Methods of making and using embryonic mesenchymal progenitor cells
阅读说明：本技术 制备和使用胚胎间充质祖细胞的方法 (Methods of making and using embryonic mesenchymal progenitor cells ) 是由 E.格施文格 R.罗德里格斯 Y.欧阳 于 2018-05-25 设计创作，主要内容包括：本公开提供了生成非聚簇的干细胞的方法。中胚层分化之前的聚簇破坏提高了hEMP和T细胞分化的产量和效率。因此,该方法允许开发改进的hEMP和T细胞分化的方法。(The present disclosure provides methods of generating non-clustered stem cells. Disruption of clustering prior to mesodermal differentiation increases the yield and efficiency of hEMP and T cell differentiation. Thus, this approach allows the development of improved methods of hEMP and T cell differentiation.)
1. A method of generating human embryonic mesenchymal progenitor (hEMP) cells, the method comprising the steps of:
(a) contacting the non-clustered stem cells with a substrate at a determined single cell density;
(b) culturing the stem cells under culture conditions that promote cell growth to a desired confluence; and
(c) altering the culture conditions to induce differentiation of the stem cells into hEMP cells within a desired incubation time;
thereby generating the hEMP cells.
2. The method of claim 1, wherein the stem cell is a human Embryonic Stem (ES) cell or an Induced Pluripotent Stem (iPS) cell.
3. The method of claim 2, wherein the ES or iPS cells are of human origin.
4. The method of claim 3, wherein the ES or iPS cells are H1 cells, H9 cells, HES3 cells, HSF1 cells, HSF6 cells, ESI-017 cells, CS02iCTR-NTn1 cells, CS03iCTR-NTn1 cells, CS80iCTR-Tn3 cells, CS179iCTR-NTn1 cells, CS201iCTR-NTn4 cells, CS202iCTR-NTn2 cells, or CS206iCTR-Tn5 cells.
5. The method of any one of the preceding claims, wherein the determined single cell density is at about 1.5x105And about 8x105Individual cell/cm2In the meantime.
6. The method of claim 5, wherein said determined single cell density is about 1.89x105Individual cell/cm2About 3.2x105Individual cell/cm2About 3.4x105Individual cell/cm2About 3.6x105Individual cell/cm2About 3.79x105Individual cell/cm2About 7.2x105Individual cell/cm2Or about 7.58x105Individual cell/cm2。
7. The method of any one of the preceding claims, wherein the substrate is used with
8. The method of any one of the preceding claims, wherein the substrate is a well plate, a cell culture dish, a membrane, a bag, a culture flask, an inverse opal, a polymer lattice, a static cell suspension, a stirred cell suspension, or a plasma-treated polymer.
9. The method of claim 8, wherein the substrate comprises a membrane.
10. The method of any one of the preceding claims, wherein the culture conditions that promote cell growth comprise culturing the stem cells in mTeSR1 medium.
11. The method of claim 10, wherein the mTeSR1 medium comprises ROCK inhibitor Y27632.
12. The method of any one of the preceding claims, wherein the desired confluence is between about 20% and about 80%.
13. The method of claim 12, wherein the confluence is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80%.
14. The method of any one of the preceding claims, wherein the altering cultureThe step of conditioning comprises adding X-VIVOTM15。
15. The method of any one of the preceding claims, wherein the incubation time is between about 2 and about 4 days.
16. The method of claim 15, wherein the incubation time is about 2.0, about 2.5, about 3.0, about 3.5, or about 4.0 days.
17. The method of claim 16, wherein the incubation time is about 3.5 days.
18. The method of any one of the preceding claims, further comprising the step of differentiating said hEMP cells into T cells.
19. The method of any one of the preceding claims, wherein the method further comprises the step of disrupting the clustering of stem cells to generate the non-clustered stem cells.
20. The method of claim 19, wherein the clustering of the stem cells is disrupted by mechanical or chemical disruption.
21. The method of claim 20, wherein the chemical disruption comprises incubation with a trypsin-like enzyme.
22. The method of claim 21, wherein said trypsin-like enzyme is trypsin, TrypLE Express, TrypLE Select, collagenase, dispase, trypsin-EDTA.
23. A human embryonic mesenchymal progenitor (hEMP) cell generated according to the method of any one of the preceding claims.
24. A composition comprising a population of human embryonic mesenchymal progenitor (hEMP) cells generated according to the method of any one of claims 1-22.
25. A method of generating T cells, the method comprising the steps of:
(a) contacting non-clustered stem cells with a matrix at a determined single cell density, wherein the stem cells do not comprise Mouse Embryonic Fibroblasts (MEFs);
(b) culturing the stem cells under culture conditions that promote cell growth to a desired confluence; and
(c) altering the culture conditions to induce differentiation of the stem cells into T cells within a desired incubation time;
thereby generating T cells from the stem cells.
26. The method of claim 25, wherein the stem cell is a human Embryonic Stem (ES) cell or an Induced Pluripotent Stem (iPS) cell.
27. The method of claim 26, wherein the ES or iPS cells are of human origin.
28. The method of claim 26 or 27, wherein the ES or iPS cells are H1 cells, H9 cells, HES3 cells, HSF1 cells, HSF6 cells, ESI-017 cells, CS02iCTR-NTn1 cells, CS03iCTR-NTn1 cells, CS80iCTR-Tn3 cells, CS179iCTR-NTn1 cells, CS201iCTR-NTn4 cells, CS202iCTR-NTn2 cells, or CS206iCTR-Tn5 cells.
29. The method of any one of claims 25-28, wherein the determined single cell density is at about 1.5x105And about 8x105Individual cell/cm2In the meantime.
30. The method of any one of claims 25-29, wherein the determined single cell density is about 1.89x105Individual cell/cm2About 3.2x105Individual cell/cm2About 3.4x105Individual cell/cm2About 3.6x105Individual cell/cm2About 3.79x105Individual cell/cm2About 7.2x105Individual cell/cm2Or about 7.58x105Individual cell/cm2。
31. The method of any one of claims 25-30, wherein the substrate is used with
32. The method of any one of claims 25-31, wherein the substrate is a well plate, a cell culture dish, a membrane, a bag, a culture flask, an inverse opal, a polymer lattice, a static cell suspension, a stirred cell suspension, or a plasma-treated polymer.
33. The method of claim 32, wherein the substrate comprises a membrane.
34. The method of any one of claims 25-33, wherein the culture conditions that promote cell growth comprise culturing the stem cells in mTeSR1 medium.
35. The method of claim 34, wherein the mTeSR1 medium comprises ROCK inhibitor Y27632.
36. The method of any one of claims 25-35, wherein the desired confluence is between about 20% and about 80%.
37. The method of claim 36, wherein the confluence is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80%.
38. The method of any one of claims 25-37, wherein the step of changing culture conditions comprises adding X-VIVOTM15。
39. The method of any one of claims 25-38, wherein the incubation time is between about 2 and about 4 days.
40. The method of claim 39, wherein the incubation time is about 2.0, about 2.5, about 3.0, about 3.5, or about 4.0 days.
41. The method of claim 40, wherein the incubation time is about 3.5 days.
42. The method of any one of claims 25-41, wherein the method further comprises the step of disrupting the clustering of stem cells to generate the non-clustered stem cells.
43. The method of claim 42, wherein the clustering of the stem cells is disrupted by mechanical or chemical disruption.
44. The method of claim 43, wherein the chemical disruption comprises incubation with a trypsin-like enzyme.
45. The method of claim 44, wherein said trypsin-like enzyme is trypsin, TrypLE Express, TrypLE Select, collagenase, dispase, trypsin-EDTA.
46. A T cell produced according to the method of any one of claims 25-45.
47. A composition comprising a population of T cells generated according to the method of any one of claims 25-45.
Background
Human cancers by their very nature comprise normal cells that have undergone genetic or epigenetic transformation to become abnormal cancer cells. At this point, the cancer cells begin to express proteins and other antigens that are different from those expressed by normal cells. These aberrant tumor antigens can be used by the body's immune system to specifically target and kill cancer cells. However, cancer cells employ various mechanisms to prevent immune cells, such as T and B lymphocytes, from successfully targeting cancer cells.
Current T cell therapies rely on enriched or modified human T cells to target and kill cancer cells in a patient. Patient or normal donor-derived T cells are limited in nature due to their lack of self-renewal. Derivation of T cells from stem cell sources would provide a potentially unlimited source of cells for therapeutic use. There is a need for efficient in vitro methods to differentiate pluripotent stem cells into embryonic mesodermal progenitor cells and mature T cells.
Summary of The Invention
The present invention fulfills this need, among others, by providing improved methods for generating human embryonic mesenchymal progenitor (hEMP) cells, derivatives thereof, and uses thereof in the efficient generation of T cells.
In one aspect, the present disclosure provides a method of generating human embryonic mesenchymal progenitor (hEMP) cells, the method comprising the steps of: contacting the non-clustered stem cells with a substrate at a determined single cell density; culturing the stem cells under culture conditions that promote cell growth to a desired confluence; and altering the culture conditions to induce differentiation of the stem cells into hEMP cells within the desired incubation time; thereby generating the hEMP cells.
In some embodiments, the non-clustered stem cells are human Embryonic Stem (ES) cells or Induced Pluripotent Stem (iPS) cells. In some embodiments, the ES or iPS cells are of human origin.
In some embodiments, the ES or iPS cell is an H1 cell, an H9 cell, an HES3 cell, an HSF1 cell, an HSF6 cell, an ESI-017 cell, a CS02iCTR-NTn1 cell, a CS03iCTR-NTn1 cell, a CS80iCTR-Tn3 cell, a CS179iCTR-NTn1 cell, a CS201iCTR-NTn4 cell, a CS202iCTR-NTn2 cell, or a CS206iCTR-Tn5 cell.
