symptom(s). Those in need of treatment include those already affected (e.g., those with cancer, those infected, etc.) as well as those for whom prevention is desired (e.g., those with an increased susceptibility to cancer, those with an increased likelihood of infection, those suspected of having cancer, those suspected of being infected, etc.).

[0104] Methods for engraftment The methods of the present invention provide improved stem cell engraftment after transplantation into a recipient. The recipient may be immunocompetent, and transplantation may be performed in the absence of myeloablative conditioning, i.e., in the absence of radiation and / or chemotherapy. The recipient is conditioned with a combination of agents selected according to cell and HLA matching. The selection of agents is shown in Table 1, which provides guidelines for optimized conditioning protocols. A "+" indicates that the agent should be included for the specified agent, HLA match, and cell source. A "-" indicates that it is not required, but may be included optionally. As disclosed above, certain agents can deplete both T cells and NK cells, and therefore, only one agent is needed for both. The timing and dosage for different agents are as described above. The conditioning regimens of the present invention selectively destroy endogenous stem cells and provide suitable, selected suppression of endogenous immune responses, thereby enabling engraftment even in mismatched recipients.

[0105] [Table 1]

[0106] After the transplant conditioning regimen, an effective amount of a cell composition containing exogenous stem cells is administered to the recipient during a period of transient immunosuppression. The stem cells may be autologous, allogeneic, or xenogeneic, including, but not limited to, allogeneic haploidentical stem cells, mismatched allogeneic stem cells, genetically engineered autologous cells, etc.

[0107] Infusion of HSPCs is a relatively simple process performed in clinical settings. Bone marrow products are generally used fresh and infused over several hours through a central vein. Autologous products are often cryopreserved, in which case they are thawed at the clinical setting and rapidly infused over a few minutes. PBMCs may be stored overnight or cryopreserved.

[0108] If the donor is allogeneic to the recipient, the HLA types of the donor and recipient may be tested for compatibility, or haploidentical cells may be used. HLA haploidentical donors can be engineered by CD34 or CD34CD90 selection. Furthermore, HLA haploidentical donors are now widely used for other indications (potentially beyond HLA matching). Regarding HLA matching, traditionally, the key loci for matching are HLA-A, HLA-B, and HLA-DR. HLA-C and HLA-DQ are now also being considered when determining donor suitability. A fully matched sibling donor is generally considered the ideal donor. For unrelated donors, a fully matched or single mismatch is considered acceptable for most transplants, although larger mismatches are tolerated in certain circumstances. Preferably, matching is both serological and molecular. When the donor is umbilical cord blood, the tolerable HLA disparity is much greater, with a match of three to four of the six HLA-A, HLA-B, and HLA-DRB1 antigens being sufficient for transplantation. Immunoreactive donor T cells can be removed using various methods to reduce or eliminate the possibility of graft-versus-host disease (GVHD) developing.

[0109] In some embodiments, the success of the procedure is determined by the presence of host-derived myeloid cells, e.g., CD15, in the recipient's circulation. + Blood marrow chimerism is monitored by determining the presence of donor-type, CD15, and / or IL-16 cells. Blood marrow chimerism is an indicator of true HSC engraftment due to the short-term survival characteristics of myeloid cells. Approximately 8 weeks after HCT, the methods described herein demonstrate measurable and sustained levels of blood marrow chimerism, e.g., at least about 1% donor-type, CD15, and / or IL-16 cells. + Cells, at least approximately 2% donor type, CD15 + Cells, at least approximately 4% donor type, CD15 + Cells, at least approximately 8% donor type, CD15 + cells, or more, providing blood-bone marrow chimerism.

[0110] The transplant conditioning agents may be provided in the absence of myeloablative radiation or chemotherapy and are administered according to the specific requirements described above. Some agents are administered to be active after administration of HSPCs, while others require a washout period.

[0111] The transient immunosuppressant is provided at a dose that reduces activated T cell activity by at least 10-fold, at least 100-fold, at least 1000-fold, at least 100,000-fold, or more. Effective amounts will depend on the individual and the particular agent, but when the agent is an antibody, the dose can be at least about 50 μg / kg body weight, at least about 250 μg / kg, at least about 500 μg / kg, at least about 750 μg / kg, at least about 1 mg / kg, and up to about 2.5 mg / kg, up to about 5 mg / kg, up to about 7.5 mg / kg, up to about 10 mg / kg, up to about 15 mg / kg, up to about 25 mg / kg, up to about 50 mg / kg, or up to about 100 mg / kg.

[0112] The transplant conditioning agent is formulated into a pharmaceutical composition. The exact dose depends on the purpose of the treatment and can be ascertained by one of ordinary skill in the art using known techniques (e.g., Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery; Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Dekker, ISBN 0824770846, 082476918X, 0824712692, 0824716981; Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); and Pickar, Dosage Calculations (1999)). As is known in the art, adjustments may be necessary based on the patient's condition, systemic versus local delivery, as well as age, weight, general health, sex, diet, time of administration, drug interactions, and severity of the condition, and can be ascertained by one of ordinary skill in the art using routine experimentation.

