Patent Application
20240024479
This disclosure relates to modified natural killer or T cells expressing hematopoietic growth factor receptors or chimeric hematopoietic growth factor receptors and methods of their use.
The Sequence Listing is submitted as an XML file in the form of the file named 4239-104153-07_Sequence_Listing.xml, which was created on Sep. 29, 2023, and is 68,693 bytes, which is incorporated by reference herein.
There currently exist a number of different pre-clinical and clinical strategies for utilizing natural killer (NK) cells as an immunotherapeutic to treat cancer. NK cells lack the requirement for prior-sensitization and can induce tumor cytotoxicity in an antigen-independent manner without causing graft-versus-host disease, making NK cells an attractive cell-based treatment option. Recent advances in genetic engineering techniques now provide the possibility to modify NK cells to enhance their efficacy in treating cancer. In this regard, several pre-clinical reports have shown that the antitumor cytotoxicity of NK cells can be improved by engineering them to target specific tumor antigens. One significant limitation of NK cell based immunotherapy is their reliance on cytokines, such as interleukin (IL)-2, IL-15, or IL-21, which enhance NK cell persistence and expansion in vitro and in vivo. At present, most clinical trials evaluating NK cell therapy in humans utilize the exogenous administration of IL-2 or IL-15 following adoptive cell transfer. Although IL-2 promotes activation, proliferation and cytotoxicity of NK cells, it also has the undesirable effect of inducing the expansion of regulatory T cells (Treg) which may suppress immune responses. Although IL-15 improves NK cell homeostasis, expansion and cytolytic capacity without expanding Tregs, it has cytokine-associated toxicities similar to those observed with IL-2, including capillary leak syndrome, hypotension, fever, and chills.
Investigators have recently explored a number of approaches to improve NK cell persistence in vivo that minimize toxicities and reduce or avoid the need for exogenous cytokine administration. Genetic modification of NK cell lines to express IL-2 or endoplasmic reticulum-retained IL-2 have been shown to result in autonomous NK cell proliferation. Likewise, expressing IL-15 or membrane-bound IL-15 on NK cells can enhance their proliferation and cytotoxicity in absence of exogenous cytokines. Recently, it has been demonstrated that NK cells derived from cord blood engineered to express anti-CD19 CAR, IL-15, and a suicide gene had improved anti-tumor activity and long-term persistence. In addition, an IL-15 superagonist complex called ALT-803 exhibited a longer half-life and better immune NK cell activation in vivo compared with wild-type IL-15.
There remains a need to improve NK cell survival, proliferation, and cytotoxicity, while decreasing undesirable or adverse effects. Disclosed herein are modified immune cells (such as NK cells or T cells) expressing hematopoietic growth factor receptors, or chimeric polypeptides, and methods of their use.
Provided herein are chimeric polypeptides including an extracellular domain of thrombopoietin receptor (c-MPL), a transmembrane domain, and an intracellular domain including an intracellular signaling domain of interleukin-2 receptor β (IL2RB), interleukin 21 receptor (IL21R), interleukin 7 receptor (IL7R), interferon alpha and beta receptor subunit 2 (IFNAR2), or interleukin 12 receptor subunit beta 2 (IL12RB2). In some aspects, the intracellular domain includes at least a portion of a c-MPL intracellular domain, such as box 1 and box 2 domains of c-MPL. In some examples, the transmembrane domain is a c-MPL transmembrane domain. In other examples, the transmembrane domain is a CD8α transmembrane domain, a CD28 transmembrane domain, a CD16 transmembrane domain, an ICOS transmembrane domain, a KIR2DS2 transmembrane domain, or a NKG2D transmembrane domain.
In some aspects, the c-MPL, the intracellular signaling domain from IL2RB, IL21R, IL7R, IFNAR2, or IL12RB2, or both are human. In some examples, the chimeric polypeptide includes at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 8 or SEQ ID NOs: 21-25. In additional examples, the chimeric polypeptide includes or consists of the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25.
Nucleic acids encoding the disclosed chimeric polypeptides are also provided. In some examples, the nucleic acid encoding the chimeric polypeptide includes at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the nucleic acid sequence of SEQ ID NO: 7 or SEQ ID NOs: 26-30. In additional examples, the nucleic acid includes consists of the nucleic acid sequence of SEQ ID NO: 7, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30. In addition, vectors including a nucleic acid encoding the disclosed chimeric polypeptides are provided. In some examples, the vector is a retroviral vector.
Also provided are modified immune cells (such as modified NK or T cells) expressing the disclosed chimeric polypeptides. In some examples, the modified NK cell or T cell is a human NK cell or T cell. In some examples, the modified NK cell or T cell also expresses a chimeric antigen receptor (CAR). In some examples, the chimeric polypeptide expressed by the modified NK cell or T cell includes at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of any one of SEQ ID NO: 8 and SEQ ID NOs: 21-25, or includes or consists of any one of SEQ ID NO: 8 and SEQ ID NOs: 21-25. In additional examples, the modified cell includes a nucleic acid encoding the chimeric polypeptide, such as a nucleic acid including at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the nucleic acid sequence of SEQ ID NO: 7 or including or consisting of SEQ ID NO: 7.
Methods of treating a subject with cancer are also provided. In some examples, the methods include administering a modified NK cell or T cell expressing a disclosed chimeric polypeptide to a subject with cancer and administering a c-MPL agonist (such as thrombopoietin, eltrombopag, or romiplostim) to the subject. In some examples, the methods also include administering IL-2 to the subject, for example, low dose IL-2 (such as about 0.5-2 million units/m2). In other examples, the subject is not administered IL-2.
The foregoing and other features of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.
(the last term representing the mean of all MPL+ and CD34+ groups from all donors). Groups were compared by paired T-test, as indicated. The significance of indicated comparisons was unaffected by data normalization (with the exception of bone marrow samples).
FGIS. 15A-15D show effects on proliferation of primary human T cells genetically modified to express EPOR or c-MPL. PBMC were transduced after 2 days of stimulation with anti-CD3 and anti-CD28 antibodies. After 4 additional days, CD3+ T-cells were examined for the transduced gene expression by flow cytometry (
Any nucleic acid and amino acid sequences listed herein or in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases and amino acids, as defined in 37 C.F.R. § 1.822. In at least some cases, only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
SEQ ID NO: 1 is the amino acid sequence of an exemplary thrombopoietin receptor (c-MPL).
SEQ ID NO: 2 is an exemplary nucleic acid sequence encoding the thrombopoietin receptor.
SEQ ID NO: 3 is the amino acid sequence of an exemplary erythropoietin receptor.
SEQ ID NO: 4 is an exemplary nucleic acid sequence encoding the erythropoietin receptor.
SEQ ID NO: 5 is the nucleic acid sequence of an exemplary vector including a nucleic acid encoding a thrombopoietin receptor.
SEQ ID NO: 6 is the nucleic acid sequence of an exemplary vector including a nucleic acid encoding an erythropoietin receptor.
SEQ ID NO: 7 is an exemplary nucleic acid sequence encoding a chimeric MPL-IL2RB receptor:
SEQ ID NO: 8 is the amino acid sequence of an exemplary chimeric MPL-IL2RB receptor (amino acids 1-582, c-MPL; amino acids 583-801, IL2RB):
SEQ ID NOs: 9-14 are amino acid sequences of exemplary transmembrane domains.
SEQ ID NO: 15 is an amino acid sequence of MPL TM and membrane proximal regions.
SEQ ID NO: 16 is an amino acid sequence of IL2RB TM and membrane proximal regions.
SEQ ID NO: 17 is an amino acid sequence of MPL-IL2RB chimera TM and membrane proximal regions.
SEQ ID NO: 18 is an amino acid sequence of MPL-IL2RBv2 chimera TM and membrane proximal regions.
SEQ ID NO: 19 is an amino acid sequence of MPL-IL2RBv3 chimera TM and membrane proximal regions.
SEQ ID NO: 20 is an amino acid sequence of MPL-IL2RBv4 chimera TM and membrane proximal regions.
SEQ ID NO: 21 is an amino acid sequence of MPL-IL2RBv5 chimera (amino acids 1-582, c-MPL; amino acids 583-604, IL2RB):
SEQ ID NO: 22 is an amino acid sequence of an exemplary MPL-IL21R chimera (amino acids 1-582, c-MPL; amino acids 583-795, IL21R):
SEQ ID NO: 23 is an amino acid sequence of an exemplary MPL-IL7R chimera (amino acids 1-582, c-MPL; amino acids 583-728, IL7R):
SEQ ID NO: 24 is an amino acid sequence of an exemplary MPL-IFNAR2 chimera (amino acids 1-582, c-MPL; amino acids 583-787, IFNAR2):
SEQ ID NO: 25 is an amino acid sequence of an exemplary MPL-IL12RB2 chimera (amino acids 1-582, c-MPL; amino acids 583-732, IL12RB2):
SEQ ID NO: 26 is an exemplary nucleic acid sequence encoding MPL-IL2RBv5 chimera:
SEQ ID NO: 27 is an exemplary nucleic acid sequence encoding MPL-IL21R chimera:
SEQ ID NO: 28 is an exemplary nucleic acid sequence encoding MPL-IL7R chimera:
SEQ ID NO: 29 is an exemplary nucleic acid sequence encoding MPL-IFNAR2 chimera:
SEQ ID NO: 30 is an exemplary nucleic acid sequence encoding MPL-IL12RB2 chimera:
Hematopoietic growth factors, such as recombinant human erythropoietin (EPO) and thrombopoietin (TPO) mimetics are FDA-approved for use in humans with anemia and thrombocytopenia and have been associated with minimal adverse events. Binding of EPO to its erythropoietin receptor (EPOR) promotes differentiation, proliferation and survival of erythroid progenitor cells. TPO, a ligand of the c-MPL receptor, is important for megakaryocyte differentiation and expansion as well as hematopoietic stem cell proliferation and maintenance. Both the EPO/EPOR and TPO/c-MPL interactions have been shown to transduce signals through three-main pathways; JAK-STAT, PI3K-AKT and MAPK pathways, having similarities to IL-2 and IL-15 signaling cascades.
