Patent Description:
Use of an allogeneic stem cell transplant (allo-SCT) as a therapeutic option for otherwise lethal diseases is continuously increasing. However, graft-versus-host disease (GVHD) remains a major complication of allo-SCT, affecting up to about <NUM>-<NUM>% of allo-SCT patients. It is believed that GVHD occurs when immune competent cells, namely, T-lymphocytes, recognize membrane antigens on the host cells. These membrane antigens include a set of host polypeptides such as major and minor histocompatibility antigens displayed by the human leukocyte antigen system. The polymorphism of these polypeptides is believed to trigger T-cell activation and ultimately tissue injury through a variety of cellular effector mechanisms. The activation of the donor immune cells is augmented also by cytokines released from the site of tissue injury associated with the intense conditioning regimen ("cytokine storm").

Acute GVHD (aGVHD) usually occurs in the first <NUM> days after transplantation, whereas onset of chronic GVHD (cGVHD) is observed later. Changes in the onset period of both acute and chronic GVHDs have been observed, with acute cases occurring about <NUM> days after transplantation and chronic cases noticed earlier than usual. These changes from traditional patterns of acute and chronic GVHD were observed especially in the context of reduced conditioning intensity and use of peripheral blood as a stem cell source. As used herein, the term "GVHD" encompasses both acute and chronic graft-versus-host-disease.

The goal of hematopoietic progenitor cell or stem cell transplantation (HSCT) is to achieve the successful engraftment of donor cells within a recipient host, such that immune and/or hematopoietic chimerism results. Such transplants typically are used in the treatment of disorders such as leukemia, bone marrow failure syndromes, and inherited disorders (e.g., sickle cell anemia, thalassemia, immunodeficiency disorders, and metabolic storage diseases such as mucopolysaccharidosis), as well as low-grade lymphoma. Chimerism is the reconstitution of the various compartments of the recipient's hematoimmune system with donor cell populations bearing major histocompatibility complex (MHC) molecules derived from an allogeneic or xenogeneic donor, and a cell population derived from the recipient or, alternatively, the recipient's hematoimmune system compartments which can be reconstituted with a cell population bearing MHC molecules derived from only the allogeneic or xenogeneic marrow donor. Chimerism may vary from <NUM>% (total replacement by allogenic or xenogeneic cells) to low levels detectable only by molecular methods. Chimerism levels may vary over time and be permanent or "temporary".

Donor leukocyte infusion's (DLI) have been used after allotransplant to treat relapsed or residual disease, to convert mixed to full donor chimerism, to restore full immune function as an 'add-back' after T-cell-depleted transplants and as a prophylaxis against relapse as preemptive therapy. The major complications after DLI include acute and chronic GVHD and infections associated with marrow aplasia or the use of immunosuppression. In most trials, up to about <NUM>% of evaluable recipients of DLI develop GVHD. GVHD correlates with GVT activity and response in some but not all studies.

Over the years, several methods for GVHD prophylaxis and treatment have been proposed, such as immunosuppressive medications, graft engineering, and cellular therapies. Indeed, there exist several approaches to minimizing GVHD after DLI to prevent or mitigate post-transplant immune deficiency or to induce graft-versus-malignancy (GVM) in residual or recurring disease. For example, one approach that appears to minimize GVHD involves administration of low-dose DLI followed by dose escalation. The conventional approach to DLI has been to infuse single "bulk" doses containing variable numbers of CD3+ T cells, but this is believed to be associated with significant incidences of acute and chronic GVHD and occasionally with death. On the other hand, transfusion of donor lymphocytes in multiple aliquots, starting at low cell numbers and escalating the dosage at variable intervals as required may reduce the incidence of GVHD. (see <NPL>). The assumption underlying the use of an escalating dose regimen is that the incidence of GVHD increases with the total cell dose administered. Thus, it is believed that identification of the minimal cell dose capable of inducing remission would reduce the risk for GVHD.

Alternatively, it is believed that GVHD may be reduced through depletion of CD8+ lymphocytes, which are thought to include most of the cells responsible for mediating GVHD (i.e. depletion of GVH effector cells). Outcomes suggest that graft-versus-leukemia activity can be retained with minimal GVHD. In small numbers of patients, the majority of responses have been sustained, although the overall clinical impact of this approach will require direct comparison to unmanipulated DLI.

It is also believed that GVHD may be reduced through inactivation of GVHD effector cells. Indeed, irradiated donor T-cell DLI is based on the hypothesis that the cells would induce GVM effects at the time of infusion but could not proliferate in response to allo-antigens. In addition, the use of donor T-cells expressing the herpes simplex thymidine kinase gene followed by ganciclovir treatment was studied for its effects pertaining to the modulation of alloreactivity occurring after bone marrow transplantation.

Calcineurin inhibitors and methotrexate (MTX) combination therapy has been used successfully to reduce the incidence and severity of GVHD and is the standard of care for GVHD prophylaxis. MTX, one of the earliest drugs used for GVHD prophylaxis, is believed to inhibit dihydrofolate reductase and production of thymidylate and purines, thereby suppressing T-cell response and proliferation as well as expression of adhesion molecules.

Although some of these strategies are effective in reducing the incidence of GVHD, these strategies often associate with a significant reduction in the GVM effect, thus jeopardizing the overall efficacy of HSCT. Wagner et al, relates to CRISPR mediated knockout as a novel suicide switch and selection tool for gene modified T cells. <NPL>, relates to the use of <NUM>-thioguanine for combined pre-conditioning and in vivo chemoselection to achieve reconstitution of normal hematopoiesis in HPRT-deficient bone marrow. <NPL>, relates to knockdown of HPRT for selection of genetically modified human hematopoietic progenitor cells. <NPL>, relates to the genetic modification of mouse bone marrow by lentiviral vector mediated delivery of hypoxanthine-guanine phosphoribosyltransferase short hairpin RNA and conferring chemoprotection against <NUM>-thioguanine. <CIT> relates to nucleases, and methods of using nucleases, for the modification of an HPRT locus, and for increasing the frequency of gene modification at a targeted locus. <CIT> relates to a method of radiation-free hematopoietic stem cell (HSC) transplantation comprising administering to a mammalian subject one or two doses of <NUM> to <NUM>/kg body weight of a purine base analog, such as 6TG as a pre-conditioning step. <CIT> relates to methods of selecting for modified stem cells in vivo, dosing schedules of 6TG and oral formulations comprising 6TG. <CIT> relates to potent short hairpin RNAs (shRNA734) directed to Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) that improves the rate of gene modified stem cell engraftment by a conditioning and in vivo selection strategy that is used to confer resistance to a clinically available guanine analog antimetabolite, 6TG, for efficient positive selection of gene modified stem cells. <CIT> relates to methods of developing genetically engineered immune cells for immunotherapy, which can be endowed with Chimeric Antigen Receptors targeting an antigen marker that is common to both the pathological cells and said CD38 immune cells, by the fact that the genes encoding said markers are inactivated in the immune cells by a rare cutting endonuclease such as TALEN, Cas9 or argonaute.

The present invention provides a composition comprising HPRT deficient lymphocytes for use in a method of preventing or mitigating post-transplant immune deficiency, the method comprising:.

The present invention also provides a composition comprising HPRT deficient lymphocytes for use in a method of treating a hematological cancer in a patient comprising:.

The invention is defined by the claims and any other aspects, configurations or embodiments set forth herein not falling within the scope of the claims are for information only.

Any references in the description to methods of treatment refer to the compositions of the present invention for use in said method of treatment.

In some embodiments the invention provides a composition comprising HPRT deficiency lymphocytes for use in a method of preventing or mitigating post-transplant immune deficiency, the method comprising: generating HPRT deficient lymphocytes from a donor sample; positively selecting for the HPRT deficient lymphocytes ex vivo to provide a population of modified lymphocytes; administering an HSC graft to the patient; administering the population of modified lymphocytes to the patient following the administration of the HSC graft; and optionally administering MTX if the side effects arise.