In some embodiments, the determined single cell density is about 1.5x105And about 8x105Individual cell/cm2In the meantime. In certain embodiments, the determined single cell density is about 1.89x105Individual cell/cm2About 3.2x105Individual cell/cm2About 3.4x105Individual cell/cm2About 3.6x105Individual cell/cm2About 3.79x105Individual cell/cm2About 7.2x105Individual cell/cm2Or about 7.58x105Individual cell/cm2。
In some embodiments, the substrate is used
In some embodiments, the culture conditions that promote cell growth comprise culturing the stem cells in mTeSR1 medium. In some embodiments, the mTeSR1 medium further comprises a ROCK inhibitor. In certain embodiments, the mTeSR1 medium further comprises ROCK inhibitor Y27632.
In some embodiments, the cells are grown to a desired confluence of between about 20% and about 80%. In some embodiments, the confluence is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80%.
In some embodiments, the step of changing culture conditions comprises changing culture conditions in X-VIVOTM15 medium.
In some embodiments, the incubation time is between about 2 and about 4 days. In some embodiments, the incubation time is about 2.0, about 2.5, about 3.0, about 3.5, or about 4.0 days. In certain embodiments, the incubation time is about 3.5 days.
In some embodiments, the method further comprises the step of differentiating the hEMP cells into T cells.
In some embodiments, the method further comprises the step of disrupting the clustering of the stem cells to generate non-clustered stem cells. In some embodiments, the clustering of stem cells is disrupted by mechanical or chemical disruption. In some embodiments, the chemical disruption comprises incubation with a trypsin-like enzyme (TrypLE). In certain embodiments, the trypsin-like enzyme is trypsin, TrypLE Express, TrypLE Select, collagenase, dispase, or trypsin-EDTA.
In some embodiments, human embryonic mesenchymal progenitor (hEMP) cells are generated according to the methods described herein.
In one aspect, the disclosure provides compositions comprising a population of human embryonic mesenchymal progenitor (hEMP) cells generated according to the methods described herein.
In one aspect, the present disclosure provides a method of generating T cells, the method comprising the steps of: contacting non-clustered stem cells with a matrix at a determined single cell density, wherein the stem cells do not comprise Mouse Embryonic Fibroblasts (MEFs); culturing the stem cells under culture conditions that promote cell growth to a desired confluence; and altering the culture conditions to induce differentiation of the stem cells into T cells within the desired incubation time; thereby generating T cells from the stem cells.
In some embodiments, the stem cell is a human Embryonic Stem (ES) cell or an Induced Pluripotent Stem (iPS) cell. In some embodiments, the ES or iPS cells are of human origin.
In some embodiments, the ES or iPS cell is an H1 cell, an H9 cell, an HES3 cell, an HSF1 cell, an HSF6 cell, an ESI-017 cell, a CS02iCTR-NTn1 cell, a CS03iCTR-NTn1 cell, a CS80iCTR-Tn3 cell, a CS179iCTR-NTn1 cell, a CS201iCTR-NTn4 cell, a CS202iCTR-NTn2 cell, or a CS206iCTR-Tn5 cell.
In some embodiments, the determined single cell density is about 1.5x105And about 8x105Individual cell/cm2In the meantime. In certain embodiments, the determined single cell density is about 1.89x105Individual cell/cm2About 3.2x105Individual cell/cm2About 3.4x105Individual cell/cm2About 3.6x105Individual cell/cm2About 3.79x105Individual cell/cm2About 7.2x105Individual cell/cm2Or about 7.58x105Individual cell/cm2。
In some embodiments, the substrate is used
In some embodiments, the culture conditions that promote cell growth comprise culturing the stem cells in mTeSR1 medium. In some embodiments, the mTeSR1 medium further comprises a ROCK inhibitor. In certain embodiments, the mTeSR1 medium further comprises ROCK inhibitor Y27632.
In some embodiments, the cells are grown to a desired confluence of between about 20% and about 80%. In some embodiments, the confluence is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80%.
In some embodiments, the step of changing culture conditions comprises changing culture conditions in X-VIVOTM15 medium.
In some embodiments, the incubation time is between about 2 and about 4 days. In some embodiments, the incubation time is about 2.0, about 2.5, about 3.0, about 3.5, or about 4.0 days. In certain embodiments, the incubation time is about 3.5 days.
In one aspect, the disclosure provides T cells generated according to the methods described herein.
In one aspect, the disclosure provides a composition comprising a population of T cells generated according to the methods described herein.
Any aspect or embodiment described herein may be combined with any other aspect or embodiment as disclosed herein. While the present disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
The patent and scientific literature referred to herein establishes knowledge available to those skilled in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. All other published references, dictionaries, documents, manuscripts, and scientific literature cited herein are hereby incorporated by reference.
Other features and advantages of the disclosure will be apparent from the accompanying drawings and from the following detailed description, including the embodiments, and from the claims.
Brief Description of Drawings
The above described and other features will be more clearly understood from the following detailed description when taken in conjunction with the accompanying drawings. However, these drawings are for illustration purposes only and are not limiting.
Fig. 1 shows exemplary flow cytometry data illustrating the variation of surface expressed CD326 and CD 56.
Fig. 2 shows an abstract view of exemplary flow cytometry data illustrating the variation of surface expressed CD326 and CD 56.
Fig. 3 shows an exemplary flow chart for generating hEMP cells from ES or iPS cells.
FIG. 4 shows exemplary flow cytometry data, which is illustrated inAnd changes in surface expression of CD326 and CD56 on vitronectin substrates.
Fig. 5 shows an exemplary flow chart for generating hEMP cells from ES or iPS cells.
Figure 6 shows flow cytometry data, which illustrates that the cells are pure induced and sorted hEMP cells. Post-sort purity was determined to be > 95% for both non-hEMP and hEMP cells.
Figure 7 shows flow cytometry data, which illustrates analysis of T cell development at week 4. The harvested cells were stained with antibodies against the following antigens: CD45, CD56, CD3, CD4, CD8, TCRab.
Definition of
In order that this disclosure may be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification.
As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.
As used herein, the term "or" should be understood to be inclusive and encompass both "or" and "unless the context clearly dictates otherwise.
The term "and/or" as used herein is to be taken as a specific disclosure of each of the two specified features or components, with or without the other. Thus, the term "and/or" as used in phrases such as "a and/or B" herein is intended to include a and B, a or B, a (alone) and B (alone). Likewise, the term "and/or" as used in phrases such as "A, B and/or C" is intended to encompass each of the following: A. b and C; A. b or C; a or C; a or B; b or C; a and C; a and B; b and C; a (alone); b (alone); and C (alone).
As used herein, the terms "such as" and "i.e.," are used by way of example only and are not intended to be limiting, and should not be construed as referring only to those items explicitly recited in the specification.
The term "or more", "at least", "over", etc., e.g., "at least one" should be understood to include, but not be limited to, at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 1920, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 105, 104, 102, 103, 102, 103, 100, 103, 33, 40, 108. 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148,
Conversely, the term "not more than" includes every value that is less than the recited value. For example, "no more than 100 nucleotides" includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7,6, 5, 4, 3, 2,1 and 0 nucleotides. Any smaller numbers or fractions therebetween are also included.
The terms "plurality", "at least two", "two or more", "at least a second", etc. should be understood to include, but are not limited to, at least 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 1920, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 103, 101, 104, 105, 106, 107, 109, 108, 106, 109, 108, 105, 106, 109, 103, 45, 47, 48, 110. 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148,
Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. It should be understood that the language "comprising" is used herein to describe aspects and also to provide other similar aspects described as "consisting of and/or" consisting essentially of.
Unless specifically stated or otherwise apparent from the context, the term "about," as used herein, refers to a value or composition within an acceptable error range for the particular value or composition, as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "consisting essentially of can mean within 1 or over 1 standard deviation as practiced in the art. "about" or "consisting essentially of may mean a range of up to 10% (i.e., ± 10%). Thus, "about" may be understood as being greater than or less than the stated value within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001%. For example, about 5mg may include any amount between 4.5mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, the term may mean values up to an order of magnitude or up to 5-fold. When a particular value or composition is provided in the present disclosure, unless otherwise stated, it should be assumed that the meaning of "about" or "consisting essentially of" is within an acceptable error range for that particular value or composition.
As used herein, unless otherwise specified, any concentration range, percentage range, ratio range, or integer range is to be understood as encompassing the value of any integer within the recited range, and where appropriate, including fractions thereof (e.g., tenths and hundredths of integers).
The units, prefixes, and symbols used herein are provided in their international system of units (SI) accepted form. Numerical ranges include the numbers defining the range.
Unless defined otherwise, 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 pertains. For example, Juo, "The circumcise Dictionary of biomedicine and Molecular Biology", 2nd ed., (2001), CRC Press; "The dictionary Cell & Molecular Biology", 5th ed., (2013), Academic Press; and "The Oxford Dictionary Of Biochemistry And Molecular Biology", Cammacack et al.
By "administering" is meant physically introducing the agent into the subject using any of a variety of methods and delivery systems known to those skilled in the art. Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, for example by injection or infusion. As used herein, the phrase "parenteral administration" means modes of administration other than enteral and topical administration, typically by injection and including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, and in vivo electroporation. In some embodiments, the formulation is administered via a non-parenteral route, such as orally. Other non-parenteral routes include topical, epidermal or mucosal routes of administration, such as intranasal, vaginal, rectal, sublingual or topical. Administration may also be performed, for example, once, multiple times, and/or over one or more extended periods.
