Rapid administration of manipulated lymphocytes

Abstract

Provided herein is a method for the rapid production of engineered lymphocytes, which has been demonstrated to be associated with favorable complete response rates and overall survival, as well as a reduced risk of persistent thrombocytopenia. The rapid production process can prepare transduced lymphocytes that have improved efficacy or reduced adverse effects in the treatment of cancer. An exemplary process includes obtaining lymphocytes from a patient via apheresis; incubating the lymphocytes with a polynucleotide vector to transduce them and produce transduced lymphocytes; culturing the transduced lymphocytes; and injecting the transduced lymphocytes into a patient to predict the likelihood of complete response, overall survival, and the risk of persistent thrombocytopenia in subjects receiving immunotherapy.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 381,507 filed on 28 October 2022, U.S. Provisional Patent Application No. 63 / 386,831 filed on 9 December 2022, and U.S. Provisional Patent Application No. 63 / 506,288 filed on 5 June 2023, each of which is incorporated herein by reference in whole. [0001.1] Sequence List This application includes a sequence listing submitted electronically in XML file format, the entirety of which is incorporated herein by reference. The XML copy, created on 11 October 2023, is named K-1143-WO-PCT_SL.xml and has a size of 28,791 bytes. [Background technology]

[0002] Chimeric antigen receptors (CARs) and engineered T cell receptors (TCRs) contain binding domains that can interact with specific tumor antigens. Such binding ability allows immune cells to target and kill cancer cells. The highly complex and time-consuming autologous cell engineering and production process presents significant challenges. During the process, lymphocytes collected from patients must be transported to the process center, while the produced cells must be cryopreserved and then transported to patients for transplantation. This highly complex process inevitably results in high costs and limitations on clinical application. Therefore, there is a strong, yet unmet, need to develop shorter-duration processes to improve patient outcomes. [Overview of the project]

[0003] Provided herein are methods for the rapid production and administration of engineered lymphocytes, which have been demonstrated to be associated with favorable complete response rates and overall survival, as well as a reduced risk of persistent thrombocytopenia. In particular, rapid production processes with shortened vein-to-vein time are associated with improved efficacy or reduced adverse effects in cancer treatment. Methods for predicting the likelihood of complete response, overall survival, and the risk of persistent thrombocytopenia in subjects receiving immunotherapy are also provided herein.

[0004] One embodiment of the present disclosure relates to a method for preparing lymphocytes having improved efficacy and / or reduced adverse effects in the treatment of cancer, comprising: obtaining lymphocytes from a patient via apheresis; incubating the lymphocytes with a polynucleotide vector to transduce the lymphocytes and produce transduced lymphocytes; culturing the transduced lymphocytes to obtain a sample of cultured lymphocytes; and injecting the sample into a patient, wherein the time taken from obtaining the lymphocytes to injecting the sample is 28 days or less.

[0005] In one embodiment of the present disclosure, the patient has a greater than 55% chance of achieving a complete response, a greater than 45% chance of achieving overall survival at 24 months, and / or a less than 30% chance of developing persistent thrombocytopenia.

[0006] In one embodiment of the present disclosure, the time taken from the acquisition of lymphocytes to the injection of the sample is 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 days or less.

[0007] In one embodiment of the present disclosure, the method further comprises administering lymphodepleting chemotherapy, which is administered within 5, 4, 3, 2, or 1 day of the infusion step.

[0008] One embodiment of the present disclosure relates to a method for preventing and / or reducing the likelihood of persistent thrombocytopenia in a patient having r / r LBCL, comprising: obtaining lymphocytes from the patient via apheresis; incubating the lymphocytes with a polynucleotide vector to transduce the lymphocytes and produce transduced lymphocytes; culturing the transduced lymphocytes to obtain a sample of cultured lymphocytes; and injecting the sample into the patient, wherein the time taken from obtaining the lymphocytes to injecting the sample is 28 days or less.

[0009] One embodiment of the present disclosure is a method for predicting the likelihood of complete response in a patient to immunotherapy, comprising determining the period from the leukocyte apheresis process of the patient to the administration of the immunotherapy to the patient, and, based on the determination of the period, classifying the patient into several groups, the first group characterized by a period of up to 28 days from the leukocyte apheresis process to the administration of the immunotherapy to the patient, the second group characterized by a period of 28 to 40 days from the leukocyte apheresis process to the administration of the immunotherapy to the patient, and the leukocyte apheresis process. The method comprises classifying a patient into one of several groups, including a third group characterized by a period of at least 40 days from the induction process to the administration of the immunotherapy to the patient, and determining the likelihood of complete response in the patient, at least partially based on which of the several groups the patient is classified into, wherein if the patient is classified into the first or second group, the patient has at least about 55% chance of complete response, and if the patient is classified into the third group, the patient has at least about 42% chance of complete response.

[0010] In some embodiments of this disclosure, if the patient is classified within the first group or the second group, the patient has approximately a 60% chance of complete response.

[0011] One embodiment of the present disclosure is a method for predicting the overall survival rate in a patient for immunotherapy, comprising determining the period from the leukocyte apheresis process of the patient to the administration of the immunotherapy to the patient, and, based on the determination of the period, classifying the patient into several groups, the first group characterized by a period of up to 28 days from the leukocyte apheresis process to the administration of the immunotherapy to the patient, the second group characterized by a period of 28 to 40 days from the leukocyte apheresis process to the administration of the immunotherapy to the patient, and the patient The method comprises classifying a patient into one of several groups, including a third group characterized by the period of at least 40 days prior to the administration of the immunotherapy to the patient, and determining the overall survival rate in the patient based at least in part on which of the several groups the patient is classified into, wherein if the patient is classified into the first group, the patient has an overall survival rate of at least about 49%, if the patient is classified into the second group, the patient has an overall survival rate of at least about 48%, and if the patient is classified into the third group, the patient has an overall survival rate of at least about 30%.

[0012] One embodiment of the present disclosure is a method for predicting the risk of thrombocytopenia in a patient receiving immunotherapy, comprising determining the period from the leukocyte apheresis process to the administration of the immunotherapy to the patient, and, based on the determination of the period, classifying the patient into several groups, the first group characterized by a period of up to 28 days from the leukocyte apheresis process to the administration of the immunotherapy to the patient, the second group characterized by a period of 28 to 40 days from the leukocyte apheresis process to the administration of the immunotherapy to the patient, and from the leukocyte apheresis process to the patient The method comprises classifying a patient into one of several groups, including a third group characterized by a period of at least 40 days prior to the administration of the immunotherapy, and determining the risk of thrombocytopenia in the patient, at least in part, based on which of the several groups the patient is classified into, wherein if the patient is classified into the first group, the patient has a risk of approximately 18% of thrombocytopenia; if the patient is classified into the second group, the patient has a risk of approximately 25% of thrombocytopenia; and if the patient is classified into the third group, the patient has a risk of approximately 34% of thrombocytopenia.

[0013] Embodiments of the present disclosure relate to a method for predicting life expectancy and quality-adjusted life years in a patient who has received immunotherapy, the method comprising: determining a period of time from the leukocyte apheresis process in the patient to the administration of the immunotherapy to the patient, which is either short-term or long-term; assigning a probability of successful infusion based on the period; and inputting patient information into a survival model to determine the patient's life expectancy and quality-adjusted life years.

[0014] In one embodiment of the present disclosure, the immunotherapy comprises one or more CARs that recognize one or more tumor antigens.

[0015] In one embodiment of this disclosure, the immunotherapy is axicaptagen silolucel or brexcaptagen autolucel.