The HPRT deficient lymphocytes are generated through knockout of the HPRT gene. In some embodiments, the positive selection comprises contacting the generated HPRT deficient lymphocytes with a purine analog (e.g. <NUM>-thioguanine (6TG), <NUM>-mercaptopurine (<NUM>-MP), or azathiopurine (AZA)). In some embodiments, the positive selection comprises contacting the generated HPRT deficient lymphocytes with a purine analog and a second agent. In some embodiments, the purine analog is 6TG. In some embodiments, an amount of 6TG is between about <NUM> to about <NUM>µg/mL. In some embodiments, the HSC graft is administered to the patient following myeloablative conditioning. In some embodiments, the modified lymphocytes are administered as a single bolus. In some embodiments, the modified lymphocytes are administered as multiple doses. In some embodiments, each dose comprises between about <NUM> x <NUM><NUM> cells/kg to about <NUM> x <NUM><NUM> cells/kg. In some embodiments, a total dosage of modified lymphocytes comprises between about <NUM> x <NUM><NUM> cells/kg to about <NUM> x <NUM><NUM> cells/kg. In some embodiments, the administration of the modified lymphocytes takes place <NUM> to <NUM> days after the administration of the HSC graft. In some embodiments, the administration of the modified lymphocytes takes place <NUM> to <NUM> weeks after the administration of the HSC graft. In some embodiments, the administration of the modified lymphocytes takes place contemporaneously with the administration of the HSC graft. In some embodiments, the MTX is optionally administered upon diagnosis of GVHD. In some embodiments, an amount of MTX administered ranges from about <NUM>/m<NUM>/infusion to about <NUM>/m<NUM>/infusion. In some embodiments, the MTX is administered in titrated doses.

In another aspect of the present invention is a composition comprising HPRT deficient lymphocytes for use in a method of treating a hematological cancer in a patient in need of treatment thereof comprising: generating HPRT deficient lymphocytes from a donor sample; positively selecting for the HPRT deficient lymphocytes ex vivo to provide a population of modified lymphocytes; inducing at least a partial graft versus malignancy effect by administering an HSC graft to the patient; administering the population of modified lymphocytes to the patient following the detection of residual disease or disease recurrence; and optionally administering at least one dose of MTX to suppress at least one symptom of GVHD. In some embodiments, an amount of MTX administered ranges from about <NUM>/m<NUM>/infusion to about <NUM>/m<NUM>/infusion. In some embodiments, the MTX is administered in an amount to maintain at least some of the GVM effect.

In some embodiments, the invention provides a composition comprising HPRT deficient lymphocytes for use in a method of preventing or mitigating post-transplant immune deficiency wherein the method comprises (i) administering modified T-cells that are HPRT-deficient to the patient (such as following an HSC graft); and (ii) administering MTX to the patient upon an onset of side effects. In some embodiments, the side effects are selected from the group consisting of aGVHD or cGVHD. In some embodiments, the modified T-cells are administered in a single dose. In some embodiments, an amount of modified T-cells administered in the single dose ranges from about <NUM> x <NUM><NUM>/kg body weight to about <NUM> x <NUM><NUM>/kg body weight. In some embodiments, the modified T-cells are administered over multiple doses. In some embodiments, an amount of modified T-cells administered per dose ranges from about <NUM> x <NUM><NUM>/kg body weight to about <NUM> x <NUM><NUM>/kg body weight. In some embodiments, the MTX is administered as a single dose. In some embodiments, multiple doses of the MTX are administered. In some embodiments, an amount of MTX administered ranges from about <NUM>/m<NUM>/infusion to about <NUM>/m<NUM>/infusion. In some embodiments, the amount of MTX administered ranges from about <NUM>/m<NUM>/infusion to about <NUM>/m<NUM>/infusion.

In some embodiments, the invention provides a composition comprising HPRT deficient lymphocytes for use in a method of treating a hematological cancer in a patient, wherein a graft-versus-malignancy effect is induced in a patient following stem cell transplantation and wherein the method comprises (i) administering modified T-cells that are HPRT-deficient to the patient (such as following an HSC graft); and (ii) monitoring the patient for an onset of side effects. In some embodiments, the side effects are selected from the group consisting of aGVHD or cGVHD. In some embodiments, the method further comprises administering MTX to the patient upon onset of the side effects. In some embodiments, the modified T-cells are administered in a single dose. In some embodiments, an amount of modified T-cells administered in the single dose ranges from about <NUM> x <NUM><NUM>/kg body weight to about <NUM> x <NUM><NUM>/kg body weight. In some embodiments, the modified T-cells are administered over multiple doses. In some embodiments, an amount of modified T-cells administered per dose ranges from about <NUM> x <NUM><NUM>/kg body weight to about <NUM> x <NUM><NUM>/kg body weight. In some embodiments, the MTX is administered as a single dose. In some embodiments, multiple doses of the MTX are administered. In some embodiments, an amount of MTX administered ranges from about <NUM>/m<NUM>/infusion to about <NUM>/m<NUM>/infusion. In some embodiments, the amount of MTX administered ranges from about <NUM>/m<NUM>/infusion to about <NUM>/m<NUM>/infusion.

In some embodiments, the invention provides a composition comprising HPRT deficient lymphocytes for use in a method of treating a hematological cancer in a patient wherein the method preserves a graft versus malignancy effect while mitigating graft versus host disease in a subject by administrating to the subject a therapeutically effective amount of modified T-cells and administering MTX upon onset of GVHD. In some embodiments, the graft versus malignancy effect is a graft versus leukemia effect.

In some embodiments, the invention provides a composition comprising HPRT deficient lymphocytes for use in treating a patient with cancer who has received an allogeneic hematopoietic cell transplant, comprising administering to the patient a therapeutically effective amount of modified T-cells (e.g. those that are HPRT deficient), the modified T-cells being HPRT-deficient; monitoring for an onset of side effects resulting from the administration of the modified T-cells; and administering MTX to suppress, reduce, or control the side effect while maintaining a graft-versus malignancy reaction effective to eliminate or reduce the number of cancer cells in the patient. In some embodiments, the method further comprises administering a therapeutically effective amount of a corticosteroid. In some embodiments, the "effective amount" is an amount of that reduces or eliminates one or more undesirable symptoms associated with graft-versus-host disease (GVHD) that arises as a consequence of DLI. In some embodiments, the modified T-cells are administered as a bolus transfusion. In other embodiments, multiple administrations of the modified T-cells are provided, i.e. multiple transfusions are administered. In some embodiments, the modified T-cells are produced according to the methods described herein, such as illustrated in <FIG>. In some embodiments, a single dosage of MTX is administered. In other embodiments, the amount of MTX administered depends upon the severity of the onset of GVHD and, in that regard, the dose (or dosages) of MTX may be titrated to achieve a desired reduction in GVHD symptoms and/or a desired level of the GVM effect.

In another aspect of the present invention is a composition comprising HPRT deficient lymphocytes for use in a method of treating a hematological cancer comprising (i) administering to a patient having cancer a therapeutically effective amount of substantially purified modified T-cells, the modified T-cells being HPRT-deficient; and (ii) monitoring the patient for the presence of cancer and for the onset of GVHD. In some embodiments, a therapeutically effective amount of MTX is administered upon onset of GVHD.

Applicant have found that a gene-modified heterogeneous T-cell population can provide a more complete immunologic reconstitution for immunocompromised transplantation patients (e.g. those having severe Chron's disease, irritable bowel syndrome, or aplastic anemia). In addition, because the antigen specificity of the GVM effector cells is not completely clear, the use of the entire T-cell repertoire is believed to be the best option for obtaining a GVM effect.

Moreover, in comparison to other "off switch" methods, cells treated according to the disclosed methods do not need to express a "suicide gene. " (see, for example, <NPL>; <NPL>; and <NPL>). Rather, the disclosed method provides for knockout of an endogenous gene that causes no undesirable effects in hematological cells and, overall, superior results. Applicant submit that due to ex vivo 6TG chemoselection of gene-modified cells, there exists a very high purity of engineered cells to permit the quantitative elimination of cells in vivo via MTX dosing. In addition, the composition comprising HPRT deficient lymphocytes for use in the treatment according to the disclosed methods provides for potentially higher doses and a more aggressive therapy of donor T-cells than therapy where a "kill switch" is not incorporated. Further, the use of MTX to regulate the number of modified T-cells is clinically compatible with existing methods of treating GVHD, i.e. where MTX is used to help alleviate GVHD symptoms in patients not receiving the disclosed modified T-cells.

Finally, Applicant submits that in comparison to donor lymphocytes transduced with the herpes simplex thymidine kinase gene, the composition comprising HPRT deficient lymphocytes for use in the treatment according to the disclosed methods mitigates limitations including immunogenicity resulting in the elimination of the cells and precluding the possibility of future infusions. (see <NPL>). Also, the composition comprising HPRT deficient lymphocyte for use in the present methods allow for use of ganciclovir for concurrent clinical conditions other than GVHD without resulting in undesired clearance of HSV-tk donor lymphocytes (e.g. ganciclovir would not be precluded from being administered to control CMV infections, which are common in the allo-HSCT setting, when the currently described methods are utilized).

The present disclosure is generally directed to preventing, treating, suppressing, controlling or otherwise mitigating side effects of T-cell therapy, the T-cell therapy designed to accelerate immune reconstitution, induce a GVM effect, and/or target tumor cells.