As used herein, an antigen binding molecule, antibody or antigen binding molecule thereof "cross-competes" with a reference antibody or antigen binding molecule thereof if the interaction between the antigen and the first binding molecule, antibody or antigen binding molecule thereof blocks, limits, inhibits or otherwise reduces the ability of the reference binding molecule, reference antibody or antigen binding molecule thereof to interact with the antigen. The cross-competition may be complete, e.g. binding of the binding molecule to the antigen completely blocks the ability of the reference binding molecule to bind to the antigen, or it may be partial, e.g. binding of the binding molecule to the antigen reduces the ability of the reference binding molecule to bind to the antigen. In certain embodiments, an antigen binding molecule that cross-competes with a reference antigen binding molecule binds to the same or overlapping epitope as the reference antigen binding molecule. In other embodiments, the antigen binding molecule that cross-competes with the reference antigen binding molecule binds to a different epitope than the reference antigen binding molecule and the reference antigen binding molecule. Many types of competitive binding assays can be used to determine whether one antigen binding molecule competes with another, for example: solid phase direct or indirect Radioimmunoassay (RIA); solid phase direct or indirect Enzyme Immunoassay (EIA); sandwich competition assays (Stahliet al, 1983, Methods in Enzymology 9: 242-253); solid phase direct biotin-avidin EIA (Kirkland et al, 1986, J.Immunol.137: 3614-3619); solid phase direct labeling assay, solid phase direct labeling sandwich assay (Harlow and Lane,1988, Antibodies, A Laboratory Manual, Cold spring Harbor Press); direct labeling of RIA using a 1-125 labeled solid phase (Morel et al, 1988, mol. Immunol.25: 7-15); solid phase direct biotin-avidin EIA (Cheung, et al, 1990, Virology 176: 546-552); and direct markers RIA (Moldenhauer et al, 1990, Scand. J. Immunol.32: 77-82).
"antigen" refers to any molecule that elicits an immune response or is capable of being bound by an antibody or antigen binding molecule. The immune response may involve antibody production or activation of specific immunocompetent cells or both. One skilled in the art will readily appreciate that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. The antigen may be expressed endogenously, i.e. from genomic DNA, or may be expressed recombinantly. The antigen may be specific for certain tissues, such as cancer cells, or it may be expressed broadly. In addition, larger molecule fragments may serve an antigenic role. In some embodiments, the antigen is a tumor antigen.
The term "allogeneic" refers to any substance derived from one individual and then introduced into another individual of the same species, such as allogeneic T cell transplantation.
The terms "transduction" and "transduced" refer to a process by which foreign DNA is introduced into cells via a viral vector (see Jones et al, "Genetics: printles and analysis," Boston: Jones & Bartlett Publ. (1998)). In some embodiments, the vector is a retroviral vector, a DNA vector, an RNA vector, an adenoviral vector, a baculovirus vector, an EB virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector, a lentiviral vector, or any combination thereof.
"cancer" refers to a large group of diseases characterized by uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors that invade adjacent tissues and can migrate to distant parts of the body through the lymphatic system or blood stream. "cancer" or "cancer tissue" may include tumors. Examples of cancers that can be treated by the methods of the present disclosure include, but are not limited to, cancers of the immune system, including lymphomas, leukemias, myelomas, and other leukocyte malignancies. In some embodiments, the methods of the present disclosure may be used to reduce tumor size of tumors derived from, for example: bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, multiple myeloma, hodgkin's disease, non-hodgkin's lymphoma (NHL), primary mediastinal large B-cell lymphoma (PMBC), diffuse large B-cell lymphoma (DLBCL), Follicular Lymphoma (FL), transformed follicular lymphoma, Splenic Marginal Zone Lymphoma (SMZL), esophageal cancer, small bowel cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urinary tract cancer, penile cancer, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, Acute Lymphoblastic Leukemia (ALL) (including non-T-cell ALL), Chronic Lymphocytic Leukemia (CLL), solid tumors of childhood, lymphocytic lymphomas, bladder cancer, renal or ureteral cancer, renal pelvis cancer, Central Nervous System (CNS) neoplasms, primary CNS lymphomas, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenomas, kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, other B-cell malignancies, and combinations of said cancers. In a specific embodiment, the cancer is multiple myeloma. A particular cancer may be responsive to chemotherapy or radiation therapy or the cancer may be refractory. Refractory cancer refers to cancer that is not amenable to modification by surgical intervention, and the cancer is either initially unresponsive to chemotherapy or radiation therapy, or the cancer becomes unresponsive over time.
As used herein, "anti-tumor effect" refers to a biological effect that can manifest as a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, a decrease in the number of metastases, an increase in overall or progression-free survival, an increase in life expectancy, or an improvement in various physiological symptoms associated with a tumor. An anti-tumor effect may also refer to the prevention of tumorigenesis, e.g. a vaccine.
As used herein, "cytokine" refers to a non-antibody protein released by one cell in response to contact with a particular antigen, where the cytokine interacts with a second cell to mediate a response in the second cell. The cytokine may be expressed endogenously by the cell or administered to the subject. Cytokines can be released by immune cells (including macrophages, B cells, T cells, and mast cells) to spread the immune response. Cytokines can induce a variety of responses in recipient cells. Cytokines may include homeostatic cytokines, chemokines, pro-inflammatory cytokines, effectors, and acute phase proteins. For example, homeostatic cytokines, including Interleukins (IL)7 and IL-15, promote immune cell survival and proliferation, while pro-inflammatory cytokines can promote inflammatory responses. Examples of homeostatic cytokines include, but are not limited to, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, and Interferon (IFN) gamma. Examples of proinflammatory cytokines include, but are not limited to, IL-1a, IL-1b, IL-6, IL-13, IL-17a, Tumor Necrosis Factor (TNF) -alpha, TNF-beta, Fibroblast Growth Factor (FGF)2, granulocyte macrophage colony stimulating factor (GM-CSF), soluble intercellular adhesion molecule 1(sICAM-1), soluble vascular adhesion molecule 1(sVCAM-1), Vascular Endothelial Growth Factor (VEGF), VEGF-C, VEGF-D, and placental growth factor (PLGF). Examples of effectors include, but are not limited to, granzyme a, granzyme B, soluble Fas ligand (sFasL), and perforin. Examples of acute phase proteins include, but are not limited to, C-reactive protein (CRP) and serum amyloid a (saa).
"chemokines" are a class of cytokines that mediate chemotaxis or directed movement of cells. Examples of chemokines include, but are not limited to, IL-8, IL-16, eotaxin-3, macrophage-derived chemokine (MDC or CCL22), monocyte chemotactic protein 1(MCP-1 or CCL2), MCP-4, macrophage inflammatory protein 1alpha (MIP-1 alpha, MIP-1a), MIP-1 beta (MIP-1b), gamma-inducible protein 10(IP-10), and thymus and activation regulated chemokine (TARC or CCL 17).
A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dose" of a therapeutic agent (e.g., an engineered CAR T cell) is any amount that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of a disease or promotes disease regression as evidenced by decreased severity of disease symptoms, increased frequency and duration of asymptomatic phase of the disease, or prevention of injury or disability due to disease affliction. The ability of a therapeutic agent to promote disease regression can be assessed using various methods known to skilled practitioners, for example in human subjects during clinical trials, in animal model systems for predicting efficacy in humans, or by assaying the activity of the agent in an in vitro assay.
As used herein, the term "lymphocyte" includes a Natural Killer (NK) cell, a T cell, or a B cell. NK cells are a class of cytotoxic (cell toxic) lymphocytes that represent a major component of the innate immune system. NK cells reject tumors as well as virus infected cells. It acts through the process of apoptosis or programmed cell death. It is called "natural killing" because it does not require activation to kill cells. T cells play a major role in cell-mediated immunity (no participation of antibodies). Its T Cell Receptor (TCR) distinguishes itself from other lymphocyte types. The thymus, a specialized organ of the immune system, is primarily responsible for the maturation of T cells. There are six types of T cells, namely: helper T cells (e.g., CD4+ cells), cytotoxic T cells (also known as TC, cytotoxic T lymphocytes, CTL, T killer cells, cytolytic T cells, CD8+ T cells, or killer T cells), memory T cells ((i) stem cell-like memory TSCM cells (e.g., naive cells) are CD45RO-, CCR7+, CD45RA +, CD62L + (L-selectin), CD27+, CD28+, and IL-7 Ra +, but they also express large amounts of CD95, IL-2R β, CXCR3, and LFA-1, and display many of the unique functional attributes of memory cells, (ii) central memory TCM cells express L-selectin and CCR7, which secrete IL-2 but not IFN γ or IL-4, and (iii) effector memory TEM cells, however, do not express L-selectin or 7, but produce effector cytokines, such as IFN γ and IL-4), regulatory T cells (Treg, suppressor T cells or CD4+ CD25+ regulatory T cells), natural killer T cells (NKT), and Gamma Delta T cells. On the other hand, B cells play a major role in humoral immunity (with antibody involvement). It generates antibodies and antigens and performs the action of Antigen Presenting Cells (APCs), and is transformed into memory B cells upon activation through antigen interaction. In mammals, immature B cells are formed in the bone marrow.
The terms "genetically engineered," "engineered," or "modified" refer to methods of modifying a cell, including, but not limited to, causing a genetic defect by deletion of coding or non-coding regions or portions thereof or by antisense technology, or increasing expression of a protein introduced into a coding region or portion thereof. In some embodiments, the modified cells are stem cells (e.g., Hematopoietic Stem Cells (HSCs), embryonic stem cells (ES), Induced Pluripotent Stem (iPS) cells), lymphocytes (e.g., T cells), which can be obtained from a patient or donor.
By "immune response" is meant the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, Natural Killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules produced by any of these cells or the liver (including abs, cytokines, and complements) that result in the selective targeting, binding, damaging, destroying, and/or eliminating invading pathogens, pathogen-infected cells or tissues, cancer cells or other abnormal cells in vertebrates, or in the case of autoimmunity or pathological inflammation, normal human cells or tissues.
