RU2795454C2 - Methods and compositions for obtaining genetically engineered cells - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the production of engineered T-cells, as well as to the stimulation of T-cells. At the same time, the stages of production and stimulation include incubation of the initial composition under stimulating conditions for 2–6 days, as well as the introduction of a nucleic acid encoding a genetically engineered recombinant receptor into a population of T-cells.

EFFECT: invention is effective for stimulation of T-cells.

79 cl, 3 dwg, 3 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority on the basis of U.S. Provisional Patent Application No. 62/543359, filed August 9, 2017, and entitled "METHODS AND COMPOSITIONS FOR PRODUCING GENETICALLY ENGINEERED CELLS", the contents of which are hereby incorporated by reference in their entirety.

SEQUENCE LISTING STATEMENT

The present application is filed with a sequence listing in electronic format. The sequence listing is provided as a file named 735042010540SEQLIST.txt, which was created on July 11, 2018 and has a size of 35434 kilobytes. The information contained in the electronically filed sequence listing is incorporated herein in its entirety.

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The present invention relates to methods for obtaining T cells for cell therapy, compositions obtained by these methods, and to methods of introducing cells to subjects. In particular, the invention relates to the production of engineered T cells, such as cells expressing engineered receptors, for example, engineered antigen receptors, such as engineered (recombinant) TCRs and chimeric antigen receptors (CARs), or other recombinant chimeric receptors. . Features of the methods include obtaining a more uniform and/or predictable T cell product, and/or a product with lower toxicity compared to other methods. The proposed methods include incubating the cells under stimulating conditions to induce proliferation or proliferation of naive-like T cells relative to non-naive-like T cells in the stimulated composition, which in turn can result in preferential transduction of cells derived from naive-like T cells. Features of the methods may also include cost reduction, reduction in the number of steps and resource consumption compared to other methods.

BACKGROUND OF THE INVENTION

There are various ways to obtain cells for therapeutic use and administration of cells. For example, methods are known for obtaining cells, including T cells, for engineering and cell therapy, including methods that include depletion or enrichment for certain subpopulations of cells. There is a need for improved methods, for example, to reduce the toxicity associated with the administration of certain adoptive cell therapies, to improve the manufacturing process, to facilitate administration, and/or to reduce costs or resources used. Methods, cells, compositions, kits and systems are provided to meet this need.

SUMMARY OF THE INVENTION

Provided herein are methods for genetically engineering T cells comprising incubating a parent composition under stimulating conditions for 2-6 days, wherein said parent composition contains a T cell population comprising naive-like T cells and non-naive T cells, wherein the stimulating conditions include the presence of a stimulating reagent capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and/or one or more intracellular signaling domains of one or more costimulatory molecules, to obtain a stimulated composition, and the introduction of a nucleic acid encoding a gene -engineered recombinant receptor, in a stimulated composition of T-cells, while the introduction is carried out during at least part of the incubation period.

Provided herein are methods for genetically engineering T cells comprising incubating a parent composition under stimulating conditions for 2-6 days, wherein said parent composition contains a T cell population comprising naive-like T cells and non-naive T cells, wherein the stimulating conditions include the presence of a stimulating reagent capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and/or one or more intracellular signaling domains of one or more costimulatory molecules to form a stimulated composition, and incubation of the original composition under stimulating conditions carried out before, during and/or after the introduction of a nucleic acid encoding a genetically engineered recombinant receptor. In some embodiments, the implementation of the incubation is carried out for at least 3 days. In some cases, the incubation is carried out for at least 4 days. In some embodiments, the implementation of the incubation is carried out for at least 5 days. In some embodiments, the implementation of the incubation is carried out for at least 6 days.

Provided herein are methods for stimulating T cells, comprising (a) incubating under stimulating conditions an initial composition containing T cells comprising a number of naïve-like T cells, or a subset of CD8+ T cells, required to initiate culture, to obtain a stimulated composition. ; and (b) introducing into the stimulated cell composition a nucleic acid encoding the genetically engineered recombinant receptor to obtain a composition produced in this way, containing T cells expressing the genetically engineered recombinant receptor. In some embodiments, the T cells comprise naïve-like T cells and non-naïve-like T cells, wherein the stimulating conditions preferentially induce proliferation or proliferation of naïve-like T cells compared to non-naïve-like T cells in the stimulated composition. In some embodiments, the administration is carried out during at least part of the incubation period or is carried out after the incubation.

In some embodiments, the number of naïve-like T cells or a subset of CD8+ T cells required to initiate culture is from, or from about 0.1×10 ⁸ to 5×10 ⁸ , from, or from about 0.1× 10 ⁸ to 4×10 ⁸ , from, or from about 0.1×10 ⁸ to 2×10 ⁸ , from, or from about 0.1×10 ⁸ to 1×10 ⁸ , from, or from about, 1×10 ⁸ to 5×10 ⁸ , from or from about 1×10 ⁸ to 4×10 ⁸ , from or from about 1×10 ⁸ to 2×10 ⁸ , from or from about 2× 10 ⁸ to 5×10 ⁸ , from, or from about 2×10 ⁸ to 4×10 ⁸ naive-like T cells or a subset of CD8+ T cells. In some instances, the number of naïve-like T cells or a subset of CD8+ T cells required to initiate a culture is at least, or at least about, or is, or is about, 0.5 x 10 ⁸ , 0.75 x 10 ⁸ , 1×10 ⁸ , 1.5×10 ⁸ , 2×10 ⁸ , or 4×10 ⁸ naive-like T cells or their subset of CD8+ T cells. In some cases, the number of naïve-like T cells or a subset of CD8+ T cells required to initiate culture is at least, or at least about, or is, or is about, 2×10 ⁸ naïve-like T cells, or a subset thereof. CD8+ T cells.

Methods for stimulation of T cells are proposed, including incubation under stimulating conditions of the initial composition containing T cells, including the number of naive-like T cells or their subset of CD8+ T cells required for culture initiation, which is from, or from about, 1 × 10 ⁸ up to 4×10 ⁸ like naive T cells or a subset of CD8+ T cells, to obtain a stimulated composition. In some aspects, T cells include naïve-like T cells and non-naïve-like T cells, wherein the stimulating conditions preferentially induce proliferation or proliferation of naïve-like T cells compared to non-naïve-like T cells in the stimulated composition.

In some embodiments, the number of naive-like T cells, or a subset of CD8+ T cells required to initiate culture, is at least, or at least about, or is, or is, about 2×10 ⁸ naive-like T cells, or their subset of CD8+ T cells. In some aspects, the number required to initiate culture is the number of naive-like CD8+ T cells.

In some such embodiments, naive-like T cells or naive-like CD8+ T cells have on their surface a T cell activation marker selected from the group consisting of CD45RA, CD27, CD28, and CCR7; and/or do not have on the surface a marker selected from the group consisting of CD25, CD45RO, CD56, CD62L, KLRG1; and/or have low CD95 expression; and/or lack intracellular expression of a cytokine selected from the group consisting of IL-2, IFN-γ, IL-4, IL-10. In some embodiments, naive-like cells or naive-like CD8+ cells have on their surface a T-cell activation marker selected from the group consisting of CD45RA, CD27, CD28, and CCR7; and/or do not have on the surface a marker selected from the group consisting of CD45RO, CD56, KLRG1; and/or have low CD95 expression. In some embodiments, the naive-like T cells or naive-like CD8+ cells are CD45RA+, CD27+, CCR7+, CD62- and/or CD45RO- cells.

In some of these embodiments, the non-naïve T cells do not have on their surface a T cell activation marker selected from the group consisting of CD45RA, CD27, CD28, and CCR7; and/or have on the surface a marker selected from the group consisting of CD25, CD45RO, CD56, CD62L, KLRG1 and perforin; and/or have intracellular expression of a cytokine selected from the group consisting of IL-2, IFN-γ, IL-4, IL-10; and/or have high expression of CD95. In some aspects, the non-naïve T cells are CD45RA-, CD27-, CCR7-, CD62+ and/or CD45RO+ cells.

In some embodiments, the cells of the original composition have not, and are not, subjected prior to incubation to a selection step based on an endogenous T-cell surface marker that distinguishes between naive-like and non-naive-like T cells.

In some embodiments, the method further comprises administering the engineered recombinant receptor to the stimulated cells to produce a method-produced composition comprising T cells expressing the engineered recombinant receptor. In some cases, the incubation of the composition under stimulating conditions is carried out before, during and/or after the administration of a nucleic acid encoding a genetically engineered recombinant receptor.

In some embodiments, the recombinant receptor is capable of binding to a target antigen that is associated with, specific to, and/or expressed on a cell or tissue associated with a disease, disorder, or condition. In some examples, the disease, disorder or condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease or tumor, or cancer. In some cases, the target antigen is a tumor antigen. In some embodiments, the target antigen is selected from αvβ6 integrin (avb6 integrin), B cell maturation antigen (BCMA), carbonic anhydrase 9 (CAIX), Her2/neu (erbB2 tyrosine kinase receptor), L1-CAM, B7-H3, B7- H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), testicular cancer antigen, testicular cancer antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), and surface antigen hepatitis B virus, antifolate receptor, cyclin, cyclin A2, chemokine ligand 1 with C-C motif (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin A2 receptor (EPHa2), erb -B2, erb-B3, erb-B4, erbB dimers, mutant epidermal growth factor receptor type III (EGFR vIII), folate binding protein (FBP), Fc receptor-like protein 5 (FCRL5; also known as Fc receptor 5 or FCRH5 homolog), embryonic acetylcholine receptor (embryonic AchR), folate binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glypican-3 ( GPC3), G protein-coupled receptor 5D (GPRC5D), Her2/neu (erbB2 tyrosine kinase receptor), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, human high molecular weight antigen associated with melanoma (HMW- MAA), hepatitis B surface antigen, human leukocyte antigen A1 (HLA-A1), human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha (IL-22R-alpha), IL-13R-alpha2 (IL- 13Rα2), kinase insert domain receptor (kdr), kappa light chain, Lewis Y, L1 cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM, member of the A family of 8 proteins containing leucine-rich repeats (LRRC8A), Lewis Y, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, mouse cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, representative D ligands natural killer group 2 (NKG2D), melan A (MART-1), neuronal cell adhesion molecule (NCAM), oncoembryonic antigen, preferentially expressed melanoma antigen (PRAME), progesterone receptor, prostate specific antigen, prostatic stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), like tyrosine kinase orphan receptor 1 (ROR1), survivin, trophoblastic glycoprotein (TPBG, also known as 5T4), tumor-associated glycoprotein 72 (TAG72), tyrosinase-dependent protein 1 (TRP1, also known as TYRP1 or gp75), tyrosinase dependent protein 2 (TRP2, also known as dopachrome tautomerase, dopachrome delta isomerase or DCT), folate receptor-α, 8H9, dual antigen, glycoprotein 100 (gp100), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGF-R2), estrogen receptor, progesterone receptor, Wilms tumor antigen 1 (WT-1), pathogen-specific or pathogen-expressed antigen, and universal marker-associated antigen.

In some embodiments, the recombinant receptor is, or contains, a functional non-TCR antigen receptor, or a TCR, or an antigen-binding fragment thereof. In some embodiments, the recombinant receptor is a chimeric antigen receptor (CAR).

In some of these embodiments, the recombinant receptor comprises an extracellular domain containing an antigen-binding domain, optionally wherein the antigen-binding domain specifically binds the target antigen. In some instances, the antigen binding domain is, or contains, an antibody, or an antibody fragment, which optionally is a single chain fragment. In some aspects, the fragment contains the variable regions of the antibody connected by a flexible linker. In some cases, the fragment is an scFv.

In some embodiments, the recombinant receptor further comprises a spacer and/or a hinge region. In some aspects, the recombinant receptor contains an intracellular signaling region. In some examples, an intracellular signaling region contains an intracellular signaling domain. In some aspects, an intracellular signaling domain is, or comprises, a basic signaling domain, a signaling domain capable of inducing a basic activation signal in a T cell, a T cell receptor (TCR) component signaling domain, and/or a signaling domain containing an immunoreceptor tyrosine activating motif. (ITAM). In some embodiments, the intracellular signaling domain is, or contains, an intracellular signaling domain of the CD3 chain, optionally the CD3 zeta chain (CD3ζ), or a signal fragment thereof.

In some embodiments, the recombinant receptor further comprises a transmembrane domain located between the extracellular domain and the intracellular signaling region. In some aspects, the intracellular signaling region further comprises a costimulatory signaling region. In some embodiments, the costimulatory signaling region comprises an intracellular signaling domain of a T-cell costimulatory molecule, or a signal fragment thereof. In some cases, the co-stimulatory signaling region contains an intracellular signaling domain of CD28, 4-1BB or ICOS, or a signaling fragment thereof. In some embodiments, the CAR comprises an scFv antigen-specific transmembrane domain, a cytoplasmic signaling domain from a co-stimulatory molecule, which optionally is, or contains, 4-1BB, and a cytoplasmic signaling domain from a core signaling ITAM-containing molecule, which, optionally is, or contains, a CD3-zeta signaling domain, and optionally further comprises a spacer between the transmembrane domain and the scFv; CAR contains, in the following order, an scFv antigen-specific transmembrane domain, a cytoplasmic signaling domain from a co-stimulatory molecule that optionally is, or contains, a 4-1BB signaling domain, and a cytoplasmic signaling domain from a core signaling ITAM-containing molecule , which is optionally a CD3-zeta signaling domain; or CAR contains, in the following order, an antigen-specific scFv, a spacer, a transmembrane domain, a cytoplasmic signaling domain from a costimulatory molecule, which optionally is, or contains, a 4-1BB signaling domain, and a cytoplasmic signaling domain from the basic signaling ITAM -containing molecule, which, optionally, is, or contains, the signal domain of CD3-zeta.

In some embodiments, the co-stimulatory signaling region is located between the transmembrane domain and the intracellular signaling region.

In some of these embodiments, the stimulatory condition includes incubation with a stimulatory reagent capable of activating T cells, CD4+ T cells, and/or CD8+ T cells; capable of inducing a signal through the TCR complex; and/or capable of inducing the proliferation of T cells, CD4+ T cells and/or CD8+ T cells. In some embodiments, the stimulatory condition includes incubation with a stimulatory reagent capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and/or one or more intracellular signaling domains of one or more costimulatory molecules. In some instances, the stimulatory reagent includes a base agent that specifically binds a component of the TCR complex, optionally that specifically binds CD3. In some embodiments, the primary agent is an antibody or antigen-binding fragment.

In some examples, the stimulant further includes an adjuvant that specifically binds a T-cell co-stimulatory molecule, optionally wherein the co-stimulatory molecule is selected from the group consisting of CD28, CD137 (4-1-BB), OX40, or ICOS. In some embodiments, the primary agent is an antibody or antigen-binding fragment. In some embodiments, the primary and secondary agents include antibodies, optionally wherein the one or more stimulatory agents include an anti-CD3 antibody and an anti-CD28 antibody.

In some embodiments, the implementation of the main and/or auxiliary means are on the surface of a solid substrate. In some cases, the solid support is, or contains, a bead. In some embodiments, the granule has a diameter greater than or greater than about 3.5 microns, but no greater than about 9 microns, or no greater than about 8 microns, or no greater than about 7 microns, or no more, than about 6 microns, or no more than about 5 microns. In some examples, the bead has a diameter of, or about, 4.5 microns. In some aspects, the granule has a diameter that is, or about the size of, a lymphocyte or an antigen presenting cell.

In some embodiments, the implementation of the granule is inert. In some instances, the bead is, or has, a polystyrene surface and optionally has a magnetic or superparamagnetic core.

In some embodiments, the stimulating condition includes incubating the cells at a bead to cell ratio of from, or about, 1:1 to 10:1, from, or about 1:1 to 8:1, from, or from about, 1:1 to 6:1, from, or from about, 1:1 to 4:1, from, or from about, 1:1 to 3:1, from, or from about, 2:1 to 4:1, from or from about 2:1 to 3:1, from or from about 1:1 to 2:1, from or from about 4:1 to 10:1, from or from about 4: 1 to 8:1, from, or from about, 4:1 to 6:1, from, or from about, 6:1 to 10:1, from, or from about, 6:1 to 8:1, from, or from about 8:1 to 10:1, from or from about 1:1 to 1:10, from or from about 1:1 to 1:8, from or from about 1:1 to 1:6, from, or from about, 1:1 to 1:4, from, or from about, 1:2 to 1:3. In some examples, the ratio of granules to cells is, or is about, 3:1. In some embodiments, the ratio of granules to cells is, or is about, 1:1.

Provided herein are methods for genetically engineering T cells comprising incubating a parent composition under stimulating conditions for 2-6 days, wherein said parent composition contains a T cell population comprising naive-like T cells and non-naive T cells, while the stimulating conditions include the presence of a stimulating reagent, including an anti-CD3 antibody and an auxiliary agent, which is an anti-CD28 antibody, which are attached to the granule, while the ratio of granules and cells during the incubation is from, or from about 1:1 to 4:1; and introducing a nucleic acid encoding the engineered recombinant receptor into the stimulated T cell composition, wherein the introduction is carried out for at least part of the incubation period.

Provided herein are methods for genetically engineering T cells comprising incubating a parent composition under stimulating conditions for 2-6 days, wherein said parent composition contains a T cell population comprising naive-like T cells and non-naive T cells, while the stimulating conditions include the presence of a stimulating reagent, including an anti-CD3 antibody and an auxiliary agent, which is an anti-CD28 antibody, which are attached to the granule, while the ratio of granules and cells during the incubation is from, or from about 1:1 to 4:1; and incubation of the initial composition under stimulating conditions is carried out before, during and/or after the administration of the nucleic acid encoding the genetically engineered recombinant receptor.

In some of these embodiments, the T cells are derived from a biological sample, optionally the biological sample is from a human subject. In some instances, the biological sample is, or contains, a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a white blood cell sample, an apheresis product, or a leukapheresis product.

In some embodiments, T cells include CD4+ and/or CD8+ cells. In some embodiments, the T cells include CD4+ and CD8+ T cells, and the ratio of CD4+ to CD8+ T cells is from, or from about 2:1 to, or up to about 1:5. In some cases, the ratio of CD4+ cells to CD8+ cells is, or is about, 1:1, 1:2, 2:1, 1:3, or 3:1. In some embodiments, naive-like T cells include naive-like CD4+ T cells and/or naive-like CD8+ T cells.

In some of these embodiments, the naive-like T cells are polyclonal. In some cases, the clonality of naïve-like T cells is determined by clonal sequencing, optionally next generation sequencing or spectrotype analysis.

In some embodiments, the presence, amount, number, or percentage of naive-like T cells is determined by flow cytometry.

In some of these embodiments, the implementation of the stimulating condition does not include N-acetylcysteine (NAC). In some embodiments, the stimulus condition does not include IL-15 and/or IL-7. In some embodiments, the stimulus condition results in or induces the death of non-naïve T cells or a subset thereof. In some aspects, the stimulatory condition results in activation-induced cell death (AICD) of non-naïve T cells or a subset thereof.

In some embodiments, the method further comprises adding DNase during incubation and/or to the stimulated composition.

In some embodiments, the incubation is for more than, or about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 days. In some embodiments, the percentage of cells in the stimulated composition derived from naïve T cells is increased by more than, or more than about, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 50 times, 100 times in comparison with the percentage of similar naive cells in the original composition. In some embodiments, the content in the stimulated composition of cells derived from naive T-like cells, relative to cells derived from non-naive T-cells, is increased by more than, or more than about, 1.5 times, 2 times, 3 times , 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 50 times, 100 times compared to the content of naive-like T cells relative to non-naive-like T cells in the original composition.

In some cases, the stimulated composition contains more than 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of cells that are descended from similar naive T -cells of the original composition. In some embodiments, the stimulated composition contains less than 10% of cells derived from non-naive T cells. In some examples, the stimulated composition contains less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of cells derived from non-naive T cells.

In some embodiments, of the cells in the original composition, a greater percentage of naive-like T cells, compared to non-naive T cells, are induced to proliferate and/or become activated. In some aspects, a greater percentage of T cells that were similar to naive in the original composition compared to the percentage of T cells that were not similar to naive in the original composition divide on day 1, 2, 3, 4, 5, 6 , 7, 8, 9 or 10 after the start of said incubation. In some cases, stimulatory conditions are able to induce proliferation of a greater percentage of cells in the naïve human T-like population compared to non-naïve human T-cell-like population on day 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 after the start of incubation under these conditions.

In some of these embodiments, non-naïve-like T cells are selected from the group consisting of effector T cells (T _EFF ), memory T cells, central memory T cells (T _CM ), effector memory T cells (T _EM ), as well as their combinations; or non-naïve-like T cells are a plurality of T cells including or consisting of effector T cells ( _TEFF ) and/or memory T cells, where the memory T cells optionally include central memory T cells ( T _CM ) and/or effector memory T cells (T _EM ).

In some embodiments, the percentage of naive-like T cells in the original composition is less than the percentage of engineered cells in the stimulated composition derived from naive-like T cells in the original composition. In some embodiments, a higher percentage of nucleic acid-introduced cells are, or are derived from, proliferation of naive-like T cells in the parent composition compared to non-naive-like T cells in the parent composition.

In some of these embodiments, the introduction is performed by transduction. In some embodiments, the nucleic acid is a viral vector. In some cases, the viral vector is a retroviral vector. In some aspects, the viral vector is a lentiviral vector or a gamma retroviral vector. In some embodiments, the introduction is performed by transposition of a transposon containing a nucleic acid molecule. In some embodiments, the content of naive-like T cells relative to non-naive-like T cells in the stimulated composition is increased by more than, or more than about, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 50 times, 100 times compared to the content of naive-like T cells relative to non-naive-like T cells in the original composition. In some embodiments, the implementation of the stimulated composition is more polyclonal or multiclonal in comparison with the original composition. In some embodiments, the method is used in vitro or ex vivo .

Provided herein are manufactured compositions obtained by any of the methods provided herein. Pharmaceutical compositions containing the described manufactured compositions are also provided. In some embodiments, the pharmaceutical composition further comprises a pharmaceutical carrier.

Provided herein are methods of treatment comprising administering to a mammalian subject a manufactured composition obtained by any of the methods described, or any of the pharmaceutical compositions described. In some embodiments, the cells are obtained from the subject to whom the cells will be administered.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are methods for incubating (eg, stimulating) cells in the process of obtaining cells for adoptive cell therapy, as well as compositions and cells obtained by these methods. In some embodiments, the cells include T cells, which in some aspects express engineered antigen receptors. In some embodiments, engineered antigen receptors include engineered or recombinant T cell receptors (TCRs) and functional non-TCR antigen receptors such as chimeric antigen receptors (CARs).

In some embodiments, methods for incubating (eg, stimulating) T cells are provided. Known methods use anti-CD3 and anti-CD28 to stimulate T cells. However, the methods currently used are not aimed at obtaining specific target populations of cells during the incubation process, and at obtaining a more desirable population of T cells and associated favorable results. The known methods also do not lead to elimination from the original T-cell population during incubation of the unwanted and specific T-cell population and associated adverse results. Moreover, the known methods are not designed to eliminate from the T-cell population during the incubation of a subpopulation that will be used to obtain a manufactured composition containing T-cells expressing a genetically engineered recombinant receptor. The known methods also lack the advantages of generating a more uniform and/or predictable T cell population useful for genetic engineering, dosing and/or clinical response.

In some aspects, the proposed embodiments relate to methods for producing a more uniform and/or predictable composition of T cells compared to other stimulation methods, and, in some aspects, there is additional reduction or elimination of unwanted T cell populations, for example, cells, the presence of which can give the final product such heterogeneity that the prediction of activity, efficacy and safety becomes a difficult task. In some embodiments, the proposed methods address the problems associated with generating engineered T cells in which a large number, or majority, of such engineered cells are derived from non-naïve T cells. In some aspects, lack of persistence, depletion, and/or toxicity problems may occur with adoptive T cell therapy using a composition containing genetically engineered cells derived from non-naïve T cells and/or in which the percentage the proportion or number of genetically engineered cells derived from non-naïve T cells exceeds a certain threshold. In some embodiments, methods that result in a composition of engineered T cells that is enriched in cells derived from naive-like cells may be characterized by an overall increase in the percentage of healthy cells and/or cells in the composition that are characterized by increased persistence compared to in other ways, in which the composition is not so rich in such similar naive cells.

Accordingly, methods for producing engineered T cells for adoptive therapy provided herein include methods using conditions that result in preferential propagation or proliferation, or genetic engineering, of cells derived from naïve-like T cells (or derived from CD27+, CD45RA+, CCR7+, CD62L-, or CD45RO- T cells) compared to cells derived from non-naive-like T cells (or derived from CD27-, CD45RA-, CCR7-, CD62L+, or CD45RO+ T cells ). In some embodiments, the methods include incubating T cells in an initial composition containing naïve-like T cells under stimulatory conditions that preferably result in a stimulated composition containing cells derived from naïve-like T cells of the original composition. In some aspects, the T cell population comprises a mixture of naive-like T cells and non-naive-like T cells. In some embodiments, the stimulus conditions preferentially induce a response in non-naive-like T cells, as compared to naive-like T cells. In some instances, the response includes preferential activation of non-naïve-like T cells, which in some embodiments may result in cell death.

In some embodiments, the method includes incubating the cells under stimulatory conditions, including incubation with a stimulatory agent capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and/or one or more intracellular signaling domains of one or more costimulatory molecules. In some instances, the primary agent specifically binds CD3 and/or a costimulatory molecule selected from the group consisting of CD28, CD137 (4-1-BB), OX40, or ICOS. For example, in some embodiments, the main agent is, or contains, anti-CD3, and the auxiliary agent is, or contains, anti-CD28. In some instances, the primary and secondary agents include antibodies and/or are on the surface of a solid support. In some examples, the solid support is a bead.

In some embodiments, the stimulatory condition comprises incubating the cells of the original composition with such stimulatory reagent, e.g., anti-CD3/anti-CD28 beads, wherein the bead to cell ratio is from, or about 1:1 to 10:1, from, or from about 1:1 to 8:1, from or from about 1:1 to 6:1, from or from about 1:1 to 4:1, from or from about 1:1 to 3:1, from, or from about, 4:1 to 10:1, from, or from about, 4:1 to 8:1, from, or from about, 4:1 to 6:1, from, or from about 6:1 to 10:1, from or from about 6:1 to 8:1, from or from about 8:1 to 10:1, from or from about 1:1 to 1: 10, from, or from about, 1:1 to 1:8, from, or from about, 1:1 to 1:6, from, or from about, 1:1 to 1:4, from, or from about, 1:2 to 1:3. In some specific examples, the ratio of granules to cells is, or is about, 3:1. In some cases, the ratio of granules and cells is, or is approximately, 1:3.

The methods generally further include the steps of genetically engineering the stimulated T cell composition. Genetic engineering can be carried out, or started, on the stimulated composition after, or at any time after, the start of the initial incubation, and/or simultaneously with the incubation. In some embodiments, cells are incubated with nucleic acids encoding genetically engineered molecules such that the nucleic acids are introduced into the cells and the genetically engineered molecules are expressed in the cells of the composition, thereby obtaining the produced composition. Engineered molecules include proteins, such as engineered antigen receptors, including chimeric antigen receptors (CARs), and other recombinant receptors, such as chimeric receptors with ligand-binding extracellular fragments and intracellular signaling fragments.

Also provided are culture initiating compositions, stimulated compositions, and manufactured compositions used in the preparation, or obtained by the methods described. Also provided are methods comprising administering such compositions and cells to subjects in need thereof, including patients suffering from cancer. Also provided are kits comprising compositions and/or cells obtained by any of the methods described herein.

The methods may be advantageous and result in a more desirable product. In some embodiments, the proposed methods allow for a more unified manufacturing process. In some embodiments, the implementation of the proposed methods allow you to get cells that are characterized by more uniform transduction and/or propagation at later stages of cell engineering. In some embodiments, a more uniform transduction and/or expansion may result in a T cell product that is more predictable during production. In some instances, the T cell product may be subdivided into more unitary doses for administration to a subject. Such features in some contexts may reduce or prevent potential toxicity and/or associated effects and symptoms in subjects following adoptive cell therapy.

All publications, including patent documents, scientific articles and databases referred to in this application, are incorporated by reference in their entirety and for all purposes to the same extent as if each individual publication were individually incorporated by reference. If the definition given herein conflicts with or is otherwise inconsistent with the definition given in patents, patent applications, published patent applications and other publications, which are incorporated herein by reference, the definition given in this document shall prevail. regarding the definition in documents that are incorporated herein by reference.

Section headings used in this document are for organizational purposes only and should not be construed as limiting the described subject matter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1D show the results for CAR+ T cell compositions prepared by the non-expanded and expanded methods using bead-based stimulation reagent (bead), bead-based stimulation reagent and incubation in basal medium (bead-basal medium), or oligomeric stimulation reagent (oligomer ). FIG. 1A shows the percentage of CD4+ and CD8+ T cells positive for both CCR7 and CD27. FIG. 1B shows the percentage of CCR7+CD27+ cells for CD4+CAR+ T cells. FIG. 1C shows the percentage of CCR7+CD27+ cells for CD8+CAR+ T cells. FIG. 1D shows the percentage of CCR7+CD27+ cells obtained from a representative donor in an expanded manner on different days of the manufacturing process, including activation on day 1 (d1 AMAT), transduction on day 2 (d2 XMAT), and at different time points after the start of culture. (d4 INOC+2, d6 INOC+4, d7 INOC+5).

FIG. 2A-2D are Kaplan-Meier survival curves for grouped subjects treated with CAR ⁺ T cell compositions containing the percentage of CCR7 ⁺ CD27 ⁺ CAR ⁺ T cells among CD4 ⁺ CAR ⁺ T cells (FIG. 2A for progression-free survival, FIG. 2C for duration of response) and among CD8 ⁺ CAR ⁺ T cells (FIG. 2B for progression-free survival, FIG. 2D for duration of response) that was above or below a certain threshold.

FIG. 3 shows T cell clonality of isolated CD4+ and CD8+ T cell compositions before engineering (CMAT) and of CD4+ and CD8+ therapeutic CAR+ T cell compositions after engineering (Shannon index applied).

I. Method of incubation (eg, stimulation) of T cells

Provided herein are methods for incubating (eg, stimulating) cells in the process of obtaining cells for adoptive cell therapy, as well as compositions and cells obtained by these methods. In some embodiments, cells typically include T cells, which in some embodiments express engineered antigen receptors, such as engineered or recombinant T cell receptors (TCRs), and functional non-TCR antigen receptors, such as as chimeric antigen receptors (CARs).

In some embodiments, a method for incubating (e.g., stimulating) T cells is provided, comprising incubating under stimulating conditions an initial composition comprising a population of T cells including naive-like T cells and non-naive T cells, wherein said initial composition comprises the number of naïve-like T cells, or a subset of CD8+ T cells thereof, necessary to initiate the culture to form a stimulated composition. In some aspects, the stimulating conditions preferentially induce proliferation or proliferation of naive-like T cells as compared to non-naive-like T cells in the stimulated composition. In some embodiments, the method further comprises incorporating into the stimulated cell composition a nucleic acid encoding the engineered recombinant receptor to provide a method-produced composition comprising T cells expressing the engineered recombinant receptor. In some aspects, the administration is carried out during at least part of the incubation period or is carried out after the incubation. In some embodiments, the cells of the original composition have not, and are not, subjected prior to incubation to a selection step based on an endogenous T-cell surface marker that distinguishes between naive-like and non-naive-like T cells.

In some embodiments of the proposed methods, administration of a nucleic acid encoding a genetically engineered recombinant receptor occurs before, during, and/or after administration of a nucleic acid encoding a genetically engineered recombinant receptor. In some embodiments, the method includes incubating the composition under stimulating conditions prior to the introduction of a nucleic acid encoding a genetically engineered recombinant receptor. In some instances, the method includes incubating the composition under stimulating conditions while introducing a nucleic acid encoding the engineered recombinant receptor. In some embodiments, the method includes incubating the composition under stimulating conditions following administration of a nucleic acid encoding a genetically engineered recombinant receptor.

In specific embodiments, the implementation of the stimulating condition includes a surface, such as a magnetic bead, attached to it by one or more agents that bind the fragment on the cell surface. In one embodiment, at least anti-CD3 antibodies are attached to the surface. In another embodiment, anti-CD3 and/or anti-CD28 antibodies are attached to the surface. In some embodiments, at least a significant portion of at least one population of T cells in the original composition is removed after incubation. In one embodiment, the ratio of cells to beads under stimulating conditions is from, or about, 50:1 to about 5:1. In specific embodiments, the ratio is from, or about, 100:1 to about 1:1. In some embodiments, the ratio is from, or about, 1:1 to 1:50. In specific embodiments, the ratio is at least about 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1: 4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10. In one particular embodiment, the ratio is about 3:1. In one particular embodiment, the ratio is about 1:3.

In some embodiments of the methods provided herein, culture conditions preferably induce proliferation, stimulation, and/or activation of non-naive-like T cells as compared to naive-like T cells. In some embodiments, the methods of incubation (eg, stimulation) result in the desired manufactured composition consisting of the desired number of cells derived from naive-like T cells of the original composition. In some embodiments, using the T cell stimulation method described herein, of the cells in the initial composition, a greater percentage of naive-like T cells, compared to non-naïve-like T cells, are induced to proliferate and/or become activated. In some aspects, the stimulated composition obtained by the stimulation methods described herein contains less than 10% of cells derived from non-naive T cells. In some examples, the stimulated composition contains less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of non-naive-like cells. T cells. In some embodiments, a greater percentage of T cells that were similar to naive in the original composition compared to the percentage of T cells that were not similar to naive in the original composition divide on day 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 after the start of said incubation. In some instances, the stimulatory conditions of the T cell stimulation method induce cell death in particular cell subpopulations. In specific examples, the stimulatory conditions of the method induce the activation of non-naïve-like T cells, thereby causing activation-induced cell death (AICD).

In one aspect, the method preferably results in the activation of non-naive T cells of the parent composition, thereby inducing cell death of non-naive T cells of the parent composition, thereby eliminating T cells derived from non-naive cells of the parent composition from the stimulated compositions. At the same time, the desired cells that remain, such as those derived from naive-like T cells of the original composition, are activated, survive, and are stimulated to proliferate, resulting in a population of activated cells, of which at least a significant proportion unwanted T-cell subpopulations has been eliminated. In addition, the incubation conditions (eg, stimulation) in the methods described herein result in the restoration of polyclonality of the T cell population for the expressed TCR genes, as evidenced by spectrotype analysis or other clonality quantification methods. In some embodiments, the signature of cells in the manufactured composition, derived from naive-like T cells of the original composition, is characterized by the expression of specific markers.

A. Original composition

In the proposed methods of incubation (eg, stimulation) of T cells, the method includes incubation under stimulating conditions of the original composition containing a population of T cells. In some aspects, the initial composition contains a population of T cells, for example, including naive-like T cells and non-naive-like T cells. In some embodiments, the T cell population includes CD4+ and/or CD8+ cells. In some embodiments, the initial composition contains the number of cells required to initiate the culture. In some cases, the number of cells required to initiate culture is based on the number of naive-like T cells or a subset of CD8+ T cells in the original composition. In some aspects, specific subsets of T cells, such as naive-like and/or non-naive-like T cells, can be identified using specific markers or signatures. In some specific examples, the expression of cell surface markers is used to evaluate and/or identify subsets of T cells. In some aspects, the clonality of the original composition can be characterized.

In some aspects, the cell composition that is incubated and/or engineered, eg, the parent composition, has not been and is not pre-selected for naïve-like T cells. In some embodiments, the implementation of the method does not include positive or negative selection, or enrichment, like naive cells of the original composition. In some aspects, the cell composition that is stimulated and/or engineered, eg, the parent composition and/or the stimulated composition, has not been and is not subject to such selection prior to incubation (eg, stimulation). In some embodiments, the cells have not and/or are not subjected prior to incubation to a selection step based on the level or presence of a T-cell surface marker to distinguish between naive-like and non-naive-like T cells, such as CD27, CD28, CD45RA, CD45RO, CD56 , CD62L, CD95, KLRG1 or CCR7. For example, in some aspects, the cells in the original composition were not selected based on expression, or based on surface expression, of such a marker prior to incubation under stimulatory conditions. In some aspects, the cells in the stimulated composition are not selected based on expression, or surface expression, of such a marker prior to genetic engineering, such as incubation with nucleic acid. In some aspects, the incubation of the original composition is carried out without selection based on the surface expression of the marker for enrichment in similar naive T cells.

In some embodiments, the methods include fewer selection steps compared to other methods, for example, do not involve selection of subsets of T cells, and thus are simpler and have cost and/or resource reduction advantages compared to multi-step selection methods. . In some embodiments, the methods do not include enrichment based on the expression of markers characteristic of memory T cells or subsets thereof and/or similar to naive T cells. In some aspects, the cell composition that is incubated (eg, stimulated), eg, the original composition and/or the stimulated composition, has not been and is not subject to such selection.

In some examples, the methods do not include positive selection based on a marker that is characteristic of non-naïve T cells or that distinguishes specific subsets of T cells, e.g., CD62L, CCR7, CD27, CD28, CD56, CD3, CD122, CD95, CD25, IL7-Ra and/or CD127. In some examples, the methods do not include positive selection or enrichment based on the expression of CD62L, CCR7, CD27, CD28, CD56, CD3, CD122, CD95, CD25, IL7-Rα and/or CD127. In some embodiments, the methods do not use samples or compositions that have been enriched or positively selected based on such markers, or enriched for specific cell subtypes.

In some embodiments, the methods do not include affinity-based selection steps to separate or distinguish between naive-like and non-naive-like T cells. In some examples, the methods do not include positive selection based on a marker that is characteristic of naïve-like or non-naïve-like cells, or that distinguishes one from another, such as CD27, CD28, CD45RO, CD45RA, CD56, CCR7, CD95, KLRG1, and/or CD62L . In some examples, the methods do not include positive selection or enrichment based on the expression of such markers. In some embodiments, the methods do not use samples or compositions enriched or positively selected based on such markers, or enriched for such subtypes.

Cells

In some embodiments, the methods provided herein include one or more steps of obtaining an initial cell composition that contains the number of naive-like T cells necessary to initiate a culture, for example, in connection with a method that includes one or more steps of stimulation, propagation , proliferation and/or genetic engineering, for example, transduction, cells. The cells are typically eukaryotic cells such as mammalian cells and are typically human cells. The original composition can be obtained or created in various ways, including the isolation or selection of cells from a biological sample. In some embodiments, the initial composition contains CD4+ and/or CD8+ cells from a biological sample, for example, obtained or isolated using one or more isolation, selection, or enrichment steps. In some specific examples, the ratio of CD4+ cells to CD8+ cells in the original culture is, or is about, 1:1, 1:2, 2:1, 1:3, or 3:1. In some embodiments, the initial composition containing isolated CD4+ and/or CD8+ T cells contains a mixture of naive-like and non-naive-like T cells.

In some embodiments, the cells of the original composition are derived from blood, bone marrow, lymphatic or lymphoid organs, are cells of the immune system, for example, cells of the innate or acquired immune system, including from samples containing myeloid or lymphoid cells, including lymphocytes, such as usually T cells. The cells are typically primary cells, eg, cells obtained directly from the subject and/or obtained from the subject and frozen. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as all T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof that differ in function, activation state, maturity, ability to differentiate, reproduction, recycling, localization and/or persistence, specificity for antigens, type of antigen receptor, presence in a particular organ or compartment, markers or cytokine secretion profile, and/or degree of differentiation. For the subject to be treated, the cells may be allogeneic and/or autologous. The methods also include available standard methods. In some embodiments, the methods include obtaining cells from a subject, preparing, processing, culturing and/or engineering them as described herein, and reintroducing them to the same patient before or after cryopreservation.

Subtypes and subpopulations of T cells and/or CD4+ and/or CD8+ T cells include naïve T cells (T _N ), effector T cells (T _EFF ), memory T cells and their subtypes such as memory T stem cells (T _SCM ), central memory T cells (T _CM ), effector memory T cells (T _EM ) or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells , mature T cells, helper T cells, cytotoxic T cells, mucosal invariant T cells (MAIT), preexisting and acquired regulatory T cells (Treg), helper T cells such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells, and delta/gamma T cells. In some embodiments, the cell is a regulatory T cell (Treg). In some embodiments, the cell also contains recombinant FOXP3 or a variant thereof.

In some embodiments, obtaining engineered cells includes one or more culturing and/or preparation steps. Cells for engineering can be isolated from a sample, such as a biological sample, for example, cells obtained from, or extracted from, a subject. In some embodiments, the subject from which the cells are obtained is a subject who has a disease or condition, or is in need of cell therapy, or who will receive cell therapy. In some embodiments, the subject is a human in need of a particular therapeutic intervention, such as adoptive cell therapy for which cells are isolated, processed and/or engineered.

Accordingly, in some embodiments, the cells are primary cells, such as primary human cells. Samples include tissues, fluids, and other samples obtained directly from the subject, as well as samples obtained after one or more processing steps such as separation, centrifugation, genetic engineering (eg, transduction with a viral vector), washing, and/or incubation. The biological sample may be a sample obtained directly from a biological source or a sample that has been processed. Biological samples include, but are not limited to, body fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.

In some aspects, the sample from which cells are obtained or isolated is blood or a blood-derived sample, or is a product of, or obtained by, apheresis or leukapheresis. Illustrative specimens include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia cells, lymphomas, lymph node, intestinal lymphoid tissue, mucosal lymphoid tissue, spleen, other lymphoid tissues, liver , lung, stomach, intestines, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsils or other organs and/or cells derived from them. Samples include, in the context of cell therapy, such as adoptive cell therapy, samples from autologous and allogeneic sources.

In some embodiments, cells are derived from cell lines, such as T cell lines. In some embodiments, the cells are obtained from a xenogeneic source, such as a mouse, rat, primate, or pig.

In some embodiments, cell isolation includes one or more preparation and/or non-affinity cell separation steps. In some examples, cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse, or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more properties such as density, adherence, size, sensitivity, and/or resistance to particular components.

In some examples, cells from the subject's circulating blood are obtained, for example, by apheresis or leukapheresis. In some aspects, the samples contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, erythrocytes and/or platelets, and in some aspects contain cells other than erythrocytes and platelets.

In some embodiments, the blood cells obtained from the subject are washed, for example, to remove a plasma fraction and to transfer the cells to an appropriate buffer or medium for subsequent processing steps. In some embodiments, cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution is free of calcium and/or magnesium and/or many or all of the divalent cations. In some aspects, the washing step is performed using a semi-automated "flow through" centrifuge (eg, Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some aspects, the washing step is carried out by the method of flow filtration along the flow (TFF) in accordance with the manufacturer's instructions. In some embodiments, cells are resuspended in various biocompatible buffers after washing, such as, for example, Ca ⁺⁺ /Mg ⁺⁺ free PBS. In specific embodiments, components of the blood cell sample are removed and the cells directly resuspended in culture medium.

In some embodiments, the methods include density-based separation techniques, such as obtaining white blood cells from peripheral blood by lysing red blood cells and centrifuging through a percoll or ficoll density gradient.

In some embodiments, isolation methods include separating different cell types based on the expression or presence of one or more specific molecules in the cell, such as surface markers, for example, surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known separation method based on such markers can be used. In some embodiments, the separation is based on affinity or immune affinity. For example, in some aspects, isolation includes separating cells and cell populations based on cellular expression, or level of expression, of one or more markers, typically cell surface markers, such as by incubation with an antibody or binding partner that specifically binds such markers, followed by , as a rule, the steps of washing and separating cells associated with the antibody or binding partner from cells not associated with the antibody or binding partner follow.

