Methods for manufacturing immunotherapy cells

Abstract

Described herein are methods for isolating highly enriched populations of target cells using either binding particles such as microbubbles or detachable beads. Also described herein are methods for producing immunotherapy cells from the isolated target cells. The methods involve labeling nontarget cells with binding agents, binding microbubbles to the labeled cells, removing the microbubble-bound cells, and collecting the unlabeled target cells. The methods also involve contacting target cells with detachable beads, removing unbound cells, and detaching the beads from the target cells. The isolated target cells demonstrate high purity, viability and functionality for applications such as CAR-T cell manufacturing.

Description

METHODS FOR MANUFACTURING IMMUNOTHERAPY CELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Application Nos. 63 / 657,655 filed on June 7, 2024, and 63 / 802,090 filed on May 8, 2025, the entire disclosures of which are herein incorporated by reference.FIELD

[0002] The present disclosure relates to the field of cell isolation and purification. More specifically, this disclosure pertains to methods for isolating target cells from complex cell mixtures such as leukapheresis samples, using binding particles such as microbubbles or detachable beads to bind cells, and methods of producing immunotherapy cells, including CAR-T cells and dual antigen CAR-T cells.BACKGROUND

[0003] The following includes information that may be useful in understanding the present invention. It is not an admission that any of the information, publications or documents specifically or implicitly referenced herein is prior art, or essential, to the presently described or claimed inventions. All publications, patents, related applications, and other written or electronic materials mentioned or identified herein are hereby incorporated herein by reference in their entirety. The information incorporated is as much a part of the application as filed as if all of the text and other content was repeated in the application and should be treated as part of the text and content of the application as filed.

[0004] Autologous chimeric antigen receptor T cells (CAR-T) products are living products that are manufactured from whole blood or leukapheresis collected from each patient, unlike many traditional pharmaceutical products. The manufacture of CAR-T cells generally consists of five major processes, with the first step being the processing of the starting material. Whole blood is processed by leukapheresis, wherein the white blood cells are separated from all other components, including red blood cells and platelets. T lymphocytes (T cells) and other white blood cell types can be isolated from the leukapheresis product. For CAR-T therapy, peripheral blood mononuclear cells (PBMCs) are usually harvested, but T cells can be directly selected for and harvested. The purification of target cells, such as, for example, CD3+ T cells, is an essential step in CAR-T manufacture.10005] Delays in cell isolation extend turnaround times and reduce manufacturing success. Starting material is generally limited and is affected by donor variability and availability. When the starting material is directly collected from a patient, the starting material may be limited.SUMMARY

[0006] Purification of target cells for CAR-T manufacture, e.g., CD3+ T cells, is often a root cause of manufacturing failure. Efficient isolation of pure, viable T cells is crucial for various therapeutic applications, including CAR-T cell therapies. However, traditional cell isolation methods often rely on magnetic beads or columns, which can be both expensive and cumbersome. Having rapid, efficient, high-quality isolation of desired target cells significantly improves the overall quality and efficacy of the final products.

[0007] In one aspect, this disclosure features innovative GMP-compliant methods to isolate target cells from complex cell mixtures using microbubbles. As featured and disclosed herein, microbubbles are small, coated gas-filled bubbles having the unique ability to specifically bind to and float microbubble-bound cells, thus enabling easy separation from unbound or unlabeled cells.

[0008] In one aspect, the methods featured herein leverage the unique properties of microbubbles to efficiently separate target cells, e.g., as a non-limiting example, to separate target T cells from nontarget cell populations, providing a fast, gentle, and cost-effective alternative to traditional methods. In some aspects, microbubbles are used to separate a target cell population from a mixture of cell populations. In some aspects, nontarget cell populations include e.g., as non-limiting examples, red blood cells, granulocytes, monocytes, B lymphocytes (B cells), natural killer (NK) cells, or platelets. In some aspects, this disclosure features open and closed cell isolation processes to shorten the time to isolate target cells, such as, for example, CD3+ T cells for making CAR-T products. Using exemplary processes featured herein, target cells, such as, for example, CD3+ T cells were enriched, expanded, and transduced with a chimeric antigen receptor (CAR) construct.

[0009] In some aspects, one or more binding agents bind moieties on nontarget cells, and microbubbles bind to the binding agent-bound nontarget cells. The microbubble-binding agentbound nontarget cells are then separated from unbound target cells to provide isolated target cells.

[0010] In some aspects, one or more binding agents bind moieties on target cells, and microbubbles bind to the binding agent-bound target cells. In some aspects microbubble-binding agent-bound target cells are separated from unbound nontarget cells, and target cells are isolated.

[0011] In some aspects, microbubbles are coated with, e.g., streptavidin, biotin, protein A, or another protein, peptide or nucleotide that binds a functionalized or non-functionalized binding agent that binds to cells. As a non-limiting example, in some aspects, microbubbles are coated with streptavidin, which binds to, for example, biotinylated binding agents, e.g. as a non-limiting example, biotinylated antibodies, e.g., nanobodies, including biotinylated antibodies or biotinylated nanobodies bound to cells. In some aspects, microbubbles bind to biotinylated camelid-derived single domain VHH antibodies, or nanobodies, having high specificity for surface proteins on target cells including, as non-limiting examples, CD3, CD4, and CD8. In some aspects, sequential microbubble isolation steps using a different binding agent in each step is performed. For example, in some embodiments, sequential isolation steps using anti-CD4 and anti-CD8 antibodies or nanobodies are performed.

[0012] This disclosure additionally features, in some aspects, innovative GMP-compliant methods to isolate target cells by using binding particles, e.g., without limitation, magnetic particles or beads and / or detachable beads, including detachable Dynabeads®. As featured and disclosed herein, detachable beads such as detachable Dynabeads® are uniform, spherical, and superparamagnetic polystyrene beads. In some aspects, the binding particles are functionalized with binding agents. In some aspects the functionalized binding particles are functionalized detachable beads, e.g., functionalized detachable beads, e.g., detachable magnetic beads. In some aspects, the Dynabeads® are functionalized, e.g., coated with a binding agent having high specificity for target surface proteins including, as non-limiting examples, CD3, CD4, and CD8. In some aspects, the functionalized binding particles, e.g., functionalized Dynabeads® are functionalized through cognate binding partner interactions. In some aspects, the cognate binding partners are, e.g., streptavidin-biotin, avidin-biotin, protein A-antibody, and / or complementary oligonucleotides. In some aspects, the functionalized Dynabeads® are functionalized with streptavidin-biotin derivative complexes coupled to binding agents. In some aspects, the binding agents comprising the complexes are, e.g., without limitation, camelid-derived single domain VHH antibodies having high specificity for surface proteins on target cells including, as non-limiting examples, CD3, CD4, and CD8. In some aspects, Dynabeads® are coated with binding agents having high specificity for surface proteins on nontarget cells including, as non-limiting examples, CD14, CD16, CD19, CD36, CD56, CD 123, or CD235a.

[0013] In one aspect, the present invention provides a method for isolating target cells from a sample comprising cells, the method comprising: (a) contacting the sample with one or more binding agents to form a mixture comprising binding agent-bound cells and unbound cells; (b) contacting the mixture comprising binding agent-bound cells and unbound cells with microbubbles that bind the binding agent to form a mixture comprising microbubble-binding agent-bound cells and unbound cells; (c) separating the microbubble-binding agent-bound cells from the unbound cells; and (d) extracting the unbound cells, thereby obtaining target cells. The binding agents, bound and unbound cells, and microbubbles may be any of the binding agents, bound and unbound cells, and microbubbles featured in this disclosure. In some aspects, sequential microbubble isolation steps using a different binding agent in each step is performed. For example, in some aspects, sequential isolation steps using anti-CD4 and anti-CD8 antibodies or nanobodies are performed.

[0014] In one aspect, the present disclosure provides a method for isolating target cells from a sample comprising cells, the method comprising: (a) contacting the sample with one or more binding agents to form a mixture comprising binding agent-bound cells and unbound cells; (b) contacting the mixture comprising binding agent-bound cells and unbound cells with binding particles, e.g. as a non-limiting example, detachable beads, e.g., detachable Dynabeads®, that bind the binding agent to form a mixture comprising detachable beadbinding agent-bound cells and unbound cells; (c) separating the detachable bead-binding agentbound cells from the unbound cells; and (d) extracting the unbound cells, thereby obtaining target cells.

[0015] In some aspects, the binding particles, e.g., detachable beads bind to binding agentbound target cells. For example, in some aspects, the detachable Dynabeads® specifically bind target cells, and cells which are not bound to beads are separated from the Dynabead®-bound target cells. In some aspects, Dynabead®-bound target cells are actively released from the Dynabeads® by contacting the Dynabead®-bound target cells with a release buffer containing a competitive binding agent that displaces a biotin derivative on the surface of the Dynabeads®. The beads are then magnetically captured and separated from the detached, desired target cells.

[0016] In some aspects, the binding particles, e.g., detachable beads bind to binding agentbound nontarget cells. In some aspects, for example, Dynabeads® specifically bind nontarget cells, and cells which are not bound to the Dynabeads® are separated from the Dynabead®- bound nontarget cells using a magnet separating nontarget Dynabead®-bound cells from the unbound target cells, to provide isolated target cells.

[0017] As featured and disclosed herein, the term “binding agent” refers to a molecule that binds a molecule of interest, such as a ligand binding its cognate receptor or antibodies recognizing cell surface markers. In some aspects of this disclosure, binding agents include, as non-limiting examples, antibodies, nanobodies, peptides, proteins, aptamers, lectins, cyclodextrins, avidin, streptavidin, or SuperAvidin™. In some aspects, binding agents directly bind to target cells of interest. In some aspects, binding agents bind to, or select nontarget cells, for use in negative selection protocols. In one aspect, binding agents are used to target specific T cell populations from PBMCs and other mixed cell populations. In another aspect, binding agents are used to select nontarget cell populations for removal from target cells, e.g., target T cell populations. In some aspects, microbubbles or binding particles, e.g., detachable beads, bind to the binding agents.

[0018] In some aspects, binding particles are, as non-limiting examples, beads e.g., functionalized beads, e.g., functionalized detachable beads, e.g., detachable Dynabeads®, and / or nanoparticles or functionalized nanoparticles. In some aspects, cognate binding partners used to functionalize particles and microparticles include, e.g., biotin, avidin, streptavidin, or SuperAvidin™, or protein A, and antibodies. In some aspects, binding particles are functionalized microbubbles. In some aspects, binding particles are functionalized beads, e.g., functionalized detachable beads. In some aspects, binding particles are functionalized microbubbles that bind antibodies bound to target or nontarget cells, including, as non-limiting examples, CD3, CD4, CD8, CD14, CD16, CD19, CD36, CD56, CD123, or CD235a, or any subcombination or combination thereof. In some aspects, binding particles are functionalized detachable beads that bind antibodies bound to target or nontarget cells, including, as nonlimiting examples, CD3, CD4, CD8, CD14, CD16, CD19, CD36, CD56, CD123, or CD235a, or any subcombination or combination thereof.

[0019] In some aspects, undesired, or nontarget cells are bound to the binding agent. The nontarget cells are then separated from the unbound, desired target cells using, e.g., a magnet, flotation, filtration, or centrifugation. The nontarget cells are removed and only the unbound target cells remain. In some aspects, the microbubble and / or binding particles bind to and remove the undesired, nontarget cell types, thereby negatively selecting for the target cells.

[0020] In some aspects, one or more binding agents are selected from an antibody, nanobody, peptide, protein, aptamer, lectin, cyclodextrin, avidin, or streptavidin, or combinations thereof. In some aspects, binding agents bind to one or more surface marker of nontarget or target cell types. In some aspects, binding agents conjugated to a moiety including, as non-limiting examples, a fluorescent dye, a fluorescent protein, an enzyme, a metal, a proteintag, an oligonucleotide tag, or a combination thereof. In some aspects, binding agents are antibodies that bind CD3, CD4, CD8, CD14, CD16, CD19, CD36, CD56, CD123, or CD235a, or a combination thereof.

[0021] As featured and disclosed herein, “functionalized” refers to a bead or microbubble with one or more binding agents or cognate binding partners bound to its exterior. In some aspects, functionalized beads or microbubbles use, as non-limiting examples, antibody binding and cognate receptor-ligand interactions, such as, e.g., streptavidin-biotin, avidin-biotin, or superavidin-biotin binding. In some aspects, functionalized microbubbles are streptavidin- coated albumin microbubbles. In some aspects, functionalized detachable beads are antibody- coated or nanobody-coated detachable beads, including, as non-limiting examples, anti-CD3+ detachable Dynabeads®, anti-CD3+ / CD28+ detachable Dynabeads®, anti-CD4+ detachable Dynabead®, or anti-CD8+ detachable Dynabeads®. In some aspects, the functionalized detachable beads are anti-CD3+ detachable Dynabeads®. In some aspects, the functionalized detachable beads are anti-CD3+ / CD28+ detachable Dynabeads®. In some aspects, the functionalized detachable beads are anti-CD4+ detachable Dynabeads®. In some aspects, the functionalized detachable beads are anti-CD8+ detachable Dynabeads®.

[0022] In some aspects, this disclosure features cell isolation processes that involve the use of microbubbles, e.g., functionalized microbubbles or detachable beads, e.g., functionalized beads, e.g., functionalized detachable beads Dynabeads®.

[0023] As featured and disclosed herein, surface markers include proteins, sugars, lipids, glycolipids, cholesterol, and phospholipids that are on, embedded in, or otherwise have a portion that extends from, the surface of a cell and is recognized by a binding agent, e.g., as a non-limiting example, an antibody or nanobody, lectin, cyclodextrin, or other binding partner featured herein. In some aspects, surface markers are proteins known as cluster of differentiation (CD) proteins, such as, e.g., CD3, CD4, CD8, CD14, CD16, CD19, CD36, CD56, CD 123, or CD235a. In some aspects, surface makers comprise one or more of CD3, CD4, CD8, CD14, CD16, CD19, CD36, CD56, CD123, or CD235a. In some aspects, surface markers can include or exclude CD3, CD4, CD8, CD14, CD16, CD19, CD36, CD56, CD123, or CD235a.

[0024] In some aspects, the one or more binding agents bind to one or more surface markers of nontarget cell types. In some aspects, the one or more binding agents bind to the CD proteins including or excluding CD3, CD4, or CD8, or a subcombination or combination thereof. In some aspects, the binding agents that bind to positively selected target cells bind to, as nonlimiting examples, CD3, CD4, or CD8, or a subcombination or combination thereof. In someaspects, the binding agents that bind to positively selected target cells bind to, as non-limiting examples, CD14, CD16, CD19, CD36, CD56, CD123, or CD235a, or a combination thereof.

[0025] In some aspects, the one or more binding agents bind to the CD proteins including or excluding CD14, CD16, CD19, CD36, CD56, CD123, or CD235a, or any subset thereof, or a combination thereof. For example, in some aspects the one or more binding agents bind to the CD proteins including or excluding CD 14 and CD 19. In some aspects, the one or more binding agents bind to the CD proteins including or excluding CD 16 and CD56. In some aspects, the one or more binding agents bind to CD45 and CD16 / 56. In some aspects, the one or more binding agents bind to CD45 and CD19. In some aspects, the one or more binding agents bind to CD45 and CD 14. In some aspects, the one or more binding agents is a cocktail of antibodies that bind to CD14, CD16, CD19, CD36, CD56, CD123, and CD235a. In some aspects, the one or more binding agents bind to one or more surface markers comprising CD3, CD4, CD8, or a subcombination or combination thereof.

[0026] As featured and disclosed herein, the term “separation” includes any means of substantially purifying one component from another, such as, e.g., by filtration or magnetic attraction. In some aspects, separation includes fractionation, centrifugation, filtration, magnetic attraction, chelation, and pelletization. As featured and disclosed herein, the terms “fractionation” or “fractionating” describe the separation of one component from another resulting in distinct component compartments, such as, e.g., a non-limiting example in which a mixture of labeled target cells, unlabeled cells, debris, and buffer are distinctly separated in a single container after centrifugation.