The term "immunotherapy" refers to the treatment of a subject having a disease or at risk of contracting a disease or suffering from a relapse of a disease by a method that includes inducing, enhancing, suppressing or otherwise modifying an immune response. Examples of immunotherapy include, but are not limited to, T cell therapy. The T cell therapy may include adoptive T cell therapy, Tumor Infiltrating Lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT)TM) And allogeneic T cell transplantation. However, one skilled in the art will appreciate that the conditioning methods disclosed herein will enhance the effectiveness of any transplanted T cell therapy. Examples of T cell therapies are described in U.S. patent publication nos. 2014/0154228 and 2002/0006409, U.S. patent No. 5,728,388, and international publication No. WO 2008/081035.
T cells for immunotherapyMay be from any source known in the art. For example, T cells can be differentiated from hematopoietic stem cell populations in vitro; induced pluripotent stem cells (iPS), embryonic stem cells (ES), or T cells may be obtained from a subject. T cells can be obtained from, for example, Peripheral Blood Mononuclear Cells (PBMCs), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. Furthermore, the T cells may be derived from one or more T cell lines available in the art. T cells can also be obtained using any number of techniques known to those skilled in the art (e.g., FICOLL)TMIsolation and/or apheresis) is obtained from a unit of blood collected from a subject. Additional methods of isolating T cells for T cell therapy are disclosed in U.S. patent publication No. 2013/0287748, which is incorporated by reference herein in its entirety.
The term "engineered autologous cell therapy" (which may be abbreviated as "eACTTM", also known as adoptive cell transfer) is the process of collecting the patient's own T cells, which are then genetically altered to recognize and target one or more antigens expressed on the cell surface of one or more specific tumor cells or malignancies. As used herein, "patient" includes any human having cancer (e.g., lymphoma or leukemia). Herein, the terms "subject" and "patient" are used interchangeably.
As used herein, the term "in vitro cell" refers to any cell cultured ex vivo. In particular, the in vitro cells may comprise T cells.
The terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound comprising amino acid residues covalently linked by peptide bonds. There is no limitation on the maximum number of amino acids that a protein or peptide contains at least two amino acids and may comprise the sequence of the protein or peptide. A polypeptide includes any peptide or protein comprising two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains (which are also commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers) and longer chains (which are commonly referred to in the art as proteins, which are of many types). "polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. The polypeptide includes a natural peptide, a recombinant peptide, a synthetic peptide, or a combination thereof.
As used herein, "stimulation" refers to a primary response induced by the binding of a stimulatory molecule to its cognate ligand, wherein the binding mediates a signaling event. A "stimulatory molecule" is a molecule on a T cell, such as the T Cell Receptor (TCR)/CD3 complex that specifically binds to a cognate stimulatory ligand present on an antigen presenting cell. A "stimulatory ligand" is a ligand that, when present on an antigen presenting cell (e.g., APC, dendritic cell, B cell, etc.), can specifically bind to a stimulatory molecule on the T cell, thereby mediating a primary response (including but not limited to activation, initiation of an immune response, proliferation, etc.) of the T cell. Stimulatory ligands include, but are not limited to, anti-CD 3 antibodies, peptide-loaded MHC class I molecules, hyperactivating anti-CD 2 antibodies, and hyperactivating anti-CD 28 antibodies.
As used herein, "co-stimulatory signal" refers to a signal that, when combined with a primary signal (e.g., TCR/CD3 linkage), results in a T cell response (e.g., without limitation, up-or down-regulation of proliferation and/or key molecules).
As used herein, "co-stimulatory ligand" includes molecules on antigen presenting cells that specifically bind to cognate co-stimulatory molecules on T cells. Binding of the co-stimulatory ligand provides a signal that mediates T cell responses including, but not limited to, proliferation, activation, differentiation, etc. The co-stimulatory ligand induces a signal in addition to the primary signal provided by the stimulatory molecule (e.g., provided by the binding of the T Cell Receptor (TCR)/CD3 complex to a Major Histocompatibility Complex (MHC) molecule loaded with a peptide). Costimulatory ligands can include, but are not limited to, 3/TR6, 4-1BB ligand, agonists or antibodies that bind Toll ligand receptors, B7-1(CD80), B7-2(CD86), CD30 ligand, CD40, CD7, CD70, CD83, herpes virus invasion mediator (HVEM), human leukocyte antigen G (HLA-G), ILT4, immunoglobulin-like transcript (ILT)3, inducible costimulatory ligand (ICOS-L), intracellular adhesion molecule (ICAM), ligands that specifically bind B7-H3, lymphotoxin beta receptor, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), OX40 ligand, PD-L2, or Programmed Death (PD) L1. Costimulatory ligands include, but are not limited to, antibodies that specifically bind to costimulatory molecules present on T cells, such as, but not limited to, 4-1BB, B7-H3, CD2, CD27, CD28, CD30, CD40, CD7, ICOS, ligands that specifically bind to CD83, lymphocyte function-associated antigen-1 (LFA-1), natural killer cell receptor C (NKG2C), OX40, PD-1, or tumor necrosis factor superfamily member 14(TNFSF14 or LIGHT).
A "costimulatory molecule" is a cognate binding partner that specifically binds to a costimulatory ligand on a T cell, thereby mediating a costimulatory response (e.g., without limitation, proliferation) of the T cell. Costimulatory molecules include, but are not limited to, 4-1BB/CD137, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD 33, CD45, CD100(SEMA4D), CD103, CD134, CD137, CD154, CD16, CD160(BY55), CD18, CD19, CD19a, CD2, CD247, CD2, CD276 (B2-H2), CD2 (alpha; delta; epsilon; gamma; zeta), CD2, CD49 2, CD 368, CD 6372, CD 72, CD2, GAI-2, CD-C-I-L2, CD-L-2, CD2, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGBl, KIRDS2, LAT, LFA-1, LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), LTBR, Ly9(CD229), lymphocyte function-associated antigen-1 (LFA-1(CDl la/CD18), MHC class I molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80(KLRF1), OX40, PAG/Cbp, PD-1, PSGL1, SELPLG (CD162), signaling lymphocyte activating molecule, SLAM (SLAMF 1; CD 150; IPO-3), SLAMF4(CD 244; 2B4), SLAMF6 (VLB-24; VLSLLA 24), SLNL 5976, TNFR 7, TNFR 596, TNFR-9, or a truncated fragment thereof.
Herein, the terms "reduce" and "reducing" are used interchangeably and mean any change less than the original. "decrease" and "decrease" are relative terms and require comparison between before and after measurement. "reduce" and "reducing" include complete consumption.
By "treating" or "treatment" of a subject is meant any type of intervention or procedure performed on the subject, or administration of an active agent to the subject, with the goal of reversing, alleviating, ameliorating, inhibiting, slowing, or preventing the onset, progression, severity, or recurrence of a symptom, complication, or condition, or biochemical indicator associated with the disease. In one embodiment, "treating" or "treatment" includes partial remission. In another embodiment, "treating" or "treatment" includes complete remission.
To calculate percent identity, the compared sequences are typically aligned in a manner that gives the greatest match between the sequences. One example of a Computer program that can be used to determine percent identity is the GCG package, which includes GAP (Devereux et al, 1984, Nucl. acid Res.12: 387; Genetics Computer Group, university of Wisconsin, Madison, Wis.). The computer algorithm GAP is used to align two polypeptides or polynucleotides for which the percentage of sequence identity is to be determined. The sequences are aligned so that their respective amino acids or nucleotides match best ("match span", determined by an algorithm). In certain embodiments, the algorithm also uses a standard comparison matrix (for
As used herein, the term "disruption" refers to mechanical, chemical and/or enzymatic dissociation of clustered cells. As used herein, the disrupted cells remain intact but loose cell-cell adhesion contacts and membrane adhesion properties.
As used herein, the term "culturing" or grammatical equivalents refers to the process of maintaining cells under conditions conducive to growth, survival, or differentiation. The terms "culture" and "cell culture" or any synonym are used interchangeably in this application.
As used herein, the term "cell density" or "single cell density" refers to the amount of cells in a volume or surface area. In some embodiments, cell density is expressed in wells of a cell/six well plate. According to the present disclosure, a standard six-well plate has about 9.5cm2Surface area of (a). In some embodiments, the cell density is expressed as cells/cm2。
As used herein, the term "culture vessel" refers to any vessel that can provide a sterile environment for culturing cells. Exemplary culture vessels include, but are not limited to, glass, plastic, or metal vessels.
Various aspects of the disclosure are described in further detail in the following subsections.
Detailed Description
In accordance with the present disclosure, HSCs or other stem cells (embryonic stem (ES) or Induced Pluripotent Stem (iPS)) can be used to generate large, possibly unlimited, quantities of engineered T cells with desired lineages. The present disclosure provides, inter alia, highly efficient methods for differentiating human Embryonic Stem (ES) or Induced Pluripotent Stem (iPS) cells into embryonic mesodermal progenitor cells (hEMP) and/or T cells, as well as compositions comprising embryonic mesodermal progenitor cells (hEMP) and/or T cells.
Without wishing to be bound by any particular theory, it is contemplated that it is more efficient to generate T cells from ES or iPS cells if prior to an induction step called "mesoderm push" or hEMP induction. Briefly, ES or iPS cells are cultured, passaged and grown until desired confluence. Subsequently, the culture conditions were changed to the hEMP induction medium. In some embodiments, the method of culturing ES or iPS cells is in
Pluripotent stem cells
Various pluripotent stem cells may be used to practice the present disclosure. For example, Hematopoietic Stem Cells (HSCs) in the bone marrow (as well as cord blood or peripheral blood) produce committed thymic progenitors in addition to all other mature blood cells. These thymic progenitors are transported to the thymus where they begin to develop into mature T cells. The signaling of Notch receptors via their ligands Delta and Jagged (in the thymus, in particular Notch1 and Delta-like 4) drives the transcriptional cascade (i.e., Tcf7, Gata3, Bcl11b, etc.) which results in the TCR locus rearrangement of the activated genes RAG1 and RAG2 by the recombinase. First, a productive rearrangement of TCRb (i.e., production of TCR protein) will generate and transport to the surface a protein that pairs with pTa. This surface transport conveys signals back to the cell, allowing it to develop further. The surface pTa-TCRb does not require interaction with MHC as occurs in mature TCRs, i.e., the survival signal can be peptide-MHC independent. The cells are then subjected to rearrangement of TCRa, carefully examined for successful alpha/beta pairing and poor recognition of the self peptide, MHC (i.e., positive and negative selection or central tolerance), and then become mature naive T cells and circulate to the periphery.