As used herein, the singular terms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and a reference to "the protein" includes reference to one or more proteins 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 to one of ordinary skill in the art to which this disclosure belongs unless clearly indicated otherwise.

The terms "comprising," "including," "having," and the like are used interchangeably and have the same meaning. Similarly, "comprises," "includes," "has," and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of "comprising" and is therefore interpreted to be an open term meaning "at least the following," and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, "a device having components a, b, and c" means that the device includes at least components a, b and c. Similarly, the phrase: "a method involving steps a, b, and c" means that the method includes at least steps a, b, and c. Moreover, while the steps and processes may be outlined herein in a particular order, the skilled artisan will recognize that the ordering steps and processes may vary.

Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e. "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of" or "exactly one of.

As used herein, the term "administration" as it applies to a subject or patient, a placebo subject, a research subject, an experimental subject, a cell, a tissue, an organ, or a biological fluid, refers, without limitation, to contact of an exogenous ligand, reagent, placebo, small molecule, pharmaceutical agent, therapeutic agent, diagnostic agent, or composition to the subject, cell, tissue, organ, or biological fluid, and the like. "Administration" can refer, e.g., to therapeutic, pharmacokinetic, diagnostic, research, placebo, and experimental methods. Treatment of a cell encompasses contact of a reagent to the cell, as well as contact of a reagent to a fluid, where the fluid is in contact with the cell. "Administration" also encompasses in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding composition, or by another cell.

"Allogeneic T-cell" refers to a T-cell from a donor having a tissue HLA type that matches the recipient. Typically, matching is performed based on variability at three or more loci of the HLA gene, and a perfect match at these loci is preferred. In some instances, allogeneic transplant donors may be related (usually a closely HLA matched sibling), syngeneic (a monozygotic 'identical' twin of the patient) or unrelated (donor who is not related and found to have very close degree of HLA matching). The HLA genes fall in two categories (Type I and Type II). In general, mismatches of the Type-I genes (i.e. HLA-A, HLA-B, or HLA-C) increase the risk of graft rejection. A mismatch of an HLA Type II gene (i.e. HLA-DR, or HLA-DQB1) increases the risk of graft-versus-host disease.

As used herein, the terms "CAR T" or "CAR-T cells" refer to a T-cell or population thereof, which has been modified through methods to express a chimeric antigen receptor (CAR) on the T-cell surface. The CAR is a polypeptide having a pre-defined binding specificity to a desired target expressed operably connected to (e.g., as a fusion, separate chains linked by one or more disulfide bonds, etc.) the intracellular part of a T-cell activation domain.

As used herein, the terms "effective amount" or "therapeutically effective amount" encompasses, without limitation, an amount that can ameliorate, suppress, control, reverse, mitigate, prevent, or diagnose a symptom or sign of a medical condition or disorder. Unless dictated otherwise, explicitly or by context, an "effective amount" is not limited to a minimal amount sufficient to ameliorate, suppress, control, or reverse a condition.

As used herein, the terms "hematopoietic cell transplant" or "hematopoietic cell transplantation" refer to bone marrow transplantation, peripheral blood stem cell transplantation, umbilical vein blood transplantation, or any other source of pluripotent hematopoietic stem cells. Likewise, the terms the terms "stem cell transplant," or "transplant," refer to a composition comprising stem cells that are in contact with (e.g. suspended in) a pharmaceutically acceptable carrier. Such compositions are capable of being administered to a subject through a catheter.

As used herein, "HPRT" is an enzyme involved in purine metabolism encoded by the HPRT1 gene. HPRT1 is located on the X chromosome, and thus is present in single copy in males. HPRT1 encodes the transferase that catalyzes the conversion of hypoxanthine to inosine monophosphate and guanine to guanosine monophosphate by transferring the <NUM>-phosphorobosyl group from <NUM>-phosphoribosyl <NUM>-pyrophosphate to the purine. The enzyme functions primarily to salvage purines from degraded DNA for use in renewed purine synthesis (see <FIG>).

As used herein, the terms "knock down" or "knockdown" when used in reference to an effect of RNAi on gene expression, means that the level of gene expression is inhibited, or is reduced to a level below that generally observed when examined under substantially the same conditions, but in the absence of RNAi.

As used herein, the terms "knock out" or "knockout" refer to partial or complete suppression of the expression of an endogenous gene. This is generally accomplished by deleting a portion of the gene or by replacing a portion with a second sequence, but may also be caused by other modifications to the gene such as the introduction of stop codons, the mutation of critical amino acids, the removal of an intron junction, etc. Accordingly, a "knockout" construct is a nucleic acid sequence, such as a DNA construct, which, when introduced into a cell, results in suppression (partial or complete) of expression of a polypeptide or protein encoded by endogenous DNA in the cell. In some embodiments, a "knockout" includes mutations such as, a point mutation, an insertion, a deletion, a frameshift, or a missense mutation.

As used herein, the term "lentiviral vector" is used to denote any form of a nucleic acid derived from a lentivirus and used to transfer genetic material into a cell via transduction. The term encompasses lentiviral vector nucleic acids, such as DNA and RNA, encapsulated forms of these nucleic acids, and viral particles in which the viral vector nucleic acids have been packaged.

As used herein, the terms "subject," or "patient," refers to a vertebrate animal, including a mammal. A human, homo sapiens, is considered a subject or patient.

As used herein, the term "T cell receptor" or "TCR" refers to a complex of membrane proteins that participate in the activation of T-cells in response to the presentation of antigen. The TCR is believed to be responsible for recognizing antigens bound to major histocompatibility complex molecules. TCR is composed of a heterodimer of an alpha (a) and beta (β) chain, although in some cells the TCR consists of gamma and delta (γ/δ) chains. TCRs may exist in alpha/beta and gamma/delta forms, which are structurally similar but have distinct anatomical locations and functions. Each chain is composed of two extracellular domains, a variable and constant domain. In some embodiments, the TCR may be modified on any cell comprising a TCR, including, for example, a helper T ceil, a cytotoxic T-cell, a memory T cell, regulatory T-cell, natural killer T-cell, and gamma delta T-cell.

As used herein, the terms "TCR-modified T cell" or "modified TCTTCR T-cells" mean T-cells that comprise altered specificity or which lack expression of a functional TCR. In some embodiments, the TCR-modified T-cells are modified such that they possess enhanced tumor-killing activity, i.e. they are modified such that they efficiently recognize antigen-bearing tumor cells.

As used herein, the term "titration" refers to the continual adjustment of a dose based on patient response. For example, dosages may be adjusted until a desired clinical effect is observed or achieved.

As used herein, the terms "transduce" or "transduction" refers to the delivery of a gene(s) using a viral or retroviral vector by means of infection rather than by transfection. For example, an anti-HIV gene carried by a retroviral vector (a modified retrovirus used as a vector for introduction of nucleic acid into cells) can be transduced into a cell through infection and provirus integration. Thus, a "transduced gene" is a gene that has been introduced into the cell via lentiviral or vector infection and provirus integration. Viral vectors (e.g., "transducing vectors") transduce genes into "target cells" or host cells.

As used herein, the terms "treatment," "treating," or "treat," with respect to a specific condition, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or can be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. "Treatment," as used herein, covers any treatment of a disease in a subject, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease and/or relieving one or more disease symptoms. "Treatment" can also encompass delivery of an agent or administration of a therapy in order to provide for a pharmacologic effect, even in the absence of a disease or condition. The term "treatment" is used in some embodiments to refer to administration of a compound of the present disclosure to mitigate a disease or a disorder in a host, preferably in a mammalian subject, more preferably in humans. Thus, the term "treatment" can include: preventing a disorder from occurring in a host, particularly when the host is predisposed to acquiring the disease but has not yet been diagnosed with the disease; inhibiting the disorder; and/or alleviating or reversing the disorder. Insofar as the methods of the present disclosure are directed to preventing disorders, it is understood that the term "prevent" does not require that the disease state be completely thwarted. Rather, as used herein, the term preventing refers to the ability of the skilled artisan to identify a population that is susceptible to disorders, such that administration of the compounds of the present disclosure can occur prior to onset of a disease. The term does not mean that the disease state must be completely avoided.

As used herein, the term "vector" refers to a nucleic acid molecule capable of mediating entry of, e.g., transferring, transporting, etc., another nucleic acid molecule into a cell. The transferred nucleic acid is generally linked to, e.g., inserted into, the vector nucleic acid molecule. A vector may include sequences that direct autonomous replication or may include sequences sufficient to allow integration into host cell DNA. As will be evident to one of ordinary skill in the art, viral vectors may include various viral components in addition to nucleic acid(s) that mediate entry of the transferred nucleic acid. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viral vectors. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors (including lentiviral vectors), and the like.