[0027] In some aspects, separating the microbubble-binding agent-bound cells from the unbound cells comprises magnetic capture, flotation, centrifugation, filtration, or a subcombination or combination thereof. In some aspects, separating the microbubble-binding agent-bound cells from the unbound cells is performed by flotation. In some aspects, separating the microbubble-binding agent-bound cells from the unbound cells is performed by centrifugation. In some aspects, separating the microbubble-binding agent-bound cells from the unbound cells is performed by filtration.

[0028] In some aspects, the microbubbles have a mean diameter between about 1-10 micrometers (pm), comprise a shell made from a material comprising, e.g., as non-limiting examples, proteins, polymers, lipids, sugars, and surfactants, and a gaseous core selected from the group consisting of air, nitrogen, carbon dioxide, and perfluorocarbons. In some aspects, the microbubbles have a mean diameter that ranges between about 1-10 pm. In some aspects, the microbubbles have a mean diameter of 0.5 pm, 1 pm, 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, 7pm, 8 pm, 9 pin, 10 pm, 11 pm, 12 pm, 13 pm, 14 pm, 15 pm, or any diameter between any two diameters referred to above or herein.

[0029] In one aspect, the present invention provides a method for isolating target cells from a sample comprising cells, the method comprising: (a) contacting the sample with one or more binding particles to form a mixture comprising binding particle-bound cells and unbound cells; (b) separating binding particle-bound cells from the unbound cells; (c) removing unbound cells; and (d) releasing the binding particles from the binding particle-bound cells thereby obtaining target cells. In some aspects, binding particles are functionalized beads, e.g., functionalized detachable beads. In some aspects, binding agents are detachable beads, e.g., functionalized detachable beads, e.g., detachable magnetic beads, e.g., functionalized Dynabeads®.

[0030] In one aspect, the present invention provides a method for isolating target cells from a sample comprising cells, the method comprising: (a) contacting the sample with one or more detachable beads to form a mixture comprising detachable bead-bound cells and unbound cells; (b) separating detachable bead-bound cells from the unbound cells; (c) removing unbound cells; and (d) releasing the binding agent from the detachable bead-bound cells thereby obtaining target cells. In some aspects, the detachable beads are functionalized detachable beads, e.g., detachable Dynabeads®. In some aspects, the detachable beads bind to CD4 only. In some aspects, the detachable beads bind to CD8 only. In some aspects, the detachable beads bind to CD3 only. In some aspects, the detachable beads bind to both CD4 and CD8.

[0031] In some aspects, binding agents bind to one or more surface marker of nontarget or target cell types. In some aspects, binding agents are functionalized microbubbles that bind antibodies bound to target or nontarget cells, including, as non-limiting examples, CD3, CD4, CD8, CD14, CD16, CD19, CD36, CD56, CD123, or CD235a, or a combination thereof. In some aspects, binding agents are functionalized detachable beads that bind antibodies bound to target or nontarget cells, including, as non-limiting examples, CD3, CD4, CD8, CD 14, CD16, CD19, CD36, CD56, CD123, or CD235a, or a combination thereof. In some aspects, binding agents are antibodies that bind CD3, CD4, CD8, CD14, CD16, CD19, CD36, CD56, CD 123, or CD235a, or a combination thereof. In some aspects, the one or more binding agents bind to one or more surface markers comprising CD3, CD4, CD8, or a combination thereof. In some aspects, the detachable beads are functionalized beads, e.g., as a non-limiting example, detachable Dynabeads®. In some aspects, the detachable beads bind to CD4 only. In some aspects, the detachable beads bind to CD 8 only. In some aspects, the detachable beads bind to both CD4 and CD 8.

[0032] In some aspects, separating the binding particle-bound cells from the unbound cells comprises flotation, centrifugation, filtration, or a subcombination or a combination thereof. In some aspects, separating the binding particle-bound cells from the unbound cells is performed by flotation. In some aspects, separating the binding particle-bound cells from the unbound cells is performed by centrifugation. In some aspects, separating the binding particle-bound cells from the unbound cells is performed by filtration. In some aspects the binding particles are microbubbles.

[0033] In some aspects, separating the detachable bead-bound cells from the unbound cells comprises magnetic capture, flotation, centrifugation, filtration, or a subcombination or a combination thereof. In some aspects, separating the detachable bead-bound cells from the unbound cells is performed by flotation. In some aspects, separating the detachable bead-bound cells from the unbound cells is performed by centrifugation. In some aspects, separating the detachable bead-bound cells from the unbound cells is performed by filtration. In some aspects, separating the detachable bead-bound cells from the unbound cells is performed by magnetic capture.

[0034] As featured and disclosed herein, target cells comprise immune cells, stem cells, progenitor cells, malignant cells, bacterial cells, and parasites. In some aspects, target cells are the immune cells T lymphocytes (T cells). In some aspects, target cells include or exclude immune cells, stem cells, progenitor cells, malignant cells, bacterial cells, and parasites. In some aspects, nontarget cells include or exclude red blood cells, granulocytes, monocytes, B cells, NK cells, or platelets. In some aspects, nontarget cells include red blood cells, granulocytes, monocytes, B cells, NK cells, or platelets. In some aspects, the target cells are immune cells. In some aspects, the target cells are T cells. In some aspects, the target cells are CD3+. In some aspects, the target cells are CD4+ and / or CD8+ T cells. In some aspects, the target cells are tumor infiltrating T cells. In some aspects, the one or more binding agents or binding particles binds to one or more surface markers of positively selected target cell types. In some aspects, the one or more binding agent or binding particles binds to one or more surface markers of positively selected nontarget cell types. In some aspects, the target cells are stem cells. In some aspects, the target cells are progenitor cells. In some aspects, the nontarget cells are malignant cells. In some aspects, the target cells are bacterial cells. In some aspects, the target cells are parasites.

[0035] As featured and disclosed herein, nontarget cells comprise immune cells, stem cells, progenitor cells, malignant cells, bacterial cells, and parasites. In some aspects, nontarget cells include or exclude immune cells, stem cells, progenitor cells, malignant cells, bacterial cells,and parasites. In some aspects, nontarget cells include or exclude red blood cells, granulocytes, monocytes, B cells, NK cells, or platelets. In some aspects, nontarget cells include red blood cells, granulocytes, monocytes, B cells, NK cells, or platelets. In some aspects, the nontarget cells red blood cells, granulocytes, monocytes, B cells, NK cells, or platelets. In some aspects, the nontarget cells are immune cells. In some aspects, the nontarget cells are CD3+ cells. In some aspects, the nontarget cells are CD4+ and / or CD8+ T cells. In some aspects, the target cells are tumor infiltrating T cells. In some aspects, the one or more binding agent binds to one or more surface markers of negatively selected target cell types. In some aspects, the one or more binding agent binds to one or more surface markers of negatively selected nontarget cell types. In some aspects, the nontarget cells are stem cells. In some aspects, the nontarget cells are progenitor cells. In some aspects, the nontarget cells are malignant cells. In some aspects, the nontarget cells are bacterial cells. In some aspects, the nontarget cells are parasites.

[0036] As featured and disclosed herein, the terms “negative selection” and “negatively selected” refer to a method of selection where a target within a sample is not bound or otherwise manipulated. As used herein, an exemplary method of negative selection refers to a target cell type within a sample being isolated by the stepwise removal, including through the use of binding agents, of nontarget cells. In some aspects, nontarget cells are initially bound by binding agents, e.g. without limitation antibodies and / or nanobodies, subsequently bound by microbubbles, separated from target cells by centrifugation, filtration, or flotation, and removed from the sample, such that the negatively selected target cells are isolated. In some aspects, nontarget cells are bound by detachable beads, separated from target cells by centrifugation or using a magnet, and removed from the sample, such that the negatively selected target cells are that remain and are isolated.

[0037] As featured and disclosed herein, the terms “positive selection” and “positively selected” refer to a method of selection where a target within a sample is directly bound or otherwise manipulated to isolate the target. As used herein, an exemplary method of positive selection refers to a target cell type within a sample being isolated by the direct binding of target cells. In some aspects, desired cells are bound by binding particles, microbubbles and / or beads, e.g., detachable beads, separated from nontarget cells by centrifugation, then nontarget cells are removed such that only the binding particle bound cells, e.g., microbubble bound target cells or bead-bound target cells are all that remain, and the detachable beads are released using a release buffer and the detached cells are separated from the released particles, e.g., by use of flotation, centrifugation, filtration, or a magnet, such that the positively selected target cells are obtained.

[0038] In some aspects, the methods of this disclosure further comprise the step of positively or negatively selecting for CD4+ or CD8+ T cell subsets.

[0039] As featured and disclosed herein, the term “cell purity” refers to the amount of cells positive for specific markers and is assessed by, as a non-limiting example, flow cytometry after staining with fluorescent antibodies against specific cellular markers. For example, T cell purity is assessable by staining for CD3, CD3, and / or CD8 and using flow cytometry or microscopy. In some aspects, the method achieves at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or at any percentage between any two percentages of target cell purity referred to above or herein. In some aspects, the method achieves at least 80% target cell purity. In some aspects, the method achieves at least 90% target cell purity. In some aspects, the method achieves at least 95% target cell purity. In some aspects, the method achieves at least 80% target cell purity and at least 50% target cell recovery.

[0040] As featured and disclosed herein, the term “cell recovery” refers to the amount of cells that are obtained after a method of cell isolation is performed. Cell recovery further refers to the percentage of cells remaining relative to the number of cells at the start of a cell insolation process. In some aspects, the method achieves at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or at any percentage between any two percentages of target cell recovery referred to above or herein. In some aspects, the method achieves at least 50% target cell recovery. In some aspects, the method achieves at least 60% target cell recovery. In some aspects, the method achieves at least 70% target cell recovery. In some aspects, the method achieves at least 80% target cell recovery. In some aspects, the method achieves at least 90% target cell recovery.

[0041] As featured and disclosed herein, the terms “closed system” and “closed process” indicate that the method is performed in an environment where the product or material is isolated from the external environment. For example, a closed system includes, e.g., a cell separation bag. Conversely, as featured and disclosed herein, the terms “open system” and open process” indicate that the method performed in an environment where the product or material is exposed to the external environment. In some aspects, the method utilizing microbubbles is performed in an open process. In some aspects, the method utilizing microbubbles is performed in a closed process. In some aspects, the method utilizing detachable beads is performed in an open process. In some aspects, the method utilizing detachable beads is performed in a closed process.

[0042] In some aspects, the method achieves at least 80% target cell purity and at least 50% target cell recovery, and the method is performed in a closed system.

[0043] In some aspects, the method for isolating target cells from a sample comprising cells is performed in a closed system comprising a cell separation bag, or in an open system comprising centrifugation. In some aspects, the method for isolating target cells from a sample comprising cells comprises: (a) contacting the sample with one or more binding agents to form a mixture comprising binding agent-bound cells and unbound cells; (b) contacting the mixture comprising binding agent-bound cells and unbound cells with microbubbles that bind the binding agent to form a mixture comprising microbubble-binding agent-bound cells and unbound cells; (c) separating the microbubble -binding agent-bound cells from the unbound cells; and (d) extracting the unbound cells, thereby obtaining target cells. In some aspects, the method for isolating target cells from a sample comprising cells, the method comprising: (a) contacting the sample with one or more binding agents to form a mixture comprising binding agent-bound cells and unbound cells; (b) contacting the mixture comprising binding agentbound cells and unbound cells with binding particles, e.g. as a non-limiting example, detachable beads, e.g., detachable Dynabeads®, that bind the binding agent to form a mixture comprising detachable bead-binding agent-bound cells and unbound cells; (c) separating the detachable bead-binding agent-bound cells from the unbound cells; and (d) extracting the unbound cells, thereby obtaining target cells. In some aspects, steps (a) through (c) are performed in a closed system comprising a cell separation bag or in an open system comprising centrifugation. In some aspects, steps (a) through (c) are performed in a closed system comprising a cell separation bag. In some aspects, wherein steps (a) through (c) are performed in an open system, further wherein separation comprises centrifugation. In some aspects, the method is performed in a closed system.

[0044] As featured and disclosed herein, the term “CD4:CD8 T cell ratio” refers to the proportion of CD4+ T cells to CD8+ T cells. In healthy individuals, the proportion of CD4+ T cells to CD8+ T cells is between about 1.0 to 3.0. In some aspects, the isolation process of this disclosure improve cell viability and / or T cell purity while, in some aspects, also maintaining CD4:CD8 T cell ratios of the isolated T cells. In some aspects, the method maintains a CD4:CD8 ratio between about 1.0: 1.0 to 3.0: 1.0. In some aspects, the CD4:CD8 ratio is between 0.5:1.0, 1.0:1.0, 1.5: 1.0, 2.0:1.0, 2.5:1.0, 3.0:1.0, 3.5:1.0, 4:1.0, 4.5:1.0, or 5.0:1.0, or at any ratio between any two ratios referred to above or herein.

[0045] In some aspects, the sample comprises, e.g., as non-limiting examples, blood, leukapheresis product, lymph, peripheral blood, bone marrow, cerebrospinal fluid, umbilical cord, umbilical cord blood, amniotic fluid, menses, pleural effusion, urine, ascites, tumor tissue, or semen. In some aspects, the sample is blood. In some aspects, the sample is aleukapheresis product. In some aspects, the sample is lymph. In some aspects, the sample is peripheral blood. In some aspects, the sample is bone marrow. In some aspects, the sample is cerebrospinal fluid. In some aspects, the sample is umbilical cord. In some aspects, the sample is umbilical cord blood. In some aspects, the sample is amniotic fluid. In some aspects, the sample is menses. In some aspects, the sample is pleural effusion. In some aspects, the sample is urine. In some aspects, the sample is ascites. In some aspects, the sample is tumor tissue. In some aspects, the sample is semen.

[0046] In some aspects, the method for isolating target cells from a sample comprising cells further comprises washing the sample prior to step (a). In some aspects, the method for isolating target cells from a sample comprising cells comprises: (a) contacting the sample with one or more binding agents to form a mixture comprising binding agent-bound cells and unbound cells; (b) contacting the mixture comprising binding agent-bound cells and unbound cells with microbubbles that bind the binding agent to form a mixture comprising microbubble-binding agent-bound cells and unbound cells; (c) separating the microbubble-binding agent-bound cells from the unbound cells; and (d) extracting the unbound cells, thereby obtaining target cells. In some aspects, the method for isolating target cells from a sample comprising cells, the method comprising: (a) contacting the sample with one or more binding agents to form a mixture comprising binding agent-bound cells and unbound cells; (b) contacting the mixture comprising binding agent-bound cells and unbound cells with binding particles, e.g. as a nonlimiting example, detachable beads, e.g., detachable Dynabeads®, that bind the binding agent to form a mixture comprising detachable bead-binding agent-bound cells and unbound cells; (c) separating the detachable bead-binding agent-bound cells from the unbound cells; and (d) extracting the unbound cells, thereby obtaining target cells.

[0047] In one aspect, the present invention provides a method of producing immunotherapy cells comprising: (a) isolating target cells from a sample comprising cells; (b) stimulating target cells with anti-CD3 / CD28-coated beads; and transducing stimulated target cells with one or more chimeric antigen receptors, or one or more T-cell receptor. In some aspects, isolating target cells comprises (i) contacting the sample with one or more binding agents to form a mixture comprising binding agent-bound cells and unbound cells; (ii) contacting the mixture comprising binding agent-bound cells and unbound cells with microbubbles that bind the binding agent to form a mixture comprising microbubble-binding agent-bound cells and unbound cells; (iii) separating the microbubble-binding agent-bound cells from the unbound cells; and (iv) extracting the unbound cells thereby obtaining target cells. In one aspect, the chimeric antigen receptor is a dual antigen chimeric antigen receptor.