Such separation steps may be based on positive selection, in which cells that have bound to the reagents are retained for later use, and/or negative selection, in which cells that have not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for later use. In some aspects, negative selection can be particularly useful when there is no antibody specifically recognizing a cell type in a heterogeneous population, such that separation is best done based on markers expressed by cells other than the desired population.

Separation need not lead to 100% enrichment or removal of specific cell populations or cells expressing a particular marker. For example, positive selection or enrichment for cells of a particular type, such as those that express a marker, means an increase in the number or percentage of such cells, but does not necessarily lead to a complete absence of cells that do not express the marker. Similarly, negative selection, removal or depletion of cells of a particular type, such as those expressing a marker, means a decrease in the number or percentage of such cells, but does not necessarily lead to the complete removal of all such cells.

In some examples, multiple rounds of separation steps are performed, with a positively or negatively selected fraction from one step being subjected to a separation step, such as a subsequent positive or negative selection. In some examples, a single separation step may result in the simultaneous depletion of cells expressing multiple markers, for example, by incubating cells with multiple antibodies or binding partners, each specific for a negative selection target marker. Similarly, multiple cell types can be simultaneously positively selected by incubating cells with multiple antibodies or binding partners specific for markers expressed on different cell types.

For example, in some aspects, specific subpopulations of T cells, such as cells having or expressing at high levels one or more surface markers, for example, CD28 ⁺ , CD62L ⁺ , CCR7 ⁺ , CD27 ⁺ , CD127 ⁺ , CD4 ⁺ , CD8 ⁺ , CD56 ⁺ , CD45RA ⁺ , CD95 ^hi , and/or CD45RO ⁺ T cells are isolated by positive and negative selection methods.

For example, CD3 ⁺ , CD28 ⁺ T cells can be positively selected using anti-CD3/anti-CD28 conjugated magnetic beads (eg, ^DYNABEADS® M-450 CD3/CD28 T Cell Expander).

In some embodiments, isolation is performed by enriching a particular cell population by positive selection, or depleting a particular cell population by negative selection. In some embodiments, positive or negative selection is made by incubating cells with one or more antibodies or other binders that specifically bind one or more surface markers expressed (marker ⁺ ), or expressed at a relatively high level (marker ^high ), positively or negatively selected cells, respectively.

In some embodiments, T cells are isolated from a PBMC sample by negative selection for markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells such as CD14 cells. In some aspects, a CD4 ⁺ or CD8 ⁺ selection step is used to separate CD4 ⁺ helper and CD8 ⁺ cytotoxic T cells. Such CD4 ⁺ and CD8 ⁺ populations can be further sorted into subpopulations by positive or negative selection for markers expressed, or relatively highly expressed, on cells from one or more subpopulations of naïve T cells, memory T cells, and/or effector T cells. cells.

In some embodiments, CD8 ⁺ cells are further enriched or depleted for naïve cells, central memory cells, effector memory cells, and/or central memory stem cells, e.g., by positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, enrichment for central memory T cells (T _CM ) is carried out to increase efficiency, for example, to increase long-term survival, expansion and/or engraftment after administration, which in some aspects is especially strong in such subpopulations. See Terakura et al. (2012) Blood. 1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, combining T _CM -rich CD8 ⁺ T cells and CD4 ⁺ T cells further improves efficiency.

In embodiments, memory T cells are present in both CD62L ⁺ and CD62L ^- subsets of CD8 ⁺ peripheral blood lymphocytes. PBMC can be enriched or depleted in CD62L ^- CD8 ⁺ and/or CD62L ⁺ CD8 ⁺ fractions, for example, using anti-CD8 and anti-CD62L antibodies.

In some embodiments, the enrichment for central memory T cells ( _TCM ) is based on the presence, or high surface expression, of CD45RO, CD62L, CCR7, CD28, CD3 and/or CD127; in some aspects it is based on negative selection for cells expressing, or highly expressing, CD45RA and/or granzyme B. In some aspects, isolation of a CD8 ⁺ population enriched in T _CM cells is performed by depletion of cells expressing CD4, CD14 , CD45RA, and positive selection, or enrichment, for cells expressing CD62L. In one aspect, enrichment for central memory T cells ( _TCM ) is performed starting with a negative fraction of cells selected based on CD4 expression, negatively selected based on CD14 and CD45RA expression, and positively selected based on CD62L. In some aspects, these types of selection are carried out simultaneously, and in other aspects, they are carried out sequentially, in any order. In some aspects, the same selection step based on CD4 expression that is used to generate a population or subset of CD8 ⁺ cells is also used to generate a population or subset of CD4 ⁺ cells, such that both the positive and negative fractions obtained from separation based on CD4 are stored and used in subsequent steps of the method, optionally after one or more additional positive or negative selection steps.

In a specific example, a PBMC sample or other white blood cell sample is selected for CD4 ⁺ cells while retaining both negative and positive fractions. The negative fraction is then negatively selected based on CD14 and CD45RA or ROR1 expression and positively selected based on a central memory T cell marker such as CD62L or CCR7, with positive and negative selection in any order.

Helper CD4 ⁺ T cells are sorted into naïve cells, central memory cells and effector cells by identifying cell populations having cell surface antigens. CD4 ⁺ lymphocytes can be obtained by standard methods. In some embodiments, the naive CD4 ⁺ T lymphocytes are CD45RO ^- , CD45RA ⁺ , CD62L ^- CD4 ⁺ T cells. In some embodiments, the central CD4 ⁺ memory cells are CD62L ⁺ and CD45RO ⁺ cells. In some embodiments, the effector CD4 ⁺ cells are CD62L ^- and CD45RO ^- cells.

In one example, for enrichment for CD4 ⁺ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is coupled to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow cells to be separated for positive and/or negative selection. For example, in some embodiments, cells and cell populations are separated or isolated using immuno-magnetic (or affinity-magnetic) separation methods (reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In vitro and In vivo , p 17-25 edited by SA Brooks and U. Schumacher © Humana Press Inc., Totowa, NJ).

In some aspects, the sample or composition of cells to be separated is incubated with a fine, magnetizable or magnetically sensitive material, such as magnetically sensitive particles or microparticles, such as paramagnetic beads (such as Dynalbeads or MACS beads). The magnetically sensitive material, such as a particle, is typically attached directly or indirectly to a binding partner, such as an antibody, that specifically binds a molecule, such as a surface marker, found on the cell, cells, or cell population that is desired to be separated, such as which it is desirable to subject to negative or positive selection.

In some embodiments, the implementation of the magnetic particle or bead contains a magnetically sensitive material associated with a specific binding agent, such as an antibody or other binding partner. In some aspects, magnetically sensitive materials used in magnetic separation methods can be used. Suitable magnetic particles include those described in Molday's US Pat. No. 4,452,773 and EP 452342 B, which are incorporated herein by reference. Other examples are colloidal particles such as those described in Owen US Pat. No. 4,795,698 and Liberti et al. U.S. Patent No. 5200084.

Incubation is generally carried out under conditions in which antibodies or binding partners, or molecules such as secondary antibodies or other reagents specifically binding such antibodies or binding partners that are bound to the magnetic particle or bead, specifically bind cell surface molecules if they are on the cells in the sample.

In some aspects, the sample is placed in a magnetic field, and those cells that are associated with magnetically sensitive or magnetizable particles will be attracted to the magnet and separated from unlabeled cells. For positive selection, cells that are attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained. In some aspects, a combination of positive and negative selection is used during the same selection step, with the positive and negative fractions retained and further processed or subjected to additional separation steps.

In specific embodiments, the magnetically sensitive particles are coated with primary antibodies or other binding partners, secondary antibodies, lectins, enzymes, or streptavidin. In specific embodiments, magnetic particles are attached to cells by coating with primary antibodies specific for one or more markers. In specific embodiments, cells, rather than beads, are labeled with a primary antibody or binding partner, and then magnetic particles coated with a cell-type-specific secondary antibody or other binding partner (eg, streptavidin) are added. In specific embodiments, streptavidin-coated magnetic particles are used in combination with biotinylated primary or secondary antibodies.

In some embodiments, magnetically sensitive particles are left attached to cells that will subsequently be incubated, cultured, and/or engineered; in some aspects, the particles are left attached to cells to be administered to a patient. In some embodiments, the magnetizable or magnetically sensitive particles are removed from the cells. Methods for removing magnetizable particles from cells may include, for example, the use of competing unlabeled antibodies, magnetizable particles, or antibodies conjugated to cleavable linkers, and so on. In some embodiments, the magnetizable particles are biodegradable.

In some embodiments, affinity-based selection is performed by activated cell magnetic sorting (MACS) (Miltenyi Biotec, Auburn, CA). Activated Cell Magnetic Sorting (MACS) systems are capable of producing highly pure cells after selection with magnetizable particles attached to them. In specific embodiments, the MACS operates in a mode in which non-target and target molecules are sequentially eluted after the application of an external magnetic field. That is, cells attached to the magnetizable particles are held in place while non-attached cells are eluted. Then, after this first elution step is completed, the cells that were held in the magnetic field and were not eluted are released in a certain way so that they can be eluted and recovered. In specific aspects, non-target cells are labeled and removed from a heterogeneous population of cells.

In specific embodiments, isolation or separation is carried out using a system, device, or instrument that performs one or more of the steps of isolation, preparation, separation, processing, incubation, culture, and/or formulation of cells. In some aspects, a system is used to perform each of these steps in a closed or sterile environment, such as to minimize errors, user manipulation, and/or contamination. In one example, the system is the system described in International PCT Publication No. WO2009/072003 or US 20110003380 A1.

In some embodiments, the system or instrument performs one or more, eg, all, of the isolation, processing, engineering, and formulation steps in an integrated or stand-alone system, and/or in automatic or programmable mode. In some aspects, the system or instrument includes a computer and/or computer program that communicates with the system or instrument to allow a user to program, control, evaluate results, and/or correct various aspects of the processing, isolation, engineering, and formulation steps.

In some aspects, the separation and/or other steps are carried out using the CliniMACS system (Miltenyi Biotec), for example, for automated separation of cells at clinically relevant levels in a closed and sterile system. Components may include an embedded microcomputer, magnetic separator, peristaltic pump and various pinch valves. In some aspects, an integrated computer controls all components of the instrument and controls the system to carry out repetitive procedures in a standardized sequence. In some aspects, the magnetic separation cell includes a movable permanent magnet and a selection column holder. The peristaltic pump controls the flow rate through the tubing set and, in conjunction with the pinch valves, provides a controlled flow of buffer through the system and a constant cell suspension.

In some aspects, the CliniMACS system uses antibody-bound magnetizable particles that are supplied in a sterile, pyrogen-free solution. In some embodiments, after labeling cells with magnetic particles, the cells are washed to remove excess particles. The cell preparation bag is then connected to a tubing set, which in turn is connected to a bag containing buffer and a cell collection bag. The tubing set consists of pre-connected sterile tubing, including pre-column and separation column, which are for single use only. Once the separation program has started, the system automatically applies the cell sample to the separation column. Labeled cells are retained on the column while unlabeled cells are removed in a series of washing steps. In some embodiments, cell populations used in the methods described herein are unlabeled and are not retained on the column. In some embodiments, cell populations used in the methods described herein are labeled and retained on a column. In some embodiments, the cell populations used in the methods described herein are eluted from the column after the magnetic field is removed and collected in a cell collection bag.

In specific embodiments, separation and/or other steps are performed using a CliniMACS Prodigy system (Miltenyi Biotec). In some aspects, the CliniMACS Prodigy system is equipped with a cell processing unit that automates the washing and fractionation of cells by centrifugation. The CliniMACS Prodigy system can also include an integrated camera and image recognition software that determines the optimal cell fractionation endpoint by recognizing macroscopic layers of the original cell product. For example, peripheral blood can be automatically separated into red blood cells, white blood cells, and plasma layers. The CliniMACS Prodigy system can also include an integrated cell culture chamber that performs cell culture protocols such as, for example, cell differentiation and expansion, antigen loading, and long-term cell culture. Entry ports can allow for sterile removal and replenishment of media, and cell monitoring can be done using an on-board microscope. See, for example, Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood.1:72-82 and Wang et al. (2012) J Immunother. 35(9):689-701.

In some embodiments, the cell population described herein is harvested and enriched (or depleted) by flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluid stream. In some embodiments, the cell population described herein is harvested and enriched (or depleted) by preparative sorting (FACS). In specific embodiments, the cell population described herein is harvested and enriched (or depleted) by using microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system (see, for example, WO 2010/033140, Cho et al. ( 2010) Lab Chip 10:1567-1573 and Godin et al (2008) J Biophoton 1(5):355-376 In both cases, cells can be labeled with multiple markers, allowing the isolation of well-defined subsets of T cells with high purity.

In some embodiments, the antibodies or binding partners are labeled with one or more detectable markers to facilitate separation in positive and/or negative selection. For example, separation may be based on binding to fluorescently labeled antibodies. In some examples, cell separation based on the binding of antibodies or other binding partners specific for one or more cell surface markers is carried out in a liquid stream, for example, by fluorescence activated cell sorting (FACS), including preparative scale (FACS) and /or using microelectromechanical systems (MEMS) chips, for example, in combination with a flow cytometric detection system. Such methods allow simultaneous positive and negative selection based on multiple markers.

In some embodiments, the preparation methods include the steps of freezing, eg, cryopreserving, cells, either before or after, isolation, incubation, and/or engineering. In some embodiments, freezing, followed by a thawing step, removes granulocytes and, to some extent, monocytes in the cell population. In some embodiments, the cells are suspended in a freezing solution, for example, after a washing step to remove plasma and platelets. In some aspects, any of various known freezing solutions and parameters may be used. One example involves the use of PBS containing 20% DMSO and 8% human serum albumin (HSA) or other suitable cell freezing medium. The suspension is then diluted 1:1 with medium so that the final concentrations of DMSO and HSA are 10% and 4%, respectively. The cells are then frozen to -80° C. at a rate of 1 per minute and stored in a liquid nitrogen vapor phase storage tank.

a. Naive-like T cells

In some embodiments, the method includes incubating a stock composition containing naive-like T cells or a certain threshold number of naive-like T cells. In one aspect of the present methods, a parent composition is provided that has been evaluated for cell subpopulations based on the expression of various markers such as CD27, CD28, CD56, CD62L, CD95, KLRG1, CD45RA, or CD45RO, cytokines (e.g., IL-2, IFN- γ, IL-4, IL-10), cytokine receptors (eg, CD25), perforin, adhesion molecules (eg, VLA-1, VLA-2, VLA-4, LPAM-1, LFA-1) and/or molecules homing (eg, L-selectin), before incubation (eg, stimulation). In some embodiments, different signatures of naive-like T cells can be used to identify naive-like T cells. In some embodiments, the expression of specific markers like naive T cells can be assessed. For example, in some cases, naive-like T cells have a marker on their surface, including T cell activation markers, selected from the group consisting of CD27, CD28, CD45RA, CD62L, and CCR7. In some aspects, naive-like T cells do not have CD56 and/or CD45RO on their surface. In some aspects, naive-like T cells do not have CD45RO on their surface, and have CD27, CD45RA and CCR7 on their surface. In some cases, naive-like T cells lack intracellular expression of a cytokine such as IL-2, IFN-γ, IL-4 and/or IL-10. In some additional examples, naive-like T cells lack the expression of CD25 and/or perforin markers. In some instances, the naive-like T cells are CD95 ^lo cells.

To evaluate the expression of naïve T cell-like markers, the method, in some embodiments, includes detecting the markers by conducting an in vitro assay. In some examples, the in vitro assay is an immune assay, an aptamer assay, a histological or cytological assay, or an mRNA expression level assay. In some cases, the in vitro assay used may be enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunophenotyping, immunoprecipitation, radioimmunoassay (RIA), immunostaining, flow cytometry, surface plasmon resonance (SPR), chemiluminescence, immunochromatographic, assay inhibition or avidity assay. In some embodiments, expression of naïve-like T cell markers is determined by RNA sequencing.

Naive-like T cells of the original composition can also be assessed based on the clonality of the T cells. In some embodiments, the assessment of the clonality of the T cell population is an assessment of the clonal diversity of the T cell population. In some embodiments, the naive-like T cells are polyclonal or multiclonal. The polyclonality of said initial T cell composition is measured based on the breadth of the population's response to a particular antigen. In some aspects, the original composition can be assessed by measuring the number of different epitopes recognized by antigen-specific cells. This can be done using standard methods for obtaining and cloning antigen-specific T cells in vitro . In some embodiments, the naive-like T cells are polyclonal (or multiclonal), with no single clonotypic population predominating in the naive-like T cell population.

In the context of a T cell population, such as a parent composition, in some aspects a polyclonal signature means that the T cell population has multiple and broad antigenic specificity. In some embodiments, polyclonality means that the T cell population has a greater diversity in the TCR repertoire. In some cases, diversity in the TCR repertoire arises from V(D)J recombination events, which in some cases are triggered by selection events for self and foreign antigens. In some embodiments, a T cell population that is diverse, or polyclonal, is a T cell population in which analysis indicates the presence of multiple different or distinct TCR transcripts or products present in the population. In some embodiments, the population of T cells exhibiting high, or relatively high, clonality is a population of T cells in which the TCR repertoire is less diverse. In some embodiments, T cells are oligoclonal if the assay indicates the presence of multiple, eg, two or three, TCR transcripts or products in the T cell population. In some embodiments, T cells are monoclonal if the assay indicates the presence of a single TCR transcript or product in the T cell population.

In some examples, the clonality of cells in the original composition, such as naive-like T cells, is determined by clonal sequencing, optionally next generation sequencing or spectrotype analysis. In some aspects, next generation sequencing techniques using genomic DNA or cDNA from T cells can be used to assess the TCR repertoire, including sequences encoding complementarity determining region 3 (CDR3). In some embodiments, whole transcriptome sequencing by RNA sequencing can be used. In some embodiments, single cell sequencing techniques may be used.

In some embodiments, the implementation of polyclonality can be assessed or determined in the analysis of spectrotypes (determination of the repertoire of hypervariable regions of Vβ, Vα, Vγ or Vδ TCR chains). A T cell population is considered polyclonal if the Vβ spectrotype profile for a particular Vβ, Vα, Vγ or Vδ TCR family has multiple peaks, typically 5 or more dominant peaks, and in most cases with a Gaussian distribution. Polyclonality can also be determined by creating and characterizing antigen-specific clones for the antigen of interest.

In some embodiments, methods for assessing clonality may include various features of the methods described in International Patent Publication Nos. WO2012/048341, WO2014/144495, WO2017/053902, WO2016044227, WO2016176322, and WO2012048340, each of which is incorporated herein by reference in its entirety. . In some embodiments, such methods can be used to obtain sequence information of a target polynucleotide of interest, such as a TCR, in a cell. Target genes can be obtained from the genomic DNA or mRNA of a cell from a sample or population of cells. The sample or population of cells may include immune cells. For example, for target TCR molecules, the genes encoding the TCR chains can be obtained from the genomic DNA or mRNA of immune cells or T cells. In some embodiments, the starting material is RNA from T cells, consisting of transcripts of genes encoding the TCR chain.

In some embodiments, Shannon's index is applied to clonality as a threshold for filtering clones ("Shannon-corrected clonality"), see Chaara et al. (2018) Front Immunol 9:1038).

In some embodiments, the implementation of the proposed methods stimulate, or lead to an increase in the polyclonality of a population of T cells or a subset of them from the original composition. In some embodiments, the implementation of the proposed methods stimulate, or lead to an increase in the diversity of the population of T cells or a subset of them from the original composition. In some embodiments, T cells, or their CD4 or CD8 subsets, of the incubated or stimulated composition have reduced or reduced clonality compared to T cells, or their CD4 or CD8 subsets, in the original composition prior to application of the methods. In some embodiments, the degree of clonality is reduced by more than, or more than about, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more.

In one aspect of the proposed methods, the population of T cells in the original composition is activated or stimulated to induce apoptosis, as described below in the section entitled "Cell Incubation", and thereby eliminate the subpopulation from the cell population. At the same time, desired cells that remain, such as naïve-like cells, are activated and stimulated to proliferate, producing an activated cell population from which at least a significant proportion of unwanted T cell subpopulations (such as non-naïve-like T cells ) was eliminated. As mentioned previously, the stimulation/activation described herein can be carried out on cells remaining after exposure directly to the cell population of pro-apoptotic compositions. Moreover, the subsequent stimulation and activation of the present invention results in the restoration of polyclonality in the T cell population for expressed TCR genes, as evidenced by the results of spectrotype analysis or sequencing.

Amount required to initiate culture

In aspects of the proposed methods, the initial composition contains the amount necessary to initiate culture for preferential activation or propagation of naive-like T cells. In some instances, the amount required to initiate a culture includes, or is determined by, or is based on the number of naive-like T cells or a subset of CD8+ T cells in the composition. Thus, in aspects of the proposed method, incubation (for example, stimulation) is carried out without taking into account the total number or percentage of non-naive T cells, provided that there is a threshold, or necessary to initiate culture, the number of naive-like T cells in the initial composition .

In some embodiments of the cell incubation (e.g., stimulation) methods provided herein, the number of naïve-like T cells or a subset of CD8+ T cells required to initiate culture is from, or about, 0.1 x 10 ⁸ to 5 x 10 ⁸ , from or from about 0.1×10 ⁸ to 4×10 ⁸ , from or from about 0.1×10 ⁸ to 2×10 ⁸ , from or from about 0.1×10 ⁸ up to 1×10 ⁸ , from or from about 1×10 ⁸ to 5×10 ⁸ , from or from about 1×10 ⁸ to 4×10 ⁸ , from or from about 1×10 ⁸ to 2 ×10 ⁸ from or from about 2x10 ⁸ to 5x10 ⁸ from or from about 2x10 ⁸ to 4x10 ⁸ naive-like T cells. In some instances, the number of naïve-like T cells or a subset of CD8+ T cells required to initiate a culture is at least, or at least about, or is, or is about, 0.5 x 10 ⁸ , 0.75 x 10 ⁸ , 1×10 ⁸ , 1.5×10 ⁸ , 2×10 ⁸ , or 4×10 ⁸ naïve-like T cells. In some examples, the number of naïve-like T cells or a subset of CD8+ T cells required to initiate culture is at least, or at least about, or is, or is about, 2×10 ⁸ cells.

In some embodiments of the incubation methods provided herein, under the stimulating conditions described herein, the initial composition containing a population of T cells, including naive-like T cells and non-naive T cells, contains the amount necessary to initiate culture , comprising from, or from about 1×10 ⁸ to 4×10 ⁸ like naive T-cells or their subset of CD8+ T-cells. In some embodiments, the methods result in a stimulated composition, wherein the stimulating conditions preferably induce expansion or proliferation of naive-like T cells compared to non-naive-like T cells in the stimulated composition. In some aspects, the number of naive-like T cells or a subset of CD8+ T cells required to initiate culture is at least, or at least about, or is, or is about, 2x10 ⁸ cells. In some embodiments, the numbers of naive-like and non-naive-like T cells in the initial composition are about the same. In some cases, the number required to initiate culture is the number of naive CD8+ T cells. In some cases, the amount required for culture initiation does not take into account the number of non-naive-like T cells in the initial composition. In some aspects, the amount required to initiate culture is determined based on the number of target non-naive-like T cells in the stimulated composition.

b. Not naive-like T-cells

In some embodiments, the method includes incubating an initial composition containing a population of T cells, including naive-like T cells and non-naive-like T cells. In some embodiments, non-naïve T cells include effector T cells (T _EFF ), memory T cells, central memory T cells (T _CM ), effector memory T cells (T _EM ), as well as combinations thereof. In one aspect of the present methods, a parent composition is provided that has been evaluated against populations of cells expressing various markers such as CD27, CD28, CD56, CD62L, CD95, KLRG1, CD45RA, or CD45RO, cytokines (e.g., IL-2, IFN-γ , IL-4, IL-10), cytokine receptors (eg, CD25), perforin, adhesion molecules (eg, VLA-1, VLA-2, VLA-4, LPAM-1, LFA-1) and/or homing molecules (eg, L-selectin), before incubation (eg, stimulation). In some embodiments, different non-naive T cell signatures can be used to identify non-naive T cells. In some embodiments, the expression of specific non-naive T cell-like markers can be assessed. For example, in some cases, non-naive T cells do not have a marker on their surface, including T cell activation markers such as CD27, CD28, CD45RA, and CCR7; and in some cases, non-naïve T cells have a marker on their surface, including CD62L. In some aspects, non-naïve T cells have CD56 and/or CD45RO on their surface. In some aspects, non-naïve T cells have CD45RO on their surface and lack CD27, CD45RA and CCR7 on their cell surface. In some cases, non-naive T cells have intracellular expression of cytokines such as IL-2, IFN-γ, IL-4 and/or IL-10. In some additional examples, non-naïve T cells have expression of CD25 and/or perforin markers. In some cases, the non-naïve T cells are CD95 ^hi cells.

To assess the expression of non-naïve T cell markers, the method, in some embodiments, includes detecting the markers by conducting an in vitro assay. In some examples, the in vitro assay is an immune assay, an aptamer assay, a histological or cytological assay, or an mRNA expression level assay. In some cases, the in vitro assay used may be enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunophenotyping, immunoprecipitation, radioimmunoassay (RIA), immunostaining, flow cytometry, surface plasmon resonance (SPR), chemiluminescence, immunochromatographic, assay inhibition or avidity assay. In some embodiments, expression of non-naïve T cell markers is determined by RNA sequencing.

The non-naïve T cells of the original composition can also be assessed based on the clonality of the T cells. In some embodiments, the non-naïve T cells are monoclonal. The clonality of said initial T cell composition is measured based on the breadth of the population's response to a particular antigen. In some embodiments, monoclonal means that the T cell population has a very low degree of diversity. In some aspects, the original composition can be assessed by measuring the number of different epitopes recognized by antigen-specific cells. This can be done using standard methods for obtaining and cloning antigen-specific T cells in vitro . In some embodiments, non-naïve T cells exhibit a predominance of one TCR gene rearrangement pattern. In some examples, the clonality of cells in the original composition, such as non-naïve-like T cells, is determined by clonal sequencing, optionally next generation sequencing or spectrotype analysis.

In some embodiments, the assessment of the clonality of the T cell population is an assessment of the clonal diversity of the T cell population. In some embodiments, a monoclonal T cell population means that the T cell population has a low degree of diversity. In the context of a T cell population, such as a parent composition, monoclonality means that a T cell population has a single specificity when defined in a spectrotype analysis (determining the Vβ, Vα, Vγ, or Vδ hypervariable region repertoire of TCR chains). A T cell population is considered monoclonal (or monospecific) if the Vβ, Vα, Vγ, and/or Vδ spectrotype profile for a particular Vβ, Vα, Vγ, and/or Vδ TCR family has one dominant peak. Spectrotype analysis allows discrimination between rearranged variable region genes of a specific size, not sequence. Thus, it is understood that a single peak may represent a population of T cells expressing any of a limited number of rearranged TCR variable region genes (Vβ, Vα, Vγ, or Vδ) containing any of the 4 potential nucleotides (adenine (a), guanine (g ), cytosine (c) or thymine (t)) or a combination of 4 nucleotides in the junction region. In particular embodiments, it may be desirable to clone and sequence a specific band to determine the sequence(s) of the rearranged variable region gene(s) present in the band corresponding to the particular length.

In some embodiments, methods for assessing clonality may include various features of the methods described in International Patent Publication Nos. WO2012/048341, WO2014/144495, WO2017/053902, WO2016044227, WO2016176322, and WO2012048340, each of which is incorporated herein by reference in its entirety. . In some embodiments, such methods can be used to obtain sequence information of a target polynucleotide of interest, such as a TCR, in a cell. Target genes can be obtained from the genomic DNA or mRNA of a cell from a sample or population of cells. The sample or population of cells may include immune cells. For example, for target TCR molecules, the genes encoding the TCR chains can be obtained from the genomic DNA or mRNA of immune cells or T cells. In some embodiments, the starting material is RNA from T cells, consisting of transcripts of genes encoding the TCR chain. In some embodiments, Shannon's index is applied to clonality as a threshold for filtering clones ("Shannon-corrected clonality"), see Chaara et al. (2018) Front Immunol 9:1038).

Accordingly, methods for incubating (e.g., stimulating) T cells to produce engineered T cells for adoptive therapy include methods using conditions that preferentially induce multiplication and proliferation of cells derived from naïve-like T cells (or derived from CD45RA+ or CD45RO- T cells), compared with cells derived from non-naïve T cells (or derived from CD45RA- or CD45RO+ T-cells). In some embodiments, the naive-like T cells are CD45RA+, CD45RO-, CD27+, and CCR7+ cells. In some embodiments, the non-naïve T cells are CD45RA-, CD45RO+, CD27- and CCR7- cells. In some embodiments, the methods include incubating T cells in a culture initiating composition under stimulating conditions that preferentially induce expansion or proliferation of naive-like T cells over non-naïve-like T cells to form a stimulated composition. Using the proposed methods, the assessment of the original composition based on the markers and signatures described above, can be used as part of the method.

B. Cell incubation

In some embodiments, the proposed methods include the steps of culturing, incubating, culturing, and/or genetically engineering the cells in an initial composition, such as an initial composition containing an amount of naïve-like T cells necessary to initiate a culture. For example, in some embodiments, methods are provided for incubating and/or engineering the amount of proposed starting compositions required to initiate a culture that contains a threshold number of the described naïve-like T cells. Incubation and/or engineering can be carried out in a culture vessel such as a block, chamber, well, column, tube, tube set, valve, vial, culture dish, bag or other container for culture or culture of cells.

In some embodiments, cells are incubated and/or cultured prior to, or concurrently with, genetic engineering, in accordance with any of the methods described in Section II. Incubation steps may include cultivation, cultivation, stimulation, activation and/or growth. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulating agent. Such conditions include conditions designed to induce proliferation, expansion, activation and/or survival of cells in a population, to mimic antigen exposure, and/or to prime cells for genetic engineering, for example, to introduce a recombinant receptor, for example, CAR.

The conditions may include one or more of the specific environment, temperature, oxygen content, carbon dioxide content, time period, means, such as nutrients, amino acids, antibiotics, ions, and/or stimulatory factors such as cytokines, chemokines, antigens, binding partners. , fusion proteins, recombinant soluble receptors, as well as any other means designed to activate cells.

In some embodiments, the stimulatory conditions or agents include one or more agents, such as a ligand, that is capable of activating the intracellular signaling domain of the TCR complex. In some aspects, the agent triggers or initiates an intracellular TCR/CD3 signaling cascade in a T cell. Such agents may include antibodies, such as those specific for TCR, such as anti-CD3 antibodies. In some embodiments, the stimulatory conditions include one or more agents, such as a ligand, that is capable of stimulating a costimulatory receptor, such as an anti-CD28 antibody. In some embodiments, such agents and/or ligands may be associated with a solid support, such as a bead, and/or may be one or more cytokines. Optionally, the propagation method may further comprise the step of adding the anti-CD3 and/or anti-CD28 antibody to the culture medium (eg, at a concentration of at least about 0.5 ng/mL).

Proposed methods generally include the presence, design, and/or use of stimulating conditions that preferentially induce proliferation or proliferation of naive-like versus non-naive-like T cells, and/or that preferably do not induce proliferation or proliferation of non-naive-like T cells. , compared to similar naive, T-cells. In some aspects, the conditions include the use of agents that induce an activating signal such that only non-naïve-like T cells present in the composition become activated such that it results in cell death. Such preferred conditions are typically used in a step prior to the introduction of nucleic acids encoding engineered molecules, such as engineered antigen receptors.

Conditions include, but are not limited to, conditions designed to induce proliferation, expansion, activation, and/or survival of cells in a population. In some embodiments, the conditions include a stimulatory, eg, activating, signal sufficient to activate and/or induce proliferation or division of non-naive T cells, or a subset thereof. Such naive T cells typically require minimal signaling through TCR/CD3 to reach a threshold for activation, eg, to achieve full activation and entry into the cell cycle. This minimum signal is generally higher than the signal required to induce activation/cell cycle entry of non-naive T cells and/or some subsets thereof. Not like naive T cells, as a rule, a much lower level of TCR/CD3 involvement is required for activation and entry into the cell cycle. In some aspects, stronger signals can induce, or increase, levels of activation-induced cell death in non-naive cells or certain subsets thereof.

Thus, in some embodiments in which the composition comprises a population of naïve-like and non-naive-like T cells, and conditions are used that induce an activating signal below the activation threshold required to activate naïve-like T cells, substantially non-naïve T- cells will become activated and susceptible to stimulating conditions that can lead to cell death. For example, under specific stimulating conditions, cell death may occur due to activation.

In some aspects, the stimulating conditions are such that activation induces cell death in non-naïve-like cells, as opposed to other conditions such as standard conditions. Thus, the present invention relates to methods for eliminating at least a significant portion of any undesirable subpopulation of T cells, for example, non-naive T cells of the original composition. In the case of the proposed methods, a significant part means at least 70% of the unwanted subpopulation of cells, for example, not similar to the naive T cells of the original composition. In specific embodiments, a significant portion means 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, of an undesirable subset of cells, e.g., not similar to the naive T cells of the original compositions. Cell elimination, e.g., due to non-naïve T cell-like cell death, can be measured using a variety of methods, including, but not limited to, flow cytometric analysis using various antibodies and/or peptide-MHC tetramers, as well as functional assays such as as chromium proliferation and release assays.

In some embodiments, an enzyme is added to the initial composition to remove residual cellular components after cell elimination. For example, in some exemplary embodiments, deoxyribonuclease (DNase) or recombinant human deoxyribonuclease I ( ^Pulmozyme® ) is added to the original composition. In some aspects, the stimulatory conditions include the use of deoxyribonuclease (DNase) or recombinant human deoxyribonuclease I ( ^Pulmozyme® ).

In some cases, the stimulus conditions do not include the use of culture components that are added to maintain specific subsets of T cells, eg, non-naïve T cells. Thus, in some cases, removal of components from culture under stimulating conditions may contribute to the elimination of non-naive-like T cells. In some aspects, stimulating conditions are, or are also used, by eliminating or reducing the concentration of culture reagents that are known or likely to reduce AICD and/or that promote the survival of older cells, such as non-naive cells. In some cases, stimulatory conditions do not include N-acetylcysteine, or include N-acetylcysteine in a lower amount or concentration. In some cases, the stimulatory conditions do not include recombinant IL-7 and/or recombinant IL-15, or include recombinant IL-7 or IL-15 at a lower amount or concentration. In some embodiments, culture supplements may be included that further facilitate or promote the removal of non-naive cells (eg, CD45RO-associated toxin).

In specific embodiments, stimulation and/or breeding periods may be 2 to 15 days, 2 to 12 days, 2 to 10 days, 2 to 8 days, 2 to 6 days, 2 to 4 days, 4 up to 12 days, 4 to 10 days, 4 to 8 days, 4 to 6 days, 6 to 12 days, 6 to 10 days, 6 to 8 days, 8 to 12 days, 8 to 10 days or 10 to 12 days. In some embodiments, cells are incubated for at least 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or more than 14 days. In one embodiment of the proposed methods, the mixture can be cultured for a period of time from 30 minutes to several hours (about 3 hours) or up to about 14 days, or for any intervening whole number of hours or minutes. In another embodiment, the mixture can be cultured for 21 days. In one embodiment, the beads and T cells are co-cultured for about eight days. In another embodiment, the beads and T cells are co-cultured for at least, or at least about, 2-3 days. In another embodiment, the beads and T cells are co-cultured for at least, or at least about 2 days, or at least, or at least about 48 hours. In some aspects, multiple cycles of stimulation may be required, so that the T cell culture time may be 60 days or more.

Incubation and/or engineering can be carried out in a culture vessel such as a block, chamber, well, column, tube, tube set, valve, vial, culture dish, bag or other container for culture or culture of cells. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulating agent. Such conditions include conditions designed to induce proliferation, expansion, activation and/or survival of cells in a population, to mimic antigen exposure, and/or to prime cells for genetic engineering, for example, to introduce a recombinant receptor.

The proposed methods typically include incubation (eg containing T-cells of the original compositions) in stimulating conditions. In the context of T cell stimulation, stimulatory conditions typically include a base agent capable of binding, ligating, crosslinking and/or inducing activation through the intracellular signaling domain of the TCR complex or a component thereof such as CD3. In some embodiments, the stimulatory conditions include a base agent that specifically binds a component of the TCR complex and an adjuvant that specifically binds a T-cell co-stimulatory molecule. In some specific examples, the parent agent specifically binds CD3 and/or the costimulatory molecule is selected from the group consisting of CD28, CD137 (4-1-BB), OX40, or ICOS.

In some embodiments, the stimulatory conditions include immobilized anti-CD3 and anti-CD28 antibodies, soluble anti-CD3 antibodies, derivatives of such antibodies, and/or other ligands that bind the TCR/CD3 complex on T cells. In some aspects, the primary agent triggers or initiates an intracellular TCR/CD3 signaling cascade in a T cell. Such agents may include binding partners such as natural ligands and/or antibodies, including antigen-binding antibody fragments such as those specific for a TCR component such as CD3. Illustrative anti-CD3 antibodies are BC3, OKT3 and G19-4.

In some embodiments, the stimulatory conditions further include incubation with an adjunct agent, such as an agent capable of inducing a T cell co-stimulatory signal in a T cell, such as a signal that, in combination with a primary signal (e.g., TCR/CD3 binding), results in proliferation and/or T cell activation. Exemplary adjuvants are agents that specifically bind, ligate, crosslink and/or induce intracellular signaling events via a T cell costimulatory or accessory molecule such as CD28, CD137 (4-1-BB), OX40, ICOS, CD40, LFA -1, DAP10 and/or CD54. Means include antibodies, including fragments thereof, natural ligands, and other binding partners. In some embodiments, the adjuvant binds CD28, eg, an anti-CD28 antibody, including antigen-binding fragments of the antibody. In some aspects, the agent is an anti-CD28 antibody. Illustrative anti-CD28 antibodies are B-T3 and XR-CD28.

In some embodiments, binding partners or agents that specifically bind a particular molecule, such as a T-cell stimulatory or costimulatory molecule, include antibodies specific for such molecules, including antigen-binding antibody fragments. In some aspects, an antibody, such as an anti-CD3 and/or anti-CD28 antibody, is included at a concentration of at least or about 0.5 ng/mL. In some aspects, the agents include one or more other binding partners for such a molecule, such as a natural binding partner. Agents may also include natural ligands and complexes, including molecules and/or complexes on antigen-presenting cells and/or superantigen ( Staphylococcus enterotoxin A (SEA), Staphylococcus enterotoxin B (SEB), toxic shock syndrome toxin 1 (TSST-1)) , endotoxin). Other exemplary agents are agents that mimic signaling via a major or co-stimulatory T cell signaling molecule, e.g., a mitogen, including a PKC activator, phorbol myristate acetate (PMA), phytohemoagglutinin (PHA), and/or a calcium ionophore, e.g., ionomycin, lipopolysaccharide (LPS), T-cell mitogen and cytokines. In some aspects, the conditions include incubation with one or more stimulatory cytokines or other factors such as IL-2 and/or IL-15. In some aspects, the cytokine concentration is at least about 10 units/mL.

In some embodiments, the primary and secondary (costimulatory) agents are associated with a solid surface or support, such as a particle, such as a bead. In some embodiments, cells may be incubated with, and/or contacted with, a stimulant capable of activating and/or proliferating T cells. In some aspects, the primary and secondary agents are associated with a solid surface, such as a particle, for example, a granule. In specific embodiments, the stimulant includes a particle, eg a bead, that is conjugated or linked to one or more agents, eg biological molecules, that are capable of activating and/or proliferating cells, eg T cells. In some embodiments, one or more agents are associated with the bead. In some embodiments, the implementation of the pellet is biocompatible, that is, consists of a material that is suitable for biological use. In some embodiments, the implementation of the beads are non-toxic to cultured cells, such as cultured T cells. In some embodiments, the beads may be any particles capable of attaching agents in a manner that allows interaction between the agent and the cell.

In some embodiments, the stimulant includes one or more agents capable of activating and/or proliferating cells, such as T cells, that are associated with or otherwise connected to the bead, such as the surface of the bead. In specific embodiments, the implementation of the granule is a non-cellular particle. In specific embodiments, the implementation of the granule may include a colloidal particle, microsphere, nanoparticle, magnetic granule or the like. In some embodiments, the beads are agarose beads. In specific embodiments, the beads are Sepharose beads.

In specific embodiments, the implementation of the stimulant includes granules that are monodisperse. In particular embodiments, beads that are monodisperse have a size distribution with a standard deviation between diameters of less than 5%.

In some embodiments, the implementation of the granule contains one or more agents, for example, an agent that is connected, conjugated or associated (directly or indirectly) with the surface of the granule. In some embodiments, the agent provided herein may include, but is not limited to, RNA, DNA, proteins (e.g., enzymes), antigens, polyclonal antibodies, monoclonal antibodies, antibody fragments, carbohydrates, lipids, lectins, or any other a biological molecule with affinity for the desired target. In some embodiments, the desired target is a T cell receptor and/or a T cell receptor component. In specific embodiments, the desired target is CD3. In a particular embodiment, the desired target is a costimulatory molecule, eg CD28. One or more agents can be attached directly or indirectly to the bead in various ways. The connection may be covalent, non-covalent, electrostatic or hydrophobic, and may be carried out by various methods of attachment, including, for example, chemical methods, mechanical methods, or enzymatic methods. In some embodiments, a biological molecule (eg, a biotinylated anti-CD3 antibody) may be attached to the bead indirectly through another biological molecule (eg, an anti-biotin antibody) that is directly linked to the bead.

In some embodiments, one or more of the agents attached to the bead is an antibody. An antibody may include a polyclonal antibody, a monoclonal antibody (including full length antibodies that have an immunoglobulin Fc region), antibody compositions with polyepitope specificity, multispecific antibodies (e.g., bispecific antibodies), diabodies, and single chain molecules, as well as antibody fragments (e.g., Fab, F(ab') ₂ and Fv). In some embodiments, the stimulatory reagent is an antibody fragment (comprising an antigen-binding fragment), eg, a Fab, Fab'-SH, Fv, scFv, or (Fab') _{2 fragment} . It should be understood that constant regions of any isotype may be used for the antibodies provided herein, including IgG, IgM, IgA, IgD, and IgE constant regions, and that such constant regions may be derived from any human or animal (e.g., mouse) antibody. . In some embodiments, the agent is an antibody that binds and/or recognizes one or more T-cell receptor components. In specific embodiments, the agent is an anti-CD3 antibody. In specific embodiments, the agent is an antibody that binds and/or recognizes a co-receptor. In some embodiments, the stimulatory reagent is an anti-CD28 antibody.

In some embodiments, the granule has a diameter greater than about 0.001 microns, greater than about 0.01 microns, greater than about 0.1 microns, greater than about 1.0 microns, greater than about 10 microns, greater than about 50 microns, greater than about 100 microns, or greater than about 1000 µm; and no more than about 1500 microns. In some embodiments, the bead has a diameter of about 1.0 µm to about 500 µm, about 1.0 µm to about 150 µm, about 1.0 µm to about 30 µm, about 1.0 µm to about 10 µm , from about 1.0 µm to about 5.0 µm, from about 2.0 µm to about 5.0 µm, or from about 3.0 µm to about 5.0 µm. In some embodiments, the implementation of the granule has a diameter of from about 3 microns to about 5 microns. In some embodiments, the granule has a diameter of at least, or at least about, or about, 0.001 µm, 0.01 µm, 0.1 µm, 0.5 µm, 1.0 µm, 1.5 µm, 2, 0 µm, 2.5 µm, 3.0 µm, 3.5 µm, 4.0 µm, 4.5 µm, 5.0 µm, 5.5 µm, 6.0 µm, 6.5 µm, 7, 0 µm, 7.5 µm, 8.0 µm, 8.5 µm, 9.0 µm, 9.5 µm, 10 µm, 12 µm, 14 µm, 16 µm, 18 µm or 20 µm. In particular embodiments, the granule has a diameter of, or about, 4.5 microns. In specific embodiments, the granule has a diameter of, or about, 2.8 microns.

