CELL POPULATIONS WITH IMPROVED PRODUCTION AND THERAPEUTIC CHARACTERISTICS

Abstract

The present invention is directed to improved methods of preparing cells and compositions for therapeutic uses.

Claims

1-87. (canceled)

88. A method of producing a population of genetically engineered target cells from a sample comprising target cells that are not terminally differentiated, comprising: a) obtaining a sample comprising said target cells together with contaminant cells, proteins or other factors that act as agents that promote the proliferation or differentiation of the target cells; b) separating the target cells from the sample obtained in step a) using a size and/or affinity based separation method to obtain an enriched population of target cells wherein the contaminants are reduced by at least 70% compared to the sample before separation and/or the ratio of contaminants to target cells is at least 70% lower than in the sample originally obtained in step a); c) genetically engineering the target cells in the enriched population of cells obtained in step b), with a nucleotide sequence to produce genetically engineered target cells with a therapeutically useful phenotype; wherein, no factors that promote, or otherwise re-direct, the proliferation or differentiation of the target cells are added more than three days before the genetic engineering of step c) is initiated.

89. The method of claim 88, wherein the target cells are T cells and the “one or more factors that promote or otherwise re-direct the proliferation or differentiation of target cells” comprises cytokines, peptides, peptide receptor complexes, antibodies either alone or in conjunction with other costimulatory molecules.

90. The method of claim 88, wherein a factor that promotes the proliferation of target cells is added within three days prior to the time when genetic engineering is initiated and wherein the target cells are not centrifuged after sample is obtained.

91. The method of claim 88, wherein the target cells are selected from the group consisting of: a) leukocytes, including neutrophils, basophils, eosinophils, lymphocytes (including B cells, T cells and natural killer cells); monocytes, macrophages, mast cells, dendritic cells; b) stem cells including: i) stem cells that develop into leukocytes such as stem cells with CD34 and/or CD38 markers and leukocyte lineage negative cells; and ii) stem cells that develop into cells other than leukocytes; c) erythroid precursor cells.

92. The method of claim 88, wherein the size based separation method is deterministic lateral displacement (DLD) on a microfluidic device.

93. The method of claim 88, wherein at least 80% of the target cells do not divide more than once from the time that they are obtained until the separation of paragraph b) is initiated.

94. The method of claim 88, wherein the sample is an apheresis or leukapheresis sample.

95. The method of claim 88, wherein, after step c), the genetically engineered cells are: d) cultured to expand their number; and e) transferred into a pharmaceutical composition for administration to a patient.

96. The method of claim 88, wherein the separation step in part b) is initiated within 5 hours after the sample comprising target cells is obtained.

97. The method of claim 88 wherein one or more agents are added to the sample of target cells before or during steps a) to c) to reversibly inhibit proliferation and/or differentiation.

98. A method of producing a population of genetically engineered target cells from a sample comprising target cells that are not terminally differentiated, comprising: a) obtaining the sample comprising target cells that are not terminally differentiated; b) separating the target cells from the sample obtained in step a) from other cells, particles or unwanted materials to obtain an enriched population of target cells; c) genetically engineering the target cells in the enriched population of cells to produce genetically engineered target cells with a therapeutically useful phenotype; wherein the proliferation and/or differentiation of target cells is minimized until initiation of genetic engineering of step c), so as to maintain the cells in the same developmental state that they were in when first obtained.

99. The method of claim 98, wherein one or more factors that promote the proliferation or differentiation of target cells are added at no more than three days before the genetic engineering of cells is initiated.

100. The method of claim 99, wherein the target cells are T cells and the “one or more factors that promote or otherwise re-direct the proliferation or differentiation of target cells” comprises cytokines, peptides, peptide-receptor complexes, antibodies either alone or in conjunction with other costimulatory molecules.

101. The method of claim 98, wherein the target cells are selected from the group consisting of: a) leukocytes, including neutrophils, basophils, eosinophils, lymphocytes (including B cells, T cells and natural killer cells); monocytes, macrophages, mast cells, dendritic cells; b) stem cells including: i) stem cells that develop into Leukocytes such as stem cells with CD34 and/or CD38 markers and leukocyte lineage negative cells; and ii) stem cells that develop into cells other than leukocytes; c) erythroid precursor cells.

102. The method of claim 98, wherein the separation in step b) is performed using a size and/or affinity based separation method to obtain an enriched population of target cells and the target cells are not centrifuged after sample has been obtained.