[0113] Administration of the agent can be done in a variety of ways, as described above, including, but not limited to, oral, subcutaneous, intravenous, intranasal, transdermal, intraperitoneal, intramuscular, or intraocular. Antibodies can be delivered by intravenous injection.

[0114] In one embodiment, the pharmaceutical composition is in a water-soluble form, e.g., as a pharmaceutically acceptable salt, which is intended to include both acid and base addition salts. "Pharmaceutically acceptable acid addition salts" refers to salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc., and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc., which retain the biological effectiveness of the free base and are not biologically or otherwise undesirable. "Pharmaceutically acceptable base addition salts" include those derived from inorganic salts such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, etc. Particularly useful are ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.

[0115] The pharmaceutical compositions may also include one or more of carrier proteins such as serum albumin, buffers, fillers such as microcrystalline cellulose, lactose, corn and other starches, binders, sweeteners and other flavors, colorants, and polyethylene glycol.

[0116] Pharmaceutical compositions can be administered in various dosage unit forms depending on the method of administration.For example, dosage unit forms suitable for oral administration include, but are not limited to, powders, tablets, pills, capsules, and lozenges.It is recognized that the compositions of the present invention should be protected from digestion when orally administered.This is typically achieved by either complexing the molecule with the composition to make it resistant to acid and enzymatic hydrolysis, or by packaging the molecule in a suitable resistant carrier, such as liposomes or a protective barrier.Means for protecting active substances from digestion are well known in the art.

[0117] Compositions for administration generally contain an antibody or other agent dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, such as buffered saline. These solutions are sterile and generally free of undesirable material. These compositions may be sterilized by conventional, well-known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, and the like, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the active agent in these formulations can vary widely and is selected primarily based on fluid volume, viscosity, body weight, and the like, in accordance with the particular mode of administration selected and the patient's needs (e.g., Remington's Pharmaceutical Science (15th ed., 1980) and Goodman & Gillman, The Pharmacological Basis of Therapeutics (Hardman et al., eds., 1996)).

[0118] Compositions containing disruptive agents, such as antibodies, soluble SIRPα, etc., can be administered for therapeutic treatment. The compositions are administered to patients in an amount sufficient to substantially destroy the targeted endogenous stem cells, as described above. An amount sufficient to achieve this is defined as a "therapeutically effective amount." Single or multiple administrations of the compositions can be administered depending on the dosage and frequency required and tolerated by the patient. The specific dosage required for treatment depends on the mammal's condition and medical history, as well as other factors, such as age, weight, sex, route of administration, and efficacy.

[0119] In the method of the present invention, the active substance is administered as a short-term treatment before transplantation. Usually, the treatment is completed at least about 1 week before transplantation, at least about 5 days before transplantation, or at least about 3 days before transplantation. The process can be repeated as needed, for example, 2, 3, 4, 5 or more times as needed to clear the niche.

[0120] Conditions for treatment Indications for stem cell transplantation vary by disease category and are influenced by factors such as chromosomal abnormalities, response to previous treatment, age and performance status of the patient, disease status (remission vs. relapse), disease-specific prognostic factors, availability of a suitable graft source, time of referral, and time of transplant.

[0121] Autologous HSCT is currently used to treat conditions such as multiple myeloma, non-Hodgkin's lymphoma, Hodgkin's disease, acute myeloid leukemia, neuroblastoma, germ cell tumors, autoimmune diseases - systemic lupus erythematosus (SLE), systemic sclerosis, and amyloidosis.

[0122] Allogeneic HSCT is currently used to treat disorders such as acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, myelodysplastic syndromes, multiple myeloma, non-Hodgkin's lymphoma, Hodgkin's disease, aplastic anemia, aplasia true, paroxysmal nocturnal hemoglobinuria, Fanconi anemia, thalassemia major, sickle cell anemia, severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome, hemophagocytic syndrome (HLH), inborn errors of metabolism such as mucopolysaccharidoses, Gaucher disease, metachromatic leukodystrophy, adrenoleukodystrophy, epidermolysis bullosa, severe congenital neutropenia, Shwachman-Diamond syndrome, Diamond-Blackfan anemia, and leukocyte adhesion deficiency.

[0123] Embodiments of the present invention include transplantation into patients suffering from inherited blood disorders, in which exogenous stem cells of normal phenotype are transplanted into the patient. Such diseases include, but are not limited to, the treatment of anemia caused by impaired hemoglobin synthesis (hemoglobinopathies). The stem cells can be allogeneic stem cells of normal phenotype, or autologous cells that have been genetically engineered to delete undesirable gene sequences and / or introduce gene sequences that correct the genetic defect.