[0048] In another aspect, the present invention provides a method of producing immunotherapy cells comprising: (a) isolating target cells from a sample comprising cells; (b) stimulating target cells with anti-CD3 / CD28-coated beads; and transducing stimulated target cells with one or more chimeric antigen receptors, or one or more T-cell receptor. In some aspects, the isolating target cells comprises (i) contacting the sample with one or more binding particles to form a mixture comprising binding particle-bound cells and unbound cells; (ii) separating particle-bound cells from the unbound cells; (iii) removing unbound cells; and (iv) releasing the binding particles from the binding particle-bound cells thereby obtaining target cells. In one aspect, the chimeric antigen receptor is a dual antigen chimeric antigen receptor.

[0049] In one aspect, the present invention provides for a composition of CD3+ T cells isolated according to the method where the CD3+ T cells have a purity greater than 90% and a viability greater than 95%. In some aspects, the CD3+ T cells have a purity greater than about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or between any percentage between any two percentages referred to above or herein. In some aspects, the CD3+ T cells have a viability greater than about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or between any percentage between any two percentages referred to above or herein. In another aspect, the present invention provides for a composition where the isolated CD3+ T cells are transduced in step (c) using a lentiviral vector encoding a chimeric antigen receptor.

[0050] In one aspect, a composition is provided comprising CD3+ T cells isolated according to any of the methods featured herein, wherein the CD3+ T cells have a purity greater than 90%, a viability greater than 95%, and are transduced with a lentiviral vector encoding a chimeric antigen receptor. In some aspects, the CD3+ T cells have a purity greater than about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or between any percentage between any two percentages referred to above or herein. In some aspects, the CD3+ T cells have a viability greater than about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or between any percentage between any two percentages referred to above or herein. In some aspects, the isolated T cells are transduced with a CAR, including, as non-limiting examples, a CD19 or B-cell maturation antigen (BCMA) CAR, or a dual antigen CAR, and further expanded. High-quality T cells with optimal viability and purity are imperative for selective delivery of CAR exclusively to T cells, ensuring effective transduction and successful CAR-T therapy.

[0051] In one aspect, separated T cells, e.g., CD3+ T cells are transduced using a lentiviral construct comprising a 5’ long terminal repeat, a sequence encoding a CAR, at least one elementselected from a linker and a tag, and a 3’ long terminal repeat, as described in U.S. provisional application 63 / 641,846 filed May 2, 2024.

[0052] In some aspects, CAR constructs and dual antigen CAR constructs can be made by methods known in the art, including, for example CD19 or B-cell maturation antigen (BCMA) CARs, (See, e.g., U.S. Pat. Nos. 12,195,514; 12,214,037; 10,357,514; and 10,647,778, and Xie et al., Cancers (Basel). 2022 Jun 30;14(13):3230, each of which is herein incorporated by reference), and used to transduce target cells. In some aspects, a CAR construct is a dual antigen CAR construct. In some embodiments, target cells, such as, for example, CD3+ T cells are transduced with a CAR construct such as, for example, a CD19 or BCMA CAR, or a dual antigen CAR construct, e.g., without limitation, CD19 / BCMA, CD19 / CD20, CD19 / CD22, CD19 / CD123, BCMA / CD38, BCMA / .GPRC5D, CD138 / CD38, CD5 / CD7, BCMA / CS1 or other dual constructs that CD 19 or BCMA and a second cancer antigen target.

[0053] It is noted that in this disclosure and particularly in the claims and / or paragraphs, terms such as "comprises", "comprised", "comprising" and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean "includes", "included", "including", and the like; and that terms such as "consisting essentially of" and "consists essentially of" have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

[0054] These and other embodiments are disclosed or are obvious from and encompassed by the following Detailed Description.BRIEF DESCRIPTION OF THE DRAWINGS

[0055] The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings.

[0056] FIG. 1 displays a schematic overview of the microbubble-based cell separation process. Undesired, target cells are bound to or labeled with a representative specific biotinylated antibody binding agent. Streptavidin-coated microbubbles are added to bind the bound, labeled target cells via the antibodies. Following incubation, the microbubble-bound cells float to the top of the sample container, allowing the nontarget unlabeled cells to be collected from the bottom.

[0057] FIG. 2 shows a flowchart of the exemplary open microbubble-based CD3+ T cell isolation protocol. Key steps include washing the starting cell sample to remove platelets,labeling non-target cells with a biotinylated antibody cocktail, capturing the labeled cells with streptavidin microbubbles, centrifuging and allowing microbubble-bound cells to separate by floatation, and collecting the unlabeled CD3+ T cells from the bottom pellet. All steps are performed in open tubes using manual pipetting.

[0058] FIG. 3 displays a flowchart of the exemplary closed microbubble-based CD3+ T cell isolation protocol. The initial cell washing step is performed using an automated Sepax device. Subsequent antibody labeling and microbubble capture steps occur within a closed bag. After incubation, the bag is hung vertically to allow microbubble-bound cells to float to the top. The bottom fraction containing purified CD3+ T cells is then welded off and collected into a new bag via the Sepax instrument.

[0059] FIG. 4 shows the starting vs recovered cells (left chart) and viability (right chart) of isolated cells in an open vs closed process. The left bar graph compares the absolute number of total nucleated cells in the starting leukapheresis sample versus the number of viable CD3+ T cells recovered after microbubble separation using either the exemplary open or closed process. Both processes achieved a similar T cell yield, representing approximately 30% of the starting cell number. The right bar graph compares the percent viability of total nucleated cells in the starting leukapheresis sample versus the viability of target CD3+ T cells recovered after microbubble isolation using either the exemplary open or closed process. Cell viability remained high (>98%) after both isolation methods, comparable to the input sample.

[0060] FIG. 5 displays a bar graph comparing the number of starting leukocytes versus the number of recovered viable T cells in the CliniMACS Prodigy system (a standard immunomagnetic bead-based cell separation device). The resulting T cell yield is similar to both the open and closed microbubble processes of FIG. 4.

[0061] FIGs. 6 A and 6B display representative flow cytometry plots characterizing the cell phenotype before and after open process microbubble separation. FIG. 6A shows the starting leukapheresis sample contained a heterogeneous mixture of B cells, T cells, NK cells and monocytes based on expression of lineage markers. FIG. 6B shows that after separation, the purified fraction was highly enriched for CD3+ T cells (82.8%) with depletion of non-target cell types to <5% each. The isolated T cells also showed expected proportions of CD4+ helper and CD8+ cytotoxic subsets.

[0062] FIG. 7 displays a quantification of cell lineage frequencies in a starting leukapheresis sample by flow cytometry using an exemplary open microbubble separation process. Values represent the percent of CD3+ T cells (total, CD4+, CD8+), CD56+ NK cells, CD14+ monocytes and CD19+ B cells among viable CD45+ leukocytes.

[0063] FIG. 8 displays process and analytical (flow cytometry-based) evaluation assessing transduction efficiency of microbubble-purified T cells using a GFP-tagged lentiviral vector encoding an anti-CD19 chimeric antigen receptor (CAR). T cells were activated, transduced at varying multiplicities of infection (MOI), and analyzed for GFP and CAR expression after 72 hours. At MOI >5, approximately 15% of T cells co-expressed GFP and CAR, indicative of successful transduction. Higher MOIs did not yield further improvements.

[0064] FIGs. 9A-9D display the quantification of cell lineage frequencies in a starting leukapheresis sample by flow cytometry. Values represent the percent of CD3+ T cells (total, CD4+, CD8+), CD56+ NK cells, CD14+ monocytes and CD19+ B cells among viable CD45+ leukocytes. FIG. 9A shows the gating strategy for sorting analysis: the initial flow cytometry counts are displayed with a gate Pl capturing about 38.54% of all cells in the sample (left); gate P2 excluding doublets and debris capturing about 99.75% of the Pl-gated cells (middle); and Q2 viable cell gating of the P2-gated cells indicating that 98.31% of sorted cells were viable (right). FIG. 9B shows the flow cytometry histograms of the viable CD45+ leukocytes (left) and CD3+ leukocytes (right). FIG. 9C shows the flow cytometry histograms of the flow cytometry counts of CD4+, CD8+ double-labeled CD3+ leukocytes (leftmost), the flow cytometry counts of CD 16 / 56+ cells (center-left), and the flow cytometry histograms of the CD14+ leukocytes (center-right) and CD19+ leukocytes (rightmost). FIG. 9D shows the summary of the percent of the cell types analyzed: 58% CD3+, 49% CD3+ / CD4+, 44% CD3+ / CD8+, 6% CD56+ / CD16+, 27% CD14+, and 5% CD19+ cells.

[0065] FIGs. 10A-10D display the quantification of residual non-T cell frequencies compared to T cell frequencies after closed process microbubble separation by flow cytometry, using the same markers as in FIGs. 9A-9D. FIG. 10A shows the gating strategy for sorting analysis: the initial flow cytometry counts are displayed with a gate Pl capturing about 71.34% of all cells in the sample (left); gate P2 excluding doublets and debris capturing about 99.91% of the Pl -gated cells (middle); and Q2 viable cell gating of the P2-gated cells indicating that 99.66% of sorted cells were viable (right). FIG. 10B shows the flow cytometry histograms of the viable CD45+ leukocytes (left) and CD3+ leukocytes (right). FIG. 10C shows the flow cytometry histograms of the flow cytometry counts of CD4+, CD8+ double-labeled CD3+ leukocytes (leftmost), the flow cytometry counts of CD16 / 56+ cells (center-left), and the flow cytometry histograms of the CD 14+ leukocytes (center-right) and CD 19+ leukocytes (rightmost). FIG. 10D shows the summary of the percent of the cell types analyzed: 95% CD3+, 52% CD3+ / CD4+, 41% CD3+ / CD8+, 3% CD56+ / CD16+, 0% CD14+, and 0% CD19+ cells.Near complete depletion of B cells, monocytes and NK cells to < 1% is achieved, while T cells are enriched to >90%.

[0066] FIG. 11 displays the summary of the flow cytometry analysis of cell phenotypes before and after the exemplary closed process microbubble T cell isolation, similar to FIG. 7. Again, starting leukapheresis samples contain various leukocyte subsets which are efficiently removed after separation, leaving a highly purified CD3+ T cell population (96% of CD45+ cells). Recovered target T cells include both CD4+ and CD8+ subsets at typical ratios.

[0067] FIGs. 12A and 12B display a comparison of key process and cost parameters between the exemplary microbubble T cell isolation method (open and closed versions) and the CliniMACS Prodigy platform. Microbubble separation offers significant advantages in terms of consumables cost ($500-600 vs $12,500 per run), processing time (2.5 vs 5.5 hours), equipment requirements and ease of use.

[0068] FIG. 13 displays a summary of T cell recovery and purity data using the exemplary open and closed microbubble processes. Both approaches achieve >40% T cell yield with >95% purity and viability from the starting leukapheresis material. The closed system performs slightly better, likely due to reduced cell loss and contamination.

[0069] FIG. 14 displays a comparison of CD3+ T cell recovery efficiency between the microbubble method (open and closed) and the CliniMACS Prodigy across multiple validation runs. All processes show comparable yields around 40% on average.

[0070] FIG. 15 displays a bar graph directly comparing T cell recovery between the exemplary open and closed microbubble processes and the CliniMACS Prodigy system. Bars represent the absolute number of starting CD3+ T cells in leukapheresis samples versus the number recovered after separation.

[0071] FIG. 16 shows the workflow of CAR-T Cell Therapy.

[0072] FIGs. 17A and 17B show representative schematics of certain embodiments of a negative selection microbubble methodology and a positive selection detachable Dynabeads® methodology. FIG. 17A is a representative schematic of a negative selection microbubble method. The negative fractions bind to the microbubbles and float on top. The positive fractions remain at the bottom and can be collected for further processing. FIG. 17B is a representative schematic of positive selection detachable Dynabeads® (CD4 / CD8) method. The positive fractions bind to magnetic Dynabeads® and then are detached from the Dynabeads® with release buffer. The negative fractions do not bind to the Dynabeads® and are removed.

[0073] FIG. 18 shows the exemplary Microbubble-Based T Cell Isolation Using Open and Closed System Designs: open process (top) and closed process (bottom), starting from frozen or fresh leukapheresis material (APH) and resulting in cells ready for CAR-T manufacturing.

[0074] FIGs. 19A-E shows the T cell purity, viability and performance results of using either the exemplary microbubble open or microbubble closed process. FIG. 19A shows that both workflows result in effective enrichment of CD3+ T cells with a significant reduction in non-target immune populations (CD14+, CD19+, CD56+). FIG. 19B shows that high postisolation viability (>90%) and consistent CD4:CD8 ratios were maintained across donors. FIGs. 19C and 19D show that subsequent lentiviral transduction with CD19 CAR demonstrated robust expansion (FIG. 19C) and CAR expression (FIG. 19D) through Day 11. The closed system design offers GMP-compliant automation while preserving performance. FIG. 19E shows the fold cell expansion resulting from the microbubble closed system.

[0075] FIG. 20 shows the Detachable Dynabeads® Isolation System Design, a closed- process design starting from frozen or fresh leukapheresis material (APH) and resulting in cells ready for CAR-T manufacturing.

[0076] FIGs. 21A-G show the T cell purity, viability and performance of using the exemplary Detachable Dynabeads® closed process. FIGs. 21A-C show that the isolation workflow results in effective enrichment of CD3+ T cells with a significant reduction in nontarget immune populations (CD14+, CD19+, CD56+). FIG. 21A shows the percentage of pre- and post-isolation immune cell population for CD3+ T cells, CD56+ NK cells, CD 14+ monocytes, CD19+ B cells, and other cell types. FIG. 21B shows the T cell purity of pre- and post-isolation by flow cytometry. FIG. 21C shows the reduction from the pre-isolation (top) to post-isolation (bottom) percentage of impurities by CD45+ CD16 / 56+ dual positive, CD45+ CD19+ dual positive and CD45+ CD14+ dual positive cell populations. FIG. 21D shows the high post-isolation viability (>90%) and consistent CD4:CD8 ratios were maintained. FIGS. 21E-F show that subsequent lentiviral transduction with CD19 CAR demonstrated robust expansion (FIG. 21E) and CAR expression (FIG. 21F) through Day 11. This system design offers GMP-compliant automation while preserving performance. FIG. 21G shows the fold cell expansion resulting from the detachable Dynabeads® closed system.DETAILED DESCRIPTION

[0077] The inventions described and claimed herein have many attributes and embodiments including, but not limited to, those set forth or described or referenced in this Detailed Description. It is not intended to be all-inclusive and the inventions described andclaimed herein are not limited to or by the features or embodiments identified in this Detailed Description, which is included for purposes of illustration only and not restriction.

[0078] Cell isolation for cellular therapies, including for chimeric antigen receptor (CAR)- T Cell Therapy Clinical Manufacturing, is a first and critical step of the process. Delays in cell isolation extend turnaround and reduce manufacturing success. There is limited starting material due to, as non-limiting examples, donor availability and donor variability. Having rapid, efficient, high-quality isolation of desired cells significantly impacts the overall quality and efficacy of the final products.

[0079] For critically ill patients, rapid and efficient isolation of T cells is a critical first step in CAR-T cell manufacturing and essential to minimize turnaround time. High-quality T cells with optimal viability and purity are imperative for selective delivery of CAR exclusively to T cells, ensuring effective transduction and successful CAR-T therapy. Although current isolation methods have addressed some challenges for CAR-T manufacturing, the methods and compositions of this disclosure provide additional opportunities to better fit for each therapy. Embodiments of the present invention include rapid T cell isolation processes that leverage microbubble and detachable beads, e.g., detachable Dynabeads® that present additional opportunities and process flexibilities. Comparing the new isolation methods, including open process microbubble, closed process microbubble, open process Dynabeads® and closed process Dynabeads® isolation methods, showed that each of the new isolation methods performs comparably with historical data from standard CAR-T processes. In some embodiments these new isolation methods offer advantages in operational time and raw material costs as viable alternatives for standard isolation methods.