In some embodiments, Embryonic Stem (ES) or Induced Pluripotent Stem (iPS) cells may be used.
The stem cells may be obtained from any source known in the art. For example, induced pluripotent stem cells (iPS) or embryonic stem cells (ES) can be obtained from commercial sources. Suitable HSC, ES cells, iPS cells and other stem cells may also be cultured immortalized cell lines or isolated directly from the patient. Various methods for isolating, developing and/or culturing stem cells are known in the art and can be used to practice the present disclosure.
Generating non-clustered stem cells
As described herein, culturing non-clustered, single-cell stem cells in suspension prior to mesoderm induction results in increased stem cell differentiation efficiencyHigh. Various methods can be used to generate non-clustered stem cell cultures. For example, ES or iPS cells can be first cultured on Mouse Embryonic Fibroblasts (MEFs),
In some embodiments, the culture conditions are adapted to promote single cell growth such that the stem cells do not form clusters. For example, stem cells can be grown in suspension.
Substrate
According to the present disclosure, non-clustered stem cells are seeded on a substrate at a determined single cell density for growth. As used herein, the term "substrate" refers to any solid or semi-solid surface or support. For example, a suitable substrate may be a layer, a microbead, a well plate, a cell culture dish, a membrane, a bag, a culture flask, a container, inverse opal, a polymer lattice, a gel, or a polymer.
In some embodiments, a suitable substrate may be treated with a desired coating. For example, can use
According to the present disclosure, non-clustered cells are seeded on a substrate at a determined single cell density. The density of single cells suitable for use in the present disclosure may range from about 1.0 to 50X10 in a standard 6-well plate6One cell/well (e.g., about 1.0-40X 10)6About 1.0-
In some embodiments, cells are passaged at a defined single cell density (e.g., about 1.8X 10)6Individual cell/well, about 3.6X106Individual cells/well, about 7.2X106Individual cells/well).
In some embodiments, in the range of about 1.5x105To about 8x105Individual cell/cm2(e.g., 1.89x105Individual cell/cm2About 3.2x105Individual cell/cm2About 3.4x105Individual cell/cm2About 3.6x105Individual cell/cm2About 3.79x105Individual cell/cm2About 7.2x105Individual cell/cm2Or about 7.58x105Individual cell/cm2) Cell density of (3) passaging the cells.
In some embodiments, the single cell density determined at the time of inoculation is expressed as percent confluence. According to the present disclosure, seeding at a determined cell density results in a surface confluence ranging from 1% to 100% (e.g., about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 99%). In some embodiments, cells are passaged at a high percentage of confluence (e.g., about 70-100%, about 80%). In some embodiments, cells are passaged at a moderate percentage of confluence (e.g., about 30-70%, about 40%). In some embodiments, cells are passaged at low percentage confluence (e.g., about 1-30%, about 20%).
In some embodiments, the cells are seeded in a growth medium. In other embodiments, the cells are seeded in an induction medium. In some embodiments, cells are seeded in induction medium according to the present disclosure, grown to the desired confluence, and replated in induction medium.
Cell culture conditions
According to the present disclosure, ES or iPS cells may be suitable for growth as single cells in suspension culture. In some embodiments, the ES or iPS cells are maintained in suspension. ES or iPS cells suspended in nutrient medium may be maintained with a circulation device that ensures that the isolated cells remain suspended in the nutrient medium.
Culture medium
Various cell culture media and conditions may be used in accordance with the present disclosure. For example, cells can be produced in cell culture media containing serum or serum-free. In some embodiments, the culture medium is a serum-free medium. In some embodiments, the culture medium is an animal-free medium, i.e., a medium lacking animal-derived components. In some embodiments, the medium is a chemically defined medium. As used herein, the term "chemically-defined nutrient medium" refers to a medium in which substantially all of the chemical components are known. In some embodiments, the chemically-defined nutrient medium is free of animal-derived components, such as serum, serum-derived proteins (e.g., albumin or fetuin), and other components. In some cases, the chemically-defined medium comprises one or more proteins (e.g., protein growth factors or cytokines). In some cases, the chemically-defined nutrient medium comprises one or more protein hydrolysates. In other instances, the chemically-defined nutrient medium is a protein-free medium, i.e., a serum-free medium that does not contain proteins, hydrolysates, or unknown components.
In some embodiments, the chemically-defined medium can be supplemented with one or more animal-derived components. Such animal-derived components include, but are not limited to, fetal bovine serum, horse serum, goat serum, donkey serum, human serum, and serum-derived proteins, such as albumin (e.g., bovine serum albumin or human serum albumin).
In certain embodiments, certain preferred attributes or growth under specific conditions may be selected for culturing the cells. Those skilled in the art will appreciate that such attributes may be determined based on known characteristics and/or properties of established lines (i.e., characterized commercially available cell lines) or by empirical evaluation. In some embodiments, cell lines may be selected for their ability to grow on a feeder layer of cells. In some embodiments, cell lines can be selected for their ability to grow as adherent monolayers of cells. In some embodiments, the methods involve culturing stem cells and/or progenitor cells in a cell culture comprising a culture medium.
According to the present disclosure, generating human embryonic mesenchymal progenitor (hEMP) cells includes a growth phase and a differentiation phase. In some embodiments, the medium during the growth phase is substantially different from the medium during the differentiation phase. In certain embodiments, mTESR1 medium is used for the growth phase, while X-VIVOTM15 medium was used for the differentiation phase.
Various media can be utilized. Illustrative, but non-limiting, media include, but are not limited to, MEM (minimum essential Medium), DMEM (Du's modified Eagle Medium),BME (Eagle basal Medium), RPMI 1640, DMEM/F-12 (Du's modified Eagle Medium: nutrient mixture F-12), DMEM/F-10 (Du's modified Eagle Medium: nutrient mixture F-10), a-MEM (a-minimal essential Medium), G-MEM (Glasgow minimal essential Medium), FMDM (Isocove modified Du's Medium), essential 8(E8) Medium, knockout DMEM, AIM V, mTeSRTM 1、X-VIVOTM15. StemSpan, CellGro dendritic cell culture media.
In some embodiments, feeder cells-free media for human embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) are used. In some embodiments, the present disclosure is used to generate non-clustered ES or iPS cells. In some embodiments, serum-free media suitable for use in the present disclosure lack animal-derived components. In some embodiments, a serum-free medium suitable for the present disclosure is a chemically-defined medium. For example, mTeSRTM1 medium can be used for cell growth. mTeSRTM1 is a highly specialized, serum-free and complete cell culture medium.
In certain embodiments, the medium is supplemented with an inhibitor of the Rho-associated protein kinase (ROCK) pathway (e.g., Y27632). ROCK inhibitors can be used to aid reprogramming, maintenance, self-renewal and/or differentiation.
Desired confluency upon Induction
According to the present disclosure, non-clustered cells are resuspended or transferred to induction medium and plated on a substrate at a determined single cell density. The density of single cells suitable for use in the present disclosure may range from about 1.0 to 50X10 in a standard 6-well plate61.0-50X10 cells/well6One cell/well (e.g., about 1.0-40X 10)6About 1.0-
In some embodiments, non-clustered cells are resuspended or transferred to induction medium and plated on a substrate at a defined single cell density (e.g., about 1.8X106Individual cell/well, about 3.6X106Individual cells/well, about 7.2X106Individual cells/well).
In some embodiments, non-clustered cells are resuspended or transferred to induction medium and plated on a substrate at a determined single cell density ranging from about 1.5x105And about 8x105Individual cell/cm2E.g., 1.89x105Individual cell/cm2About 3.2x105Individual cell/cm2About 3.4x105Individual cell/cm2About 3.6x105Individual cell/cm2About 3.79x105Individual cell/cm2About 7.2x105Individual cell/cm2Or about 7.58x105Individual cell/cm2)。
In some embodiments, the single cell density determined at induction is expressed as percent confluence. According to the present disclosure, non-clustered cells are resuspended or transferred to an induction medium and plated on a substrate at a determined cell density such that the surface confluence ranges from 1% to 100% (e.g., about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 99%). In some embodiments, cells are induced with a high percentage of confluence (e.g., about 70-100%, about 80%). In some embodiments, cells are induced at a moderate percentage of confluence (e.g., about 30-70%, about 40%). In some embodiments, cells are induced at low percentage confluence (e.g., about 1-30%, about 20%).
Stage of growth and differentiation
In some embodiments, the cells are cultured at a temperature in the range of about 30-37 ℃ (e.g., about 31-37 ℃, about 32-37 ℃, about 33-37 ℃, about 34-37 ℃, about 35-37 ℃, about 36-37 ℃). In some embodiments, the cells are cultured at a temperature of about 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃ or 37 ℃. Any of the temperatures described herein may be used for the growth and/or differentiation stages. In some embodiments, the cells are cultured at different temperatures during the growth phase and the differentiation phase. In some embodiments, the cells are cultured at substantially the same temperature during the growth phase and the differentiation phase. Any of the media pH described herein can be used in the growth and/or differentiation stages. In some embodiments, the pH of the medium used in the growth and differentiation stages is different. In some embodiments, the pH of the medium used in the growth and differentiation stages is substantially the same.