[0124] Sickle cell disease includes HbS disease, sickle cell anemia, and meniscocytosis. Chronic hemolytic anemia occurs almost exclusively in blacks and is characterized by sickle-shaped RBCs caused by homozygous inheritance of HbS. Homozygotes have sickle cell anemia, while heterozygotes are not anemic but have demonstrable sickle tendency (sickleemia) in vitro. In HbS, valine is substituted for glutamic acid at the sixth amino acid of the beta chain. Deoxy-HbS is much less soluble than deoxy-HbA and forms rod-shaped tactoids, a semisolid gel, causing RBCs to sickle at sites of low PO2. The distorted, inelastic RBCs adhere to the vascular endothelium and block small arterioles and capillaries, leading to occlusion and infarction. Sickled RBCs are too fragile to withstand the mechanical trauma of the circulation, resulting in hemolysis after entering the circulation. In homozygotes, clinical symptoms are caused by anemia and vaso-occlusive events, leading to tissue ischemia and infarction. Growth and development are impaired and susceptibility to infection is increased. The anemia is usually severe but varies widely among individuals. Anemia may be exacerbated in children by acute accumulation of sickle red blood cells in the spleen.

[0125] Thalassemia is a group of chronic, hereditary, microcytic anemias characterized by impaired hemoglobin synthesis and ineffective red blood cell production, particularly common in individuals of Mediterranean, African, and Southeast Asian ancestry. Thalassemia is the most common inherited hemolytic disorder. It results from unbalanced hemoglobin synthesis caused by reduced production of at least one globin polypeptide chain (β, α, γ, δ).

[0126] Aplastic anemia results from a loss of RBC precursors, either due to abnormalities in the stem cell pool or to impairments in the bone marrow supportive microenvironment, and is often associated with upper-normal MCV values. The term aplastic anemia generally refers to generalized bone marrow hypoplasia accompanied by leukopenia and thrombocytopenia.

[0127] Combined immunodeficiencies are a group of disorders characterized by congenital and usually inherited deficiencies of both B- and T-cell systems, lymphoid hypoplasia, and thymic dysplasia. Combined immunodeficiencies include severe combined immunodeficiency, Swiss-type agammaglobulinemia, combined immunodeficiency with adenosine deaminase or nucleoside phosphorylase deficiency, and combined immunoglobulin deficiency (Nezelof syndrome). Most patients experience early onset of infections due to thrush, pneumonia, and diarrhea. Without treatment, most patients die by the age of two. Most patients have severe B-cell and immunoglobulin deficiencies. Lymphocyte hypoplasia, low or absent T-cell levels, poor proliferative responses to mitogens, cutaneous anergy, absent thymus, and loss of lymphoid tissue are common. Pneumocystis pneumonia and other opportunistic infections are common. [Example]

[0128] Example 1 Non-genotoxic conditioning regimens for haploidentical hematopoietic stem cell transplantation Materials and Methods Mice. All donor and recipient mice were 8 to 12 weeks old. Donor mice were AKR×Hz F1 mice bred by Shizuru lab. AKR×Hz F1 mice are double-positive for 45.1 and 45.2, and H2Kb and H2Kk. Recipient mice were CB6F1 mice from JAX. CB6F1 mice are single-positive for 45.2 and double-positive for H2Kb and H2Kd. All procedures were approved by the International Animal Care and Use Committee. Mouse strains were maintained at the Research Animal Facility at Stanford University.

[0129] Antibodies. All antibodies for in vivo transplant conditioning were purchased from Bio X Cell and included anti-CD47 (clone 3 / clone mIAP410), anti-CD117 (clone ACK2), anti-CD40L (clone MR-1), and anti-CD122 (clone TM-b1).

[0130] BM transplantation. Recipient CB6F1 mice received a prime dose of 100 μg of anti-CD47 intraperitoneally on day -8. On day -6, mice received a retro-orbital injection of 500 μg of anti-CD117. Prior to anti-CD117 treatment, mice received an intraperitoneal injection of Benadryl. From day -6 to day -2, mice received an additional daily intraperitoneal injection of 500 μg of anti-CD47. On day -2, mice received up to 250 μg of anti-CD122. On day 0, 500 μg of anti-CD40L was given several hours before transplant.

[0131] For transplantation, whole bone marrow was harvested from 8-12 week-old AKR × Hz mice. Whole bone marrow was harvested from the tibia, femur, hip, and spine. Red blood cells were lysed, and the remaining cells were counted and appropriately resuspended prior to injection. Cells were delivered via retroorbital injection.

[0132] Chimerism Confirmation. Recipient mice are periodically bled using retroorbital puncture to measure donor chimerism. Blood is stained with fluorescent antibodies against CD45.1, CD45.2, CD3, CD19, CD11b, and Gr-1.

[0133] result As shown in Figures 1A-1F, the combination of antibodies specific for c-kit, CD47, CD40L, and CD122 with the above protocol enabled effective engraftment of haploidentical whole bone marrow into immunocompetent animals. The percentage of mice that were chimeric per cohort, as well as the mean levels of total donor, T cell, B cell, and granulocyte chimerism, are shown in Figure 2. As shown in Figure 3, low doses of NK cell (NK cell) depletion using anti-CD122 are required.