The data presented herein demonstrate that genetic manipulation of NK cells or T cells to exogenously express hematopoietic growth factor receptors can provide adjuvant signals to improve their proliferative capacity, tumor cytotoxicity, and survival while simultaneously reducing their requirement for IL-2. In the presence of relatively low doses of IL-2, the cell yields of both EPOR and c-MPL expressing NK-92 NK cells were improved when cells were cultured in the presence of EPO or TPO respectively. Similar effects were also observed in the KHYG-1 cell line, albeit these effects were less pronounced, perhaps due to its higher baseline proliferative rate and greater dependence on higher concentrations of IL-2. In particular examples, enhanced tumor cytotoxicity and an augmentation in proliferation in primary human NK cells transduced to express the c-MPL receptor was observed.
In the data presented herein, TPO concentrations analogous to those obtained pharmacologically in humans were utilized. Remarkably, proliferation was augmented in c-MPL+ transduced NK cells cultured in TPO plus 25 U/ml of IL-2 to a level comparable to that observed in NK cell populations cultured in 20-fold higher doses of IL-2 (500 U/ml). Although EPO administration enhanced the proliferative capacity of EPOR transduced NK-92 cells, it did not augment the proliferation of EPOR transduced primary NK cells, possibly due to limitations in the experiments shown herein, where surface expression of the EPOR on primary NK cells appeared to not be induced to the higher levels achieved with the c-MPL receptor. In receptor transduced NK92 cells, TPO or EPO ligands clearly showed additive or synergistic effects on proliferation, survival, and cytotoxicity when combined with IL-2.
Stimulating c-MPL and IL-2 receptor together on transduced NK-92 cells augmented the anti-tumor function against Raji cells, which remarkably was superior to IL-2 alone, even when cells were cultured in high doses of this cytokine (200 U/ml). Primary NK cells efficiently recognize K562 targets, and MPL-transduced human NK cells co-incubated with K562 showed striking functional increases in degranulation and cytokine production in response to TPO administration. These data suggest that MPL ligation in NK cells with transgenic c-MPL expression could facilitate NK cell activation and tumor killing.
An unexpected finding was that primary NK cells engineered to express c-MPL and treated with TPO showed higher percentages of the lymphoid tissue homing receptor CD62L (L-selectin). IL-2 activation and ex vivo expansion have previously been shown to down-regulate CD62L.
The results presented herein in immunodeficient mice indicate that c-MPL-transduced primary NK cells have superior in vivo survival in response to systemic TPO administration compared with control populations. The numbers of MPL NK cells recovered in TPO-treated mice approached those detected in mice administered IL-2. These data may be translated to a clinical trial setting, including infusion of genetically modified c-MPL expressing NK cells followed by administration of an FDA approved TPO-mimetic (e.g. Eltrombopag, Romiplostim) and combined with low dose IL-2. Such regimens might avoid the substantial side effects caused by intermediate or high-dose IL-2, while inducing a selective cellular proliferative effect that is restricted to the genetically modified NK cells, avoiding the induction of proliferation of regulatory T-cells which inhibit the antitumor effects of NK and T-cells.
Administration of IL-2 or IL-15 in many adoptive immunotherapy regimens causes serious dose-dependent toxicities. These include capillary leak syndrome, which can be associated with hypotension, renal failure, and rarely death. These IL-2 side effects endanger patients, cause patient suffering, and limit the doses of IL-2 to those that may sub-optimally stimulate the function of administered immunotherapeutic cells. IL-2 also has the undesirable effect of inducing the expansion of regulatory T cells which inhibit the antitumor effects of NK and T-cells. To reduce or preclude the necessity for IL-2 use, a chimeric protein containing the extracellular, transmembrane, and membrane-proximal signaling regions of MPL fused with a membrane-distal intracellular portion of IL2RB (the resulting molecule listed herein as MPL-IL2RB) was developed. In the presence of FDA-approved specific pharmaceutical ligands eltrombopag or romiplostim, primary human NK cells retrovirally transduced to express MPL-IL2RB showed increased proliferation and augmented cell numbers in vitro compared to NK cells expressing MPL or unmodified NK cells. When expressed in human NK cells, both MPL and MPL-IL2RB increased NK cell anti-tumor functions (lytic granule and cytokine release) and showed appropriate signal transduction events (STAT5 phosphorylation and mTOR activation) in response to drug ligands. After transfer of MPL-IL2RB-expressing human NK cells into immunodeficient mice, administration of eltrombopag enhanced NK cell persistence. These data support use of MPL-IL2RB expressed in NK cells or T cells as a strategy to specifically augment NK and/or T-cell proliferation and anti-tumor immunity.
Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Lewin's Genes X, ed. Krebs et al., Jones and Bartlett Publishers, 2009 (ISBN 0763766321); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Publishers, 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Wiley, John & Sons, Inc., 1995 (ISBN 0471186341); and George P. Rédei, Encyclopedic Dictionary of Genetics, Genomics, Proteomics and Informatics, 3rd Edition, Springer, 2008 (ISBN: 1402067534), and other similar references.
Unless otherwise explained, 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 disclosure belongs. The singular terms “a,” “an,” and “the” include plural referents unless the context clearly indicates otherwise. “Comprising A or B” means including A, or B, or A and B. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description.
Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety, as are the GenBank Accession numbers, as present on the filing date of this application. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
In order to facilitate review of the various aspects of the disclosure, the following explanations of specific terms are provided:
Autologous: Refers to tissues, cells or nucleic acids taken from an individual's own tissues. For example, in an autologous transfer or transplantation of NK cells or T cells, the donor and recipient are the same person. Autologous (or “autogeneic” or “autogenous”) is related to self, or originating within an organism itself.
Erythropoietin (EPO): Erythropoietin is a hematopoietic growth factor that stimulates red blood cell production in the bone marrow. It is produced by the kidneys and secreted into the blood. EPO expression is upregulated by hypoxia, leading to increased red blood cell production and oxygen carrying capacity in the blood. Exemplary human EPO sequences include GenBank Accession No. NM_000799 (nucleic acid) and NP_000790 (amino acid). However, one of ordinary skill in the art can identify additional EPO sequences.
Erythropoietin receptor (EPOR): EPOR is the receptor for erythropoietin. It is expressed primarily in bone marrow, but is also expressed in other tissues, including kidney, thyroid, adrenal, and lung. Binding of EPO to EPOR appears to stimulate erythroid proliferation and/or survival. It also has neuroprotective effects in vitro and in models of neurodegenerative diseases. Exemplary human EPOR sequences include GenBank Accession No. NM_000121 (nucleic acid) and NP_000112 (amino acid). However, one of ordinary skill in the art can identify additional EPOR sequences.
“Erythropoietin receptor agonists” refers to a group of compounds that bind to and activate the erythropoietin receptor. EPO is the endogenous erythropoietin receptor agonist. Erythropoietin receptor agonists also include asialo-erythropoietin and carbamoylated erythropoietin (CEPO), ARA 290, which have non-erthryopoietic activities. Additional EPO receptor agonists include darbepoetin alfa (ARANESP®), Epoetin alfa (EPOGEN®, PROCRIT®, or RETACRIT®), and methoxy polyethylene glycol-Epoetin beta (MIRCERA®).
Heterologous: Originating from a different genetic source. A nucleic acid molecule or protein that is heterologous to a cell originates from a genetic source other than the cell in which it is expressed, or is not normally expressed in the cell. In one specific, non-limiting example, a heterologous nucleic acid molecule encoding a protein, such as a thrombopoietin receptor, an erythropoietin receptor, or a chimeric polypeptide is expressed in a cell, such as a mammalian cell. Methods for introducing a heterologous nucleic acid molecule in a cell or organism are well known in the art, for example transformation with a nucleic acid, including electroporation, lipofection, particle gun acceleration, and homologous recombination.
Interleukin 2 receptor subunit beta (IL2RB): An interleukin 2 receptor that forms a heterodimer with the IL2RA subunit, or a heterotrimer with the IL2RA and IL2RG subunits. Both the heterodimer and the heterotrimer are involved in IL2-mediated processes, including receptor-mediated endocytosis and transduction of mitogenic signals in immune cells. IL2RB sequences are publicly available and include GenBank Accession Nos. NM_000878.5, NM_001346222,1, and NM_001346223.2 (nucleic acids) and NP_000869.1, NP_001333151.1, and NP_001333152.1 (amino acids). However, one of ordinary skill in the art can identify additional IL2RB sequences.
Interleukin 7 receptor (IL7R): A receptor for interleukin 7 (IL7). This protein has been shown to play a role in V(D)J recombination during lymphocyte development. Human IL7R sequences are publicly available and include GenBank Accession Nos. NM_002185 and NM_001410734 (nucleic acid) and NP_002176 and NP_001397663 (amino acid). However, one of ordinary skill in the art can identify additional IL7R sequences.
Interleukin 21 receptor (IL21R): A type I cytokine receptor for interleukin 21 (IL21). This receptor transduces the growth promoting signal of IL21, and is important for the proliferation and differentiation of T cells, B cells, and natural killer (NK) cells. The ligand binding of this receptor leads to the activation of multiple downstream signaling molecules, including JAK1, JAK3, STAT1, and STAT3. Human IL21R sequences are publicly available and include GenBank Accession Nos. NM_181078, NM_181079, and NM_021798 (nucleic acid) and NP_851564, NP_851565, and NP_068570 (amino acid). However, one of ordinary skill in the art can identify additional IL21R sequences.