In some embodiments, the ES or iPS cells are grown and maintained in a growth phase. In some embodiments, ES or iPS cells are seeded and grown to a desired confluence (e.g., about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% confluence). In other embodiments, the ES or iPS cells are not seeded prior to the differentiation stage. In certain embodiments, the ES or iPS cells are resuspended in differentiation media and plated at a desired confluence (e.g., about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% confluence). In some embodiments, the ES or iPS cells are during the growth phase
In some embodiments, the incubation time for the growth phase is about 1-6 days (e.g., about 1-5 days, about 1-4 days, about 1-3 days, about 1-2 days, about 1 day, about 2 days, about 2.5 days, about 3 days, about 3.5 days, about 4 days). In some embodiments, the incubation time of the growth phase occurs in two steps, wherein the culture is inoculated. In some embodiments, each step of the growth phase is about 1-6 days (e.g., about 1-5 days, about 1-4 days, about 1-3 days, about 1-2 days, about 1 day, about 2 days, about 2.5 days, about 3 days, about 3.5 days, about 4 days). In some embodiments, the differentiation stage lasts for about 2, 3, 4, 5, 6, or 7 days.
In some embodiments, the incubation time for the differentiation stage is about 1-6 days (e.g., about 1-5 days, about 1-4 days, about 1-3 days, about 1-2 days, about 1 day, about 2 days, about 2.5 days, about 3 days, about 3.5 days, about 4 days). In some embodiments, the incubation time for the differentiation stage occurs in two steps, wherein the culture is replated. In some embodiments, each step of the differentiation stage is about 1-6 days (e.g., about 1-5 days, about 1-4 days, about 1-3 days, about 1-2 days, about 1 day, about 2 days, about 2.5 days, about 3 days, about 3.5 days, about 4 days). In some embodiments, the differentiation stage lasts for about 2, 3, 4, 5, 6, or 7 days.
Stem cell differentiation
The hEMP cells generated using the single cell non-clustering method according to the present invention can be further differentiated into various cell types.
Mesoderm induction
The earliest CD326-CD56+ hEMP cells were generated from hescs or ipscs in the presence of activin A, BMP4, VEGF and FGF2, representing a pluripotent mesodermally committed progenitor cell population. CD326-CD56+ progenitor cells are unique in their ability to generate all mesodermal lineages, including hematopoietic, endothelial, mesenchymal (bone, cartilage, fat, fibroblasts), smooth muscle and cardiomyocytes, despite the lack of pluripotency of hescs or ipscs. CD326-CD56+ hEMP cells are precursors to more lineage-restricted mesodermal progenitors.
CD326-CD56+ hEMP cells can be produced with a combination of BMP4, VEGF and bFGF and a brief exposure to activin a.
Selection of hEMP cells
The conversion of ESCs or iPSCs to hEMPs was characterized by the loss of CD326 EPCAM and the gain of CD56NCAM (CD326-CD56 +). The epithelial marker CD326 is uniformly expressed at high levels in undifferentiated cells from human embryonic stem cell lines (e.g., H9, H1, and HES3) or ipscs, whereas CD56 is not expressed in undifferentiated hescs or ipscs. Following differentiation under mesendoderm-induced conditions, populations marked with loss of CD326 expression and gain of CD56 (CD326-CD56+) were clearly detectable.
Furthermore, E-cadherin, CD 326/TACTD 1, Claudin 3, Claudin6, Claudin 7, Syndecano 1, Syndecano 2, Beta-catenin, Occludin, Nanog, Sox 2 and/or OCT4 can be down-regulated following hEMP differentiation. Following hEMP differentiation, Snail-1, Snail2/Slug, Twist 1, LEF1, ZEB1, MMP9, fibronectin, vimentin and/or ZEB2 can be upregulated.
Modulation of hEMP cell markers can be determined by techniques known in the art, including protein detection methods (e.g., flow cytometry, FACS, Western blotting, ELISA, HPLC, LC/MS, protein immunoprecipitation, immunoelectrophoresis, protein immunostaining, etc.) and nucleic acid detection methods (e.g., mRNA transcript analysis, Northern blotting, cDNA, DNA microarray analysis, polymerase chain reaction, gene expression profiling, etc.).
Differentiation and selection of T cells
The hEMP cells have the potential to differentiate into blood endothelial (endothelium) cells (e.g., blood, endothelium), cardiovascular (e.g., endothelium, cardiomyocytes, smooth muscle) and mesenchymal (e.g., smooth muscle, fibroblasts, bone, cartilage, fat). Lymphoid cells include T cells, B cells and natural killer cells. T cells originate from hematopoietic stem cells in the bone marrow. Hematopoietic progenitor cells (e.g., hEMP cells) from hematopoietic stem cells accumulate in the thymus and expand through cell division to produce large quantities of immature thymocytes. The earliest thymocytes expressed neither CD4 nor CD8, however they developed through their development, they became Double Positive (DP) thymocytes (CD4+ CD8+), and finally matured into Single Positive (SP) (CD4+ CD 8-or CD4-CD8+) thymocytes, which were then released from the thymus into the surrounding tissues.
Increased efficiency and yield of hEMP cells according to the present disclosure results in rapid and robust T lineage commitment. In some embodiments, T cells can be differentiated from the hEMP cells in an ATO system. In some embodiments, T cells can be differentiated from the hEMP cells using a lentiviral transduction method. In some embodiments, T cells can be differentiated from the hEMP cells using a matrix monolayer (stromalmonolayer).
The appearance of CD4+ CD 3-Immature Single Positive (ISP) cells and CD4+ CD8+ (DP) cells indicates differentiation of the hEMP cells into T cells. More mature CD3+ TCR α β + cells appeared and increased over time. Consistent with the positive selection in ATO, smaller fractions of CD3+ TCR γ δ + T cells may also be generated, gradually maturing to CD8SP and to a lesser extent to CD4SP T cells.
Flow cytometric analysis of thymus and ATO-derived T cell progenitors can be used to assess the following surface phenotypes: early thymic progenitor cells (ETP; CD34+ CD7-CD1a-), CD1a-pro-T (CD34+ CD7+ CD1a-), and CD1a + pro-T (CD34+ CD7+ CD1a +); or CD5-pro-T (pro-T1; CD34+ CD7+ CD5-) and CD5+ pro-T (pro-T2; CD34+ CD7+ CD5 +). Thymus and ATO derived T cells and their precursors are defined as CD14-CD56-, in combination with the following phenotypes: total T lineage cells (CD7+ CD5+), double negative (DN; CD4-CD8-), immature single positive for CD 4(CD 4 ISP; CD5+ CD4+ CD3-), double positive (DP; CD4+ CD8+), CD8SP (CD3+ TCR. alpha. beta. + CD8+ CD4-), CD4SP (CD3+ TCR. alpha. beta. + CD8-CD4+), immature naive (CD 45RA-CD45RO +) of CD8SP or CD4SP, mature naive (CD 45RA + CD45RO-) of CD8SP or CD4 SP). Immature and mature initial phenotypes were confirmed by co-staining for CD1a, CD27, CD28 and CCR 7.
Artificial Thymus Organoid (ATO)
Genetically modified mouse models in vivo, humanized mice and in vitro systems such as OP9-DLL1 or recently described Artificial Thymus Organoids (ATO) have shown a variety of pathways by which stem cells can be modified or cultured to generate desired mature T cells, including mature T cells with antigen receptors for anti-cancer antigens.
Pluripotent stem cells and/or hemps according to the present disclosure may be further differentiated in OP9-DLL1 or Artificial Thymus Organoid (ATO) cell culture systems. ATO is a serum-free 3-dimensional cell culture technique that recapitulates T cell differentiation. ATO technology has the potential to generate ready engineered T cells to treat cancer and other diseases.
Suitable Artificial Thymic Organoid (ATO) systems support efficient in vitro differentiation and positive selection of native and TCR-engineered human T cells from umbilical cord blood, bone marrow and peripheral blood HSPCs. ATO-derived T cells exhibit an initial phenotype, diverse TCR composition, and TCR-dependent activation and proliferation. ATO-derived engineered T cells also matured to the initial phenotype and showed antigen-specific tumor killing in vitro and in vivo. Thus, ATO represents an efficient method for generating mature naive and potentially non-alloreactive engineered T cells for adoptive cell therapy. Exemplary methods for producing engineered T cells using an ATO culture system are described in, e.g., set CS, He C, Bethune MT, et al, generation of format T cells from human hematogenic stem/promoter cells in characterization chemical organisms, nature methods, 2017; 14(5) 521-530.doi 10.1038/nmeth.4237, the contents of which are incorporated herein by reference.
The high purity T cell population is readily collected from ATO by mechanical dissociation and can be further purified by standard methods to remove < 0.5% of contaminating stromal cells. The ATO cell yield per stem or progenitor cell is inversely proportional to the number of seeded cells (e.g., hEMP cells) and the ratio of input cells to stromal cells.
The ATO system is able to support differentiation and positive selection of human T cells from progenitor cells (e.g., hEMP cells) while retaining key transforming medical properties such as standardized composition, reproducibility and scalability of generation of T cells suitable for therapeutic applications. The present disclosure provides a means for significant fidelity of T cell differentiation in ATO compared to human thymus, which ultimately achieves the appearance of true naive T cells similar to those found in thymus and blood.
Providing an ATO with progenitor cells according to the present disclosure supports robust in vitro differentiation, positive selection, and maturation of human T cells. ATO-derived mature T cells exhibit an antigenic initial phenotype, diverse TCR composition, and activation/proliferation in response to antigenic stimulation. ATO also supports efficient differentiation of TCR-engineered antigen-specific T cells from progenitor cells specific for tumor-associated antigens.