103. The method of claim 98, wherein the target cells are T cells and antibodies or other factors are added to the sample or elsewhere during the process that block or reversibly inhibit the action of costimulators needed for the activation of the target T cells.

104. The method of claim 98, wherein an agent is added to the sample or during the processing of target cells that reversibly blocks the activation of T cells by inhibiting the binding of, and/or signaling from, the T cell receptor in response to antigen binding.

105. The method of claim 98, wherein the method reduces by at least 20% the amount of cells in the biological sample not capable of entering into cell division and increases the percentage of cells that effectively integrate nucleic acids and/or different forms of RNA, including miRNA and tRNA.

106. The method of claim 98, wherein at least 80% of the target cells have not been activated at the time that the separation of paragraph b) is completed.

107. A method of treating or preventing a disease or condition in a patient comprising administering to said patient a therapeutically effective amount of a pharmaceutical composition comprising cells made by the method of claim 98.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0040] The text below provides guidance regarding methods disclosed herein and information that may aid in the making and use of devices involved in carrying out those methods.

I. The Processing of Sample to Remove Platelets and Other Factors

[0041] The methods described herein are characterized, in part, by the removal of platelets from blood samples, or samples derived from blood, soon after cells are first collected and by processing cells in a way that controls the number of cell divisions that they undergo. The most preferred purification method is by microfluidic separation. This not only rapidly removes small factors that may be detrimental to a high yield of therapeutic cells, including T memory stem cells and central memory cells, but may also be used to wash cells. It can also be used in the rapid removal of reagents and other factors that may be introduced in the processing of cells.

[0042] The methods disclosed herein, especially DLD, should preferably be capable of removing about 3.5 logs of virus in one pass as opposed to about 2 logs expected with most other approaches. Through the removal of platelets and other detrimental factors, 2-13× more central memory T cells (Tcm) cells should preferably be obtained. The ability to process cells within an hour of collection may limit degradation that might otherwise occur in this process, and may be done with a minimal dilution of the sample. Although DLD is preferred, other methods of separation that are applied very rapidly after cell collection and which rapidly separate desired cells from platelets and small detrimental factors may be employed.

[0043] In addition to eliminating detrimental factors, the invention may include the use of factors that direct cells to a therapeutically desirable phenotype. These may include: T cell activators; proteins (including affinity reagents, proteins, protein constructs, growth factors, specific antigens, engineered constructs); nucleic acids; nanomatrixes; micro-RNA; promoters; feedback inhibitors; and other agents that control division or promote integration of genetic content.

[0044] Separation Methods

[0045] The invention includes methods in which there is genetic engineering of a population of target cells. This is done by isolating the target cells from a crude fluid composition by performing a separation method, preferably a microfluidic method such as Deterministic Lateral Displacement (DLD) or an affinity based method.

[0046] An especially preferred separation method is DLD. In this type of separation, microfluidic devices are characterized by the presence of at least one channel which extends from a sample inlet to one or more fluid outlets, and which is bounded by a first wall and a second wall opposite from the first wall. An array of obstacles is arranged in rows in the channel, with each subsequent row of obstacles being shifted laterally with respect to a previous row. The obstacles are disposed in a manner such that, when a crude fluid composition is applied to an inlet of the device and passed through the channel, target cells flow to one or more collection outlets where an enriched product is collected, and contaminant cells or particles flow to one more waste outlets that are separate from the collection outlets.

[0047] Once the target cells have been purified using the device, they may be transfected or transduced with nucleic acids designed to impart upon the cells a desired phenotype, e.g., to express a chimeric molecule that makes the cells of therapeutic value. The population of cells may then be expanded by culturing in vitro.

[0048] In a preferred embodiment, the crude fluid composition is blood or, more preferably, a preparation of leukocytes that has been obtained by performing apheresis or leukapheresis on the blood of a patient. Preferred target cells include T cells, B-cells, NK-cells, monocytes and progenitor cells, with T cells being the most preferred. Apart from leukocytes, other types of cells, e.g., dendritic cells or stem cells, may also serve as target cells.

[0049] In general, crude fluid compositions containing target cells should be processed without freezing (at least up until the time that they are genetically engineered), and, preferably, at the site of collection. The crude fluid composition will preferably be the blood of a patient, and more preferably be a composition containing leukocytes obtained as the result of performing apheresis or leukapheresis on such blood. However, the term “crude fluid composition” also includes bodily fluids such as lymph or synovial fluid as well as fluid compositions prepared from bone marrow or other tissues. The crude fluid composition may also be derived from tumors or other abnormal tissue.