Interleukin12 receptor subunit beta 2 (IL12RB2): A type I transmembrane protein identified as a subunit of the interleukin 12 receptor complex. The coexpression of this and IL12RB1 proteins leads to the formation of high-affinity IL12 binding sites and reconstitution of IL12 dependent signaling. The expression of IL12RB2 is up-regulated by interferon gamma in Th1 cells, and plays a role in Th1 cell differentiation. Human IL12RB2 sequences are publicly available and include GenBank Accession Nos. NM_001258214, NM_001319233, NM_001258216, NM_001258215, NM_001374259, and NM_001559 (nucleic acid) and NP_001245143, NP_001306162, NP_001245145, NP_001245144, NP_001361188, and NP_001550 (amino acid). However, one of ordinary skill in the art can identify additional IL12RB2 sequences.
Interferon alpha and beta receptor subunit 2 (IFNAR2): A type I membrane protein that forms one of the two chains of a receptor for interferons alpha and beta and is a type II cytokine receptor family member. Binding and activation of the receptor stimulates Janus protein kinases, which in turn phosphorylate several proteins, including STAT1 and STAT2. Human IFNAR2 sequences are publicly available and include GenBank Accession Nos. NM_000874, NM_001289125, NM_001289128, NM_207584, NM_207585, NM_001385055, NM_001385054, NM_001289126 (nucleic acid) and NP_000865, NP_001276054, NP_001276057, NP_997467, NP_997468, NP_001371984, NP_001371983, NP_001276055 (amino acid). However, one of ordinary skill in the art can identify additional IFNAR2 sequences.
Isolated: A biological component (such as a nucleic acid or protein, or cell) that has been substantially separated, produced apart from, or purified away from other biological components, for example other nucleic acids, proteins, and/or cells. Thus, isolated nucleic acids and proteins include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids. An isolated nucleic acid, protein, or cell can be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure.
Natural Killer (NK) cells: Cells of the immune system that kill target cells in the absence of a specific antigenic stimulus and without restriction according to MHC class. Target cells can be tumor cells or cells harboring viruses. NK cells are characterized by the presence of CD56 and the absence of CD3 surface markers. NK cells typically comprise approximately 10 to 15% of the mononuclear cell fraction in normal peripheral blood. Historically, NK cells were first identified by their ability to lyse certain tumor cells without prior immunization or activation. NK cells are thought to provide a “back up” protective mechanism against viruses and tumors that might escape the CTL response by down-regulating MHC class I presentation. In addition to being involved in direct cytotoxic killing, NK cells also serve a role in cytokine production, which can be important to control cancer and infection.
In some examples, a “modified NK cell” is a NK cell transduced with a heterologous nucleic acid (such as one or more of the nucleic acids or vectors disclosed herein) or expressing one or more heterologous proteins. The terms “modified NK cell” and “transduced NK cell” are used interchangeably in some examples herein.
Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers of use are known to those of ordinary skill in the art. Remington: The Science and Practice of Pharmacy, 22nd ed., London, UK: Pharmaceutical Press, 2013, describes compositions and formulations suitable for pharmaceutical delivery of the disclosed agents. In general, the nature of the carrier will depend on the particular mode of administration being employed. For example, parenteral formulations usually include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, added preservatives (such as non-natural preservatives), and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. In particular examples, the pharmaceutically acceptable carrier is sterile and suitable for parenteral administration to a subject for example, by injection. In some aspects, the active agent and pharmaceutically acceptable carrier are provided in a unit dosage form such as in a selected quantity in a vial. Unit dosage forms can include one dosage or multiple dosages (for example, in a vial from which metered dosages of the agent can selectively be dispensed).
Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified protein or cell preparation is one in which the protein or cell is more enriched than in its original environment. In one aspects, a preparation is purified such that the protein or cells represent at least 50% of the total peptide or protein content of the preparation.
Recombinant: A recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished by chemical synthesis or, more commonly, by the manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques. In several aspects, a recombinant protein is encoded by a heterologous (for example, recombinant) nucleic acid that has been introduced into a host cell, such as a bacterial or eukaryotic cell. The nucleic acid can be introduced, for example, on an expression vector having signals capable of expressing the protein encoded by the introduced nucleic acid, or the nucleic acid can be integrated into the host cell chromosome.
Subject: Living multi-cellular vertebrate organisms, a category that includes human and non-human mammals, such as non-human primates, pigs, sheep, cows, dogs, cats, rodents, and the like. In an example, a subject is a human.
T cells: A white blood cell (lymphocyte) that is an important mediator of the immune response. T cells include, but are not limited to, CD4+ T cells and CD8+ T cells. A CD4+ T cell is an immune cell that carries a marker on its surface known as “cluster of differentiation 4” (CD4). These cells, also known as helper T cells, help orchestrate the immune response, including antibody responses as well as killer T cell responses. CD8+ T cells carry the “cluster of differentiation 8” (CD8) marker. In one aspects, a CD8+ T cell is a cytotoxic T lymphocyte (CTL). In another aspects, a CD8+ cell is a suppressor T cell.
Activated T cells can be detected by an increase in cell proliferation and/or expression of or secretion of one or more cytokines (such as IL-2, IL-4, IL-6, IFNγ, or TNFα). Activation of CD8+ T cells can also be detected by an increase in cytolytic activity in response to an antigen.
A natural killer T (NK-T) cell is a class of T cell that expresses both T-cell receptors (TCR) characteristic of adaptive immunity, and surface receptors for NK cells (e.g. the cell-surface marker NK1.1 normally associated with NK cells along with an α/β T-cell receptor). See, e.g., Tupin et al., Nat Rev Microbiol 5:405-417, 2007.
In some examples, a “modified T cell” is a T cell transduced with a heterologous nucleic acid (such as one or more of the nucleic acids or vectors disclosed herein) or expressing one or more heterologous proteins. The terms “modified T cell” and “transduced T cell” are used interchangeably in some examples herein. Similarly, a “modified NK-T cell” is an NK-T cell transduced or transfected with a heterologous nucleic acid (such as one or more of the nucleic acids or vectors disclosed herein) or expressing one or more heterologous proteins.
Thrombopoietin (TPO): Thrombopoietin is a hematopoietic growth factor that promotes megakaryocyte proliferation and maturation and platelet production (thrombopoiesis). TPO is primarily produced by the liver and kidneys. Exemplary human TPO sequences include GenBank Accession No. NM_000460 (nucleic acid) and NP_000451 (amino acid). However, one of ordinary skill in the art can identify additional TPO sequences.
“Thrombopoietin receptor agonists” refers to a group of compounds that bind to and activate the thrombopoietin receptor. TPO is the endogenous thrombopoietin receptor agonist. The peptide romiplostim is also a thrombopoietin receptor agonist. Two small molecule thrombopoietin receptor agonists are eltrombopag and avatrombopag. Romiplostim, eltrombopag, and avatrombopag are all used clinically in the treatment of thrombocytopenias.
Thrombopoietin receptor (c-MPL): c-MPL is the receptor for thrombopoietin. Binding of TPO to c-MPL appears to stimulate megakaryocyte proliferation and platelet formation. Exemplary human c-MPL sequences include GenBank Accession No. NM_005373 (nucleic acid) and NP_005364 (amino acid). However, one of ordinary skill in the art can identify additional c-MPL sequences.
Transduced or transformed: A transformed cell is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. As used herein, the term transformed and the like (e.g., transformation, transfection, transduction, etc.) encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transduction with viral vectors, transformation with plasmid vectors, and introduction of DNA by electroporation, lipofection, and particle gun acceleration.
Treating or inhibiting a disorder: “Inhibiting” a condition refers to inhibiting the full development of a condition or disease, for example, cancer or a tumor. Inhibition of a condition can span the spectrum from partial inhibition to substantially complete inhibition of the disease. In some examples, the term “inhibiting” refers to reducing or delaying the onset or progression of a condition. “Treatment” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or condition after it has begun to develop. A subject to be administered an effective amount of the disclosed T cells or NK cells can be identified by standard diagnosing techniques for such a disorder, for example, presence of the disease or disorder or risk factors to develop the disease or disorder.
Vector: A nucleic acid molecule (such as a DNA or RNA molecule) including a promoter(s) that is operably linked to the coding sequence of a protein of interest and can express the coding sequence. Non-limiting examples include a naked or packaged (lipid and/or protein) DNA, a naked or packaged RNA, a subcomponent of a virus or bacterium or other microorganism that may be replication-incompetent, or a virus or bacterium or other microorganism that may be replication-competent. A vector is sometimes referred to as a construct. Recombinant DNA vectors are vectors having recombinant DNA. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements. In some aspects, a vector includes a nucleic acid molecule encoding a thrombopoietin receptor or an erythropoietin receptor. In some examples, the vector is a bacterial vector. In other examples, the vector is a viral vector, such as a nucleic acid vector having at least some nucleic acid sequences derived from one or more viruses. In some aspects, the viral vector is a retroviral vector, such as a lentiviral vector.
Provided herein are modified immune cells that express a heterologous hematopoietic growth factor receptor, for example, a heterologous thrombopoietin receptor (c-MPL) or a heterologous erythropoietin receptor (EPOR). Also provided are modified immune cells expressing a chimeric polypeptide including a c-MPL extracellular domain and a heterologous intracellular domain (see Section IV). In some aspects, the modified immune cells are NK cells, T cells, or NK-T cells. In further aspects, the modified immune cells are NK cells, T cells, or NK-T cells that also express a chimeric antigen receptor (CAR), such as CAR-NK cells, CAR-T cells, or CAR-NK-T cells.
In some aspects, modified immune cells (e.g., NK cells, T cells, NK-T cells, CAR-NK cells, CAR-T cells, or CAR-NK-T cells) expressing a heterologous thrombopoietin receptor (c-MPL) are provided. In particular non-limiting examples, modified NK cells expressing a heterologous c-MPL are provided. In some examples, the heterologous thrombopoietin receptor expressed in the modified cells has at least 95% sequence identity (such as at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to the amino acid sequence of SEQ ID NO: 1. In other examples, the heterologous thrombopoietin receptor expressed in the modified cells includes or consists of the amino acid sequence of SEQ ID NO: 1. In other examples, the nucleic acid encoding the heterologous thrombopoietin receptor has at least 95% sequence identity (such as at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to the nucleic acid sequence of SEQ ID NO: 2. In other examples, the heterologous thrombopoietin receptor expressed in the modified cells is encoded by a nucleic acid that includes or consists of the nucleic acid sequence of SEQ ID NO: 2.