[0134] Example 2 Antibody conditioning enables MHC-mismatched hematopoietic stem cell transplantation and organ graft tolerance Replacing a patient's diseased blood system through hematopoietic cell transplantation (HCT) can treat or cure inherited disorders of the blood and immune system, including leukemia, autoimmune diseases, and immunodeficiencies. In HCT, a patient's blood and immune system are typically destroyed using a toxic "conditioning regimen" (chemotherapy and / or radiation) and then replaced with donor cells containing hematopoietic stem cells (HSCs) to regenerate a healthy blood system. While HCT is a foundational treatment, its use and safety are hindered by graft-versus-host disease (which can be overcome by transplanting purified HSCs uncontaminated by donor T cells) and lethal toxicity caused by the conditioning regimen. Therefore, a critical goal is to achieve HCT conditioning using more specific and safer agents (e.g., monoclonal antibodies) that eliminate the need for toxic chemotherapy or radiation.

[0135] Here, we demonstrate that a combination of six monoclonal antibodies can safely and specifically deplete host HSCs, T cells, and NK cells in immunocompetent mice, allowing foreign (allogeneic) HSC engraftment. The engrafted donor HSCs were mismatched at either half (haploidentical) or all MHC genes; in both cases, they generated donor blood and immune systems that stably coexisted with host blood cells. These chimeric immune systems were functional, demonstrating tolerance to the HSC donor lineage and rejection of the third-party heart. These studies demonstrate an antibody-based pre-transplant treatment that can be applied as a regenerative medicine platform for purified human HSC transplantation, facilitating applications including foreign organ transplantation and the treatment of a variety of blood and immune system disorders.

[0136] Many inherited blood and immune system disorders can be treated with hematopoietic cell transplantation (HCT). Examples include thalassemia, sickle cell anemia, Fanconi anemia, inherited immunodeficiencies, autoimmune diseases (e.g., multiple sclerosis), and metabolic storage diseases. These disorders can be corrected when an individual's blood system is replaced with healthy, transplanted blood cells stably derived from rare transplanted hematopoietic stem cells (HSCs) in the HCT graft. After regeneration of the donor-derived blood and immune system, the HCT recipient becomes immunologically tolerant to organ transplants from the HSC donor. While any monogenic or polygenic inherited disorder of the blood system can be cured by allogeneic HCT, treatment of nonmalignant hematologic or immunologic disorders accounted for less than 6% of total HCT cases reported in Europe in 2015.

[0137] To overcome the infrequent use of HCT to treat nonmalignant hematologic disorders and expand its scope, two key challenges must be addressed: safety concerns and donor availability. Currently, allogeneic HCT results in clinical or subclinical graft-versus-host disease (GvHD) caused by contaminating donor-derived T cells. However, GvHD can be overcome by transplanting purified HSCs that lack T cells. Furthermore, HCT conditioning regimens require chemotherapy and radiation, which can cause life-threatening side effects.

[0138] Another challenge facing HCT for inherited blood disorders is the current need for fully matched donors at the human leukocyte antigen (HLA, also known as the major histocompatibility complex [MHC]) locus. While 75% of Caucasian Americans currently have matched donors, finding fully matched donors is significantly more difficult for Black Americans (currently 16–19% matched) or other minority groups. If HCT could be safely performed using haploidentical donors (matched at half the HLA locus), this would significantly expand donor availability, theoretically allowing any individual to receive HCT from 75% of their parents, children, or siblings. Finally, if fully HLA-mismatched HSCs could be safely transplanted, this would dramatically open up the available donor pool, with the added benefit that recipients may be immunologically tolerant to foreign organs or tissues obtained from the same donor. This would enable HLA-mismatched organ transplants without the lifelong immunosuppression typically required to prevent rejection of vital organ transplants.

[0139] The safety of HCT would improve significantly if toxic conditioning regimens (chemotherapy and / or radiation) were replaced with more specific agents that deplete components of the immune system, such as monoclonal antibodies. While previous antibody conditioning regimens allow for the transplantation of minor histocompatibility antigen-mismatched HSCs (see, e.g., Patent Publication WO 2016 / 033201), the transplantation of MHC-mismatched HSCs using antibody-based conditioning has not previously been demonstrated.

[0140] Here, we demonstrate that conditioning with six monoclonal antibodies enables wild-type mice to receive partially (haploidentical) or fully MHC-mismatched HSCs, thus enabling hematopoietic replacement and induction of tolerance to mismatched donor organs without resorting to chemotherapy or radiation.

[0141] For haploidentical transplantation experiments, AKR × C57BL / 6F1 (hereinafter referred to as AB6F1) mice were used as bone marrow or HSC donors, and BALB / C × C57BL / 6F1 (CB6F1) (Figure 4a) mice served as recipients. These mouse strains are H2 b Only matched with haplotype, H2 k and H2 d (i.e., half of the major histocompatibility complex [MHC] haplotype) (Fig. 4b). We sought to determine whether conventional transplant conditioning could be replaced with monoclonal antibodies (mAbs). We previously demonstrated that immunodeficient mice can be pretreated with anti-Kit antibodies to allow syngeneic HSC engraftment, whereas equivalent transplant conditioning of immunocompetent mice requires dual administration of anti-Kit and anti-CD47 blocking agents. CD47 blockade prevents macrophages from binding to antibody-bound (opsonized) cells, e.g., KIT opsonized by anti-c-KIT antibodies. + Allows HSCs to phagocytose.