Cells
The cells of the present disclosure can be obtained by any source known in the art. For example, T cells can be differentiated in vitro from a population of hematopoietic stem cells or pluripotent stem cells, or can be obtained from a subject. T cells can be obtained from, for example, peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. Furthermore, the T cells may be derived from one or more T cell lines available in the art. Any number of techniques known to those skilled in the art (e.g., FICOLL) may also be usedTMIsolation and/or apheresis) T cells are obtained from blood units collected from a subject. In certain embodiments, cells collected by apheresis are washed to remove plasma fractions and placed in an appropriate buffer or medium for subsequent processing. In some embodiments, the cells are washed with PBS. As should be appreciated, this can be accomplished, for example, by using a semi-automatic non-countercurrent centrifuge (e.g., Cobe)TM2991 cell processor, Baxter CytoMateTMEtc.) to use a washing step. In some embodiments, the washed cells are resuspended in one or more biocompatible buffers, or other salt solutions with or without buffers. In certain embodiments, undesired components of the apheresis sample are removed. Additional methods of isolating T cells for T cell therapy are disclosed in U.S. patent publication No. 2013/0287748, which is incorporated by reference herein in its entirety.
In certain embodiments, the monocytes are depleted by lysing the red blood cells (e.g., via PERCOLL)TMGradient, isolated by using centrifugation) to isolate stem cells from PBMCs. In some embodiments, specific subpopulations of T cells, such as CD4+, CD8+, CD28+, CD45RA +, and CD45RO + T cells, may be further isolated by positive or negative selection techniques known in the art. For example, the Chinese medicinal composition can be used for treating pudendumEnrichment of the T cell population by negative selection is accomplished by antibody combinations of surface markers unique to sexually selected cells. In some embodiments, cell sorting and/or selection via negative magnetic immunoadhesion or flow cytometry (which uses a mixture of monoclonal antibodies directed against cell surface markers present on negatively selected cells) may be used. For example, to enrich for CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD8, CD11b, CD14, CD16, CD20, and HLA-DR. In certain embodiments, flow cytometry and cell sorting are used to isolate cell populations of interest for use in the present disclosure.
In some embodiments, CD8+ cells are further sorted into naive, stem, central, effector, and effector cells by identifying cell surface antigens associated with each of naive, stem, central, effector, and effector types of CD8+ cells. In some embodiments, expression of the phenotypic markers of central memory T cells comprises CCR7, CD3, CD28, CD45RO, CD62L, and CD127 and is granzyme B negative. In some embodiments, the central memory T cells are CD8+, CD45RO +, and CD62L + T cells. In some embodiments, effector T cells are CCR7, CD28, CD62L, and CD127 negative and are granzyme B and perforin positive. In certain embodiments, the CD4+ T cells are further sorted into subpopulations. For example, CD4+ T helper cells can be sorted into naive, central memory and effector cells by identifying cell populations with cell surface antigens.
In some embodiments, the immune cells, e.g., T cells, are genetically modified after isolation using known methods, or the immune cells are activated and expanded (or, in the case of progenitor cells, differentiated) in vitro before being genetically modified. In another embodiment, the pluripotent stem cells are modified prior to induction to hemps. In another embodiment, the transduced hemps are treated by ATO. In another embodiment, immune cells, such as T cells, are genetically modified (e.g., with a chimeric antigen receptor or TCR comprising one or more codes forViral vector transduction of the nucleotide sequence of the CAR or TCR), and then activated and/or amplified in vitro. Methods for activating and expanding T cells are known in the art and are described, for example, in U.S. patent nos. 6,905,874, 6,867,041, and 6,797,514; and PCT publication No. WO 2012/079000, the contents of which are incorporated herein by reference in their entirety. In general, such methods involve contacting PBMCs or isolated T cells with stimulating and co-stimulating agents (e.g., anti-CD 3 and anti-CD 28 antibodies, typically adhered to beads or other surfaces) in media with appropriate cytokines (e.g., IL-2). anti-CD 3 and anti-CD 28 antibodies attached to the same beads serve as "replacement" Antigen Presenting Cells (APCs). One example is
In certain embodiments, the T cells are obtained from a donor subject. In some embodiments, the donor subject is a human patient having a cancer or tumor. In other embodiments, the donor subject is a human patient that does not have a cancer or tumor.
Other aspects of the disclosure relate to compositions comprising a polynucleotide described herein, a vector described herein, a polypeptide described herein, or an in vitro cell described herein. In some embodiments, the composition comprises a pharmaceutically acceptable carrier, diluent, solubilizer, emulsifier, preservative, and/or adjuvant. In some embodiments, the composition comprises an excipient.
In other embodiments, the composition is selected for parenteral delivery, for inhalation, or for delivery through the digestive tract, such as oral administration. It is within the ability of those skilled in the art to prepare such pharmaceutically acceptable compositions. In certain embodiments, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically in the pH range of about 5 to about 8. In certain embodiments, when parenteral administration is contemplated, the compositions are in the form of pyrogen-free, parenterally acceptable aqueous solutions comprising the compositions described herein with or without additional therapeutic agents in a pharmaceutically acceptable vehicle. In certain embodiments, the vehicle for parenteral injection is sterile distilled water, wherein the compositions described herein (with or without at least one additional therapeutic agent) are formulated as sterile, isotonic solutions that are suitable for storage. In certain embodiments, the preparation involves a formulation of the desired molecule with a polymeric compound (e.g., polylactic acid or polyglycolic acid), beads, or liposomes that provides controlled or sustained release of the product, which formulation is then delivered via depot injection (depot injection). In certain embodiments, the desired molecules or cells are introduced using an implantable drug delivery device.
Cancer treatment
The methods of the present disclosure can be used to treat cancer, reduce tumor size, kill tumor cells, prevent tumor cell proliferation, prevent tumor growth, eliminate tumors from a patient, prevent tumor recurrence, prevent tumor metastasis, induce remission in a patient, or any combination thereof in a subject. In certain embodiments, the method induces a complete response. In other embodiments, the method induces a partial response.
Cancers that may be treated include tumors that are not vascularized, have not substantially vascularized, or have vascularized. Cancer may also include solid or non-solid tumors. In some embodiments, the cancer is a hematologic cancer. In some embodiments, the cancer is a cancer of leukocytes. In other embodiments, the cancer is a cancer of plasma cells. In some embodiments, the cancer is leukemia, lymphoma, or myeloma. In certain embodiments, the cancer is Acute Lymphoblastic Leukemia (ALL) (including non-T-cell ALL), Acute Lymphoid Leukemia (ALL) and Hemophagocytic Lymphohistiocytosis (HLH), B-cell prolymphocytic leukemia, B-cell acute lymphoid leukemia ("BALL"), blastic plasmacytoid dendritic cell neoplasms, burkitt's lymphoma, Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), Chronic Myeloid Leukemia (CML), chronic or acute granulomatous disease, chronic or acute leukemia, diffuse large B-cell lymphoma (DLBCL), Follicular Lymphoma (FL), hairy cell leukemia, hemophagic cell syndrome (macrophage activating syndrome (MAS)), hodgkin's disease, large cell granuloma, granulomatous, lymphomatosis, leukocyte adhesion deficiency, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, Monoclonal Gammopathy of Unknown Significance (MGUS), multiple myeloma, myelodysplasia, and myelodysplastic syndrome (MDS), myeloid diseases including, but not limited to, Acute Myeloid Leukemia (AML), non-Hodgkin's lymphoma (NHL), plasmacytoid disorders (e.g., asymptomatic myeloma (smotic multiple myeloma or indolent myeloma), plasmacytoma-dendritic cell neoplasm, plasmacytoma (e.g., plasmacytoma; monocytogenes; unigenic plasmacytoma; extramedullary plasmacytoma; and multiple plasmacytoma), POEMS syndrome (Crow-Fukase syndrome; Takatsuki disease; PEP syndrome), primary mediastinal large B cell lymphoma (PMBC), Small or large cell follicular lymphoma, Splenic Marginal Zone Lymphoma (SMZL), systemic amyloid light chain amyloidosis, T-cell acute lymphoid leukemia ("TALL"), T-cell lymphoma, transformed follicular lymphoma, or waldenstrom's macroglobulinemia, or a combination thereof.
In one embodiment, the cancer is myeloma. In a specific embodiment, the cancer is multiple myeloma. In another embodiment, the cancer is leukemia. In one embodiment, the cancer is acute myeloid leukemia.
In some embodiments, the method further comprises administering a chemotherapeutic agent. In certain embodiments, the chemotherapeutic agent of choice is a lymphodepleting (preconditioning) chemotherapeutic agent. Beneficial preconditioning treatment regimens and related beneficial biomarkers are described in U.S. provisional patent applications 62/262,143 and 62/167,750, which are incorporated herein by reference in their entirety. These describe e.g. the requirement for conditioningA method of treating a patient with cellular therapy comprising administering to the patient a prescribed beneficial dose of cyclophosphamide (200 mg/m)2Daily and 2000mg/m2Between/day) and the indicated dose of fludarabine (20 mg/m)2Daily and 900mg/m2Between/day). One such dosage regimen involves treating the patient, including administering to the patient about 500mg/m per day2Cyclophosphamide per day and about 60mg/m2Fludarabine/day for 3 days, and then administering a therapeutically effective amount of the engineered T cells to the patient.
In other embodiments, the antigen binding molecule, transduced (or otherwise engineered) cells (e.g., CARs or TCRs), and the chemotherapeutic agent are each administered in an amount effective to treat the disease or condition in the subject.