In other aspects, modified immune cells (e.g., NK cells, T cells, NK-T cells, CAR-NK cells, CAR-T cells, or CAR-NK-T cells) expressing a heterologous erythropoietin receptor (EPOR) are provided. In particular non-limiting examples, modified NK cells or modified T cells expressing a heterologous EPOR are provided. In some examples, the heterologous EPOR expressed in the modified cells has at least 95% sequence identity (such as at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to the amino acid sequence of SEQ ID NO: 3. In other examples, the heterologous EPOR expressed in the modified cells includes or consists of the amino acid sequence of SEQ ID NO: 3. In other examples, the nucleic acid encoding the heterologous EPOR has at least 95% sequence identity (such as at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to the nucleic acid sequence of SEQ ID NO: 4. In other examples, the heterologous EPOR expressed in the modified cells is encoded by a nucleic acid that includes or consists of the nucleic acid sequence of SEQ ID NO: 4.
In additional aspects, modified immune cells (e.g., NK cells, T cells, NK-T cells, CAR-NK cells, CAR-T cells, or CAR-NK-T cells) expressing a chimeric polypeptide comprising a c-MPL extracellular domain and an IL2RB intracellular domain are provided. In particular non-limiting examples, the modified cells express a chimeric polypeptide including a c-MPL extracellular domain linked to an IL-2RB intracellular domain (such as a chimeric polypeptide described in Section IV). In some non-limiting examples, the chimeric polypeptide expressed in the modified cells has at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 21. In one example, the chimeric polypeptide includes or consists of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 21. In other non-limiting examples, the modified cells include a nucleic acid encoding the chimeric polypeptide with at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 7. In one example, the chimeric polypeptide expressed in the modified cells is encoded by a nucleic acid that includes or consists of the nucleic acid sequence of SEQ ID NO: 7.
In further examples, modified immune cells (e.g., NK cells, T cells, NK-T cells, CAR-NK cells, CAR-T cells, or CAR-NK-T cells) expressing a chimeric polypeptide comprising a c-MPL extracellular domain and a heterologous intracellular domain selected from an IL21R intracellular domain (such as amino acids 583-795 of SEQ ID NO: 22), an IL7R intracellular domain (such as amino acids 583-728 of SEQ ID NO: 23), an IFNAR2 intracellular domain (such as amino acids 583-787 of SEQ ID NO: 24), and an IL12RB2 intracellular domain (such as amino acids 583-732 of SEQ ID NO: 25) are provided. In some non-limiting examples, the chimeric polypeptide expressed in the modified cells has at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 22-25. In one example, the chimeric polypeptide includes or consists of the amino acid sequence of any one of SEQ ID NOs: 22-25.
In some examples, the immune cells (such as T cells or NK cells) are obtained from peripheral blood (such as peripheral blood mononuclear cells), lymph node, thymus, bone marrow, tumor tissue, adipose tissue, human embryonic stem cells (hESC), induced pluripotent stem cells (iPSC), or umbilical cord blood. In some aspects, the cells are autologous to a subject being treated. In other examples, the cells are allogeneic to the subject.
In some aspects, the immune cells (e.g., T cells or NK cells) are transduced with a vector including a heterologous nucleic acid encoding a thrombopoietin receptor or erythropoietin receptor, including, but not limited to a vector including SEQ ID NO: 2 or SEQ ID NO: 4. In some non-limiting examples, the vector includes or consists of the nucleic acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6. In other aspects, the immune cells (e.g., T cells or NK cells) are transduced with a vector including a nucleic acid encoding a chimeric polypeptide, such as a vector encoding an amino acid sequence selected from SEQ ID NOs: 8 and 21-25. In one example, the vector includes the nucleic acid sequence of SEQ ID NO: 7. In some examples, the vector is a viral vector, such as a lentiviral vector.
Viral vectors suitable for gene delivery to NK cells or T cells include retrovirus, adenovirus, adeno-associated virus, vaccinia virus, fowlpox, and lentivirus vectors. In particular non-limiting examples disclosed herein, NK cells or T cells are transduced with lentiviral vectors including a heterologous nucleic acid encoding a thrombopoietin receptor or erythropoietin receptor or a chimeric polypeptide described herein. Some advantages of using a lentiviral system include long-term expression of the transgene, the ability to transduce both dividing cells and non-dividing cells, the ability to deliver complex genetic elements, lack of expression of viral proteins after transduction, low probability of insertional mutagenesis in human cells, high titer production, and ease of vector manipulation and production.
In particular examples, the nucleic acid encoding the thrombopoietin receptor or erythropoietin receptor or chimeric MPL polypeptide is included in a lentiviral gene transfer vector. The receptor nucleic acid in the transfer vector is operably linked to one or more expression control elements, such as a promoter. Exemplary promoters include constitutive promoters such as cytomegalovirus (CMV), SV40, phosphoglycerate kinase (PGK), ubiquitin C (UBC), elongation factor-1 (EFS), chicken β-actin short promoter (CBH), EF-1 alpha (EF1a) promoter, or EF1a short promoter, a hybrid promoter (such as a CMV enhancer fused to chicken β-actin promoter (CAG)), or an inducible or tissue-specific promoter.
Additional expression control elements that may be included in the transfer vector include sequences that control or regulate transcription and/or translation of a nucleic acid, such as enhancers, leader sequences, transcription terminators, start and/or stop codons, internal ribosome entry sites (IRES), splicing signals, and polyadenylation signals. In examples where the vector or construct includes two (or more) heterologous nucleic acids of interest, the nucleic acids are operably linked, for example, separated by an IRES or other multicistronic element such as a P2A and/or T2A element. The vector may also contain additional elements such as packaging signals (e.g., lentivirus w packaging signal), a central polypurine tract (cPPT), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and/or a Rev Response element (RRE). In some examples, the lentivirus vector is self-inactivating.
Lentivirus vectors including one or more nucleic acids of interest can be prepared by one of ordinary skill in the art utilizing conventional molecular biology techniques. For example, the nucleic acid of interest can be cloned into a lentivirus transfer vector. Lentivirus plasmid systems (such as 3 or 4 plasmid systems) are commercially available, for example from Clontech (Mountain View, CA), ThermoFisher Scientific (Waltham, MA), or Addgene (Cambridge, MA).
In some aspects, modified (e.g., transduced) T cells expressing an EPOR or c-MPL or chimeric MPL polypeptide are produced by obtaining a population of lymphocytes (such as a population of PBMCs) from a subject, for example by apheresis. Naïve or quiescent T cells in the population of lymphocytes are activated prior to transduction, for example, by contacting the lymphocytes with one or more cytokines (such as one or more of IL-2, IL-4, IL-6, IL-7, IL-12, IL-15, and IL-23). In some examples, the lymphocytes are contacted with anti-CD3 antibody and IL-2 for 1-4 days (such as 1 day, about 2 days, about 3 days, or about 4 days) to produce activated T cells. In some examples, the lymphocytes are contacted with 30 ng/ml anti-CD3 antibody and 300 IU/ml IL-2 for 2 or 3 days. In some examples, the lymphocytes are contacted with 1-10 μg/ml (e.g., 5 μg/ml) of surface bound anti-CD3 antibody with 1-5 μg/ml (e.g., 2 μg/ml) anti-CD28 antibody for 2-5 days (e.g., 2 days).
The activated T cells are transduced or transfected with a vector encoding an EPOR or c-MPL protein or a chimeric polypeptide disclosed herein. In some examples, the transduced T cells are enriched and/or expanded. The transduced T cells can be expanded by culturing the transduced T cells with anti-CD3 (e.g., about 30 ng/ml), anti-CD28 (e.g., about 30 ng/ml), and/or IL-2 (e.g., about 300 IU/ml) for a period of time (such as about 7-14 days or 9-11 days). In some examples, the transduced T cells are expanded by culture on irradiated PBMC cells.
In other aspects, modified (e.g., transduced) NK cells expressing an EPOR or c-MPL or chimeric MPL polypeptide are produced by obtaining a population of lymphocytes (such as a population of PBMCs) from a subject, for example by apheresis. In some examples, the NK cells are activated by culturing isolated NK cells with one or more cytokines for a period of time prior to transduction. In some examples, the NK cells are cultured in culture medium including IL-2, IL-15, and/or IL21 for 1-14 days prior to transduction. In some non-limiting examples, the NK cells are activated in culture medium including 500 IU/ml IL-2 or 1000 IU/ml IL-2 for 1-6 days, 2-3 days, or 3-5 days. In some examples, the NK cells are activated in the presence of feeder cells, such as EBV-LCLs (TM-LCL, SMI-LCL), allogeneic or autologous PBMCs, Wilms tumor cell line HFWT, and K562 cells (such as genetically modified K562 cells, for example, K562-mb15-41BBL or K562-mbIL-21-41BBL cells).
The activated NK cells are transduced or transfected with a vector encoding an EPOR or c-MPL protein or chimeric MPL polypeptide. The transduced NK cells can also be enriched and/or expanded, for example by culturing the transduced NK cells in a medium including IL-2 (such as 500 IU/ml IL-2) and/or one or more additional cytokines. In some examples, the NK cells are expanded on feeder cells, such as EBV-LCLs (TM-LCL, SMI-LCL), allogeneic or autologous PBMCs, Wilms tumor cell line HFWT, and K562 cells (such as genetically modified K562 cells, for example, K562-mb15-41BBL or K562-mbIL-21-41BBL cells).