The present invention involves a method of producing HPRT-deficient T-cells (also referred to herein as "modified T-cells"). With reference to <FIG>, cells, namely lymphocytes (T-cells), are first collected from a donor (step <NUM>). In embodiments where hematopoietic stem cells (HSC) are also collected from a donor, the T-cells may be collected from the same donor from which the HSC graft is collected or from a different donor. In these embodiments, the cells may be collected at the same time or at a different time as the cells for the HSC graft. In some embodiments, the cells are collected from the same mobilized peripheral blood HSC harvest. In some embodiments, this could be a CD34-negative fraction (CD34-positive cells collected as per standard of care for donor graft), or a portion of the CD34-positive HSC graft if a progenitor T-cell graft is envisaged.

The skilled artisan will appreciate that the cells may be collected by any means. For example, the cells may be collected by apheresis, leukapheresis, or merely through a simple venous blood draw. In embodiments where the HSC graft is collected contemporaneously with the cells for modification, the HSC graft is cryopreserved so as to allow time for manipulation and testing of the T-cells collected.

Following collection of the cells, T-cells are isolated (step <NUM>). The T-cells may be isolated from the aggregate of cells collected by any means known to those of ordinary skill in the art. For example, CD3+ cells may be isolated from the collected cells via CD3 microbeads and the MACS separation system (Miltenyi Biotec). It is believed that the CD3 marker is expressed on all T-cells and is associated with the T-cell receptor. It is believed that about <NUM> to about <NUM>% of human peripheral blood lymphocytes and about <NUM>-<NUM>% of thymocytes are CD3+. In some embodiments, the CD3+ cells are magnetically labeled with CD3 MicroBeads. Then the cell suspension is loaded onto a MACS Column which is placed in the magnetic field of a MACS Separator. The magnetically labeled CD3+ cells are retained on the column. The unlabeled cells run through and this cell fraction is depleted of CD3+ cells. After removal of the column from the magnetic field, the magnetically retained CD3+ cells can be eluted as the positively selected cell fraction.

Alternatively, CD62L+ T-cells may be isolated from the collected cells is via an IBA life sciences CD62L Fab Streptamer Isolation Kit. Isolation of human CD62L+ T-cells is performed by positive selection. PBMCs are labeled with magnetic CD62L Fab Streptamers. Labeled cells are isolated in a strong magnet where they migrate toward the tube wall on the side of the magnet. This CD62L positive cell fraction is collected and cells are liberated from all labeling reagents by addition of biotin in a strong magnet. The magnetic Streptamers migrate toward the tube wall and the label-free cells remain in the supernatant. Biotin is removed by washing. The resulting cell preparation is highly enriched with CD62L+ T-cells with a purity of more than <NUM>%. No depletion steps and no columns are needed.

In alternative embodiments, T-cells are not isolated at step <NUM>, but rather the aggregate of cells collected at step <NUM> are used for subsequent modification. While in some embodiments the aggregate of cells may be used for subsequent modification, in some instances the method of modification may be specific for a particular cell population within the total aggregate of cells. This could be done in a number of ways; for example, targeting genetic modification to a particular cell type by targeting gene vector delivery.

Following isolation of the T-cells, the T-cells are treated to decrease HPRT activity (step <NUM>), i.e. to crease expression of the HPRT gene. The T-cells may be modified according to several methods.

In some embodiments, a gene editing approach may be used to knockout HPRT. For example, isolated cells may be treated with a HPRT-targeted CRISPR/Cas9 RNP. In some embodiments, the HPRT-targeted CRISP/Cas9 RNP may be formulated within a nanocapsule. <NPL>), describe various gene editing techniques, including CRISPR/Cas9 nuclease mediated methods. Gene editing tools may also be delivered via any method known to those of ordinary skill in the art including by way of AAV vectors, non-integrating and non-reverse transcribed lentiviral vectors, and other physical delivery methods (e.g. electroporation, cell squeezing, sonoporation, etc.). Transfection methods including calcium phosphate, lipofectamine, fugene, dendrimers, liposomes (usually cationic liposomes), and other cationic polymers (e.g., DEAE or PEI) may also be utilized. There also other exist other particle-based methods including nanoparticle delivery systems, which may be biologically or chemically functionalized to increase delivery, or may be used in physical methods of delivery, e.g., magnofection, or particle bombardment.

In some embodiments, electroporation is used to introduce nucleic acids into eukaryotic cells, such as by opening transient pores in the cell member to allow the uptake of material. Electroporation is a method whereby DNA (or RNA) is introduced into cells by passing an electric current across the cell membrane.

Other gene editing techniques using certain nucleases are described in <CIT> and <CIT>. In some embodiments, a zinc-finger protein (ZFP) that binds to a target site in an HPRT gene in a genome may be utilized, wherein the ZFP comprises one or more engineered zinc-finger binding domains. In some embodiments, ZFPs are used as a pair of zinc-finger nucleases (ZFNs) that dimerize and then cleave a target genomic region of interest, wherein the ZFNs comprise one or more engineered zinc-finger binding domains and a nuclease cleavage domain or cleavage half-domain. In some embodiments, a TALE protein (Transcription activator like effector) that binds to target site in an HPRT gene in a genome may be utilized, wherein the TALE comprises one or more engineered TALE DNA binding domains. In some embodiments, the TALE is a nuclease (TALEN) that cleaves a target genomic region of interest, wherein the TALEN comprises one or more engineered TALE DNA binding domains and a nuclease cleavage domain or cleavage half-domain. Cleavage domains and cleavage half domains of ZFNs and/or TALENs can be obtained, for example, from various restriction endonucleases and/or homing endonucleases. In some embodiments, the cleavage half-domains are derived from a Type IIS restriction endonuclease (e.g., Fok I).

After the T-cells are modified at step <NUM>, the population of HPRT-deficient T-cells is selected for and/or expanded (step <NUM>). In some embodiments, the culture may concurrently select for and expand cells with enhanced capacity for engraftment (e.g. central memory or T stem cell phenotype). In some embodiments, the culture period is less than <NUM> days. In some embodiments, the culture period is less than <NUM> days.

In some embodiments, the step of selecting for and expanding cells comprises treating the population of HPRT-deficient T-cells ex vivo with a guanosine analog antimetabolite (such as <NUM>-thioguanine (6TG), <NUM>-mercaptopurine (<NUM>-MP), or azathiopurine (AZA). In some embodiments, the T-cells are cultured in the presence of <NUM>-thioguanine ("6TG"), thus killing cells which have not been modified at step <NUM>. 6TG is a guanine analog that can interfere with dGTP biosynthesis in the cell. Thio-dG can be incorporated into DNA during replication in place of guanine, and when incorporated, often becomes methylated. This methylation can interfere with proper mis-match DNA repair and can result in cell cycle arrest, and/or initiate apoptosis. 6TG has been used clinically to treat patients with certain types of malignancies due to its toxicity to rapidly dividing cells. In the presence of 6TG, HPRT is the enzyme responsible for the integration of 6TG into DNA and RNA in the cell, resulting in blockage of proper polynucleotide synthesis and metabolism (see <FIG>). On the other hand, the salvage pathway is blocked in HPRT-deficient cells (see <FIG>). Cells thus use the de novo pathway for purine synthesis (see <FIG>). However, in HPRT wild type cells, cells use the salvage pathway and 6TG is converted to 6TGMP in the presence of HPRT. 6TGMP is converted by phosphorylation to thioguanine diphosphate (TGDP) and thioguanine triphosphate (TGTP). Simultaneously deoxyribosyl analogs are formed, via the enzyme ribonucleotide reductase. Given that 6TG is highly cytotoxic, it can be used as a selection agent to kill cells with a functional HPRT enzyme.

The generated HPRT-deficient cells are then contacted with a purine analog ex vivo.

For the knockout approach, it is believed that HPRT may be totally eliminated or near totally eliminated from HPRT-knockout cells and the generated HPRT-deficient cells will be highly tolerant to purine analogs. The concentration of purine analogs used for ex vivo selection in this case ranges from about <NUM> to about <NUM>. In some embodiments, the concentration ranges from about <NUM> to about <NUM>. In other embodiments, the concentration ranges from about <NUM> to about <NUM>. In yet other embodiments, the concentration ranges from about <NUM> to about <NUM>.

In other embodiments, modification of the cells (e.g. through knockout of HPRT) may be efficient enough such that ex vivo selection for the HPRT-deficient cells is not necessary, i.e. selection with 6TG or other like compound is not required.