[0016] In one embodiment of the present disclosure, the patient has relapsed or refractory (r / r) large B-cell lymphoma (LBCL). [Modes for carrying out the invention]

[0017] definition To facilitate understanding of this disclosure, certain terms are first defined below. Further definitions of these terms and other terms are provided throughout this specification.

[0018] As used herein, unless otherwise specified or evident from the context, the term “or” is understood to be inclusive and encompasses both “or” and “and.”

[0019] As used herein, the term "and / or" should be interpreted as a specific disclosure of each of two designated features or components, with or without the other. Accordingly, as used herein in phrases such as "A and / or B," the term "and / or" is intended to include A and B, A or B, A (alone), and B (alone). Similarly, as used in phrases such as "A, B, and / or C," the term "and / or" is intended to include each of the following embodiments: A, B, and C, A, B, or C, A or C, A or B, B or C, A and C, A and B, B and C, A (alone), B (alone), and C (alone).

[0020] Unless specifically stated or evident from the context, the term “about” refers to a value or composition that falls within an acceptable margin of error for a particular value or composition as determined by those skilled in the art, and this depends to some extent on how that value or composition is measured or determined, i.e., on the limits of the measuring system. For example, “about” or “essentially from” may mean within a range of 1 or more than 1 standard deviation by the practice of the art. “About” or “essentially from” may mean a range of up to 10% (i.e., ±10%). Thus, “about” may be understood to be greater or less than the stated value by 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001%. For example, about 5 mg may include any amount between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, the term may mean a value of up to one order of magnitude or up to five times the stated value. Where specific values ​​or compositions are presented in this disclosure, unless otherwise specified, the meaning of “approximately” or “essentially consisting of” should be assumed to be within an acceptable margin of error for those specific values ​​or compositions.

[0021] "Administering" refers to the physical delivery of a drug, such as the modified T cells disclosed herein, to a subject using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration for the formulations disclosed herein include, for example, intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral administration routes by injection or infusion. The term "parenteral administration" means a mode of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intralymphatic, intrafocal, intracapsular, intraorbital, intracardiac, intratracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions, as well as in vivo electroporation. In some embodiments, the formulation is administered via a non-parenteral route, for example, orally. Other non-parenteral routes include topical, epidermal, or mucosal administration routes, such as intranasal, intravaginal, rectal, sublingual, or topical. The administration may also be carried out, for example, once, multiple times, and / or over a longer period of time.

[0022] The term "homogeneous" refers to any material that originates from one individual and is then introduced into another individual of the same species.

[0023] The term "antibody" (Ab) includes, but is not limited to, glycoprotein immunoglobulins that specifically bind to an antigen. Generally, an antibody may comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding molecule thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL contains three CDRs and four FRs arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of Ab can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Generally, human antibodies are approximately 150 kD tetramers composed of two identical heavy (H) chain polypeptides (approximately 50 kD each) and two identical light (L) chain polypeptides (approximately 25 kD each) that associate with each other in a structure commonly referred to as a "Y-shape". The heavy and light chains are linked or connected to each other by a single disulfide bond, and two other disulfide bonds connect the hinge regions of the heavy chain, resulting in the dimers being linked to each other to form a tetramer. Naturally produced antibodies are also glycosylated, for example, on this CH2 domain.

[0024] The terms “antigen-binding molecule,” “antigen-binding moiety,” “antigen-binding fragment,” or “antibody fragment” refer to any molecule containing the antigen-binding moiety (e.g., CDR) of an antibody from which the molecule originates. Antigen-binding molecules may contain an antigen complementarity-determining region (CDR). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, dAb, linear antibodies, scFv antibodies, and multispecific antibodies formed from antigen-binding molecules. Peptibodies (i.e., Fc fusion molecules containing peptide-binding domains) are another example of a suitable antigen-binding molecule. In some embodiments, the antigen-binding molecule binds to an antigen on tumor cells. In some embodiments, the antigen-binding molecule binds to an antigen on cells involved in hyperproliferative diseases, or to a viral or bacterial antigen. In certain embodiments, the antigen-binding molecule is a chimeric antigen receptor (CAR) or an engineered T-cell receptor (TCR).

[0025] The terms “variable region” and “variable domain” are used interchangeably. A variable region typically refers to a portion of an antibody, generally a portion of the light or heavy chain, typically the amino-terminal approximately 110–120 amino acids of the mature heavy chain and approximately 90–115 amino acids of the mature light chain, which vary significantly in sequence between antibodies and are used for the binding and specificity of a particular antibody to a particular antigen. Sequence variability is concentrated in a region called the complementarity-determining region (CDR), while more highly conserved regions within the variable domain are called the framework region (FR). While we do not wish to be bound to any particular mechanism or theory, the CDRs of the light and heavy chains are considered to be primarily responsible for the antibody's interaction with and specificity to the antigen. In certain embodiments, the variable region is the human variable region. In certain embodiments, the variable region includes rodent or mouse CDRs and human framework regions (FRs). In certain embodiments, the variable region is the primate (e.g., non-human primate) variable region. In certain embodiments, the variable region includes rodent or mouse CDR and primate (e.g., non-human primate) framework regions (FR).

[0026] The terms "VL" and "VL domain" are used interchangeably to refer to the variable region of the light chain of an antibody or its antigen-binding molecule.

[0027] The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody or its antigen-binding molecule.

[0028] Many definitions of CDRs are commonly used: Kabat numbering, Chothia numbering, AbM numbering, or Contact numbering. The AbM definition is a compromise between the two, used by Oxford Molecular's AbM antibody modeling software. The Contact definition is based on the analysis of available composite crystal structures.

[0029] The term “autologous” refers to any material derived from the same individual that is later reintroduced. For example, the engineered autologous cell therapy (eACT®) method described herein comprises collecting lymphocytes from a patient, which are then engineered to express, for example, a CAR construct, and then administered to the same patient.

[0030] A “chimeric antigen receptor” or “CAR” refers to a molecule engineered to include a binding motif and means for activating immune cells (e.g., T cells such as naive T cells, central memory T cells, effector memory T cells, or combinations thereof) upon antigen binding. CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immune receptors. In some embodiments, a CAR includes a binding motif, an extracellular domain, a transmembrane domain, one or more costimulatory domains, and an intracellular signaling domain. T cells genetically engineered to express a chimeric antigen receptor may be referred to as CAR T cells. An “extracellular domain” (or “ECD (extracellular domain)”) refers to a portion of a polypeptide that, if the polypeptide is present on the cell membrane, is understood to be located outside the cell membrane, in the extracellular space.

[0031] "T cell receptors" or "TCRs" refer to antigen-recognizing molecules present on the surface of T cells. During normal T cell development, each of the four TCR genes—α, β, γ, and δ—can rearrange to produce a wide variety of TCR proteins.

[0032] The term "heterogeneous" means that a sequence originates from any source other than those found in nature. For example, a heterogeneous sequence included as part of a costimulatory protein is an amino acid that does not exist naturally as part of the wild-type human costimulatory protein, i.e., it does not align with the wild-type human costimulatory protein. For example, a heterogeneous nucleotide sequence refers to a nucleotide sequence other than the nucleotide sequence of the wild-type human costimulatory protein coding sequence.

[0033] The term "identity" refers to the overall relationship between polymer molecules, for example, between nucleic acid molecules (e.g., DNA and / or RNA molecules), and / or polypeptide molecules. Methods for calculating the identity percentage between two given polypeptide sequences are known. For example, the calculation of the identity percentage between two nucleic acid or polypeptide sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps may be introduced into one or both of the first and second sequences for optimal alignment, and non-identical sequences may be ignored for comparison purposes). Then, nucleotides or amino acids at corresponding positions are compared. If a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The identity percentage between two sequences is optionally a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap, which may need to be introduced for optimal alignment of the two sequences. The comparison or alignment of sequences and the determination of the percentage of identity between two sequences can be achieved using mathematical algorithms such as BLAST (a basic local alignment search tool). In some embodiments, polymer molecules are considered "homologous" to each other if their sequences are identical by at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%).