In some aspects of the disclosed methods, the transduced NK cells or T cells are cultured with a thrombopoietin receptor agonist (if the modified cells express c-MPL or c-MPL extracellular domain) or an erythropoietin receptor agonist (if the modified cells express EPOR) for example, prior to administering to a subject. In some examples, the transduced cells are cultured with about 10-250 ng/ml of a thrombopoietin receptor agonist (e.g., about 10-50 ng/ml, 25-75 ng/ml, 50-100 ng/ml, 75-150 ng/ml, 125-175 ng/ml, 150-200 ng/ml, 175-225 ng/ml, or 200-250 ng/ml). In one particular example, the transduced cells are cultured with about 50 ng/ml TPO. In other examples, the transduced cells are cultured with about 1-50 U/ml of an EPOR agonist (e.g., about 1-5 U/ml, about 2.5-7.5 U/ml, about 5-10 U/ml, about 7.5-15 U/ml, about 10-20 U/ml, about 15-25 U/ml, about 25-40 U/ml, or about 35-50 U/ml). In a particular example, the transduced cells are cultured with about 5 U/ml EPO. The transduced cells are cultured with the thrombopoietin receptor agonist or erythropoietin receptor agonist for about 7-28 days (e.g., about 7-10 days, about 8-12 days, about 10-15 days, about 12-18 days, about 14-21 days, about 16-24 days, or about 21-28 days), for example, prior to cryopreserving or administering the cells to a subject. In particular examples, the transduced cells are cultured with the thrombopoietin receptor agonist or erythropoietin receptor agonist for about 10-17 days (such as about 10, 11, 12, 13, 14, 15, 16, or 17 days).
In some aspects, the transduced NK cells or T cells are cultured with at least one cytokine in addition to the thrombopoietin receptor agonist or erythropoietin receptor agonist. In some examples, the transduced NK cells or T cells are cultured with IL-2 in addition to the thrombopoietin receptor agonist or erythropoietin agonist. In particular examples the amount of IL-2 is a reduced amount of IL-2 (“low dose”), which in some examples, is about 1-50 U/ml IL-2 (for example, about 1-5 U/ml, about 2-10 U/ml, about 5-15 U/ml, about 10-25 U/ml, about 20-40 U/ml, about 25-50 U/ml, or about 1-25 U/ml).
In additional aspects, the transduced NK cells or T cells include one or more additional modifications. In some examples, the NK cells or T cells are modified to eliminate or reduce expression of one or more proteins. In one example, NK cells are modified to eliminate or reduce expression of CD38, for example, when modified NK cells are to be used in combination with an anti-CD38 antibody, such as daratumumab. Suitable methods for reducing expression of proteins are known, and include, but are not limited to gene editing methods, such as CRISPR.
Methods of treating cancer with the modified immune cells disclosed herein are provided. In some aspects, the methods include administering modified immune cells (e.g., modified NK cells, T cells, NK-T cells, CAR-NK cells, CAR-T cells, or CAR-NK-T cells) expressing a hematopoietic growth factor receptor (such as a thrombopoietin receptor or an erythropoietin receptor) or expressing a chimeric polypeptide disclosed herein to a subject with cancer. In some aspects, the subject is also administered an agonist for the hematopoietic growth factor receptor expressed by the modified cells (e.g., a thrombopoietin receptor agonist or erythropoietin receptor agonist). In other aspects a subject is administered modified cells expressing a disclosed chimeric polypeptide and a thrombopoietin receptor agonist.
In some aspects, the subject has a hematological malignancy. In some examples, the subject is administered modified NK cells or T cells expressing c-MPL or EPOR or chimeric MPL polypeptide. In other examples, the subject is administered modified CAR-NK cells or CAR-T cells expressing c-MPL or EPOR or a disclosed chimeric polypeptide and a CAR targeting the hematological malignancy. Examples of hematological malignancies include leukemias, including acute leukemias (such as 11q23-positive acute leukemia, acute lymphocytic leukemia (ALL), T-cell ALL, acute myelocytic leukemia, acute myelogenous leukemia (AML), and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia (CML), and chronic lymphocytic leukemia (CLL)), lymphoblastic leukemia, polycythemia vera, lymphoma, diffuse large B cell lymphoma, Burkitt lymphoma, T cell lymphoma, follicular lymphoma, mantle cell lymphoma, Hodgkin disease, non-Hodgkin lymphoma, multiple myeloma, Waldenstrom macroglobulinemia, heavy chain disease, myelodysplastic syndrome (MDS), hairy cell leukemia, and myelodysplasia. In particular examples, the subject has a leukemia or lymphoma, such as AML, ALL, CLL, multiple myeloma, MDS, a B cell lymphoma, or a T cell lymphoma.
In other examples, the subject has a solid tumor. In some examples, the subject is administered modified NK cells or T cells expressing c-MPL or EPOR or a disclosed chimeric polypeptide. In other examples, the subject is administered modified CAR-NK cells or CAR-T cells expressing c-MPL or EPOR or a chimeric polypeptide disclosed herein and a CAR targeting the solid tumor. Examples of solid tumors, include sarcomas (such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas), synovioma, mesothelioma, Ewing sarcoma, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, peritoneal cancer, esophageal cancer, pancreatic cancer, breast cancer (including basal breast carcinoma, ductal carcinoma and lobular breast carcinoma), lung cancer, ovarian cancer, prostate cancer, liver cancer (including hepatocellular carcinoma), gastric cancer, squamous cell carcinoma (including head and neck squamous cell carcinoma), basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms tumor, cervical cancer, fallopian tube cancer, testicular tumor, seminoma, bladder cancer, kidney cancer (such as renal cell cancer), melanoma, and CNS tumors (such as a glioma, glioblastoma, astrocytoma, medulloblastoma, craniopharyrgioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma and retinoblastoma). Solid tumors also include tumor metastases (for example, metastases to the lung, liver, brain, or bone). In particular examples, the subject has renal cell cancer, breast cancer, colon cancer, bladder cancer, or a uroepithelia carcinoma.
The modified immune cells (such as T cells, NK cells, NK-T cells, CAR-T cells, CAR-NK cells, or CAR-NK-T cells) expressing a thrombopoietin receptor or a erythropoietin receptor or chimeric polypeptide described herein can be incorporated into pharmaceutical compositions. Such compositions typically include a population of cells and a pharmaceutically acceptable carrier. A “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration (see, e.g., Remington: The Science and Practice of Pharmacy, 22nd ed., London, UK: Pharmaceutical Press, 2013). Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, balanced salt solutions, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. Supplementary active compounds can also be incorporated into the compositions. Actual methods for preparing administrable compositions include those provided in Remington: The Science and Practice of Pharmacy, 22nd ed., London, UK: Pharmaceutical Press, 2013.
In some examples, the composition includes about 104 to 1012 of the modified immune cells (for example, about 104-108 cells, about 106-108 cells, or about 106-1012 cells). For example, the composition may be prepared such that about 104 to 1010 modified NK cells or modified T cells cells/kg (such as about 104, 105, 106, 107, 108, 9, or 1010 cells/kg) are administered to a subject. In specific examples, the composition includes at least 104, 105, 106, or 107 modified immune cells (e.g., modified NK cells, T cells, NK-T cells, CAR-NK cells, CAR-T cells, or CAR-NK-T cells). The population of modified cells is typically administered parenterally, for example intravenously; however, other routes of administration can also be used. Appropriate routes of administration can be determined based on factors such as the subject, the condition being treated, and other factors.
Multiple doses of the population of modified immune cells can be administered to a subject. For example, the cells can be administered daily, every other day, twice per week, weekly, every other week, every three weeks, monthly, or less frequently. A skilled clinician can select an administration schedule based on the subject, the condition being treated, the previous treatment history, and other factors.
In particular aspects, the subject is also administered a thrombopoietin agonist, if the modified cells express a thrombopoietin receptor or a chimeric polypeptide disclosed herein. In particular examples, the thrombopoietin receptor agonist is thrombopoietin. In other examples, the thrombopoietin receptor agonist is romiplostim, eltrombopag, or avatrombopag. In some examples, the subject is administered 0.3-2.4 μg/kg of TPO, about 1-10 μg/kg of romiplostim per week, about 25-150 mg of eltrombopag per day, or about 20-100 mg of avatrombopag per day. However, one of ordinary skill in the art will recognize that these dosages and frequencies can be varied, for example, as a result of clinical trials, the particular subject being treated, or other factors. In some examples, the thrombopoietin receptor agonist is administered to the subject prior to, substantially simultaneously with (e.g., at approximately the same time), or subsequent to administration of the modified cells. In particular examples, the thrombopoietin receptor agonist is administered to the subject for up to 6 months, or in other examples for about 7-30 days.
In other aspects, the subject is also administered an erythropoietin receptor agonist, if the modified cells express an erythropoietin receptor. In particular examples, the erythropoietin receptor agonist is erythropoietin. In other examples, the erythropoietin receptor agonist is asialo-erythropoietin, carbamoylated erythropoietin (CEPO), or ARA 290. In still further examples, the EPO receptor agonist is darbepoetin alfa (ARANESP®), epoetin alfa (EPOGEN®, PROCRIT®, or RETACRIT®), or methoxy polyethylene glycol-epoetin beta (MIRCERA®). In some examples, the subject is administered 2-4 mg of ARA 290, about 0.45 μg/kg to 5 mg/kg of darbepoietin alfa, about 50-600 U/kg of epoietin alfa, or 0.6 μg/kg or more of epoietin beta. However, one of ordinary skill in the art will recognize that these dosages can be varied, for example, as a result of clinical trials, the particular subject being treated, or other factors. In some examples, the erythropoietin receptor agonist is administered to the subject prior to, substantially simultaneously with (e.g., at approximately the same time), or subsequent to administration of the modified cells. In particular examples, the erythropoietin receptor agonist is administered to the subject for up to 6 months, or in other examples for about 7-30 days.