In some embodiments, the granules have a density greater than 0.001 g/cm ³ , greater than 0.01 g/cm ³ , greater than 0.05 g/cm ³ , greater than 0.1 g/cm ³ , greater than 0.5 g/cm ³ , more than 0.6 g/ ^cm3 , more than 0.7 g/ ^cm3 , more than 0.8 g/ ^cm3 , more than 0.9 g/ ^cm3 , more than 1 g/ ^cm3 , more than 1.1 g/cm ³ , more than 1.2 g/cm ³ , more than 1.3 g/cm ³ , more than 1.4 g/cm ³ , more than 1.5 g/cm ³ , more than 2 g/cm ³ , more than 3 g/cm ³ , more than 4 g/cm ³ or more than 5 g/cm ³ . In some embodiments, the granules have a density of from about 0.001 g/cm ³ to about 100 g/cm ³ , from about 0.01 g/cm ³ to about 50 g/cm ³ , from about 0.1 g/cm ³ to about 10 g/cm ³ from about 0.1 g/cm ³ to about 0.5 g/cm ³ from about 0.5 g/cm ³ to about 1 g/cm ³ from about 0.5 g/cm ³ to about 1.5 g/cm ³ , from about 1 g/cm ³ to about 1.5 g/cm ³ , from about 1 g/cm ³ to about 2 g/cm ³ , or from about 1 g/cm ³ up to about 5 g/cm ³ . In some embodiments, the granules have a density of about 0.5 g/cm ³ , about 0.6 g/cm ³ , about 0.7 g/cm ³ , about 0.8 g/cm ³ , about 0.9 g/cm 3 ³ , about 1.0 g/cm ³ , about 1.1 g/cm ³ , about 1.2 g/cm ³ , about 1.3 g/cm ³ , about 1.4 g/cm ³ , about 1, 5 g/cm ³ , about 1.6 g/cm ³ , about 1.7 g/cm ³ , about 1.8 g/cm ³ , about 1.9 g/cm ³ , or about 2.0 g/cm ³ . In specific embodiments, the implementation of the granules have a density of about 1.6 g/cm ³ . In specific embodiments, the implementation of the granules, or particles, have a density of about 1.5 g/cm ³ . In particular embodiments, the particles have a density of about 1.3 g/cm ³ .

In specific embodiments, the plurality of granules have a uniform density. In specific embodiments, uniform density means that the density standard deviation is less than 10%, less than 5%, or less than 1% of the average density of the granules.

In some embodiments, the granules have a surface area from about 0.001 m ² per gram of particles (m ² /g) to about 1,000 m ² /g, from about 0.010 m ² /g to about 100 m ² /g, from about 0, 1 m ² /g to about 10 m ² /g, from about 0.1 m ² /g to about 1 m ² /g, from about 1 m ² /g to about 10 m ² /g, from about 10 m ² /g to about 100 m ² /g, from about 0.5 m ² /g to about 20 m ² /g, from about 0.5 m ² /g to about 5 m ² /g or from about 1 m ² / g to about 4 m ² /g. In some embodiments, the particles, or granules, have a surface area of from about 1 m ² /g to about 4 m ² /g.

In some embodiments, the implementation of the granule contains at least one material on the surface, or near the surface, of the granule, which can be connected, associated or conjugated with the agent. In some embodiments, the surface of the bead is functionalized, that is, contains functional groups that are capable of forming a covalent bond with a binding molecule, such as a polynucleotide or polypeptide. In specific embodiments, the implementation of the granule has exposed on the surface of the carboxyl, amino, hydroxyl, tosyl, epoxy and/or chloromethyl groups. In specific embodiments, the beads have surface-exposed agarose and/or sepharose. In specific embodiments, the surface of the bead contains attached stimulatory reagents that can bind or attach binding molecules. In specific embodiments, the biological molecules are polypeptides. In some embodiments, the beads have surface-exposed Protein A, Protein G, or biotin.

In some embodiments, the implementation of the pellet is responsive to a magnetic field. In some embodiments, the implementation of the bead is a magnetic bead. In some embodiments, the magnetic bead is paramagnetic. In particular embodiments, the magnetic bead is superparamagnetic. In particular embodiments, the beads do not exhibit any magnetic properties when they are not subjected to a magnetic field.

In particular embodiments, the bead has a magnetic core, a paramagnetic core, or a superparamagnetic core. In some embodiments, the implementation of the magnetic core contains a metal. In some embodiments, the metal may be, but is not limited to, iron, nickel, copper, cobalt, gadolinium, manganese, tantalum, zinc, zirconium, or any combination thereof. In particular embodiments, the magnetic core comprises metal oxides (eg, iron oxides), ferrites (eg, manganese ferrites, cobalt ferrites, nickel ferrites, and so on), hematite, and metal alloys (eg, CoTaZn). In some embodiments, the magnetic core comprises one or more of ferrite, metal, metal alloy, iron oxide, or chromium dioxide. In some embodiments, the implementation of the magnetic core contains elemental iron or an iron compound. In some embodiments, the magnetic core comprises one or more of magnetite (Fe3O4), maghemite (γFe2O3), or greigite (Fe3S4). In some embodiments, the implementation of the inner core contains iron oxide (eg, Fe ₃ O ₄ ).

In specific embodiments, the implementation of the granule contains a magnetic, paramagnetic and/or superparamagnetic core, which has a shell, or coating, with a functionalized surface. In some embodiments, the implementation of the coating may contain a material that may include, but is not limited to, a polymer, polysaccharide, silicon dioxide, fatty acid, protein, carbohydrate, agarose, sepharose, or a combination thereof. In some embodiments, the polymer may be polyethylene glycol, poly(lactic-co-glycolic acid), polyglutaraldehyde, polyurethane, polystyrene, or polyvinyl alcohol. In specific embodiments, the implementation of the outer shell, or cover, contains polystyrene. In specific embodiments, the implementation of the surface of the outer coating is functionalized.

In some embodiments, the stimulant reagent includes a bead that has a metal oxide core (e.g., an iron oxide core) and a coating, the metal oxide core contains at least one polysaccharide (e.g., dextran), and the coating contains at least one polysaccharide (eg aminodextran), at least one polymer (eg polyurethane) and silicon dioxide. In some embodiments, the metal oxide core is a colloidal iron oxide core. In specific embodiments, one or more agents comprise an antibody, or an antigen-binding fragment thereof. In specific embodiments, one or more of the agents comprise an anti-CD3 antibody and an anti-CD28 antibody. In some embodiments, the stimulatory reagent includes an anti-CD3 antibody, an anti-CD28 antibody, and an anti-biotin antibody. In some embodiments, the implementation of the stimulating reagent includes an antibody against biotin. In some embodiments, the implementation of the granule has a diameter of from about 3 microns to about 10 microns. In some embodiments, the implementation of the granule has a diameter of from about 3 microns to about 5 microns. In specific embodiments, the implementation of the granule has a diameter of about 3.5 microns.

In some embodiments, the stimulating agent includes one or more agents that are attached to a bead containing a metal oxide core (eg, an iron oxide inner core) and a coating (eg, a protective coating), wherein the coating comprises polystyrene. In specific embodiments, the beads are monodisperse, superparamagnetic beads having a superparamagnetic iron core, eg, a core containing magnetite (Fe ₃ O ₄ ) and/or maghemite (γFe ₂ O ₃ ), and a polystyrene coating or sheath. In some embodiments, the granule is non-porous. In some embodiments, the beads have a functionalized surface to which one or more agents are attached. In specific embodiments, one or more agents are covalently attached to the surface of the beads. In some embodiments, one or more agents comprise an antibody, or an antigen-binding fragment thereof. In some embodiments, the one or more agents comprise an anti-CD3 antibody and an anti-CD28 antibody. In particular embodiments, the granules have a density of about 1.5 g/cm ³ and a surface area of from about 1 m ² /g to about 4 m ² /g. In particular embodiments, the beads are monodisperse, superparamagnetic beads that have a diameter of about 4.5 microns and a density of about 1.5 g/cm ³ . In some embodiments, the beads are monodisperse, superparamagnetic beads that have an average diameter of about 2.8 microns and a density of about 1.3 g/cm ³ .

When isolating different T-cell populations, the exposure time of the particles can be varied. For example, in one preferred embodiment, T cells are isolated by incubation with 3x28 beads such as ^DYNABEADS® M-450 or ^Dynabeads® CD3/CD28 CTS™ for a period of time sufficient to positively select the desired T cells. In one embodiment, the time period is about 30 minutes. In a further embodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In another preferred embodiment, the time period is 10-24 hours or more. In one preferred embodiment, the incubation period is 24 hours. When obtaining T cells from patients with cancer, the use of longer periods of incubation, for example, 24 hours, can increase the yield of cells.

When binding to a surface, the agents may be bound to the same surface (ie, in the "cis" position) or to separate surfaces (ie, in the "trans" position). Alternatively, one agent may be associated with the surface and the other agent may be in solution. In one embodiment, the agent providing the costimulatory signal is associated with the surface, and the agent providing the main activating signal is in solution or associated with the surface. In a preferred embodiment, the two agents are immobilized on the beads, either on the same bead, i.e. "cis", or on separate beads, i.e. "trans". As an example, the agent providing the main activating signal is an anti-CD3 antibody, and the agent providing the costimulatory signal is an anti-CD28 antibody; and both agents are co-immobilized on the same bead in equivalent molecular amounts. In one embodiment, a 1:1 ratio is used for each bead-bound antibody to expand CD4+ T cells and grow T cells. In particular aspects of the present invention, a ratio of anti-CD3:CD28 bead-bound antibodies is used such that an increase in T cell expansion is observed compared to that observed using a 1:1 ratio. In one particular embodiment, there is from, or from about 0.5-fold to about 3-fold increase in comparison with the reproduction observed when using the ratio of 1:1. In one embodiment, the ratio of anti-CD3:CD28 bead-bound antibodies is in the range of 100:1 to 1:100, including all integer values in between. In one aspect of the present invention, more anti-CD28 antibody is bound to the particles than anti-CD3 antibody, ie, the ratio of anti-CD3:CD28 is less than one. In specific embodiments, the ratio of anti-CD28 antibody to anti-CD3 antibody associated with beads is greater than 2:1. In one specific embodiment, a 1:200 ratio of anti-CD3:CD28 is used for bead-bound antibodies. In one specific embodiment, a 1:150 ratio of anti-CD3:CD28 is used for bead-bound antibodies. In one specific embodiment, a 1:100 ratio of anti-CD3:CD28 is used for bead-bound antibodies. In another embodiment, for bead-bound antibodies, an anti-CD3:CD28 ratio of 1:75 is used. In a further embodiment, a 1:50 ratio of anti-CD3:CD28 is used for bead-bound antibodies. In another embodiment, for bead-bound antibodies, an anti-CD3:CD28 ratio of 1:45 is used. In another embodiment, a 1:40 ratio of anti-CD3:CD28 is used for bead-bound antibodies. In another embodiment, for bead-bound antibodies, an anti-CD3:CD28 ratio of 1:35 is used. In another embodiment, a 1:30 ratio of anti-CD3:CD28 is used for bead-bound antibodies. In another embodiment, a 1:25 ratio of anti-CD3:CD28 is used for bead-bound antibodies. In another embodiment, a 1:20 ratio of anti-CD3:CD28 is used for bead-bound antibodies. In another embodiment, a 1:15 ratio of anti-CD3:CD28 is used for bead-bound antibodies. In one embodiment, a 1:10 ratio of anti-CD3:CD28 is used for bead-bound antibodies. In another embodiment, a 1:5 ratio of anti-CD3:CD28 is used for bead-bound antibodies. In another embodiment, a 1:4 ratio of anti-CD3:CD28 is used for bead-bound antibodies. In another embodiment, a 1:3 ratio of anti-CD3:CD28 is used for bead-bound antibodies. In another embodiment, a 3:1 ratio of anti-CD3:CD28 is used for bead-bound antibodies.

In some embodiments, the agent, such as an antibody, is added in soluble form to the culture or composition. In some embodiments, one agent is included in soluble form and the other agent is included bound to a solid support. In some aspects, when two or more agents are associated with a solid support(s), the two agents are associated with the same support or particle, for example, incubation is performed with anti-CD3/anti-CD28 beads, the beads contain antibodies that recognize CD3 and CD28. In other aspects, two or more agents are associated with separate supports, such as separate beads. For example, in some aspects, anti-CD3 and anti-CD28 beads are added to the culture separately.

In some embodiments, culture conditions include artificial antigen-presenting cells. For example, in some aspects, the surface is an artificial antigen-presenting cell loaded with an agent or agents capable of potentiating a signal through the TCR complex, such as anti-CD3 and/or anti-CD28 antibodies. Exemplary antigen presenting cells are genetically modified cells, such as myeloid cells (eg, K562 or U937), engineered to express Fc receptors such as medium affinity CD32 or high affinity CD64 Fc receptors. Such cells can be loaded with anti-CD3 and/or anti-CD28 antibodies recognized by Fc receptors and incubated with T cells to signal in cell culture. Exemplary artificial APCs and their ratios, as well as methods of using them under culture conditions, are described, for example, in Suhoski et al., Molecular Therapy (2007) 15 5, 981-988; Thomas et al., Clin Immunol (2002) 105(3): 259-72; Kim et al., Nature Biotechnology 22, 403-410 (2004). In some embodiments, culture conditions include antigen-presenting cells, such as PBMCs, loaded with antigen, such as an antigen recognized by the TCR complex or other receptor on cells to be transduced, such as T cells specific for a particular tumor antigen.

In some embodiments, preferential expansion or proliferation of naïve-like versus non-naïve-like T cells is achieved by using stimulatory conditions designed to induce a signal of a particular strength, e.g. to induce cell death in non-naïve-like T cells. In some aspects, a stronger signal induces cell death of non-naive T cells of the parent composition, while a weaker signal does not induce cell death of non-naive T cells of the parent composition and/or promotes survival of non-naive T cells, in compared to the stronger signal. Thus, in some embodiments, a stronger signal preferentially activates or induces cell death in non-naïve-like T cells.

In some aspects, preferential expansion or proliferation of naïve-like versus non-naïve-like T cells is achieved by a particular ratio of granules to cells. In some embodiments, any stimulatory condition that promotes the multiplication or survival of naive-like T cells more than non-naive-like T cells can be used. In some embodiments, stimulatory conditions include incubation in the presence of bead reagents providing a primary and/or auxiliary signal for T cell activation, e.g., anti-CD3/anti-CD28 bead reagents present in an amount that is favorable for proliferation and/ or survival of naïve-like cells, and/or which preferably induces activation-induced cell death (AICD) of non-naive-like cells. In some instances, such bead reagents are incubated with cells at a bead to cell ratio of 1:1 or higher, which in some aspects may induce more AICD and increase the percentage of surviving naïve-like cells, see e.g. Kalamasz et al., J Immunother. (2004) 27(5):405-418 and US Pat. Nos. 7,977,095 and 9,528,088. In one embodiment, the ratio is from, or about, 50:1 to about 5:1. In specific embodiments, the ratio is from, or about, 100:1 to about 1:1. In one embodiment, the ratio is at least about 45:1. In specific embodiments, the ratio is at least about 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1 or 1:1. In some aspects, a bead to cell ratio of less than 5:1 or less than 3:1 is used. In one particular embodiment, the ratio is about 3:1.

In one embodiment of the proposed methods, the bead:cell ratio can be adjusted to achieve the desired T cell phenotype. In one specific embodiment, bead:cell ratios can be varied to selectively expand or remove subsets of T cells. In one embodiment, the particular bead:cell ratio used selectively induces cell death of non-naive T cells or cells derived from non-naive T cells of the parent composition. In a further embodiment, the particular bead:cell ratio used results in selective expansion of naive-like T cells or cells derived from naive-like T cells. In some embodiments, any ratio may be used, as long as the desired expansion or removal of subsets of T cells occurs. Thus, the compositions and methods described herein can be used to expand specific populations of T cells, or to remove specific populations of T cells, which find use in a wide variety of immunotherapeutic situations described herein.

Stimulating conditions suitable for the method of incubation (eg, stimulation) of T cells are also provided. Conditions suitable for culturing T cells include an appropriate medium (e.g., OpTmizer™ (Gibco) or minimal maintenance medium, or RPMI 1640 medium, or X-vivo 15, (BioWhittaker)), which may contain factors necessary for proliferation and viability, including serum (eg, fetal bovine or human serum), serum substitutes or interleukin-2 (IL-2), insulin, or any other cell growth supplement. The medium may include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15 and X-Vivo 20, with added amino acids and vitamins, or without serum, or with the addition of an appropriate amount of serum (or plasma) or serum substitute, or with the addition of a certain set of hormones and / or cytokine(s) in an amount sufficient for the growth and reproduction of T-cells. Antibiotics, such as penicillin and streptomycin, are only included in experimental cultures, not in cell cultures to be administered to a subject. The target cells are maintained under conditions necessary for growth, eg at the appropriate temperature (eg 37° C.) and atmosphere (eg air plus 5% CO ₂ ).

In another embodiment, the time of exposure to stimulants, such as anti-CD3/anti-CD28 coated (ie, 3x28) beads, can be changed or adjusted to obtain the desired T cell phenotype. It may be desirable to obtain a larger population of helper T cells (TH), typically CD4+ as opposed to CD8+ cytotoxic or regulatory T cells, since expansion of TH cells may lead to improvement or restoration of overall immune reactivity. While many specific immune responses are mediated by CD8+ antigen-specific T cells that can directly lyse or kill target cells, most immune responses require the assistance of CD4+ T cells that express important immune regulatory molecules such as GM-CSF. , CD40L and IL-2 for example. If CD4-mediated assistance is preferred, a method such as described herein that maintains or increases the CD4:CD8 ratio may be particularly useful. Elevated numbers of CD4+ T cells can lead to an increase in the amount of CD40L cells expressed when administered to patients, potentially improving target cell visibility (improved APC function). Similar effects can be observed by increasing the number of infused cells expressing GM-CSF or IL-2, all of which are predominantly expressed by CD4+ T cells. Similarly, in some applications it may be desirable to use a population of regulatory T cells (e.g., Autoimmun Rev. 2002 August; 1(4):190-7; Curr Opin Immunol. 2002 December; 14(6):771-8), which can be obtained and propagated using the methods described in this document. Alternatively, in situations where CD4 help is less needed and higher numbers of CD8+ T cells are needed, the XCELLERATE™ approaches described herein can also be used, for example, by preselection for CD8+ cells prior to stimulation and/or culture. Such situations may occur when it is preferable to have elevated levels of IFN-γ or increased cytolysis of target cells. It is also possible to modify the duration and type of exposure to stimulants to proliferate T cells with the desired TCR repertoire, eg expressing the desired Vβ family genes.

In some embodiments, the other constituents of the stimulus conditions also include one or more of the specific environment, temperature, oxygen content, carbon dioxide content, time period, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors such as cytokines. , chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other means designed to activate cells.

In some embodiments, cells or compositions are evaluated and/or adjusted during the incubation steps. For example, the evaluation and/or adjustment can be made at any time after the start of incubation or culture, for example, during incubation. The evaluation may include making one or more measurements on a composition or container containing cells, for example, to evaluate cells for proliferation rate, survival rate, phenotype, for example, the expression of one or more surface or intracellular markers such as proteins or polynucleotides, and/ or to evaluate the composition, or capacity, in relation to temperature, component(s) of the environment, oxygen or carbon dioxide content, and/or the presence or absence, or amount, or relative amount, of one or more factors, agents, components and/or cellular types, including subtypes. The evaluation may also include evaluation for a measure or predictor of toxic outcome, for example using in vitro or ex vivo systems as described herein.

In some aspects, the assessment is carried out automatically, for example, using the device described herein, and/or scheduled in advance to determine at specific points in time during the incubation process. In some aspects, the result of the evaluation indicates that an adjustment is needed.

The adjustment may include adjusting any factor or parameter of the cell culture, such as temperature, duration (time) of each incubation, or its stage, (incubation duration), cell feeding, addition and/or removal of one or more components in the incubated composition, for example, medium or buffer, or components thereof, agents such as nutrients, amino acids, antibiotics, ions and/or stimulatory factors such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, or cells or cell types , or populations of cells. In some aspects, the removal or addition of various components, or other adjustment, is carried out automatically, for example, using the device or system described herein. In some embodiments, the system is programmed so that the adjustment starts automatically based on a certain result of the interim assessment. For example, in some cases, a system or device is programmed to perform one or more evaluations at specific times; in such cases, the system or device can be further programmed such that a particular result of such evaluation, such as a particular ratio of two cell types, triggers a particular adjustment, such as adding one or more cell types.

In some aspects, adjustment is made by adding or removing components in a manner that does not disturb the closed system containing cells and compositions, for example, using inlet and/or outlet valves designed to add or remove components while maintaining sterility, such as those found in one or more of the devices or systems described herein. Various adjustments to stimulus conditions to achieve responses and/or outcomes in specific cell types are described in, for example, US Pat. No. 8,617,884 and US Patent Application Publication No. US 20030235908 A1.

In some embodiments, cells are incubated for, or for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or for, or for approximately, 1, 2, 3, 4 or more weeks, either cumulatively or prior to engineering. In some examples, the incubation is for more than, or about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 days.

In some aspects, the incubation is carried out in accordance with the methods described in US patent No. 6040177 issued by Riddell et al., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood. 1:72-82 and/or Wang et al. (2012) J Immunother. 35(9):689-701.

In some embodiments, the stimulating conditions include the addition of nurse cells, such as non-dividing peripheral blood mononuclear cells (PBMC) (e.g., such that the resulting cell population contains at least about 5, 10, 20, or 40 or more, PBMC nurse cells per each T-lymphocyte in the original breeding population); and incubating the culture (eg, for a period of time sufficient to increase the number of T cells). In some aspects, non-dividing nurse cells may be gamma irradiated PBMC nurse cells. In some embodiments, PBMCs are irradiated with gamma radiation in the range of about 3000-3600 rad to prevent cell division. In some aspects, feeder cells are added to the culture medium prior to the addition of T cell populations.

In some embodiments, the stimulating conditions include a temperature suitable for the growth of human T lymphocytes, such as at least about 25 degrees Celsius, typically at least about 30 degrees, and typically exactly or about 37 degrees Celsius. Optionally, the incubation may further include the addition of non-dividing EBV-transformed lymphoblastoid cells (LCL) as nurse cells. LCLs can be exposed to gamma radiation in the range of about 6000-10000 rads. In some aspects, LCL feeder cells are provided in any suitable amount such that the ratio of LCL feeder cells to parental T lymphocytes is at least about 10:1.

In embodiments, antigen-specific T cells, such as antigen-specific CD4+ and/or CD8+ T cells, are obtained by stimulating naive or antigen-specific T lymphocytes with antigen. For example, antigen-specific T cell lines or clones can be generated for cytomegalovirus antigens by obtaining T cells from infected subjects and stimulating the cells in vitro with the same antigen.

Incubation and/or engineering. can be carried out in a culture vessel such as a block, chamber, well, column, tube, tube set, valve, vial, culture dish, bag or other container for culture or cell culture.

Also provided are culture initiating compositions used in the method, such as compositions containing T cells, e.g., human primary T cells, stimulating conditions, e.g., various agents at concentrations/ratios designed to preferentially activate or expand non-naive T cells. .

In some embodiments, when cells are being engineered, for example to introduce a genetically engineered antigen receptor, incubation in the presence of one or more stimulants is continued during the engineering phase.

Bead to cell ratios ranging from 1:500 to 500:1, including any integer values in between, can be used to stimulate T cells or other target cells. In some cases, the ratio of beads to cells may depend on the size of the particles compared to the target cell. For example, small granules may bind only a few cells, while larger granules may bind a large number of cells. In specific embodiments, the ratio of cells to particles is in the range of 1:100 to 100:1, including any intermediate integer values, and in further embodiments, the ratio is from 1:50 to 50:1, including any intermediate integer values. In another embodiment, a ratio of cells to particles ranging from 1:9 to 9:1, including any integer values in between, can also be used to stimulate T cells. The ratio of anti-CD3 and anti-CD28 bound particles to T cells that results in T cell stimulation may vary as described herein, however specific preferred values include at least 1:150, 1:125, 1:100, 1:75, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1: 4, 1:3, 1:2.5, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9: 1, 10:1 and 15:1, with one preferred ratio being at least 1:1 particles and T cells. In one particular embodiment, the preferred ratio of granules to cells is 3:1. In some cases, the ratio of granules and cells is 1:3.

In the following embodiments, the implementation of the ratio of particles and cells may vary depending on the day of stimulation. For example, in one embodiment, the ratio of particles to cells is from 1:1 to 10:1 on the first day, and then additional particles are added to the cells every day or every other day for up to 10 days, with a final ratio ranging from 1:1: 1 to 1:10 (based on the number of cells on the day of addition). In another embodiment, the ratio of particles to cells is at least about 1:2.5 on the first day, and additional particles are added to the cells on day 5 in a ratio of about 1:10, 1:25, 1:50, or 1: 100, on day 7 with a ratio of 1:10, 1:25, 1:50, or 1:100, and on day 9 with a ratio of 1:10, 1:25, 1:50, or 1:100. In one specific embodiment, the particle to cell ratio is 1:1 on the first day of stimulation and adjusted to 1:5 on the third and fifth days of stimulation. In another embodiment, the particles are added daily or every other day, with a final ratio of 1:1 on the first day and 1:5 on the third and fifth days of stimulation. In another embodiment, the particle to cell ratio is 2:1 on the first day of stimulation and is adjusted to 1:10 on the third and fifth days of stimulation. In another embodiment, the particles are added daily or every other day, with a final ratio of 1:1 on the first day and 1:10 on the third and fifth days of stimulation. In some aspects, various other ratios may be suitable for use in the present invention. In particular, the ratios will vary with particle size as well as cell size and type.

One aspect of the present invention relates to the fact that the use of different ratios of beads (eg, anti-CD3/anti-CD28 beads) and cells can lead to different expansion of antigen-specific T cells. In particular, bead to cell ratios can be varied to selectively expand or remove antigen-specific or antigen-exposed T cells (such as memory cells or effector or activated cells) as opposed to other cell types such as naive or naive-like , T cells. In one embodiment, the specific bead to cell ratio used results in the selective removal of antigen-specific T cells. In particular, bead to cell ratios such as bead to cell ratios of or greater than 1:1 and/or higher bead to cell ratios such as at least or about 3:1, 5:1 , 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, and higher, can lead to the removal of antigen-specific T cells . Without wishing to be bound by a particular theory, it is believed that antigen-specific T cells are sensitized for further stimulation. Thus, in some embodiments, the strength of an activating signal delivered to a T cell by a reagent or composition may influence the expansion and/or death of one or more specific subsets of T cells. In some aspects, selective expansion of memory T cells (antigen-specific T cells) can occur with signals through TCRs and/or co-receptors that are weak compared to others; while in some embodiments, selective removal of memory T cells and/or other antigen-exposed T cells, while retaining naïve cells, may occur after incubation with signaling reagents that are relatively stronger. In some aspects, the number of CD3/TCR (and CD28) receptors associated with ligands, among other factors, may determine or contribute to the determination of signal strength. Thus, stimulation using high bead-to-cell ratios in some contexts can provide a high concentration of stimulatory antibody (i.e., a “strong” signal), leading to overstimulation of antigen-specific T cells, causing their death, either due to apoptosis, or through other mechanisms. Thus, in this regard, the granule compositions described herein may, in some contexts and/or in the case of specific compositions, act as pro-apoptotic compositions. In some aspects, the signal should not be too strong so as not to lead to the death of similar naive T cells as well, for example, death induced by activation. In some embodiments, the proportion of stimulatory bead reagent, eg, anti-CD3/anti-CD28 bead reagent, is less than 10:1.

In addition, in specific embodiments, such a reagent or composition used as proapoptotic conditions, for example, in relation to specific cell types (for example, a surface with an agent attached to it that stimulates a molecule on the cell surface, for example, the composition of the granules described herein document) are used to expand the remaining cell populations for use in the various immunotherapeutic situations described herein. In a further embodiment, the particular bead to cell ratio used results in the selective expansion of naive-like T cells. The specific ratio can be adjusted to achieve the desired expansion of specific T cells or removal of specific T cells. Thus, the compositions and methods described herein can be used to expand specific populations of T cells in the population of the original composition, or to remove specific populations of T cells in the population of the original composition, for use in a wide variety of immunotherapeutic situations described in this document.

In addition, in specific embodiments, the same reagent or substance or composition used as a proapoptotic composition, for example, in relation to specific cell types (for example, a surface with an agent attached to it that stimulates a molecule on the cell surface, for example, compositions beads described herein) are used to expand the remaining cell populations for use in the various immunotherapeutic situations described herein. The use of lower bead-to-cell ratios provides a stimulatory signal to antigen-specific T cells that does not over-stimulate them but induces rapid proliferation of these cells. In a further embodiment, the particular bead to cell ratio used results in the selective expansion of antigen-specific T cells. In some aspects, any ratio may be used as long as the desired expansion or removal of cells occurs. Thus, the compositions and methods described herein can be used to expand specific T cell populations, or to remove specific T cell populations, for use in a wide variety of immunotherapeutic situations described herein.

Using certain methodologies, it may be beneficial to maintain long-term stimulation of the T cell population after initial activation and stimulation by separating T cells from the stimulus over a period of 2 to about 14 days. The proliferation rate of T cells is monitored periodically (eg, daily), for example, by determining the size or volume of T cells, for example, using a Coulter counter. In this respect, a resting T cell has an average diameter of approximately 6.8 microns, and after initial activation and stimulation in the presence of a stimulatory ligand, the average T cell diameter increases to over 12 microns by day 4 and then begins to decrease around day 6. When the average T cell diameter is reduced to about 8 microns, the T cells can be reactivated and restimulated to induce further T cell proliferation. Alternatively, the rate of T cell proliferation and the time for T cell restimulation can be determined by analyzing the presence on the cell surface of molecules such as CD154, CD54, CD25, CD137, CD134 that are induced on activated T cells.

France)), несколько раз для получения популяции CD4+ или CD8+ клеток, превосходящей по размеру от, или от примерно, 10 до примерно 1000 раз оригинальную T-клеточную популяцию. Например, в одном варианте осуществления настоящего изобретения T-клетки стимулируют, как описано, 2-3 раза. В следующих вариантах осуществления T-клетки стимулируют, как описано, 4 или 5 раз. С использованием настоящей методологии возможно получать T-клетки с повышенной поликлональностью, количество которых от, или от примерно, 100 до примерно 100000 раз превышает количество до стимуляции. Более того, T-клетки, размноженные способом по настоящему изобретению, секретируют на значительных уровнях цитокины (например, IL-2, IFN-γ, IL-4, GM-CSF и TNF-α) в супернатанты клеточных культур. Например, в сравнении с стимуляцией IL-2, CD4+ T-клетки, размноженные с использованием совместной стимуляции анти-CD3 и анти-CD28 антителами, секретируют большие количества GM-CSF и TNF-α в среду для культивирования. Эти цитокины можно очищать из супернатантов клеточных культур, или супернатанты можно использовать непосредственно для поддержания клеток в культуре. Аналогично, T-клетки, размноженные способом по настоящему изобретению, совместно с супернатантом клеточных культур и цитокинами можно вводить субъекту для поддержания роста клеток in vivo.To induce long-term stimulation of a population of CD4+ and/or CD8+ T cells, it may be necessary to reactivate and restimulate the T cells with a stimulant such as an anti-CD3 antibody and an anti-CD28 antibody (eg, B-T3, XR-CD28 (Diaclone ,

France) several times to obtain a population of CD4+ or CD8+ cells larger than, or about 10 to about 1000 times the original T cell population. For example, in one embodiment of the present invention, T cells are stimulated as described 2-3 times. In further embodiments, T cells are stimulated as described 4 or 5 times. Using the present methodology, it is possible to obtain T-cells with increased polyclonality, the number of which is from, or from about 100 to about 100,000 times the amount before stimulation. Moreover, T cells expanded by the method of the present invention secrete significant levels of cytokines (eg, IL-2, IFN-γ, IL-4, GM-CSF and TNF-α) into cell culture supernatants. For example, compared to IL-2 stimulation, CD4+ T cells expanded using co-stimulation with anti-CD3 and anti-CD28 antibodies secrete large amounts of GM-CSF and TNF-α into the culture medium. These cytokines can be purified from cell culture supernatants, or the supernatants can be used directly to maintain the cells in culture. Similarly, T cells expanded by the method of the present invention, together with cell culture supernatant and cytokines, can be administered to a subject to support cell growth in vivo .

In one embodiment, T cells are stimulated with, for example, anti-CD3 and anti-CD28 antibodies co-immobilized on beads (3x28 beads) for a period of time sufficient to return the cells to a quiescent state (low, or without, proliferation) (approximately 8-14 days after initial stimulation). The stimulus signal is then removed from the cells, the cells are washed and re-infused into the patient. Cells at the end of the stimulation phase become "super-inducible" using the methods of the present invention, as evidenced by their ability to respond to antigens, as well as the ability of these cells to display a phenotype similar to that of memory cells, as demonstrated in the examples. Accordingly, upon re-stimulation, either exogenously or with in vivo antigen after infusion, activated T cells exhibit a strong response characterized by unique phenotypic properties such as persistent CD154 expression, increased cytokine production, and so on.

In further embodiments of the present invention, cells, such as T cells, are combined with coated or agent-conjugated beads, then the cells are separated, after which the cells are cultured. In an alternative embodiment, the coated or agent-conjugated beads and cells are not separated prior to culture but cultured together. In a further embodiment, the beads and cells are first concentrated by force, resulting in ligation of the cell surface markers, thereby inducing cell stimulation and/or polarization of the activating signal.

As an example, when T cells are the target cell population, cell surface molecules can be ligated by bringing anti-CD3 and anti-CD28 antibody bound paramagnetic beads (3x28 beads) into contact with prepared T cells. In one embodiment, cells (eg, 10 ⁴ -10 ⁹ T cells) and beads (eg, ^DYNABEADS® M-450 CD3/CD28 T paramagnetic beads in a 1:1 ratio) are pooled in a buffer, preferably PBS (free of divalent cations). such as calcium and magnesium). In some aspects, any concentration of cells can be used. For example, target cells may be very rare in a sample, representing only 0.01% of the sample, or the entire sample (ie, 100%) may be target cells of interest. Accordingly, any number of cells is provided in the context of the present invention. In particular embodiments, it may be desirable to greatly reduce the volume in which particles and cells are mixed together (ie, increase the concentration of cells) to maximize cell-particle contact. For example, in one embodiment, a concentration of about 2 billion cells/mL is used. In another embodiment, a concentration of more than 100 million cells/ml is used. In a further embodiment, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/mL is used. In yet another embodiment, a cell concentration of 75, 80, 85, 90, 95, or 100 million cells/mL is used. In further embodiments, concentrations of 125 or 150 million cells/mL can be used. The use of high concentrations can lead to increased cell yield, cell activation and cell proliferation. In addition, the use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells. Such cell populations may be of therapeutic value and it may be desirable to obtain them. For example, the use of a high concentration of cells allows more efficient selection of CD8+ T cells, which usually have a weaker expression of CD28.

In a related embodiment, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T-cells and particles, the interaction between particles and cells is minimized. This allows the selection of cells that express in large quantities the desired antigens that bind to the particles. For example, CD4+ T cells express CD28 at a higher level and are more efficiently captured and stimulated than CD8+ T cells at low concentrations. In one embodiment, the cell concentration used is about 5×10 ⁶ /ml. In other embodiments, the concentration used may be from, or about, 1×10 ⁵ /ml to about 1×10 ⁶ /ml, including any value in between.

The buffer in which the cells are suspended may be any buffer suitable for a particular cell type. When using certain types of cells, the buffer may contain other components, for example 1-5% serum, necessary to maintain the integrity of the cells during the process. In another embodiment, the cells and beads can be combined in a cell culture medium. Cells and beads can be mixed, for example, by rotation, agitation or the use of any means for mixing, for a period of time from one minute to several hours. The granules and cells are then concentrated in the container by applying force, for example by placing the container in a magnetic field. The medium and unbound cells are removed, and the cells bound to the beads or other surface are washed, for example by pumping through a peristaltic pump, and then resuspended in a medium suitable for cell culture.

In one embodiment of the present invention, the mixture can be cultured for a period of time from 30 minutes to several hours (about 3 hours) or up to about 14 days, or for any intervening whole number of hours or minutes. In another embodiment, the mixture can be cultured for 21 days. In one embodiment, the beads and T cells are co-cultured for about eight days. In another embodiment, the beads and T cells are co-cultured for 2-3 days. As described above, it may be desirable to use multiple cycles of stimulation so that the T cell culture time may be 60 days or more. Conditions suitable for culturing T cells include an appropriate medium (e.g., minimal maintenance medium or RPMI 1640 or X-vivo 15, (BioWhittaker)), which may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum) or interleukin-2 (IL-2), insulin, or any other cell growth supplement. The medium may include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15 and X-Vivo 20, with added amino acids and vitamins, either without serum or supplemented with an appropriate amount of serum (or plasma), or with the addition of a certain set of hormones and/or cytokine(s) in an amount sufficient for the growth and reproduction of T-cells. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures, not in cell cultures to be administered to a subject. The target cells are maintained under conditions necessary for growth, eg at the appropriate temperature (eg 37° C.) and atmosphere (eg air plus 5% CO ₂ ).

In one embodiment of the present invention, the bead:cell ratio can be adjusted to achieve the desired T cell phenotype. In one specific embodiment, the bead:cell ratio can be varied to selectively expand or remove antigen-specific (memory) T cells. In one embodiment, the particular bead:cell ratio used results in the selective removal of antigen-specific T cells. In a further embodiment, the particular bead:cell ratio used results in the selective expansion of antigen-specific T cells. In some aspects, any ratio may be used, as long as the desired expansion or removal of antigen-specific T cells occurs. Thus, the compositions and methods described herein can be used to expand specific populations of T cells, or to remove specific populations of T cells, which find use in a wide variety of immunotherapeutic situations described herein.

In another embodiment, the time of exposure to stimulants, such as anti-CD3/anti-CD28 antibody coated (ie, 3×28) beads, can be altered or adjusted to obtain the desired T cell phenotype. Alternatively, the desired population of T cells can be selected using any of the selection methods prior to stimulation. It may be desirable to obtain a larger population of helper T cells (TH), typically CD4+ as opposed to CD8+ cytotoxic or regulatory T cells, since expansion of TH cells may lead to improvement or restoration of overall immune reactivity. While many specific immune responses are mediated by CD8+ antigen-specific T cells that can directly lyse or kill target cells, most immune responses require the assistance of CD4+ T cells that express important immune regulatory molecules such as GM-CSF. , CD40L and IL-2 for example. If CD4-mediated assistance is preferred, a method such as described herein that maintains or increases the CD4:CD8 ratio may be particularly useful. Elevated numbers of CD4+ T cells can lead to an increase in the amount of CD40L cells expressed when administered to patients, potentially improving target cell visibility (improved APC function). Similar effects can be observed by increasing the number of infused cells expressing GM-CSF or IL-2, all of which are predominantly expressed by CD4+ T cells. Similarly, in some applications it may be desirable to use a population of regulatory T cells (e.g., Autoimmun Rev. 2002 August; 1(4):190-7; Curr Opin Immunol. 2002 December; 14(6):771-8), which can be obtained and propagated using the methods described in this document. Alternatively, in situations where CD4 help is less needed and higher numbers of CD8+ T cells are needed, the XCELLERATE™ approaches described herein can also be used, for example, by preselection for CD8+ cells prior to stimulation and/or culture. Such situations may occur when it is preferable to have elevated levels of IFN-γ or increased cytolysis of target cells. It is also possible to modify the duration and type of exposure to stimulants to proliferate T cells with the desired TCR repertoire, eg expressing the desired Vβ family genes.

When isolating different T-cell populations, the exposure time of the particles can be varied. For example, in one preferred embodiment, T cells are isolated by incubation with 3×28 beads such as ^DYNABEADS® M-450 for a period of time sufficient to positively select the desired T cells. In one embodiment, the time period is about 30 minutes. In a further embodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In another preferred embodiment, the time period is 10-24 hours or more. In one preferred embodiment, the incubation period is 24 hours. When obtaining T cells from patients with cancer, the use of longer periods of incubation, for example, 24 hours, can increase the yield of cells.

In specific embodiments, the total periods of stimulation and/or reproduction can be from 2 to 15 days, from 2 to 12 days, from 2 to 10 days, from 2 to 8 days, from 2 to 6 days, from 2 to 4 days, from 4 to 12 days 4 to 10 days 4 to 8 days 4 to 6 days 6 to 12 days 6 to 10 days 6 to 8 days 8 to 12 days 8 to 10 days or 10 to 12 days, all ranges are inclusive. In some embodiments, cells are incubated and/or incubated with a stimulating reagent (e.g., the particle described herein) for at least, or at least about, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days or more than 14 days. If T cell stimulation is given for shorter periods of time, the T cell population may not increase in number, may increase slightly in number, may decrease in number, or may remain the same, but the population will contain more viable and healthy activated T cells that can continue to proliferate in vivo and/or more faithfully reproduce the natural pool of effector T cells. For example, if T cell stimulation is performed for shorter periods of time, the T cell population may contain a higher percentage and/or proportion of naive or naive-like T cells, or engineered T cells, compared to a parallel process in which stimulation of T cells is carried out for a longer period of time. In some embodiments, a shorter period of time can be characterized as a total incubation time, in some embodiments, a total incubation time with a stimulant that is less than or less than about 6 days, less than or less than about 5 days, less than or less about 4 days, less or less than about 3 days or less, or less than about 2 days. Because the availability of T cell assistance is often the limiting factor in antibody production in response to protein antigens, the ability to selectively expand or selectively infuse CD4+ rich T cell populations into a subject is very valuable. The added benefit of such enriched populations is clear because activated helper T cells that recognize antigens presented by B lymphocytes provide two types of stimuli, physical contact and cytokine production, leading to B cell proliferation and differentiation.

In some cases, the stimulating conditions do not include cell culture components or agents that are added to maintain specific subsets of T cells, eg, non-naïve T cells. Thus, in some cases, removal of components or agents from culture under stimulating conditions may promote the elimination of non-naïve-like T cells. In some embodiments, the stimulatory conditions do not include agents such as N-acetylcysteine, which can be used to modulate and/or fine-tune the intensity of TCR/CD3 signaling. In some aspects, such agents are removed or reduced in amounts/proportions to increase the strength of the activating signal. In some aspects, stimulating conditions are, or are also used, by eliminating or reducing the concentration of culture reagents that are known or likely to reduce AICD and/or that promote the survival of older cells, such as non-naive cells. In some cases, stimulatory conditions do not include N-acetylcysteine, or include N-acetylcysteine in a lower amount or concentration. In some cases, the stimulatory conditions do not include recombinant IL-7 and/or recombinant IL-15, or include recombinant IL-7 or IL-15 at a lower amount or concentration. In some embodiments, culture supplements may be included that further facilitate or promote the removal of non-naive cells (eg, CD45RO-associated toxin).