[0034] Immune cells for immunotherapy may be derived from any source known in the art. For example, immune cells can be differentiated in vitro from a population of hematopoietic stem cells, or immune cells can be obtained from a subject. Immune cells may be obtained, for example, from peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from an infection site, ascites, pleural fluid, splenic tissue, and tumors. Furthermore, immune cells may be derived from one or more immune cell lines available in the art. Immune cells may also be obtained from blood units taken from a subject using various techniques known to those skilled in the art, such as FICOLL® isolation and / or apheresis. Further methods for isolating immune cells for immunotherapy are disclosed in U.S. Patent Application Publication 2013 / 0287748, which is incorporated herein by reference in its entirety.

[0035] "Patient" includes any human being suffering from cancer (e.g., lymphoma or leukemia). The terms "Subject" and "Patient" are used interchangeably herein.

[0036] The term "pharmaceutically acceptable" refers to a molecule or composition that, when administered to a recipient, is not harmful to that recipient or whose benefits to the recipient outweigh any harmful effects. With respect to carriers, diluents, or excipients used to formulate the compositions disclosed herein, a pharmaceutically acceptable carrier, diluent, or excipient must be compatible with the other components of the composition and must not be harmful to the recipient or whose benefits to the recipient outweigh any harmful effects. The term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulation material, that is involved in the transport or delivery of a drug from one part of the body to another (for example, from one organ to another). Each carrier present in a pharmaceutical composition must be compatible with the other components of the formulation and "acceptable" in the sense that it is not harmful to the patient, or whose benefits to the recipient outweigh any harmful effects. Some examples of materials that can function as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose, and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth powder; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffer solutions; polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic, suitable substances used in pharmaceutical formulations.

[0037] The term "pharmaceutical composition" refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose amount suitable for administration in a therapeutic regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to the relevant subjects or population. In some embodiments, the pharmaceutical composition may be formulated for administration in solid or liquid form, but is not limited to, forms adapted for: oral administration, e.g., liquid (aqueous or non-aqueous or suspension), tablets, e.g., buccal, sublingual, and those targeting systemic absorption, bolus, powder, granules, paste for application to the tongue; parenteral administration, e.g., sterile solution or suspension, or as a sustained-release formulation, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection; topical administration, e.g., as a cream, ointment, or sustained-release patch or spray applied to the skin, lungs, or oral cavity; intravaginal or rectal, e.g., as a pessary, cream, or foam; sublingual; ocular; transdermal; or transnasal, lung, and other mucosal surfaces.

[0038] The terms “reduce” and “decrease” are used interchangeably herein and refer to any change that becomes less than the original. “Reduce” and “decrease” are relative terms and require a comparison between before and after measurement. “Reduce” and “decrease” include complete depletion.

[0039] The term “reference” describes the standard or control on which the comparison is performed. For example, in some embodiments, the drug, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control which is a drug, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested, measured, and / or determined substantially simultaneously with the test, measurement, or determination of interest. In some embodiments, the reference or control is a reference or control from the past that is optionally embodied in a tangible medium. Generally, the reference or control is determined or characterized under conditions or circumstances equivalent to those under evaluation, where the similarity is sufficient to justify the dependence on and / or comparison with the selected reference or control.

[0040] The “therapeutic effective dose,” “effective dose,” “effective amount,” or “therapeutic effective dosage” of a therapeutic agent, such as engineered CAR T cells, is any amount that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of the disease or promotes disease regression, as demonstrated by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or the prevention of disability or impairment resulting from the onset of the disease. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems predicting efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0041] The terms “transduction” and “transduced” refer to the process by which foreign nucleic acids are introduced into cells via a viral vector (see Jones et al., “Genetics: principles and analysis,” Boston: Jones & Bartlett Publ. (1998)). In some embodiments, the vector is a retroviral vector, DNA vector, RNA vector, adenovirus vector, baculovirus vector, Epstein-Barr virus vector, papovavirus vector, vaccinia virus vector, herpes simplex virus vector, adenovirus-associated vector, lentiviral vector, or any combination thereof.

[0042] "Treatment" or "treating" a subject means any type of intervention or process performed on the subject, or administration of an activator to the subject, with the aim of reversing, alleviating, improving, inhibiting, slowing or preventing the onset, progression, development, severity or relapse of symptoms, complications or conditions, or biochemical signs associated with the disease. In one embodiment, "treatment" or "treating" includes partial remission. In another embodiment, "treatment" or "treating" includes complete remission. In some embodiments, treatment may be the treatment of a subject that does not show signs of the related disease, disorder and / or condition, and / or a subject that shows only initial signs of the disease, disorder and / or condition. In some embodiments, such treatment may be the treatment of a subject that shows one or more definitive signs of the related disease, disorder and / or condition. In some embodiments, treatment may be the treatment of a subject that has been diagnosed with the related disease, disorder and / or condition. In some embodiments, treatment may be the treatment of a subject that is known to have one or more susceptibility factors that are statistically correlated with an increased risk of developing the related disease, disorder and / or condition.

[0043] The term “vector” refers to a recipient nucleic acid molecule that contains or has been modified to incorporate a provided nucleic acid sequence. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA molecule into which additional DNA can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors can autonomously replicate in the host cell into which they are introduced (e.g., bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) may be incorporated into the host cell's genome upon introduction into the host cell, thereby replicating with the host genome. Furthermore, certain vectors contain sequences that direct the expression of the inserted gene into which they are operably linked. Such vectors may be referred to herein as “expression vectors.” Standard techniques can be used for vector manipulation and are found, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), which is incorporated herein by reference.

[0044] One embodiment of the present disclosure relates to a method for preparing lymphocytes having improved efficacy and / or reduced adverse effects in the treatment of cancer, comprising: obtaining lymphocytes from a patient via apheresis; incubating the lymphocytes with a polynucleotide vector to transduce the lymphocytes and produce transduced lymphocytes; culturing the transduced lymphocytes to obtain a sample of cultured lymphocytes; and injecting the sample into a patient. In such an embodiment, the time elapsed from the acquisition of lymphocytes to the injection of the sample is 28 days or less.

[0045] One embodiment of the present disclosure relates to a method for preventing and / or reducing the likelihood of persistent thrombocytopenia in a patient having r / r LBCL, comprising: obtaining lymphocytes from the patient via apheresis; incubating the lymphocytes with a polynucleotide vector to transduce the lymphocytes and produce transduced lymphocytes; culturing the transduced lymphocytes to obtain a sample of cultured lymphocytes; and injecting the sample into the patient. In such an embodiment, the time elapsed from lymphocyte acquisition to sample injection is 28 days or less.

[0046] One embodiment of the present disclosure relates to any of the methods discussed above, wherein the patient has a greater than 55% chance of achieving a complete response, a greater than 45% chance of achieving overall survival at 24 months, and / or a less than 30% chance of developing persistent thrombocytopenia.

[0047] One embodiment of the present disclosure relates to any of the methods discussed above, wherein the time taken from the acquisition of lymphocytes to the injection of the sample is 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 days or less.

[0048] One embodiment of the present disclosure further comprises administering lymphocyte depletion chemotherapy, wherein the lymphocyte depletion chemotherapy is administered within 5, 4, 3, 2, or 1 day of the infusion step, relating to any of the methods discussed above.