In additional examples, the subject is also administered at least one cytokine (such as IL-2). In some examples, the cytokine supports survival and/or proliferation of the modified cells. In specific, non-limiting examples, the subject is administered “low dose” IL-2. In some examples, low dose IL-2 is about 0.5-4 million units/m2 (e.g., about 0.5-1 million units/m2, about 0.75-1.25 million units/m2, about 1-1.5 million units/m2, about 1.25-1.75 million units/m2, about 1.5-2 million units/m2, about 2.25-2.75 million units/m2, about 2.5-3 million units/m2, about 3-3.5 million units/m2, about 3.25-3.75 million units/m2, or about 3.5-4 million units/m2. In other examples, the subject is administered about 0.5-2 million units/m2 of IL-2, or less than about 2 million units/m2 of IL-2, or less than about 1 million units/m2 of IL-2. The IL-2 may be administered before, after, or substantially simultaneously (e.g., at approximately the same time) with the modified cells. In some non-limiting examples, IL-2 is administered one or more times prior to or substantially simultaneously with administration of the modified cells. In particular examples, IL-2 is administered to the subject once or twice daily for 2-14 days, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days. In other examples, the subject is administered IL-2 for about 7 days. In further examples, the subject is not administered IL-2 in combination with the modified cells (for example, prior to, substantially simultaneously with, and/or subsequent to administration of the modified cells).
In some examples, the subject is also treated with one or more of surgery, radiation therapy, chemotherapeutic agents, monoclonal antibodies, or other therapies. In one particular example, the subject has multiple myeloma and is also treated with daratumumab. A skilled clinician can select appropriate additional therapies for the subject, depending on factors such as the subject, the cancer being treated, treatment history, and other factors.
Provided herein are chimeric polypeptides that include an extracellular domain of c-MPL, a transmembrane domain, and an intracellular domain including a heterologous intracellular signaling domain. In some examples, the chimeric polypeptides include a heterologous intracellular signaling domain from interleukin-2 receptor β (IL2RB), interleukin 21 receptor (IL21R), interleukin 7 receptor (IL7R), interferon alpha and beta receptor subunit 2 (INFAR2), or interleukin 12 receptor subunit beta 2 (IL12RB2). The chimeric peptides bind TPO or a TPO agonist and activate signal transduction via the intracellular signaling domain. In some examples, the chimeric polypeptide includes a c-MPL transmembrane domain and membrane-proximal regions, including box 1 and box 2 domains. The c-MPL membrane proximal box 1 and box 2 domains bind kinase Jak2. Jak kinase activation and phosphorylation are key initial steps in cytokine and growth factor signaling. Other residues of MPL between the transmembrane domain and box 1 are reported to prevent continuous activity of the MPL receptor.
In some aspects, the disclosed chimeric polypeptides include an extracellular domain of c-MPL, such as a thrombopoietin binding domain. In some examples, the extracellular domain includes amino acids corresponding to amino acids 26-491 of SEQ ID NO: 8. In additional examples, the chimeric polypeptides include a signal peptide, which in some examples, is amino acids 1-25 of SEQ ID NO: 8. The chimeric polypeptides also include a transmembrane domain located between the extracellular domain and the intracellular domain. In some aspects, the transmembrane domain is from c-MPL, for example, amino acids corresponding to amino acids 492-513 of SEQ ID NO: 8. In other aspects, the transmembrane domain is from CD8 (e.g., IYIWAPLAGTCGVLLLSLVIT; SEQ ID NO: 9), CD28 (e.g., FWVLVVVGGVLACYSLLVTVAFIIFWV; SEQ ID NO: 10), CD16A (e.g., VSFCLVMVLLFAVDTGLYFSV; SEQ ID NO: 11), ICOS (e.g., FWLPIGCAAFVVVCILGCILI; SEQ ID NO: 12), KIR2DS2 (e.g., VLIGTSVVKIPFTILLFFLL; SEQ ID NO: 13), or NKG2D (e.g., PFFFCCFIAVAMGIRFIIMVT; SEQ ID NO: 14). Additional transmembrane domains can be selected by one of ordinary skill in the art, such as those described in U.S. Pat. App. Publ. No. 2021/0015864.
In additional aspects, the chimeric polypeptides also include at least a portion of an intracellular domain of c-MPL. In some examples, the chimeric polypeptides include membrane proximal portions of the intracellular domain of c-MPL, such as box 1 and/or box 2 domains, which are known to interact with Jak2 kinase and initiate signal transduction events upon ligand engagement (see, e.g.,
In some aspects, the disclosed chimeric polypeptides include an intracellular domain including at least a portion of an intracellular domain of IL2RB, such as an intracellular signaling domain. In some examples, the intracellular signaling domain of IL2RB includes amino acids corresponding to amino acids 583-801 of SEQ ID NO: 8. In other examples, the intracellular signaling domain of IL2RB includes amino acids corresponding to amino acids 583-604 of SEQ ID NO: 21.
In other aspects, the disclosed chimeric polypeptides include an intracellular domain including at least a portion of an intracellular domain of IL21R, such as an intracellular signaling domain. In some examples, the intracellular signaling domain of IL21R includes amino acids corresponding to amino acids 583-795 of SEQ ID NO: 22.
In further aspects, the disclosed chimeric polypeptides include an intracellular domain including at least a portion of an intracellular domain of IL7R, such as an intracellular signaling domain. In some examples, the intracellular signaling domain of IL7R includes amino acids corresponding to amino acids 583-728 of SEQ ID NO: 23.
In additional aspects, the disclosed chimeric polypeptides include an intracellular domain including at least a portion of an intracellular domain of IFNAR2, such as an intracellular signaling domain. In some examples, the intracellular signaling domain of IFNAR2 includes amino acids corresponding to amino acids 583-787 of SEQ ID NO: 24.
In still further aspects, the disclosed chimeric polypeptides include an intracellular domain including at least a portion of an intracellular domain of IL12RB2, such as an intracellular signaling domain. In some examples, the intracellular signaling domain of IL12RB2 includes amino acids corresponding to amino acids 583-732 of SEQ ID NO: 25.
In some aspects, the disclosed chimeric polypeptides have at least 90% (such as at least 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to the amino acid sequence of any one of SEQ ID NO: 8 and SEQ ID NOs: 21-25. In some examples, the chimeric polypeptide includes or consists of the amino acid sequence of any one of SEQ ID NO: 8 and SEQ ID NOs: 21-25. Also provided are nucleic acids encoding the disclosed chimeric polypeptides. In some examples, the chimeric polypeptide is encoded by a nucleic acid with at least 90% (such as at least 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to the nucleic acid sequence of any one of SEQ ID NO: 7 and SEQ ID NOs: 26-30. In some examples, the chimeric polypeptide is encoded by a nucleic acid including or consisting of the nucleic acid sequence of any one of SEQ ID NO: 7 and SEQ ID NOs: 26-30. Vectors including nucleic acids encoding the chimeric polypeptides are also provided, such as the vectors described in Section II.
The following examples are provided to illustrate certain particular features and/or aspects. These examples should not be construed to limit the disclosure to the particular features or aspects described.
Materials and Methods
Cell culture and cell lines: NK-92, Raji, 721.221, and K562 cell lines were purchased from ATCC (VA, USA). The KHYG-1 cell line was purchased from Leibniz Institute DSMZ (Germany). NK-92 cells were cultured in RPMI medium supplemented with 12.5% heat-inactivated fetal bovine serum (FBS), 12.5% horse serum and 2 mM L-glutamine (Life Technologies). The KHYG-1 cells were cultured in RPMI medium containing 10% FBS, penicillin/streptomycin (100 μg/ml) and 2 mM L-glutamine. Both NK-92 and KHYG-1 were supplemented with 200 U/ml recombinant human (rh) IL-2 (Roche). Raji, K562, and 721.221 cells were also cultured in RPMI medium with 10% FBS, while 293T cells were maintained in Dulbecco's Modified Eagle's medium (DMEM) with 10% FBS and 2 mM L-glutamine. All cell cultures were at 37° C. with 5-6.5% CO2.
Lentiviral production and transduction: Lentivirus gene expression vectors encoding EGFP-P2A linker-human EPOR or EGFP-P2A-human c-MPL were cloned according to our design by VectorBuilder (TX, USA). Plasmid DNA for the above and pCMV-dR8.2 dvpr and pCMV-VSV-G packaging plasmids was purified using ZymoPUREII-EndoZero plasmid maxiprep kit (Zymo Research, CA). Lentiviral particles were generated with HEK293T cells and Lipofectamine3000 reagent (Thermo Fisher Scientific, MA) following the manufacturers' instructions. Viral supernatant was harvested at 24 and 48 hours, pooled, and concentrated with lenti-X concentrator (Takara Bio, Japan) or by centrifugation at 18,600 rpm for 2 hours at 4° C. The target cells were transduced with lentivirus at a multiplicity of infection (MOI) of 20 using retronectin (Takara Bio, Japan) coated plate protocol. After 5 days post-transduction, cells were stained and sorted with fluorescence-activated cell sorting (FACS) using a BD FACSAria II to purify GFP+EPOR+ and GFP+c-MPL+ NK cell populations.
In vitro cell expansion cultures: The NK-92 (5×104 cell/ml) and KHYG-1 (1×104 cell/ml) cells were cultured in 1 ml of complete medium in 24-well plate. The NK cell lines were supplemented with different IL-2 doses, 5 U/ml rhEPO (R&D) and/or 50 ng/ml rhTPO (R&D) depending on experiments. Trypan blue negative cells were counted every other day. On day 4, the medium was changed and replaced with fresh IL-2/EPO/TPO as their initial conditions. The short-term cell expansion assay was performed in similar fashion, except cells were initially cultured without IL-2 overnight before adding different cytokines and enumerating the cell number. For competitive primary NK and T cell expansion assay, all transduced cells (5×105/ml for NK and 4×105/ml for T cell) containing a mixture of GFP positive (transduced) and GFP negative (untransduced) cells were plated on day 0. Fresh medium with appropriate supplements was replaced every other day for NK cells and only on day 3 for T cell experiment. Viable cells were counted and percentage of GFP+ cells was acquired on indicated days (NK: day 4 and 6, T: day 3 and 6). The fold change of GFP+ cells was calculated by multiplying the counted cell number times the fraction GFP+.