[0080] A. Certain Exemplary Definitions

[0081] Before the present compounds, compositions, articles, devices, and / or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods or specific recombinant biotechnology methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0082] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like.

[0083] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed. For example, if the value “10” is disclosed the “less than or equal to 10” as well as “greater than or equal to 10” is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as values between 10 and 15. For example, it is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed. In this application, if a data point range is disclosed, it is understood that each unit from the lowest data point to the highest stated datapoint, including the first (lowest) and last (highest) data point is disclosed. For example, if a data point range 1-20 is disclosed, it is understood that data points 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, and each unit between any two particular units in the range are also disclosed. It is also understood that whenever a series of values are disclosed, that any range falling between any two of the recited values is also understood to be included.

[0084] In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:

[0085] “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes embodiments where the event or circumstance occurs and embodiments where it does not.

[0086] As described herein, any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited rangeand, when appropriate, fractions thereof (including one-tenth and one-hundredth of an integer), unless otherwise indicated.

[0087] As used herein, the term “substantially the same” means an amount of expression, level, and / or activity of a gene or gene product within 90% of baseline expression, level, and / or activity as in a subject unaffected by a disease. “Substantially the same” can also mean an amount of expression, level, and / or activity of a gene or gene product within 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, to 100% of the baseline expression, level, and / or activity as in a subject unaffected by unaffected by a disease.

[0088] As used herein, the term “separation”, includes any means of substantially purifying one component from another, such as, e.g., by flotation, centrifugation, filtration or magnetic attraction. In some embodiments, separation includes, e.g, fractionation, centrifugation, filtration, magnetic attraction, chelation, and pelletization. As used herein, the terms “fractionation” or “fractionating” describe the separation of one component from another resulting in distinct component compartments, such as, e.g., a non-limiting example in which a mixture of labeled target cells, unlabeled cells, debris, and buffer are distinctly separated in a single container after centrifugation.

[0089] As used herein, the term “isolation” or “isolating”, includes the extraction, purification, and separation of cells from a biological sample, wherein the target cells are removed from non-target cells and other cellular components, contaminants, or impurities, to obtain a preparation that is suitable for downstream applications such as culture, activation and transduction.

[0090] As used herein, the term “microbubbles” refers to small gas-filled spheres, typically ranging in diameter from 0.1 to 100 microns, more commonly 1 to 50 microns, and frequently 2 to 30 microns. Microbubbles, including albumin-coated microbubbles, can be obtained from commercial sources (e.g., Akadeum Life Sciences®) or made by methods known in the art (Khan, A. et al., Kinetics of albumin microbubble dissolution in aqueous media. Soft Matter, 2020,16, 2149-2163; J. L. Chen, A. H. Dhanaliwala, A. J. Dixon, A. L. Klibanov and J. A. Hossack, Synthesis of albumin microbubbles using a microfluidic device for real-time imaging and therapeutics, 2013 IEEE International Ultrasonics Symposium (IUS), Prague, Czech Republic, 2013, pp. 1150-1153, doi: 0.1109 / ULTSYM.2013.0294; Upadhyay, A. et al., Synthesis, characterization and stability of BSA-encapsulated microbubbles, RSC Adv., 2016,6, 15016-15026; Sirsi, S. R., & Borden, M. A. (2009). Microbubble compositions, properties and biomedical applications. Bubble Science, Engineering & Technology, 1(1-2),3-17. doi.org / 10.1179 / 175889709X446507; and Borrelli, M. et al., Production of uniformly sized serum albumin and dextrose microbubbles, Ultrasonics Sonochemistry, (19) 1, 2012, 198-208, doi.org / 10.1016 / j.ultsonch.2011.05.010, each of which is herein incorporated by reference). In some embodiments, the microbubbles have a mean diameter between about 1-10 micrometers (pm), comprise a shell made from a material comprising, e.g., as non-limiting examples, proteins, polymers, lipids, sugars, and surfactants, and a gaseous core selected from the group consisting of air, nitrogen, carbon dioxide, and perfluorocarbons. In some embodiments, the microbubbles have a mean diameter that ranges between about 1-10 pm. In some embodiments, the microbubbles have a mean diameter of 0.5 pm, 1 pm, 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, 7 pm, 8 pm, 9 pm, 10 pm, 11 pm, 12 pm, 13 pm, 14 pm, 15 pm, or any diameter between any two diameters referred to above or herein. In some embodiments, microbubbles are coated with affinity molecules such as antibodies to enable specific binding and isolation of target cells or other analytes from complex samples. In some embodiments, microbubbles are composed of different materials such as glass, lipids, or polymers to enhance stability and binding efficiency. In some embodiments, microbubbles are coated or functionalized with a member of cognate binding pair, such as streptavidin, avidin, superavidin, biotin, or protein A, facilitating microbubble binding to a binding agent-bound cells, for example, cells bound to, or labeled with a binding agent, e.g., antibodies, nanobodies, biotinylated antibodies or biotinylated nanobodies. In some embodiments, one or more binding agents bind moieties on target cells, and microbubble-binding agent-bound target cells are separated from unbound nontarget cells. In embodiments, one or more binding agents bind moieties on nontarget cells, and microbubble-binding agent-bound nontarget cells are separated from unbound target cells. In some embodiments, microbubbles bind to binding agent-bound target cells. In some embodiments, microbubbles bind to binding agent-bound nontarget cells. In some embodiments, microbubbles bind to biotinylated camelid-derived single domain VHH antibodies, or nanobodies having high specificity for surface proteins on target cells including, as non-limiting examples, CD3, CD4, and CD8. In some embodiments, sequential microbubble isolation steps using a different binding agent in each step is performed. For example, in some embodiments, sequential isolation steps using anti-CD4 and anti-CD8 antibodies or nanobodies are performed.

[0091] The term "analyte" is used herein to refer to any molecule, cell or other entity that is the subject of isolation, enrichment or detection using the affinity microbubble compositions and methods of the present invention. In particular embodiments, the analyte is a specific cell type such as a T lymphocyte or T cell subset, e.g., a CD4+ population or a CD8+ population.In other embodiments, the analyte is a protein, peptide, nucleic acid, receptor, ligand, carbohydrate, lipid, drug or any other biological or non-biological molecule that is bound by an affinity molecule coated on the microbubble surface. In certain embodiments, the analyte is an individual molecular species or a higher-order complex or assembly such as an intracellular organelle, virus or whole cell. In certain applications, the analyte is a particular antibody or class of antibodies that is captured using microbubbles functionalized with an appropriate antigen or other binding partner.

[0092] As used herein, the term “surface marker” and “cell surface marker” are used interchangeably and refer to proteins, sugars, lipids, cholesterol, glycolipids, and phospholipids that are on, embedded in, or otherwise have a portion that extends from, the surface of a cell and is recognized by a binding agent, e.g., as a non-limiting example, an antibody. In some embodiments, surface markers are proteins known as cluster of differentiation (CD) proteins, such as, e.g., CD3, CD4, CD8, CD14, CD16, CD19, CD36, CD56, CD123, or CD235a. In some embodiments, surface makers comprise one or more of CD3, CD4, CD8, CD14, CD16, CD19, CD36, CD56, CD123, or CD235a. In some embodiments, surface markers can include or exclude CD3, CD4, CD8, CD14, CD16, CD19, CD36, CD56, CD123, or CD235a.

[0093] As used herein, the term “binding agent” refers to a molecule that binds a molecule of interest, such as a ligand binding its cognate receptor or antibodies recognizing cell surface markers. As used herein, binding agents include, as non-limiting examples, antibodies, peptides, proteins, aptamers, nanoparticles, lectins, cyclodextrins, avidin, streptavidin, or SuperAvidin™. In some embodiments, binding agents bind to one or more surface marker of nontarget or target cell types. In some embodiments, binding agents are conjugated to a moiety including, as non-limiting examples, a fluorescent dye, a fluorescent protein, an enzyme, a metal, a protein tag, an oligonucleotide tag, or a combination thereof. In one embodiment, binding agents are used to target specific T cell populations from PBMCs and other mixed cell populations. In another embodiment, binding agents are used to select nontarget cell populations for removal.

[0094] Functionalized, as used herein, refers to a binding particle, e.g, bead, e.g, detachable bead, or microbubble, with one or more binding agents bound to its exterior. In some embodiments, functionalized beads or microbubbles use, as non-limiting examples, antibody or nanobody binding and cognate receptor- ligand interactions, which can include or exclude, e.g., streptavidin-biotin, avidin-biotin, superavidin-biotin, or protein A-antibody binding. In some embodiments, functionalized microbubbles are streptavidin-coated albumin microbubbles. In some embodiments, functionalized detachable beads are antibody-coated ornanobody-coated detachable beads, including, as non-limiting examples, anti-CD3+ detachable Dynabeads®, anti-CD3+ / CD28+ detachable Dynabeads®, anti-CD4+ detachable Dynabead®, or anti-CD8+ detachable Dynabeads®. In some embodiments, the functionalized detachable beads are anti-CD3+ detachable Dynabeads®. In some embodiments, the functionalized detachable beads are anti-CD3+ / CD28+ detachable Dynabeads®. In some embodiments, the functionalized detachable beads are anti-CD4+ detachable Dynabeads®. In some embodiments, the functionalized detachable beads are anti-CD8+ detachable Dynabeads®.

[0095] As used herein, the term “detachable bead” refers to a bead of uniform size and spherical shape having superparamagnetic property and specific surface chemistry, that is used to bind specific proteins of interest and is later detached from the same proteins of interest. For example, a detachable bead is a polystyrene bead that is coated with any one or more binding agents featured herein, e.g. without limitation, antibodies or nanobodies specific for, e.g., as a non-limiting example, CD3, CD4, and / or CD8 or any combination or subcombination thereof. In some embodiments, the detachable bead is a functionalized detachable bead, e.g., a detachable magnetic bead, e.g., a Dynabead®.

[0096] As used herein, the terms “negative selection” and “negatively selected” refer to a method of selection where a target within a sample is not bound or otherwise manipulated. As used herein, an exemplary method of negative selection refers to a target cell type within a sample being isolated by the stepwise removal, including through the use of binding agents or binding particles, of nontarget cells. In some embodiments, nontarget cells are initially bound by antibodies or nanobodies, subsequently bound by microbubbles, separated from target cells by centrifugation, filtration, or flotation, and removed from the sample, such that the negatively selected target cells are all that remain. In some embodiments, nontarget cells are bound by binding particles such as detachable beads, separated from target cells by centrifugation or using a magnet, and removed from the sample, such that the negatively-selected target cells are all that remain.

[0097] As used herein, the terms “positive selection” and “positively selected” refer to a method of selection where a target within a sample is directly bound or otherwise manipulated to isolate the target. As used herein, an exemplary method of positive selection refers to a target cell type within a sample being isolated by the direct binding of target cells. As a non-limiting example, desired cells are bound by binding particles, e.g., microbubbles or beads, e.g., detachable beads, separated from nontarget cells by, e.g., flotation, filtration, centrifugation, or magnetic capture, then nontarget cells are removed such that only the binding particle-boundtarget cells, e.g. the microparticle-bound target cells or bead-bound target cells, are all that remain, and the detachable beads are released using a release buffer and bound a magnet, such that the positively-selected target cells are obtained.

[0098] As used herein, target cells comprise immune cells, stem cells, progenitor cells, malignant cells, bacterial cells, and parasites. In some embodiments, target cells are the immune cells T lymphocytes (T cells). In some embodiments, the target cells are CD3+. In some embodiments, the target cells are CD4+ and / or CD8+ T cells. In some embodiments, the target cells are tumor infiltrating T cells. In some embodiments, nontarget cells include or exclude immune cells, stem cells, progenitor cells, malignant cells, bacterial cells, and parasites. In some embodiments, nontarget cells include or exclude red blood cells, granulocytes, monocytes, B cells, NK cells, or platelets. In some embodiments, nontarget cells include red blood cells, granulocytes, monocytes, B cells, NK cells, or platelets. In some embodiments, the one or more binding agents binds to one or more surface markers of positively selected target cell types. In some embodiments, the one or more binding agent binds to one or more surface markers of positively selected nontarget cell types. In some embodiments, the target cells are stem cells. In some embodiments, the target cells are progenitor cells. In some embodiments, the nontarget cells are malignant cells. In some embodiments, the target cells are bacterial cells. In some embodiments, the target cells are parasites.

[0099] As used herein, nontarget cells comprise immune cells, stem cells, progenitor cells, malignant cells, bacterial cells, and parasites. In some embodiments, nontarget cells include or exclude immune cells, stem cells, progenitor cells, malignant cells, bacterial cells, and parasites. In some embodiments, nontarget cells include or exclude red blood cells, granulocytes, monocytes, B cells, NK cells, or platelets. In some embodiments, nontarget cells include red blood cells, granulocytes, monocytes, B cells, NK cells, or platelets. In some embodiments, the nontarget cells red blood cells, granulocytes, monocytes, B cells, NK cells, or platelets. In some embodiments, the nontarget cells are immune cells. In some embodiments, the nontarget cells are CD3+. In some embodiments, the nontarget cells are CD4+ and / or CD8+ T cells. In some embodiments, the target cells are tumor infiltrating T cells. In some embodiments, the one or more binding agent binds to one or more surface markers of negatively selected target cell types. In some embodiments, the one or more binding agent binds to one or more surface markers of negatively selected nontarget cell types. In some embodiments, the nontarget cells are stem cells. In some embodiments, the nontarget cells are progenitor cells.In some embodiments, the nontarget cells are malignant cells. In some embodiments, the nontarget cells are bacterial cells. In some embodiments, the nontarget cells are parasites.

[0100] As used herein, the terms “leukapheresis sample” and “leukapheresis product” interchangeably refer to the leukocytes (also known as “white blood cells”) separated from other blood components through the process of leukapheresis.

[0101] As used herein, the term “stimulation” refers to activation of a cell type. As a nonlimiting example, T cells are stimulated using anti-CD3 / anti-CD28-coated beads or anti- CD3 / CD28 antibodies or nanobodies leading to changes in gene expression and triggering cell expansion. In some embodiments, activation may also include contacting T cells with cytokines, e.g., without limitation IL-2, IL-7, IL-4, IL- 15, or a subcombination or a combination thereof.

[0102] As used herein, the term “cell purity” refers to the amount of cells positive for specific markers and is assessed by, as a non-limiting example, flow cytometry after staining with fluorescent antibodies against specific cellular markers. For example, T cell purity is assessable by staining for CD3, CD3, and / or CD8 and using flow cytometry or microscopy. In some embodiments, the method achieves at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or at any percentage between any two percentages of target cell purity referred to above or herein. In some embodiments, the methods disclosed herein achieve at least 80% target cell purity. In some embodiments, the method achieves at least 90% target cell purity. In some embodiments, the method achieves at least 95% target cell purity. In some embodiments, the method achieves at least 80% target cell purity and at least 50% target cell recovery.

[0103] As used herein, the term “cell recovery” refers to the amount of cells that are obtained after a method of cell isolation is performed. Cell recovery further refers to the percentage of cells remaining relative to the number of cells at the start of a cell insolation process. In some embodiments, the method achieves at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or at any percentage between any two percentages of target cell recovery referred to above or herein. In some embodiments, the method achieves at least 50% target cell recovery. In some embodiments, the method achieves at least 60% target cell recovery. In some embodiments, the method achieves at least 70% target cell recovery. In some embodiments, the method achieves at least 80% target cell recovery. In some embodiments, the method achieves at least 90% target cell recovery.