[0049] One embodiment of the present disclosure relates to any of the methods discussed above, but does not involve the cryopreservation of cultured lymphocytes.

[0050] One embodiment of the present disclosure relates to any of the methods discussed above, wherein transduced lymphocytes are cultured for 72 hours, 48 ​​hours, or less than 36 hours.

[0051] One embodiment of the present disclosure relates to any of the methods discussed above, wherein incubation is carried out in a closed system.

[0052] One embodiment of the present disclosure is a closing system that is at least 1500 cm 2 This relates to one of the methods discussed above, having the following internal surface area.

[0053] One embodiment of the present disclosure relates to any of the methods discussed above, wherein the closed system has an inner surface coated with recombinant human fibronectin, and the coating is carried out with a solution containing approximately 1 to 10 μg / ml of recombinant human fibronectin.

[0054] One embodiment of the present disclosure relates to any of the methods discussed above, wherein the inner surface is further in contact with a second solution containing a polynucleotide vector, and the second solution has a volume of about 200 mL.

[0055] One embodiment of the present disclosure relates to any of the methods discussed above, wherein the coating further includes a drain for a second solution.

[0056] One embodiment of the present disclosure is a closed system in which the sample is at least 1.5 × 10 8 This relates to any of the methods discussed above, which involve having individual lymphocytes.

[0057] One embodiment of the present disclosure is that the sample is at least 4 × 10 8 This relates to any of the methods discussed above, which involve having individual lymphocytes.

[0058] One embodiment of the present disclosure relates to any of the methods discussed above, wherein the lymphocytes are peripheral blood mononuclear cells (PBMCs) or T cells.

[0059] One embodiment of the present disclosure relates to any of the methods discussed above, wherein the sample comprises CD4+ T cells and CD8+ T cells.

[0060] One embodiment of the present disclosure relates to any of the methods discussed above, wherein a total of 10,000 to 1,000,000 cultured lymphocytes per kilogram of the patient are administered to the patient.

[0061] One embodiment of the present disclosure relates to any of the methods discussed above, wherein a total of 20,000 to 400,000 cultured lymphocytes per kilogram of the patient are administered to the patient.

[0062] One embodiment of the present disclosure relates to any of the methods discussed above, wherein at least 15% of cultured lymphocytes are transduced with a vector.

[0063] One embodiment of the present disclosure relates to any of the methods discussed above, wherein the polynucleotide vector is a viral vector.

[0064] One embodiment of the present disclosure relates to any of the methods discussed above, wherein the viral vector is a retroviral vector or a lentiviral vector.

[0065] One embodiment of the present disclosure relates to any of the methods discussed above, wherein the vector encodes one or more chimeric antigen receptors (CARs) or one or more T cell receptors (TCRs).

[0066] One embodiment of the present disclosure relates to any of the methods discussed above, wherein one or more CARs have intracellular co-stimulatory domains.

[0067] One embodiment of the present disclosure includes an intracellular costimulatory domain containing DAP-10, CD28, OX-40, 4-1BB (CD137), CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD11a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), tumor necrosis factor superfamily member 14, TNFSF14, LIGHT), NKG2C, Ig alpha (CD79a), Fc gamma receptor, MHC class I molecule, TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, and signaling lymphocytic activation molecule.(molecule, SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, CDS, GITR, BAFFR, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD (CD11d), ITGAE (CD103), ITGAL (CD11a), ITGAM (CD11b), ITGAX (CD11c), ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, TN This relates to one of the methods discussed above, concerning the signaling region of a protein selected from the group consisting of ligands that specifically bind to FR2, TRANCE (RANKL), DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG (Cbp), CD19a, CD83, and combinations thereof.

[0068] One embodiment of the present disclosure relates to any of the methods discussed above, wherein the intracellular co-stimulatory domain is a signaling region of CD28.

[0069] One embodiment of the present disclosure relates to any of the methods discussed above, wherein one or more CARs recognize one or more tumor antigens.

[0070] One embodiment of the present disclosure relates to any of the methods discussed above, wherein the target antigen is CD19.

[0071] One embodiment of the present disclosure relates to any of the methods discussed above, wherein the CAR-containing lymphocytes are axicapbutagen silolucel or brexcapbutagen autolucel.

[0072] One embodiment of the present disclosure relates to any of the methods discussed above, in which a patient has relapsed or refractory (r / r) large B-cell lymphoma (LBCL).

[0073] One embodiment of the present disclosure relates to any of the methods discussed above, wherein the tumor antigen is CD19 and CD20.

[0074] One embodiment of the present disclosure is a method for predicting the likelihood of a complete response in a patient to immunotherapy, the method comprising: determining the time from the patient's leukocyte apheresis process to the administration of immunotherapy to the patient; classifying the patient into one of several groups based on the determination of the time, the groups including a first group characterized by a maximum period of 28 days from the leukocyte apheresis process to the administration of immunotherapy to the patient; a second group characterized by a period of 28 to 40 days from the leukocyte apheresis process to the administration of immunotherapy to the patient; and determining the likelihood of a complete response in the patient, at least in part, based on which of the several groups the patient is classified into. In such embodiments, if a patient is classified into the first or second group, the patient has at least about a 55% chance of complete response, and if a patient is classified into the third group, the patient has at least about a 42% chance of complete response.

[0075] One embodiment of the present disclosure relates to a method for predicting the likelihood of complete response in a patient to the immunotherapy discussed above, wherein if the patient is classified into a first group or a second group, the patient has approximately a 60% chance of achieving a complete response.

[0076] One embodiment of the present disclosure relates to a method for predicting the likelihood of complete response in a patient to the immunotherapy discussed above, wherein the immunotherapy comprises one or more CARs that recognize one or more tumor antigens.

[0077] One embodiment of the present disclosure relates to a method for predicting the likelihood of complete response in a patient to the immunotherapy discussed above, wherein the tumor antigen is CD19.

[0078] One embodiment of the present disclosure relates to a method for predicting the likelihood of complete response in a patient to the immunotherapy discussed above, wherein the tumor antigens are CD19 and CD20.

[0079] One embodiment of the present disclosure relates to a method for predicting the likelihood of complete response in a patient to the immunotherapy discussed above, wherein the immunotherapy is axicapbutagen silolucel or brexcapbutagen autolucel.

[0080] One embodiment of the present disclosure relates to a method for predicting the likelihood of complete response in a patient to the immunotherapy discussed above, wherein the patient has relapsed or refractory (r / r) large B-cell lymphoma (LBCL).

[0081] One embodiment of the present disclosure is a method for predicting the overall survival rate in a patient for immunotherapy, comprising: determining the time from a patient's leukocyte apheresis procedure to the administration of immunotherapy to the patient; classifying the patient into one of several groups based on the determination of the time, the groups including a first group characterized by a maximum period of 28 days from the leukocyte apheresis procedure to the administration of immunotherapy to the patient; a second group characterized by a period of 28 to 40 days from the leukocyte apheresis procedure to the administration of immunotherapy to the patient; and a third group characterized by a period of at least 40 days from the leukocyte apheresis procedure to the administration of immunotherapy to the patient; and determining the overall survival rate in the patient at least in part based on which of the several groups the patient is classified into. In such embodiments, if a patient is classified into the first group, the patient has an overall survival rate of at least about 49%; if a patient is classified into the second group, the patient has an overall survival rate of at least about 48%; and if a patient is classified into the third group, the patient has an overall survival rate of at least about 30%.

[0082] One embodiment of the present disclosure relates to a method for predicting overall survival in a patient for the immunotherapy discussed above, wherein the immunotherapy comprises one or more CARs that recognize one or more tumor antigens.