[0050] Although it is not essential that target cells be bound to a carrier before being genetically engineered, either before or after separation is first performed, they may be bound to one or more carriers provided that the carriers do not activate the cells. The exact means by which this occurs is not critical to the invention but binding should preferably be done “in a way that promotes DLD separation.” This term, as used in the present context, means that the method must ultimately result in binding that exhibits specificity for a particular target cell type, that provides for an increase in size of the complex relative to the unbound cell of at least 2 μm (and alternatively at least 20, 50, 100, 200, 500 or 1000% when expressed as a percentage) and, in cases where therapeutic or other uses require free target cells, that allow the target cell to be released from complexes by chemical or enzymatic cleavage, chemical dissolution, digestion, due to competition with other binders, by physical shearing, e.g., using a pipette to create shear stress, or by other means.

[0051] In one embodiment, the carriers have on their surface an affinity agent (e.g., an antibody) that allows the carriers to bind directly to the target cells with specificity. As used in this context, the word “specificity” means that at least 100 (and preferably at least 1000) target cells will be bound by carrier in the crude fluid composition relative to each non-target cell bound. In cases where the carrier binds after target cells in samples are separated, the binding may occur either before the target cells are genetically engineered or after.

[0052] Making of CAR T Cells

[0053] Methods for making and using CAR T cells are well known in the art. Procedures have been described in, for example, U.S. Pat. Nos. 9,629,877; 9,328,156; 8,906,682; US 2017/0224789; US 2017/0166866; US 2017/0137515; US 2016/0361360; US 2016/0081314;US 2015/0299317; and US 2015/0024482; each of which is incorporated by reference herein in its entirety.

[0054] In general, CAR T cells may be made by obtaining a crude fluid composition comprising T cells and performing DLD on the composition using a microfluidic device. Generally, the crude fluid composition comprising T cells will be an apheresis or leukapheresis product derived from the blood of a patient and containing leukocytes.

[0055] The microfluidic device should preferably have at least one channel extending from a sample inlet to one or more fluid outlets, wherein the channel is bounded by a first wall and a second wall opposite from the first wall. An array of obstacles is preferably arranged in rows in the channel, each subsequent row of obstacles being shifted laterally with respect to a previous row. These obstacles are disposed in a manner such that, when the crude fluid composition comprising T cells is applied to an inlet of the device and fluidically passed through the channel, the T cells flow to one or more collection outlets where an enriched product is collected and other cells (e.g., red blood cells, and platelets) or other particles of a different (generally smaller) size than the T cells flow to one more waste outlets that are separate from the collection outlets. Once obtained, the T cells are genetically engineered to produce chimeric antigen receptors (CARs) on their surface using procedures well established in the art. These receptors should generally bind antigens that are on the surface of a cell associated with a disease or abnormal condition. For example, the receptors may bind antigens that are unique to, or overexpressed on, the surface of cancer cells. In this regard, CD19 may sometimes be such an antigen.

[0056] Treating Cancer, Autoimmune Disease or Infectious Disease Using Cells In another aspect, the invention is directed to a method of treating a patient for a disease using cells prepared using the methods described herein. For example, CAR T cells may be used to treat an autoimmune disease, an infectious disease or cancer by administering the cells to a patient. Generally the patent treated should be the same patient that gave the blood from which the T cells were isolated.

II. Designing Microfluidic Plates

[0057] Cells, particularly cells in compositions prepared by apheresis or leukapheresis, may be isolated using microfluidic devices. The preferred method is DLD using a device that contains a channel through which fluid flows from an inlet at one end of the device to outlets at the opposite end. Basic principles of size based microfluidic separations and the design of obstacle arrays for separating cells have been provided elsewhere (see, US 2014/0342375; US 2016/0139012; U.S. Pat. Nos. 7,318,902 and 7,150,812, which are hereby incorporated herein in their entirety) and are also summarized in the sections below.