Flow cytometry, reagents and pathway analysis: Fluorochrome-conjugated monoclonal antibodies were used as follows: BD Biosciences: CD56-PE-Cy7 (NCAM16.2), CD3-PE (UCHT1), c-MPL-PE (1.6.1), STAT5(pY694)-Alexa Fluor647 (47/stat5(pY694)), NKp46-APC (9E2), NKp44-PE (p44-8), FAS-PE (DX2) CD62L-V450 (DREG-56); R&D systems: EPOR-PE (FAB307P); Biolegend: Bc1-2-PE (100), TRAIL-APC (RIK-2), CXCR4-BV421 (12G5), KIR3DL1-BV421 (DX9); Abcam: Bcl-xL-PE (ab208747); Cell signaling technology: P-p44/42 MAPK-Alexa Fluor647 (E10) and p-S6 ribosomal protein-PE (D57.2.2E); Beckman Coulter: NKG2A-PE (Z199), KIR2DL1/S1-PE (EB6B), KIR2DL2/3-PE/Cy5.5 (GL183). Staining was performed in phosphate-buffered saline (PBS) with 2% FBS and 2 mM EDTA and analyzed on a BD LSRFortessa machine. If intracellular staining was required, BD cytofix/cytoperm (BD Biosciences) was applied for cell fixation and permeabilization after surface staining. The apoptosis assay was performed using PE annexinV apoptosis detection kit (BD Biosciences). The data were analyzed on FlowJo software.
For measurement of cell signaling and anti-apoptotic proteins, cells were cultured overnight without IL-2 then incubated in appropriate medium supplemented with different IL-2/EPO/TPO conditions for 15 minutes for phospho-protein detection and 24 hours for anti-apoptotic protein studies. Expression of target proteins was evaluated afterward by flow cytometry as described above.
Flow cytometry based killing assay: The effector cells were prepared by culturing EPOR+ NK-92 and c-MPL+ NK-92 cells in different IL-2/EPO/TPO concentrations for 24-40 hours before the experiment. The target cells were labeled with 1 μM cell trace violet in some experiments (Thermo Fisher Scientific). The effector and target cells were incubated together in duplicate wells at specific E:T ratio in complete medium without cytokines for 4 hours (total cell number: 200,000 to 250,000 per well in 96-well plate). 7-AAD and Sphero AccuCount Fluorescent particles (Spherotech, IL) were added before analysis by flow cytometry. The absolute number of viable 7-AAD negative target cells was calculated according to bead numbers added (Cooper et al., Leukemia 32:1970-1983, 2018). The percentage of dead cells was calculated relative to target cell only control wells.
Primary NK cell activation and transduction: Peripheral blood NK cells and T cells from healthy volunteers were isolated from de-identified buffy-coat samples. NK cells were separated by immune-density negative selection and density gradient centrifugation using RosetteSep human NK cell enrichment cocktail (Stemcell Technologies, Canada) and lymphocyte separation medium (MP Biomedicals, CA). The primary NK cells were activated with irradiated Epstein-Barr virus (EBV) transformed lymphoblastic cell line (SMI-LCL) (Berg et al., Cytotherapy 11:341-355, 2009) and maintained in X-Vivo20 medium with 10% heat-inactivated human serum, 2 mM L-glutamine and 500 U/ml IL-2. After 4-5 days of NK cell activation, NK cells were transduced with the previously described lentiviral particles (MOI=20) in combination with 1.5 μM BX795 (InvivoGen, CA). On day 3 post-transduction, cells were evaluated for their expression of CD3, GFP, CD56, EPOR and c-MPL expression. The transduction efficiency (measured as GFP positive NK cells) was 4-57%. Both activated GFP+ and GFP− NK cells were utilized in the competitive cell expansion assay.
Degranulation assay and cytokine production: Transduced human NK cells or control untransduced cells were cultured with IL-2 and/or TPO for 4 days, then mixed at a 1:1 ratio with K562 target cells (2.5×105 total cells per well of 96 well plate) in medium lacking added cytokines. To examine degranulation, cells were washed after two hours and stained with anti-CD107a (H4A3) (BioLegend). To assay cytokine production, GolgiPlug and GolgiStop (BD Biosciences) were added at manufacturer's recommended concentrations within the first hour of co-culture with K562. After 4 hours, cells were washed and stained for cell surface markers, then stained intracellularly with mAb recognizing TNF-α (Mab 11) and IFN-γ (B27) (BD Biosciences).
Persistence of transduced human NK cells in immunodeficient mice: NK cells from human PBMC were transduced with matched lentiviral vectors encoding GFP/MPL or GFP/CD34 then mixed at a 1:1 ratio and injected intravenously into NSG mice (2.2×107 total cells/mouse). Mice received daily intraperitoneal injections of human TPO (50 μg/kg/mouse) or human IL-2 (10,000 IU/mouse) for 4 days. After euthanasia, blood was drawn by cardiac puncture, bone marrow was harvested by flushing the tibia, and spleen, liver, and lung fragments were processed by grinding over 70 μm nylon mesh. Blood, bone marrow, lung, and spleen samples received 1-2 rounds of 5 minute incubations with ACK lysis buffer (Quality biological). Liver samples were purified by centrifugation over 35% Percoll (Sigma) solution in RPMI. The presence of transduced human cells was quantified in various organs via flow cytometry using antibodies to human MHC class I (W6/32) (BioLegend), c-MPL (1.6.1), CD34 (581) (BD BioSciences), GFP-fluorescence, and Live/Dead Aqua viability dye (Thermo Fisher).
Primary T cell activation and transduction: Peripheral blood mononuclear cells (PBMC) were obtained from healthy donor buffy-coat samples as specified previously and were separated by density gradient centrifugation. The PBMC were activated with the CD3 antibody (OKT3, Invitrogen)-coated plate and soluble CD28 antibody (CD28.2, Biolegend) and maintained in RPMI with 10% FBS and 300 U/ml IL-2. After 2 days of T cell activation, cells were transduced with the previously described lentiviral particles (MOI=10). On day 4 post-transduction, cells were evaluated for the expression of CD3, GFP, EPOR and c-MPL expression, in each case showing >95% CD3+ cells. The transduction efficiency (measured as GFP positive T cells) was 40-60%. Both activated GFP+ and GFP− T cells were utilized in the competitive cell expansion assay.
Statistical analysis: Data were analyzed and presented using GraphPad Prism software. The mean, standard deviation (SD) and p-value were displayed with statistical tests as indicated.
Exogenous Expression of EPOR or c-MPL in Human NK Cell Lines Enhances their Proliferative Response to Low-Dose IL-2 in the Presence of EPO or TPO
To investigate the functional effects of exogenously expressing EPOR or c-MPL in NK cells, the IL-2-dependent human NK cell line NK-92 was transduced using lentiviral vectors to stably express EPOR or c-MPL along with an EGFP marker (
Introduction of soluble EPO to cultures of EPOR+ NK-92 supported the growth of NK cells independent of IL-2, with a 20-fold expansion occurring by day 8 (
In a similar fashion, c-MPL+ NK-92 showed IL-2-independent proliferation in cultures supplemented with TPO alone (7-fold expansion on day 8) (
EPOR and c-MPL Expressing NK-92 Cells Up-Regulate the Anti-Apoptotic Proteins Bcl-2 and Bcl-xL Upon EPO or TPO Stimulation Enhancing their Survival
EPOR and c-MPL+ NK-92 cells were used for subsequent studies to characterize in more detail the consequences of ligating these exogenous receptors. Simultaneous short-term cell expansion and apoptosis assays examined the mechanisms underlying EPOR and c-MPL-induced increases in NK cell numbers (
Anti-apoptotic proteins, especially Bc1-xL, have been shown to be regulated by EPO/EPOR and TPO/c-MPL signaling in erythroid progenitors and megakaryocytic cells, respectively. Therefore, transduced NK-92 cells were queried for down-stream signaling pathways including Bcl-2 and Bcl-xL. EPOR and c-MPL have been shown to signal via three main pathways: JAK-STAT, PI3K-AKT and MAPK, which can be assessed using antibodies recognizing pSTAT5, pS6 and phospho-p44/42 MAPK. No alteration of phospho-p44/42 MAPK expression was detected in EPOR+ and c-MPL+ NK-92 cells when cultures were supplemented with IL-2, EPO or TPO (
The Bc1-2 and Bc1-xL proteins were investigated as potential target proteins of EPOR and c-MPL signaling in NK cells. As shown in
These results suggest that hematopoietic growth factor receptors exogenously expressed in NK-92 cells can signal to up-regulate the anti-apoptotic proteins Bc1-2 and Bc1-xL, which together prolong cell survival. In EPOR+ NK-92 cells, this signaling may proceed through STAT5, while MPL+ NK-92 cells may utilize both STAT5 and AKT-S6 pathways. Similar results were obtained in experiments querying other time points of the cell culture (
Expression of c-MPL in NK-92 Cells Augments their Killing of Raji Tumor Cells in the Presence of TPO
In order to test the functional consequences of expressing EPO or TPO receptors, transgenic EPOR and c-MPL+ NK-92 cells were expanded in the presence of EPO or TPO and cytotoxicity assays were performed with three different tumor cell lines (Raji, K562, and 721.221). Compared to parental controls, no deficit in the killing capacity of transduced NK-92 cells expanded in presence of EPO or TPO was observed (data not shown). Compared to LD IL-2 alone, there was a trend towards higher Raji killing by EPOR+ NK-92 cells stimulated with EPO and LD IL-2, while killing between LD IL-2 and EPO stimulated EPOR+ NK-92 cells against 721.221 or K562 tumor cell lines was similar (
Transgenic Expression of c-MPL in Primary NK Cells Promotes Proliferation in Presence of TPO
Data from the above experiments indicated proliferative, survival, and functional benefits of over-expressing EPOR or c-MPL in NK 92 cells in the presence of EPO or TPO and LD IL-2. Therefore, using the same lentiviral constructs as used to transduce NK cell lines, 4-37% of primary NK cells (activated before transduction with IL-2 plus an EBV-transformed lymphoblastic cell line) were successfully transduced to express either EPOR/GFP or c-MPL/GFP (
c-MPL-Transduced Primary NK Cells Show Increased Anti-Tumor Function Upon TPO Stimulation