[0083] One embodiment of the present disclosure relates to a method for predicting overall survival in a patient receiving the immunotherapy discussed above, wherein the tumor antigen is CD19.

[0084] One embodiment of the present disclosure relates to a method for predicting overall survival in patients receiving the immunotherapy discussed above, wherein the tumor antigens are CD19 and CD20.

[0085] One embodiment of the present disclosure relates to a method for predicting overall survival in a patient to the immunotherapy discussed above, wherein the immunotherapy is axicaptagen silolucel or brexcaptagen autolucel.

[0086] One embodiment of the present disclosure relates to a method for predicting overall survival in a patient to the immunotherapy discussed above, wherein the patient has relapsed or refractory (r / r) large B-cell lymphoma (LBCL).

[0087] One embodiment of the present disclosure is a method for predicting the risk of thrombocytopenia in a patient receiving immunotherapy, comprising: determining the time from the patient's leukocyte apheresis process to the administration of immunotherapy to the patient; classifying the patient into one of several groups, based on the determination of said time, including a first group characterized by a maximum period of 28 days from the leukocyte apheresis process to the administration of immunotherapy to the patient; a second group characterized by a period of 28 to 40 days from the leukocyte apheresis process to the administration of immunotherapy to the patient; and a third group characterized by a period of at least 40 days from the leukocyte apheresis process to the administration of immunotherapy to the patient; and determining the risk of thrombocytopenia in the patient, at least in part, based on which of the several groups the patient is classified into. In such embodiments, if a patient is classified into the first group, the patient has a risk of approximately 18% of thrombocytopenia; if a patient is classified into the second group, the patient has a risk of approximately 25% of thrombocytopenia; and if a patient is classified into the third group, the patient has a risk of approximately 34% of thrombocytopenia.

[0088] One embodiment of the present disclosure relates to a method for predicting the risk of thrombocytopenia in a patient receiving the immunotherapy discussed above, wherein the immunotherapy comprises one or more CARs that recognize one or more tumor antigens.

[0089] One embodiment of the present disclosure relates to a method for predicting the risk of thrombocytopenia in patients receiving the immunotherapy discussed above, wherein the tumor antigen is CD19.

[0090] One embodiment of the present disclosure relates to a method for predicting the risk of thrombocytopenia in patients receiving the immunotherapy discussed above, wherein the tumor antigens are CD19 and CD20.

[0091] One embodiment of the present disclosure relates to a method for predicting the risk of thrombocytopenia in patients receiving the immunotherapy discussed above, wherein the immunotherapy is axicaptagen silolucel or brexcaptagen autolucel.

[0092] One embodiment of the present disclosure relates to a method for predicting the risk of thrombocytopenia in a patient receiving the immunotherapy discussed above, wherein the patient has relapsed or refractory (r / r) large B-cell lymphoma (LBCL).

[0093] Embodiments of the present disclosure relate to a method for predicting life expectancy and quality-adjusted life years in a patient receiving immunotherapy, the method comprising: determining a period from the patient's leukocyte apheresis process to the administration of immunotherapy to the patient, which is either short-term or long-term; assigning a probability of successful infusion based on that period; and inputting patient information into a survival model to determine the patient's life expectancy and quality-adjusted life years.

[0094] One embodiment of the present disclosure relates to a method for predicting life expectancy and quality-adjusted life years in patients who have received the immunotherapy discussed above, wherein the short term is approximately 24 days or less.

[0095] One embodiment of the present disclosure relates to a method for predicting life expectancy and quality-adjusted life years in patients who have received the immunotherapy discussed above, wherein the long term is approximately 54 days or longer.

[0096] One embodiment of the present disclosure relates to a method for predicting life expectancy and quality-adjusted life years in patients who have received the immunotherapy discussed above, wherein the long term is approximately 37 days or longer.

[0097] One embodiment of the present disclosure relates to a method for predicting life expectancy and quality-adjusted life years in a patient who has received the immunotherapy discussed above, wherein a short-term method indicates that the patient's increase in life expectancy and quality-adjusted life years will be greater than 5 years, and a long-term method indicates that the patient's increase in life expectancy and quality-adjusted life years will be less than 5 years.

[0098] One embodiment of the present disclosure relates to a method for predicting life expectancy and quality-adjusted life years in patients who have received the immunotherapy discussed above, wherein the immunotherapy comprises one or more CARs that recognize one or more tumor antigens.

[0099] One embodiment of the present disclosure relates to a method for predicting life expectancy and quality-adjusted survival years in patients who have received the immunotherapy discussed above, wherein the tumor antigen is CD19.

[0100] One embodiment of the present disclosure relates to a method for predicting life expectancy and quality-adjusted survival years in patients who have received the immunotherapy discussed above, wherein the tumor antigens are CD19 and CD20.

[0101] One embodiment of the present disclosure relates to a method for predicting life expectancy and quality-adjusted life years in patients who have received the immunotherapy described above, wherein the immunotherapy is axicapbutagen silolucel or brexcapbutagen autolucel.

[0102] One embodiment of the present disclosure relates to a method for predicting life expectancy and quality-adjusted life years in a patient who has received the immunotherapy described above, wherein the patient has relapsed or refractory (r / r) large B-cell lymphoma (LBCL).

[0103] Rapid production of manipulated lymphocytes A typical autologous CAR-T cell manufacturing process begins with leukocyte apheresis. The patient's apheresis material is collected at the treatment center. This process takes approximately 3-4 hours, during which about 10-20 liters of blood are recirculated and about 100-500 mL of apheresis material is collected. The apheresis material collection bags are then transported at low temperature to a central manufacturing facility.

[0104] T cells are enriched from an apheresis substance and then transduced with a retroviral vector containing the CAR gene. The transduced cells are cultured until they reach the target dose, at which point the cells are washed and cryopreserved for return to the treatment center. Each batch undergoes a series of tests to ensure it meets specific standards before shipment.

[0105] Once the treatment center receives CAR-T cell products from the manufacturing facility, the patient receives lymphodepleting (LD) chemotherapy. Lymphodepleting for axicapbutagensilolucel is administered with fludarabine (30 mg / m²). 2 ) and cyclophosphamide (500 mg / m²) 2 The regimen consists of three days (days -5, -4, and -3). Currently, the center typically admits patients before specific CAR-T cell infusions. CAR-T cells are administered three days after the completion of lymphocyte depletion (day 0), for example, 2 × 10¹⁶ cells in the case of axicaptagensilolucel. 6 The target dose of individual CAR-T cells / kg is injected.

[0106] The entire vein-to-vein process, from apheresis to injection, may take a median of 28 days for axicapbutagensilolucel, 45 days for tisagenlecleucel, and 37 days for lysocabbutagenmaralucel.

[0107] The vein-to-vein time refers to the time from leukocyte apheresis to infusion. The vein-to-vein time may be affected by factors including, but not limited to, the time from leukocyte apheresis to delivery of the product to an authorized treatment center, the time from leukocyte apheresis to product shipment, transportation time, the time from leukocyte apheresis to product release from the manufacturing site, and the time required for product manufacturing.

[0108] As Example 1 demonstrates, shorter venous-to-venous times are associated with improved complete response (CR) rates and overall survival (OS), as well as a reduced risk of persistent thrombocytopenia. Therefore, such findings highlight the importance of developing and deploying rapid CAR-T manufacturing processes.

[0109] Non-limitingly, in one embodiment, the time from vein to vein can be reduced by shortening the time required for transport. The time for transporting the apheresis substance to the manufacturing facility and returning the transduced cells to the treatment center can be shortened in some embodiments by using faster transport or by using a manufacturing facility closer to the treatment center.