In some embodiments, the generated HPRT-deficient cells are contacted with both a purine analog and with allopurinol , which is an inhibitor of xanthine oxidase (XO). By inhibiting XO, more available 6TG to be metabolized by HPRT. When 6TG is metabolized by HPRT it forms 6TGNs which are the toxic metabolites to the cells (6TGN encompasses <NUM>-TG monophosphate (6TGMP), diphosphate (<NUM>-TGDP) and triphosphate (6TGTP)). (see <FIG>). (see, for example, <NPL>; <NPL>; <NPL>; and <NPL>).

In some embodiments, allopurinol is introduced to the generated HPRT-deficient cells prior to introduction of the purine along. In other embodiments, allopurinol is introduced to the generated HPRT-deficient cells simultaneously with the introduction of the purine along. In yet other embodiments, allopurinol is introduced to the generated HPRT-deficient cells following the introduction of the purine along.

Following selection and expansion, the modified T-cell product is tested. In some embodiments, the modified T-cell product is tested according to standard release testing (e.g. activity, mycoplasma, viability, stability, phenotype, etc.; see <NPL>).

In other embodiments, the modified T-cell product is tested for sensitivity to MTX or mycophenolic acid (MPA). Both MTX and MPA inhibit de novo synthesis of purines but have different mechanisms of action. It is believed that MTX competitively inhibits dihydrofolate reductase (DHFR), an enzyme that participates in tetrahydrofolate (THF) synthesis. DHFR catalyzes the conversion of dihydrofolate to active tetrahydrofolate. Folic acid is needed for the de novo synthesis of the nucleoside thymidine, required for DNA synthesis. Also, folate is essential for purine and pyrimidine base biosynthesis, so synthesis will be inhibited. Mycophenolic acid (MPA) is potent, reversible, non-competitive inhibitor of inosine-<NUM>'-monophosphate dehydrogenase (IMPDH), an enzyme essential to the de novo synthesis of guanosine-<NUM>'-monophosphate(GMP) from inosine-<NUM>'-monophosphate (IMP).

MTX or MPA, therefore inhibits the synthesis of DNA, RNA, thymidylates, and proteins. MTX or MPA blocks the de novo pathway by inhibiting DHFR. In HPRT-/- cell, there is no salvage or de novo pathway functional, leading to no purine synthesis, and therefore the cells die. However, the HPRT wild type cells have a functional salvage pathway, their purine synthesis takes place and the cells survive. In some embodiments, the modified T-cells are HPRT-deficient. In some embodiments, at least <NUM>% of the modified T-cells population is sensitive to MTX or MPA. In other embodiments, at least <NUM>% of the modified T-cells population is sensitive to MTX or MPA. In yet other embodiments, at least <NUM>% of the modified T-cells population is sensitive to MTX or MPA.

Given the sensitivity of the modified T-cells produced according to steps <NUM> through <NUM> to MTX or MPA, MTX or MPA may be used to selectively eliminate HPRT-deficient cells, as described herein.

The composition comprising the HPRT deficient lymphocytes for use in a method of preventing or mitigating post-transplant immune deficiency as defined in claim <NUM>, or for use in a method of treating a hematological cancer in a patient, as defined in claim <NUM> (i.e. the modified T-cells prepared according to steps <NUM> to <NUM>) are administered to a patient (step <NUM>). In some embodiments, the modified T-cells are provided to the patient in a single administration (e.g. a single bolus, or administration over a set time period, for example and infusion over about <NUM> to <NUM> hours or more). In other embodiments, multiple administrations of the modified T-cells are made. If multiple doses of the modified T-cells are administered, each dose may be the same or different (e.g. escalating doses, decreasing doses).

In some embodiments, an amount of the dose of modified T-cells is determined based on the CD3-positive T-cell content/kg of the subject's body weight. In some embodiments, the total dose of modified T-cells ranges from about <NUM> x <NUM><NUM>/kg body weight to about <NUM> x <NUM><NUM>/kg body weight. In other embodiments, the total dose of modified T-cells ranges from about <NUM> x <NUM><NUM>/kg body weight to about <NUM> x <NUM><NUM>/kg body weight. In yet other embodiments, the total dose of modified T-cells ranges from about <NUM> x <NUM><NUM>/kg body weight to about <NUM> x <NUM><NUM>/kg body weight. In further embodiments, the total dose of modified T-cells ranges from about <NUM> x <NUM><NUM>/kg body weight to about <NUM> x <NUM><NUM>/kg body weight. In yet further embodiments, the total dose of modified T-cells ranges from about <NUM> x <NUM><NUM>/kg body weight to about <NUM> x <NUM><NUM>/kg body weight.

Where multiple doses are provided, the frequency of dosing may range from about <NUM> week to about <NUM> weeks. Likewise, where multiple doses are provided, each dose of modified T-cells ranges from about <NUM> x <NUM><NUM>/kg body weight to about <NUM> x <NUM><NUM>/kg body weight. In other embodiments, each dose of modified T-cells ranges from about <NUM> x <NUM><NUM>/kg body weight to about <NUM> x <NUM><NUM>/kg body weight. In other embodiments, each dose of modified T-cells ranges from about <NUM> x <NUM><NUM>/kg body weight to about <NUM> x <NUM><NUM>/kg body weight. In other embodiments, each dose of modified T-cells ranges from about <NUM> x <NUM><NUM>/kg body weight to about <NUM> x <NUM><NUM>/kg body weight. In other embodiments, each dose of modified T-cells ranges from about <NUM> x <NUM><NUM>/kg body weight to about <NUM> x <NUM><NUM>/kg body weight. Other dosing strategies are described by <NPL>).

The modified T-cells may be administered alone or as part of an overall treatment strategy. In some embodiments, the modified T-cells are administered following an HSC transplant, such as about <NUM> to about <NUM> weeks after the HSC transplant. For example, in some embodiments, the modified T-cells are administered after administration of a HSC transplant to help prevent or mitigate post-transplant immune deficiency. It is believed that the modified T-cells may provide a short term (e.g. about <NUM> to about <NUM> month) immune reconstitution and/or protection. As another example, and in other embodiments, the modified T-cells are administrated as part of cancer therapy to help induce a graft-versus-malignancy (GVM) effect or a graft-versus-tumor (GVT) effect. Administration of the modified T-cells according to each of these treatment avenues are described in more detail herein. Of course, the skilled artisan will appreciate that other treatments for any underlying condition may occur prior to, subsequent to, or concurrently with administration of the modified T-cells.

Administration of T-cells to a patient may result in unwanted side effects, including those recited herein. For example, graft-versus-host disease may occur after a patient is treated with T-cells, including modified T-cells (e.g. via knockdown or knockout of HPRT). In some aspects of the present disclosure, following administration of the modified T-cells at step <NUM>, the patient is monitored for the onset of any side effects, including, but not limited to, GVHD. Should any side effects arise, such as GVHD (or symptoms of GVHD), MTX or MPA is administered to the patient (in vivo) at step <NUM> to remove at least a portion of the modified T-cells in an effort to suppress, reduce, control, or otherwise mitigate side effects, e.g. GVHD. In some embodiments, MTX or MPA is administered in a single dose. In other embodiments, multiple does of MTX and/or MPA are administered.

It is believed that the modified T-cells of the present disclosure (once selected for ex vivo and administered to the patient or mammalian subject), may serve as a modulatable "on" / "off" switch given their sensitivity to MTX (or MPA). The modulatable switch allows for regulation of immune system reconstitution by selectively killing at least a portion of the modified T-cells in vivo through the administration of MTX to the patient should any side effects occur. This modulatable switch may be further regulated by administering further modified T-cells to the patient following MTX administration to allow further immune system reconstitution after side effects have been reduced or otherwise mitigated. Likewise, the modulatable switch allows for regulation of a graft-versus-malignancy effect by selectively killing at least a portion of the modified T-cells in vivo through the administration of MTX should any side effects occur. Again, the GVM effect may be fine-tuned by subsequently dosing further aliquots of modified T-cells to the patient once side effects are reduced or otherwise mitigated. The person of ordinary skill in the art will appreciate that any medical professional overseeing treatment of a patient can balance immune system reconstitution and/or the GVM effect while keeping side effects at bay or within tolerable or acceptable ranges. By virtue of the above, patient treatment may be enhanced while mitigating adverse effects.