C. Stimulated Composition

Also proposed is a stimulated composition obtained by the described methods of incubation (eg, stimulation). In some embodiments, cells of the stimulated composition are further engineered, for example, to introduce a genetically engineered antigen receptor, after incubation of the original composition. In some embodiments, the method further comprises incubation in the presence of one or more stimulants during the engineering phase.

Cells of a stimulated composition that have not undergone apoptosis using the incubation (eg, stimulation) methods described herein may have increased polyclonality of said remaining T cell population, as measured based on the breadth of the population's response to a particular antigen. Recovery or increase in polyclonality can be determined based on the breadth of response to a particular antigen of interest, for example, by measuring the number of different epitopes recognized by antigen-specific cells. This can be done using standard methods for obtaining and cloning antigen-specific T cells in vitro .

In some embodiments of the methods provided herein, culture conditions preferably induce multiplication, proliferation, and/or survival of non-naïve-like T cells as compared to naïve-like T cells. Preferential expansion, proliferation, and/or survival of a first cell type or population over a second cell type or population means that the relative expansion, proliferation and/or survival is greater for the first cell type or population than for the second type. This situation may include a scenario in which the percentage of the first type or population of cells that have multiplied, proliferated and/or survived is greater, and/or that the degree (e.g., overall or average for cells of the population or type) to which the cells multiplied, proliferated and/or survived is greater in the case of cells of the first population or type than in the case of the second.

In some embodiments, preferred multiplication, proliferation and/or survival is expressed by comparing the percentage of naive-like cells in the original composition with the percentage of engineered cells in the stimulated composition that originated from naive-like cells in the original original composition. In some examples, the percentage of engineered cells in the stimulated composition that are derived from naive-like cells in the original original composition is generally greater under the stimulation conditions described herein. For example, in one embodiment, the percentage of cells in the stimulated composition that are descended from naive-like cells in the original parent composition is greater than the percentage of naive cells in the culture initiator composition. In one aspect, the starting composition (or the T cells therein) contains, or contains, about 70% naive-like T cells and 30% non-naive T cells, with more than 50%, e.g., at least 70%, engineered cells in the resulting stimulated composition are derived from naive-like cells in the original composition. In some instances, the use of other conditions, such as stimulation with an agent(s) that induces a strong signal through the TCR complex, would result in less than 50%, such as being, or about 5-10% of the cells in the stimulated composition occurring from not similar to naive cells. In some embodiments, the stimulating conditions induce a strong signal that causes activation-induced cell death of non-naïve T cells of the parent composition.

In some embodiments, the percentage of naive-like T cells, or cells derived from naive-like T cells of the parent composition, in the stimulated composition is increased by more than, or more than about, 1.5-fold, 2-fold, 3-fold, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 50 times, 100 times, in comparison with the original composition. In some cases, the ratio of cells derived from naive-like T cells of the original composition to cells derived from non-naive-like T cells of the original composition, or the ratio of naive-like T cells to non-naive-like T cells in the stimulated composition is increased by more than , or more than about 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 50 times, 100 times in comparison with the ratio of similar naive T cells and non-naive-like T cells in the original composition. In some embodiments, the stimulated composition contains more than 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of cells derived from similar naive T -cells of the original composition.

In some embodiments, using the T cell stimulation method described herein, of the cells in the initial composition, a greater percentage of naive-like T cells, compared to non-naïve-like T cells, are induced to proliferate and/or become activated. In some aspects, the stimulated composition obtained by the stimulation methods described herein contains less than 10% of cells derived from non-naive T cells. In some cases, the stimulated composition contains less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of cells derived from non-naive T -cells. In some embodiments, a greater percentage of T cells that were similar to naive in the original composition compared to the percentage of T cells that were not similar to naive in the original composition divide on day 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 after the start of said incubation. In some cases, stimulating conditions induce cell death. In specific examples, the stimulatory conditions of the method induce the activation of non-naïve-like T cells, thereby causing activation-induced cell death (AICD).

In some embodiments, the method includes stimulatory conditions capable of inducing proliferation of a greater percentage of cells in a population of naïve-like T cells versus non-naïve T-cell-like, on day 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 after the start of incubation under these conditions.

In some embodiments, the stimulus conditions result in a stimulated composition, wherein the stimulus conditions preferably induce the expansion of naive-like T cells to produce a target number of T cells in the stimulated composition derived from naive-like T cells of the original composition. In some embodiments, the stimulated composition is further adjusted to achieve a preferred ratio of CD4:CD8 T cells. In some cases, specific cell populations can be removed from the stimulated composition to achieve a preferred ratio of CD4:CD8 cells.

In some embodiments, stimulatory conditions, such as those under which the parent compositions are incubated, preferably induce multiplication, proliferation, and/or survival of naive-like T cells or a subset thereof, as compared to non-naive-like T cells or a subset thereof. In some aspects, the stimulatory conditions strongly activate non-naive T cells of the parent composition, thereby activating cell death, especially non-naive T cells of the parent composition. In some aspects, this advantageously promotes the survival of naïve-like T cells. In some aspects, such stimulating conditions result in a reduction in the level of toxicity and/or toxicity-related outcome(s) or symptom(s) following administration to subjects of cells and compositions obtained by these methods.

Whether a particular response has been induced, or preferably induced, in one population versus another population can be determined in a variety of ways. For example, whether a cell has been induced to enter the cell cycle can be determined by flow cytometry methods, including those that use dyes and other agents such as CFSE and other chelating agents to evaluate cell division, followed by flow cytometric evaluation, evaluation incorporation of tritiated (H3) thymidine and the like, and/or cell counting. Comparisons can be made by evaluating different pure test populations, comparing naive and non-naive T cell populations separately under specific conditions. Activation can be determined, for example, based on secretion of various cytokines and/or upregulation or expression of various activation markers, including CD25, CD69, and/or cell size (eg, based on direct light scatter as measured by flow cytometry). Survival and/or apoptosis can be assessed by various methods, including flow cytometric methods, incorporation of various stains, including propidium iodide, and staining, for example staining with annexin V and the like.

In some embodiments, the percentage of naive-like T cells in the original composition is less than the percentage of T cells in the stimulated composition derived from naive-like T cells in the original composition. In some embodiments, the methods provided herein result in a higher percentage of nucleic acid-introduced cells that are, or are derived from, proliferation of naïve-like T cells in the parent composition compared to non-naïve-like T cells. in the original composition. Various options for adjusting stimulus conditions to promote responses and/or outcomes for specific cell types are disclosed, for example, in US Pat. No. 8,617,884 and in US Patent Application Publication No. US 20030235908 A1.

In some embodiments, the implementation of the stimulated composition contains cells that express, or are derived from cells that express, specific markers like naive T cells. For example, the stimulated composition obtained by the proposed methods comes from cell populations that have on their surface a T-cell activation marker selected from the group consisting of CD27, CD28, CD45RA, and CCR7. In some cases, the stimulated composition obtained by the proposed methods comes from cell populations that do not have a T-cell activation marker on their surface, such as CD62L. In some aspects, the cells of the stimulated composition are, or are derived from, cells that do not have CD56 and/or CD45RO on their surface. In some specific embodiments, the implementation of the stimulated composition obtained by the proposed methods, comes from populations of cells that are CD27+, CD45RA+, CD45RO- and CCR7+ cells. In some instances, the cells of the stimulated composition are or are derived from cells that lack intracellular expression of a cytokine such as IL-2, IFN-γ, IL-4 and/or IL-10. In some additional examples, the cells of the stimulated composition are or are derived from cells that lack the expression of CD25 and/or perforin markers. In some instances, the cells of the stimulated composition are or are derived from cells that are CD95 ^lo cells.

In some cases, the stimulated composition contains cells that are derived from at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of T cells, having a T-cell activation marker such as CD27, CD28, CD45RA and CCR7 on their surface and not having CD62L on their surface. In some embodiments, the stimulated composition contains cells that are derived from at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of T cells. that do not have CD56 and/or CD45RO on their surface. In some aspects, the stimulated composition contains cells that are derived from at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of T cells, not having CD45RO on the surface and having CD27, CD45RA and CCR7 on the cell surface. In some examples, the stimulated composition contains cells that are derived from at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of T cells, lacking intracellular cytokine expression such as IL-2, IFN-γ, IL-4, IL-10. In some aspects, the stimulated composition contains cells that are derived from at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of T cells, devoid of expression of CD25 and/or perforin markers. In some cases, the stimulated composition contains cells that are derived from at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of T cells, representing CD95 ^lo cells.

In specific embodiments, the implementation of the stimulated composition is more polyclonal, or multiclonal, in comparison with the original composition. In some embodiments, the implementation of the stimulated composition is more diverse in comparison with the original composition. In some embodiments, this increase in polyclonality is a shift from mono- to oligoclonality, or to polyclonality, of the T-cell population, as determined based on the Vβ, Vα, Vγ, or Vδ spectrotype profile of at least one gene of the Vβ, Vα, Vγ family, or Vd. Stimulation and activation of the remaining cells that survived, proliferated, proliferated using the proposed methods, can lead to an increase in the polyclonality of these remaining T cells of the stimulated composition, as measured based on the breadth of the response of the population to a particular antigen. Recovery or increase in polyclonality of the stimulated composition can be determined based on the breadth of response to a particular antigen of interest, for example, by measuring the number of different epitopes recognized by antigen-specific cells. This can be done using standard methods for obtaining and cloning antigen-specific T cells in vitro .

II. Methods of genetic engineering of cells

In some embodiments, the method further comprises introducing a genetically engineered recombinant receptor, e.g., a chimeric receptor such as a chimeric antigen receptor (CAR), into a stimulated T cell composition to provide a manufactured composition comprising T cells expressing the genetically engineered recombinant receptor. In some cases, the incubation of the original composition under stimulating conditions is carried out before, during and/or after the introduction of a nucleic acid encoding a genetically engineered recombinant receptor. In some examples, the introduction is performed by transduction. In some embodiments, the proposed methods result in a stimulated cell composition that can be uniformly transduced. In some embodiments, the nucleic acid is a viral vector. In some cases, the viral vector is a retroviral vector. In some examples, the viral vector is a lentiviral vector or a gamma retroviral vector. In some embodiments, the implementation of the method, including the introduction of the nucleic acid, is used in vitro or ex vivo .

Thus, the methods provided herein include one or more steps in preparing cells for genetic engineering. In specific embodiments, one or more of the steps include isolating cells from a biological sample, stimulating the original cell composition, and preparing the cell composition for genetic engineering. Also provided are populations of such cells, compositions containing such cells and/or enriched in such cells, for example, in which cells expressing a recombinant receptor, for example, a chimeric receptor, are at least 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or more, percent of the total number of cells in the composition, or cells of a certain type, such as T cells or CD8+ or CD4+ cells. The compositions include pharmaceutical compositions and preparations for administration, for example, for adoptive cell therapy. Also provided are methods for engineering, obtaining, or creating such cells, methods for administering cells and compositions to subjects, such as patients, and methods for detecting, selecting, isolating, or separating such cells. Thus, genetically engineered cells expressing recombinant receptors, such as CAR, are provided.

In some embodiments, cells contain one or more genetically engineered nucleic acids and, as a result, express recombinant or genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous, i.e., not normally found in a cell or cell sample, e.g., derived from another organism or cell, e.g., not normally found in a cell being engineered and/or in an organism from which the cell was obtained. In some embodiments, the nucleic acids are non-natural, that is, the nucleic acids are not naturally occurring, including nucleic acids containing chimeric combinations of nucleic acids encoding different domains from many different cell types.

A. Genetic engineering

1. Recombinant antigen receptors

In some embodiments, engineered cells, such as T cells, are provided that express CAR with specificity for a particular antigen (or marker or ligand), such as an antigen expressed on the surface of a particular cell type. In some embodiments, the antigen is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen is selectively expressed or overexpressed on cells associated with a disease or condition, such as tumor or pathogenic cells, compared to normal or non-target cells or tissues. In other embodiments, the antigen is expressed on normal cells and/or is expressed on engineered cells.

In specific embodiments, the recombinant receptor, such as a chimeric receptor, comprises an intracellular signaling region including a cytoplasmic signaling domain (also interchangeably referred to as an intracellular signaling domain), such as a cytoplasmic (intracellular) region that is capable of inducing a major activating signal in a T cell, e.g. , a component of the cytoplasmic signaling domain of the T cell receptor (TCR) (e.g., the cytoplasmic signaling domain of the CD3 zeta chain (CD3ζ), or a functional variant or signaling region thereof), and/or which contains an immunoreceptor tyrosine activating motif (ITAM).

In some embodiments, the chimeric receptor also contains an extracellular ligand-binding domain that specifically binds a ligand (eg, an antigen). In some embodiments, the chimeric receptor is a CAR that contains an extracellular antigen recognition domain that specifically binds an antigen. In some embodiments, a ligand, such as an antigen, is a protein expressed on the surface of cells. In some embodiments, the CAR is a TCR-like CAR and the antigen is a truncated peptide antigen, such as an intracellular protein peptide antigen, which, as with TCR, is recognized on the cell surface in the context of a major histocompatibility complex (MHC) molecule.

Exemplary antigen receptors, including CARs, as well as methods for constructing and introducing such receptors into cells, include those described, for example, in International Patent Application Publication Nos. , WO2013/071154, WO2013/123061, US Patent Publication Nos. US2002131960, US2013287748, US20130149337, US Patent Nos. 6451995, 7446190, 8252592, 8339645, 8398 282, 7446179, 6410319, 7070995, 7265209, 7354762, 7446191, 8324353 and 8479118 , and European patent application number EP2537416, and/or those described in Sadelain et al., Cancer Discov. April 2013; 3(4): 388-398; Davila et al. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 March 18(2): 160-75. In some aspects, antigen receptors include the CARs described in US Pat. No. 7,446,190 and those described in International Patent Application Publication No. WO/2014055668 A1. Examples of CARs include those disclosed in any of the above publications such as WO2014031687, US 8339645, US 7446179, US 2013/0149337, US Patent No. 7446190, US Patent No. 8389282, Kochenderfer et al. , 2013, Nature Reviews Clinical Oncology, 10, 267-276 (2013); Wang et al. (2012) J. Immunother. 35(9): 689-701; and Brentjens et al., Sci Transl Med. 2013 5(177). See also WO2014031687, US 8339645, US 7446179, US 2013/0149337, US Pat. No. 7,446,190 and US Pat. No. 8,389,282.

In some embodiments, the CAR is designed with specificity for a particular antigen (or marker or ligand), such as an antigen expressed on a particular cell type that is targeted by adoptive therapy, such as a cancer marker and/or an antigen designed to elicit a damping response, for example, an antigen expressed on normal or non-disease-associated cells. Thus, a CAR typically contains in its extracellular portion one or more antigen-binding molecules, for example, one or more antigen-binding fragments, domains or parts, or one or more antibody variable domains and/or antibody molecules. In some embodiments, the CAR comprises an antigen-binding fragment, or fragments, of an antibody molecule, such as a single chain antibody fragment (scFv) derived from the heavy (VH) and variable light (VL) regions of a monoclonal antibody (mAb).

In some embodiments, the antibody, or antigen-binding fragment thereof, is expressed on cells as part of a recombinant receptor, such as an antigen receptor. Antigen receptors include functional non-TCR antigen receptors such as chimeric antigen receptors (CARs). Generally, CARs containing an antibody or antigen-binding fragment that exhibits TCR-like specificity for peptide-MHC complexes may also be referred to as TCR-like CARs. In some embodiments, the MHC-peptide specific extracellular antigen-binding domain of a TCR-like CAR is linked to one or more intracellular signaling components, in some aspects via linkers and/or transmembrane domain(s). In some embodiments, such molecules generally can mimic or approximate a signal through a natural antigen receptor, such as a TCR, and optionally a signal through such a receptor in combination with a co-stimulatory receptor.

In some embodiments, a recombinant receptor, such as a chimeric receptor (eg, CAR), contains a ligand-binding domain that binds, eg, specifically binds, an antigen (or ligand). Antigens targeted by chimeric receptors include antigens expressed in the context of the disease, condition, or cell type that is targeted in adoptive cell therapy. Diseases and conditions include proliferative, neoplastic, and malignant diseases and disorders, including cancers and tumors, including hematologic cancers, immune system cancers such as lymphomas, leukemias, and/or myelomas, such as B, T, and myeloid leukemias. , lymphomas and multiple myelomas.

In some embodiments, the antigen (or ligand) is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen (or ligand) is selectively expressed or overexpressed on cells associated with the disease or condition, such as tumor or pathogenic cells, over normal or non-target cells or tissues. In other embodiments, the antigen is expressed on normal cells and/or is expressed on engineered cells.

In some embodiments, the CAR contains an antibody, or an antigen-binding fragment (e.g., scFv) that specifically recognizes an antigen, such as an intact antigen expressed on the cell surface.

In specific embodiments, the antigen is an αvβ6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), cancer testicular antigen, cancer - testicular antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), cyclin, cyclin A2, chemokine ligand 1 with C-C motif (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), mutated epidermal growth factor receptor type III (EGFR) vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin A2 receptor (EPHa2), estrogen receptor Fc receptor-like protein 5 (FCRL5; also known as the Fc receptor homologue 5 or FCRH5), embryonic acetylcholine receptor (embryonic AchR), folate binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp100), glypican-3 (GPC3 ), G protein-coupled receptor 5D (GPRC5D), Her2/neu (erbB2 tyrosine kinase receptor), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, human high molecular weight melanoma-associated antigen (HMW-MAA ), hepatitis B surface antigen, human leukocyte antigen A1 (HLA-A1), human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha (IL-22Rα), IL-13 alpha 2 receptor (IL-13Rα2) , kinase insert domain receptor (kdr), kappa light chain, L1 cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM, member of the A family of 8 proteins containing leucine-rich repeats (LRRC8A), Lewis Y, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, mouse cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 D ligands ( NKG2D), melan A (MART-1), neuronal cell adhesion molecule (NCAM), oncoembryonic antigen, preferably expressed melanoma antigen (PRAME), progesterone receptor, prostate specific antigen, prostate stem cell antigen (PSCA), prostate specific membrane antigen ( PSMA), tyrosine kinase receptor orphan receptor 1 (ROR1), survivin, trophoblastic glycoprotein (TPBG, also known as 5T4), tumor-associated glycoprotein 72 (TAG72), tyrosinase-dependent protein 1 (TRP1, also known as TYRP1 or gp75 ), tyrosinase-dependent protein 2 (TRP2, also known as dopachrome tautomerase, dopachrome delta isomerase or DCT), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms tumor antigen 1 (WT- 1), a pathogen-specific or pathogen-expressed antigen, or an antigen associated with a universal marker, and/or biotinylated molecules, and/or molecules expressed by HIV, HCV, HBV, or other pathogens. In some embodiments, the antigens targeted by the receptors include antigens associated with B-cell malignancies, for example, any of a variety of known B-cell markers. In some embodiments, the antigen is, or includes, CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Ig-kappa, Ig-lambda, CD79a, CD79b, or CD30.

In some embodiments, the antigen is, or includes, a pathogen-specific or pathogen-expressed antigen. In some embodiments, the antigen is a viral antigen (such as a viral antigen from HIV, HCV, HBV, and so on), bacterial antigens, and/or parasitic antigens.

In some embodiments, the antigen of the antigen-binding domain is CD19. In some embodiments, the scFv comprises VH and VL derived from an antibody or antibody fragment specific for CD19. In some embodiments, the antibody, or antibody fragment, that binds CD19 is a mouse antibody, such as FMC63 and SJ25C1. In some embodiments, the antibody, or antibody fragment, is a human antibody, such as that described in US Patent Publication No. US 2016/0152723.

In some embodiments, the scFv is derived from FMC63. FMC63 is a mouse monoclonal IgG1 antibody raised against Nalm-1 and -16 cells expressing human CD19 (Ling, NR, et al . (1987). Leucocyte typing III . 302). The FMC63 antibody contains CDRH1 and H2 with the sequences of SEQ ID NO: 38, 39, respectively, and CDRH3 with the sequence of SEQ ID NO: 40 or 54; as well as CDRL1 with the sequence of SEQ ID NO: 35, CDRL2 with the sequence of SEQ ID NO: 36 or 55, and CDRL3 with the sequence of SEQ ID NO: 37 or 34. The FMC63 antibody contains a heavy chain variable region (V _H ) containing the amino acid sequence of SEQ ID NO: 41, and a light chain variable region (V _L ) containing the amino acid sequence of SEQ ID NO: 42. In some embodiments, the implementation of svFv contains a light chain variable region containing CDRL1 with the sequence of SEQ ID NO: 35, CDRL2 with the sequence of SEQ ID NO: 36 and CDRL3 with the sequence of SEQ ID NO: 37; and/or a heavy chain variable region comprising a CDRH1 of SEQ ID NO: 38, a CDRH2 of SEQ ID NO: 39, and a CDRH3 of SEQ ID NO: 40. In some embodiments, the scFv comprises the FMC63 heavy chain variable region shown in SEQ ID NO: 41, and the FMC63 light chain variable region shown in SEQ ID NO: 42. In some embodiments, the heavy chain variable region and the light chain variable region are linked by a linker. In some embodiments, the implementation of the linker has the sequence of SEQ ID NO: 56. In some embodiments, the scFv contains, in the following order, V _H , the linker and V _L . In some embodiments, the implementation of the scFv contains, in the following order, V _L , a linker and V _H . In some embodiments, the svFc is encoded by the nucleotide sequence shown in SEQ ID NO: 57, or by a sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 57. In some embodiments, the scFv contains the amino acid sequence shown in SEQ ID NO: 43, or a sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 43.

In some embodiments, the scFv is derived from SJ25C1. SJ25C1 is a mouse monoclonal IgG1 antibody raised against Nalm-1 and -16 cells expressing human CD19 (Ling, NR, et al . (1987). Leucocyte typing III . 302). The SJ25C1 antibody contains CDRH1, H2 and H3 with the sequences of SEQ ID NOs: 47-49, respectively, and CDRL1, L2 and L3 with the sequences shown in SEQ ID NOs: 44-46, respectively. The SJ25C1 antibody comprises a heavy chain variable region (V _H ) comprising the amino acid sequence of SEQ ID NO: 50 and a light chain variable region (V _L ) comprising the amino acid sequence of SEQ ID NO: 51. In some embodiments, the svFv comprises a light chain variable region containing CDRL1 with the sequence of SEQ ID NO: 44, CDRL2 with the sequence of SEQ ID NO: 45 and CDRL3 with the sequence of SEQ ID NO: 46; and/or a heavy chain variable region comprising a CDRH1 of SEQ ID NO: 47, a CDRH2 of SEQ ID NO: 48, and a CDRH3 of SEQ ID NO: 49. In some embodiments, the scFv comprises an SJ25C1 heavy chain variable region of SEQ ID NO: 50 and the SJ25C1 light chain variable region of SEQ ID NO: 51. In some embodiments, the heavy chain variable region and the light chain variable region are linked by a linker. In some embodiments, the implementation of the linker has the sequence of SEQ ID NO: 52. In some embodiments, the scFv contains, in the following order, V _H , the linker and V _L . In some embodiments, the implementation of the scFv contains, in the following order, V _L , a linker and V _H . In some embodiments, the scFv contains the amino acid sequence shown in SEQ ID NO: 53, or a sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 53.

In some embodiments, the antigen of the antigen-binding domain is BCMA. In some embodiments, the scFv comprises VH and VL derived from an antibody or antibody fragment specific for BCMA. In some embodiments, the antibody or antibody fragment that binds BCMA is or contains the VH and VL of the antibody or antibody fragment described in International Patent Application Publication Numbers WO 2016/090327 and WO 2016/090320.

In some embodiments, the antigen of the antigen-binding domain is GPRC5D. In some embodiments, the scFv comprises VH and VL derived from an antibody or antibody fragment specific for GPRC5D. In some embodiments, the antibody or antibody fragment that binds GPRC5D is or contains the VH and VL of the antibody or antibody fragment described in International Patent Application Publications WO 2016/090329 and WO 2016/090312.

In some embodiments, the CAR comprises a TCR-like antibody, such as an antibody, or an antigen-binding fragment (e.g., scFv) that specifically recognizes an intracellular antigen, such as a tumor-associated antigen, presented on the cell surface as an MHC-peptide complex. In some embodiments, the antibody, or antigen-binding fragment thereof, that recognizes the MHC-peptide complex can be expressed on cells as part of a recombinant receptor, such as an antigen receptor. Antigen receptors include functional non-TCR antigen receptors such as chimeric antigen receptors (CARs). In general, a CAR containing an antibody or antigen-binding fragment that exhibits TCR-like specificity against peptide-MHC complexes may also be referred to as a TCR-like CAR.

The term "major histocompatibility complex" (MHC) means a protein, usually a glycoprotein, containing a polymorphic peptide-binding site, or binding groove, which in some cases is capable of complexing with peptide antigens of polypeptides, including peptide antigens processed by the cell machinery. In some cases, MHC molecules may be displayed or expressed on the cell surface, including as a complex with a peptide, i.e., an MHC-peptide complex, to present the antigen in a conformation recognized by an antigen receptor on T cells, such as TCR or TCR. -like antibody. Typically, class I MHC molecules are heterodimers having a membrane-spanning α-chain, in some cases with three α-domains, and a non-covalently bound β2-microglobulin. Typically, class II MHC molecules are composed of two transmembrane glycoproteins, α and β, both of which tend to pass through the membrane. The MHC molecule may include an effective portion of the MHC that contains the antigen-binding site, or sites, for binding the peptide and the sequence required for recognition by the appropriate antigen receptor. In some embodiments, class I MHC molecules deliver peptides from the cytosol to the cell surface, where the MHC-peptide complex is recognized by T cells, typically CD8 ⁺ T cells, but in some cases, CD4+ T cells. In some embodiments, class II MHC molecules deliver peptides from the vesicular system to the cell surface, where they are normally recognized by CD4 ⁺ T cells. Typically, MHC molecules are encoded by a group of related loci that are collectively named H-2 in the mouse and human leukocyte antigen (HLA) in humans. Thus, in general, human MHC can also be referred to as human leukocyte antigen (HLA).

The term "MHC-peptide complex", or "peptide-MHC complex", or variations thereof, refers to the complex or association of a peptide antigen and an MHC molecule, for example, typically through non-covalent interactions of the peptide in the binding groove, or cleft, of the MHC molecule. . In some embodiments, the MHC-peptide complex is present or exposed on the cell surface. In some embodiments, the MHC-peptide complex may be specifically recognized by an antigen receptor, such as a TCR, a TCR-like CAR, or antigen-binding fragments thereof.

In some embodiments, a peptide, such as a peptide antigen or an epitope, from a polypeptide can bind to an MHC molecule, for example, for recognition by an antigen receptor. Typically, a peptide is derived from, or based on, a fragment of a larger biological molecule, such as a polypeptide or protein. In some embodiments, the implementation of the peptide, as a rule, has a length of from about 8 to about 24 amino acids. In some embodiments, the implementation of the peptide has a length of from, or from about 9 to 22 amino acids for recognition in the MHC class II complex. In some embodiments, the peptide is from, or about 8 to 13 amino acids long, for recognition in an MHC class I complex. In some embodiments, after recognition of the peptide in the context of an MHC molecule, e.g., an MHC-peptide complex, an antigen receptor such as a TCR or a TCR-like CAR that produces, or initiates, an activating signal for a T cell that induces a T cell response, such as T cell proliferation, cytokine production, a cytotoxic T cell response, or other response.

In some embodiments, a TCR-like antibody, or antigen-binding fragment, can be generated (see, for example, US Published Application Nos. US 2002/0150914; US 2003/0223994; US 2004/0191260; US 2006/0034850; US 2007/ 00992530; US20090226474; US20090304679; and PCT International Publication No. WO 03/068201).

In some embodiments, an antibody, or antigen-binding fragment thereof, that specifically binds an MHC-peptide complex can be produced by immunizing the host with an effective amount of an immunogen containing the MHC-peptide complex. In some cases, the peptide of the MHC-peptide complex is an epitope of an antigen capable of binding to MHC, such as a tumor antigen, for example, a universal tumor antigen, myeloma antigen, or other antigen described below. In some embodiments, an effective amount of the immunogen is then administered to the host to elicit an immune response, wherein the immunogen retains its three-dimensional form for a period of time sufficient to elicit an immune response against the three-dimensionally presented peptide in the binding groove of the MHC molecule. Serum obtained from the host is then analyzed to determine if the desired antibodies have been obtained that recognize the 3D peptide in the binding groove of the MHC molecule. In some embodiments, the resulting antibodies can be evaluated to confirm that the antibody is capable of distinguishing an MHC-peptide complex from a single MHC molecule, a single peptide of interest, and from an MHC complex with a foreign peptide. The desired antibodies can then be isolated.

In some embodiments, an antibody, or antigen-binding fragment thereof, that specifically binds an MHC-peptide complex can be generated by methods using an antibody display library, such as an antibody display library in phage. In some embodiments, phage display libraries of mutant Fab, scFv, or other forms of antibodies, for example, in which library members have mutated one or more residues of the CDR region or CDR regions, can be generated. See, for example, US Published Application Nos. US20020150914, US2014/0294841; and Cohen CJ. et al . (2003) J Mol. Recogniz . 16:324-332.

In this document, the term "antibody" is used in the broadest sense and covers polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including antigen-binding (Fab) fragments, F(ab') ₂ fragments, Fab' fragments , Fv fragments, recombinant IgG (rIgG) fragments, heavy chain variable regions (V _H ) capable of specifically binding antigen, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibody fragments (e.g., sdAb, sdFv, nanobody). The term encompasses engineered and/or otherwise modified forms of immunoglobulins such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies and heteroconjugate antibodies, multispecific, e.g. bispecific, antibodies, diabodies, tribodies and tetrabodies, tandem di-scFvs, tandem tri-scFv. Unless otherwise indicated, the term "antibody" should be understood to encompass functional fragments of antibodies. The term also encompasses intact or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD.

In some embodiments, antigen-binding proteins, antibodies, and antigen-binding fragments thereof specifically recognize an antigen recognized by a full-length antibody. In some embodiments, the heavy and light chains of the antibody may be full length, or may be an antigen binding fragment (Fab, F(ab') ₂ , Fv or single chain Fv fragment (scFv)). In other embodiments, the antibody heavy chain constant region is selected from, e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE, in particular is selected from, e.g., IgG1, IgG2, IgG3, and IgG4, more specifically , IgG1 (for example, human IgG1). In another embodiment, the antibody light chain constant region is selected from, for example, kappa or lambda, in particular kappa.

Suggested antibodies include antibody fragments. The term "antibody fragment" means a molecule, other than an intact antibody, which contains a portion of the intact antibody that binds an antigen that binds the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ ; diabody; linear antibodies; heavy chain variable regions (V _H ), single chain antibody molecules such as scFv and single domain V _H antibodies; as well as multispecific antibodies formed from antibody fragments. In specific embodiments, the antibodies are single chain antibody fragments containing a heavy chain variable region and/or a light chain variable region, such as scFv.

The term "variable region" or "variable domain" means the heavy or light chain domain of an antibody that is involved in the binding of an antibody to an antigen. The heavy chain and light chain variable domains (V _H and V _L , respectively) of a natural antibody generally have similar structures, with each domain containing four conserved framework regions (FRs) and three CDR regions. (See, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007). A single V _H or V _L domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind a particular antigen can be isolated using the V _H or V _L domain from an antigen-binding antibody to screen for a library of complementary V _L or V _H domains, respectively See, for example, Portolano et al., J. Immunol 150:880 -887 (1993); Clarkson et al., Nature 352:624-628 (1991).

Single domain antibodies are antibody fragments containing all or part of the heavy chain variable domain, or all or part of the light chain variable domain of an antibody. In specific embodiments, the single domain antibody is a human single domain antibody. In some embodiments, the CAR comprises an antibody heavy chain domain that specifically binds an antigen, such as a cancer marker or cell surface antigen, of target cells or diseased tissues, e.g., tumor cells or cancer cells, such as any of the target antigens described in this document or known in the art.

Antibody fragments can be obtained by various methods, including, but not limited to, proteolytic cleavage of intact antibodies, as well as production by recombinant host cells. In some embodiments, the antibodies are recombinantly produced fragments, such as fragments containing constructs not naturally occurring, such as constructs with two or more antibody regions or chains linked by synthetic linkers, such as peptide linkers, and/or constructs which cannot be obtained by enzymatic cleavage of the natural intact antibody. In some embodiments, the antibody fragments are scFv.

A "humanized" antibody is an antibody in which all or substantially all of the CDR amino acid residues are derived from non-human CDRs and all or substantially all of the FR amino acid residues are derived from human FR. The humanized antibody may optionally comprise at least a portion of the antibody constant region derived from a human antibody. A "humanized form" of a non-human antibody means a variant of a non-human antibody that has been humanized, typically to reduce immunogenicity in humans while retaining the specificity and affinity of the original non-human antibody. In some embodiments, some FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (eg, an antibody from which the CDR residues are derived), for example, to restore or increase the specificity or affinity of the antibody.

Thus, in some embodiments, the implementation of the chimeric antigen receptor, including TCR-like CAR, contains an extracellular fragment containing the antibody or antibody fragment. In some embodiments, the antibody or fragment comprises an scFv. In some aspects, the chimeric antigen receptor contains an extracellular fragment containing the antibody, or fragment, and an intracellular signaling region. In some embodiments, the implementation of the intracellular signaling region contains an intracellular signaling domain. In some embodiments, the intracellular signaling domain is, or contains, a basic signaling domain, a signaling domain capable of inducing a basic activating signal in a T cell, a T cell receptor (TCR) signaling domain component, and/or a signaling domain containing an immunoreceptor tyrosine activating motive (ITAM).

In some embodiments, the recombinant receptor, such as CAR, e.g., an antibody fragment thereof, further comprises a spacer, which may be, or include, at least a portion of an immunoglobulin constant region, or a variant or modified version thereof, e.g., a hinge region, e.g. , IgG4 hinge region, and/or C _H 1/C _L , and/or Fc region. In some embodiments, the recombinant receptor further comprises a spacer and/or a hinge region. In some embodiments, the implementation of the constant region, or part thereof, is derived from human IgG, for example, IgG4 or IgG1. In some aspects, a portion of the constant region serves as a spacer region between the antigen recognition component, eg, scFv, and the transmembrane domain. The spacer may be of a length that provides for an increased responsiveness of the cell after antigen binding, as compared to the situation in the absence of the spacer. In some examples, the spacer is, or is, about 12 amino acids in length, or is no more than 12 amino acids in length. Exemplary spacers include those having at least about 10-229 amino acids, about 10-200 amino acids, about 10-175 amino acids, about 10-150 amino acids, about 10-125 amino acids, about 10-100 amino acids, about 10-75 amino acids, about 10-50 amino acids, about 10-40 amino acids, about 10-30 amino acids, about 10-20 amino acids, or about 10-15 amino acids, including any integer value between the endpoints of the specified ranges. In some embodiments, the spacer region contains about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less. Exemplary spacers include a single IgG4 hinge, an IgG4 hinge associated with CH2 and CH3 domains, or an IgG4 hinge associated with a CH3 domain. Illustrative spacers include, but are not limited to, those described in Hudecek et al . (2013) Clin. Cancer Res ., 19:3153 or International Patent Application Publication No. WO2014031687. In some embodiments, the spacer has the sequence given in SEQ ID NO: 1 and is encoded by the sequence given in SEQ ID NO: 2. In some embodiments, the spacer has the sequence given in SEQ ID NO: 3. In some embodiments, the spacer has the sequence shown in SEQ ID NO: 4.

In some embodiments, the constant region, or fragment, is from IgD. In some embodiments, the spacer has the sequence shown in SEQ ID NO: 5. In some embodiments, the spacer has an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity with any of SEQ ID NOs: 1, 3, 4, and 5. In some embodiments, the spacer has the sequence shown in SEQ ID NO: 23-31. In some embodiments, the spacer has an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99% or more sequence identity with any of SEQ ID NOs: 27-31, 58, 59.

The antigen recognition domain is typically associated with one or more intracellular signaling components, such as signaling components that mimic activation through an antigen receptor complex, such as the TCR complex in the case of CAR, and/or a signal through another cell surface receptor. Thus, in some embodiments, an antigen-binding component (eg, an antibody) is associated with one or more transmembrane and intracellular signaling regions. In some embodiments, the transmembrane domain is fused to the extracellular domain. In one embodiment, a transmembrane domain is used that is naturally associated with one of the domains in the receptor, such as CAR. In some instances, a transmembrane domain is chosen, or modified by amino acid substitution, to avoid binding such domains to transmembrane domains of the same or different surface membrane proteins to minimize interactions with other constituents of the receptor complex.

In some embodiments, the transmembrane domain is obtained from a natural or synthetic source. In some aspects, if the source is natural, the domain is from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e., contain at least the transmembrane region(s)) T cell receptor alpha, beta, or zeta chain, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD 154. Alternatively, in some embodiments, the transmembrane domain is synthetic. In some aspects, the synthetic transmembrane domain predominantly contains hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be present at each end of the synthetic transmembrane domain. In some embodiments, communication is via linkers, spacers, and/or transmembrane domain(s).

Intracellular signaling regions include those that mimic or approximate a signal via a natural antigen receptor, a signal via such a receptor in combination with a costimulatory receptor, and/or a signal via a costimulatory receptor alone. In some embodiments, there is a short oligo- or polypeptide linker, such as a linker between 2 and 10 amino acids in length, such as a linker containing glycine and serine residues, such as a glycine-serine doublet, which is the link between the transmembrane domain and the cytoplasmic signaling domain CAR.

A receptor, such as a CAR, typically contains at least one intracellular signaling component, or components. In some embodiments, the receptor contains an intracellular component of the TCR complex, such as the CD3 TCR chain, which mediates T cell activation and cytotoxicity, such as the CD3 zeta chain. Thus, in some aspects, the ROR1 binding antibody is associated with one or more signaling modules of the cell. In some embodiments, cell signaling modules include a CD3 transmembrane domain, CD3 intracellular signaling domains, and/or other CD transmembrane domains. In some embodiments, the implementation of the receptor, for example, CAR, also contains part of one or more additional molecules, such as Fc receptor γ, CD8, CD4, CD25 or CD16. For example, in some aspects, a CAR comprises a chimeric molecule from CD3-zeta (CD3-ζ) or Fc receptor γ and CD8, CD4, CD25, or CD16.

In some embodiments, upon CAR binding, the cytoplasmic domain, or intracellular signaling region, of CAR activates at least one of the normal effector functions, or responses, of an immune cell, such as a T cell engineered to express CAR. For example, in some contexts, CAR induces a T cell function such as cytolytic activity or T helper activity, such as the secretion of cytokines or other factors. In some embodiments, a truncated fragment of an intracellular antigen receptor signaling region component or co-stimulatory molecule is used in place of an intact immunostimulatory chain, for example if it signals an effector function. In some embodiments, intracellular signaling regions, e.g., containing an intracellular domain or domains, contain cytoplasmic T cell receptor (TCR) sequences, and in some aspects also cytoplasmic co-receptor sequences, which in a natural context act in concert with such a receptor to initiate signaling after antigen receptor binding, and/or any derivative or variant of such molecules, and/or any synthetic sequence having the same functionality.

In the context of a natural TCR, full activation typically requires not only signaling through the TCR, but also a costimulatory signal. Thus, in some embodiments, a component for generating an auxiliary or costimulatory signal is also included to stimulate full activation in the CAR. In other embodiments, the CAR does not contain a component for generating a costimulatory signal. In some aspects, the additional CAR is expressed in the same cell and provides a component for generating an accessory or costimulatory signal.

In some aspects, it is described that T-cell activation is mediated by two kinds of cytoplasmic signal sequences: those that initiate antigen-dependent primary activation via TCRs (basic cytoplasmic signal sequences) and those that act in an antigen-independent manner, providing an auxiliary or co-stimulatory signal (auxiliary cytoplasmic signal sequences). In some aspects, the CAR contains one or both of these signaling components.

In some aspects, the CAR contains a basic cytoplasmic signal sequence that regulates the basic activation of the TCR complex. The main cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs, which are known as immunoreceptor tyrosine activating motifs, or ITAMs. Examples of ITAM-containing major cytoplasmic signal sequences include those derived from TCR or CD3-zeta, FcR-gamma or FcR-beta. In some embodiments, the implementation of the cytoplasmic signaling molecule(s) in CAR contains(at) a cytoplasmic signaling domain, part or sequence of CD3-zeta.

In some embodiments, the implementation of the CAR contains the signaling region and/or the transmembrane part of the costimulatory receptor, such as CD28, 4-1BB, OX40, DAP10 and ICOS. In some aspects, the same CAR contains both a signaling region and co-stimulatory components.

In some embodiments, the signaling region is included in one CAR while the co-stimulatory component is provided to other CARs recognizing a different antigen. In some embodiments, a CAR includes an activating or stimulatory CAR and a costimulatory CAR, both of which are expressed on the same cell (see WO2014/055668).

In specific embodiments, the intracellular signaling region comprises a CD28 transmembrane and signaling domain associated with an intracellular CD3 domain (eg, CD3 zeta). In some embodiments, the intracellular signaling region comprises CD28 and CD137 chimeric co-stimulatory domains (4-1BB, TNFRSF9) associated with the CD3-zeta intracellular domain.

In some embodiments, the implementation of the CAR contains one or more, for example, two or more co-stimulatory domains and an activating domain, for example, the main activating domain, in the cytoplasmic part. An exemplary CAR contains the intracellular components CD3-zeta, CD28, and 4-1BB.

In some cases, CARs are referred to as first, second and/or third generation CARs. In some aspects, a first generation CAR is a CAR that, upon antigen binding, provides a CD3 chain-induced signal exclusively; in some aspects, a second generation CAR is a CAR that provides such a signal and a costimulatory signal, for example, a CAR containing an intracellular signaling domain from a costimulatory receptor such as CD28 or CD137; in some aspects, a third generation CAR is a CAR that contains multiple costimulatory domains from different costimulatory receptors.

In some embodiments, the implementation of the chimeric antigen receptor contains an extracellular fragment containing the antibody, or a fragment described in this document. In some aspects, the chimeric antigen receptor contains an extracellular fragment containing an antibody, or a fragment described herein, and an intracellular signaling domain. In some embodiments, the antibody or fragment comprises an scFv or single domain V _H antibody and the intracellular domain comprises ITAM. In some aspects, the intracellular signaling domain includes the CD3 zeta chain (CD3ζ) signaling domain. In some embodiments, the implementation of the chimeric antigen receptor contains a transmembrane domain located between the extracellular domain and the intracellular signaling region.

In some aspects, the transmembrane domain contains a CD28 transmembrane fragment. The extracellular domain and the transmembrane domain can be linked directly or indirectly. In some embodiments, the extracellular domain and the transmembrane domain are linked by a spacer, such as any of the spacers described herein. In some embodiments, the chimeric antigen receptor comprises an intracellular domain of a T-cell co-stimulatory molecule, for example, between a transmembrane domain and an intracellular signaling domain. In some aspects, the T cell costimulatory molecule is CD28 or 4-1BB.

In some embodiments, the CAR comprises an antibody, e.g., an antibody fragment, a transmembrane domain that is, or contains, a CD28 transmembrane fragment, or a functional variant thereof, and an intracellular signaling domain containing a CD28 signal fragment, or a functional variant thereof, and a signal fragment CD3-zeta, or its functional variant. In some embodiments, the CAR comprises an antibody, e.g., an antibody fragment, a transmembrane domain that is, or contains, a CD28 transmembrane fragment, or a functional variant thereof, and an intracellular signaling domain containing the 4-1BB signal fragment, or a functional variant thereof, and signal fragment of CD3-zeta, or its functional variant. In some such embodiments, the receptor also comprises a spacer containing a fragment of an Ig molecule, such as a human Ig molecule, eg, an Ig hinge, eg, an IgG4 hinge, eg, a spacer containing only a hinge.