[0104] As used herein, the terms “closed system” and “closed process” indicate that the method is performed in an environment where the product or material is isolated from the external environment. For example, a closed system includes, e.g., a cell separation bag.Conversely, as featured and disclosed herein, the terms “open system” and open process” indicate that the method performed in an environment where the product or material is exposed to the external environment. In some embodiments, the method utilizing microbubbles is performed in an open process. In some embodiments, the method utilizing microbubbles is performed in a closed process. In some embodiments, the method utilizing detachable beads is performed in an open process. In some embodiments, the method utilizing detachable beads is performed in a closed process.

[0105] As used herein, the term “CD4:CD8 T cell ratio” refers to the proportion of CD4+ T cells to CD8+ T cells. In healthy individuals, the proportion of CD4+ T cells to CD8+ T cells is between about 1.0 to 3.0. In some embodiments, the isolation process of this disclosure improves cell viability and / or T cell purity while, in some embodiments, also maintaining CD4:CD8 T cell ratios of the isolated T cells. In some embodiments, the method maintains a CD4:CD8 ratio between about 1.0: 1.0 to 3.0: 1.0. In some embodiments, the CD4:CD8 ratio is between 0.5:1.0, 1.0:1.0, 1.5: 1.0, 2.0:1.0, 2.5:1.0, 3.0:1.0, 3.5:1.0, 4:1.0, 4.5:1.0, or 5.0:1.0, or at any ratio between any two ratios referred to above or herein.

[0106] B. Certain Exemplary Methods

[0107] The present disclosure features GMP-compliant methods to isolate target cells from samples using microbubbles. In some embodiments, microbubbles are small gas-filled bubbles, having the unique ability to specifically bind to and float microbubble -bound cells, thus enabling easy separation from unbound or unlabeled cells. In some embodiments, microbubbles are coated in streptavidin, facilitating microbubble binding to a binding agentbound cells, for example, cells labeled with biotinylated antibodies. In some embodiments, one or more binding agents bind moieties on target cells, and microbubble-binding agent-bound target cells are separated from unbound nontarget cells. In some embodiments, one or more binding agents bind moieties on nontarget cells, and microbubble-binding agent-bound nontarget cells are separated from unbound target cells. In some embodiments, microbubbles bind to binding agent-bound target cells. In some embodiments, microbubbles bind to binding agent-bound nontarget cells. In some embodiments, microbubbles bind to biotinylated camelid- derived single domain VHH antibodies, or nanobodies, having high specificity for surface proteins on target cells including, as non-limiting examples, CD3, CD4, and CD8. In some embodiments, sequential microbubble isolation steps using a different binding agent in each step is performed. For example, in some embodiments, sequential isolation steps using anti- CD4 and anti-CD8 antibodies or nanobodies are performed.

[0108] In some embodiments, microbubbles are used to separate a target cell population from a mixture of cell populations. In some embodiments, microbubbles are used to separate target T cells from nontarget cell populations such as, e.g., as non-limiting examples, red blood cells, granulocytes, monocytes, B lymphocytes (B cells), natural killer (NK) cells, or platelets.

[0109] The present disclosure further relates to methods, compositions and kits for isolating CD3+ T lymphocytes from heterogeneous cell populations using glass microbubbles coated with anti-CD3 antibodies. The methods exploit the high surface area, buoyancy and ease of functionalization of the microbubbles to enable rapid, specific and gentle affinity-based cell separation.

[0110] The present disclosure also features innovative GMP-compliant methods to isolate target cells by using other binding particles, e.g., as a non-limiting example, detachable beads, including detachable Dynabeads®. As featured and disclosed herein, detachable beads such as detachable Dynabeads® are uniform, spherical, and superparamagnetic polystyrene beads. In some embodiments, the Dynabeads® are coated with or functionalized with a binding agent having high specificity for target surface proteins including, as non-limiting examples, CD3, CD4, and CD8.

[0111] In some embodiments, the binding particles, e.g., detachable beads bind to binding agent-bound target cells. In some embodiments, the binding particles, e.g., the detachable beads bind to binding agent-bound nontarget cells. In some embodiments the binding particles are functionalized.

[0112] In some embodiments, the functionalized binding particles, e.g., detachable magnetic beads, e.g., functionalized Dynabeads® are functionalized through cognate binding partner interactions. In some embodiments, the cognate binding partners are, e.g., streptavidinbiotin, avidin-biotin, protein A-antibody, and / or complementary oligonucleotides. In some embodiments, the functionalized Dynabeads® are functionalized with streptavidin-biotin derivative complexes coupled to binding agents. In some embodiments, the binding agents comprising the complexes are, e.g., without limitation, camelid-derived single domain VHH antibodies, or nanobodies, having high specificity for surface proteins on target cells including, as non-limiting examples, CD3, CD4, and CD8. In some embodiments, Dynabeads® are coated with binding agents having high specificity for surface proteins on nontarget cells including, as non-limiting examples, CD14, CD16, CD19, CD36, CD56, CD123, or CD235a.

[0113] In some embodiments, the detachable Dynabeads® specifically bind target cells, and cells which are not bound to beads are separated from the Dy nabead® -bound target cells. In some embodiments, Dynabead®-bound target cells are actively released from theDynabeads® by contacting the Dynabead®-bound target cells with a release buffer containing a competitive binding agent that displaces a biotin derivative on the surface of the Dynabeads®. The beads are then magnetically captured and separated from the desired target cells. In some embodiments, the Dynabeads® specifically bind nontarget cells, and cells which are not bound to the Dynabeads® are separated from the Dynabead®-bound nontarget cells using a magnet separating nontarget Dynabead®-bound cells from the unbound target cells.

[0114] In some embodiments, this disclosure features cell isolation processes using the binding particles, e.g., microbubbles or detachable beads, e.g., functionalized detachable beads, e.g., detachable Dynabeads®. In some embodiments, this disclosure features open and closed cell isolation processes to shorten the time to isolate target cells, such as, for example, CD3+ T cells for making CAR-T products. Using the process, target cells, such as, for example, CD3+ T cells were enriched, expanded, and transduced with a CAR construct. In some embodiments, target cells, such as, for example, CD3+ T cells, are enriched, expanded, and transduced with a CAR construct or a dual antigen CAR construct.

[0115] Preparation of glass microbubbles

[0116] Glass microbubbles are more chemically and mechanically stable than albumin microbubbles. Their preparation requires several washing and surface modification steps. In some embodiments, borosilicate glass microparticles with a density around 0.6 g / cc and sizes around 30 pm are the starting material (e.g. 3M Scotchlite KI glass). In some embodiments, the particles are first treated with a strong acid or base solution (e.g. 0.5-2 N hydrochloric acid or sodium hydroxide for 10-24 hrs at 50-100°C) to clean the surface and generate silanol groups. In an embodiment, sonication or flotation in water or alcohol is used to remove residual acid / base and fractionate intact microparticles from fragments.

[0117] In an embodiment, the glass microparticles are silanized to introduce functional amine, epoxy, aldehyde or cis-diol groups for covalent antibody coupling. For example, 3- aminopropyl triethoxysilane (APTES) treatment produces an amine-functionalized surface, while 3-glycidoxypropyl trimethoxy silane (GPTMS) yields epoxy groups that can be further converted to aldehydes or cis-diols by acid hydrolysis. In an embodiment, the silanization reaction is performed in dry acetone, ethanol or toluene with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15% silane, or any percentage of silane between any two percentages or silane referred to above or herein, for 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4 hours, or for any amount of time between any number of hours between the hours referred to above or herein at 20-60°C. In an embodiment, the silanization reaction is in dry acetone, ethanol or toluene with 1-10% silane for 0.5-3 hr at 20-60°C. In one embodiment, unreacted silanes are removed by extensivewashing with the same solvent followed by drying. Silane-modified glass microbubbles are generally stable for several weeks to months when stored dry at room temperature.

[0118] In some embodiments, the type and density of functional groups on the glass microbubbles are regulated by the silane reagent used and the reaction conditions. High surface densities (>1 group per nm2) promote multivalent antibody coupling and target binding, but also increase the risk of non-specific interactions and aggregation. Conversely, very low densities (<0.01 per nm2) are insufficient for stable attachment and cell capture. Optimal values need to be determined for each application.

[0119] Antibody conjugation

[0120] As used herein, the term “antibody” (Ab) includes, without limitation, a glycoprotein immunoglobulin which binds specifically to an antigen. In general, an antibody can comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CHI, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain, CL. The VH and VL regions are further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the Abs may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. An immunoglobulin may derive from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG and IgM. IgG subclasses also include but are not limited to human IgGl, IgG2, IgG3 and IgG4. “Isotype” refers to the Ab class or subclass (e.g., IgM or IgGl) that is encoded by the heavy chain constant region genes. The term “antibody” includes or excludes both naturally occurring and non-naturally occurring Abs; monoclonal and polyclonal Abs; chimeric and humanized Abs; human or nonhuman Abs; wholly synthetic Abs; and single chain Abs. In certain embodiments, a nonhuman Ab is humanized by recombinant methods to reduce its immunogenicity in man. The term “antibody” also includes an antigen-binding fragment or an antigen-binding portion of any of the aforementioned immunoglobulins, and includes amonovalent and a divalent fragment or portion, and a single chain Ab, camelid-derived single domain VHH antibody or nanobody.

[0121] In some embodiments, monoclonal or polyclonal antibodies specific for the human CD3, CD4, and / or CD8 antigen are used to functionalize the albumin and glass microbubbles for T cell isolation. The antibodies can be in intact or fragmented (e.g. Fab) formats, derived from any species (e.g. mouse, rat, goat), and of any subtype (e.g. IgG, IgM). In an embodiment, hybridoma-produced murine anti-CD3, anti-CD4, and / or anti-CD8 IgGs are employed. In one embodiment, antibodies are purified from ascites fluid or cell culture supernatant by protein A / G chromatography prior to use.

[0122] In certain embodiments, antibodies are coupled to albumin microbubbles via either non-covalent biotin-avidin / streptavidin interactions or covalent chemical crosslinking. For the former approach, albumin microbubbles are first biotinylated by reacting surface lysine groups with NHS-biotin. In some embodiments, antibodies are separately modified with NHS-biotin at a molar ratio giving 2-6 biotins per antibody. In an embodiment, the biotinylated microbubbles are then coated with streptavidin, enabling them to capture biotinylated antibodies. In another embodiment, native antibodies are directly coupled to albumin microbubbles using reagents such as SMCC or SIA that react with lysine and cysteine groups. In an embodiment, a two-step biotinylation approach is used. In some embodiments albumin is human serum albumin or bovine serum albumin. Microbubbles may be made by methods known to those of skill in the art or obtained commercially.

[0123] In some embodiments, antibodies are conjugated to glass microbubbles via covalent reaction with surface amine, epoxy, aldehyde or cis-diol groups introduced by silanization. In one embodiment, amide, imine and / or secondary amine bonds are formed with the antibody's own amine and carboxyl groups. In some embodiments, reaction conditions including as nonlimiting examples, pH, ionic strength, and temperature, and antibody: microbubble ratios are optimized to achieve high coupling efficiency while preserving antibody binding activity. In certain embodiments, sodium cyanoborohydride or similar reducing agents is used to stabilize acid-labile imine bonds. In an embodiment, blocking reagents including as non-limiting examples, ethanolamine and glycine are used to quench residual reactive sites. In some embodiments, biotin / streptavidin linking is used to functionalize glass microbubbles with biotinylated antibodies.

[0124] In some embodiments, the presence and activity of antibodies on the microbubbles are verified by immunofluorescence staining with fluorophore-labeled anti-Fc antibodies or antigen proteins. In an embodiment, quantitation is be done by spectrofluorimetry or flowcytometry. In an embodiment, antibody surface densities of 1,000-100,000 per pm2are achieved for cell capture.

[0125] T cell isolation

[0126] Standard leukapheresis can be performed using instruments available from various manufacturers, including Haemonetics, Fenwall, and Cobe and following the manufacturers' instructions. In an embodiment, a sample containing the target CD3+ T cells, such as, for example, blood, leukapheresis product or tissue single cell suspension, is first processed to remove irrelevant solids (e.g. by density gradient centrifugation and / or red blood cell lysis). In an embodiment, the cell suspension is combined with an excess of anti-CD3 antibody-coated albumin or glass microbubbles, with a typical microbubble: target cell ratio of 10:1 to 100:1. In an embodiment, the mixture is incubated at room temperature for 15-30 min with gentle agitation to allow microbubble-cell binding. In an embodiment, the microbubbles and captured cells are then separated from the liquid phase by 1) natural buoyancy / creaming; 2) low speed centrifugation (<300 ref); or 3) passage through a flotation column, filtration, or microfluidic device. In an embodiment, the separated microbubbles are washed by resuspension and recentrifugation in buffer to remove weakly bound cells.

[0127] In one embodiment, the methods featured herein leverage the unique properties of microbubbles to efficiently separate target T cells from nontarget cell populations, providing a fast, gentle, and cost-effective alternative to traditional methods. In some embodiments, this disclosure features open and closed cell isolation processes to shorten the time to isolate target cells, such as, for example, CD3+ T cells for making CAR-T products. Using the exemplary processes detailed herein, target cells, such as, for example, CD3+ T cells were enriched, expanded, and transduced with a CAR construct.

[0128] In one embodiment, the present invention provides a method for isolating target cells from a sample comprising cells, the method comprising: (a) contacting the sample with one or more binding agents to form a mixture comprising binding agent-bound cells and unbound cells; (b) contacting the mixture comprising binding agent-bound cells and unbound cells with microbubbles that bind the binding agent to form a mixture comprising microbubblebinding agent-bound cells and unbound cells; (c) separating the microbubble-binding agentbound cells from the unbound cells; and (d) extracting the unbound cells, thereby obtaining target cells.

[0129] In one embodiment, the present invention provides a method for isolating target cells from a sample comprising cells, the method comprising: (a) contacting the sample with one or more binding agents to form a mixture comprising binding agent-bound cells andunbound cells; (b) contacting the mixture comprising binding agent-bound cells and unbound cells with binding particles, e.g., detachable beads that bind the binding agent to form a mixture comprising detachable bead-binding agent-bound cells and unbound cells; (c) separating the detachable bead-binding agent-bound cells from the unbound cells; and (d) extracting the unbound cells, thereby obtaining target cells.

[0130] In some embodiments, undesired, or nontarget cells are bound to the binding agent. The nontarget cells are then separated from the unbound desired, or target cells using, e.g., a magnet, centrifugation, or flotation. The nontarget cells are removed and only the unbound target cells remain and are isolated. The embodiments using binding particles, e.g., microbubbles or detachable beads to bind and remove the undesired, nontarget cell types are negatively selecting for the target cells.

[0131] In some embodiments, one or more binding agents are selected from an antibody, e.g., nanobody, peptide, protein, aptamer, lectin, cyclodextrin, avidin, or streptavidin, or subcombinations or a combination thereof. In some embodiments, the one or more binding agents bind to one or more surface markers of nontarget cell types. In some embodiments, the one or more binding agents bind to the CD proteins including or excluding CD3, CD4, or CD8, or a combination thereof. In some embodiments, the binding agents that bind to positively selected target cells bind to, as non-limiting examples, CD3, CD4, or CD8, or a combination thereof. In some embodiments, the binding agents that bind to positively selected target cells bind to, as non-limiting examples, CD14, CD16, CD19, CD36, CD56, CD123, or CD235a, or a combination thereof. In some embodiments, binding agents are antibodies, e.g., nanobodies that bind CD3, CD4, CD8, CD14, CD16, CD19, CD36, CD56, CD123, or CD235a, or any subcombination or combination thereof. In some embodiments, binding agents bind to one or more surface marker of nontarget or target cell types. In some embodiments, binding agents conjugated to a moiety including, as non-limiting examples, a fluorescent dye, a fluorescent protein, an enzyme, a metal, a protein tag, an oligonucleotide tag, or a combination thereof.