[0142] To engraft allogeneic HSCs that are mismatched at the MHC locus, it may be necessary to suppress or eliminate both T cells and NK cells, which reject cells that express foreign major and minor histocompatibility antigens or lack "self" MHC. To eliminate host NK cells, we targeted CD122 / Il2Rβ (expressed throughout human and mouse NK cell development) using the anti-CD122 mAb Tm-β1 to deplete these cells. To prevent T cell-mediated rejection, we targeted CD40L (also known as CD154), a costimulatory cell surface molecule expressed by activated T cells, and CD40 + It is required for signaling with antigen-presenting cells. Interference with the CD40-CD40L axis can help induce tolerance to hematopoietic cells and skin grafts, and importantly, does not deplete all T cells because CD40L is upregulated on activated T cells. We blocked CD40L using the anti-CD40L antibody MR1.

[0143] Mice were treated with four monoclonal antibodies (anti-CD122, anti-CD40L, anti-Kit, and anti-CD47, herein referred to as the four-antibody conditioning regimen) over an 8-day period (Figure 4c) and then transplanted with 30 million whole bone marrow (WBM) cells. Chimerism was periodically measured by CD45 allele differentiation (Figure 8a), and multilineage mixed chimerism was observed in all animals receiving the four-antibody conditioning regimen (Figures 8b-d). Importantly, mixed chimerism was also observed in the long-term HSC (LT-HSC) compartment (Figure 4d), indicating that donor chimerism was actively maintained by donor stem cells rather than resulting from the engraftment of long-term surviving mature immune cells.

[0144] To identify the minimum required components of this cocktail, we tested each antibody individually (Figure 9) and then in various combinations of the four antibodies. The minimum required cocktail for engraftment of 30 million WBM cells was anti-CD47, anti-c-KIT, and anti-CD40L (Figure 4e-g). However, only 75% of mice in the group lacking anti-CD122 were chimeric. In the group receiving the full four-antibody conditioning regimen, 100% of mice were chimeric. Interestingly, engrafted animals from both groups showed similar levels of multilineage chimerism over 20 weeks. Additionally, the four-antibody conditioning regimen did not induce granulocytopenia before transplantation (Figure 4h).

[0145] We tested the lowest dose of WBM that could result in engraftment by titrating the dose of WBM while adjusting the use of anti-CD122. As the amount of transplanted bone marrow decreased, the number of chimeric mice decreased (Figure 4i). At 3 million WBM cells, 80% of mice were chimeric in this cell dose group with NK depletion, while 20% of mice without anti-CD122 were chimeric.

[0146] To rule out the possibility of GvHD, we next transplanted enriched HSC populations (as opposed to WBM). In these experiments, we transplanted Lineage 1 (LS1) cells, which are highly enriched for HSCs and are multipotent progenitor (MPP) cells. - Sca1 + Kit + Kit-enriched and LSK cells (Figure 5a) were transplanted into irradiated controls. Both Kit-enriched and LSK cells were administered at doses corresponding to their abundance in 30 million WBM cells (Figure 5b). All three types of grafts demonstrated complete, long-term multilineage chimerism in irradiated controls. Surprisingly, mice pretreated with the four antibodies successfully engrafted long-term with WBM but were not reconstituted by Kit-enriched or LSK transplants (Figure 5b). This suggests that additional pretreatment antibodies may be required for the enriched HSC population to engraft successfully.

[0147] To facilitate LSK engraftment, we attempted to provide additional immunosuppression by eliminating T cells using anti-CD4 and anti-CD8 depleting antibodies (Figure 5c). The addition of anti-CD4 and anti-CD8 antibodies to the four-antibody regimen strongly depleted T cells from the peripheral blood, spleen, and bone marrow (Figure 5d and Figure 10). The use of this six-antibody cocktail, termed the six-antibody conditioning regimen (anti-CD122, anti-CD40L, anti-Kit, anti-CD47, anti-CD4, and anti-CD8 mAbs), induced long-term chimerism in recipients transplanted with 9,000 LSK cells (Figure 5e). This cell dose corresponds to approximately 360,000 LSK cells / kg, well below the HSC dose seen in preclinical studies of allografts in mice and clinical use of autologous transplants in humans. In summary, the 6-antibody transplant conditioning treatment allows mice to engraft with low doses of cells, e.g., purified HSCs, without resorting to chemotherapy or radiation.

[0148] To determine whether all six components of this cocktail are necessary, we used a reductive process to identify unnecessary antibodies. Removal of anti-CD40L, anti-CD4, and anti-CD8 resulted in fewer chimeric animals and lower chimerism within each cohort compared with the full six-antibody conditioning cohort (Figures 5f and 11). However, removal of anti-CD122 did not significantly change the percentage of chimeric animals compared with the control cohort. Unlike the four-antibody conditioning regimen, CD122 may be less necessary in the six-antibody conditioning regimen due to the loss of NK cell dependence for T cell activation due to the near-complete depletion of T cells in the six-antibody conditioning regimen.