In some embodiments, the transmembrane domain of the receptor, e.g., CAR, is the transmembrane domain of human CD28, or a variant thereof, e.g., the 27 amino acid transmembrane domain of human CD28 (Accession No: P10747.1), or is a transmembrane domain containing the amino acid sequence , shown in SEQ ID NO: 8, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 8; in some embodiments, the transmembrane domain-containing recombinant receptor fragment comprises the amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence having at least, or about, 85%, 86%, 87%, 88%, 89%, 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with it.

In some embodiments, the implementation of the chimeric antigen receptor contains the intracellular domain of a T-cell costimulatory molecule. In some aspects, the T cell costimulatory molecule is CD28 or 4-1BB.

In some embodiments, the intracellular signaling region comprises a human CD28 intracellular costimulatory signaling domain, or a functional variant or fragment thereof, e.g., a 41 amino acid domain and/or such domain with an LL to GG substitution at positions 186-187 of the native CD28 protein. In some embodiments, the intracellular signaling domain may comprise the amino acid sequence shown in SEQ ID NO: 10 or 11, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity with SEQ ID NO: 10 or 11. In some embodiments, the intracellular region contains an intracellular co-stimulatory signaling domain 4-1BB, or a functional variant or fragment thereof, such as the 42 amino acid cytoplasmic domain of human 4-1BB (Accession No. Q07011.1), or a functional variant or fragment thereof, such as the amino acid sequence shown in SEQ ID NO: 12, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99% or more sequence identity to SEQ ID NO: 12.

In some embodiments, the intracellular signaling region comprises a human CD3 chain, optionally a CD3-zeta stimulatory signaling domain, or a functional variant thereof, e.g. -zeta, as described in US Pat. %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity with SEQ ID NO : 13, 14 or 15.

In some aspects, the spacer comprises only an IgG hinge, e.g., only an IgG4 or IgG1 hinge, e.g., a hinge-only spacer of SEQ ID NO: 1. In other embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge associated with domains C _H 2 and/or C _H 3. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to the C _H 2 and C _H 3 domains, e.g., to the sequence of SEQ ID NO: 3. In some embodiments, the implementation the spacer is an Ig hinge, such as an IgG4 hinge, linked only to a C _H 3 domain, such as SEQ ID NO: 4. In some embodiments, the spacer is, or contains, a glycine-serine residue-rich sequence or other flexible linker , such as known flexible linkers.

2. Chimeric Autoantibody Receptors (CAAR)

In some embodiments, the recombinant receptors expressed by the engineered cells used in conjunction with the proposed methods, uses, articles, and compositions include a chimeric autoantibody receptor (CAAR). In some embodiments, the CAAR is specific for an autoantibody. In some embodiments, a cell expressing CAAR, such as a T cell engineered to express CAAR, can be used to specifically bind and kill cells expressing autoantibodies, but not cells expressing normal antibodies. In some embodiments, CAAR-expressing cells can be used to treat an autoimmune disease associated with the expression of self antigens, such as autoimmune diseases. In some embodiments, CAAR-expressing cells can be targeted to B cells that eventually produce autoantibodies and display autoantibodies on their cell surfaces, making these B cells disease-specific targets for therapeutic intervention. In some embodiments, CAAR-expressing cells can be used to effectively target and kill pathogenic B cells in autoimmune diseases by targeting disease-causing B cells with an antigen-specific autoantibody chimeric receptor. In some embodiments, the recombinant receptor is a CAAR as described in US Patent Application Publication No. US 2017/0051035.

In some embodiments, the CAAR comprises an autoantibody-binding domain, a transmembrane domain, and an intracellular signaling region. In some embodiments, the implementation of the intracellular signaling region contains an intracellular signaling domain. In some embodiments, the intracellular signaling domain is, or contains, a basic signaling domain, a signaling domain capable of inducing a basic activating signal in a T cell, a T cell receptor (TCR) signaling domain component, and/or a signaling domain containing an immunoreceptor tyrosine activating motive (ITAM). In some embodiments, the implementation of the intracellular signaling region contains an auxiliary or costimulatory signaling region (auxiliary intracellular signaling regions).

In some embodiments, the autoantibody-binding domain comprises a self-antigen or fragment thereof. The choice of autoantigen may depend on the type of autoantibody being targeted. For example, an autoantigen may be selected because it recognizes an autoantibody on a target cell, such as a B cell, associated with a particular disease state, eg, an autoimmune disease, such as an autoantibody-mediated autoimmune disease. In some embodiments, the autoimmune disease includes pemphigus vulgaris (PV). Illustrative autoantigens include desmoglein 1 (Dsg1) and Dsg3.

3. Multiple targeting

In some embodiments, cells used in conjunction with the proposed methods, uses, articles, and compositions include cells used in a multiple targeting strategy. In some embodiments, the cells express multi-chain chimeric antigen receptors (CARs) or express two or more engineered receptors on the cell, each of which recognizes an epitope of different antigens and typically each contains a different intracellular signaling component. Such multiple targeting strategies are described, for example, in International Patent Application Publication No. WO 2014055668 A1 (which describes combinations of activating and costimulatory CARs, e.g. targeting two different antigens, present separately on non-targeted, e.g., normal cells, but present together only on cells associated with the disease or condition to be treated) and Fedorov et al., Sci. Transl. Medicine, 5(215) (2013) (which describes cells expressing both an activating and an inhibitory CAR, such as those in which the activating CAR binds a single antigen expressed on both normal or non-disease-associated cells and cells associated disease or condition to be treated and the inhibitory CAR binds another antigen expressed only on normal cells or cells that do not require treatment).

For example, in some embodiments, the cells contain a first engineered antigen receptor (e.g., CAR or TCR) that is capable of inducing an activating or stimulatory signal in the cell, typically upon specific binding to an antigen recognized by the first receptor, e.g., the first antigen. In some embodiments, the cell further comprises a second engineered antigen receptor (e.g., CAR or TCR), e.g., a chimeric costimulatory receptor, that is capable of inducing a costimulatory signal in an immune cell, typically upon specific binding to a second antigen recognized by the second receptor. In some embodiments, the first antigen and the second antigen are the same. In some embodiments, the first antigen and the second antigen are different.

In some embodiments, the first and/or second engineered antigen receptor (eg, CAR or TCR) is capable of inducing an activating signal in a cell. In some embodiments, the implementation of the receptor contains an intracellular signaling component containing ITAM or ITAM-like motifs. In some embodiments, the activation induced by the first receptor includes signal transduction or a change in protein expression in the cell resulting in the initiation of an immune response, such as ITAM phosphorylation and/or initiation of an ITAM-mediated signal transduction cascade, formation of an immune synapse, and/or clustering of molecules near the bound receptor. (eg CD4 or CD8, etc.), activation of one or more transcription factors such as NF-κB and/or AP-1, and/or induction of gene expression of factors such as cytokines, proliferation and/or survival.

In some embodiments, the first and/or second receptor comprise intracellular signaling domains or regions of co-stimulatory receptors, eg, CD28, CD137 (4-1BB), OX40, and/or ICOS. In some embodiments, the implementation of the first and second receptors contain intracellular signaling domains of co-stimulatory receptors, which are different. In one embodiment, the first receptor contains the CD28 costimulatory signaling region and the second receptor contains the 4-1BB costimulatory signaling region, or vice versa.

In some embodiments, the first and/or second receptor comprise both an intracellular signaling domain containing ITAM or ITAM-like motifs and an intracellular signaling domain of a costimulatory receptor.

In some embodiments, the first receptor contains an intracellular signaling domain containing ITAM or ITAM-like motifs, and the second receptor contains an intracellular signaling domain of a costimulatory receptor. The costimulatory signal, combined with an activating signal induced in the same cell, results in an immune response, such as a strong and persistent immune response, such as increased gene expression, secretion of cytokines, and other factors, and T-cell mediated effector functions, such as killing cells.

In some embodiments, neither binding of the first receptor alone nor binding of the second receptor alone induces a strong immune response. In some aspects, when binding to only one receptor, the cell becomes tolerant or non-responsive to the antigen, or inhibited, and/or does not begin to proliferate or secrete factors, or perform effector functions. However, in some such embodiments, if multiple receptors are bound, e.g., by interaction with a cell expressing the first and second antigens, the desired response is achieved, such as complete immune activation or stimulation, e.g., as evidenced by secretion of one or more cytokines, proliferation, persistence and/or performing an immune effector function such as cytotoxic killing of a target cell.

In some embodiments, two receptors induce, respectively, an activating and inhibitory signal in a cell such that binding of one of the receptors to its antigen activates the cell or induces a response, but binding of the second inhibitory receptor to its antigen induces a signal that suppresses or dampens that response. Examples are combinations of an activating CAR and an inhibitory CAR, or iCAR. For example, such a strategy can be used in which an activating CAR binds an antigen expressed in a disease or condition, but which is also expressed on normal cells, and an inhibitory receptor binds a single antigen, which is expressed on normal cells, but not on cells associated with disease or condition.

In some embodiments, a multiple targeting strategy is used when an antigen associated with a particular disease or condition is expressed on a non-disease-associated cell and/or is expressed on the genetically engineered cell itself, either transiently (e.g., after stimulation due to a gene engineering), or permanently. In such cases, by requiring binding of two separate, and with separate specificity, antigen receptors, specificity, selectivity and/or efficacy can be increased.

In some embodiments, multiple antigens, such as the first and second antigens, are expressed on a cell or tissue associated with the target disease or condition, such as a cancer cell. In some aspects, the cell or tissue associated with the disease or condition is a multiple myeloma or multiple myeloma cell. In some embodiments, one or more of the multiple antigens is typically also expressed on a cell that should not be the target of cell therapy, such as a normal or non-disease-associated cell or tissue and/or the genetically engineered cells themselves. In such embodiments, specificity and/or potency can be increased by requiring multiple receptors to bind to achieve a response in a cell.

4. T cell receptor

In some embodiments, engineered cells are also provided, such as T cells expressing a T cell receptor (TCR), or an antigen-binding fragment thereof, that recognizes a peptide epitope or a T cell epitope of a target polypeptide, such as an antigen of a tumor, viral or autoimmune protein.

In some embodiments, a "T cell receptor" or "TCR" is a molecule containing α and β variable chains (also known as TCRα and TCRβ, respectively) or γ and δ variable chains (also known as TCRγ and TCRδ, respectively), or antigen-binding fragments thereof, which is capable of specific binding to a peptide associated with an MHC molecule. In some embodiments, the implementation of the TCR has the form αβ. Typically, TCRs that exist in the αβ and γδ forms generally have a similar structure, but the T cells expressing them may have different anatomical locations or different functions. TCR is found on the surface of cells or in soluble form. Typically, the TCR occurs on the surface of T cells (or T lymphocytes), where it is usually responsible for recognizing antigens associated with major histocompatibility complex (MHC) molecules.

Unless otherwise indicated, the term "TCR" shall encompass all TCRs as well as their antigen-binding fragments or antigen-binding portions. In some embodiments, the TCR is intact or full length TCR, including TCR in αβ form or γδ form. In some embodiments, the TCR is an antigen-binding fragment that is smaller than a full-length TCR but that binds a particular peptide associated with an MHC molecule, eg, binds an MHC-peptide complex. In some cases, an antigen-binding fragment, or TCR fragment, may contain only a subset of the structural domains of the full-length or intact TCR, but still be able to bind a peptide epitope, such as an MHC-peptide complex, that binds the full-length TCR. In some instances, the antigen-binding fragment contains TCR variable domains, eg, the TCR variable α chain and the TCR variable β chain, sufficient to form a binding site for binding a specific MHC-peptide complex. Typically, TCR variable chains contain complementarity-determining regions involved in peptide, MHC and/or MHC-peptide recognition.

In some embodiments, the TCR variable domains contain hypervariable loops, or complementarity determining regions (CDRs), which are generally considered to play a major role in antigen recognition and binding, as well as specificity for antigen. In some embodiments, the CDRs in a TCR, or combinations thereof, form all, or substantially all, of the antigen-binding sites of a particular TCR molecule. The various CDRs in the variable region of the TCR chain are usually separated by framework regions (FRs), which, in general, have less variability among TCR molecules compared to CDRs (see, for example, Jores et al., Proc. Nat'l Acad. Sci. USA 87:9138, 1990; Chothia et al., EMBO J. 7:3745, 1988; see also Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). In some embodiments, the CDR3 is the primary CDR responsible for antigen binding or specificity, or is the most important of the three CDRs in the variable region of a particular TCR for antigen recognition and/or interaction with the truncated peptide portion of the peptide-MHC complex. In some contexts, the alpha chain CDR1 may interact with the N-terminal portion of certain antigenic peptides. In some contexts, the CDR1 of the beta chain may interact with the C-terminal portion of the peptide. In some contexts, CDR2 contributes the most, or is the primary CDR responsible for interaction with, or recognition of, the MHC portion of the MHC-peptide complex. In some embodiments, the β-chain variable region may contain an additional hypervariable region (CDR4 or HVR4) that is generally involved in superantigen binding but not antigen recognition (Kotb (1995) Clinical Microbiology Reviews, 8:411-426) .

In some embodiments, the TCR may also contain a constant domain, a transmembrane domain, and/or a short cytoplasmic tail (see, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd Ed., Current Biology Publications, p 4:33, 1997). In some aspects, such a TCR chain may have one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic "tail" at the C-terminus. In some embodiments, the TCR is associated with invariant CD3 complex proteins involved in signaling mediation.

In some embodiments, the TCR chain contains one or more constant domains. For example, an extracellular fragment of a particular TCR chain (eg, α-chain or β-chain) may contain two immunoglobulin-like domains, such as variable domains (eg, Vα or Vβ; typically amino acids 1-116 according to the Kabat Kabat et al. al., "Sequences of Proteins of Immunological Interest", US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5th ed.), and a constant domain (e.g., an α-chain constant domain or Cα, as generally positions 117-259 in the Kabat numbering chain, or the β-chain constant domain, or Cβ, typically positions 117-295 in the Kabat numbering chain), near the cell membrane. For example, in some cases, an extracellular fragment of a TCR formed by two chains contains two constant domains proximal to the membrane and two variable domains distal to the membrane, with each of the variable domains containing CDR regions. The TCR constant domain may contain short junction sequences in which a cysteine residue forms a disulfide bond, thereby linking two TCR strands. In some embodiments, the TCR may have an additional cysteine residue in each of the α and β chains such that the TCR contains two disulfide bonds in the constant domains.

In some embodiments, the TCR chains contain a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chain contains a cytoplasmic tail. In some cases, the structure allows the TCR to bind to other molecules such as CD3 and its subunits. For example, a TCR containing constant domains with a transmembrane region can anchor the protein in the cell membrane and bind to invariant signaling subunits or the CD3 complex. The intracellular tails of the CD3 signaling subunits (eg, CD3γ, CD3δ, CD3ε, and CD3ζ chains) contain one or more immunoreceptor tyrosine activating motifs, or ITAMs, that are involved in signaling the TCR complex.

In some embodiments, the TCR may be a two chain heterodimer of α and β (or optionally γ and δ), or it may be a single chain TCR construct. In some embodiments, the TCR is a heterodimer containing two separate chains (α and β chains or γ and δ chains) that are linked, for example, by a disulfide bond or disulfide bonds.

In some embodiments, the TCR may be derived from known TCR sequence(s), such as the Vα,β chain sequences, for which a substantially full length coding sequence is readily available. Techniques for obtaining full length TCR sequences, including V chain sequences, from cellular sources can be used. In some embodiments, TCR-encoding nucleic acids can be obtained from various sources, such as polymerase chain reaction (PCR) amplification of TCR-encoding nucleic acids residing within, or isolated from, a particular cell or cells, or by synthesis of commonly known DNA sequences. TCR.

In some embodiments, the TCR is derived from a biological source, such as cells, such as T cells (eg, cytotoxic T cells), T cell hybridomas, or other publicly available source. In some embodiments, T cells can be obtained from in vivo isolated cells. In some embodiments, the TCR is thymus-selected TCR. In some embodiments, the TCR is a neoepitope-restricted TCR. In some embodiments, the T cells may be a cultured T cell hybridoma or a clone. In some embodiments, a TCR, or an antigen-binding fragment thereof, or an antigen-binding portion thereof, can be synthesized based on the sequence information of the TCR.

In some embodiments, the TCR is derived from a TCR identified or selected by screening a candidate TCR library against a target polypeptide antigen or epitope thereof that is targeted by T cells. TCR libraries can be obtained by amplifying the Vα and Vβ repertoire from T cells obtained from a subject, including cells present in PBMC, spleen, or other lymphoid organ. In some cases, T cells can be amplified from tumor infiltrating lymphocytes (TILs). In some embodiments, TCR libraries can be obtained from CD4+ or CD8+ cells. In some embodiments, TCRs can be amplified from a T cell source from a normal or healthy subject, ie, a library of normal TCRs. In some embodiments, TCRs can be amplified from a T-cell source from a diseased subject, ie, a library of disease-associated TCRs. In some embodiments, degenerate primers are used to amplify the Vα and Vβ gene repertoire, such as by RT-PCR, in samples such as human T cells. In some embodiments, scTv libraries can be assembled from naïve Vα and Vβ libraries in which the amplified products are cloned or assembled such that they are separated by a linker. Depending on the source of the subject and the cells, the libraries may be specific for HLA alleles. Alternatively, in some embodiments, TCR libraries can be obtained by mutagenesis or diversification of the original, or framework, TCR molecule. In some aspects, the TCR is subjected to directed evolution, such as by mutagenesis, such as α or β chains. In some aspects, specific residues in the CDR regions of the TCR are altered. In some embodiments, the selected TCR may be modified by affinity maturation. In some embodiments, antigen-specific T cells can be selected, for example, by screening to evaluate CTL activity for a peptide. In some aspects, the TCR, eg, found on antigen-specific T cells, can be selected, eg, based on binding activity, eg, specific affinity or avidity for the antigen.

In some embodiments, the TCR, or antigen-binding fragment thereof, is a TCR that has been modified or engineered. In some embodiments, directed evolution techniques are used to generate TCRs with altered properties, such as higher affinity for a particular MHC-peptide complex. In some embodiments, directed evolution is performed by display methods, including but not limited to yeast display (Holler et al. (2003) Nat Immunol, 4, 55-62; Holler et al. (2000) Proc Natl Acad Sci USA, 97, 5387-92), phage display (Li et al. (2005) Nat Biotechnol, 23, 349-54), or T-cell display (Chervin et al. (2008) J Immunol Methods, 339, 175-84). In some embodiments, display approaches involve constructing or modifying a known, original, or reference TCR. For example, in some instances, wild-type TCRs may be used as a template to generate a mutant TCR in which one or more residues of the CDR regions are mutated, and mutants with a desired altered property, such as higher affinity for the desired target antigen, are selected.

In some embodiments, the target polypeptide peptides for use in making or creating a TCR of interest are known or readily identifiable. In some embodiments, peptides suitable for use in the production of TCRs, or antigen-binding fragments, can be determined based on the presence of an HLA-restricted motif in a target polypeptide of interest, such as the target polypeptide described below. In some embodiments, peptides are identified using available computer prediction models. In some embodiments, for predicting class I MHC binding sites, such models include, but are not limited to, ProPred1 (Singh and Raghava (2001) Bioinformatics 17(12):1236-1237), and SYFPEITHI (see Schuler et al. (2007) Immunoinformatics Methods in Molecular Biology, 409(1): 75-93 2007). In some embodiments, the MHC-restricted epitope is HLA-A0201, which is expressed in about 39-46% of all white Caucasians and thus is a suitable MHC antigen variant for use in the production of a TCR or other MHC peptide-binding molecule. .

HLA-A0201 binding motifs and cleavage sites for proteasomes and immunoproteasomes obtained using predictive computer models can be used. For prediction of MHC class I binding sites, such models include, but are not limited to, ProPred1 (described in more detail in Singh and Raghava, ProPred: prediction of HLA-DR binding sites. BIOINFORMATICS 17(12):1236-1237 2001), and SYFPEITHI (see Schuler et al. SYFPEITHI, Database for Searching and T-Cell Epitope Prediction, in: Immunoinformatics Methods in Molecular Biology, vol 409(1): 75-93 2007).

In some embodiments, the TCR, or antigen-binding fragment thereof, may be a recombinantly produced natural protein, or a mutant form thereof, in which one or more properties, such as binding characteristics, have been altered. In some embodiments, the TCR may be from one of a variety of animal species, such as a human, mouse, rat, or other mammal. The TCR may be associated with the cell or be in a soluble form. In some embodiments, for the purposes of the proposed methods, the TCR is in a cell-bound form expressed on the cell surface.

In some embodiments, the TCR is a full length TCR. In some embodiments, the TCR is an antigen binding fragment. In some embodiments, the TCR is a dimeric TCR (dTCR). In some embodiments, the TCR is a single chain TCR (scTCR). In some embodiments, the dTCR or ssTCR have the structures described in WO 03/020763, WO 04/033685, WO 2011/044186.

In some embodiments, the TCR contains a sequence corresponding to a transmembrane sequence. In some embodiments, the implementation of the TCR does not contain a sequence corresponding to the cytoplasmic sequence. In some embodiments, the TCR is capable of complexing the TCR with CD3. In some embodiments, any of the TCRs, including dTCR or ssTCR, can be associated with signaling domains, resulting in an active TCR on the T cell surface. In some embodiments, the TCR is expressed on the surface of cells.

In some embodiments, the dTCR comprises a first polypeptide wherein a sequence corresponding to the TCR α chain variable region sequence is fused to the N-terminus of a sequence corresponding to the TCR α chain extracellular constant region sequence, and a second polypeptide wherein the sequence corresponding to the TCR α chain variable region sequence region of the β-chain of TCR, fused to the N-terminus of the sequence corresponding to the extracellular sequence of the constant region of the β-chain of TCR, while the first and second polypeptides are linked by a disulfide bond. In some embodiments, the bond may correspond to a naturally occurring interchain disulfide bond found in a natural dimeric αβ TCR. In some embodiments, interchain disulfide bonds are not present in natural TCR. For example, in some embodiments, one or more cysteine residues may be included in the extracellular constant region sequences of a pair of dTCR polypeptides. In some cases, both natural and non-natural disulfide bonds may be desirable. In some embodiments, the implementation of the TCR contains a transmembrane sequence for anchoring in the membrane.

In some embodiments, the dTCR comprises a TCR α-chain containing an α-variable domain, a constant α-domain and a first dimerization motif linked to the C-terminus of the constant α-domain, and a TCR β-chain containing a variable β-domain, constant β -domain and a second dimerization motif linked to the C-terminus of the constant β-domain, wherein the first and second dimerization motifs readily interact to form a covalent bond between the amino acid in the first dimerization motif and the amino acid in the second dimerization motif, linking together the α-strand TCR and β-chain of TCR.

In some embodiments, the TCR is a scTCR. Typically, ssTCR can be prepared using methods known to those skilled in the art, see, for example, Soo Hoo, WF et al. PNAS (USA) 89, 4759 (1992); Wülfing, C. and Plückthun, A., J. Mol. Biol. 242, 655 (1994); Kurucz, I. et al. PNAS (USA) 90 3830 (1993); PCT International Publication Application Nos. WO 96/13593, WO 96/18105, WO99/60120, WO99/18129, WO 03/020763, WO2011/044186; and Schlueter, CJ et al. J. Mol. Biol. 256, 859 (1996). In some embodiments, the ssTCR contains an introduced non-natural interchain disulfide bond to facilitate binding of the TCR chains (see, for example, International PCT Published Application No. WO 03/020763). In some embodiments, the ssTCR is a non-disulfide truncated TCR in which heterologous leucine zippers fused to its C-terminus provide chain binding (see, for example, PCT International Published Application No. WO99/60120). In some embodiments, the scTCR comprises a TCR α chain variable domain covalently linked to a TCR β chain variable domain via a peptide linker (see, for example, PCT International Published Application No. WO99/18129).

In some embodiments, the scTCR comprises a first segment consisting of an amino acid sequence corresponding to the variable region of the TCR α chain, a second segment consisting of an amino acid sequence corresponding to the variable region of the TCR β chain fused to the N-terminus of the amino acid sequence corresponding to the extracellular constant sequence a TCR β-chain domain, and a linker sequence linking the C-terminus of the first segment to the N-terminus of the second segment.

In some embodiments, the scTCR comprises a first segment consisting of an α-chain variable region sequence fused to the N-terminus of an extracellular α-chain constant domain sequence and a second segment consisting of a β-chain variable region sequence fused to the N-terminus of an extracellular a β-chain constant and transmembrane sequence; and, optionally, a linker sequence linking the C-terminus of the first segment to the N-terminus of the second segment.

In some embodiments, the scTCR comprises a first segment consisting of the TCR β-chain variable region sequence fused to the N-terminus of the extracellular β-chain constant domain sequence and a second segment consisting of the α-chain variable region sequence fused to the N-terminus an extracellular constant and transmembrane α-chain sequence; and, optionally, a linker sequence linking the C-terminus of the first segment to the N-terminus of the second segment.

In some embodiments, the scTCR linker that binds the first and second segments of the TCR may be any linker capable of forming a single polypeptide chain while retaining TCR binding specificity. In some embodiments, the linker sequence may, for example, have the formula -P-AA-P-, where P is proline and AA is an amino acid sequence in which the amino acids are glycine and serine. In some embodiments, the first and second segments are paired such that their variable region sequences are oriented for such binding. Thus, in some cases, the linker is long enough to create a space between the C-terminus of the first segment and the N-terminus of the second segment, or vice versa, but not too long to block the decrease in scTCR binding to the target ligand. In some embodiments, the implementation of the linker may contain from, or from about 10 to 45 amino acids, such as 10-30 amino acids or 26-41 amino acid residues, such as 29, 30, 31 or 32 amino acids. In some embodiments, the linker has the formula -PGGG-(SGGGG)5-P-, where P is proline, G is glycine, and S is serine (SEQ ID NO: 23). In some embodiments, the implementation of the linker has the sequence GSADDAKKDAAKKDGKS (SEQ ID NO: 24).

In some embodiments, the ssTCR comprises a covalent disulfide bond linking a remainder of the immunoglobulin α chain constant domain region to a remainder of the immunoglobulin β chain constant domain region. In some embodiments, there is no interchain disulfide bond in natural TCR. For example, in some embodiments, one or more cysteine residues may be included in the extracellular sequences of the constant regions of the first and second segments of the scTCR polypeptide. In some cases, both natural and non-natural disulfide bonds may be desirable.

In some embodiments, dTCRs or ssTCRs containing introduced interchain disulfide bonds do not have natural disulfide bonds. In some embodiments, one or more naturally occurring cysteine residues forming natural interchain disulfide bonds are replaced by another residue, such as serine or alanine. In some embodiments, the introduced disulfide bond may be formed by mutating residues other than cysteine in the first and second segments to cysteine. Exemplary non-natural TCR disulfide bonds are described in PCT International Published Application No. WO2006/000830.

In some embodiments, the TCR, or antigen-binding fragment thereof, has an affinity with an equilibrium binding constant for the target antigen of from, or about, 10 ^-5 to 10 ^-12 M, including all individual values and ranges in between. In some embodiments, the target antigen is an MHC-peptide or ligand complex.

In some embodiments, the TCR-encoding nucleic acid or nucleic acids, such as the α and β chains, can be PCR amplified, cloned, or subjected to other appropriate procedures and cloned into a suitable expression vector or vectors. The expression vector may be any suitable recombinant expression vector, and may be used to transform or transfect any suitable host. Suitable vectors include vectors intended for propagation or expansion or expression or both, such as plasmids and viruses.

In some embodiments, the vector may be a pUC series (Fermentas Life Sciences), pBluescript series (Stratagene, LaJolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), or pEX series (Clontech, Palo Alto, Calif.). In some cases, bacteriophage-based vectors such as λG10, λGT11, λZapII (Stratagene), λEMBL4 and λNM1149 can also be used. In some embodiments, plant expression vectors can be used, which include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). In some embodiments, animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). In some embodiments, a viral vector is used, such as a retroviral vector.

In some embodiments, the implementation of recombinant expression vectors can be obtained using standard methods of recombinant DNA. In some embodiments, vectors may contain regulatory sequences, such as transcription and translation initiation sequences, and termination codons, that are specific to the type of host (e.g., bacteria, fungi, plants, or animals) into which the vector is to be introduced, as needed and with considering whether the vector is a DNA-containing or RNA-containing vector. In some embodiments, the vector may contain a non-natural promoter operably linked to a nucleotide sequence encoding a TCR or an antigen binding fragment (or other MHC peptide binding molecule). In some embodiments, the promoter may be a non-viral promoter or a viral promoter such as the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter, and a promoter present in the long terminal repeat of mouse stem cell virus. Other known promoters are also contemplated.

In some embodiments, to obtain a vector encoding a TCR, the α and β chains are PCR amplified from a total cDNA isolated from a T cell clone expressing the TCR of interest and cloned into an expression vector. In some embodiments, the α and β chains are cloned into the same vector. In some embodiments, the α and β chains are cloned into different vectors. In some embodiments, the generated α and β chains are introduced into a retroviral, eg, lentiviral, vector.

B. Nucleic acids and vectors

Also provided are one or more polynucleotides (eg, nucleic acid molecules) encoding recombinant receptors, vectors for genetic engineering of cells for expression of receptors, and methods for producing genetically engineered cells. In some aspects, the recombinant receptor is, or contains, a chimeric antigen receptor (CAR). In some aspects, the recombinant receptor is, or contains, a T cell receptor (TCR), such as a transgenic TCR.

In some cases, the nucleotide sequence encoding a recombinant receptor, for example, a chimeric antigen receptor (CAR), contains a signal sequence that encodes a signal peptide. Non-limiting illustrative examples of signal peptides include, for example, the GMCSFR alpha chain signal peptide of SEQ ID NO: 26 encoded by the nucleotide sequence shown in SEQ ID NO: 25 or the CD8 alpha chain signal peptide of SEQ ID NO: 18.

In some cases, when nucleic acid molecules encode two or more different polypeptide chains, each of the polypeptide chains may be encoded by a separate nucleic acid molecule. For example, two separate nucleic acids are provided and each can be individually transferred or introduced into a cell for expression in the cell.

In some embodiments, such as those in which the polynucleotide comprises first and second nucleotide sequences, the coding sequences encoding each of the different polypeptide chains may be operably linked to promoters that may be the same or different. In some embodiments, the implementation of the nucleic acid molecule may contain a promoter that controls the expression of two or more different polypeptide chains. In some embodiments, such nucleic acid molecules may be multicistronic (bicistronic or tricistronic, see, for example, US Pat. No. 6,060,273). In some embodiments, transcription units can be designed as a bicistronic unit containing an IRES (internal ribosome binding site) that allows co-expression of gene products from a single promoter template. Alternatively, in some cases, a single promoter may direct expression of RNA that contains two or three genes in a single open reading frame (ORF), separated from each other by sequences encoding a self-cleaving peptide (e.g., 2A sequences) or a protease recognition site (e.g., furin ). Thus, the ORF encodes a single polypeptide, which, either during the process (in the case of 2A) or after translation, is separated into separate proteins. In some cases, a peptide such as T2A can cause a ribosome skip (ribosome skip) to synthesize a peptide bond at the C-terminus of the 2A element, resulting in a separation between the end of the 2A sequence and the downstream peptide (see, e.g., de Felipe. Genetic Vaccines and Ther 2:13 (2004) and de Felipe et al Traffic 5:616-626 (2004)). Various 2A elements are known. Examples of 2A sequences that can be used in the methods and systems disclosed herein include, but are not limited to, 2A sequences from foot-and-mouth disease virus (F2A, e.g., SEQ ID NO: 22), equine rhinitis A virus (E2A, e.g., SEQ ID NO: 21), Thosea asigna virus (T2A, e.g. SEQ ID NO: 6 or 17) and porcine teshovirus-1 (P2A, e.g. SEQ ID NO: 19 or 20), as described in US Patent Publication No. 20070116690.

In some embodiments, external marker genes may, in certain cases, be used in combination with therapeutic engineered cells to enable cell discovery or selection, and in some cases also to promote cell self-destruction. Exemplary surrogate markers may include truncated forms of cell surface polypeptides, e.g., truncated forms that are non-functional and do not, or are unable to, transmit the signal or signal normally conveyed by the full-length form of the cell surface polypeptide and/or are not subject to, or are unable to undergo internalization. Exemplary truncated cell surface polypeptides include truncated forms of growth factor receptors or other receptors such as truncated human epidermal growth factor receptor 2 (tHER2), truncated epidermal growth factor receptor (tEGFR, illustrative sequence of tEGFR is given in SEQ ID NO: 7 or 16), or prostate specific membrane antigen (PSMA), or a modified form thereof. The tEGFR may contain an epitope recognized by the cetuximab ( ^Erbitux® ) or other therapeutic anti-EGFR antibody or binding molecule that can be used to identify or select cells that have been engineered with the tEGFR construct and the exogenous protein encoded, and/or to eliminate or separation of cells expressing the encoded exogenous protein. See US Pat. No. 8,802,374 and Liu et al., Nature Biotech. April 2016; 34(4): 430-434). In some aspects, the marker, eg, a surrogate marker, contains all or part (eg, a truncated form) of CD34, NGFR, CD19, or a truncated CD19, eg, truncated non-human CD19, or an epidermal growth factor receptor (eg, tEGFR).

In some embodiments, the marker is, or contains, a fluorescent protein, e.g., green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP) such as superfolded GFP (sfGFP), red fluorescent protein (RFP) such as tdTomato, mCherry, mStrawberry, AsRed2, DsRed or DsRed2, blue fluorescent protein (CFP), blue-green fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), and yellow fluorescent protein (YFP), and variants thereof, including species variants, monomeric variants; and codon-optimized and/or enhanced variants of fluorescent proteins. In some embodiments, the marker is, or contains, an enzyme such as luciferase, E. coli lacZ gene, alkaline phosphatase, secreted embryonic alkaline phosphatase (SEAP), chloramphenicol acetyltransferase (CAT). Exemplary light emitting reporter proteins include luciferase (luc), β-galactosidase, chloramphenicol acetyltransferase (CAT), β-glucuronidase (GUS), or variants thereof.

In some embodiments, the marker is a selectable marker. In some embodiments, the selectable marker is, or contains, a polypeptide that confers resistance to exogenous agents or drugs. In some embodiments, the selectable marker is an antibiotic resistance gene. In some embodiments, an antibiotic resistance selectable marker confers antibiotic resistance on a mammalian cell. In some embodiments, the selectable marker is, or comprises, a puromycin resistance gene, a hygromycin resistance gene, a blasticidin resistance gene, a neomycin resistance gene, a geneticin resistance gene, or a zeocin resistance gene, or a modified form thereof.

In some embodiments, a nucleic acid encoding a marker is operably linked to a polynucleotide encoding a linker sequence, such as a cleavable linker sequence, such as T2A. For example, the marker and optionally the linker sequence may be any of those described in PCT Publication No. WO2014031687. For example, the marker may be a truncated EGFR (tEGFR), which is optionally linked to a linker sequence, such as a cleavable T2A linker sequence. An exemplary truncated EGFR polypeptide (e.g., tEGFR) contains the amino acid sequence shown in SEQ ID NO: 7 or 16, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity with SEQ ID NO: 7 or 16. In some examples, a truncated epidermal growth factor receptor (EGFRt), for example, with the sequence of SEQ ID NO: 7 or 16, in some cases, can be co-expressed with the transgene of interest (CAR or TCR) in transduced cells (see, for example, US patent No. 8802374). EGFRt may contain an epitope recognized by the cetuximab antibody (Erbitux^®) or another therapeutic anti-EGFR antibody or binding molecule that can be used to identify or select cells that have been engineered with an EGFRt construct and another recombinant receptor such as a chimeric antigen receptor (CAR) and/or to eliminate or select cells, expressing the receptor. See U.S. Patent No. 8802374 and Liu Publicationet al., nature biotech. April 2016; 34(4): 430-434).

Also provided are vectors or constructs containing such nucleic acids and/or polynucleotides. In some embodiments, the vectors or constructs contain one or more promoters operably linked to a nucleic acid encoding a recombinant receptor to direct its expression. In some embodiments, the promoter is operably linked to one or more nucleic acid molecules or polynucleotides. Thus, vectors are also provided, such as those containing any of the polynucleotides provided herein.

In some instances, the vector is a viral vector such as a retroviral vector such as a lentiviral vector or a retroviral gamma vector. Compositions containing such vectors, or a combination of vectors, are also provided. In some embodiments, a set, or combination, of vectors is used together to construct cells. In some embodiments, the first and second vectors in the set are administered simultaneously or sequentially, in any order, to the construction cell.

In some embodiments, the vectors include viral vectors, e.g., retroviral or lentiviral, non-viral vectors or transposons, e.g., the sleeping beauty transposon system, vectors derived from simian virus 40 (SV40), adenoviruses, adeno-associated virus (AAV), lentiviral vectors or retroviral vectors such as retroviral gamma vectors, Moloney mouse leukemia virus (MoMLV) derived retroviral vector, myeloproliferative sarcoma virus (MPSV), mouse embryonic stem cell virus (MESV), mouse stem cell virus (MSCV), necrosis virus spleen (SFFV) or adeno-associated virus (AAV).

C. Genetic Engineering Vectors and Methods

Various methods of introducing genetically engineered components, such as recombinant receptors such as CAR or TCR, are well known. Illustrative methods include methods for transferring receptor-encoding nucleic acids using viral, eg, retroviral or lentiviral, transduction, transposons, and electroporation. In some embodiments, surface glycan expression is evaluated in a cell composition that was harvested before, during, or immediately after the engineering process. In some embodiments, surface glycan expression is assessed and compared between cell compositions at appropriate stages in the process of introducing genetically engineered components. In some embodiments, the compositions are or have been designed to express the same recombinant receptor but by different methods of introducing genetic material.

In some embodiments, gene transfer is performed by first stimulating the cell, for example by combining it with a stimulus that induces a response such as proliferation, survival, and/or activation, for example as measured by expression of a cytokine or an activation marker, followed by transduction of the activated cells, and propagating them in culture to quantities sufficient for clinical use.

In some embodiments, recombinant nucleic acids are transferred into cells using recombinant infectious viral particles, such as, for example, vectors derived from simian virus 40 (SV40), adenoviruses, adeno-associated virus (AAV). In some embodiments, recombinant nucleic acids are transferred into T cells using recombinant lentiviral vectors or retroviral vectors such as retroviral gamma vectors (see e.g. Koste et al. Gene Therapy doi: 10,1038/gt.2014.25 (2014) ; Carlens et al. Exp Hematol . , 28(10): 1137-46 (2000); Alonso-Camino et al. Mol Ther Nucl Acids, 2, e93 (2013); Park et al., Trends Biotechnol. , November 29 (11): 550-557 (2011) In some embodiments, the virus is an adeno-associated virus (AAV).

In some embodiments, the retroviral vector contains a long terminal repeat (LTR) sequence, e.g., a retroviral vector derived from Moloney mouse leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), mouse embryonic stem cell virus (MESV), mouse stem cell virus (MSCV), spleen necrosis virus (SFFV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, retroviruses include those derived from any avian or mammalian cell. Retroviruses are generally amphotropic, meaning that they are capable of infecting host cells of several species, including humans. In one embodiment, the gene to be expressed replaces the gag, pol and/or env retroviral gene sequences. Various illustrative retroviral systems have been described (see, for example, US Pat . ); Scarpa et al. Virology , 180:849-852 (1991); Burns et al. Proc. Natl. Acad. Sci. USA, 90:8033-8037 (1993); and Boris-Lawrie and Temin, Cur. Opin Genet Develop 3:102-109 (1993).

Methods for lentiviral transduction are known. Illustrative methods are described, for example, in Wang et al., J. Immunother., 35(9): 689-701 (2012); Cooper et al. Blood . 101:1637-1644 (2003); Verhoeyen et al., Methods Mol Biol ., 506: 97-114 (2009); and Cavalieri et al., Blood., 102(2): 497-505 (2003).

In some embodiments, recombinant nucleic acids are transferred into T cells by electroporation (see e.g., Chicaybam et al, PLoS ONE 8(3): e60298 (2013) and Van Tedeloo et al. Gene Therapy 7(16): 1431-1437 (2000)). In some embodiments, recombinant nucleic acids are transferred into T cells by transposition (see, e.g., Manuri et al. Hum Gene Ther 21(4): 427-437 (2010); Sharma et al. Molec Ther Nucl Acids 2, e74 ( 2013); and Huang et al. Methods Mol Biol 506: 115-126 (2009)). Other methods for introducing and expressing genetic material in immune cells include calcium phosphate transfection (eg, as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York. NY), protoplast fusion, cationic liposome-mediated transfection; bombardment with tungsten microparticles (Johnston, Nature , 346: 776-777 (1990)); and coprecipitation of DNA with strontium phosphate (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).

Other approaches and vectors for the transfer of nucleic acids encoding recombinant products are described, for example, in International Patent Application Publication No. WO2014055668 and US Patent No. 7446190.

In some embodiments, cells, such as T cells, may be transfected either during or after expansion, such as with a T cell receptor (TCR) or a chimeric antigen receptor (CAR). This transfection to introduce the desired receptor gene can be performed, for example, using any suitable retroviral vector. The population of genetically modified cells can then be released from the initial stimulus (a CD3/CD28 stimulus, for example), followed by stimulation with a second type of stimulus, for example, via a de novo introduced receptor). This second type of stimulus may include an antigenic stimulus in the form of a peptide/MHC molecule, a cognate (cross-linking) ligand of a genetically introduced receptor (e.g., a natural CAR ligand), or any ligand (such as an antibody) that directly binds within the framework of the novel receptor (e.g. , due to the recognition of constant regions in the receptor). See, for example, Cheadle et al, Methods Mol Biol . 907:645-66 (2012); or Barrett et al., Chimeric Antigen Receptor Therapy for Cancer Annual Review of Medicine, Vol. 65:333-347 (2014).

In some cases, a vector may be used that does not require cells, such as T cells, to be activated. In some of these cases, cells may be selected and/or transduced prior to activation. Thus, cells can be constructed before or after cell culture, and in some cases simultaneously, or during at least part of the culture process.

In some aspects, the cells are further engineered to stimulate the expression of cytokines or other factors. Additional nucleic acids, eg, genes to be introduced, include those that increase the efficacy of therapy, eg, by stimulating the viability and/or function of the cells into which they are transferred; marker genes for selection and/or evaluation of cells, for example, for evaluation of survival or localization in vivo ; genes to improve safety, for example, allowing negative selection of cells in vivo , as described in Lupton SD et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319-338 (1992); see also PCT/US91/08442 and PCT/US94/05601 by Lupton et al. which describe the use of bifunctional selective fusion genes resulting from the fusion of a dominant positive selectable marker with a negative selectable marker. See, for example, Riddell et al., US Pat. No. 6,040,177, columns 14-17.

D. Signs of the produced composition

In specific embodiments, the implementation of the methods proposed herein, allow you to get or create a composition of cells containing genetically engineered cells, for example, manufactured composition. In specific embodiments, the composition produced is a composition of cells that is obtained using some or all of the steps to obtain genetically engineered cells. In specific embodiments, the implementation of the produced composition is obtained by a method of obtaining genetically engineered cells from the original cell composition. In specific embodiments, the implementation of the method includes one or more steps of activation, transduction or transfection, expansion and/or collection of cells, such as cells that were obtained from the original cell composition. In specific embodiments, the cell composition produced contains cells that have been genetically engineered. In specific embodiments, the cells of the produced composition have been put through all steps of the genetic engineering method.