[0132] In some embodiments, binding particles are functionalized microbubbles. In some embodiments, binding particles are functionalized beads, e.g., functionalized detachable beads. In some embodiments, binding particles are functionalized microbubbles that bind biding agents, e.g., antibodies, e.g., nanobodies bound to target or nontarget cells, including, as nonlimiting examples, CD3, CD4, CD8, CD14, CD16, CD19, CD36, CD56, CD123, or CD235a, or a combination thereof. In some embodiments, binding particles are functionalized detachable beads that bind antibodies, e.g., nanobodies bound to target or nontarget cells, including, as non-limiting examples, CD3, CD4, CD8, CD14, CD16, CD19, CD36, CD56,CD 123, or CD235a, or a combination thereof. In some embodiments, the one or more binding agents or binding particles bind to the CD proteins including or excluding CD14, CD16, CD19, CD36, CD56, CD 123, or CD235a, or any subset thereof, or a combination thereof. In some embodiments, the one or more binding agents or binding particles bind to the CD proteins including or excluding CD14 and CD19. In some embodiments, the one or more binding agents bind to the CD proteins including or excluding CD 16 and CD56. In some embodiments, the one or more binding agents or binding particles bind to CD45 and CD 16 / 56. In some embodiments, the one or more binding agents or binding particles bind to CD45 and CD19. In some embodiments, the one or more binding agents or binding particles bind to CD45 and CD 14. In some embodiments, the one or more binding agents or binding particles is a cocktail of antibodies that bind to CD14, CD16, CD19, CD36, CD56, CD123, and CD235a. In some embodiments, the one or more binding agents or binding particles bind to one or more surface markers comprising CD3, CD4, CD8, or a combination thereof.

[0133] In some embodiments, the binding agents are detachable beads are functionalized beads, e.g., as a non-limiting example, detachable Dynabeads® that bind to markers on cells. In some embodiments, the detachable beads bind to CD3 only. In some embodiments, the detachable beads bind to CD3 and CD28. In some embodiments, the detachable beads bind to CD4 only. In some embodiments, the detachable beads bind to CD8 only. In some embodiments, the detachable beads bind to both CD4 and CD 8.

[0134] In some embodiments, T cell isolation is performed using a microbubble method in which 1) microbubbles are mixed with the sample, 2) microbubbles capture target cells, and 3) target cells float to the surface for removal. In some embodiments, the microbubble T cell isolation is performed in an open process where the frozen or fresh leukapheresis material (APH) are processed by thawing and warming APH in a Plasmatherm; centrifuging and washing cells; enriching for T cells using isolation tubes; resuspending and recentrifuging the cells; and transferring the enriched T cells into a G-Rex bioreactor for CAR-T manufacturing.

[0135] In some embodiments, the microbubble T cell isolation is performed in a closed process where the frozen or fresh leukapheresis material (APH) are processed by thawing and warming APH in a Plasmatherm; using a Sepax C-pro to concentrate and clean (wash) cells; transferring the cells into a transfer bag to enrich for T cells; using to a Sepax C-pro to concentrate and clean (wash) the enriched T cells; and transferring the enriched T cells into a G-Rex bioreactor for CAR-T manufacturing. In some embodiments, the closed process microbubble design offers GMP-compliant automation while preserving performance.

[0136] In certain embodiments, the microbubbles bound to cells are ruptured by sonication or pressure change to recover viable T cells. In other embodiments, the cells are detached by cleaving the biotin- streptavidin linkages with brief protease treatment. In an embodiment, residual microbubbles or their fragments are readily removed by filtration or differential centrifugation. In an embodiment, the isolated T cells are resuspended in the appropriate medium for downstream analysis or culture.

[0137] In certain embodiments, the microbubble separation parameters, e.g., CD3 microbubble separation parameters are optimized to maximize T cell yield, purity and recovery. In an embodiment, higher microbubble doses and more vigorous mixing increase capture efficiency. In an embodiment, incubation times vary to optimize yield and cell viability. In certain embodiments, multiple rounds of separation are performed to further enrich T cells to >99% purity if desired. In certain embodiments, 80% purity or more and 50% recovery or more are often be obtained after a single separation cycle under well-optimized conditions. In certain embodiments, 90% purity or more and 70% recovery or more are often be obtained after a single separation cycle under well-optimized conditions.

[0138] In an embodiment, the microbubble approach is readily scalable from microliter to multi-liter volumes and from millions to billions of target cells. It is compatible with manual, automated and closed system (e.g. blood bag) formats. In an embodiment, albumin and glass microbubble separations perform similarly in terms of T cell yield and purity. In an embodiment, glass microbubble separation are used for large-scale processing to optimize stability and durability.

[0139] In some embodiments, CD4 / CD8 Detachable Dynabeads® are used for T cell isolation. In some embodiments, the CD4 / CD8 Detachable Dynabeads® isolation is an open process. In some embodiments, the CD4 / CD8 Detachable Dynabeads® isolation is a closed process. In certain embodiments the CD4 / CD8 Detachable Dynabeads® closed process isolation includes one or more, or all of the following steps: thawing and warming APH in a Plasmatherm; using a Sepax C-pro to concentrate and clean (wash) cells; using a DynaMag™ Magnet to enrich for T cells; using to a Sepax C-pro to concentrate and clean (wash) the enriched T cells; and transferring the enriched T cells into a G-Rex bioreactor for CAR-T manufacturing. In some embodiments, the CD4 / CD8 Detachable Dynabeads® closed isolation process offers GMP-compliant automation while preserving performance.

[0140] In one embodiment, the present invention provides a method for isolating target cells from a sample comprising cells, the method comprising: (a) contacting the sample with one or more binding particles to form a mixture comprising binding particle-bound cells andunbound cells; (b) separating binding particle-bound cells from the unbound cells; (c) removing unbound cells; and (d) releasing the binding particles from the binding particle-bound cells thereby obtaining target cells. In some embodiments, binding particles are functionalized beads, e.g., functionalized detachable beads. In some embodiments, binding agents are detachable beads, e.g., functionalized detachable beads, e.g., detachable magnetic beads, e.g., Dynabeads®.

[0141] In one embodiment, the present invention provides a method for isolating target cells from a sample comprising cells, the method comprising: (a) contacting the sample with one or more detachable beads to form a mixture comprising detachable bead-bound cells and unbound cells; (b) separating detachable bead-bound cells from the unbound cells; (c) removing unbound cells; and (d) releasing the binding agent from the detachable bead-bound cells thereby obtaining target cells. In some embodiments, the detachable beads are functionalized detachable beads, e.g., detachable Dynabeads®. In some embodiments, the detachable beads bind to CD3 only. In some embodiments, the detachable beads bind to CD3 and CD28. In some embodiments, the detachable beads bind to CD4 only. In some embodiments, the detachable beads bind to CD8 only. In some embodiments, the detachable beads bind to both CD4 and CD 8.

[0142] In some embodiments, binding particles bind to one or more surface marker of nontarget or target cell types. In some embodiments, binding particles are functionalized microbubbles that bind antibodies bound to target or nontarget cells, including, as non-limiting examples, CD3, CD4, CD8, CD14, CD16, CD19, CD36, CD56, CD123, or CD235a, or a combination thereof. In some embodiments, binding particles are functionalized detachable beads that bind antibodies bound to target or nontarget cells, including, as non-limiting examples, CD3, CD4, CD8, CD14, CD16, CD19, CD36, CD56, CD123, or CD235a, or a combination thereof. In some embodiments, binding agents to which microbubbles bind are antibodies that bind CD3, CD4, CD8, CD14, CD16, CD19, CD36, CD56, CD123, or CD235a, or a combination thereof. In some embodiments, the one or more binding agents or binding particles bind to one or more surface markers comprising CD3, CD4, CD8, or a combination thereof.

[0143] In some embodiments, separating the binding particle-bound cells from the unbound cells comprises magnetic capture, flotation, centrifugation, filtration, or a subcombination or a combination thereof. In some embodiments, separating the binding agentbound cells from the unbound cells is performed by flotation. In some embodiments, separating the binding particle-bound cells from the unbound cells is performed by centrifugation. In someembodiments, separating the binding particle-bound cells from the unbound cells is performed by filtration. In some embodiments, the binding particles are microbubbles.

[0144] In some embodiments, separating detachable bead-bound cells from the unbound cells comprises magnetic capture, flotation, centrifugation, filtration, or a subcombination or a combination thereof. In some embodiments, separating detachable bead-bound cells from the unbound cells is performed by flotation. In some embodiments, separating detachable beadbound cells from the unbound cells is performed by centrifugation. In some embodiments, separating detachable bead-bound cells from the unbound cells is performed by filtration. In some embodiments, separating the detachable bead-bound cells from the unbound cells is performed by magnetic capture.

[0145] In some embodiments, the methods of this disclosure further comprise the step of positively or negatively selecting for CD4+ or CD8+ T cell subsets.

[0146] In some embodiments, the method achieves at least 80% target cell purity and at least 50% target cell recovery, and the method is performed in a closed system.

[0147] In some embodiments, the method for isolating target cells from a sample comprising cells is performed in a closed system comprising a cell separation bag, or in an open system comprising centrifugation. In some embodiments, the method for isolating target cells from a sample comprising cells further comprises washing the sample prior to step (a). In some embodiments, the method for isolating target cells from a sample comprising cells comprises: (a) contacting the sample with one or more binding agents to form a mixture comprising binding agent-bound cells and unbound cells; (b) contacting the mixture comprising binding agent-bound cells and unbound cells with microbubbles that bind the binding agent to form a mixture comprising microbubble-binding agent-bound cells and unbound cells; (c) separating the microbubble -binding agent-bound cells from the unbound cells; and (d) extracting the unbound cells, thereby obtaining target cells. In some embodiments, the method for isolating target cells from a sample comprising cells, the method comprising: (a) contacting the sample with one or more binding agents to form a mixture comprising binding agent-bound cells and unbound cells; (b) contacting the mixture comprising binding agent-bound cells and unbound cells with binding particles, e.g. as a nonlimiting example, detachable beads, e.g., detachable Dynabeads®, that bind the binding agent to form a mixture comprising detachable bead-binding agent-bound cells and unbound cells; (c) separating the detachable bead-binding agent-bound cells from the unbound cells; and (d) extracting the unbound cells, thereby obtaining target cells. In some embodiments, steps (a) through (c) are performed in a closed system comprising a cell separation bag or in an opensystem comprising centrifugation. In some embodiments, steps (a) through (c) are performed in a closed system comprising a cell separation bag. In some embodiments, wherein steps (a) through (c) are performed in an open system, further wherein separation comprises centrifugation. In some embodiments, the method is performed in a closed system. In some embodiments, the method further comprises washing the sample prior to step (a).

[0148] In some embodiments, the sample comprises, e.g., as non-limiting examples, blood, leukapheresis product, lymph, peripheral blood, bone marrow, cerebrospinal fluid, umbilical cord, umbilical cord blood, amniotic fluid, menses, pleural effusion, urine, ascites, tumor tissue, or semen. In some embodiments, the sample is blood. In some embodiments, the sample is a leukapheresis product. In some embodiments, the sample is lymph. In some embodiments, the sample is peripheral blood. In some embodiments, the sample is bone marrow. In some embodiments, the sample is cerebrospinal fluid. In some embodiments, the sample is umbilical cord. In some embodiments, the sample is umbilical cord blood. In some embodiments, the sample is amniotic fluid. In some embodiments, the sample is menses. In some embodiments, the sample is pleural effusion. In some embodiments, the sample is urine. In some embodiments, the sample is ascites. In some embodiments, the sample is tumor tissue. In some embodiments, the sample is semen.

[0149] Isolated T cell characteristics

[0150] In some embodiments, this disclosure provides methods for analyzing the quantity and quality of T cells before and after microbubble isolation. In an embodiment, cell counting is done by hemocytometer or automated cell counter. In an embodiment, purity is assessed by flow cytometry after staining with fluorescent antibodies against CD3 and other lymphocyte markers such as, for example, CD4, CD8, CD19, and CD56. In an embodiment, viability of the isolated T cells is confirmed by dye exclusion. In an embodiment, viability of the isolated T cells is 90% or more.

[0151] This disclosure features methods to examine the isolated T cells' morphology and receptor expression using, for example, light microscopy and electron microscopy. In an embodiment, functional competence is verified by measuring proliferation (e.g. thymidine uptake or CFSE dilution) and cytokine secretion (ELISA or ELISpot) after stimulation with mitogens or antigens. In an embodiment, antigen- specific T cells are detected by MHC tetramer staining. In an embodiment, sequential isolation of CD4+ and CD8+ subsets are achieved using microbubbles bearing respective antibodies. In some embodiments, CD3+ T cells are enriched by positive selection, removing CD56+ NK cells, CD14+ monocytes, CD19+ B cells. In some embodiments, CD45+ CD16 / 56+ dual positive, CD45+ CD19+ dual positive and CD45+CD 14+ dual positive cell populations are removed. In some embodiments, CD4:CD8 T cell ratios are maintained during CD3+ T cell enrichment.

[0152] Bioprocessing and clinical applications

[0153] In one embodiment, the microbubble methods featured herein are applied to the isolation of T cells for various in vitro and in vivo applications. In another embodiment, the microbubble methodology facilitates T cell immunophenotyping, antigen- specific T cell detection, and in vitro expansion of T cells for adoptive immunotherapy.

[0154] In one embodiment, robust and economical large-scale T cell separations that were previously challenging are enabled. In an embodiment, up to 1010CD3+ T cells at 95% purity or more are be recovered from a standard leukapheresis sample (~1010total white blood cells, WBCs) after a single microbubble processing cycle. In some embodiments, the T cell purity in a microbubble closed process results in about >95% purity, persisting up to at least 11 days. In some embodiments, the T cell purity resulting from a microbubble closed process results in about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or any percentage of purity between any two percentages referred to above or herein. In another embodiment, the T cell purity in a detachable Dynabeads® closed process results in about >80% purity that improves up to >95% and persists up to at least 11 days. In some embodiments, the T cell purity resulting from a microbubble closed process results in about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or any percentage of purity between any two percentages referred to above or herein. In some embodiments, the T cell purity is maintained for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 days, or more than 11 days, or any or for any time range between any two of the number of days referred to above or herein.

[0155] In one embodiment, the methods of this disclosure are applied to micro-scale separations, such as, for example, the isolation of rare antigen- specific T cells from peripheral blood for single-cell genomic and functional analyses.

[0156] In some embodiments, the methods of this disclosure produce T cells used in activation, expansion and transduction processes for further applications. In some embodiments, the isolated T cells are used in vector transduction processes. In some embodiments, the vectors are viral vectors, e.g., without limitation lentiviral vectors. In one embodiment, the T cells isolated from a microbubble closed process are transduced using a vector, e.g., without limitation, a lentiviral vector encoding a CAR, e.g., without limitation, a CD 19 CAR or a BCMA CAR. In some embodiments the transduced T cells demonstrate robust expansion through Day 11, with about >55-fold cell expansion. In some embodiments, the microbubble closed process transduced cells are expanded about 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70-fold expansion, or any fold expansion between any two of the numbers of fold expansion referred to above or herein. In an embodiment, the T cells isolated from a detachable Dynabeads® closed process were transduced using lentivirus with a CD19 CAR and demonstrated robust expansion through Day 11, with about >55-fold cell expansion. In some embodiments, the detachable Dynabeads® closed process transduced cells are expanded about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70-fold expansion, or any fold expansion between any two of the numbers of fold expansion referred to above or herein.