[0058] During DLD, a fluid sample containing cells is introduced into a device at an inlet and is carried along with fluid flowing through the device to outlets. As cells in the sample traverse the device, they encounter posts or other obstacles that have been positioned in rows and that form gaps or pores through which the cells must pass. Each successive row of obstacles is displaced relative to the preceding row so as to form an array direction that differs from the direction of fluid flow in the flow channel. The “tilt angle” defined by these two directions, together with the width of gaps between obstacles, the shape of obstacles, and the orientation of obstacles forming gaps are primary factors in determining a “critical size” for an array. Cells having a size greater than the critical size travel in the array direction, rather than in the direction of bulk fluid flow and particles having a size less than the critical size travel in the direction of bulk fluid flow. In devices used for blood, apheresis or leukapheresis compositions, array characteristics may be chosen that result in white blood cells being diverted in the array direction whereas red blood cells and platelets continue in the direction of bulk fluid flow. In order to separate a chosen type of leukocyte from others having a similar size, a carrier may then be used that binds to that cell in a way that promotes DLD separation and which thereby results in a complex that is larger than uncomplexed leukocytes. It may then be possible to carry out a separation on a device having a critical size smaller than the complexes but bigger than the uncomplexed cells.

[0059] The obstacles used in devices may take the shape of columns or be triangular, square, rectangular, diamond shaped, trapezoidal, hexagonal or teardrop shaped. In addition, adjacent obstacles may have a geometry such that the portions of the obstacles defining the gap are either symmetrical or asymmetrical about the axis of the gap that extends in the direction of bulk fluid flow.

III. Making and Operating Microfluidic Devices

[0060] General procedures for making and using microfluidic devices that are capable of separating cells on the basis of size are well known in the art. Such devices include those described in U.S. Pat. Nos. 5,837,115; 7,150,812; 6,685,841; 7,318,902; 7,472,794; and 7,735,652; all of which are hereby incorporated by reference in their entirety. Other references that provide guidance that may be helpful in the making and use of devices for the present invention include: U.S. Pat. Nos. 5,427,663; 7,276,170; 6,913,697; 7,988,840; 8,021,614; 8,282,799; 8,304,230; 8,579,117; US 2006/0134599; US 2007/0160503; US 20050282293; US 2006/0121624; US 2005/0266433; US 2007/0026381; US 2007/0026414; US 2007/0026417; US 2007/0026415; US 2007/0026413; US 2007/0099207; US 2007/0196820; US 2007/0059680; US 2007/0059718; US 2007/005916; US 2007/0059774; US 2007/0059781; US 2007/0059719; US 2006/0223178; US 2008/0124721; US 2008/0090239; US 2008/0113358; and WO2012094642 all of which are also incorporated by reference herein in their entirety. Of the various references describing the making and use of devices, U.S. Pat. No. 7,150,812 provides particularly good guidance and U.S. Pat. No. 7,735,652 is of particular interest with respect to microfluidic devices for separations performed on samples with cells found in blood (in this regard, see also US 2007/0160503).

[0061] A device can be made using any of the materials from which micro- and nano-scale fluid handling devices are typically fabricated, including silicon, glasses, plastics, and hybrid materials. A diverse range of thermoplastic materials suitable for microfluidic fabrication is available, offering a wide selection of mechanical and chemical properties that can be leveraged and further tailored for specific applications.

[0062] Techniques for making devices include Replica molding, Softlithography with PDMS, Thermoset polyester, Embossing, Injection Molding, Laser Ablation and combinations thereof. Further details can be found in “Disposable microfluidic devices: fabrication, function and application” by Fiorini, et al. (BioTechniques 38:429-446 (March 2005)), which is hereby incorporated by reference herein in its entirety. The book “Lab on a Chip Technology” edited by Keith E. Herold and Avraham Rasooly, Caister Academic Press Norfolk UK (2009) is another resource for methods of fabrication, and is hereby incorporated by reference herein in its entirety.

[0063] To reduce non-specific adsorption of cells or compounds, e.g., released by lysed cells or found in biological samples, onto the channel walls, one or more walls may be chemically modified to be non-adherent or repulsive. The walls may be coated with a thin film coating (e.g., a monolayer) of commercial non-stick reagents, such as those used to form hydrogels. Additional examples of chemical species that may be used to modify the channel walls include oligoethylene glycols, fluorinated polymers, organosilanes, thiols, poly-ethylene glycol, hyaluronic acid, bovine serum albumin, poly-vinyl alcohol, mucin, poly-HEMA, methacrylated PEG, and agarose. Charged polymers may also be employed to repel oppositely charged species. The type of chemical species used for repulsion and the method of attachment to the channel walls can depend on the nature of the species being repelled and the nature of the walls and the species being attached. Such surface modification techniques are well known in the art.

[0064] All references cited herein are fully incorporated by reference. Having now fully described the invention, it will be understood by one of skill in the art that the invention may be performed within a wide and equivalent range of conditions, parameters and the like, without affecting the spirit or scope of the invention or any embodiment thereof.