In some embodiments, the manufactured composition comprises cells containing one or more genetically engineered nucleic acids and, as a result, expressing recombinant or genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous, that is, not normally found in a cell or sample derived from a cell, e.g., derived from another organism or cell, e.g., not normally found in a cell being engineered and/or in an organism from which the cell is derived. In some embodiments, the nucleic acids are not naturally occurring, for example, the nucleic acid is not naturally occurring, including nucleic acid containing chimeric combinations of nucleic acids encoding different domains from several different cell types.

In some embodiments, the implementation of the manufactured composition contains cells that have been subjected to genetic engineering. In specific embodiments, the cell composition produced contains engineered T cells. In some embodiments, engineered T cells include engineered CD4+ T cells and engineered CD8+ T cells. In specific embodiments, the composition produced contains or includes at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% , at least 99.5%, at least 99.9%, or 100%, or about 100%, engineered T cells. In specific embodiments, the genetically engineered cells express the recombinant receptor. In specific embodiments, the composition produced contains or includes at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% , at least 99.5%, at least 99.9%, or 100%, or about 100% of T cells expressing the recombinant receptor. In some embodiments, the implementation of the recombinant receptor is a TCR or CAR. In specific embodiments, the implementation of the recombinant receptor is a CAR.

III. Compositions and preparations

Provided herein are compositions or preparations containing cells obtained by the incubation (eg, stimulation) methods described herein. In some embodiments, the compositions and methods described herein can be used to eliminate at least a portion of cell populations, such as non-naïve-like T cells, from a population of cells. Also provided are compositions containing populations of cells that no longer contain unwanted cells, or contain significantly fewer unwanted cells in the stimulated composition, such as cells derived from non-naïve T cells, and uses thereof. The compositions and methods provided herein are also used to selectively propagate a population of cells from which unwanted subpopulations have been removed for use in therapy. Also provided herein is a manufactured, or enriched, stimulated composition prepared by any of the T cell stimulation methods described herein.

In some embodiments, cells obtained using any of the incubation (e.g., stimulation) methods described herein, e.g., recombinant receptor genetically engineered cells (e.g., CAR-T cells), are provided as compositions, including pharmaceutical compositions and formulations, such as unit dosage form compositions containing cells to be administered in a specific dose or portion thereof. Pharmaceutical compositions and formulations typically contain one or more optional pharmaceutically acceptable carriers or excipients. In some embodiments, the implementation of the composition contains at least one additional therapeutic agent.

In some embodiments, the implementation of the cell composition receive, or produce, for the purposes of cell therapy. In some embodiments, the implementation of the cell composition is a pharmaceutical composition or drug. Such compositions can be used in accordance with the proposed methods, for example, for evaluation at their release for use in the prevention or treatment of diseases, conditions and disorders, or in methods of detection, diagnosis and prognosis.

The term "pharmaceutical preparation" means a preparation in a form that provides effective biological activity of the active ingredient contained in the preparation, and which does not contain any additional components that are unacceptably toxic to the subject to whom the preparation will be administered. In some embodiments, the methods provided herein can be used to compare the surface expression of a glycan in cell compositions consisting of the same genetically engineered cells but in different pharmaceutical formulations.

A "pharmaceutically acceptable carrier" means an ingredient in a pharmaceutical preparation, other than the active ingredient, that is non-toxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative. In specific embodiments, the methods provided herein can be used to compare the surface expression of a glycan in cell compositions consisting of the same engineered cells but with different pharmaceutically acceptable carriers.

In some embodiments, a T cell therapeutic, such as engineered T cells (eg, CAR T cells), is formulated with a pharmaceutically acceptable carrier. In some aspects, the choice of carrier is determined in part by the particular cell and/or route of administration. Accordingly, there are various suitable preparations. For example, the pharmaceutical composition may contain preservatives. Suitable preservatives may include, for example, methyl paraben, propyl paraben, sodium benzoate and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservatives, or mixtures thereof, are typically present in an amount of from about 0.0001% to about 2% by weight of the total weight of the composition. Carriers are described, for example, in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations used, and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG).

In some aspects, buffering agents are included in the composition. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffer agents is used. Buffers, or mixtures thereof, are typically present in an amount of from about 0.001% to about 4% by weight of the total weight of the composition. Methods for preparing injectable pharmaceutical compositions are known. Illustrative methods are described in more detail, for example, in Remington: Science and Practice of Pharmacy, Lippincott Williams &Wilkins; 21st Edition (May 1, 2005).

Formulations may include aqueous solutions. The preparation or composition may also contain more than one active ingredient useful in the prevention of a particular disorder, disease or condition by cells, including one or more active ingredients whose activities are complementary to those of the cells and/or the respective activities do not mutually negatively influence . Such active ingredients are preferably present in combination in amounts that are effective for the intended purpose. Thus, in some embodiments, the pharmaceutical composition further comprises other pharmaceutically active agents or drugs such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine and so on.

In some embodiments, the pharmaceutical composition contains cells in amounts effective to treat or prevent a disease or condition, such as therapeutically effective or prophylactically effective amounts. In some embodiments, therapeutic or prophylactic efficacy is monitored by periodic evaluation of treated subjects. In the case of repeated administrations over several days or more, depending on the condition, the treatment is repeated until the desired suppression of the symptoms of the disease is achieved. However, other dosing regimens may be useful and may be determined. The desired dose may be delivered by a single bolus of the composition, by multiple boluses of the composition, or by administering the composition as a continuous infusion.

Cells may be formulated for administration using standard administration methods, formulations and/or devices. Formulations and devices such as syringes and vials are provided for storing and administering the compositions. When applied to cells, administration may be autologous or heterologous. For example, immunoreactive or progenitor cells may be obtained from one subject and administered to the same subject or another compatible subject. Peripheral blood-derived immunoreactive cells or their progeny (eg, derived in vivo , ex vivo or in vitro ) can be administered by localized injection, including catheter, systemic injection, localized injection, intravenous injection, or parenteral administration. When a therapeutic composition (eg, a pharmaceutical composition containing genetically modified immunoreactive cells) is administered, it will typically be formulated in a unit injectable dosage form (such as a solution, suspension, emulsion).

Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the agent or cell populations are administered parenterally. As used herein, the term "parenteral" administration includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the agent or cell populations are administered to a subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

In some embodiments, compositions are provided as sterile liquid preparations, eg, isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which in some aspects can be buffered to a selected pH. Liquid formulations are generally easier to prepare than gels, other viscous formulations, and solid formulations. In addition, liquid formulations are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within a suitable viscosity range to provide longer periods of contact with particular tissues. Liquid or viscous compositions may contain carriers, which may be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, a polyol (eg, glycerol, propylene glycol, liquid polyethylene glycol), as well as their respective mixtures.

Sterile injectable solutions can be obtained by incorporating the cells into a solvent, for example as a mixture with an appropriate carrier, diluent or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions may also be lyophilized. The compositions may contain adjuvants such as wetting, dispersing or emulsifying agents (e.g. methylcellulose), pH buffering agents, gelling or viscosity enhancing agents, preservatives, flavoring agents, coloring agents, and the like, depending on the route of administration and the desired formulation. In some aspects, you can refer to standard guidelines for the preparation of appropriate drugs.

Various additives may be added to enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of an injectable pharmaceutical form can be achieved through the use of agents that delay absorption, such as aluminum monostearate and gelatin.

The preparations used for in vivo administration are generally sterile. Sterility can be achieved, for example, by filtration through membranes for sterilization by filtration.

For the prevention or treatment of a disease, the appropriate dose may depend on the type of disease to be treated, the type of agent or agents, the type of cell or recombinant receptor, the severity and course of the disease, whether the agent or cells are being administered prophylactically or therapeutically, previous therapy, the subject's clinical history and response to the agent or cells, and the judgment of the attending physician. In some embodiments, the implementation of the composition is preferably administered to the subject once or in a series of procedures.

Also provided are methods and uses for cells and compositions, such as those present in the manufactured composition described herein, in the treatment of diseases, conditions, and disorders in which an antigen recognized by a recombinant receptor (eg, CAR) is expressed. Also provided herein are methods of treatment comprising administering to a subject a manufactured, or enriched, manufactured composition obtained by any incubation (eg, stimulation) method described. In some embodiments, the method includes obtaining engineered T cells using any of the methods described herein, and introducing genetically engineered T cells obtained by the methods described herein.

Methods are provided for administering engineered cells and compositions, as well as using such engineered cells and compositions to treat or prevent diseases, conditions, and disorders, including cancer. In some embodiments, cell therapy is, or includes, the administration of cells, such as immune cells, such as T cells, that are directed to a molecule expressed on the surface of a lesion, such as a tumor or cancer. Suggested methods and uses include methods and uses for adoptive cell therapy. In some embodiments, the methods include introducing engineered cells, or a composition comprising cells, such as cells from the described manufactured composition, into a tissue or cell of a subject, such as suffering from, or at risk of developing, or suspected of suffering from, a disease, condition, or disorder. . In some embodiments, cells, populations, and compositions are administered to a subject having a particular disease or condition to be treated, for example, by an adoptive cell therapy, such as adoptive T cell therapy. In some embodiments, cells or compositions are administered to a subject, such as a subject suffering from, or at risk of developing, a disease or condition, to alleviate one or more symptoms of the disease or condition, for example, to reduce the tumor burden in a form of cancer in which an antigen is expressed. , recognizable by engineered T cells.

In some aspects, the disease or condition being treated may be any one in which antigen expression is associated with, characteristic of, and/or occurs on a cell or tissue associated with the disease, disorder, or condition, and/or is involved in the etiology of a disease, condition, or disorder, for example, causes, aggravates, or is otherwise involved in such disease, condition, or disorder. Exemplary diseases and conditions may include diseases or conditions associated with malignancy or cell transformation (eg, cancer), an autoimmune or inflammatory disease, or an infectious disease, eg, caused by a bacterial, viral, or other pathogen. Exemplary antigens, including antigens associated with various diseases and conditions that may be treated, are described above. In specific embodiments, an immunomodulatory polypeptide and/or a recombinant receptor, such as a chimeric antigen receptor or TCR, specifically binds an antigen associated with a disease or condition. In some embodiments, the subject has a disease, disorder or condition, optionally cancer, tumor, autoimmune disease, disorder or condition, or infectious disease.

In some embodiments, the disease, disorder, or condition includes tumors associated with various forms of cancer. In some embodiments, the cancer may be any form of cancer in the subject's body, such as, but not limited to, cancer affecting the head and neck, breast, liver, colon, ovary, prostate, pancreas, brain, cervix, bones, skin, eyes, bladder, stomach, esophagus, peritoneum, or lungs. For example, an anticancer agent can be used to treat colon cancer, cervical cancer, central nervous system cancer, breast cancer, bladder cancer, anal carcinoma, head and neck cancer, ovarian cancer, endometrial cancer, small cell lung cancer, non-small cell lung carcinoma. , neuroendocrine cancer, soft tissue carcinoma, penile cancer, prostate cancer, pancreatic cancer, gastric cancer, gallbladder cancer, or esophageal cancer. In some cases, the cancer may be blood cancer. In some embodiments, the disease, disorder, or condition is a tumor, such as a solid tumor, lymphoma, leukemia, a hematopoietic tumor, a metastatic tumor, or another type of cancer or tumor. In some embodiments, the disease, disorder, or condition is selected from colon, lung, liver, breast, prostate, ovarian, skin, melanoma, bone, brain, ovarian, epithelial, renal cell, pancreatic adenocarcinoma , cervical carcinoma, colorectal cancer, glioblastoma, neuroblastoma, Ewing's sarcoma, medulloblastoma, osteosarcoma, synovial sarcoma and/or mesothelioma.

Diseases, conditions and disorders include tumors, including solid tumors, blood cancers and melanomas, and including localized and metastatic tumors, infectious diseases such as infection caused by a virus or other pathogen, such as HIV, HCV, HBV, CMV, HPV, parasitic diseases, and autoimmune and inflammatory diseases. In some embodiments, the disease, disorder, or condition is a tumor, cancer, malignancy, or other proliferative disease or disorder. Such diseases include, but are not limited to, leukemia, lymphoma, e.g., acute myeloid (or myelogenous) leukemia (AML), chronic myeloid (or myelogenous) leukemia (CML), acute lymphocytic (or lymphoblastic) leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone cell lymphoma, Burkitt's lymphoma, Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), anaplastic large cell lymphoma (ALCL), follicular lymphoma, refractory follicular lymphoma, diffuse large B-cell lymphoma (DLBCL), and multiple myeloma (MM); The B-cell malignancy is selected from acute lymphoblastic leukemia (ALL), adult ALL, chronic lymphoblastic leukemia (CLL), non-Hodgkin's lymphoma (NHL), and diffuse large B-cell lymphoma (DLBCL).

In some embodiments, the disease or condition is an infectious disease or condition such as, but not limited to, viral, retroviral, bacterial, and protozoan infection, immunodeficiency, cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus , BK polyomavirus. In some embodiments, the disease or condition is an autoimmune or inflammatory disease or condition, such as arthritis, e.g., rheumatoid arthritis (RA), type I diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, psoriasis, scleroderma, autoimmune thyroid disease glands, Graves' disease, Crohn's disease, multiple sclerosis, asthma, and/or a transplant related disease or condition.

In some embodiments, the antigen associated with the disease or disorder is selected from the group consisting of orphan tyrosine kinase receptor ROR1, B cell maturation antigen (BCMA), carbonic anhydrase 9 (CAIX), tEGFR, Her2/neu (erbB2 tyrosine kinase receptor), L1-CAM, CD19, CD20, CD22, mesothelin, CEA, hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 ( EPG-40), aphrin A2 receptor (EPHa2), Her2/neu (erb-B2 tyrosine kinase receptor), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, mutant epidermal growth factor receptor type III (EGFR vIII ), folate binding protein (FBP), FCRL5, Fc receptor-like protein 5 (FCRL5; also known as the Fc receptor 5 homologue or FCRH5), embryonic acetylcholine receptor, GD2 ganglioside, G protein-coupled 5D receptor ganglioside GD3 (GPCR5D), HMW-MAA, IL-22R-alpha, IL-13R-alpha 2, kinase insert domain receptor (kdr), kappa light chain, member of the A family of 8 proteins containing leucine-rich repeats (LRRC8A), Lewis Y, L1 cell adhesion molecules (L1-CAM), melanoma associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, preferably expressed melanoma antigen (PRAME), survivin, TAG72, B7-H6, receptor alpha 2 IL- 13 (IL-13Ra2), CA9, GD3, HMW-MAA, CD171, G250/CAIX, human leukocyte antigen A1 (HLA-A1), MAGE A1, HLA-A2, NY-ESO-1, PSCA, folate receptor-α , CD44v6, CD44v7/8, αvβ6 integrin (avb6 integrin), 8H9, NCAM, VEGF receptors, 5T4, embryonic AchR, natural killer group 2 D ligands (NKG2D), CD44v6, dual antigen, testicular cancer antigen, mesothelin, mouse CMV, mucin 1 (MUC1), MUC16, prostatic stem cell antigen (PSCA), NKG2D, cancer-testicular antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2), MART-1, glycoprotein 100 (gp100 ), oncoembryonic antigen, ROR1, trophoblastic glycoprotein (TPBG, also known as 5T4), TAG72, VEGF-R2, carcinoembryonic antigen (CEA), Her2/neu, estrogen receptor, progesterone receptor, ephrin B2, CD123, c-Met, GD -2, O-acetylated GD2 (OGD2), CE7, Wilms tumor antigen 1 (WT-1), cyclin, cyclin A2, C-C motif chemokine ligand 1 (CCL-1), CD138, pathogen-specific antigen and antigen associated with a universal marker, and/or biotinylated molecules, and/or molecules expressed by HIV, HCV, HBV or other pathogens.

In some embodiments, the antigen or ligand is a tumor antigen or cancer marker. In some embodiments, the antigen or ligand is, or includes, an αvβ6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), cancer testicular antigen, cancer testicular antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), cyclin, cyclin A2, C-C motif chemokine ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), mutant epidermal growth receptor growth factor type III (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin A2 receptor (EPHa2), estrogen receptor, Fc receptor-like protein 5 (FCRL5; also known as the Fc receptor homologue 5 or FCRH5), embryonic acetylcholine receptor (embryonic AchR), folate binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp100) , glypican-3 (GPC3), G protein-coupled receptor 5D (GPRC5D), Her2/neu (erbB2 tyrosine kinase receptor), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, human macromolecular antigen, coupled with melanoma (HMW-MAA), hepatitis B surface antigen, human leukocyte antigen A1 (HLA-A1), human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha (IL-22Rα), IL-2 receptor alpha 13 (IL-13Rα2), kinase insert domain receptor (kdr), kappa light chain, L1 cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM, member of the A family of 8 proteins containing leucine-rich repeats (LRRC8A), Lewis Y, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, mouse cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, representative ligands Natural killer group 2 D (NKG2D), melan A (MART-1), neuronal cell adhesion molecule (NCAM), oncoembryonic antigen, preferentially expressed melanoma antigen (PRAME), progesterone receptor, prostate specific antigen, prostate stem cell antigen (PSCA) , prostate specific membrane antigen (PSMA), tyrosine kinase receptor-like orphan receptor 1 (ROR1), survivin, trophoblastic glycoprotein (TPBG, also known as 5T4), tumor-associated glycoprotein 72 (TAG72), tyrosinase-dependent protein 1 (TRP1, also known as TYRP1 or gp75), tyrosinase-dependent protein 2 (TRP2, also known as dopachrome tautomerase, dopachrome delta isomerase or DCT), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), antigen Wilms tumor 1 (WT-1), a pathogen-specific or pathogen-expressed antigen, or an antigen associated with a universal marker, and/or biotinylated molecules, and/or molecules expressed by HIV, HCV, HBV or other pathogens.

In some embodiments, the disease or condition is a B-cell malignancy. In some embodiments, the B-cell malignancy is a leukemia or lymphoma. In some aspects, the disease or condition is acute lymphoblastic leukemia (ALL), adult ALL, chronic lymphoblastic leukemia (CLL), non-Hodgkin's lymphoma (NHL), or diffuse large B-cell lymphoma (DLBCL). In some instances, the disease or condition is NHL, such as or including NHL, which is aggressive NHL, diffuse large B cell lymphoma (DLBCL), NOS ( de novo and degenerated from slow growing), primary mediastinal large B-cell cellular lymphoma (PMBCL), large B-cell lymphoma rich in T-cells/histiocytes (TCHRBCL), Burkitt's lymphoma, mantle cell lymphoma (MCL) and/or follicular lymphoma (FL), optionally stage 3B follicular lymphoma (FL3B) . In some aspects, a recombinant receptor, such as CAR, specifically binds an antigen associated with a disease or condition, or expressed in cells surrounding a disease associated with a B-cell malignancy. In some embodiments, the antigens targeted by the receptors include antigens associated with B-cell cancer, for example, any of a variety of known B-cell markers. In some embodiments, the receptor target antigen is CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Ig-kappa, Ig-lambda, CD79a, CD79b, or CD30.

In some embodiments, the disease or condition is a myeloma, such as multiple myeloma. In some aspects, a recombinant receptor, such as CAR, specifically binds an antigen associated with a disease or condition, or expressed in cells surrounding a disease associated with multiple myeloma. In some embodiments, the receptor-targeted antigens include multiple myeloma-associated antigens such as GPRC5d or BCMA.

In some embodiments, the antigen is a pathogen-specific or pathogen-expressed antigen. In some embodiments, the antigen is a viral antigen (such as a viral antigen from HIV, HCV, HBV, and so on), bacterial antigens, and/or parasitic antigens.

In some embodiments, immune cells express a T cell receptor (TCR) or other antigen-binding receptor. In some embodiments, the immune cells express a recombinant receptor, such as a transgenic TCR or a chimeric antigen receptor (CAR). In some embodiments, the cells are autologous to the subject. In some embodiments, the cells are allogeneic to the subject.

Methods for introducing engineered cells for adoptive cell therapy are known and can be used in combination with the proposed methods and compositions. For example, adoptive T cell therapy techniques are described, for example, in US Patent Application Publication No. 2003/0170238 by Gruenberg et al. ; US patent No. 4690915 issued to Rosenberg; Rosenberg Publications (2011) Nat Rev Clin Oncol. 8(10):577-85). See, for example, Themeli et al. , (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. , (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. , (2013) PLoS ONE 8(4): e61338.

In some embodiments, cell therapy, such as adoptive T cell therapy, is performed by autologous transfer, in which cells are isolated and/or otherwise obtained from a subject to receive cell therapy, or from a sample obtained from such a subject. Thus, in some aspects, cells are obtained from a subject, such as a patient in need of treatment, and the cells, after isolation and processing, are administered to the same subject.

In some embodiments, cell therapy, such as adoptive T-cell therapy, is performed by allogeneic transfer, in which cells are isolated and/or otherwise obtained from a subject other than the subject to be, or who ultimately receives, cell therapy. , for example, the first subject. In such embodiments, the cells are then administered to another subject, such as a second subject of the same species. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses molecules of the same class or supertype of HLA as the first subject.

In specific embodiments, cells, or distinct populations of cell subtypes, are administered to a subject in a range of amounts from about one million to about 100 billion cells, and/or a number of cells per kilogram of body weight, such as, for example, from 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or in the range determined by any two of the listed values), e.g., about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells , about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or in the range of any two of the above values), and in some cases from about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value in between these ranges, and/or per kilogram of body weight. Doses may vary depending on the characteristics of the disease or disorder and/or the patient and/or other treatments.

In some embodiments, for example, when the subject is human, the dose comprises less than about 5×10 ⁸ of all recombinant receptor (eg, CAR) expressing cells, T cells, or peripheral blood mononuclear cells (PBMCs). In some embodiments, for example, when the subject is a human, the dose comprises less than about ^1x108 of all recombinant receptor (e.g., CAR) expressing cells, T cells, or peripheral blood mononuclear cells (PBMCs), e.g., in the range of about 1x 10 ⁶ to 1x10 ⁸ such cells, for example 2x10 ⁶ , 5x10 ⁶ , 1x10 ⁷ , 5x10 ⁷ , or 1x10 ⁸ such cells in total, or a range between any two of the above.

In some embodiments, the dose of engineered cells is from, or about, 1x10 ⁵ to 5x10 ⁸ total CAR-expressing T cells, 1x10 ⁵ to 2.5x10 ⁸ total CAR-expressing T -cells, 1×10 ⁵ to 1×10 ⁸ total CAR-expressing T cells, 1×10 ⁵ to 5×10 ⁷ total CAR-expressing T cells, 1×10 ⁵ to 2.5×10 ⁷ total CAR-expressing T cells, 1x10 ⁵ to 1x10 ⁷ total CAR-expressing T cells, 1x10 ⁵ to 5x10 ⁶ total CAR-expressing T cells, 1x10 ⁵ up to 2.5×10 ⁶ total CAR-expressing T cells, 1×10 ⁵ to 1×10 ⁶ total CAR-expressing T cells, 1×10 ⁶ to 5×10 ⁸ total CAR-expressing T cells , 1×10 ⁶ to 2.5×10 ⁸ total CAR-expressing T cells, 1×10 ⁶ to 1×10 ⁸ total CAR-expressing T cells, 1×10 ⁶ to 5×10 ⁷ total CAR-expressing T cells, from 1x10 ⁶ to 2.5x10 ⁷ total CAR-expressing T cells, from 1x10 ⁶ to 1x10 ⁷ total CAR-expressing T cells, from 1x10 ⁶ up to 5×10 ⁶ total CAR-expressing T cells, from 1×10 ⁶ to 2.5×10 ⁶ total CAR-expressing T cells, from 2.5×10 ⁶ to 5×10 ⁸ total CAR-expressing T -cells, from 2.5×10 ⁶ to 2.5×10 ⁸ total CAR-expressing T-cells, from 2.5×10 ⁶ to 1×10 ⁸ total CAR-expressing T-cells, from 2.5× 10 ⁶ to 5x10 ⁷ total CAR-expressing T cells, 2.5x10 ⁶ to 2.5x10 ⁷ total CAR-expressing T cells, 2.5x10 ⁶ to 1x10 ⁷ total CAR-expressing T cells, 2.5×10 ⁶ to 5×10 ⁶ total CAR-expressing T cells, 5×10 ⁶ to 5×10 ⁸ total CAR-expressing T cells, 5×10 ⁶ up to 2.5x10 ⁸ total CAR-expressing T cells, 5x10 ⁶ to 1x10 ⁸ total CAR-expressing T cells, 5x10 ⁶ to 5x10 ⁷ total CAR-expressing T cells , 5×10 ⁶ to 2.5×10 ⁷ total CAR-expressing T cells, 5×10 ⁶ to 1×10 ⁷ total CAR-expressing T cells, 1×10 ⁷ to 5×10 ⁸ total CAR-expressing T cells, 1×10 ⁷ to 2.5×10 ⁸ total CAR-expressing T cells, 1×10 ⁷ to 1×10 ⁸ total CAR-expressing T cells, 1×10 ⁷ up to 5×10 ⁷ total CAR-expressing T cells, from 1×10 ⁷ to 2.5×10 ⁷ total CAR-expressing T cells, from 2.5×10 ⁷ to 5×10 ⁸ total CAR-expressing T -cells, from 2.5×10 ⁷ to 2.5×10 ⁸ total CAR-expressing T-cells, from 2.5×10 ⁷ to 1×10 ⁸ total CAR-expressing T-cells, from 2.5× ¹⁰⁷ to ^5x107 total CAR-expressing T cells, ^{5x107 to} 5x108 total CAR-expressing T cells, ^5x107 to ^2.5x108 total CAR-expressing T -cells, 5×10 ⁷ to 1×10 ⁸ total CAR-expressing T cells, 1×10 ⁸ to 5×10 ⁸ total CAR-expressing T cells, 1×10 ⁸ to 2.5×10 ⁸ total CAR-expressing T cells or 2.5×10 ⁸ to 5×10 ⁸ total CAR-expressing T cells.

In some embodiments, the dose of genetically engineered cells contains at least, or at least about, 1x10 ⁵ CAR-expressing cells, at least, or at least about 2.5x10 ⁵ CAR-expressing cells, at least, or at least about, 5x10 ⁵ CAR-expressing cells, at least, or at least about 1x10 ⁶ CAR-expressing cells, at least, or at least about 2, 5×10 ⁶ CAR-expressing cells, at least, or at least about 5×10 ⁶ CAR-expressing cells, at least, or at least about 1×10 ⁷ CAR-expressing cells, at least , or at least about 2.5×10 ⁷ CAR-expressing cells, at least, or at least about 5×10 ⁷ CAR-expressing cells, at least, or at least about 1×10 ⁸ CAR-expressing cells, at least, or at least about 2.5×10 ⁸ CAR-expressing cells, or at least, or at least about 5×10 ⁸ CAR-expressing cells.

In some embodiments, cell therapy comprises administering a dose containing a number of cells from, or about, 1×10 ⁵ to 5×10 ⁸ total recombinant receptor expressing cells, total T cells, or total peripheral blood mononuclear cells (PBMCs) , from, or from about 5×10 ⁵ to 1×10 ⁷ total expressing recombinant receptor cells, total T-cells, or total peripheral blood mononuclear cells (PBMC), or from, or from about 1×10 ⁶ to 1 x10 ⁷ total recombinant receptor expressing cells, total T cells, or total peripheral blood mononuclear cells (PBMCs), including all ranges. In some embodiments, cell therapy comprises administering a dose containing at least, or at least about, 1 x ¹⁰⁵ total recombinant receptor expressing cells, total T cells, or total peripheral blood mononuclear cells (PBMCs), for example, at least, or at least about, 1×10 ⁶ at least, or at least about 1×10 ⁷ at least, or at least about, 1×10 ⁸ such cells. In some embodiments, the number refers to the total number of CD3+ or CD8+, in some cases, also expressing the recombinant receptor (eg, CAR+) cells. In some embodiments, cell therapy comprises administering a dose containing a number of cells from, or about, 1×10 ⁵ to 5×10 ⁸ CD3+ or CD8+ total T cells, or CD3+ or CD8+ recombinant receptor-expressing cells, from, or from about 5×10 ⁵ to 1×10 ⁷ CD3+ or CD8+ total T cells, or CD3+ or CD8+ recombinant receptor-expressing cells, or from or from about 1×10 ⁶ to 1×10 ⁷ CD3+ or CD8+ total T -cells, or CD3+ or CD8+ cells expressing the recombinant receptor, including all ranges. In some embodiments, cell therapy comprises administering a dose containing a cell count of from, or from about 1×10 ⁵ to 5×10 ⁸ total CD3+/CAR+ or CD8+/CAR+ cells, from, or from about 5×10 ⁵ up to 1x10 ⁷ total CD3+/CAR+ or CD8+/CAR+ cells, or from or from about 1x10 ⁶ to 1x10 ⁷ total CD3+/CAR+ or CD8+/CAR+ cells, including all ranges.

In some embodiments, the T cell dose includes CD4+ T cells, CD8+ T cells, or CD4+ and CD8+ T cells.

In some embodiments, for example, when the subject is human, the dose of CD8+ T cells included in a dose containing CD4+ and CD8+ T cells is from about 1×10 ⁶ to 5×10 ⁸ of the total expression of the recombinant receptor (e.g., CAR ) CD8+ cells, for example, in the range from about 5x10 ⁶ to 1x10 ⁸ such cells, 1x10 ⁷ , 2.5x10 ⁷ , 5x10 ⁷ , 7.5x10 ⁷ , 1x10 ⁸ or 5×10 ⁸ such cells, or in the range between any two of the above values. In some embodiments, multiple doses are administered to a patient, and each of the doses, or the total dose, may be within the above ranges. In some embodiments, a dose of cells is administered from, or about, 1 x 10 ⁷ to 0.75 x 10 ⁸ total expressing the recombinant CD8+ receptor T cells, from 1 x 10 ⁷ to 2.5 x 10 ⁷ total expressing the recombinant receptor CD8+ T cells, from, or from about 1×10 ⁷ to 0.75×10 ⁸ total expressing recombinant CD8+ receptor T cells, including all ranges. In some embodiments, a dose of cells is administered that is, or is about, 1×10 ⁷ , 2.5×10 ⁷ , 5×10 ⁷ 7.5×10 ⁷ , 1×10 ⁸ , or 5×10 ⁸ total recombinant receptor expressing CD8+ T cells.

In some embodiments, a dose of cells, e.g., expressing a recombinant T cell receptor, is administered to a subject in a single dose or is administered only once for two weeks, one month, three months, six months, 1 year, or more.

In some aspects, pharmaceutical compositions and formulations are provided as unit dosage form compositions containing the number of cells to be administered in a particular dose or portion thereof. In some embodiments, the proposed methods allow cells to be obtained at predictable times for dosing, compared to other methods of incubation (eg, stimulation) of cells. In some cases, the dose of cells to be administered is determined based on the number of naive-like cells in the original composition. In some embodiments, a dose containing cells obtained using a method including a shorter stimulation period (e.g., in some embodiments, the incubation period is, or is about, 2 days, 3 days, 4 days, 5 days, or 6 days of cell incubation with a stimulant) may be lower than the dose containing cells obtained by a method involving a longer stimulation period. In some embodiments, a dose containing cells obtained using a method including a shorter stimulation period may contain a higher percentage or proportion of naive-like cells and may contain a lower total number of cells compared to a dose containing cells obtained with using a method including a longer stimulation period.

In some embodiments, the implementation of the dose of cells can be administered by introducing several compositions, or solutions, for example, the first and second or, optionally, more, each of which contains a certain dose of cells. In some aspects, multiple compositions, each containing different cell populations and/or subtypes, are administered separately or independently, optionally, over a period of time. For example, cell populations or subtypes may include CD8 ⁺ and CD4 ⁺ T cells, respectively, and/or CD8+ and CD4+ enriched populations, respectively, e.g., CD4+ and/or CD8+ T cells, separately including genetically engineered cells. to express the recombinant receptor. In some embodiments, administering a dose comprises administering a first composition containing a dose of CD8+ T cells or a dose of CD4+ T cells and administering a second composition containing a different dose of CD4+ T cells and CD8+ T cells.

In some embodiments, administering a composition or dose, for example, administering multiple cell compositions, comprises administering the cell compositions separately. In some aspects, separate administration is performed simultaneously or sequentially, in any order. In some embodiments, the dose comprises a first composition and a second composition, and the first composition and second composition are administered at 0-12 hour intervals, 0-6 hour intervals, or 0-2 hour intervals. In some embodiments, the start of administration of the first composition and the start of administration of the second composition are at intervals of no more than 2 hours, no more than 1 hour, or no more than 30 minutes, no more than 15 minutes, no more than 10 minutes, or no more than 5 minutes. In some embodiments, the start and/or end of the administration of the first composition and the end and/or start of the administration of the second composition are at intervals of no more than 2 hours, no more than 1 hour, or no more than 30 minutes, no more than 15 minutes, no more than 10 minutes, or no more than 5 minutes.

In some instances, the first composition, eg, the first dose composition, contains CD4+ T cells. In some instances, the first composition, eg, the first dose composition, contains CD8+ T cells. In some embodiments, the implementation of the first composition is administered before the introduction of the second composition.

In some embodiments, the dose or composition of cells has a specific or target ratio of CD4+ cells expressing the recombinant receptor to CD8+ cells expressing the recombinant receptor and/or a ratio of CD4+ cells to CD8+ cells, optionally about 1:1, or ranging from about 1:3 to about 3:1, such as about 1:1. In some aspects, administering a composition or dose at a target or desired ratio of different cell populations (e.g., a CD4+:CD8+ ratio or a CAR+CD4+:CAR+CD8+ ratio of, for example, 1:1) comprises administering a cell composition comprising one of populations, followed by the introduction of a separate cell composition containing another of the populations, while the introduction is carried out at, or at about, the target or desired ratio. In some aspects, administration of a dose, or composition, of cells at a specific ratio results in increased proliferation, persistence, and/or antitumor activity of therapeutic T cells.

In some embodiments, the subject receives multiple doses, such as two or more doses, or multiple consecutive doses, of cells. In some embodiments, two doses are administered to the subject. In some embodiments, the subject receives the next dose, e.g., the second dose given at about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days after the first dose. In some embodiments, several consecutive doses are administered after the first dose, for example, an additional dose or doses are administered after the next dose is administered. In some aspects, the number of cells administered to a subject in an additional dose is the same or similar to the number in the first dose and/or the next dose. In some embodiments, the additional dose or doses are greater than the previous doses.

In some aspects, the size of the first and/or next dose is determined based on one or more criteria, such as the subject's response to prior treatment, such as chemotherapy, the subject's burden of disease, such as tumor burden, weight, size, or magnitude, or grade, or type of metastasis, stage of disease, and/or the likelihood or frequency of the subject developing toxic effects, e.g., CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and/or host immune response against injected cells and/or recombinant receptors.

In some aspects, the time period between the first dose and the next dose is about 9 to about 35 days, about 14 to about 28 days, or 15 to 27 days. In some embodiments, the next dose is administered at a time point no more than about 14 days after, and less than about 28 days after, the first dose. In some aspects, the time period between the first and next dose is about 21 days. In some embodiments, an additional dose or doses, such as subsequent doses, is administered after the next dose is administered. In some aspects, the additional next dose or doses are administered at least about 14, and less than about 28, days after the previous dose. In some embodiments, an additional dose is administered less than about 14 days after the previous dose, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 days after the previous dose. In some embodiments, doses are not administered less than about 14 days after the previous dose and/or doses are not administered more than about 28 days after the previous dose.

In some embodiments, the dose of cells, e.g., recombinant receptor-expressing cells, comprises two doses (e.g., a double dose) comprising a first dose of T cells and a subsequent dose of T cells, wherein administration of one or both of the first dose and the second dose is is the introduction of a divided dose of T-cells.

In some embodiments, the cells are administered as part of an additional combination therapy, e.g., simultaneously, or sequentially, in any order, with another therapeutic intervention, e.g., using an antibody or engineered cell, or a receptor, or an agent such as a cytotoxic or therapeutic agent. . For example, in some embodiments, an anticancer agent or an immunomodulatory agent can be used in combination therapy with an adoptive cell therapy with engineered cells expressing a recombinant receptor, such as CAR. In some contexts, cells are co-administered with another therapeutic agent close enough in time that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, cells are administered prior to the administration of one or more additional therapeutic agents. In some embodiments, cells are administered following the administration of one or more additional therapeutic agents.

In some embodiments, the implementation of one or more additional therapeutic agents include a cytokine, such as IL-2, for example, to increase persistence. In some embodiments, the methods include administering a chemotherapeutic agent. In some embodiments, one or more additional therapeutic agents include one or more lymphodepletion agents, for example, before or simultaneously with the start of the engineered cells. In some embodiments, lymphodepletion therapy includes the administration of a phosphamide, such as cyclophosphamide. In some embodiments, lymphodepletion therapy may include the administration of fludarabine. In some embodiments, fludarabine is excluded from lymphodepletion therapy. In some embodiments, lymphodepletion therapy is not used.

In some embodiments, the methods include administering to the subject a preconditioning agent, such as a lymphodepletion or chemotherapeutic agent such as cyclophosphamide, fludarabine, or combinations thereof, prior to initiation of cell therapy. For example, a subject can be administered a preconditioning agent at least 2 days before, for example, at least 3, 4, 5, 6, or 7 days before starting cell therapy. In some embodiments, the subject is administered a preconditioning agent no more than 7 days before, for example, no more than 6, 5, 4, 3, or 2 days before the start of cell therapy.

In some embodiments, the subject receives the preconditioning agent cyclophosphamide at a dose of from, or about, 20 mg/kg to 100 mg/kg, for example, from, or from about 40 mg/kg to 80 mg/kg. In some aspects, the subject receives the preconditioning agent cyclophosphamide at a dose, or a dose of about 60 mg/kg. In some embodiments, cyclophosphamide may be administered in a single dose or may be administered in multiple doses, such as daily, every other day, or every three days. In some embodiments, cyclophosphamide is administered once a day for one or two days. In some embodiments, when the lymphodepletion agent is cyclophosphamide, the subject is administered cyclophosphamide at a dose of from, or from about 100 mg/m ² to 500 mg/m ² , for example, from, or from about 200 mg/m ² to 400 mg/m ² , or 250 mg/m ² to 350 mg/m ² inclusive of all ranges. In some cases, the subject is administered about 300 mg/m ² cyclophosphamide. In some embodiments, cyclophosphamide may be administered in a single dose or may be administered in multiple doses, such as daily, every other day, or every three days. In some embodiments, the implementation of cyclophosphamide is administered daily, for example, for 1-5 days, for example, from 3 to 5 days. In some cases, the subject is administered approximately 300 mg/m ² cyclophosphamide daily for 3 days prior to cell therapy.

In some embodiments, when the lymphodepletion agent is fludarabine, the subject is administered fludarabine at a dose of from, or about, 1 mg/m ² to 100 mg/m ² , for example, from, or from about 10 mg/m ² to 75 mg/m ² , 15 mg/m ² to 50 mg/m ² , 20 mg/m ² to 40 mg/m ^{2 ,} or 24 mg/m ² to 35 mg/m ² inclusive of all ranges. In some cases, the subject is administered about 30 mg/m ² fludarabine. In some embodiments, fludarabine may be administered in a single dose or may be administered in multiple doses, such as daily, every other day, or every three days. In some embodiments, fludarabine is administered daily, eg, for 1-5 days, eg, 3 to 5 days. In some cases, the subject is administered approximately 30 mg/m ² fludarabine daily for 3 days prior to cell therapy.

In some embodiments, the lymphodepletion agent includes a combination of agents, such as a combination of cyclophosphamide and fludarabine. Thus, the combination of agents may include cyclophosphamide at any dose or schedule, eg, as described above, and fludarabine, at any dose or schedule, eg, as described above. For example, in some aspects, the subject is administered 60 mg/kg (~2 g/m ² ) cyclophosphamide and 3-5 doses of 25 mg/m ² fludarabine prior to the first or next dose.

Cells can be administered by any suitable means. Cells are administered at a specific dosage regimen to achieve a therapeutic effect, such as a reduction in tumor burden. Doses and administration may depend in part on the regimen of administration of the immunomodulatory compound, which may be administered before, after and/or simultaneously with the initiation of T cell therapy. Various dosing regimens for T-cell therapy include, but are not limited to, single and multiple injections over various periods of time, bolus administration and pulsed infusion.

IV. Kits and products

Also provided are articles, such as kits and devices, for administering cells to subjects in accordance with the proposed methods for adoptive cell therapy, as well as for storing and administering cells and compositions, such as the original compositions described or manufactured compositions.

In some embodiments, a kit is provided that includes the compositions described above and instructions for use. In some embodiments, kits are provided that include a composition comprising a therapeutically effective amount of any of the engineered cells stimulated in accordance with any of the methods described herein. In some embodiments, kits are provided that include a composition comprising a therapeutically effective amount of any of the engineered cells described herein and instructions for administering them to a subject for the treatment of a disease or condition. In some aspects, the instructions are for selecting or enriching cells expressing an antigen receptor containing an antigen-binding domain specifically recognized by a stimulatory reagent from a population of cells. In some instances, the instructions are for propagating cells expressing an antigen receptor containing an antigen-binding domain specifically recognized by a stimulatory reagent from a population of cells.

In some embodiments, the kit further includes instructions for administering to a subject, for the purpose of treating a disease or condition, engineered cells stimulated using the methods described herein in combination therapy for the treatment of the disease or condition. In some examples, the kit further includes a therapeutic agent. In some embodiments, the therapeutic agent is an immunomodulatory agent, a cytotoxic agent, an anticancer agent, or a radiotherapeutic agent.

Products are also offered. In some embodiments, the articles of manufacture include any of the engineered cells, compositions, polynucleotides, polynucleotide arrays, a composition comprising a polynucleotide array, vectors, a vector array, a composition comprising a vector array, or kits provided herein.

The articles or kits include one or more containers, typically multiple containers, a packaging material, and a label or package insert placed on or associated with the container or containers and/or packaging, typically containing instructions for administering the cells to a subject. Kits and articles may include a container and a label or package insert placed on or associated with the container.

In some embodiments, the containers contain cells to be administered, for example, one or more standard doses of cells. The product typically includes several containers, each containing a standard dose of cells. A unit dose may be the number, or number, of cells to be administered to a subject at the first dose, or twice the number (or more) of cells to be administered at the first, or any one or more of the following doses. It may be the lowest dose, or the lowest possible dose, of cells that can be administered to a subject in combination with the route of administration. In some embodiments, a unit dose is the minimum number of cells, or the minimum number of reference units, or target reference units, or reference units in a reference range, that would be administered in a single dose to any subject having a particular disease or condition, or to any subject within in accordance with the methods described in this document.

Suitable containers include, for example, bottles, vials, syringes, bags for IV solutions, and so on. The containers can be made from various materials such as glass or plastic. In some embodiments, the container contains a composition that may, alone, or in combination with another composition, be effective in treating, preventing, and/or diagnosing a condition. In some embodiments, the container has a sterile access port. Exemplary containers include bags for intravenous solutions, vials, including vials with injection needle stoppers, or bottles or vials for orally administered agents. The label, or package insert, may indicate that the composition is used to treat a disease or condition. The product may further include a package insert indicating that the compositions can be used to treat a particular condition. Alternatively, or additionally, the product may further include a different, or the same, container containing a pharmaceutically acceptable buffer. It may further include other materials, such as other buffers, diluents, filters, needles and/or syringes.