[0110] In another embodiment, the time from vein to vein can be shortened by better coordination, which helps eliminate the need for cryopreservation. For example, if the patient is ready for infusion when the cells are ready, cryopreservation may not be necessary.

[0111] In another embodiment, the ability to produce products that meet specification requirements (within specifications) can reduce the time from vein to vein.

[0112] The vein-to-vein transfer time can also depend on the quality and quantity of the starting apheresis material. If the starting apheresis material contains fewer or lower-quality lymphocytes, a longer vein-to-vein transfer time may be required. Therefore, considering the improved performance associated with shorter vein-to-vein transfer times, it may be possible to shorten the time to expand lymphocytes, even if this may result in a reduction in the number of transduced cells for injection.

[0113] A key part of the manufacturing process involves lymphocyte transduction and expansion. Through several years of refinement, a transduction / expansion process taking 7 days (after the enrichment step) has been developed, as described below. In addition to these successes, a further refined process has been developed that can be completed within 5 days or even 3 days (counting after the enrichment step), which is also described below. As described herein, the 5-day process includes a transduction preparation and execution step in which a larger number of lymphocytes come into contact with a vector immobilized on recombinant fibronectin coated on the inner surface of a closed system. Such an improved transduction procedure allows for a significantly shortened post-transduction cell expansion step. Transduced cells prepared from the 5-day process have been biased towards immature cells, resulting in better in vivo antitumor efficacy.

[0114] In some embodiments, when post-transduction expansion is eliminated, the transduction and proliferation (post-enrichment) procedure can be completed in as little as three days. Compared to a five-day process, this three-day process produces a cell population with a higher percentage of immature cells and a lower percentage of more mature, differentiated, and activated cells. Thus, not only did the cell product from the three-day process show the best in vivo antitumor efficacy, but such greatly improved efficacy could be achieved even at much lower doses.

[0115] The following describes exemplary processes for 7-day, 5-day, and 3-day lymphocyte production.

[0116] 7-day lymphocyte production process In some embodiments, the therapeutic cell product is produced after a 7-day process in which lymphocytes transduced with a polynucleotide vector, such as a viral vector encoding a therapeutic protein, are prepared. The prepared lymphocytes may be useful in treating various diseases, including cancer, particularly if the therapeutic protein is a chimeric antigen receptor (CAR) or T cell receptor (TCR) designed to target cancer cells.

[0117] When used throughout, the terms “7-day process” and “7-day lymphocyte production process” are interchangeable and refer to a CAR cell production process that takes approximately 7 days after the initial enrichment and activation step. The 7-day process is one in which the length from the initial enrichment and activation step to the harvesting step is at least 8 days, and if the enrichment and activation step is included, the total may be 8 to 11 days.

[0118] Apheresis collection. Leukocytes can be collected (leukocyte apheresis) using standard apheresis equipment such as Cobe® Spectra, Spectra Optia®, Fenwal® Amicus®, or equivalent. The leukocyte apheresis process typically yields approximately 200–400 mL of apheresis product from a patient. The apheresis product can be subjected to the manufacturing process in-situ or, optionally, transported to a facility at 1–10°C for manufacturing at a different location. Further processing steps may be carried out in an ISO 7 cell culture process suite (or a similar cleanroom-type environment).

[0119] Volume reduction. If necessary, the volume reduction process can be performed using cell processing equipment such as Sepax® 2 laboratory instruments (Biosafe SA, Houston, TX) or equivalents, and can be carried out using a standard sterile tube kit. Considering the variability of the number of cells from each subject and the volume of incoming source material (approximately 200-400 mL), the volume reduction process is designed to standardize the cell volume to approximately 120 mL. If the apheresis volume is less than 120 mL, the volume reduction process is not necessary, and the cells are transported directly to the lymphocyte enrichment process. The volume reduction process standardizes the volume of cells received from each subject, preserves mononuclear cells, achieves consistent cell yield and high cell viability, and maintains a closed system to minimize the risk of contamination.

[0120] Lymphocyte enrichment. After the volume reduction step, cells can be subjected to Ficol-based separation using cell processing equipment such as Sepax® 2 or equivalent, using a standard sterile tube kit and separation protocol developed and recommended by the equipment manufacturer (NeatCell Program). The lymphocyte enrichment step reduces product-related impurities such as RBCs and granulocytes, enriches and concentrates mononuclear cells, washes and reduces process-related residues such as Ficol, formulates cells in growth medium for cell activation preparation, and achieves consistent cell yield and high cell viability. The closed system minimizes environmental contamination.

[0121] The process may be carried out at ambient temperature within an ISO7 area, and all connections may be made using a sterile pipe welding machine or within an ISO5 laminar flow hood.

[0122] Lymphocyte activation. The lymphocyte activation process can be performed using either newly processed cells from lymphocyte enrichment or pre-frozen cells. If cryopreserved cells are used, they can be thawed using a protocol developed prior to use.

[0123] The lymphocyte activation process selectively activates lymphocytes to make them receptive to retroviral vector transduction, reducing the population of all other cell types that survive, achieving consistent cell yield and high lymphocyte viability, while maintaining a closed system and minimizing the risk of contamination. Lymphocyte activation can be achieved using lymphocyte stimulants such as anti-CD3 antibodies and IL-2. In some embodiments, the lymphocyte activation process is performed at least 700 cm². 2 It is carried out within a closed system having an internal surface area.

[0124] Washing 1. After the lymphocyte activation step, cells can be washed with fresh culture medium in a standard sterile kit using a cell processing device such as Sepax® 2 or an equivalent, according to a protocol developed by the manufacturer. In preparation for retroviral vector transduction, cells were optionally concentrated to a final volume of approximately 100 mL. Washing 1 reduces process-related residues such as anti-CD3 antibody, used growth medium, and cell debris, achieving consistent cell yield and high T cell viability, maintaining a closed system to minimize the risk of contamination, and concentrating and delivering a sufficient number of viable T cells in a small volume suitable for initiating transduction.

[0125] Transduction. Activated cells from a single washing step in fresh cell growth medium can be transferred to a pre-prepared cell culture bag (Origen Biomedical PL240 or equivalent) by first coating the bag with recombinant fibronectin such as RetroNectin® (Takara Bio, Japan) or a fragment thereof. The activated cells can then be incubated with a retroviral vector according to the prescribed procedure before transduction. RetroNectin® coating (10 μg / mL) can be carried out at a temperature of 2-8°C for 20 ± 4 hours, washed with dilution buffer, and then incubated with the thawed retroviral vector at 37 ± 1°C and 5 ± 0.5% CO2 for approximately 180-210 minutes. After adding the cells to the bag, transduction can be carried out at 37 ± 1°C and 5 ± 0.5% CO2 for 20 ± 4 hours. The retroviral transduction process involves culturing activated T cells in the presence of a retroviral vector under controlled conditions to enable efficient transduction, achieving consistent cell yield and high cell viability, and maintaining a closed system to minimize the risk of contamination.

[0126] Washing 2. After the retroviral transduction step, the cells can be washed with fresh growth medium using a cell processing device such as Sepax® 2 or an equivalent in a standard sterile kit using a protocol developed by the manufacturer, and the cells were concentrated to a final volume of approximately 100 mL for preparation for the expansion step. Washing 2 reduces process-related residues such as retroviral vector particles, vector production process residues, used growth medium, and cell debris, enabling consistent cell yield and high cell viability, maintaining a closed system to minimize the risk of contamination, and allowing used growth medium to be replaced with fresh medium having a target number of cells in a specific volume suitable for starting the expansion step.