To investigate the effects on NK cell function, primary NK cells were transduced with a c-MPL/GFP encoding lentiviral vector, cultured for four days in different cytokine combinations, then were co-cultured with K562 target cells. CD107a degranulation and cytokine production were investigated in GFP+ transduced cells and untransduced controls. There was a significantly higher amount of degranulation in GFP+ MPL+ cells cultured in media with TPO and 25 U/ml IL-2 (
The overall NK cell phenotype of the c-MPL-transduced primary NK cells was also investigated using a number of antibodies recognizing NK cell receptors and molecules important for NK cell function. With the exception of CD62L (L-selectin), no phenotypic differences were observed in gated GFP+ (MPL expressing) NK cells when TPO was added to media containing 25 U/ml of IL-2 (
Primary Human NK Cells Genetically Modified to Express c-MPL Preferentially Persist Upon Adoptive Transfer into Immunodeficient Mice Receiving TPO
The capacity for exogenous c-MPL receptor expression to support adoptively transferred NK cells in vivo was examined. Primary human NK cells transduced with lentiviral constructs encoding GFP and either c-MPL or truncated surface expressed human CD34 (as a control) were mixed at a 1:1 ratio, then were injected intravenously into NOD SCID IL2rg−/− (NSG) immunodeficient mice. Following NK cell infusion, mice received daily intraperitoneal injections of TPO (50 μg/kg) or human IL-2 (10,000 IU/mouse; a dose previously reported to represent “low dose IL-2” treatment in vivo) (Granzin et al., Oncoimmunology 5:e1219007, 2016). After 4 days, GFP+ MPL+ cells represented a significantly higher proportion of human cells detected in all the organs of mice that had received TPO (
Primary T Cells Transduced to Express EPOR and c-MPL have Augmented Cell Numbers in the Presence of Ligand
The effects of transgenic expression of EPOR and c-MPL were also evaluated in primary human T cells. Efficient transduction of EPOR/GFP or c-MPL/GFP in activated T cells from healthy donors was observed, ranging from 40 to 60% positive (
c-MPL-Expressing Human NK Cells Proliferate in Response to Drug Mimetics of Thrombopoietin
NK cells from PBMC from three blood donors were stimulated and transduced to express GFP and c-MPL, then were cultured for 4-5 days with thrombopoietin, romiplostim, or eltrombopag. Numbers of GFP cells were enumerated and expressed as a percentage of numbers obtained from parallel cultures containing 500 U/mL IL-2 (
Construction and Testing of Chimeric MPL Proteins
Lentiviral and retroviral vectors expressing MPL-IL2RB and other chimeric MPL receptors: Lentiviral vectors were created by Vectorbuilder (IL, USA) by insertion of DNA sequences encoding EGFP and chimeric molecules including extracellular, transmembrane, and in some cases, proximal intracellular regions of human MPL (NM_005373.3) fused with partial intracellular domains from human IL2RB (NM_000878.5) using the linkages as shown in
In vitro cell culture experiments with human NK cells expressing MPL-IL2RB: Approximately 2-4 days after transduction, NK cells were placed in culture with varied concentrations of eltrombopag (Selleckchem, TX, USA) or romiplostim (Amgen, CA, USA); where concentrations are not indicated, 0.33 ug/mL eltrombopag or 1.5 ng/mL romiplostim were used. Medium and drugs were refreshed by addition of new medium or by changing half the culture volumes every 2-4 days. Numbers of GFP+ cells were quantitated after 4-6 days (or indicated durations) using a BD Fortessa flow cytometer (BD Biosciences, NJ, USA) and compared with initial numbers to determine the fold expansion. In other experiments, GFP+ cell numbers are expressed as a percentage of IL-2 culture by comparing with GFP+ cell numbers in paired cultures supplemented with 500 U/mL IL-2. Alternatively, transduced human NK cells were incubated overnight without added cytokines, then exposed to eltrombopag, romiplostim, and/or IL-2 for varied durations before intracellular staining to assess phospho-STAT5 or phosphor-S6 using antibodies described above.
Adoptive transfer of NK cells expressing MPL-IL2RB into mice: Human NK cells were transduced with a retroviral vector encoding both firefly luciferase and MPL-IL2RB then intravenously injected into NOD Prkdcscid Il2rgnull (NSG) immunodeficient mice (Jackson Laboratory, Maine USA). Animals received daily oral eltrombopag by gavage, intraperitoneal IL-2, or no drug. NK cell persistence was measured by whole body luminescence imaging, normalizing to values observed after initial injection of the same animal.
When expressed in NK cells, the human MPL receptor resulted in increased NK cell numbers and augmented NK cell function upon ligation with specific ligands. However, effects of MPL were often less than those obtained when IL-2 was used in vitro at high doses. Of note, these high doses of IL-2 used in vitro likely could not be achieved in patients due to prohibitive toxicity of IL-2.
Aiming to improve the potency of MPL when expressed in NK cells, by making its intracellular signal transduction more resemble that induced by IL-2, several chimeric molecules containing extracellular ligand-binding and trans-membrane (TM) domains of human MPL fused with portions of the intracellular signaling domains of the human IL-2 receptor beta chain, IL2RB were produced (
Each of these chimeric molecules was expressed in primary human NK cells using lentiviral vectors. When transferred into culture medium lacking cytokines but containing varied concentrations of the pharmaceutical MPL ligands eltrombopag or romiplostim, NK cells expressing MPL showed greater cell numbers after 4 days, compared with untransduced NK cells (
Survival and/or proliferation of NK cells expressing MPL-IL2RB chimeric receptor were examined over a wider time course in vitro. In this case and following, MPL-IL2RB or MPL were expressed in NK cells using retroviral vectors. When stimulated with varied concentrations of eltrombopag (
Effects of MPL-IL2RB on functional activity of human NK cells were examined. NK cells expressing either MPL-IL2RB or MPL showed augmented degranulation of cytotoxic granules in response to K562 target cells after 4-day exposure to eltrombopag or romiplostim, reaching levels comparable to those obtained by culture with IL-2 (
Both thrombopoietin and IL-2 can trigger STAT5 and mTOR signaling pathways in cells normally sensitive to these factors. Therefore, these signals were examined over time in NK cells transduced to express MPL or MPL-IL2RB chimeric receptors. Eltrombopag stimulation of these cells resulted in increased phosphorylated STAT5 which tended to be prolonged (at least 24 hours) although lesser in magnitude than that induced by 5 U/mL IL-2 (
Additivity of the effects of IL-2 and eltrombopag was also examined for their potential to maintain and expand receptor-expressing NK cell numbers in culture. Human NK cells transduced to express MPL-IL2RB or MPL were placed into culture with a matrix of concentrations of IL-2 and eltrombopag, before measuring cell numbers after 6 days. At low concentrations of IL-2, MPL-IL2RB-expressing NK cells showed greater numbers than those expressing MPL (
Maintenance of MPL-IL2RB-expressing NK cells was examined after adoptive transfer into immunodeficient mice. NK were transduced with a construct expressing both MPL-IL2RB and firefly luciferase, facilitating cell tracking by luminescent imaging. When compared with mice lacking drug administration, mice receiving 25 or mg/kg of eltrombopag exhibited dose dependent preservation of transferred NK cells (
Additional chimeric molecules including intracellular domains of IL21R, IL7R, IL12RB2, IFNAR2, or a truncated IL2RB intracellular domain (MPL-IL2RBv5) were expressed in primary human NK cells using retroviral vectors, as described above. Cells were transferred into culture medium lacking cytokines but containing varied concentrations of the pharmaceutical MPL ligands—eltrombopag or romiplostim (
Testing of MPL-IL2RB-NK Cells in a Mouse Model of Multiple Myeloma
Human NK cells were stimulated with LCL feeder cells and IL-2 and transduced with retroviral particles encoding MPL or MPL-IL2RB chimera (as described in Example 10). Two days subsequently, NK cells were washed with Hyclone MaxCyte electroporation buffer, mixed with precomplexed gRNA targeting CD38 (10 μM) (Synthego Corporation, Redwood City, CA) and purified Cas9 (2 μM) and electroporated with the MaxCyte ATX Transfection System (MaxCyte, Rockville, MD) using the NK-04 electroporation protocol (as described in Clara et al, J Immunother Cancer 2022;10:e003804). NOD Prkdcscid Il2rgnull (NSG) immunodeficient mice (Jackson Laboratories) received 2×106 firefly luciferase-transduced MM.1S cells by intravenous injection. After two days, mice received 107 modified NK cells intravenously along with 10 μg intraperitoneal daratumumab (Janssen Pharmaceuticals, Beerse, Belgium). Depending on the experimental group, mice were supported by daily oral gavage of eltrombopag (50 mg/kg) for 15 days or intraperitoneal IL-2 (50,000 IU/mouse) for three days starting from the day of NK cell injection. Tumor progression was followed by bioluminescent imaging. A schematic illustration of the experimental protocol is shown in
treatment group. At day 8 after tumor injection, the control group (no treatment) exhibited rapid expansion, while mice treated with daratumumab, untransduced NK cells or MPL-NK cells, and EPAG did not show expansion (
It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described aspects of the disclosure. We claim all such modifications and variations that fall within the scope and spirit of the claims below.
This application is a continuation-in-part of co-pending U.S. patent application Ser. No. 17/821,703, filed Aug. 23, 2022, which is a continuation of International Application No. PCT/US2021/019288, filed Feb. 23, 2021, which claims the benefit of U.S. Provisional Application No. 62/980,854, filed Feb. 24, 2020, each of which is incorporated herein by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62980854 | Feb 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2021/019288 | Feb 2021 | US |
| Child | 17821703 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17821703 | Aug 2022 | US |
| Child | 18480306 | US |