[0149] Importantly, six-antibody conditioning followed by HSC transplantation induced central immunological tolerance to the donor transgenic line. Central tolerance refers to thymic re-education of the host immune system to allow donor cell engraftment. To measure central tolerance in these animals, we measured the presence of the Vbeta 6 (Vb6) TCR chain in peripheral blood. Vb6 is reactive to the Mtv-7 provirus-encoded superantigen present in the AKR lineage. Therefore, for AB6F1 HSCs to coexist in CB6F1, CB6F1 endogenous Vb6 T cells must be clonally deleted. In both WBM- and LSK-transplanted animals, chimeric animals showed a loss of host Vb6 T cells (Figure 6a-b). Interestingly, in the WBM cohort conditioned with anti-Kit, anti-CD47, and anti-CD40L, the only animal with a normal Vb6 T cell frequency never achieved chimerism (Figure 6b).

[0150] Surprisingly, we found that 6-antibody conditioned mice engrafted with MHC-mismatched donor HSCs were immunologically tolerant to organs from the same donor strain. To achieve this, we used 6-antibody conditioned mice engrafted with MHC-mismatched donor HSCs, either from the HSC donor (AB6F1) or from a third party (DBA / 1J strain, H2 qHeart grafts from neonatal pups (homozygous for AB6F1 / J) were transplanted into the ear pinnae of naive and LSK-antibody-conditioned chimeric animals (Figure 6c). In naive, unconditioned, untransplanted mice, both AB6F1 and DBA1 / J hearts were rapidly rejected (Figure 6d). In six-antibody-conditioned chimeric mice, the DBA1 / J heart was rejected within 14 days, whereas active, beating AB6F1 hearts persisted for at least 115 days. Representative ear-heart grafts were harvested on day 34 and analyzed by immunohistochemistry. Macroscopic examination revealed that the AB6F1 heart was visible within the pinnae, whereas the DBA / 1J heart was no longer visible (Figure 6e). H&E analysis showed that troponin+ cardiac tissue devoid of immune cells infiltrated the AB6F1-engrafted pinnae. However, by this time point, there was no cardiac or troponin+ tissue within the ear containing the DBA / 1J heart (Fig. 6e), thus demonstrating that MHC-mismatched donor HSCs can induce immunological tolerance in six-antibody conditioned mice to cardiac grafts from syngeneic donors.

[0151] Finally, we demonstrated that a six-antibody conditioning regimen allows for the successful engraftment of fully MHC-mismatched HSCs. q ) mice as donors and CB6F1(H2 b / d ) hosts were used (Fig. 7a). After transplantation of 9000 DBA1 / J LSK cells, we observed high donor-host chimerism in all six-antibody-conditioned CB6F1 mice by week 8 (Fig. 7b). Mice transplanted with 3 million WBM cells alone failed to establish donor chimerism (confirming the need for transplant conditioning), whereas 40% of four-antibody-conditioned mice receiving WBM achieved low levels of chimerism. 80% of irradiated CB6F1 mice transplanted with WBM died by week 9 posttransplant (Fig. 7c), likely due to GvHD, which was not observed with LSK transplants.

[0152] In summary, we have now developed a method for transplanting semi-(haploidentical) and fully MHC-mismatched hematopoietic cell compositions, including purified HSCs, into immunocompetent animals. Importantly, this is accomplished without the use of chemotherapy and / or radiation and without the GvHD that occurs in most, if not all, other types of HCT transplants.

[0153] These findings are relevant to the clinical use of hematopoietic cell transplantation, for example, for the treatment of blood and immune system disorders. First, this antibody-based conditioning regimen combined with purified HSC transplantation improves the safety of blood and immune system replacement by obviating the need for chemotherapy / radiation and eliminating GvHD. Second, facilitating the transplantation of haploidentical, HLA-mismatched HSCs greatly increases the donor pool, allowing a majority of recipients to find a match, even if their age or clinical condition would have prevented HCT under previous protocols. For example, patients with Fanconi anemia are highly sensitive to DNA damage, and therefore, conventional conditioning regimens pose significant risks to this cohort.

[0154] Finally, the ability to induce immunological tolerance to a foreign organ opens opportunities for all patients in need of life-saving organ transplants, particularly by eliminating lifelong immunosuppression in patients receiving foreign organ transplants. Specifically, following antibody conditioning, the immune system in donor HSC-transplanted animals is tolerant to the donor (but not third-party) heart. Coexistence of donor and host T cells in these partially chimeric animals can provide MHC-restricted T cells to both donor and host tissues.

[0155] Today, donors for organ, tissue, or HSC transplants are living or recently deceased individuals. The goal of regenerative medicine is to differentiate pluripotent (embryonic or induced pluripotent) stem cell lines into HSCs and other needed tissue stem cells (e.g., neural, bone, and cartilage, or hepatic) either in vitro or in vivo in large animal hosts (e.g., pigs). This would alleviate the need for humans to forgo HSCs and organs for others. Antibody conditioning, followed by co-transplantation of pluripotent stem cell-derived HSCs and tissue stem cells, can deliver life-saving organs to patients without relying on long-term immunosuppression.