[0127] Lymphocyte expansion. Cells from the two washing steps can be aseptically transferred to culture bags (Origen Biomedical PL325 or equivalent), diluted with fresh cell growth medium, and cultured for approximately 72 hours at 37±1°C and 5±0.5% CO2. Cell density was measured daily, starting on day 5. Since the doubling time of T cells may vary slightly from subject to subject, further growth time beyond 72 hours (i.e., 3-6 days) may be required if the total cell count is insufficient to deliver the target dose of CAR-positive T cells / subject body weight kg. The lymphocyte expansion step is designed to culture cells under controlled conditions to produce a sufficient number of transduced cells to deliver an effective dose, maintain a closed system to minimize the risk of contamination, and achieve consistent cell yield and high cell viability. One such effective dose or target dose is 2 × 10⁶ cells produced via transduction with either the MSGV-FMC63-28Z retroviral vector or the MSGV-FMC63-CD828BBZ retroviral vector, respectively. 6 The sample contains individual FMC63-28Z CAR-positive or FMC63-CD828BBZ CAR-positive T cells per kg of body weight (±20%), both of which are described in detail in Kochenderfer et al., J Immunother. 2009 September;32(7):689-702.

[0128] Washing 3 and Concentration. After the lymphocyte expansion step, the cells can be washed with 0.9% saline using cell processing equipment such as Sepax® 2 or equivalent in a standard sterile kit using a protocol developed by the manufacturer, and the cells were concentrated to a final volume of approximately 35 mL for formulation and cryopreservation preparation. Washing 3 is designed to reduce process-related residues such as retrovirus production process residues, used growth medium, and cell debris, to achieve consistent cell yield and high cell viability, and to maintain a closed system to minimize the risk of contamination.

[0129] When cells are concentrated and washed in 0.9% saline, an appropriate cell dose can be formulated for the preparation of the final cryopreserved product.

[0130] The embodiments described herein provide for the efficient production of lymphocytetherapy that is manipulated within 7 days.

[0131] 5-day lymphocyte production process In some embodiments, the therapeutic cell product is produced after a 5-day process of preparing lymphocytes transduced with a polynucleotide vector, such as a viral vector encoding a therapeutic protein. The prepared lymphocytes can be useful for treating various diseases, such as cancer, particularly when the therapeutic protein is a chimeric antigen receptor (CAR) or a T cell receptor (TCR) designed to target cancer cells. Such a 5-day process is based on the 7-day process described above.

[0132] When used throughout, the terms "5-day process" and "5-day lymphocyte production process" are used interchangeably and refer to a CAR cell production process that takes approximately 5 days after the initial enrichment and activation steps. The 5-day process is 6 days in length from the initial enrichment and activation steps to the harvest step and can be 6 to 9 days in total when including the enrichment and activation steps.

[0133] During the 7-day process, lymphocytes are enriched and activated on day 0, the transduction bag is coated with recombinant fibronectin on day 1, viral transduction is performed on day 2, the transduced lymphocytes are washed, then expanded on days 3 and 4, the expansion is continued by changing the medium daily on days 5 and 6, and the final cell product is harvested on day 7. From approximately 1.2×10 9 lymphocytes obtained from apheresis, approximately 2.4×10 8 lymphocytes are incubated with a viral vector for transduction.

[0134] In the 5-day process, no changes were made to the process on day 0. However, larger bags were used on days 1 and 2. Instead of the Origen Biomedical PL240 bag used for transduction, an Origen Biomedical PL325 bag, or more preferably a PL750 bag, was used. The larger bags allowed for a larger volume of vector (200 mL instead of 100 mL) and more lymphocytes (2.4 × 10⁶). 8 Instead of 3.2 × 10 8 ~6×10 8 It became possible to use (individual) in the transduction process.

[0135] Interestingly, increasing the transduction volume with more vector-derived lymphocytes did not result in an unacceptable reduction in transduction efficiency (54%–35.15%) or cell viability (92%–92.4%). Therefore, the cell expansion step, which required 4 days in a 7-day process, could be reduced to 2 days, allowing for the harvesting of the final cell product on the 5th day.

[0136] However, the slight reduction in transduction rates did not correlate with clinical efficacy or patient safety. Importantly, cell products from the 5-day process were found to include an increased percentage of immature cells in both the CD4+ T cell and CD8+ T cell populations, which is thought to be related to improved therapeutic efficacy. It should be noted that these variations are within the historical range of donor runs collected from the 7-day process.

[0137] The shortened 5-day process met the specification requirements for transduction efficiency, therapeutic efficacy, and safety. On the other hand, the 5-day product showed a more immature phenotype within the historical range.

[0138] 3-day lymphocyte production process In some embodiments, therapeutic cell products are produced after a three-day process of preparing lymphocytes transduced with a polynucleotide vector, such as a viral vector encoding a therapeutic protein. The prepared lymphocytes may be useful in treating various diseases, including cancer, particularly if the therapeutic protein is a chimeric antigen receptor (CAR) or T-cell receptor (TCR) designed to target cancer cells. The three-day process is based on the seven-day and five-day processes described above.

[0139] When used throughout, the terms "3-day process" and "3-day lymphocyte production process" are interchangeable and refer to a CAR cell production process that takes approximately 3 days from the initial enrichment and activation step. The 3-day process is approximately 4 days long from the initial enrichment and activation step to the harvesting step. The 3-day process does not include a cell expansion step that includes more than one day after the transduction step and before the harvesting step.

[0140] The 3-day process is similar to the 5-day process in that the steps for days 0-1 are the same, and on day 2, it includes fibronectin coating of a larger bag (e.g., Origen Biomedical PL325 or preferably PL750) for subsequent transduction. However, on day 2, in some embodiments, approximately 4.8 × 10 8 Only 6 × 10 lymphocytes may be used in the transduction step along with the same amount of viral vector (200 mL). Alternatively, in some embodiments, approximately 6 × 10 lymphocytes may be used. 8 A number of lymphocytes may be used in the transduction step along with 200 mL of viral vector. Alternatively, in some embodiments, approximately 4.8 × 10 8 Only a few lymphocytes may be used in the transduction process along with 100 mL of viral vector. Another important difference is that, unlike the 5-day and 7-day processes, a shortened T-cell expansion process is performed. Instead, transduced lymphocytes can be harvested on day 3, and the entire process can be completed within 3 days from the initial enrichment and activation step.

[0141] Considering the absence of a specific T cell expansion step in this 3-day process, the harvested cell product contained a slightly lower percentage of T cells (CD3+). However, importantly, the product on day 3 may contain an even higher percentage of immature (naive) T cells compared to the 7-day process.

[0142] Despite not using lymphocytes from the same donor for testing, the data also showed that the percentage of naive T cells from the 3-day process was significantly higher than that from the 5-day process. In CD4+ T cells, the 3-day process produced approximately 55.75% naive T cells compared to approximately 40.65% in the 5-day process, and in CD8+ T cells, the 3-day process produced approximately 37.35% naive T cells compared to approximately 3.93% in the 5-day process.

[0143] In other words, the 3-day process may result in an approximately 1.4-fold increase in the percentage of CD4+ naive T cells compared to the percentage observed in the 5-day process, and an approximately 9.5-fold increase in the percentage of CD8+ naive T cells compared to the percentage observed in the 5-day process. Furthermore, the 3-day process may result in an approximately 3.0-fold increase in the percentage of CD4+ naive T cells compared to the historical mean observed in the 7-day process, and an approximately 18.0-fold increase in the percentage of CD8+ naive T cells compared to the historical mean observed in the 7-day process.