In some embodiments, an amount of MTX administered ranges from about <NUM>/m<NUM>/infusion to about <NUM>/m<NUM>/infusion. In some embodiments, an amount of MTX administered ranges from about <NUM>/m<NUM>/infusion to about <NUM>/m<NUM>/infusion. In some embodiments, an amount of MTX administered ranges from about <NUM>/m<NUM>/infusion to about <NUM>/m<NUM>/infusion. In some embodiments, an amount of MTX administered ranges from about <NUM>/m<NUM>/infusion to about <NUM>/m<NUM>/infusion. In some embodiments, an amount of MTX administered ranges from about <NUM>/m<NUM>/infusion to about <NUM>/m<NUM>/infusion. In some embodiments, an amount of MTX administered ranges from about <NUM>/m<NUM>/infusion to about <NUM>/m<NUM>/infusion. In some embodiments, an amount of MTX administered ranges from about <NUM>/m<NUM>/infusion to about <NUM>/m<NUM>/infusion. In some embodiments, an amount of MTX administered ranges from about <NUM>/m<NUM>/infusion to about <NUM>/m<NUM>/infusion. In some embodiments, an amount of MTX administered ranges from about <NUM>/m<NUM>/infusion to about <NUM>/m<NUM>/infusion. In some embodiments, an amount of MTX administered ranges from about <NUM>/m<NUM>/infusion to about <NUM>/m<NUM>/infusion. In some embodiments, an amount of MTX administered ranges from about <NUM>/m<NUM>/infusion to about <NUM>/m<NUM>/infusion. In other embodiments, an amount of MTX administered ranges from about <NUM>/m<NUM>/infusion to about <NUM>/m<NUM>/infusion. In yet other embodiments, an amount of MTX administered is about <NUM>/m<NUM>/infusion. In yet further embodiments, an amount of MTX administered is about <NUM>/m<NUM>/infusion.

In some embodiments, between <NUM> and <NUM> infusions are made, and the infusions may each comprise the same dosage or different dosages (e.g. escalating dosages, decreasing dosages, etc.). In some embodiments, the administrations may be made on a weekly basis, or a bi-monthly basis.

In yet other embodiments, the amount of MTX administered is titrated such that uncontrolled side effects, e.g. GVHD, is resolved, while preserving at least some modified T-cells and their concomitant effects on reconstituting the immune system, targeting cancer, or inducing the GVM effect. In this regard, it is believed that at least some of the benefit of the modified T-cells may still be recognized while ameliorating side effects, e.g. GVHD. In some embodiments, additional modified T-cells are administered following treatment with MTX, i.e. following resolution, suppression, or control of the side effects, e.g. GVHD.

In some embodiments, the subject receives doses of MTX prior to administration of the modified T-cells, such as to control or prevent side effects after HSC transplantation. In some embodiments, existing treatment with MTX is halted prior to administration of the modified T-cells, and then resumed, at the same or different dosage (and using a same or different dosing schedule), upon onset of side effects following treatment with the modified T-cells. In this regard, the skilled artisan can administer MTX on an as-need basis and consistent with the standards of care known in the medical industry.

In some embodiments, an alternative agent may be used in place of either MTX or MPA, including, but not limited to ribavarin (IMPDH inhibitor); VX-<NUM> (IMPDH inhibitor) (see <NPL>); lometrexol (DDATHF, LY249543) (GAR and/or AICAR inhibitor); thiophene analog (LY254155) (GAR and/or AICAR inhibitor), furan analog (LY222306) (GAR and/or AICAR inhibitor) (see <NPL>); DACTHF (GAR and/or AICAR inhibitor) (see <NPL>); AG2034 (GAR and/or AICAR inhibitor) (see <NPL>); LY309887 (GAR and/or AICAR inhibitor) ((<NUM>)-<NUM>-[[<NUM>-[<NUM>-[(6R)-<NUM>-amino-<NUM>-oxo-<NUM>,<NUM>,<NUM>,<NUM>-tetrahydro-<NUM>-pyrido[<NUM>,<NUM>-d]pyrimidin-<NUM>-yl]ethyl]thiophene-<NUM>-carbonyl]amino]pentanedioic acid); alimta (LY231514) (GAR and/or AICAR inhibitor) (see <NPL>); dmAMT (GAR and/or AICAR inhibitor), AG2009 (GAR and/or AICAR inhibitor); forodesine (Immucillin H, BCX-<NUM>; trade names Mundesine and Fodosine) (inhibitor of purine nucleoside phosphorylase [PNP]) (see <NPL>); and immucillin-G (inhibitor of purine nucleoside phosphorylase [PNP]).

Although hematopoietic stem cell transplantation from human leukocyte antigen (HLA) matched siblings has become a standard treatment modality for many hematological diseases (malignant and non-malignant), allogeneic HSC transplantation (allo-HSCT) remains the only proven curative therapy for chronic myeloid leukemia. The pluripotent hematopoietic stem cells required for this procedure are usually obtained from the bone marrow or peripheral blood of a related or unrelated donor. Historically, the best results of allogeneic HCT have been obtained when the stem cell donor is a HLA-matched sibling. However, any given sibling pair has only about a <NUM>% chance of inheriting the same HLA haplotypes from their parents. This means that only about <NUM>% of patients will have such a match. Consequently, attention has focused on other sources of stem cells. For patients who lack an HLA-matched sibling, alternative sources of donor grafts include suitably HLA-matched adult unrelated donors, umbilical cord blood stem cells, and partially HLA-mismatched, or HLA-haploidentical, related donors. The decision of which donor source to utilize depends, to a large degree, upon the clinical situation and the approaches employed at the individual transplant center. However, it is believed that almost all patients have at least one HLA-haploidentical mismatched family member (parent, child or sibling), who is immediately available as donor.

The major challenge of HLA-haploidentical HSCT is intense bi-directional alloreactivity leading to high incidences of graft rejection and GVHD. Advances in graft engineering and in pharmacologic prophylaxis of GVHD have reduced the risks of graft failure and GVHD after HLA-haploidentical HCT, and have made this stem cell source a viable alternative for patients lacking an HLA-matched sibling. However, it is believed that both of these approaches may lead to periods of post-transplant immunodeficiency rendering the recipient susceptible to infection, which is the primary cause of mortality not related to graft failure. It is believed that donor lymphocytes can play a central therapeutic role in the induction of immune reconstitution, especially in the subset of T-cell depleted matched transplants and in the context of partially mismatched transplants. Indeed, it is believed that DLI may be used after stem cell transplantation to prevent or mitigate infections and to establish full donor chimerism. The addition of mature T-cells which exhibit a broad repertoire of T-cell immunity against viral, fungal and other opportunistic infections might provide a clinical benefit (see, for example, <NPL>; and <NPL>).

As noted herein, GVHD may occur after a patient is treated with a stem cell transplant. To combat this, the present invention provides a composition comprising HPRT deficient lymphocytes for use in a method of preventing or mitigating post-transplant immune deficiency and a pharmacological approach to reducing, suppressing or controlling GVHD should it arise. It is believed that the disclosed approach integrates with the practice of HLA-haploidentical HSCT described above. In some embodiments, the method utilizes an infusion of HPRT-deficient modified T-cells in patients post-allogenic HSCT to accelerate immune reconstitution and provide at least some immunity for the host while concomitantly being able to suppress or control GVHD via dosing with MTX.

<FIG> illustrates one method of reducing, suppressing, or controlling GVHD upon onset of symptoms. Initially, cells are collected from a donor at step <NUM>. The cells may be collected from the same donor that provided the HSC for grafting (see step <NUM>) or from a different donor. Lymphocytes are then isolated from the collected cells (step <NUM>) and treated such that they become HPRT-deficient (step <NUM>). Methods of treating the isolated cells are set forth herein. To arrive at a population of modified T-cells that are HPRT deficient, the treated cells are positively selected for and expanded (step <NUM>), such as described herein. The modified T-cells are then stored for later use.

Prior to receiving the HSC graft (step <NUM>), patients are treated with myeloablative conditioning as per the standard of care (step <NUM>) (e.g. high-dose conditioning radiation, chemotherapy, and/or treatment with a purine analog; or low-dose conditioning radiation, chemotherapy, and/or treatment with a purine analog).

In some embodiments, the patient is treated with the HSC graft (step <NUM>) between about <NUM> and about <NUM> hours following treatment with the conditioning regimen. In other embodiments, the patient is treated with the HSC graft between about <NUM> and about <NUM> hours following treatment with the conditioning regimen. In yet other embodiments, the patient is treated with the HSC graft between about <NUM> and about <NUM> hours following treatment with the conditioning regimen. In some embodiments, the HSC graft comprises a minimum of <NUM> x <NUM><NUM> CD34+ cells/kg, with a target of greater than <NUM> x <NUM><NUM> CD34+ cells/kg.