[0157] Producing immunotherapy cells

[0158] In one embodiment, the present invention provides a method of producing immunotherapy cells comprising: (a) isolating target cells from a sample comprising cells; (b) stimulating target cells with anti-CD3 / CD28-coated beads; and transducing stimulated target cells with one or more chimeric antigen receptors, or one or more T-cell receptor. In some embodiments, isolating the target cells comprises (i) contacting the sample with one or more binding agents to form a mixture comprising binding agent-bound cells and unbound cells; (ii) contacting the mixture comprising binding agent-bound cells and unbound cells with microbubbles that bind the binding agent to form a mixture comprising microbubble-binding agent-bound cells and unbound cells; (iii) separating the microbubble-binding agent-bound cells from the unbound cells; and (iv) extracting the unbound cells thereby obtaining target cells. In one embodiment the one or more chimeric antigen receptors are dual antigen chimeric antigen receptors.

[0159] In another embodiment, the present invention provides a method of producing immunotherapy cells comprising: (a) isolating target cells from a sample comprising cells; (b) stimulating target cells with anti-CD3 / CD28-coated beads; and transducing stimulated target cells with one or more chimeric antigen receptors, or one or more T-cell receptor. In some embodiments, isolating the target cells comprises (i) contacting the sample with one or more binding particles to form a mixture comprising binding particle-bound cells and unbound cells; (ii) separating binding particle-bound cells from the unbound cells; (iii) removing unbound cells; and (iv) releasing the binding particles from the binding particle-bound cells, thereby obtaining target cells. In one embodiment the one or more chimeric antigen receptors are dual antigen chimeric antigen receptors.

[0160] In one embodiment, the present invention provides for a composition of immunotherapy cells produced by the method where step (c) comprises transducing stimulated target cells with a dual antigen chimeric antigen receptor.

[0161] In one embodiment, the present invention provides for a composition of CD3+ T cells isolated according to the method where the CD3+ T cells have a purity greater than 90% and a viability greater than 95%. In some embodiments, the CD3+ T cells have a purity greater than about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or between any percentage between any two percentages referred to above or herein. In some embodiments, the CD3+ T cells have a viability greater than about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or between any percentage between any two percentages referred to above or herein. In another embodiment, the present invention provides for a composition where the isolated CD3+ T cells are transduced in step (c) using a lentiviral vector encoding a chimeric antigen receptor.

[0162] In one embodiment, a composition is provided comprising CD3+ T cells isolated according to the method, wherein the CD3+ T cells have a purity greater than 90%, a viability greater than 95%, and are transduced with a lentiviral vector encoding a chimeric antigen receptor. In some embodiments, the CD3+ T cells have a purity greater than about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or between any percentage between any two percentages referred to above or herein. In some embodiments, the CD3+ T cells have a viability greater than about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or between any percentage between any two percentages referred to above or herein. In some embodiments, the isolated T cells are transduced with a CAR, including, as non-limiting examples, a CD19 or B-cell maturation antigen (BCMA) CAR, and further expanded. High-quality T cells with optimal viability and purity are imperative for selective delivery of CAR exclusively to T cells, ensuring effective transduction and successful CAR-T therapy.

[0163] In one embodiment, separated T cells, e.g., CD3+ T cells are transduced using a lentiviral construct comprising a 5’ long terminal repeat, a sequence encoding a CAR, at least one element selected from a linker and a tag, and a 3’ long terminal repeat, as described in U.S. provisional application 63 / 641,846 filed May 2, 2024.

[0164] In some embodiments, CAR constructs and dual antigen CAR constructs can be made by methods known in the art, including, for example CD19 or BCMA CARs, (See, e.g., U.S. Pat. Nos. 12,195,514; 12,214,037; 10,357,514; and 10,647,778, and Xie et al., Cancers (Basel). 2022 Jun 30;14(13):3230, each of which is herein incorporated by reference), and used to transduce target cells. In some embodiments, a CAR construct is a dual antigen CAR construct. In some embodiments, target cells, such as, for example, CD3+ T cells are transduced with a CAR construct such as, for example, a CD 19 or B-cell maturation antigen(BCMA) CAR, or a dual antigen CAR construct, e.g., without limitation, CD19 / BCMA, CD19 / CD20, CD19 / CD22, CD19 / CD123, BCMA / CD38, BCMA / .GPRC5D, CD138 / CD38, CD5 / CD7, BCMA / CS1 or other dual constructs that CD19 or BCMA and a second cancer antigen target.

[0165] The present invention will be further illustrated in the following Examples which are given for illustration purposes only and are not intended to limit the invention in any way.EXAMPLES

[0166] The specific methods and compositions described herein are representative of preferred embodiments and are exemplary and not intended as limitations on the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification, and are encompassed within the spirit of the invention as defined by the scope of the claims. Thus, for example, in each instance herein, and in embodiments or examples of the present invention, any of the terms “comprising”, “consisting essentially of’, and “consisting of’ may be replaced with either of the other two terms in the specification. The methods and processes illustratively described herein suitably may be practiced in differing orders of steps, and that they are not necessarily restricted to the orders of steps indicated herein or in the claims. It is also that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Under no circumstances may the patent be interpreted to be limited to the specific examples or embodiments or methods specifically disclosed herein. Under no circumstances may the patent be interpreted to be limited by any statement made by any Examiner or any other official or employee of the Patent and Trademark Office unless such statement is specifically and without qualification or reservation agreed to and expressly adopted in a responsive writing by Applicants.

[0167] The invention is further illustrated by the following non-limiting examples:Example 1: Isolation of CD3+ T cells from leukapheresis samples using an open microbubble process

[0168] A leukapheresis sample containing 1.40 x 109total mononuclear cells at 76.3% viability was obtained from a healthy donor. Cells were washed twice in PBS containing 0.2% human serum albumin (HSA) to remove platelets. The washed cells were incubated with a binding agent, e.g., a cocktail of biotinylated monoclonal antibodies against the surface markers CD14, CD16, CD19, CD36, CD56, CD123, and CD235 a at a concentration of 1 pg / mL each for 30 min at room temperature to label non-target cells. Streptavidin-coated albuminmicrobubbles were then added at a ratio of 10 microbubbles per target cell and incubated for 15 min with gentle rocking. The mixture was centrifuged at lOOxg for 10 min to separate the floating microbubble-bound cells from the unlabeled T cells in the pellet. The T cell pellet was recovered by pipetting and washed twice more in buffer.

[0169] Flow cytometry analysis (FIG. 7) showed the starting leukapheresis sample contained 55.8% CD3+ T cells (48% CD4+, 18% CD8+), along with contaminating monocytes (24.0% CD14+), B cells (9.7% CD19+), and NK cells (5.8% CD56+). After microbubble separation, the open process yielded a highly enriched CD3+ fraction (82.8% of viable cells) with 63.8% CD4+ and 29.1% CD8+ subsets. Non-target cell types were depleted to <5% each. Immunomagnetic bead-based separation (Miltenyi CliniMACS Prodigy) of the same sample produced similar results (FIGs. 6A and 6B). The microbubble process recovered 2.5 x 108viable CD3+ T cells (FIG. 4), representing a 41% yield from the 6.05 x 10A8 estimated T cells in the starting 1.40 x 109total cells (FIG. 13). T cell viability remained high at 93.5% (FIG. 5).Example 2: Isolation of CD3+ T cells from leukapheresis samples using a closed microbubble process

[0170] To minimize open processing steps, an exemplary closed microbubble T cell isolation method was developed using a sterile bag and tubing set (FIG. 3). A leukapheresis sample containing 1.50 x 10A9 total cells at 98.3% viability was washed using an automated Sepax II device. Non-target cells were labeled by incubating the washed cells with a binding agent, e.g., an biotinylated antibody cocktail in a transfer bag for 30 min. Streptavidin microbubbles were then added via a spike port and the bag was agitated for 15 min to allow binding. The bag was hung vertically for 15 min to allow the microbubble-cell complexes to rise to the top. The bottom cell fraction was then welded off and transferred into a new bag using the Sepax.

[0171] The flow cytometry analyses shown in FIGs. 9A-9D, and FIGs. 10A-10D show the gating strategy and sorting analysis of cells from a LeukoPak (FIGs. 9A-9D) and cells enriched by the closed microbubble process (FIGs. 10A-10D). The chart shown in FIG. 11 summarizes that the closed process achieved even higher T cell purity than the open process, with 96% CD3+ cells (52% CD4+, 41% CD8+) and <1% residual monocytes, B cells and NK cells. Viability was an exceptional 99.6%. T cell recovery (FIG. 4) was also slightly better at 3.82 x 108viable cells (45% yield). The Sepax automated steps reduced cell loss and contamination compared to manual pipetting. FIGs. 6A and 6B shows the closed microbubble process yielded similar numbers of highly pure T cells as the Prodigy while substantially reducing processing time (2.5 vs 5 hrs) and consumable cost ($600 vs $12,500) (FIG. 12).Example 3: Expansion and lentiviral transduction of microbubble-isolated T cells

[0172] Next, T cells isolated by the microbubble process were tested for effective activation, expansion and transduction for further applications. CD3+ T cells were isolated from a healthy donor leukapheresis sample using the exemplary open process as in Example 1. After resting overnight, 1 x 10A6 T cells were seeded per well in a 48-well plate and stimulated with anti-CD3 / CD28-coated beads (Dynabeads®, ThermoFisher™) at a 2: 1 bead:cell ratio in X-VIVO 15 medium with 5% human AB serum and 100 U / mL IL-2. On day 2, cells were transduced with a lentiviral vector encoding an anti-CD19 CAR with a GFP reporter at MOIs ranging from 1 to 50. Transductions were performed by spinning the plates at lOOOxg for 1 hr at 32°C in medium containing 10 pg / mL polybrene. After 3 more days of expansion, cells were counted and analyzed for GFP and CAR expression by flow cytometry. Total cell numbers increased by 5.3 ± 0.4 fold (mean ± SD, n=3). Transduction efficiency, as measured by the percentage of GFP+ CAR+ double positive cells (FIG. 8), reached a plateau of approximately 15% at MOI >5. This level of efficiency is comparable to that typically seen with immunomagnetic bead-isolated T cells. Higher MOIs did not improve transduction rate but negatively impacted cell expansion (3.1 ± 0.5 fold at MOI 50).

[0173] Mock-transduced cells expanded the most (6.2 ± 0.3 fold) and maintained >90% viability. Increasing MOI led to more activation-induced cell death, with viability dropping to 62 + 5% at MOI 50. CAR surface expression levels also peaked at MOI 5 based on median fluorescence intensity.

[0174] These results indicate that CD3+ T cells isolated with microbubbles can be efficiently expanded and lentivirally transduced using standard protocols to express a CAR or other desired transgene. An MOI of 5 provides the best balance of transduction efficiency, cell yield and viability. The microbubble isolation process does not appear to impair T cell proliferative potential or transducability.Example 4: Functional characterization of microbubble-isolated T cells

[0175] To confirm that target T cells isolated with the exemplary microbubble process retain normal functional capabilities, a series of in vitro assays was performed. CD3+ T cells were isolated from 3 healthy donor leukapheresis samples using either the exemplary open microbubble process, the closed microbubble process, or immunomagnetic beads (Prodigy). An aliquot of each pre-isolation sample was also analyzed for comparison. Cells were rested overnight in X-VIVO 15 with 5% human AB serum, then plated in triplicate at 1 x 10A5 per well in round bottom 96- well plates.

[0176] To assess antigen-specific recall responses, cells were stimulated with a peptide pool spanning the cytomegalovirus (CMV) pp65 protein (1 pg / mL, Miltenyi Biotec) or a lysate of CMV-infected cells (10 pg / mL, Microbix Biosystems). After 24 hours, culture supernatants were harvested and assayed for IFN-y secretion by ELISA. All T cell preparations contained CMV-reactive cells capable of responding to both pp65 and viral lysate, with no significant differences in IFN-y levels between isolation methods. Median net values were 216 pg / mL for pp65 and 452 pg / mL for lysate vs < 10 pg / mL for unstimulated cells. This indicates that antigenspecific memory T cells are preserved during the microbubble isolation process.

[0177] Polyclonal T cell activation was evaluated by stimulating cells with anti-CD3 (OKT3, 1 pg / mL) and anti-CD28 (CD28.2, 1 pg / mL) antibodies for 72 hours, then measuring proliferation by tritiated thymidine incorporation during an additional 18-hour pulse. Parallel wells were stimulated with 5 pg / mL phytohemagglutinin (PHA) or 20 ng / mL each of phorbol myristate acetate (PM A) and ionomycin (I). All T cell preparations proliferated robustly and equivalently to TCR (>50-fold over unstimulated), PHA (> 100-fold) and PMA / I (>200-fold) stimulation. There were no significant differences based on isolation method. Cell viability after 72 hours was also similar across all conditions (82-96%).

[0178] Finally, cytokine production was assessed in response to TCR or PMA / I stimulation for 6 hours in the presence of brefeldin A. Cells were then fixed, permeabilized and stained intracellularly with fluorescent antibodies to IFN-y, TNF-a and IE-2. Again, results were highly comparable between pre- and post-isolation samples regardless of separation method. PMA / I activation elicited the strongest cytokine induction, with 55-65% of CD4+ and CD8+ T cells expressing IFN-y, 60-70% TNF-a, and 45-55% IE-2 (polyfunctionality index >0.7, calculated as in Earsen et al. PEoS One 7:e42403). TCR stimulation evoked similar cytokine patterns but with lower frequencies (30-40% IFN-y, 40-50% TNF-a, 25-35% IE-2, poly functionality index 0.4-0.5).

[0179] Collectively, these functional data confirm that closed and open microbubble isolation methods recover T cells equivalent in quality to a standard cell separation system. The process does not induce any detectable T cell activation or alter the frequency of memory T cells. Isolated cells remain fully responsive to antigen- specific and polyclonal stimuli and are capable of multilineage cytokine expression.Example 5: CD4+ and CD8+ T cell subset isolation using sequential microbubble separations

[0180] Depending on the downstream application, it is desirable to further isolate target purified CD4+ helper or CD8+ cytotoxic T cell subsets from the total CD3+ population. To demonstrate the feasibility of subset-specific microbubble separations, nontarget CD8+ and CD56+ cells were first depleted from a leukapheresis sample using the exemplary closed process with binding agents, e.g., biotinylated anti-CD8 and anti-CD56 in addition to the standard antibody cocktail. The resulting negatively selected cells were then positively selected for target CD4+ T cells using microbubbles directly conjugated with anti-CD4 antibody at 1 pg / mg microbubble. Conversely, target CD8+ T cells were isolated by negatively depleting nontarget CD4+ and CD56+ cells followed by positive selection with target anti-CD8 microbubbles.

[0181] Flow cytometric analysis of the resulting populations revealed highly pure CD4+ (97.8 ± 0.6%) and CD8+ (98.1 ± 0.4%) T cell fractions with minimal cross-contamination (<0.5%) or residual NK cells (<0.2%). Yields were also excellent, with an average of 45% of each subset recovered relative to the starting frequency in the pre-purification sample. The CD4+ to CD8+ ratio was a typical 2.3 in the CD3+ fraction, 385 in the CD4+ fraction, and 0.002 in the CD8+ fraction, representing > 1000-fold enrichment of each subset.

[0182] Functional testing of the microbubble-purified CD4+ and CD8+ T cells demonstrated preserved subset-specific cytokine signatures. CD4+ T cells preferentially produced IL-2, TNF-a and IFN-y in response to PMA / I or SEB stimulation, while CD8+ T cells primarily expressed IFN-y and exhibited stronger cytolytic activity against anti-CD3- redirected P815 target cells. There were no significant phenotypic or functional differences between T cell subsets isolated by microbubbles versus immunomagnetic beads.