In particular embodiments, the containers are bags, such as flexible bags such as those suitable for infusing cells into subjects, such as flexible plastic or PVC bags, and/or bags for IV solutions. In some embodiments, the bags are resealable and/or can be sterilized to ensure solution sterility and sterile delivery of cells and compositions. In some embodiments, containers, such as bags, have a capacity of, or about, or at least, or at least about, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 or 1000 ml e.g. from or from about 10 and up to or up to about 100 or from or from about 10 and up to or up to about 500 ml including all range values. In some embodiments, the containers, such as bags, are made and/or made from a material that is stable and/or allows for stable storage and/or maintenance of cells at one or more of different temperatures, such as refrigeration temperatures, such as below than, or lower than about, or at, or at about -20°C, -80°C, -120°C, 135°C and/or temperatures suitable for cryopreservation and/or other temperatures, such as temperatures suitable for cell thawing, and body temperature, eg at, or at about 37° C., eg for thawing, eg, at the subject's location or for treatment, eg, at the bedside, just prior to treatment.

The containers can be made from various materials such as glass or plastic. In some embodiments, the container has one or more ports, such as sterile access ports, such as for tubing or cannulation to one or more tubing, such as for intravenous or other infusion and/or connection for transfer to and from other containers such as cell culture and/or storage containers, or other containers. Exemplary containers include infusion bags, bags for intravenous administration of solutions, vials, including vials with injection needle stoppers.

The product may further include a package insert or label with one or more pieces of identifying information and/or instructions for use. In some embodiments, the information or instructions indicate that the content can, or should, be used to treat a particular condition or disease and/or provide instructions to do so. The label, or package insert, may indicate that the contents of the product are for use in the treatment of a disease or condition. In some embodiments, the label, or package insert, provides instructions for treating a subject, e.g., the subject from which the cells were obtained, by a method comprising administering a first and one or more subsequent doses of cells, for example, in accordance with any of the embodiments of the proposed ways. In some embodiments, the instructions describe the administration, in the first dose, of one unit dose, e.g., the contents of one individual container in the article, followed by one or more subsequent doses at a specified time point or within a specified time window and/or upon detection of the presence or absence of , or the amount or level of one or more factors or outcomes in a subject.

In some embodiments, the instructions describe administering one or more unit doses to a subject.

In some embodiments, the label or package insert or package contains identifying information indicating the specific subject from which the cells were obtained and/or to which the cells are to be administered. In the case of autologous transfer, the identity of the subject from whom the cells were obtained is the same as the identity of the subject to whom the cells will be injected. Thus, the identification information may indicate that the cells are to be administered to a particular patient, eg, the one from whom the cells were originally obtained. Such information may be on the packaging material and/or label in the form of a barcode or other encoded identifier, or may indicate the name and/or other identifying characteristics of the subject.

In some embodiments, the article of manufacture includes one or more, usually more than one, containers containing compositions containing cells, for example, their separate unit doses, and also includes one or more additional containers containing a composition that contains an additional agent, such as a cytotoxic or other therapeutic agent, for example, to be administered in combination with cells, for example, simultaneously or sequentially, in any order. Alternatively or additionally, the product may also include a different or the same container containing a pharmaceutically acceptable buffer. It may also include other materials such as other buffers, diluents, filters, tubing, needles and/or syringes.

As used herein, the term "package insert" means the instructions commonly included in commercial packages of therapeutic products that contain information about the approximate indications for use, doses, administration, combination therapy, contraindications and / or warnings related to the use of such therapeutic drugs.

V Definitions

Unless otherwise indicated, all technical and scientific terms, notation systems and other technical and scientific terms or terminology used in this document should have the same meaning as they are usually given by experts in the field to which the claimed subject matter belongs. In some cases, terms with conventional meanings are defined herein for clarity and/or for easy reference, and the inclusion of such definitions herein does not necessarily mean that they differ materially from the definitions generally accepted in the art.

As used herein, the term "subject" refers to a mammal, such as a human or other animal, and generally refers to a human. In some embodiments, the subject, eg, the patient, to whom the immunomodulatory polypeptides, engineered cells, or compositions are administered is a mammal, typically a primate, such as a human. In some embodiments, the primate is a monkey or a great ape. The subject may be male or female, and may be of any age, including infant, child, adolescent, adult, or elderly subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent.

As used herein, the term "treatment" (and its grammatical variations such as "treat" or "treatment") means the total or partial alleviation or amelioration of a disease or condition, or disorder, or symptom, adverse effect, or outcome, or associated with them. phenotype. The desired effects of treatment include, but are not limited to, prevention of the development or recurrence of the disease, relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, attenuation or temporary relief of the disease state, and remission or better prognosis. The terms do not imply complete cure of a disease or complete elimination of any symptom, or effect(s) on all symptoms or outcomes.

As used herein, "delaying the development of a disease" means delaying, hindering, slowing down, inhibiting, stabilizing, suppressing, and/or delaying the development of a disease (such as cancer). This delay may vary in length depending on the history of the disease and/or the individual being treated. Obviously, a sufficient or significant delay may, in fact, include prevention, since the individual does not develop the disease. For example, late stage cancer, such as the development of metastases, can be delayed.

As used herein, the term "prevention" includes providing prophylaxis against the development or recurrence of a disease in a subject who may be predisposed to the disease but has not yet been diagnosed with the disease. In some embodiments, the provided cells and compositions are used to delay the development of a disease or slow the progression of a disease.

As used herein, the term "suppression" of function or activity means a decrease in function or activity relative to otherwise the same conditions, except for the condition or parameter of interest, or, alternatively, relative to another condition. For example, cells that inhibit tumor growth reduce the rate of tumor growth compared to the rate of tumor growth in the absence of cells.

An "effective amount" of an agent, e.g., pharmaceutical, cell, or composition, in the context of administration, means an amount effective, at the required doses/amounts and for the necessary periods of time, to achieve the desired result, such as a therapeutic or prophylactic result.

"Therapeutically effective amount" of an agent, such as a pharmaceutical product or engineered cells, means an amount effective, at the required doses and for the required periods of time, to achieve the desired therapeutic result, for example, to treat a disease, condition or disorder, and/or pharmacokinetic or pharmacodynamic effect of treatment. The therapeutically effective amount may vary depending on such factors as disease state, age, sex and body weight of the subject, as well as administered immunomodulatory polypeptides or engineered cells. In some embodiments, the proposed methods include the introduction of immunomodulatory polypeptides, engineered cells or compositions in effective amounts, for example, therapeutically effective amounts.

"Prophylactically effective amount" means an amount effective, at the required doses and for the necessary periods of time, to achieve the desired prophylactic result. Typically, but not necessarily, as long as a prophylactic dose is used in subjects prior to, or at an early stage in the development of, the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

The term "pharmaceutical preparation" means a preparation in a form that provides effective biological activity of the active ingredient contained in the preparation, and which does not contain any additional components that are unacceptably toxic to the subject to whom the preparation will be administered.

A "pharmaceutically acceptable carrier" means an ingredient in a pharmaceutical preparation, other than the active ingredient, that is non-toxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, a statement that nucleotide or amino acid positions "correspond" to nucleotide or amino acid positions in a disclosed sequence, such as that shown in a sequence listing, refers to nucleotide or amino acid positions identified when aligned with the disclosed sequence to achieve maximum identity using a standard an alignment algorithm such as the GAP algorithm. By aligning sequences, appropriate residues can be identified, for example, using conserved and identical amino acid residues as a guide. Typically, to identify relevant positions, amino acid sequences are aligned to achieve the highest order match (see, for example: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW , ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje , G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; Carrillo et al. (1988) SIAM J Applied Math 48: 1073 ).

As used herein, the singular form of nouns includes the corresponding plural form, unless the context clearly dictates otherwise. For example, "one" means "at least one" or "one or more". It is understood that aspects and variations described herein include "consisting" and/or "consisting essentially of" aspects and variations.

Throughout the text of this application, various aspects of the claimed subject matter are presented in range format. It should be understood that the description in range format is used solely for convenience and brevity, and should not be taken as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range is to be understood as specifically disclosing all possible sub-ranges as well as individual numerical values within a given range. For example, if a range of values is specified, it is understood that every intermediate value between the upper and lower limit of this range, as well as any other claimed or intermediate value in this specified range, is included in the scope of the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also within the scope of the claimed subject matter, subject to any specifically excluded limit in the claimed range. If the specified range includes one or both of these limits, ranges excluding one or both of these included limits are also included in the claimed subject matter. This rule applies regardless of the latitude of the range.

As used herein, the term "about" refers to the usual range of error for the respective value, well known to those skilled in the art. As used herein, the use of the term "about" in relation to a value or parameter includes (and describes) embodiments that address that value or parameter as such. For example, a description referring to "about X" includes a description of "X". In specific embodiments, "about" the specified value means a value within ±25%, ±20%, ±10%, ±5%, ±1%, ±0.1%, or ±0.01% of the specified value.

As used herein, "composition" means any mixture of two or more products, substances, or compounds, including cells. It may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous formulation, or any combination thereof.

As used herein, a statement that a cell or population of cells is "positive" for a particular marker means the detectable presence on, or in, a cell of a particular marker, typically a surface marker. When referring to a surface marker, the term means the expression of the marker on the surface as determined by flow cytometry, e.g. staining determined by the same procedure with isotype control under otherwise identical conditions, and/or at a level substantially similar to that of a cell known to contain a given marker, and/or at a level substantially higher than that of a cell, which is not known to contain the given token.

As used herein, a statement that a cell or population of cells is "negative" for a particular marker means the absence of significant detectable presence on, or in, the cell of a particular marker, typically a surface marker. When referring to a surface marker, the term means the absence of expression of the marker on the surface, as determined by flow cytometry, for example, by staining with an antibody that specifically binds the marker and detecting said antibody, and the staining cannot be detected by flow cytometry at a level significantly higher than the level of staining determined by carrying out the same procedure with isotype control under otherwise identical conditions, and/or at a level significantly lower than the level for a cell known to contain a given marker, and/or at a level substantially similar to level for a cell known not to contain a token.

As used herein, the term "vector" means a nucleic acid molecule capable of spreading another nucleic acid to which it is linked. The term includes a vector as a self-replicating nucleic acid structure, as well as a vector inserted into the genome of the host cell into which it has been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked. In this document, such vectors are called "expression vectors".

The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include the primary transformed cell and its progeny, regardless of the number of passages. The progeny may not be completely identical to the parent cell in terms of nucleic acid content, and may contain mutations. Mutant progeny having the same function or biological activity that was used in the screening or selection of the original transformed cell is also included in the present invention.

VI. Illustrative Embodiments

Suggested embodiments include the following:

1. The method of genetic engineering of T-cells, including:

(a) incubating the initial composition under stimulating conditions for 2-6 days, wherein said initial composition contains a population of T cells, including naive-like T cells and non-naive T-cells, wherein the stimulating conditions include the presence of a stimulating reagent, capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and/or one or more intracellular signaling domains of one or more costimulatory molecules to form a stimulated composition, and

(b) introducing a nucleic acid encoding a genetically engineered recombinant receptor into the stimulated T cell composition, wherein the introduction is carried out for at least part of the incubation period.

2. The method of genetic engineering of T-cells, including:

(a) incubating the parent composition under stimulating conditions for 2-6 days, wherein said parent composition contains a T cell population comprising naive-like T cells and non-naive-like T cells to form a stimulated composition, and

(b) introducing a nucleic acid encoding a genetically engineered recombinant receptor into the stimulated T cell composition, wherein the introduction is carried out for at least part of the incubation period.

3. The method of embodiment 2, wherein the stimulatory conditions comprise incubation of the parent composition with a stimulatory reagent capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and/or one or more intracellular signaling domains of one or more costimulatory molecules.

4. The method of genetic engineering of T cells, including the incubation of the original composition in stimulating conditions for 2-6 days, while the specified initial composition contains a population of T cells, including naive-like T cells and non-naive T-cells, while :

stimulatory conditions include the presence of a stimulatory reagent capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and/or one or more intracellular signaling domains of one or more co-stimulatory molecules to form a stimulated composition, and

incubation of the initial composition under stimulating conditions is carried out before, during and/or after the administration of the nucleic acid encoding the genetically engineered recombinant receptor.

5. The method according to any one of embodiments 1-4, wherein the incubation is carried out for at least 3 days.

6. The method according to any one of embodiments 1-4, wherein the incubation is carried out for at least 4 days.

7. The method according to any one of embodiments 1-4, wherein the incubation is carried out for at least 5 days.

8. The method according to any one of embodiments 1-4, wherein the incubation is carried out for at least 6 days.

9. A method for stimulating T cells, comprising:

(a) incubation under stimulatory conditions of the initial composition containing T cells, including the number of naive-like T cells, or a subset of CD8+ T cells, necessary for culture initiation, to obtain a stimulated composition; And

(b) introducing into the stimulated cell composition a nucleic acid encoding the genetically engineered recombinant receptor to obtain a composition produced by the method, containing T cells expressing the genetically engineered recombinant receptor.

10. The method of Embodiment 9, wherein the T cells comprise naïve-like T cells and non-naïve-like T cells, wherein the stimulatory conditions preferably induce multiplication or proliferation of naïve-like T cells versus non-naïve-like T cells in a stimulated composition.

11. The method of Embodiment 9 or Embodiment 10, wherein the administration is carried out during at least part of the incubation period or is carried out after the incubation.

12. The method of any one of embodiments 9-11, wherein the number of naive-like T cells or a subset of CD8+ T cells required to initiate culture is from, or about, 0.1 x 10 ⁸ to 5 x 10 ⁸ . from, or from about 0.1×10 ⁸ to 4×10 ⁸ , from, or from about 0.1×10 ⁸ to 2×10 ⁸ , from, or from about 0.1×10 ⁸ to 1 ×10 ⁸ , from or from about 1×10 ⁸ to 5×10 ⁸ , from or from about 1×10 ⁸ to 4×10 ⁸ , from or from about 1×10 ⁸ to 2×10 ⁸ , from, or from about 2x10 ⁸ to 5x10 ⁸ , from, or from about 2x10 ⁸ to 4x10 ⁸ naive-like T cells or a subset of CD8+ T cells.

13. The method of any one of embodiments 9-12, wherein the number of naïve-like T cells or a subset of CD8+ T cells required to initiate culture is at least, or at least about, or is, or is about, 0 .5×10 ⁸ , 0.75×10 ⁸ , 1×10 ⁸ , 1.5×10 ⁸ , 2×10 ⁸ , or 4×10 ⁸ naïve-like T cells or their subset of CD8+ T cells.

14. The method of any one of embodiments 9-13, wherein the number of naïve-like T cells or a subset of CD8+ T cells required to initiate culture is at least, or at least about, or is, or is about, 2 ×10 ⁸ naive-like T cells or a subset of CD8+ T cells.

15. A method for stimulating T cells, comprising incubating under stimulating conditions an initial composition containing T cells, including the number of naive-like T cells or their subset of CD8+ T cells required for culture initiation, which is from, or from about, 1 × 10 ⁸ to 4×10 ⁸ like naive T cells or a subset of CD8+ T cells, to obtain a stimulated composition.

16. The method of embodiment 15, wherein the T cells comprise naive-like T cells and non-naive-like T cells, wherein the stimulatory conditions preferably induce multiplication or proliferation of naive-like T cells versus non-naive-like T cells in a stimulated composition.

17. The method of embodiment 15 or embodiment 16, wherein the number of naïve-like T cells or a subset of CD8+ T cells required to initiate culture is at least, or at least about, or is, or is about, 2 ×10 ⁸ naive-like T cells or a subset of CD8+ T cells.

18. The method of any one of embodiments 1-17, wherein the amount required to initiate the culture is the number of naive-like CD8+ T cells.

19. The method of any one of embodiments 1-18, wherein naive-like T cells or naive-like CD8+ T cells:

have on their surface a T cell activation marker selected from the group consisting of CD45RA, CD27, CD28 and CCR7; and/or

do not have on the surface a marker selected from the group consisting of CD25, CD45RO, CD56, CD62L, KLRG1; and/or

have low expression of CD95; and/or

lack intracellular expression of a cytokine selected from the group consisting of IL-2, IFN-γ, IL-4, IL-10.

20. The method of any one of embodiments 1-19, wherein naive-like cells or naive-like CD8+ cells:

have on their surface a T cell activation marker selected from the group consisting of CD45RA, CD27, CD28 and CCR7; and/or

do not have on the surface a marker selected from the group consisting of CD45RO, CD56, KLRG1; and/or

have low CD95 expression.

21. The method of any one of embodiments 1-20, wherein the naive-like T cells or naive-like CD8+ cells are CD45RA+, CD27+, CCR7+ and/or CD45RO- cells.

22. The method of any of embodiments 1-17 and 16-21, wherein non-naive T cells are:

do not have on the surface a T-cell activation marker selected from the group consisting of CD45RA, CD27, CD28 and CCR7; and/or

have on their surface a marker selected from the group consisting of CD25, CD45RO, CD56, CD62L, KLRG1 and perforin; and/or

have intracellular expression of a cytokine selected from the group consisting of IL-2, IFN-γ, IL-4, IL-10; and/or

have high expression of CD95.

23. The method of any of embodiments 1-14 and 16-22, wherein the non-naïve-like T cells are CD45RA-, CD27-, CCR7- and/or CD45RO+ cells.

24. The method of any one of embodiments 1-23, wherein the cells of the original composition have not, and are not, subjected prior to incubation to a selection step based on an endogenous T-cell surface marker that distinguishes between naive-like and non-naive-like T cells.

25. The method of any one of embodiments 15-24, further comprising introducing the engineered recombinant receptor into the stimulated cells,

with obtaining by this method the produced composition containing T-cells expressing the genetically engineered recombinant receptor.

26. The method of embodiment 25, wherein the composition is incubated under stimulating conditions before, during and/or after administration of a nucleic acid encoding a genetically engineered recombinant receptor.

27. The method of any one of embodiments 1-14 and 25-26, wherein the recombinant receptor is capable of binding to a target antigen that is associated with, specific for, and/or expressed on a cell or tissue associated with a disease, disorder, or condition.

28. The method of embodiment 27, wherein the disease, disorder, or condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, or a tumor, or cancer.

29. The method of Embodiment 27 or Embodiment 28, wherein the target antigen is a tumor antigen.

30. The method of any one of embodiments 27-29, wherein the target antigen is selected from αvβ6 integrin (avb6 integrin), B cell maturation antigen (BCMA), carbonic anhydrase 9 (CAIX), Her2/neu (erbB2 tyrosine kinase receptor), L1-CAM, B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), testicular cancer antigen, testicular cancer antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2 ), carcinoembryonic antigen (CEA) and hepatitis B surface antigen, antifolate receptor, cyclin, cyclin A2, chemokine ligand 1 with C-C motif (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123, CD133, CD138, CD171, proteoglycan 4 chondroitin sulfate (CSPG4), epidermal growth factor protein (EGFR), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin A2 receptor (EPHa2), erb-B2, erb-B3, erb-B4, erbB dimers, mutant epidermal growth factor receptor type III (EGFR vIII), folate-binding protein (FBP) like Fc receptor protein 5 (FCRL5; also known as Fc receptor 5 or FCRH5 homolog), embryonic acetylcholine receptor (embryonic AchR), folate binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glypican-3 ( GPC3), G protein-coupled receptor 5D (GPRC5D), Her2/neu (erbB2 tyrosine kinase receptor), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, human high molecular weight antigen associated with melanoma (HMW- MAA), hepatitis B surface antigen, human leukocyte antigen A1 (HLA-A1), human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha (IL-22R-alpha), IL-13R-alpha2 (IL- 13Rα2), kinase insert domain receptor (kdr), kappa light chain, Lewis Y, L1 cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM, member of the A family of 8 proteins containing leucine-rich repeats (LRRC8A), Lewis Y, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, mouse cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, representative D ligands natural killer group 2 (NKG2D), melan A (MART-1), neuronal cell adhesion molecule (NCAM), oncoembryonic antigen, preferentially expressed melanoma antigen (PRAME), progesterone receptor, prostate specific antigen, prostatic stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), like tyrosine kinase orphan receptor 1 (ROR1), survivin, trophoblastic glycoprotein (TPBG, also known as 5T4), tumor-associated glycoprotein 72 (TAG72), tyrosinase-dependent protein 1 (TRP1, also known as TYRP1 or gp75), tyrosinase dependent protein 2 (TRP2, also known as dopachrome tautomerase, dopachrome delta isomerase or DCT), folate receptor-α, 8H9, dual antigen, glycoprotein 100 (gp100), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGF-R2), estrogen receptor, progesterone receptor, Wilms tumor antigen 1 (WT-1), pathogen-specific or pathogen-expressed antigen, and universal marker-associated antigen.

31. The method of any one of embodiments 1-14 and 25-30, wherein the recombinant receptor is, or comprises, a functional non-TCR antigen receptor or TCR, or an antigen-binding fragment thereof.

32. The method of any one of embodiments 1-14 and 25-30, wherein the recombinant receptor is a chimeric antigen receptor (CAR).

33. The method of any one of embodiments 1-14 and 25-32, wherein the recombinant receptor comprises an extracellular domain containing an antigen-binding domain, optionally wherein the antigen-binding domain specifically binds the target antigen.

34. The method of embodiment 33, wherein the antigen binding domain is, or contains, an antibody, or an antibody fragment, which is optionally a single chain fragment.

35. The method of embodiment 33, wherein the fragment comprises antibody variable regions joined by a flexible linker.

36. The method of Embodiment 34 or Embodiment 35, wherein the fragment is an scFv.

37. The method of any of embodiments 1-14 and 25-36, wherein the recombinant receptor further comprises a spacer and/or a hinge region.

38. The method of any one of embodiments 1-14 and 25-37, wherein the recombinant receptor contains an intracellular signaling region.

39. The method of embodiment 38, wherein the intracellular signaling region comprises an intracellular signaling domain.

40. The method of embodiment 39, wherein the intracellular signaling domain is, or comprises, a basic signaling domain, a signaling domain capable of inducing a basic activation signal in a T cell, a T cell receptor (TCR) component signaling domain, and/or a signaling domain a domain containing an immunoreceptor tyrosine activating motif (ITAM).

41. The method of embodiment 40, wherein the intracellular signaling domain is, or comprises, an intracellular signaling domain of the CD3 chain, optionally CD3 zeta chain (CD3ζ), or a signal fragment thereof.

42. The method of any one of embodiments 38-41, wherein the recombinant receptor further comprises a transmembrane domain located between the extracellular domain and the intracellular signaling region.

43. The method of any one of embodiments 38-42, wherein the intracellular signaling region further comprises a costimulatory signaling region.

44. The method of embodiment 43, wherein the costimulatory signaling region comprises an intracellular signaling domain of a T cell costimulatory molecule, or a signal fragment thereof.

45. The method of embodiment 43 or embodiment 44, wherein the costimulatory signaling region comprises an intracellular signaling domain of CD28, 4-1BB, or ICOS, or a signal fragment thereof.

46. The method of any one of embodiments 43-45, wherein the co-stimulatory signaling region is between the transmembrane domain and the intracellular signaling region.

47. The method of any one of embodiments 1-46, wherein the stimulating condition comprises incubation with a stimulating reagent capable of activating T cells, CD4+ T cells, and/or CD8+ T cells; able to induce a signal through the TCR complex; and/or capable of inducing the proliferation of T cells, CD4+ T cells and/or CD8+ T cells.

48. The method of any one of embodiments 1-47, wherein the stimulatory condition comprises incubation with a stimulatory reagent capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and/or one or more intracellular signaling domains of one or more costimulatory molecules. .

49. The method of Embodiment 47 or Embodiment 48, wherein the stimulatory reagent comprises a base agent that specifically binds a component of the TCR complex, optionally that specifically binds CD3.

50. The method of Embodiment 49, wherein the stimulant further comprises an adjuvant that specifically binds a T-cell co-stimulatory molecule, optionally, the co-stimulatory molecule is selected from the group consisting of CD28, CD137 (4-1-BB), OX40 or ICOS.

51. The method of any one of embodiments 47-50, wherein the primary and secondary agents comprise antibodies, optionally, wherein the one or more stimulatory agents comprise an anti-CD3 antibody and an anti-CD28 antibody.

52. The method of Embodiment 50 or Embodiment 51, wherein the primary and/or secondary means are on the surface of the solid support.

53. The method of Embodiment 52, wherein the solid support is, or contains, a bead.

54. The method of Embodiment 53, wherein the bead has a diameter greater than or greater than about 3.5 microns, but no greater than about 9 microns, or no greater than about 8 microns, or no greater than about 7 microns, or not more than about 6 microns, or not more than about 5 microns.

55. The method of Embodiment 53 or Embodiment 54, wherein the pellet has a diameter of, or is, about 4.5 microns.

56. The method of any one of embodiments 53-55, wherein the bead has a diameter of, or about the size of, a lymphocyte or an antigen presenting cell.

57. The method of any one of embodiments 53-56, wherein the pellet is inert.

58. The method of any one of embodiments 53-57, wherein the bead is, or has, a polystyrene surface and optionally has a magnetic or superparamagnetic core.

59. The method of any one of embodiments 53-58, wherein the stimulating condition comprises incubating cells at a bead to cell ratio of from, or about 1:1 to 10:1, from, or about 1:1 to 8:1, from, or from about, 1:1 to 6:1, from, or from about, 1:1 to 4:1, from, or from about, 1:1 to 3:1, from, or from about 2:1 to 4:1, from or from about 2:1 to 3:1, from or from about 1:1 to 2:1, from or from about 4:1 to 10: 1, from, or from about, 4:1 to 8:1, from, or from about, 4:1 to 6:1, from, or from about, 6:1 to 10:1, from, or from about, 6:1 to 8:1, from, or from about, 8:1 to 10:1, from, or from about, 1:1 to 1:10, from, or from about, 1:1 to 1:8, from, or from about 1:1 to 1:6, from, or from about 1:1 to 1:4, from, or from about 1:2 to 1:3.

60. The method of genetic engineering of T-cells, including:

(a) incubating the initial composition under stimulating conditions for 2-6 days, wherein said initial composition contains a population of T cells including naive-like T cells and non-naive T-cells, wherein the stimulating conditions include the presence of a stimulating reagent, comprising an anti-CD3 antibody and an anti-CD28 antibody adjuvant, which are attached to the bead, wherein the bead to cell ratio during incubation is from, or about, 1:1 to 4:1; And

(b) introducing a nucleic acid encoding a genetically engineered recombinant receptor into the stimulated T cell composition, wherein the introduction is carried out for at least part of the incubation period.

61. A method for genetic engineering of T cells, including incubation of the initial composition under stimulating conditions for 2-6 days, while the specified initial composition contains a population of T cells, including naive-like T cells and non-naive T-cells, while :

stimulating conditions include the presence of a stimulating reagent comprising an anti-CD3 antibody and an anti-CD28 antibody adjuvant, which are attached to the bead, wherein the bead to cell ratio during incubation is from, or about, 1:1 to 4: 1; And

incubation of the initial composition under stimulating conditions is carried out before, during and/or after the administration of the nucleic acid encoding the genetically engineered recombinant receptor.

62. The method of embodiment 59 wherein the granule to cell ratio is, or is about, 3:1.

63. The method of embodiment 59 wherein the granule to cell ratio is, or is about, 1:1.

64. The method of any one of embodiments 1-63, wherein the T cells are derived from a biological sample, optionally the biological sample is derived from a human subject.

65. The method of embodiment 64, wherein the biological sample is, or contains, a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a white blood cell sample, an apheresis product or a leukapheresis product.

66. The method of any one of embodiments 1-65, wherein the T cells include CD4+ and/or CD8+ cells.

67. The method of any one of embodiments 1-66, wherein the T cells include CD4+ and CD8+ T cells, and the ratio of CD4+ to CD8+ T cells is from, or from about 2:1 to, or up to about, 1 :5.

68. The method of embodiment 55 wherein the ratio of CD4+ cells to CD8+ cells is, or is about, 1:1, 1:2, 2:1, 1:3, or 3:1.

69. The method of any one of embodiments 1-68, wherein naive-like T cells include naive-like CD4+ T cells and/or naive-like CD8+ T cells.

70. The method of any one of embodiments 1-69, wherein the naive-like T cells are polyclonal.

71. The method of embodiment 70 wherein clonality of naïve-like T cells is determined by clonal sequencing, optionally next generation sequencing or spectrotype analysis.

72. The method of any one of embodiments 1-71, wherein the presence, number, number, or percentage of naive-like T cells is determined by flow cytometry.

73. The method of any of embodiments 1-72, wherein the stimulus condition does not include N-acetylcysteine (NAC).

74. The method of any of embodiments 1-72, wherein the stimulus condition does not include IL-15 and/or IL-7.

75. The method of any one of embodiments 1-72, wherein the stimulus condition results in or induces the death of non-naïve T cells or a subset thereof.

76. The method of any one of embodiments 1-75, wherein the stimulatory condition results in activation-induced cell death (AICD) of non-naïve-like T cells or a subset thereof.

77. The method of any one of embodiments 1-76, further comprising adding DNase during incubation and/or to the stimulated composition.

78. The method of any one of embodiments 1-77, wherein the incubation is for more than, or about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 days.

79. The method of any one of embodiments 1-78, wherein the percentage of cells in the stimulated composition derived from similar naive T cells is increased by more than, or more than about, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 50 times, 100 times compared to the percentage of naive-like cells in the original composition.

80. The method of any one of embodiments 1-79, wherein the content in the stimulated composition of cells derived from naive T-like cells, relative to cells derived from non-naive T cells, is increased by more than, or more than about, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, 100-fold compared to naive-like versus non-naive-like T cells T cells in the original composition.

81. The method according to any one of embodiments 1-80, wherein the stimulated composition contains more than 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% of cells that originated from similar naive T-cells of the original composition.

82. The method of any one of embodiments 1-81, wherein the stimulated composition contains less than 10% of cells derived from non-naive T cells.

83. The method of any one of embodiments 1-82, wherein the stimulated composition contains less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of cells derived from non-naive T cells.

84. The method of any one of embodiments 1-83, wherein, of the cells in the original composition, a greater percentage of naive-like T cells, compared to non-naïve-like T cells, are induced to proliferate and/or become activated.

85. The method of any one of embodiments 1-84, wherein a greater percentage of T cells that were similar to naive in the original composition compared to the percentage of T cells that were not similar to naive in the original composition divide per day 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 after the start of said incubation.

86. The method of any one of embodiments 1-85, wherein the stimulating conditions are capable of inducing proliferation of a greater percentage of cells in the naïve human T cell-like population as compared to non-naïve human T cell-like, on day 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 after the start of incubation under these conditions.

87. The method of any one of embodiments 1-86, wherein: (i) non-naïve-like T cells are selected from the group consisting of effector T cells (T _EFF ), memory T cells, central memory T cells ( T _CM ), effector memory T-cells (T _EM ), as well as their combinations; or (ii) the non-naïve T cells are a plurality of T cells including or consisting of effector T cells (T _EFF ) and/or memory T cells, wherein the memory T cells optionally include central T cells. memory cells (T _CM ) and/or effector memory T cells (T _EM ).

88. The method of any one of embodiments 1-87, wherein:

the percentage of naive-like T cells in the original composition is less than the percentage of engineered cells in the stimulated composition derived from naive-like T cells in the original composition.

89. The method of any one of embodiments 1-14 and 25-88, wherein a large percentage of cells with the introduced nucleic acid are, or are derived from, proliferation of naïve-like T cells in the original composition, compared to non-naïve T-like -cells in the original composition.

90. The method of any one of embodiments 1-14 and 25-89, wherein the administration is by transduction.

91. The method of any of embodiments 1-14 and 25-90, wherein the nucleic acid is a viral vector.

92. The method of Embodiment 91, wherein the viral vector is a retroviral vector.

93. The method of Embodiment 91 or Embodiment 92, wherein the viral vector is a lentiviral vector or a gamma retroviral vector.

94. The method according to any one of embodiments 1-14 and 25-89, wherein the introduction is performed by transposition of a transposon containing a nucleic acid molecule.

95. The method of any one of embodiments 1-94, which is used in vitro or ex vivo .

96. A manufactured composition obtained by the method of any one of embodiments 1-83.

97. A pharmaceutical composition containing the manufactured composition of embodiment 96.

98. The pharmaceutical composition of Embodiment 97 further comprising a pharmaceutical carrier.

99. A method of treatment comprising administering to a mammalian subject a manufactured composition obtained by the method of any of embodiments 1-95, or a pharmaceutical composition of embodiment 97 or embodiment 98.

100. The method of embodiment 99, wherein the cells are obtained from the subject to whom the cells will be injected.

VII. Examples

The following examples are provided for illustrative purposes only and should not limit the scope of the present invention.

Example 1 Evaluation of naïve-like cell markers in engineered T cell compositions containing CAR+ T cells.

Engineered primary T cell compositions containing T cells expressing chimeric antigen receptors (CARs) were generated in parallel by two methods that used a stimulatory reagent consisting of paramagnetic polystyrene-coated beads with attached anti-CD3 and anti-CD28 antibodies to activate T cells before being transduced with a viral vector. In both methods, cells were engineered by lentiviral transduction to express the same anti-BCMA CAR. The CAR contained an scFv antigen-binding domain specific for BCMA, a CD28 transmembrane region, a costimulatory 4-1BB signaling region, and a CD3-zeta-derived intracellular signaling domain. The methods differed in the duration of the process and the conditions for cell propagation. The resulting T cell compositions were evaluated for cell surface markers.

In both methods, individual compositions of CD4+ and CD8+ cells were selected from PBMCs isolated from a leukapheresis sample from a human donor and the selected cell compositions were frozen. The individual compositions of CD4+ and CD8+ T cells were then thawed and viable CD4+ T cells were mixed with viable CD8+ T cells in a 1:1 ratio. Approximately 300x10 ⁶ T cells (150x10 ⁶ CD4+ and 150x10 6 CD4+ and 150x10 ⁶ CD8+ T cells) of the mixed stock cell composition were stimulated by incubating the cells for 18-30 hours in the presence of anti-CD3/anti-CD28 conjugated beads antibodies at a ratio of granules and cells of 1:1 in a serum-free medium. The medium also contained recombinant IL-2, IL-7 and IL-15. After stimulation, the cells were washed and resuspended in serum-free medium containing additives, as well as recombinant IL-2, IL-7 and IL-15.

In one method (hereinafter referred to as "non-expanded") cells from the stimulated cell composition are then transduced with a lentiviral vector encoding an anti-BCMA CAR by spinoculation. After spinoculation, the cells were washed and resuspended in serum-free medium containing recombinant IL-2, IL-7 and IL-15. The cells of the resuspended composition were incubated at about 37.0° C. in an incubator. Approximately 96 hours after the start of stimulation, cells were washed twice in a magnetic field to remove anti-CD3/anti-CD28 conjugated paramagnetic beads, and formulated in a solution containing 10% DMSO. The formulated cell composition was transferred into a bag or vial and stored at about -80°C.

In another method (hereinafter referred to as "extended") after incubation, approximately 100×10 ⁶ viable cells from the stimulated cell composition were concentrated in serum-free medium containing recombinant IL-2, IL-7 and IL-15. Cells were transduced with a lentiviral vector encoding the same anti-BCMA CAR described above by spinoculation at about 1600 g for 60 minutes. After spinoculation, the cells were resuspended in serum-free medium containing recombinant IL-2, IL-7 and IL-15 and incubated for about 18-30 hours at about 37°C. The cells were then cultured for expansion by transfer to a bioreactor (e.g., a shaking bioreactor) in about 500 ml of exemplary serum-free medium containing IL-2, IL-7, and IL-15 at a concentration twice that used during the incubation and transduction steps. . After reaching the established density of viable cells, perfusion was started, while the medium was replaced by the method of semi-continuous perfusion with constant agitation. Cells were cultured the next day in the bioreactor until a threshold cell density of approximately 3×10 ⁶ cells/ml was reached, which typically occurred after 6-7 days of expansion. Paramagnetic beads conjugated with anti-CD3 and anti-CD28 antibodies were removed from the cell composition under the influence of a magnetic field. The cells were then harvested, formulated and frozen as described above.

T cell compositions from the extended and non-expanded constructs were stained with antibodies recognizing surface markers including CD4, CD8, CCR7 and CD27 and quantified by flow cytometry. The percentages of CD4+CAR+ and CD8+CAR+ T cells staining positive for both CCR7 and CD27 are shown in FIG. 1A. FIG. 1B and FIG. 1C shows the percentages of CCR7+CD27+ cells for CD4+CAR+ T cells or CD8+CAR+ T cells, respectively, in the resulting T cell composition compared to the percentage of CCR7+CD27+ cells in the original composition prior to incubation with stimulatory reagent ( CMAT), which are granules conjugated with anti-CD3/anti-CD28 antibodies. As shown, a higher percentage of CCR7+CD27+ was observed in T-cell compositions obtained by the non-expanded design method compared to the expanded design method.

Additional analysis of cells obtained in an expanded way from cells of a representative donor, on various days during the production process, including during activation (AMAT), transduction (XMAT) or at various points in time after the start of cultivation (inoc +2, inoc + 4 or inoc +5) showed that cell expansion is associated with a more differentiated phenotype, as determined by a reduced percentage of CCR7+ CD27+ cells (FIG. 1D).

Example 2 Association of Naive-Like Cell Phenotype with Donor Response to CAR Expressing T Cells

Received exemplary therapeutic T-cell compositions containing autologous T-cells expressing a chimeric antigen receptor (CAR) specific for CD19. The anti-CD19 CAR contained an anti-CD19 scFv derived from a mouse antibody (variable region derived from FMC63), an immunoglobulin derived spacer, a transmembrane domain derived from CD28, a costimulatory region derived from 4-1BB, and an intracellular signaling domain of CD3-zeta .

To obtain cell compositions for administration, autologous cells were obtained from subjects by leukapheresis. Leukapheresis samples were processed by the method to obtain CAR-expressing cells. The method included cell washing using an automated washing system and immune affinity-based selection to purify CD4 ⁺ and CD8 ⁺ T cells, resulting in two compositions enriched in CD8 ⁺ (among which, on average, 99% of cells, with an interquartile latitude (IQR) 98-100%, were CD8 ⁺ cells) and CD4 ⁺ (among which, on average, 99% of the cells, with an IQR of 99-100%, were CD4 ⁺ cells) cells, respectively.

Cells enriched in CD4 ⁺ and CD8 ⁺ compositions were activated with anti-CD3/anti-CD28 paramagnetic beads, and then separately subjected to lentiviral transduction with a vector encoding an anti-CD19 CAR with a 4-1BB costimulatory domain. The transduced populations were then separately incubated in the presence of stimulatory reagents for cell expansion. The expanded CD8 ⁺ and CD4 ⁺ cells were separately formulated, cryopreserved and stored until administration. To minimize variation between batches and/or cell compositions obtained from different patients, for example, those that had patient-specific characteristics of parameters that are indicators of cellular health, cells were maintained at constant volumes for all parties. The cell products had a narrow range of viable cell concentrations (based on the results of the evaluation of cell compositions for one group of subjects, CD8 ⁺ : average number of 31×10 ⁶ cells/ml, IQR 28-40×10 ⁶ cells/ml, N=38; CD4 ⁺ : average number 35×10 ⁶ cells/ml, IQR 31-40×10 ⁶ , N=36).

The therapeutic CAR ⁺ T cell composition described above was administered to subjects with relapsed or refractory (R/R) aggressive non-Hodgkin's lymphoma (NHL) in a clinical study. In particular, a group of adults with R/R NHL, including diffuse large B-cell lymphoma (DLBCL), de novo or degenerated from slow-growing lymphoma (NOS), high-grade B-cell lymphoma (including "double/triple hit" ), DLBCL degenerated from chronic lymphocytic leukemia (CLL) or marginal zone cell lymphomas (MZL), primary mediastinal large B-cell lymphoma (PMBCL), and stage 3b follicular lymphoma (FLG3B) were administered anti-CD19 CAR-expressing T -cells. Outcomes were separately assessed for a major subgroup of subjects in the overall cohort (excluding subjects in poor general condition (ECOG 2) having DLBCL degenerated from marginal zone cell lymphoma (MZL) and/or chronic lymphocytic leukemia (CLL, Richter), and excluding subjects with primary mediastinal large B-cell lymphoma (PMBCL) and stage 3b follicular lymphoma (FLG3B) (main group)). The main cohort included subjects with DLBCL, NOS, and degenerated follicular lymphoma (tFL) or high grade B-cell lymphoma (“double/triple hit”) or high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements, with DLBCL ("double/triple hit") histology and with an Eastern Joint Oncology Group (ECOG PS) overall score of 0 or 1. The time-point analysis presented in this example is based on an evaluation of a total of 91 subjects in the full group (88 (65 of the main group) who were evaluated for response and 91 subjects (67 of the main group) who were evaluated for safety) who were injected with anti-CD19 CAR-expressing cells.

Cryopreserved cell compositions containing anti-CD19 CAR-expressing cells were thawed prior to intravenous administration. The therapeutic dose of T cells was administered as a defined cell composition by separately administering the formulated CD4 ⁺ CAR ⁺ cell population and the formulated CD8 ⁺ CAR ⁺ population, which were administered in a target ratio of approximately 1:1. Subjects were administered a single or double dose of CAR-expressing T cells (each single dose by separate infusions of CD4 ⁺ CAR-expressing T cells and CD8 ⁺ CAR-expressing T cells, respectively) as follows: single dose of Dose Level 1 (DL1) containing 5x10 ⁷ total CAR-expressing T cells, or one dose level 2 (DL2) containing 1x10 ⁸ total CAR-expressing T cells. In some cases, subjects were administered a double dose of DL1, with each dose administered approximately fourteen (14) days apart, administered on Day 1 and Day 14, including one subject who accidentally received two doses of DL2 in a two-dose regimen due to dosing errors. The dosage level and target numbers of T-cell subsets of the administered formulation for DL1 and DL2 are shown in Table E1. In the main group, 34 subjects were administered DL1 and 27 subjects were administered DL2.

Table E1. Target dose level and number of T cell subsets for cell compositions containing anti-CD19 CAR T cells Dose level Dose of Helper T Cells ( _TH )
(CD4 ⁺ CAR ⁺ ) Dose of cytotoxic T cells ( _TC )
(CD8 ⁺ CAR ⁺ ) Total dose of T cells
(CD3 ⁺ CAR ⁺ ) 1 25×10 ⁶ 25×10 ⁶ 50×10 ⁶ 2 50×10 ⁶ 50×10 ⁶ 100×10 ⁶

Table E2 shows the overall results of the assessment of response and safety for the full group and the main group at two dose levels. The objective response rate (ORR) was 74%, including 52% of subjects who had a complete response (CR). The incidence of cytokine release syndrome (CRS) of any grade was 35%, with 1% of severe CRS cases; and the incidence of neurotoxicity (NT) of any degree was 19%, with 1% of severe NT.

Table E2. Response and safety rates after CAR administration ⁺ cells COMPLETE MAIN All dose levels All dose levels ^a DL1 DL2 Best overall response (BEO), n ^b 88 65 34 27 OR, % (95% CI) 74 (63.83) 80(68, 89) 77 (59.89) 82 (62, 94) PO, % (95% CI) 52 (41.63) 55(43, 68) 47 (30, 65) 63 (42, 81) Security, n ^c 91 67 34 29 Any SCV, % (95% CI) 35 (25, 46) 36 (24, 48) 41 (25, 59) 24 (10, 44) tSVP (grade 3-4), % (95% CI) 1 (0, 6) 1 (0.8) 38 (0.15) 0 Any NT, % (95% CI) 19 (11.28) 21 (12, 33) 24 (11, 41) 17 (6, 36) tNT (grade 3-4), % (95% CI) 12 (6, 21) 15 (7, 26) 21 (9, 38) 7 (1, 23)

^a Four patients treated with DL1D (dose level 1, two-dose regimen) with identical results.