Following HSC grafting, the modified T-cells from step <NUM> are administered to the patient according to standard transfusion protocols (step <NUM>). In some embodiments, the modified T-cells are administered between about <NUM> to about <NUM> weeks after the HSC graft. In other embodiments, the modified T-cells are administered between about <NUM> to about <NUM> weeks after the HSC graft. In yet other embodiments, the modified T-cells are administered between about <NUM> to about <NUM> weeks after the HSC graft. In some embodiments, the modified T-cells are administered between about <NUM> day and about <NUM> days after the HSC graft. In some embodiments, the modified T-cells are administered between about <NUM> day and about <NUM> days after the HSC graft. In some embodiments, the modified T-cells are administered between about <NUM> day and about <NUM> days after the HSC graft. In some embodiments, the modified T-cells are administered between about <NUM> days and about <NUM> days after the HSC graft. In some embodiments, the modified T-cells are administered contemporaneously with the HSC graft or within a few hours of the HSC graft (e.g. <NUM>, <NUM>, <NUM>, or <NUM> hours after the HSC graft).

The modified T-cells may be transfused in a single administration. Alternatively, the modified T-cells may be transfused over a course of multiple administrations. In embodiments where multiple administrations of modified T-cells are made, the same or different amounts of modified T-cells may be transfused at each administration, such as described herein.

Following administration of the modified T-cells, the patient is monitored for the onset of GVHD. Should symptoms arise, MTX may be administered (step <NUM>) to reverse, suppress, or control GVHD. MTX may be administered in a single dose or in multiple doses. If multiple MTX administrations are made, the dosage may be titrated so as to balance GVHD while maintaining some of the protections afforded to the immune system by the modified T-cells.

In some embodiments, between <NUM> and <NUM> infusions are made, and the infusions may each comprise the same dosage or different dosages (e.g. escalating dosages, decreasing dosages, etc.). In other embodiments, between <NUM> and <NUM> infusions are made. In some embodiments, the administrations may be made on a weekly basis, or a bi-monthly basis.

Treatment of hematological malignancies, including leukemia, lymphoma and myeloma, usually involves one or more forms of chemotherapy and/or radiation therapy. These treatments destroy the malignant cells, but also destroy the body's healthy blood cells as well. Allogeneic bone marrow transplantation (BMT) is an effective therapy useful in the treatment of many hematologic malignancies. In allogeneic BMT, bone marrow (or, in some cases, peripheral blood) from an unrelated or a related (but not identical twin) donor is used to replace the healthy blood cells in the cancer patient. The bone marrow (or peripheral blood) contains stem cells, which are the precursors to all the different cell types (e.g., red cells, phagocytes, platelets and lymphocytes) found in blood. It is believed that allogeneic BMT has both a restorative effect and a curative effect. The restorative effect arises from the ability of the stem cells to repopulate the cellular components of blood. The curative properties of allogeneic BMT derive largely from a graft-versus-malignancy (GVM) effect (also referred to as a graft-versus-tumor effect (GVT)). The hematopoietic cells from the donor (specifically, the T lymphocytes) are believed to attack the cancerous cells, enhancing the suppressive effects of the other forms of treatment. Essentially, the GVM effect comprises an attack on the residual tumor cells by the blood cells derived from the BMT, making it less likely that the malignancy will return after transplant.

The efficacy of allogeneic hematopoietic stem cell transplantation for hematologic malignancies is limited by the difficulty in suppressing graft-versus-host disease without compromising graft-versus-malignancy effects. DLI has been used after allotransplant to treat relapsed or residual disease, to convert mixed to full donor chimerism, to restore full immune function as an 'add-back' after T-cell-depleted transplants and to prophylax against relapse as preemptive therapy. Indeed, donor lymphocyte infusion has provided a dramatic example of the potency of GVM, which can induce complete and sustained remissions in many patients even when all cytotoxic therapy has failed. While DLI can be a much safer alternative option than second allogeneic HSCT, GVHD is a common complication resulting in significant morbidity and mortality (see <NPL>; <NPL>). Unfortunately, and as noted herein, acute GVHD has contributed to death in almost <NUM>% of patients. Indeed, in some cases DLI-induced GVHD may be quite severe and, it is believed that between about <NUM>% to about <NUM>% of DLI recipients may develop grade III to IV acute GVHD. As such, it can be said that controlling the GVM effect prevents escalation of the GVM effect into GVHD. Therefore, managing the threat of GVHD while maximizing the beneficial GVM effect would broaden the scope and usefulness of allogenic BMT procedures.

As noted herein, conventional methods of reducing GVHD comprise controlling the number T-cells administered during donor lymphocyte infusion. This method, however, may not only result in a decreased GVM effect and a slower immune recovery, but may also cause increased rates of graft rejection. To combat this, in some aspects of the present invention, are compositions comprising HPRT deficient lymphocytes for use in a method of treating a hematological cancer by stimulating or encouraging a GVM effect by administering lymphocytes to the patient that have been modified so as to be at least partially HPRT-deficient, and then monitoring for the onset of GVHD. At the onset of GVHD, one or more therapeutically effective doses of MTX may be administered to suppress, reduce, control, or otherwise mitigate GVHD. In some embodiments, a single dosage of MTX is administered. In other embodiments, the amount of MTX administered depends upon the severity of the onset of GVHD and, in that regard, the dose (or dosages) of MTX may be titrated to achieve a desired reduction in GVHD symptoms (again, with the intent to balance any GVM effect). In some embodiments, the GVM is a graft-versus-leukemia effect (GVL). In some embodiments, the modified T-cells are provided during a single administration. In other embodiments, multiple administrations of the modified T-cells are provided. In some embodiments, the modified T-cells are produced according to the methods (steps <NUM> through <NUM>) described herein, such as illustrated in <FIG>.

<FIG> illustrates one method of reducing, suppressing, or controlling GVHD upon onset of symptoms. Initially, cells are collected from a donor at step <NUM>. The cells may be collected from the same donor that provided the HSC for grafting (see step <NUM>) or from a different donor. Lymphocytes are then isolated from the collected cells (step <NUM>) and treated such that they become HPRT-deficient (step <NUM>). Methods of treating the isolated cells are set forth herein. To arrive at a population of modified T-cells that are HPRT deficient, the treated cells are selected for and expanded (step <NUM>), such as described herein. The modified T-cells are then stored for later use.

A patient having a hematological cancer, may be treated according to the standard of care available to the patient at the time of presentation and staging of the cancer (e.g. radiation and/or chemotherapy, including biologics) (step <NUM>). The patient may also be a candidate for HSC transplantation and, if so, a conditioning regimen (step <NUM>) is implemented (e.g. by high-dose conditioning radiation or chemotherapy). It is believed that for malignancy, one wishes to "wipe out" the blood system completely, or as close to completely as possible, thus to killing off as many malignant cells as possible. The goals of such a conditioning regimen being to treat the cancer cells intensively, thereby making a cancer recurrence less likely, inactivate the immune system to reduce the chance of a stem cell graft rejection, and enable donor cells to travel to the marrow. In some embodiments, conditioning includes administration of one or more of cyclophosphamide, cytarabine (AraC), etoposide, melphalan, busulfan, or high-dose total body irradiation. The patient is then treated with an allogenic HSC graft (step <NUM>). In some embodiments, the allogenic HSC graft induces at least a partial GVM, GVT, or GVL effect.

Following grafting, the patient is monitored (step <NUM>) for residual or recurrent disease. Should such residual or recurrent disease present itself, the modified T-cells (produce at step <NUM>) are administered to the patient (step <NUM>) such that a GVM, GVT, or GVT effect may be induced. The modified T-cells may be infused in a single administration of over a course of several administrations. In some embodiments, the modified T-cells are administered between about <NUM> day and about <NUM> days after the HSC graft. In some embodiments, the modified T-cells are administered between about <NUM> day and about <NUM> days after the HSC graft. In some embodiments, the modified T-cells are administered between about <NUM> day and about <NUM> days after the HSC graft. In some embodiments, the modified T-cells are administered between about <NUM> days and about <NUM> days after the HSC graft. In some embodiments, the modified T-cells are administered contemporaneously with the HSC graft or within a few hours of the HSC graft (e.g. <NUM>, <NUM>, <NUM>, or <NUM> hours after the HSC graft).

Should symptoms of GVHD arise, MTX is administered to the patient, either in a single dose or over multiple doses. In some embodiments, the amount of MTX administered depends upon the severity of the onset of GVHD and, in that regard, the dose (or dosages) of MTX may be titrated to achieve a desired reduction in GVHD symptoms and/or a desired level of the GVM, GVT, or GVL effect.

CAR-T cells are produced by infecting the cells with the CAR construct in tandem with the shRNA to HPRT. This is in a single lentiviral vector with the CAR and shRNA driven by different promoters (Pol II and Pol III respectively). It is believed that if the shRNA targeting HPRT is within a miRNA framework, it could also be expressed from a Pol II promoter (maybe even the same promoter).