[0183] Volumes of starting material and reagents, including buffer types and other process parameters, are the key input influencing CAR-T cell expansion response in the target cell (e.g. CD3+ T cells) isolation process. The modifications presented in this study are contemplated for implementation in in an already established protocol to significantly improve CAR-T products’ quality and success rate.Example 6: Comparison of T Cell Isolation Methods from Leukapheresis

[0184] Delays in cell isolation extend turnaround and reduce manufacturing success. Cell isolation for cellular therapies such as CAR-T Cell Therapy Clinical Manufacturing is a first and critical step of the process. There is limited starting material due to, as non-limiting examples, donor variability and donor availability. Having rapid, efficient, high-qualityisolation of desired cells significantly impacts the overall quality and efficacy of the final products.

[0185] Rapid and efficient isolation of T cells is a critical first step in chimeric antigen receptor - T cells (CAR-T) manufacturing and essential to minimize turnaround time, particularly for critically ill patients. The workflow of CAR-T Cell Therapy is shown in FIG. 16. High-quality T cells with optimal viability and purity are imperative for selective delivery of CAR exclusively to T cells, ensuring effective transduction and successful CAR-T therapy. Although standard isolation methods have addressed some challenges for CAR-T manufacturing, scientific innovations have brought additional opportunities to better fit for each therapy. This study evaluates two rapid T cell isolation processes, implemented in both open and closed systems, and compares their performance to a standard GMP-compliant T cell isolation method.

[0186] Two new T cell isolation processes were designed based on new methodologies, Microbubble (Akadeum Life Sciences®) and CD4 / CD8 Detachable Dynabeads® (ThermoFisher Scientific™). Representative schematics of the microbubble and detachable Dynabeads® methods are shown in FIGs. 17 A and 17B. For the representative microbubble method (FIG. 17A), the negative fractions bind to the microbubbles and float on top and the positive fractions remain at the bottom and can be collected for further processing. For the representative detachable Dynabeads® (CD4 / CD8) method, the positive fractions bind to magnetic Dynabeads® and then are detached from the Dynabeads® with release buffer. The negative fractions do not bind to the Dynabeads® and are removed.

[0187] The Microbubble-Based T Cell Isolation Using Open and Closed System Designs are shown in FIG. 18: open process (top) and closed process (bottom). The Detachable Dynabeads® Isolation System Design, a closed-process design, is shown in FIG. 20. Both processes can start from frozen or fresh leukapheresis material (APH) and resulting in cells ready for CAR-T manufacturing. In this example, healthy donor leukapheresis was used as starting materials for each T cell isolation process.

[0188] Cells pre- and post-isolation were evaluated for cell viability, T cell purity, CD4:CD8 ratio and impurities to assess efficiency of the isolation processes. The T cell purity, viability and performance results of using either the exemplary microbubble open or microbubble closed process are shown in FIGs. 19A-E. Both workflows using microbubbles resulted in effective enrichment of target CD3+ T cells with a significant reduction in nontarget immune populations (CD14+, CD19+, CD56+) (FIG. 19A). T cells were effectively enriched for both processes: CD3+ increased from 56% to 83% using the exemplary openprocess, and CD3+ increased from 58% to 96% using the exemplary closed process. High postisolation viability (>90%) and consistent CD4:CD8 ratios were maintained across donors (FIG. 19B). The CD4:CD8 ratio was maintained for both processes, and the cell viability remained at >95% for both processes.

[0189] The isolated target T cells were transduced with a lentiviral vector encoding a CD19-CAR and expanded to further assess their performance for CAR-T applications. T cell viability, purity, transduction efficiency, and expansion capacity were analyzed, and results were evaluated against historical CAR-T baseline from standard T cell isolations. The subsequent lentiviral transduction with CD 19 CAR demonstrated robust expansion (FIG. 19C) and CAR expression (FIG. 19D) through Day 11. The closed process microbubble design offers GMP-compliant automation while preserving performance. In particular, the fold cell expansion resulting from the microbubble closed system (FIG. 19E).

[0190] The T cell purity, viability and performance results of using the exemplary Detachable Dynabeads® closed process are shown in FIGs. 21A-G. The isolation workflow using Detachable Dynabeads® results in effective enrichment of target CD3+ T cells with a significant reduction in non-target immune populations (CD14+, CD19+, CD56+) (FIGs. 21A- C). The percentages of pre- and post-isolation immune cell population for target CD3+ T cells, nontarget CD56+ NK cells, nontarget CD14+ monocytes, nontarget CD19+ B cells, and other cell types are shown in FIG. 21 A. The T cell purity of pre- and post-isolation by flow cytometry is shown in FIG. 21B. The reduction from the pre-isolation (top) to post-isolation (bottom) percentage of impurities by nontarget CD45+ CD16 / 56+ dual positive, CD45+ CD19+ dual positive and CD45+ CD14+ dual positive cell populations are shown in FIG. 21C. T cells were effectively enriched with the designed process: T cell purity increased from 35% to 83%, and monocytes reduced from 35% to 5%. The high post-isolation viability (>90%) and consistent CD4:CD8 ratios were maintained as shown in FIG. 21D. Notably the cell viability after isolation remained above >95%.

[0191] Moreover, the isolated T cells were transduced with a lentiviral vector encoding a CD19-CAR and expanded to further assess their performance for CAR-T applications. T cell viability, purity, transduction efficiency, and expansion capacity were analyzed, and results were evaluated against historical CAR-T baseline from standard T cell isolations. The subsequent lentiviral transduction with CD19 CAR demonstrated robust expansion (FIG. 21E) and CAR expression (FIG. 21F) through Day 11. This system design offers GMP-compliant automation while preserving performance. In particular, FIG. 21G shows the fold cell expansion resulting from the detachable Dynabeads® closed system.Example 7: Additional Indices

[0192] Methods for open process detachable beads, including an open process detachable Dynabeads® method are contemplated herein. It is expected that such an isolation method, similar to the new isolation methods described herein in the above Examples, would perform comparably with standard CAR-T processes.

[0193] The methods described herein are contemplated for the production immunotherapy cells targeting, or targeting the minimal residual disease of, hematological cancers including, as non-limiting examples, a leukemia, lymphoma, or myeloma.

[0194] The methods described herein may be used to produce immunotherapy cells targeting solid tumor cancers, or targeting the minimal residual disease of solid tumor cancers, including, as non-limiting examples, lung cancer, breast cancer, adrenocortical carcinoma, bladder urothelial carcinoma, cervical cancer, endocervical cancer, cholangiocarcinoma, colorectal cancer, esophageal cancer, brain cancer, head and neck cancers, kidney cancer, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancers, stomach cancer, testicular cancer, thyroid cancer, and uterine cancers.

[0195] The methods described herein are also contemplated for the isolation of tumor infiltrating lymphocytes from both hematological cancers, including, as non-limiting examples leukemia, lymphoma, myeloma, and for solid tumor cancers, including, as non-limiting examples lung cancer, breast cancer, adrenocortical carcinoma, bladder urothelial carcinoma, cervical cancer, endocervical cancer, cholangiocarcinoma, colorectal cancer, esophageal cancer, brain cancer, head and neck cancers, kidney cancer, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancers, stomach cancer, testicular cancer, thyroid cancer, and uterine cancers.

[0196] Immunotherapy cells produced by the methods described herein, such as, e.g., T cells, are contemplated for transduction with a CAR, including any CAR or dual antigen CAR featured herein, as non-limiting examples, a CD19 or B-cell maturation antigen (BCMA) CAR, or a dual antigen CAR, e.g., without limitation, CD19 / BCMA, CD19 / CD20, CD19 / CD22, CD19 / CD123, BCMA / CD38, BCMA / .GPRC5D, CD138 / CD38, CD5 / CD7, BCMA / CS1 or other dual constructs that CD 19 or BCMA and a second cancer antigen target. Immunotherapy CAR-T cells produced by the methods described herein are contemplated for use in patients and for use in patients with cancer recurrence.

[0197] Overall, the designs described above resulted in high cell viability and T cell purity throughout the processes with transduction efficiencies and expansion within historical ranges.

[0198] This study highlights two rapid T cell isolation processes suitable for GMP- compliant CAR-T manufacturing. Both isolation processes achieved > 80% T cell purity with reduction in impurities from leukapheresis. T cells isolated from both processes maintained >90% viability and CD4:CD8 ratios. They demonstrated comparable performance in viral transduction and expansion compared to historical data from standard CAR-T processes. The new isolation methods may offer advantages in operational time and raw material costs. These findings support the use of the new rapid cell isolation process as a viable alternative for automated, closed system. CAR-T cell manufacturing, offering additional opportunities for clinical applications besides traditional isolation solutions.

[0199] The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention. Thus, it will be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

[0200] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0201] The inventions described and claimed herein have many attributes and embodiments including, but not limited to, those set forth or described or referenced in this Detailed Disclosure. It is not intended to be all-inclusive and the inventions described and claimed herein are not limited to or by the features or embodiments identified in this Detailed Disclosure, which is included for purposes of illustration only and not restriction. A person having ordinary skill in the art will readily recognize that many of the components and parameters may be varied or modified to a certain extent or substituted for known equivalents without departing from the scope of the invention. It should be appreciated that suchmodifications and equivalents are herein incorporated as if individually set forth. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of the steps or features.

[0202] All patents, publications, scientific articles, web sites, and other documents and materials referenced or mentioned herein are indicative of the levels of skill of those skilled in the art to which the invention pertains, and each such referenced document and material is hereby incorporated by reference to the same extent as if it had been incorporated by reference in its entirety individually or set forth herein in its entirety. Applicants reserve the right to physically incorporate into this specification any and all materials and information from any such patents, publications, scientific articles, web sites, electronically available information, and other referenced materials or documents. Reference to any applications, patents and publications in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0203] The specific methods and compositions described herein are representative of preferred embodiments and are exemplary and not intended as limitations on the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification, and are encompassed within the spirit of the invention as defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed herein as essential. Thus, for example, in each instance herein, in embodiments or examples of the present invention, any of the terms “comprising”, “consisting essentially of’, and “consisting of’ may be replaced with either of the other two terms in the specification. Also, the terms “comprising”, “including”, and containing”, are to be read expansively and without limitation. The methods and processes illustratively described herein suitably may be practiced in differing orders of steps, and that they are not necessarily restricted to the orders of steps indicated herein or in the claims. It is also that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Under no circumstances may the patent be interpreted to be limited to the specific examples or embodiments or methods specifically disclosed herein. Under no circumstances may the patent be interpreted to belimited by any statement made by any Examiner or any other official or employee of the Patent and Trademark Office unless such statement is specifically and without qualification or reservation expressly adopted in a responsive writing by Applicants. Furthermore, titles, headings, or the like are provided to enhance the reader’s comprehension of this document, and should not be read as limiting the scope of the present invention. Any examples of aspects, embodiments or components of the invention referred to herein are to be considered nonlimiting.

[0204] Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims

CLAIMSWhat is claimed is:

1. A method of producing immunotherapy cells comprising: a. isolating target cells from a sample comprising cells by: i. contacting the sample with one or more binding agents to form a mixture comprising binding agent-bound cells and unbound cells; ii. contacting the mixture comprising binding agent-bound cells and unbound cells with microbubbles that bind the binding agent to form a mixture comprising microbubble-binding agent-bound cells and unbound cells; iii. separating the microbubble-binding agent-bound cells from the unbound cells; and iv. extracting the unbound cells thereby obtaining target cells; b. stimulating target cells with anti-CD3 / CD28-coated beads; and c. transducing stimulated target cells with one or more chimeric antigen receptors, or one or more T-cell receptor.

2. A method of producing immunotherapy cells comprising: a. isolating target cells from a sample comprising cells by: i. contacting the sample with one or more binding particles to form a mixture comprising binding particle-bound cells and unbound cells; ii. separating binding particle-bound cells from the unbound cells; iii. removing unbound cells; and iv. releasing the binding particle from the binding particle-bound cells thereby obtaining target cells; b. stimulating target cells with anti-CD3 / CD28-coated beads; and c. transducing stimulated target cells with one or more chimeric antigen receptors, or one or more T-cell receptors.

3. A composition comprising CD3+ T cells produced according to the method of claim 1 or 2, wherein the CD3+ T cells have a purity greater than 90% and a viability greater than 95%.

4. The composition of claim 1 or 2, wherein the chimeric antigen receptor is a dual antigen chimeric antigen receptor.

5. The composition of claim 4, wherein the CD3+ T cells are transduced in step (c) using a lentiviral vector encoding a chimeric antigen receptor.

6. A method for isolating target cells from a sample comprising cells, the method comprising: a. contacting the sample with one or more binding agents to form a mixture comprising binding agent-bound cells and unbound cells; b. contacting the mixture comprising binding agent-bound cells and unbound cells with microbubbles that bind the binding agent to form a mixture comprising microbubble-binding agent-bound cells and unbound cells; c. separating the microbubble-binding agent-bound cells from the unbound cells; and d. extracting the unbound cells, thereby obtaining target cells.

7. The method of claim 6, wherein the one or more binding agents are selected from an antibody, peptide, protein, aptamer, lectin, cyclodextrin, avidin, or streptavidin, or combinations thereof.

8. The method of claim 6, wherein the one or more binding agents bind to one or more surface markers of non-target cell types.

9. The method of claim 8, wherein the non-target cell types comprise red blood cells, granulocytes, monocytes, B cells, natural killer cells, or platelets.

10. The method of claim 8, wherein the one or more surface markers comprise one or more of CD14, CD16, CD19, CD36, CD56, CD123, or CD235a.

11. The method of claim 6, wherein separating the microbubble-binding agent-bound cells from the unbound cells comprises magnetic capture, flotation, centrifugation, filtration, or a subcombination or a combination thereof.

12. The method of claim 6, wherein the microbubbles have a mean diameter between 1-10 micrometers (pm), comprise a shell made from a material selected from the group consisting of proteins, polymers, lipids, sugars, and surfactants, and a gaseous core selected from the group consisting of air, nitrogen, carbon dioxide, and perfluorocarbons.

13. A method for isolating target cells from a sample comprising cells, the method comprising: a. contacting the sample with one or more binding particles to form a mixture comprising binding particle-bound cells and unbound cells; b. separating binding particle-bound cells from the unbound cells; c. removing unbound cells; and d. releasing the binding particle from the binding particle-bound cells thereby obtaining target cells.

14. The method of claim 13, wherein the one or more binding particles are functionalized detachable beads.

15. The method of claim 14, wherein the one or more binding particles bind to one or more surface markers comprising CD3, CD4, CD8, or a combination thereof.

16. The method of claim 6 or 13, wherein the one or more binding agents are conjugated to a fluorescent dye, a fluorescent protein, an enzyme, a metal, a protein tag, an oligonucleotide tag, or a combination thereof.

17. The method of claim 6 or 13, wherein the target cells comprise immune cells, stem cells, progenitor cells, malignant cells, bacterial cells, or parasites.

18. The method of claim 17, wherein the immune cells are T lymphocytes.

19. The method of claim 18, further comprising the step of positively or negatively selecting for CD4+ or CD8+ T cell subsets.

20. The method of claim 6 or 13, wherein the method achieves at least 80% target cell purity and at least 50% target cell recovery.

21. The method of claim 6 or 13, wherein the method maintains a CD4:CD8 ratio between about 1.0 to 3.0.

22. The method of claim 6 or 13, wherein steps (a) through (c) are performed in a closed system comprising a cell separation bag.

23. The method of claim 6 or 13, wherein steps (a) through (c) are performed in an open system, further wherein separation comprises centrifugation.

24. The method of claim 6 or 13, wherein the method is performed in a closed system.

25. The method of claim 6 or 13, wherein the sample comprises blood, leukapheresis product, lymph, cerebrospinal fluid, umbilical cord, umbilical cord blood, amniotic fluid, menses, pleural effusion, urine, ascites, tumor tissue, or semen.

26. The method of claim 25, wherein sample is a leukapheresis product.

27. The method of claim 26, further comprising washing the sample prior to step (a).

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