^b Includes patients with PT, death, or 28-day re-staging scan. One patient is missing scan results for restaging.

^c Includes all subjects who received at least one dose of an appropriate CAR-expressing cell preparation within 28 days prior to data collection or prior to death.

A. Relationship between cellular characteristics of anti-CD19 CAR-expressing T cells and response

The relationship between certain phenotypic characteristics of CAR ⁺ T cells in therapeutic compositions and parameters associated with clinical responses was assessed. Correlations between memory cell phenotype in composition and function translated into an observed positive correlation between central memory cell subset composition and peaks in in vivo proliferation of CAR ⁺ cells (ρ=0.42, P =0.002) and progression-free survival (PFS) (score Kaplan-Meier survival, P = 0.0164). FIG. 2A-2D are Kaplan-Meier survival curves for subjects treated with CAR ⁺ T cell compositions divided into groups administered compositions with a frequency of occurrence of CCR7 ⁺ CD27 ⁺ CAR ⁺ T cells among CD4 ⁺ CAR ⁺ T cells (FIG. 2A for progression-free survival, FIG. 2C for duration of response) and among CD8 ⁺ CAR ⁺ T cells (FIG. 2B for progression-free survival, FIG. 2D for duration of response) that was above or below a certain threshold. It was found that a greater number of CCR7 ⁺ CD27 ⁺ memory cells in the composition correlated with longer progression-free survival.

Example 3 Assessment of T cell clonality using sequencing and analytical methods

T cell compositions were assessed for relative abundance of T cell clones by single cell sequencing of T cell receptor (TCR) pairs before and after genetic engineering for chimeric antigen receptor (CAR) expression.

Autologous T cells were generated from leukapheresis samples from subjects by immune affinity based selection to purify CD4 ⁺ and CD8 ⁺ T cells, resulting in two compositions enriched in CD8 ⁺ and CD4+ T cells, respectively. Cells enriched in CD4 ⁺ and CD8 ⁺ compositions were activated with anti-CD3/anti-CD28 paramagnetic beads, and then separately subjected to lentiviral transduction with a vector encoding the anti-CD19 CAR. The anti-CD19 CAR contained an anti-CD19 scFv derived from a mouse antibody (variable region derived from FMC63), an immunoglobulin derived spacer, a transmembrane domain derived from CD28, a costimulatory region derived from 4-1BB, and an intracellular signaling domain of CD3-zeta . The transduced populations were then separately incubated in the presence of stimulatory reagents for cell expansion. Propagated compositions of CD8 ⁺ and CD4 ⁺ cells containing CAR-expressing T cells were separately formulated, cryopreserved and stored.

The T-cell clonality of the isolated pre-engineered CD4+ and CD8+ T-cell compositions (CMAT) and of the post-engineered therapeutic CD4+ and CD8+ CAR+ T-cell compositions was assessed by the method described above. To evaluate T-cell clonality, cells were subjected to single-cell αβ-TCR sequencing, generally as described in WO2016044227, WO2016176322 and WO2012048340. Based on single cell sequencing of the TCR genes using barcodes, the T cell clonality and diversity of the identified clones in the cell population was determined. In some cases, the Shannon index was used as a threshold value for filtering clones ("Shannon-corrected clonality"), which allows you to save connections between samples, eliminating the inherent noise of the samples. See Chaara et al. (2018) Front Immunol 9:1038).

FIG. 3 shows the clonality of each sample after applying the Shannon index. As shown in FIG. 3, the clonality of CD4 and CD8 cells differed between pre- and post-engineering T cell compositions, with CAR+ engineered T cell compositions having reduced clonality compared to T cells in compositions prior to the cell engineering process.

The genetically engineered therapeutic CD4+ and CD8+ CAR+ T cell compositions were sorted by flow cytometry based on the expression of a factor indicative of apoptosis, e.g. based on the expression of various surface markers including CD45RA, CCR7, CD27, CD4 and CD8. The cell phenotype correlated with the degree of cell clonality. It was found that, in both CD4+ and CD8+ therapeutic CAR+ T cell compositions, the presence of apoptosis marker negative cells, which were CCR7 ^- /CD27 ^- cells, positively correlated highly with the clonality of the cells in the composition. Similarly, the presence of apoptosis marker negative cells, which were CCR7+ or CCR7+/CD27+ cells, was negatively correlated with cell clonality in each of the CD4+ and CD8+ therapeutic CAR+ T cell formulations. These results are consistent with the fact that cell clonality may be inversely correlated with the presence of less differentiated naive-like cells, as determined by the presence of CCR7 and/or CD27.

The scope of the present invention is not to be limited by the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the described compositions and methods will become apparent from the description and ideas set forth herein. Such variations may be practiced without departing from the scope and spirit of the invention and should be within the scope of the present invention.

SEQUENCES

No. SUBSEQUENCE DESCRIPTION 1 ESKYGPPCPPCP spacer (IgG4 hinge) (AK)
Homo sapiens 2 GAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCT spacer (IgG4 hinge) (nt)
Homo sapiens 3 ESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK spacer hinge-CH3 Homo sapiens 4 ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK spacer hinge-CH2-CH3 Homo sapiens 5 RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLC EVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDH IgD-Hinge-Fc Homo sapiens 6 LEGGGEGRGSLTCGDVEENPGPR T2A, artificial 7 MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGC WGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM tEGFR, artificial 8 FWVLVVVGGVLACYSLLVTVAFIFWV CD28 (amino acids 153-179 in sequence accession no. P10747) Homo sapiens 9 IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP FWVLVVVGGVLACYSLLVTVAFIIFWV CD28 (amino acids 114-179 in sequence accession number P10747) Homo sapiens 10 RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS CD28 (amino acids 180-220 in P10747) Homo sapiens eleven RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS CD28 (LL to GG) Homo sapiens 12 KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 4-1BB (amino acids 214-255 in Q07011.1) Homo sapiens 13 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD3-zeta Homo sapiens 14 RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD3-zeta Homo sapiens 15 RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD3-zeta Homo sapiens 16 RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDK CNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM tEGFR, artificial 17 EGRGSLLTCGDVEENPGP T2A, artificial 18 MALPVTALLLPLALLLHA signal peptide CD8-alpha 19 GSGATNFSLLKQAGDVEENPGP P2A 20 ATNFSLLKQAGDVEENPGP P2A 21 QCTNYALLKLAGDVESNPGP E2A 22 VKQTLNFDLLKLAGDVESNPGP F2A 23 PGGG-(SGGGG)5-P- where P is proline, G is glycine and S is serine linker 24 GSADDAKKDAAKKDGKS linker 25 atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatccca GMCSFR alpha chain signal sequence 26 MLLLVTSLLLCELPHPAFLLIP GMCSFR alpha chain signal sequence 27 Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Hinge 28 ELKTPLGDTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP Hinge 29 Glu Ser Lys Tyr Gly Pro Cys Pro Ser Cys Pro Hinge thirty Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Hinge 31 Tyr Gly Pro Pro Cys Pro Pro Cys Pro Hinge 32 Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Hinge 33 Glu Val Val Val Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Hinge 34 QQGNTLPYT FMC63 LC-CDR3 35 RASQDISKYLN FMC63 CDR L1 36 SRLHSGV FMC63 CDR L2 37 GNTLPYTFG FMC63 CDR L3 38 DYGVS FMC63 CDR H1 39 VIWGSETTYYNSALKS FMC63 CDR H2 40 YAMDYWG FMC63 CDR H3 41 EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS FMC63 VH 42 DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT FMC63 VL 43 DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTI IKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS FMC63 scFv 44 KASQNVGTNVA SJ25C1 CDR L1 45 SATYRNS SJ25C1 CDR L2 46 QQYNRYPYT SJ25C1 CDR L3 47 SYWMN SJ25C1 CDR H1 48 QIYPGDGDTNYNGKFKG SJ25C1 CDR H2 49 KTISSVVDFYFDY SJ25C1 CDR H3 50 EVKLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCARKTISSVVDFYFDYWGQGTTVTVSS SJ25C1 VH 51 DIELTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKLEIKR SJ25C1 VL 52 GGGGSGGGGSGGGGS Linker 53 EVKLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCARKTISSVVDFYFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQ SPKPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKLEIKR SJ25C1 scFv 54 HYYYGGSYAMDY FMC63HC-CDR3 55 HTSRLHS FMC63 LC-CDR2 56 GSTSGSGKPGSGEGSTKG Linker 57 gacatccagatgacccagaccacctccagcctgagcgccagcctgggcgaccgggtgaccatcagctgccgggccagccaggacatcagcaagtacctgaactggtatcagcagaagcccgacggcaccgtcaagctgctgatctaccacaccagccggctgcacagcggcgtgcccagccggtttagcggcagcggctccggcaccgactac agcctgaccatctccaacctggaacaggaagatatcgccacctacttttgccagcagggcaacacactgccctacacctttggcggcggaacaaagctggaaatcaccggcagcacctccggcagcggcaagcctggcagcggcgagggcagcaccaagggcgaggtgaagctgcaggaaagcggccctggcctggtggcccccagccagagcct gagcgtgacctgcaccgtgagcggcgtgagcctgcccgactacggcgtgagctggatccggcagccccccaggaagggcctggaatggctggggcgtgatctggggcagcgaccacctactacaacagcgccctgaagagccggctgaccatcatcaaggacaacagcaagagccaggtgttcctgaagatgaacagcctgcagaccg acgacaccgccatctactactactgcgccaagcactactactacggcggcagctacgccatggactactggggccagggcaccagcgtgaccgtgagcagc scFv encoding sequence 58 X1PPX2P
X1 is glycine, cysteine or arginine
X2 is cysteine or threonine Hinge 59 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Hinge

--->

SEQUENCE LIST

<110> JUNO THERAPEUTICS, INC.

BONYHADI, Mark L.

<120> METHODS AND COMPOSITIONS FOR OBTAINING GENETICALLY ENGINEERED CELLS

<130> 735042010540

<140> Not yet defined

<141> Along with this

<150> 62/543.359

<151> 2017-08-09

<160> 59

<170> FastSEQ for Windows version 4.0

<210> 1

<211> 12

<212> PROTEIN

<213> Homo sapiens

<220>

<223> Spacer (IgG4 hinge)

<400> 1

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro

1 5 10

<210> 2

<211> 36

<212> DNA

<213> Homo sapiens

<220>

<223> Spacer (IgG4 hinge)

<400> 2

gaatctaagt acggaccgcc ctgcccccct tgccct 36

<210> 3

<211> 119

<212> PROTEIN

<213> Homo sapiens

<220>

<223> Spacer Hinge-CH3

<400> 3

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gln Pro Arg

1 5 10 15

Glu Pro Gln Val Tyr Thr Leu Pro Ser Gln Glu Glu Met Thr Lys

20 25 30

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

35 40 45

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

50 55 60

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

65 70 75 80

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

85 90 95

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

100 105 110

Leu Ser Leu Ser Leu Gly Lys

115

<210> 4

<211> 229

<212> PROTEIN

<213> Homo sapiens

<220>

<223> Spacer Hinge-CH2-CH3

<400> 4

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe

1 5 10 15

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

20 25 30

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

115 120 125

Gln Val Tyr Thr Leu Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

130 135 140

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

145 150 155 160

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

180 185 190

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly Lys

225

<210> 5

<211> 282

<212> PROTEIN

<213> Homo sapiens

<220>

<223> IgD-hinge-Fc

<400> 5

Arg Trp Pro Glu Ser Pro Lys Ala Gln Ala Ser Ser Val Pro Thr Ala

1 5 10 15

Gln Pro Gln Ala Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro Ala

20 25 30

Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys

35 40 45

Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro

50 55 60

Ser His Thr Gln Pro Leu Gly Val Tyr Leu Leu Thr Pro Ala Val Gln

65 70 75 80

Asp Leu Trp Leu Arg Asp Lys Ala Thr Phe Thr Cys Phe Val Val Gly

85 90 95

Ser Asp Leu Lys Asp Ala His Leu Thr Trp Glu Val Ala Gly Lys Val

100 105 110

Pro Thr Gly Gly Val Glu Glu Gly Leu Leu Glu Arg His Ser Asn Gly

115 120 125

Ser Gln Ser Gln His Ser Arg Leu Thr Leu Pro Arg Ser Leu Trp Asn

130 135 140

Ala Gly Thr Ser Val Thr Cys Thr Leu Asn His Pro Ser Leu Pro Pro

145 150 155 160

Gln Arg Leu Met Ala Leu Arg Glu Pro Ala Ala Gln Ala Pro Val Lys

165 170 175

Leu Ser Leu Asn Leu Leu Ala Ser Ser Asp Pro Pro Glu Ala Ala Ser

180 185 190

Trp Leu Leu Cys Glu Val Ser Gly Phe Ser Pro Pro Asn Ile Leu Leu

195 200 205

Met Trp Leu Glu Asp Gln Arg Glu Val Asn Thr Ser Gly Phe Ala Pro

210 215 220

Ala Arg Pro Pro Pro Gln Pro Gly Ser Thr Thr Phe Trp Ala Trp Ser

225 230 235 240

Val Leu Arg Val Pro Ala Pro Pro Ser Pro Gln Pro Ala Thr Tyr Thr

245 250 255

Cys Val Val Ser His Glu Asp Ser Arg Thr Leu Leu Asn Ala Ser Arg

260 265 270

Ser Leu Glu Val Ser Tyr Val Thr Asp His

275 280

<210> 6

<211> 24

<212> PROTEIN

<213> Artificial sequence

<220>

<223> T2A

<400> 6

Leu Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp

1 5 10 15

Val Glu Glu Asn Pro Gly Pro Arg

20

<210> 7

<211> 357

<212> PROTEIN

<213> Artificial sequence

<220>

<223>tEGFR

<400> 7

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly

20 25 30

Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe

35 40 45

Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala

50 55 60

Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu

65 70 75 80

Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile

85 90 95

Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu

100 105 110

Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala

115 120 125

Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu

130 135 140

Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr

145 150 155 160

Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys

165 170 175

Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly

180 185 190

Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu

195 200 205

Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys

210 215 220

Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu

225 230 235 240

Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met

245 250 255

Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala

260 265 270

His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val

275 280 285

Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His

290 295 300

Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro

305 310 315 320

Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala

325 330 335

Thr Gly Met Val Gly Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly

340 345 350

Ile Gly Leu Phe Met

355

<210> 8

<211> 27

<212> PROTEIN

<213> Homo sapiens

<220>

<223>CD28

<300>

<308> UniProt P10747

<309> 1989-07-01

<400> 8

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

20 25

<210> 9

<211> 66

<212> PROTEIN

<213> Homo sapiens

<220>

<223>CD28

<300>

<308> UniProt P10747

<309> 1989-07-01

<400> 9

Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn

1 5 10 15

Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu

20 25 30

Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly

35 40 45

Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe

50 55 60

Trp Val

65

<210> 10

<211> 41

<212> PROTEIN

<213> Homo sapiens

<220>

<223>CD28

<300>

<308> UniProt P10747

<309> 1989-07-01

<400> 10

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 11

<211> 41

<212> PROTEIN

<213> Homo sapiens

<220>

<223>CD28

<400> 11

Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 12

<211> 42

<212> PROTEIN

<213> Homo sapiens

<220>

<223> 4-1BB

<300>

<308> UniProt Q07011.1

<309> 1995-02-01

<400> 12

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 13

<211> 112

<212> PROTEIN

<213> Homo sapiens

<220>

<223> CD3-zeta

<400> 13

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 14

<211> 112

<212> PROTEIN

<213> Homo sapiens

<220>

<223> CD3-zeta

<400> 14

Arg Val Lys Phe Ser Arg Ser Ala Glu Pro Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 15

<211> 112

<212> PROTEIN

<213> Homo sapiens

<220>

<223> CD3-zeta

<400> 15

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 16

<211> 335

<212> PROTEIN

<213> Artificial sequence

<220>

<223>tEGFR

<400> 16

Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu

1 5 10 15

Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile

20 25 30

Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe

35 40 45

Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr

50 55 60

Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn

65 70 75 80

Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg

85 90 95

Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile

100 105 110

Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val

115 120 125

Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp

130 135 140

Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn

145 150 155 160

Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu

165 170 175

Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser

180 185 190

Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu

195 200 205

Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln

210 215 220

Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly

225 230 235 240

Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro

245 250 255

His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr

260 265 270

Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His

275 280 285

Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro

290 295 300

Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala

305 310 315 320

Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met

325 330 335

<210> 17

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> T2A

<400> 17

Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro

1 5 10 15

GlyPro

<210> 18

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CD8-alpha signal peptide

<400> 18

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala

<210> 19

<211> 22

<212> PROTEIN

<213> Artificial sequence

<220>

<223> P2A

<400> 19

Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val

1 5 10 15

Glu Glu Asn Pro Gly Pro

20

<210> 20

<211> 19

<212> PROTEIN

<213> Artificial sequence

<220>

<223> P2A

<400> 20

Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn

1 5 10 15

Pro Gly Pro

<210> 21

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<223>E2A

<400> 21

Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser

1 5 10 15

Asn Pro Gly Pro

20

<210> 22

<211> 22

<212> PROTEIN

<213> Artificial sequence

<220>

<223> F2A

<400> 22

Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val

1 5 10 15

Glu Ser Asn Pro Gly Pro

20

<210> 23

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Linker

<220>

<221> REPEAT

<222> (5)...(9)

<223> SGGGG repeated 5 times

<400> 23

Pro Gly Gly Gly Ser Gly Gly Gly Gly Pro

1 5 10

<210> 24

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Linker

<400> 24

Gly Ser Ala Asp Asp Ala Lys Lys Asp Ala Ala Lys Lys Asp Gly Lys

1 5 10 15

Ser

<210> 25

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> GMCSFR alpha chain signal sequence

<400> 25

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

atcca 66

<210> 26

<211> 22

<212> PROTEIN

<213> Artificial sequence

<220>

<223> GMCSFR alpha chain signal sequence

<400> 26

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro

20

<210> 27

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Hinge

<400> 27

Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro

1 5 10

<210> 28

<211> 61

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Hinge

<400> 28

Glu Leu Lys Thr Pro Leu Gly Asp Thr His Thr Cys Pro Arg Cys Pro

1 5 10 15

Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu

20 25 30

Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu Pro

35 40 45

Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro

50 55 60

<210> 29

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Hinge

<400> 29

Glu Ser Lys Tyr Gly Pro Cys Pro Ser Cys Pro

1 5 10

<210> 30

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Hinge

<400> 30

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro

1 5 10

<210> 31

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Hinge

<400> 31

Tyr Gly Pro Pro Cys Pro Pro Cys Pro

15

<210> 32

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Hinge

<400> 32

Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro

1 5 10

<210> 33

<211> 14

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Hinge

<400> 33

Glu Val Val Val Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro

1 5 10

<210> 34

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FMC63 LC-CDR3

<400> 34

Gln Gln Gly Asn Thr Leu Pro Tyr Thr

15

<210> 35

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FMC63 CDR L1

<400> 35

Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn

1 5 10

<210> 36

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FMC63 CDR L2

<400> 36

Ser Arg Leu His Ser Gly Val

15

<210> 37

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FMC63 CDR L3

<400> 37

Gly Asn Thr Leu Pro Tyr Thr Phe Gly

15

<210> 38

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FMC63 CDR H1

<400> 38

Asp Tyr Gly Val Ser

15

<210> 39

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FMC63 CDR H2

<400> 39

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser

1 5 10 15

<210> 40

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FMC63 CDR H3

<400> 40

Tyr Ala Met Asp Tyr Trp Gly

15

<210> 41

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FMC63 VH

<400> 41

Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln

1 5 10 15

Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu

65 70 75 80

Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 42

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FMC63 VL

<400> 42

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr

100 105

<210> 43

<211> 245

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FMC63 scFv

<400> 43

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Ser Thr Ser Gly

100 105 110

Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Lys

115 120 125

Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser

130 135 140

Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser

145 150 155 160

Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile

165 170 175

Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu

180 185 190

Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn

195 200 205

Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr

210 215 220

Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

225 230 235 240

Val Thr Val Ser Ser

245

<210> 44

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> SJ25C1 CDR L1

<400> 44

Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala

1 5 10

<210> 45

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> SJ25C1 CDR L2

<400> 45

Ser Ala Thr Tyr Arg Asn Ser

15

<210> 46

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> SJ25C1 CDR L3

<400> 46

Gln Gln Tyr Asn Arg Tyr Pro Tyr Thr

15

<210> 47

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> SJ25C1 CDR H1

<400> 47

Ser Tyr Trp Met Asn

15

<210> 48

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> SJ25C1 CDR H2

<400> 48

Gln Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys

1 5 10 15

gly

<210> 49

<211> 13

<212> PROTEIN

<213> Artificial sequence

<220>

<223> SJ25C1 CDR H3

<400> 49

Lys Thr Ile Ser Ser Val Val Asp Phe Tyr Phe Asp Tyr

1 5 10

<210> 50

<211> 122

<212> PROTEIN

<213> Artificial sequence

<220>

<223> SJ25C1 VH

<400> 50

Glu Val Lys Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr

20 25 30

Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gln Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Gln Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Gly Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Lys Thr Ile Ser Ser Val Val Asp Phe Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 51

<211> 108

<212> PROTEIN

<213> Artificial sequence

<220>

<223> SJ25C1 VL

<400> 51

Asp Ile Glu Leu Thr Gln Ser Pro Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Pro Leu Ile

35 40 45

Tyr Ser Ala Thr Tyr Arg Asn Ser Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Asn Val Gln Ser

65 70 75 80

Lys Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Arg Tyr Pro Tyr

85 90 95

Thr Ser Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105

<210> 52

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Linker

<400> 52

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 53

<211> 245

<212> PROTEIN

<213> Artificial sequence

<220>

<223> SJ25C1 scFv

<400> 53

Glu Val Lys Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr

20 25 30

Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gln Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Gln Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Gly Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Lys Thr Ile Ser Ser Val Val Asp Phe Tyr Phe Asp Tyr Trp

100 105 110

Gly Glyn Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser

130 135 140

Pro Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Val Thr Cys

145 150 155 160

Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala Trp Tyr Gln Gln Lys

165 170 175

Pro Gly Gln Ser Pro Lys Pro Leu Ile Tyr Ser Ala Thr Tyr Arg Asn

180 185 190

Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe

195 200 205

Thr Leu Thr Ile Thr Asn Val Gln Ser Lys Asp Leu Ala Asp Tyr Phe

210 215 220

Cys Gln Gln Tyr Asn Arg Tyr Pro Tyr Thr Ser Gly Gly Gly Thr Lys

225 230 235 240

Leu Glu Ile Lys Arg

245

<210> 54

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FMC63 HC-CDR3

<400> 54

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr

1 5 10

<210> 55

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FMC63 LC-CDR2

<400> 55

His Thr Ser Arg Leu His Ser

15

<210> 56

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Linker

<400> 56

Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr

1 5 10 15

Lys Gly

<210> 57

<211> 735

<212> DNA

<213> Artificial sequence

<220>

<223> scFv encoding sequence

<400> 57

gacatccaga tgacccagac cacctccagc ctgagcgcca gcctgggcga ccgggtgacc 60

atcagctgcc gggccagcca ggacatcagc aagtacctga actggtatca gcagaagccc 120

gacggcaccg tcaagctgct gatctaccac accagccggc tgcacagcgg cgtgcccagc 180

cggtttagcg gcagcggctc cggcaccgac tacagcctga ccatctccaa cctggaacag 240

gaagatatcg ccacctactt ttgccagcag ggcaacacac tgccctacac ctttggcggc 300

ggaacaaagc tggaaatcac cggcagcacc tccggcagcg gcaagcctgg cagcggcgag 360

ggcagcacca agggcgaggt gaagctgcag gaaagcggcc ctggcctggt ggcccccagc 420

cagagcctga gcgtgacctg caccgtgagc ggcgtgagcc tgcccgacta cggcgtgagc 480

tggatccggc agccccccag gaagggcctg gaatggctgg gcgtgatctg gggcagcgag 540

accacctact acaacagcgc cctgaagagc cggctgacca tcatcaagga caacagcaag 600

agccaggtgt tcctgaagat gaacagcctg cagaccgacg acaccgccat ctactactgc 660

gccaagcact actactacgg cggcagctac gccatggact actggggcca gggcaccagc 720

gtgaccgtga gcagc 735

<210> 58

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Hinge

<220>

<221> OPTION

<222> (1)...(1)

<223> Xaa1 is glycine, cysteine or arginine

<220>

<221> OPTION

<222> (4)...(4)

<223> Xaa4 is cysteine or threonine

<400> 58

Xaa Pro Pro Xaa Pro

15

<210> 59

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Hinge

<400> 59

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

<---

Claims (110)

1. The method of genetic engineering of T-cells, including: (a) incubating the parent composition under stimulating conditions for 2-6 days, wherein said parent composition contains a T cell population comprising naive-like T cells and non-naive-like T cells, wherein: (i) the stimulating conditions include the presence of a stimulating reagent containing an anti-CD3 antibody primary agent and an anti-CD28 antibody auxiliary agent, (ii) the naive-like T cells comprise a number of naive-like T cells required to initiate culture of at least or at least about 0.5 x 10 ⁸ of naive-like T cells, wherein the naive-like T cells are CD45RA+, CD27+, CCR7+ and/or CD45RO- cells, and (iii) non-naïve-like T cells are CD45RA-, CD27-, CCR7- and/or CD45RO+ cells; And (b) introducing a nucleic acid encoding a genetically engineered recombinant receptor into a population of T cells, wherein the introduction is carried out during at least part of the incubation period. 2. The method according to p. 1, while the incubation is carried out for at least 3 days. 3. The method according to p. 1, while the incubation is carried out for at least 4 days. 4. The method according to p. 1, while the incubation is carried out for at least 5 days. 5. The method according to p. 1, while the incubation is carried out for at least 6 days. 6. A method for stimulating T cells, comprising: (a) incubation of the initial composition under stimulating conditions for 2-6 days, wherein the specified initial composition contains T cells, including the number of naive-like T cells, or a subset of CD8+ T cells, necessary for culture initiation, to obtain a stimulated composition , wherein: (i) stimulating conditions include the presence of a stimulating reagent containing an anti-CD3 antibody primary agent and an anti-CD28 antibody auxiliary agent; And (ii) the number of naive-like T cells or a subset of CD8+ T cells required to initiate culture is at least or is at least about 0.5 x 10 ⁸ of naive-like T cells or a subset of CD8+ T cells thereof, wherein naive-like T cells, or a subset of CD8+ T cells thereof, are CD45RA+, CD27+, CCR7+ and/or CD45RO cells, and (b) introducing into the stimulated cell composition a nucleic acid encoding a genetically engineered recombinant receptor to obtain a composition produced in this way, containing T cells expressing the genetically engineered recombinant receptor. 7. The method according to any one of paragraphs. 1-6, wherein the number of naïve-like T cells or a subset of CD8+ T cells required to initiate culture is or is about 1x10 ⁸ to 5x10 ⁸ naïve-like T cells or a subset of CD8+ T cells. 8. The method according to any one of paragraphs. 1-7, wherein the number of naive-like T cells or a subset of CD8+ T cells required to initiate culture is or is about 2x10 ⁸ of naive-like T cells or a subset of CD8+ T cells. 9. The method according to any one of paragraphs. 6-8, wherein the T cells include naive-like T cells and non-naive-like T cells, wherein the stimulating conditions preferentially induce multiplication or proliferation of naive-like T cells compared to non-naive-like T cells in the stimulated composition. 10. The method according to any one of paragraphs. 6-9, wherein the administration is carried out during at least part of the incubation period or is carried out after the incubation. 11. A method for stimulating T cells, comprising: (a) incubation of the initial composition under stimulating conditions for 2-6 days, while the specified initial composition contains T-cells, including the number of naive-like T-cells or their subset of CD8+ T-cells necessary for culture initiation, constituting or constituting about 0 .5×10 ⁸ naive-like T cells, or a subset of CD8+ T cells thereof, to obtain a stimulated composition, wherein: (i) the stimulating conditions include the presence of a stimulating reagent containing an anti-CD3 antibody primary agent and an anti-CD28 antibody auxiliary agent, (ii) naive-like T cells or a subset of CD8+ T cells comprising from about 1×10 ⁸ to about 4×10 ⁸ naive-like T cells or a subset of CD8+ T cells thereof, wherein naive-like T cells or naive CD8+ T cells are CD45RA+, CD27+, CCR7+ and/or CD45RO- cells. 12. The method of claim 11, wherein the T cells comprise naïve-like T cells and non-naïve-like T cells, wherein the stimulatory conditions preferably induce multiplication or proliferation of naïve-like T cells versus non-naïve-like T cells in a stimulated composition. 13. The method according to claim 11 or 12, wherein the number of naive-like T cells or a subset of CD8+ T cells required to initiate the culture is at least about 2×10 ⁸ like naive T-cells or a subset of CD8+ T-cells . 14. The method according to any one of paragraphs. 6-13, with the amount needed to initiate culture being the number of naive-like CD8+ T cells. 15. The method according to any one of paragraphs. 1-14, with naive-like T cells or naive-like CD8+ T cells: do not have on the surface a marker selected from the group consisting of CD25, CD45RO, CD56, CD62L, KLRG1; have low expression of CD95; or lack intracellular expression of a cytokine selected from the group consisting of IL-2, IFN-γ, IL-4, IL-10. 16. The method according to any one of paragraphs. 1-14, with naive-like cells or naive-like CD8+ cells: do not have on the surface a marker selected from the group consisting of CD45RO, CD56, KLRG1; or have low CD95 expression. 17. The method according to any one of paragraphs. 1-16, wherein naive-like T cells or naive-like CD8+ cells are CD27+ and CCR7+ cells. 18. The method according to any one of paragraphs. 1-5, 9-10 and 12-17, while not similar to naive T-cells: do not have on the surface of the T-cell marker CD28; have on their surface a marker selected from the group consisting of CD25, CD56, CD62L, KLRG1 and perforin; have intracellular expression of a cytokine selected from the group consisting of IL-2, IFN-γ, IL-4, IL-10; or have high expression of CD95. 19. The method according to any one of paragraphs. 1-18, wherein the cells of the original composition were not and are not subjected prior to incubation to a selection step based on an endogenous T-cell surface marker that distinguishes between naive-like and non-naive-like T cells. 20. The method according to any one of paragraphs. 11-19, further comprising introducing a genetically engineered recombinant receptor into stimulated cells, with obtaining by this method the produced composition containing T-cells expressing the genetically engineered recombinant receptor. 21. The method according to claim 20, wherein the incubation of the composition under stimulating conditions is carried out before, during or after the introduction of a nucleic acid encoding a genetically engineered recombinant receptor. 22. The method according to any one of paragraphs. 1-10 and 20-21, wherein the recombinant receptor is capable of binding a target antigen that is associated with, specific for, or expressed on a cell or tissue associated with the disease, disorder, or condition. 23. The method of claim 22, wherein the disease, disorder or condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease or tumor, or cancer. 24. The method of claim 22 or 23, wherein the target antigen is a tumor antigen. 25. The method according to any one of paragraphs. 22-24, wherein the target antigen is selected from αvβ6 integrin (avb6 integrin), B cell maturation antigen (BCMA), carbonic anhydrase 9 (CAIX), Her2/neu (erbB2 tyrosine kinase receptor), L1-CAM, B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), testicular cancer antigen, testicular cancer antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), and hepatitis B surface antigen, antifolate receptor, cyclin, cyclin A2, chemokine ligand 1 with C-C motif (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin A2 receptor (EPHa2) , erb-B2, erb-B3, erb-B4, erbB dimers, mutant epidermal growth factor receptor type III (EGFR vIII), folate-binding protein (FBP), Fc receptor-like protein 5 (FCRL5, also known as the Fc homolog -receptor 5 or FCRH5), embryonic acetylcholine receptor (embryonic AchR), folate binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glypican-3 (GPC3) associated with G proteins of 5D receptor (GPRC5D), Her2/neu (erbB2 tyrosine kinase receptor), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, human high molecular weight melanoma-associated antigen (HMW-MAA), surface antigen hepatitis B virus, human leukocyte antigen A1 (HLA-A1), human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha (IL-22R-alpha), IL-13R-alpha2 (IL-13Rα2), receptor domain kinase insert (kdr), kappa light chain, Lewis Y, L1 cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM, member of the A family of 8 proteins containing leucine-rich repeats (LRRC8A), Lewis Y, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, mouse cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 D ligands ( NKG2D), melan A (MART-1), neuronal cell adhesion molecule (NCAM), oncoembryonic antigen, preferably expressed melanoma antigen (PRAME), progesterone receptor, prostate specific antigen, prostate stem cell antigen (PSCA), prostate specific membrane antigen ( PSMA), tyrosine kinase receptor orphan receptor 1 (ROR1), survivin, trophoblastic glycoprotein (TPBG, also known as 5T4), tumor-associated glycoprotein 72 (TAG72), tyrosinase-dependent protein 1 (TRP1, also known as TYRP1 or gp75 ), tyrosinase-dependent protein 2 (TRP2, also known as dopachrome tautomerase, dopachrome delta isomerase or DCT), folate receptor-α, 8H9, dual antigen, glycoprotein 100 (gp100), vascular endothelial growth factor receptor (VEGFR), receptor vascular endothelial growth factor 2 (VEGF-R2), estrogen receptor, progesterone receptor, Wilms tumor antigen 1 (WT-1), pathogen-specific or pathogen-expressed antigen, and universal marker-associated antigen. 26. The method according to any one of paragraphs. 1-10 and 20-25, wherein the recombinant receptor is, or contains, a functional non-TCR antigen receptor or TCR, or an antigen-binding fragment thereof. 27. The method according to any one of paragraphs. 1-10 and 20-26, wherein the recombinant receptor is a chimeric antigen receptor (CAR). 28. The method according to any one of paragraphs. 1-10 and 20-27, while the recombinant receptor contains an extracellular domain containing an antigen-binding domain that specifically binds the target antigen, and an intracellular signaling domain containing ITAM. 29. The method of claim 28, wherein the antigen binding domain is, or contains, an antibody or antibody fragment, which optionally is a single chain fragment. 30. The method according to claim 28 or 29, wherein the intracellular signaling domain is, or contains, an intracellular signaling domain of the CD3 chain, optionally a CD3 zeta chain (CD3ζ) or a signal fragment thereof. 31. The method according to any one of paragraphs. 28-30, wherein the intracellular signaling region further comprises a costimulatory signaling region. 32. The method of claim 31, wherein the costimulatory signaling region comprises an intracellular signaling domain of a T cell costimulatory molecule or a signal fragment thereof. 33. The method according to claim 31 or 32, wherein the co-stimulatory signaling region contains an intracellular signaling domain of CD28, 4-1BB or ICOS or a signal fragment thereof. 34. The method according to any one of paragraphs. 27-33, while: CAR contains an scFv antigen-specific transmembrane domain, a cytoplasmic signaling domain from a co-stimulatory molecule, which is optionally, or contains, 4-1BB, and a cytoplasmic signaling domain from a core signaling ITAM-containing molecule, which is optionally , or contains, the CD3-zeta signaling domain, and optionally also contains a spacer between the transmembrane domain and scFv; CAR contains, in the following order, an scFv antigen-specific transmembrane domain, a cytoplasmic signaling domain from a co-stimulatory molecule, which optionally is, or contains, a 4-1BB signaling domain, and a cytoplasmic signaling domain from a core signaling ITAM-containing molecule , which is optionally a CD3-zeta signaling domain; or The CAR contains, in the following order, an antigen-specific scFv, a spacer, a transmembrane domain, a cytoplasmic signaling domain from a costimulatory molecule that optionally is, or contains, a 4-1BB signaling domain, and a cytoplasmic signaling domain from the basic signaling ITAM- containing a molecule, which, optionally, is, or contains, the signal domain of CD3-zeta. 35. The method according to any one of paragraphs. 1-34, while the main or auxiliary agent is on the surface of the solid substrate. 36. The method of claim 35, wherein the solid support is, or contains, a bead. 37. The method of claim 36, wherein the bead has a diameter greater than about 3.5 microns but not greater than about 9 microns. 38. The method according to claim 36 or 37, wherein the granule has a diameter of about 4.5 microns. 39. The method according to any one of paragraphs. 36-38, wherein the granule is approximately the size of a lymphocyte or an antigen presenting cell in diameter. 40. The method according to any one of paragraphs. 36-39, wherein the pellet is inert. 41. The method according to any one of paragraphs. 36-40, wherein the pellet is, or has, a polystyrene surface. 42. The method according to any one of paragraphs. 36-41, wherein the granule has a magnetic or superparamagnetic core. 43. The method according to any one of paragraphs. 35-42, wherein the stimulating condition comprises incubating cells at a bead to cell ratio of about 1:1 to about 10:1. 44. The method of genetic engineering of T-cells, including: (a) incubating the parent composition under stimulating conditions for 2-6 days, wherein said parent composition contains a T cell population comprising naive-like T cells and non-naive-like T cells, wherein: (i) stimulating conditions include the presence of a stimulating reagent comprising an anti-CD3 antibody primary agent and an anti-CD28 antibody auxiliary agent, which are attached to the bead, wherein the bead-to-cell ratio during incubation is or is about from 1:1 to 4:1; And (ii) the naïve-like T cells comprise a number of naïve-like T cells required for culture initiation of at least or at least about 0.5 x 10 ⁸ of naïve-like T cells, wherein the naïve-like T cells are CD45RA+, CD27+, CCR7+ and/or CD45RO- cells, (iii) non-naïve-like T cells are CD45RA-, CD27-, CCR7- and/or CD45RO+ cells; And (b) introducing a nucleic acid encoding a genetically engineered recombinant receptor into a population of T cells, wherein the introduction is carried out during at least part of the incubation period. 45. The method of claim 44, wherein the number of naïve-like T cells or a subset of CD8+ T cells required to initiate the culture is or is about 1×10 ⁸ to 5×10 ⁸ of naïve-like T cells or a subset of CD8+ T cells. T cells. 46. The method of claim 44, wherein the number of naive-like T cells or a subset of CD8+ T cells required to initiate the culture is or is about 2x10 ⁸ of naive-like T cells or a subset of CD8+ T cells. 47. The method according to any one of paragraphs. 44-46, while naive T cells are CD27+ and CCR7+ cells. 48. The method according to any one of paragraphs. 43-47, with a granule to cell ratio of approximately 3:1. 49. The method according to any one of paragraphs. 43-47, while the ratio of granules and cells is approximately 1:1. 50. The method according to any one of paragraphs. 1-49, wherein the T cells are derived from a biological sample, optionally wherein the biological sample is derived from a human subject. 51. The method of claim 50, wherein the biological sample is, or contains, a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a white blood cell sample, an apheresis product or a leukapheresis product. 52. The method according to any one of paragraphs. 1-51, wherein the T cells include CD4+ and/or CD8+ cells. 53. The method according to any one of paragraphs. 1-52, wherein the T cells include CD4+ and CD8+ T cells and the ratio of CD4+ to CD8+ T cells is from about 2:1 to about 1:5. 54. The method according to any one of paragraphs. 1-53, wherein the T cells include CD4+ and CD8+ T cells and the ratio of CD4+ to CD8+ T cells is or is about 1:1, 1:2, 2:1, 1:3, or 3:1. 55. The method according to any one of paragraphs. 1-54, wherein naive-like T cells include naive-like CD4+ T cells and/or naive-like CD8+ T cells. 56. The method according to any one of paragraphs. 1-55, with naive-like T cells being polyclonal. 57. The method according to any one of paragraphs. 1-56, wherein the stimulus condition does not include N-acetylcysteine (NAC). 58. The method according to any one of paragraphs. 1-57, wherein the stimulus condition does not include IL-15 or does not include IL-7. 59. The method according to any one of paragraphs. 1-58, wherein the stimulus condition results in or induces the death of non-naïve T cells or a subset thereof. 60. The method according to any one of paragraphs. 1-59, wherein the stimulatory condition results in activation-induced cell death (AICD) of non-naïve-like T cells or a subset thereof. 61. The method according to any one of paragraphs. 1-60, further comprising adding DNase during incubation or to the stimulated composition. 62. The method according to any one of paragraphs. 6-58, wherein the incubation is for more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 days. 63. The method according to any one of paragraphs. 1-62, wherein after incubation under stimulatory conditions, the percentage of cells derived from similar naive T cells increases by more than about 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times , 8 times, 9 times, 10 times, 50 times, 100 times compared to the percentage of naive-like cells in the original composition. 64. The method according to any one of paragraphs. 1-63, while after incubation under stimulating conditions, the content of cells derived from naive T-like cells, relative to cells derived from non-naive T-cells like, increases by more than about 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 50 times, 100 times compared to the content of naive-like T cells relative to non-naive-like T cells in the original composition. 65. The method according to any one of paragraphs. 1-64, while after incubation under stimulating conditions, the composition contains more than 75% of cells that originated from naive-like T cells of the original composition. 66. The method according to any one of paragraphs. 1-65, while after incubation under stimulating conditions, the composition contains less than 10% of cells derived from non-naive T cells. 67. The method according to any one of paragraphs. 1-66, while after incubation under stimulating conditions, the composition contains less than 9% of cells derived from non-naive T cells. 68. The method according to any one of paragraphs. 1-67 wherein, of the cells in the original composition, a higher percentage of naive-like T cells, as compared to non-naive T-cells, are induced to proliferate or become activated. 69. The method according to any one of paragraphs. 1-68, with a large percentage of T cells that were similar to naive in the original composition, compared to the percentage of T cells that were not similar to naive in the original composition, divided on day 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 after the start of said incubation. 70. The method according to any one of paragraphs. 1-69, wherein the stimulating conditions are able to induce the proliferation of a greater percentage of cells in the naïve human T-like population compared to non-naïve human T cell population on days 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 after the start of incubation under these conditions. 71. The method according to any one of paragraphs. 1-70, wherein: (i) the non-naïve T cells are selected from the group consisting of effector T cells (T _EFF ), memory T cells, central memory T cells (T _CM ), effector T cells memory (T _EM ), as well as their combinations; or (ii) the non-naïve T cells are a plurality of T cells including or consisting of effector T cells (T _EFF ) and/or memory T cells, wherein the memory T cells optionally include central T cells. memory cells (T _CM ) and/or effector memory T cells (T _EM ). 72. The method according to any one of paragraphs. 1-71, while the percentage of naive-like T cells in the original composition is less than the percentage of engineered cells in the composition, after incubation under stimulating conditions, derived from naive-like T cells in the original composition. 73. The method according to any one of paragraphs. 1-10 and 20-72, wherein a large percentage of cells with the introduced nucleic acid are or are derived from the proliferation of naive-like T cells in the original composition compared to non-naïve-like T cells in the original composition. 74. The method according to any one of paragraphs. 1-10 and 20-73, wherein the introduction is performed by transduction with a viral vector containing a nucleic acid encoding a recombinant receptor. 75. The method of claim 74, wherein the viral vector is a retroviral vector. 76. The method of claim 74 or 75, wherein the viral vector is a lentiviral vector or a gamma retroviral vector. 77. The method according to any one of paragraphs. 1-76, wherein the ratio of naive-like T cells to non-naïve-like T cells in the composition after incubation under stimulating conditions increases by more than about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold , 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, 100-fold compared to the ratio of naive-like T cells to non-naive-like T cells in the original composition. 78. The method according to any one of paragraphs. 1-77, while the composition after incubation under stimulating conditions is more polyclonal or multiclonal in comparison with the original composition. 79. The method according to any one of paragraphs. 1-78, which is used in vitro or ex vivo .

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