The transduced CAR-T shHPRT cells are then infused into a leukemic patient and anti-leukemia response monitored while if needed, expanding the CAR-T shHPRT cells with 6TG. Once the effect is impacting, a turn-off strategy using methotrexate to kill the transduced CAR-T shHPRT cells can be contemplated. This kill-off strategy is put into place if an inflammatory response or undue clonal proliferation of the CAR-T shHPRT cells is seen. It should be noted that some anti-leukemia antigens are also present on normal healthy cells and may give an untoward effect. Thus, applying this selection/suicide strategy increases efficacy/safety profile.

In allogeneic bone marrow transplant for hematological malignancy, donor T-cells are included with bone marrow transplant for an anti-tumor effect. This is important to eliminate residual disease following pre-transplant conditioning. In this example the donor T-cells are transduced with a lentiviral vector containing shRNA to HPRT before infusion and once infused the donor T-cell impact is assessed. As the Graft vs Leukemia (GVL) effect is monitored, if there is consequent Graft versus host disease (GVHD) this can be ameliorated using the "kill" switch with methotrexate. This allows GVL without consequent GVHD.

In allogeneic bone marrow transplantation there is a delayed immune recovery with a risk of adventitious agent infection. To guard against this and maintain T-cell activity, donor T-cells are given that have been transduced with a lentiviral vector containing HPRT. Over time this will provide ancillary control over potential infections until T-cells derived from the bone marrow transplanted stem cell reconstitute the hematopoietic system. If there is untoward inflammatory response or any other donor T-cell related AE they are eliminated using methotrexate. This allows anti-infection immune control without GVHD.

A patient has a leukemia. His own or matched allogeneic T-cells are taken and grown in tissue culture with growth supporting cytokines, e.g. IL2 or IL7, during which time they are transduced (infected leading to transgene expression) with a self-inactivating lentiviral vector that contains three elements i.e. tumor targeting, cell lysis machinery and a vector including components to knockdown HPRT. These gene-modified cells at 1x10<NUM> to 2x10<NUM> cells/sq meter are infused into the patient after a dose of IV Cytoxan, e.g. at <NUM>/sq meter IV (to make space for the introduced CAR-T cells). In this example the gene-modified CAR-T cells have some effect on leukemia. The leukemic cell burden is monitored, e.g. by differential blood counts, and if the physician desires more tumor cell killing, <NUM>/kg 6TG is given to the patient IV to increase the relative number of tumor-targeted CAR-T cells by selecting for these cells. If the CAR-T cells exert their positive anti-leukemia effect but there is an "over activation" leading to, e.g. inflammatory cytokine storm, then the reverse can occur in which the CAR-T cells are killed off using IV infusion of methotrexate, e.g. at <NUM> total dose.

K562 cells were transduced with a vector including a nucleic acid sequence designed to knockdown HPRT and a nucleic acid sequence encoding the green fluorescent protein (GFP) (MOI=<NUM>/<NUM>/<NUM>); or were transfected with a nanocapsule including CRISPR/Cas9 and a sgRNA to HPRT (100ng/5x10<NUM> cells) at day zero (<NUM>). <NUM>-TG was added into the medium from day <NUM> through day <NUM>. The medium was refreshed every <NUM> to <NUM> days. GFP was analyzed on flow machine and InDel% as analyzed with T7E1 assay. <FIG> illustrates that the GFP+ population of transduced K562 cells increased from day <NUM> to day <NUM> under treatment of 6TG; while the GFP+ population was almost steady without <NUM>-TG treatment. <FIG> illustrates that HPRT knockout population of K562 cells increased from day <NUM> to <NUM> under treatment of 6TG and higher dosages (<NUM>) of 6TG led to faster selection as compared with a dosage of <NUM>/<NUM> of 6TG. It should be noted that 6TG selection process occurred much faster on HPRT knockout cells as compared with the HPRT knockdown cells (MOI=<NUM>) at the same concentration of <NUM> of 6TG from day <NUM> to day <NUM>. The difference between knockdown and knockout could be explained by some level of residual HPRT by the RNAi knockdown approach as compared with the full elimination of HPRT by the knockout approach. Therefore, HPRT-knockout cells were believed to have a much higher tolerance against 6TG and are believed to grow much faster at higher dosages of 6TG (<NUM>) compared with HPRT-knockdown cells.

CEM cells were transduced with a vector including a nucleic acid sequence designed to knockdown HPRT and a nucleic acid sequence encoding the green fluorescent protein or transfected with a nanocapsule including CRISPR/Cas9 and a sgRNA to HPRT at day <NUM>. <NUM>-TG was added into the medium from day <NUM> to day <NUM>. The medium was refreshed every <NUM> to <NUM> days. GFP as analyzed on flow machine and InDel% is analyzed by T7E1 assay. <FIG> illustrates that the GFP+ population of transduced K562 cells increased from day <NUM> to day <NUM> under treatment of 6TG while GFP+ population was almost steady without <NUM>-TG. <FIG> shows that HPRT knockout population of CEM cells increased from day <NUM> to <NUM> under treatment of 6TG and that a higher dosage (<NUM>) of 6TG leads to a faster selection as compared with a dosage of <NUM>/<NUM> of 6TG. It should be noted that 6TG selection process occurred faster on HPRT knockout cells rather than HPRT knockdown cells (MOI=<NUM>) at the same concentration of 6TG from day <NUM> to day <NUM>.

Transduced or transfected K562 cells (such as those from Example <NUM>) were cultured with or without MTX from day <NUM> to day <NUM>. The medium was refreshed every <NUM> to <NUM> days. GFP was analyzed on flow machine and InDel% was analyzed by T7E1 assay. <FIG> shows that the GFP- population of transduced K562 cells decreased under the treatment of <NUM> of MTX the population of cells was steady without MTX. <FIG> illustrates that the transfected K562 cells were eliminated under treatment with <NUM> of MTX at a faster pace as compared with the HPRT-KD population.

Transduced or transfected CEM cells (such as those from Example <NUM>) were cultured with or without MTX from day <NUM> to day <NUM>. The medium was refreshed every <NUM> to <NUM> days. GFP was analyzed on flow machine and InDel% was analyzed by T7E1 assay. 121A shows the GFP- population of transduced K562 decreased under the treatment of <NUM> of MPA or <NUM> of MTX or <NUM> of MPA while the population of cells was steady for the untreated group. FIG> 12B illustrates that the HPRT knockout population of CEM cells were eliminated at a faster pace under the treatment of <NUM> of MPA or <NUM> of MTX or <NUM> of MPA.

K562 cells were transduced with either TL20cw-GFP virus soup at dilution factor of <NUM>, TL20cw-Ubc/GFP-7SK/sh734 (one sequentially encoding GFP and a shRNA designed to knockdown HPRT) virus soup at dilution factor of <NUM> and TL20cw-7SK/sh734-UBC/GFP (one sequentially encoding a shRNA designed to knockdown HPRT and GFP) virus soup at dilution factor of <NUM>, respectively (see <FIG>). All cells were cultured with medium containing <NUM> of MTX <NUM> days later. Also shown in <FIG> are K562 cells which were transduced by TL20cw-7SK/sh734-UBC/GFP (one encoding a nucleic acid encoding a shRNA designed to knockdown HPRT) virus soup at dilution factor of <NUM> one month earlier and where GFP-sh734-transduced cells were positively selected with <NUM> of 6TG <NUM>-TG was selection during that time to reach more than <NUM>% of GFP+ population. As illustrated in <FIG>, starting from ><NUM>% of GFP+ population, GFP or GFP-sh734 transduced cells did not show a reduction in the GFP+ population while the sh734-GFP-transduced cells at high dilution and low dilution levels showed deselection of the GFP+ population. The relative sh734 expression per VCN for sh734-GFP-transduced cells and GFP-sh73-transduced cells were measured. The results suggested that methotrexate could only deselect cells transduced with sh734-high-expression lentiviral vector (TL20cw-7SK/sh734-UBC/GFP) not and not with the sh734-low-expression lentiviral vector (TL20cw-UBC/GFP-7SK/sh734). This example demonstrated that different vector designs (even those having the same shRNA) had an impact on the expression of the shRNA hairpin and could determine whether transduced cells could be deselected or not by MTX.

Claim 1:
A composition comprising HPRT deficient lymphocytes for use in a method of preventing or mitigating post-transplant immune deficiency, the method comprising:
(a) generating HPRT deficient lymphocytes from a donor sample, wherein the HPRT deficient lymphocytes are generated through knockout of the HPRT gene;
(b) positively selecting for the generated HPRT deficient lymphocytes ex vivo to provide a population of modified lymphocytes; and
(c) administering the population of modified lymphocytes to the patient contemporaneously with or after an administration of an allogenic HSC graft.