[0156] method Animals. All experiments were performed in accordance with guidelines established by the Stanford University Administrative Panel on Laboratory Animal Care. AKR x C57BL / 6F1 donors were mated and housed indoors. CB6F1 and DBA1 / J recipients were purchased from the Jackson Laboratory. DBA1 / J pregnant females were purchased from Taconic Biosciences for ear-heart transplantation.

[0157] Antibodies. Anti-CD47 (mIAP410), anti-c-KIT (ACK2), anti-CD122 (Tm-β1), anti-CD40L (MR1), anti-CD4 (GK1.5), and anti-CD8 (YTS169.4) were purchased from BioXCell. Anti-CD47 was administered intraperitoneally on day -8 as a 100 μg dose, followed by subsequent 500 μg doses throughout the transplant conditioning process. Retroorbital anti-c-KIT and intraperitoneal anti-CD40L were both administered as a single 500 μg bolus. Anti-CD122 was administered intraperitoneally as a 250 μg dose, while anti-CD4 and anti-CD8 were administered as 100 μg intraperitoneal doses. Mice receiving anti-c-KIT antibodies received 400 μg of diphenhydramine intraperitoneally 15 minutes before injection. Anti-CD25 (PC-61.5.3) was purchased from BioXCell and administered as a single 100 μg intraperitoneal injection.

[0158] Graft preparation and transplantation. Whole bone marrow was extracted from the tibia, femur, hip, and spine of donor mice. The bones were crushed and filtered, followed by red blood cell (RBC) lysis. For c-Kit-enriched transplantation, RBC-lysed whole bone marrow was bound to Miltenyi CD117 microbeads according to the manufacturer's instructions and collected after magnetic separation. For LSK cell transplantation, RBC-lysed whole bone marrow was bound to Miltenyi Lineage Cell Depletion Kit cocktail according to the manufacturer's instructions. The flow-through from the magnetic separation column was collected and stained in PBS containing 2% FBS using optimal concentrations of antibodies for CD3PE (17A2), CD4PE (GK1.5), CD5PE (53-7.3), CD8aPE (53-6.7), B220PE (RA3-6B2), Gr-1PE (RB6-8C5), Mac-1PE (M1 / 70), Ter119PE (TER119), SCA1Pe-Cy7 (D7), and CD117APC (2B8). Propidium iodide was added as a viability stain immediately before sorting on the BD Aria. All cells for transplantation were resuspended at the desired concentration in PBS containing 2% FBS. Irradiated control mice were lethally irradiated with two doses of 6.5 Gy prior to transplantation. All mice were anesthetized using isoflurane and then implanted with 100 uL of cell suspension via retro-orbital injection.

[0159] Peripheral blood chimerism. Mice were periodically bled via retroorbital bleed into EDTA tubes. Blood was then incubated with 5 mM EDTA in 1% dextran at 37°C for 1 hour. Supernatant from each tube was extracted, lysed, and stained with optimal concentrations of CD3APC (17A2), CD19PE-Cy7 (ebio103), Gr-1BV421 (RB6-8C5), Mac-1APC-Cy7 (M1 / 70), CD45.1FITC (A20), and CD45.2PE (104). Samples were analyzed on a BD Fortessa, and donor-versus-host chimerism was identified based on CD45 allelic differences.

[0160] Ear-heart grafts. Neonatal mice were euthanized 1-2 days after birth, and their hearts were harvested. Recipient mice were prepared by making a small incision on the dorsal surface of the ear near the skull. A pouch was then created by tunneling from the incision site to the tip of the ear pinna using a trocar. The neonatal heart was delivered at the distal end of the pouch using a trocar. The tunnel was closed by gently pushing the elevated skin back into the dermis. Cardiac viability was monitored for beating by visualizing the graft through a dissecting microscope.

[0161] References Lv et al.Autoimmune hematological diseases following haploidentical donor hematopoietic stem cell Transplant compared with matched sibling and unrelated donor.Oncotarget.2017;8(16):26505-26514.

[0162] Gandy and Weissman.Tolerance of allogeneic heart grafts in mice simultaneously reconstituted with purified allogeneic hematopoietic stem cells.Transplantation.1998;65(3):295-304.

[0163] Shizuru et al.Purified hematopoietic stem cell grafts induce tolerance to alloantigens and can mediate positive and negative T cell selection.Proc Natl Acad Sci USA.2000;97(17):9555-60.