In certain embodiments, a composition comprising an immune effector cell expressing a CAR and/or TCR disclosed herein can be administered in combination with any number of chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents (alkylating agents), such as thiotepa and cyclophosphamide (cycloxan)TM) (ii) a Alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines (aziridines), such as benzodidopa (benzodipa), carboquone (carboquone), metodopa (medeopa) and urodopa (uredopa); ethyleneimines and methylmelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimetylomelamine; nitrogen mustards such as chlorambucil (chlorambucil), chlorambucil (chlorenaphazine), cholorophosphamide (cholorophosphamide), estramustine (estramustine), ifosfamide (ifosfamide), mechlorethamine (mechlorethamine), mechlorethamine hydrochloride (mechlorethamine oxide hydrochloride), melphalan (melphalan), neomustard (novembichin), benzene mustard cholesterol (phenylesterine), prednimustine (prednimustine), triamcinolone (trofosfamide), uracil mustard (uracil mustard); nitrosoureas (nitrosureas), such as carmustine (ca)rmustine), chlorozotocin (chlorozotocin), fotemustine (fotemustine), lomustine (lomustine), nimustine (nimustine), ramustine (ranimustine); antibiotics such as aclacinomycin (aclacinomycin), actinomycin (actinomycin), auroramycin, azaserine (azaserine), bleomycin (bleomycin), actinomycin C (cactinomycin), calicheamicin (calicheamicin), carbamicin, carminomycin (carminomycin), actinomycin D (danomymycin), daunorubicin (daunorubicin), ditorexin (detorubicin), 6-diaza-5-oxo-L-norleucine, doxorubicin (doxorubicin), epirubicin (epirubicin), elsubicin (orubicin), idarubicin (idarubicin), sesamomycin (mitomycin), mitomycin (mitomycin), streptomycin (actinomycin), streptomycin (streptomycin), streptomycin (gentamycin), daunorubicin (gentamycin), doxorubicin (dapubicin), sisomicin (degluubicin), sisomicin (mitomycin), streptomycin (gentamycin), streptomycin (streptomycin), streptomycin (streptomycin), and streptomycin (streptomycin), streptomycin (streptomycin, streptomyc, Streptozocin (streptozocin), tubercidin (tubicidin), ubenimex (ubenimex), azinostatin (zinostatin), zorubicin (zorubicin); antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteroyltriglutamic acid (pteropterin), trimetrexate (trimetrexate); purine analogs, such as fludarabine (fludarabine), 6-mercaptopurine (mercaptoprine), thiamiprine (thiamiprine), thioguanine (thioguanine); pyrimidine analogs such as ancitabine (ancitabine), azacitidine (azacitidine), 6-azauridine, carmofur (carmofur), cytarabine (cytarabine), dideoxyuridine (dideoxyuridine), doxifluridine (doxifluridine), enocitabine (enocitabine), floxuridine (floxuridine), 5-FU; androgens such as carotinone (calusterone), dromostanolone propionate, epitioandrostanol (epitiostanol), mepiquitane (mepiquitane), testolactone (testolactone); anti-adrenal agents, such as aminoglutethimide (aminoglutethimide), mitotane (mitotane), trilostane (trilostane); folic acid supplements, e.g. folinic acid (folinic aci)d) (ii) a Acetoglucurolactone (acegultone); an aldophosphamide glycoside (aldophosphamideglycoside); aminolevulinic acid (aminolevulinic acid); amsacrine (amsacrine); tabularil (bestrabucil); bisantrene; edatrexate (edatraxate); defofamine; dimecorsine (demecolcine); diazaquinone (diaziqutone); elformithine; ammonium etitanium acetate; etoglut (etoglucid); gallium nitrate; hydroxyurea (hydroxyurea); lentinan (lentinan); lonidamine (lonidamine); mitoguazone (mitoguzone); mitoxantrone (mitoxantrone); mopidamol (mopidamol); diamine nitracridine (nitrarine); pentostatin (pentostatin); methionine mustard (phenamett); pirarubicin (pirarubicin); podophyllinic acid (podophyllic acid); 2-ethyl hydrazide (ethylhydrazide); procarbazine (procarbazine);
In some embodiments, the chemotherapeutic agent is administered simultaneously with or within a week after the administration of the engineered cell or nucleic acid. In other embodiments, the chemotherapeutic agent is administered 1 to 4 weeks, 1 week to 1 month, 1 week to 2 months, 1 week to 3 months, 1 week to 6 months, 1 week to 9 months, or 1 week to 12 months after administration of the engineered cell or nucleic acid. In some embodiments, the chemotherapeutic agent is administered at least 1 month prior to administration of the cell or nucleic acid. In some embodiments, the method further comprises administering two or more chemotherapeutic agents.
A variety of additional therapeutic agents may be used in combination with the compositions described herein. For example, potentially useful additional therapeutic agents include PD-1 inhibitors, such as nivolumab
Additional therapeutic agents suitable for use in combination with the present disclosure include, but are not limited to, ibrutinib (ibrutinib)Oxamumumab (ofatumumab)
In additional embodiments, a composition comprising a CAR and/or TCR immunization is administered with an anti-inflammatory agent. Anti-inflammatory agents or drugs may include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrogenCortisone, methylprednisolone, prednisolone (prednisone), prednisone (prednisone), and triamcinolone (triamcinolone)), non-steroidal anti-inflammatory drugs (NSAIDS) including aspirin (aspirin), ibuprofen (ibuprofen), naproxen (naproxen), methotrexate, sulfasalazine, leflunomide (leflunomide), anti-TNF drugs, cyclophosphamide, and mycophenolate mofetil (mycophenolate). Exemplary NSAIDs include ibuprofen, naproxen sodium, Cox-2 inhibitors, and sialylate. Exemplary analgesics include tramadol (hydrochloride) of paracetamol (acetaminophen), oxycodone (oxycodone), and propoxyphene hydrochloride. Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors such as TNF antagonists, (e.g., etanercept)
In certain embodiments, the compositions described herein are administered in combination with a cytokine. As used herein, "cytokine" means a protein released by one cell population that acts on another cell as an intercellular mediator. Examples of cytokines are lymphokines, monokines, and traditional polypeptide hormones. The cell factor includes growth hormone, such as human growth hormone, N-methionyl human growth hormone and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; (ii) prorelaxin; glycoprotein hormones such as Follicle Stimulating Hormone (FSH), Thyroid Stimulating Hormone (TSH), and Luteinizing Hormone (LH); hepatic Growth Factor (HGF); fibroblast Growth Factor (FGF); prolactin; placental lactogen; mullerian-inhibiting substances (mullerian-inhibiting substance); mouse gonadotropin-related peptides; a statin; an activin; vascular endothelial growth factor; an integrin; thrombopoietin (TPO); nerve Growth Factor (NGF) such as NGF-beta; platelet growth factor; transforming Growth Factors (TGF) such as TGF-alpha and TGF-beta; insulin-like growth factors-I and-II; erythropoietin (EPO); an osteoinductive factor; interferons such as interferon-alpha, beta and-gamma; colony Stimulating Factors (CSFs), such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (IL), such as IL-1, IL-1alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, tumor necrosis factors such as TNF-alpha or TNF-beta; and other polypeptide factors, including LIF and Kit Ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture, as well as biologically active equivalents of the native sequence cytokines.
Another aspect of the present disclosure relates to a method of inducing immunity against a tumor comprising administering to a subject an effective amount of a modified T cell disclosed herein. Another aspect of the present disclosure relates to a method of inducing an immune response in a subject comprising administering an effective amount of an engineered immune cell of the present application. In some embodiments, the immune response is a T cell mediated immune response. In some embodiments, the T cell-mediated immune response is directed against one or more target cells. In some embodiments, the engineered immune cell comprises a CAR or TCR, wherein the CAR or TCR comprises a THD described in the present disclosure. In some embodiments, the target cell is a tumor cell.
Another aspect of the present disclosure relates to a method for treating or preventing a malignant tumor, the method comprising administering to a subject in need thereof an effective amount of at least one immune cell, wherein the immune cell comprises at least one CAR or TCR.
Another aspect of the present disclosure relates to a method of treating cancer in a subject in need thereof, comprising administering to the subject a polynucleotide, vector, CAR or TCR, cell or composition disclosed herein. In one embodiment, the method comprises administering a polynucleotide encoding a CAR or a TCR. In another embodiment, the method comprises administering a vector comprising a polynucleotide encoding a CAR or a TCR. In another embodiment, the method comprises administering a CAR or a TCR encoded by a polynucleotide disclosed herein. In another embodiment, the method comprises administering a cell comprising a polynucleotide encoding a CAR or a TCR or a vector comprising a polynucleotide encoding a CAR or a TCR.
In some embodiments, donor T cells for use in T cell therapy are obtained from a patient (e.g., for autologous T cell therapy). In other embodiments, donor stem cells to be differentiated into T cells for use in T cell therapy are obtained from a subject other than the patient.
The T cells may be administered in a therapeutically effective amount. For example, a therapeutically effective amount of T cells can be at least about 104At least about 10 cells5At least about 10 cells6At least about 10 cells7At least about 10 cells8At least about 10 cells9Individual cell or at least about 1010And (4) cells. In another embodiment, the therapeutically effective amount of T cells is about 104One cell, about 105One cell, about 106One cell, about 107One cell or about 108And (4) cells. In a specific embodiment, the therapeutically effective amount of CAR T cells or TCR T cells is about 2X106Individual cell/kg, about
Immune tolerance
The methods of the present disclosure may be used to treat an immune tolerance disorder in a subject. In certain embodiments, the method induces a complete response. In other embodiments, the method induces a partial response.
Inadequate central or peripheral tolerance can cause autoimmune diseases, leading to syndromes such as systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes, autoimmune multiple endocrine syndrome type 1 (APS-1), and immune dysregulated multiple endocrine disorder enteropathy X-linked syndrome (IPEX), and may lead to asthma, allergy, and inflammatory bowel disease. Immune tolerance can also be problematic in transplant rejection (e.g., stem cell transplantation, kidney transplantation, liver transplantation, etc.).
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. However, citation of a reference herein shall not be construed as an admission that such reference is prior art to the present disclosure. To the extent that any definition or term provided in a reference, which is incorporated by reference, differs from the term and discussion provided herein, the term and definition of the invention shall control.
The disclosure is further illustrated by the following examples, which should not be construed as further limiting. The contents of all references cited in this application are expressly incorporated herein by reference.
Examples