[0164] Passweg et al.Use of haploidentical stem cell transplantation continues to increase:the 2015 European Society for Blood and Marrow Transplant activity survey report.Bone Marrow Transplant.2017;52(6):811-817。

[0165] Gragert et al.HLA match likelihoods for hematopoietic stem cell grafts in the U.S.registry.N Engl J Med.2014;371(4):339-48。

[0166] Cobbold SP,Martin G,Qin S,Waldmann H.Monoclonal antibodies to promote marrow engraftment and tissue graft tolerance.Nature.1986;323(6084):164-6。

[0167] Palchaudhuri et al.Non-genotoxic conditioning for hematopoietic stem cell transplantation using a hematopoietic cell-specific internalizing immunotoxin.Nat Biotechnol.2016;34(7):738-45。

[0168] Czechowicz et al.Efficient transplantation via antibody-based clearance of hematopoietic stem cell niches.Science.2007;318(5854):1296-9。

[0169] Chhabra et al.Hematopoietic stem cell transplantation in immunocompetent hosts without radiation or chemotherapy.Sci Transl Med.2016;8(351):351ra105。

[0170] Ruggeri et al.Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants.Science.2002;295(5562):2097-100。

[0171] Durham et al.Cutting edge:administration of anti-CD40 ligand and donor bone marrow leads to hemopoietic chimerism and donor-specific tolerance without cytoreductive conditioning.J Immunol.2000;165(1):1-4。

[0172] Wekerle et al.Allogeneic bone marrow transplantation with co-stimulatory blockade induces macrochimerism and tolerance without cytoreductive host treatment.Nat Med.2000;6(4):464-9。

[0173] Markees et al.Prolonged survival of mouse skin allografts in recipients treated with donor splenocytes and antibody to CD40 ligand.Transplantation.1997;64(2):329-35。

[0174] Shizuru et al.Transplantation of purified hematopoietic stem cells:requirements for overcoming the barriers of allogeneic engraftment.Biol Blood Marrow Transplant.1996;2(1):3-14.

[0175] Negrin et al.Transplantation of highly purified CD34+Thy-1+hematopoietic stem cells in patients with metastatic breast cancer.Biol Blood Marrow Transplant.2000;6(3):262-71.

[0176] Tsao et al.Purified hematopoietic stem cell allografts reconstitute immunity superior to bone marrow.Proc Natl Acad Sci USA.2009;106(9):3288-93.

[0177] Each publication cited herein is hereby incorporated by reference in its entirety for all purposes.

[0178] It is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, animal species or genera, and reagents described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which may be limited only by the appended claims.

[0179] As used herein, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a cell" includes a plural of such cells, and a reference to "the culture" includes a reference to one or more cultures and equivalents thereof known to those skilled in the art, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs unless clearly specified otherwise.

[0180] cross reference This application claims priority to U.S. Provisional Patent Application No. 62 / 452,218, filed January 30, 2017, which is incorporated herein by reference in its entirety.

Claims

1. 1. A pharmaceutical composition for use in a method of providing engraftment of HLA-mismatched hematopoietic stem cells in an immunocompetent human subject, comprising an antibody that specifically binds to CD117 on endogenous hematopoietic stem cells, The method comprises: step (a): simultaneously contacting the subject with (i) an antibody that specifically binds to CD117 and (ii) an agent that blocks the interaction between CD47 and SIRPα and is selected from a soluble SIRPα polypeptide, an antibody specific for CD47, an antibody specific for SIRPα, and a soluble CD47 polypeptide, in amounts effective to target endogenous hematopoietic stem cells to be destroyed from the bone marrow of the subject; Step (b): introducing into said subject a cell composition comprising allogeneic exogenous hematopoietic stem cells that are mismatched at one or more MHC loci, after a washout period sufficient to reduce serum levels of (i) and (ii) in said subject to non-toxic levels; step (c): contacting the subject with an agent (iii) that induces transient immunosuppression and is selected from an antibody specific for CD40 or CD40L, mycophenolic acid, cyclosporin A, rapamycin, FK506, and a corticosteroid; and a conditioning regimen comprising: step (c) is carried out before or simultaneously with step (b); the exogenous hematopoietic stem cells engraft in the absence of myeloablative conditioning; Pharmaceutical compositions.

2. The pharmaceutical composition of claim 1 , wherein the subject is haploidentical to the exogenous hematopoietic stem cells.

3. The method comprises: contacting the subject with an antibody (iv) that depletes one or both of T cells and NK cells, selected from: (a) an antibody that depletes T cells and NK cells and is specific for one or more of CD2, CD52, CD45, or is antithymocyte globulin (ATG); (b) an antibody that selectively depletes T cells and is specific for one or more of CD3, CD4, and CD8; and (c) an antibody that selectively depletes NK cells and is specific for one or more of CD122 and CD56; The antibody (iv) is administered prior to or simultaneously with the introduction of the cell composition. The pharmaceutical composition of claim 1.

4. The cell composition comprises CD34 + 10. The pharmaceutical composition of claim 1, comprising hematopoietic stem cells selected from bone marrow, umbilical cord blood, or peripheral blood for expression.

5. The cell composition comprises at least 50% CD34 + The pharmaceutical composition of claim 1 , comprising cells.

6. The pharmaceutical composition of claim 1 , wherein the cell composition comprises hematopoietic stem cells derived in vitro from pluripotent cells.

7. The cell composition comprises at least 10 cells per kg of recipient body weight. 5 CD34 + The pharmaceutical composition of claim 1 , comprising cells.

8. The pharmaceutical composition of claim 1 , wherein the antibody (i) that specifically binds to CD117 is a bispecific antibody.

9. The pharmaceutical composition according to claim 1 , wherein the antibody (i) that specifically binds to CD117 is an antibody fragment having antigen-binding ability.

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