[0144] Conversely, in CD4+ T cells, the 3-day process can only generate about 4.35% of effector memory T cells, while the 5-day process can generate about 8.55%. Similarly, in CD8+ T cells, the 3-day process can only generate about 9.85% of effector memory T cells, while the 5-day process can generate about 15.85%.

[0145] In other words, the 3-day process may result in approximately a 2.0-fold decrease in the percentage of CD4+ effector memory T cells compared to the percentage observed in the 5-day process, and approximately a 3.6-fold decrease in the percentage of CD8+ effector memory T cells compared to the percentage observed in the 5-day process. Furthermore, the 3-day process may result in approximately a 6.0-fold decrease in the percentage of CD4+ effector memory T cells compared to the historical mean observed in the 7-day process, and approximately a 3.5-fold decrease in the percentage of CD8+ effector memory T cells compared to the historical mean observed in the 7-day process.

[0146] Cell viability measurements showed that cell viability remained high (>90%) throughout the 3-day process, but the washing step on day 2 caused the greatest decrease in cell viability. In contrast, the 5-day and 7-day processes included additional washing steps, each contributing to an additional decrease in cell viability.

[0147] Accordingly, according to one embodiment of the present disclosure, a method is provided for preparing transduced lymphocytes having improved efficacy or reduced adverse effects in the treatment of cancer. In some embodiments, the method involves obtaining lymphocytes from a patient via apheresis, incubating the lymphocytes with a polynucleotide vector to transduce the lymphocytes and produce transduced lymphocytes, culturing the transduced lymphocytes, and injecting the transduced lymphocytes into a patient. In some embodiments, the method involves shortening the time required to produce the therapeutic cell product (i.e., transduced lymphocytes) by using one of the above 7-day, 5-day, or 3-day production processes.

[0148] In some embodiments, the time from obtaining lymphocytes to injecting transduced lymphocytes (vein-to-vein time) is reduced. Vein-to-vein time can be determined by several factors, including the efficiency of cell transduction and expansion, as well as the quality and quantity of the starting apheresis material. In some embodiments, vein-to-vein time can be predicted by taking such factors into account. According to one embodiment of the art, the predicted vein-to-vein time is reduced by at least one day. In another embodiment, the predicted vein-to-vein time is reduced by at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or twenty-one days.

[0149] In some embodiments, the time from vein to vein is 28 days or less. In some embodiments, the time from obtaining lymphocytes to injecting transfected lymphocytes is 27 days or less. In some embodiments, the time from vein to vein is 26 days or less. In some embodiments, the time from obtaining lymphocytes to injecting transfected lymphocytes is 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 days or less.

[0150] The time from vein to vein can also depend on the time from the start of LD chemotherapy to infusion. Therefore, in some embodiments, the time from vein to vein can be shortened by reducing the time from the start of LD chemotherapy to infusion. In some embodiments, the time from the start of LD chemotherapy to infusion is 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day or less. In some embodiments, the time from the start of LD chemotherapy to infusion is 5 days or less.

[0151] Due to such shortened vein-to-vein time, the method can produce transduced lymphocytes such that patients receiving the infusion may have a complete response (CR) rate of more than 55%. In some embodiments, patients may have a complete response rate of more than 51%, or 52%, 53%, 54%, 56%, 57%, 58%, 59%, or 60%.

[0152] In some embodiments, patients may have an overall survival (OS) of more than 45%, as measured at 24 months (post-infusion). In some embodiments, patients may have an overall survival (OS) of more than 39%, or 40%, 41%, 42%, 43%, 44%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, or 53% at 24 months (post-infusion).

[0153] In some embodiments, patients may develop persistent thrombocytopenia in less than 30% of cases. In some embodiments, patients may develop persistent thrombocytopenia in less than 32%, 31%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, or 19% of cases.

[0154] The term "complete response" (CR) refers to a treatment outcome in which the treated patient has an evaluable but immeasurable disease and all evidence of the tumor and disease has disappeared. The CR rate can be determined by methods known in the art (e.g., Cheson et al., J Clin Oncol, 2014). In some embodiments, the CR rate may be based on response assessments obtained after the initial administration of the product and before any subsequent treatments that may be given due to relapse or disease progression (e.g., retreatment with the product, subsequent hematopoietic stem cell transplantation, and / or other anticancer therapies).

[0155] The term "overall survival" means that, at the time of measurement, the patient has not died from any cause.

[0156] Thrombocytopenia is a condition characterized by abnormally low levels of platelets (also known as thrombocytes) in the blood. Normal human platelet counts range from 150,000 to 450,000 per microliter of blood. In patients with thrombocytopenia, the platelet count can be less than 50,000 per microliter. Persistent thrombocytopenia refers to a condition in which a patient has thrombocytopenia for at least 30 days after the initial infusion.

[0157] According to one embodiment of the present disclosure, a rapid manufacturing process includes an improved transduction / culture procedure. In some embodiments, the transduction / culture procedure involves incubating a lymphocyte sample with a polynucleotide vector to transduce lymphocytes to produce transduced lymphocytes, and culturing the sample containing the transduced lymphocytes before the lymphocytes are harvested to produce the harvested sample.

[0158] In some embodiments, the culture process is shortened compared to the conventional process which takes about 4 days. In some embodiments, the culture process is completed within 96 hours, or within 72 hours, 60 hours, 50 hours, 48 ​​hours, 42 hours, 36 hours, 30 hours, 29 hours, 28 hours, 27 hours, 26 hours, 25 hours, 24 hours, 23 hours, 22 hours, 21 hours, 20 hours, 19 hours, 18 hours, 17 hours, 16 hours, 15 hours, 14 hours, 13 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, or 4 hours.

[0159] In some embodiments, the time for the culture process is counted from the completion of the transduction process (e.g., removal of cells from the system with the immobilized vector) to the harvesting of cells for storage, transport, or clinical use.

[0160] The culture of transduced lymphocytes may be carried out in media and conditions known in the art. In some embodiments, the culture of transduced lymphocytes may be carried out at a certain temperature and / or in the presence of CO2. In certain embodiments, the temperature may be about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, or about 39°C. In certain embodiments, the temperature may be about 34–39°C. In certain embodiments, a predetermined temperature may be from about 35–37°C. In certain embodiments, a preferred predetermined temperature may be from about 36–38°C. In certain embodiments, a predetermined temperature may be about 36–37°C or more preferably about 37°C.

[0161] In some embodiments, the culture of transfected lymphocytes may be carried out in the presence of a predetermined level of CO2. In certain embodiments, the predetermined level of CO2 may be 1.0–10% CO2. In certain embodiments, the predetermined level of CO2 may be about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10.0% CO2. In certain embodiments, the predetermined level of CO2 may be about 4.5–5.5% CO2. In certain embodiments, the predetermined level of CO2 may be about 5% CO2. In certain embodiments, the predetermined level of CO2 may be about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, or about 6.5% CO2. In some embodiments, the step of expanding the population of transfected T cells may be carried out in any combination of a predetermined temperature and / or the presence of a predetermined level of CO2. For example, in one embodiment, the step of expanding a population of transduced T cells may include a predetermined temperature of about 36-38°C and the presence of a predetermined level of CO2 of about 4.5-5.5%.

[0162] Any suitable culture medium, such as a T cell growth medium, may be used to culture cells in a suspension. For example, the T cell growth medium may, but is not limited to, a sterile low-glucose solution containing suitable amounts of buffer, magnesium, calcium, sodium pyruvate, and sodium bicarbonate. In one embodiment, the culture medium is OpTmizer® (Life Technologies), but those skilled in the art will understand how to produce a similar medium.

[0163] The incubation (and / or transduction) process may be performed in a closed system, but is not limited to these. In certain embodiments, the closed system is a closed bag culture system using any suitable cell culture bag (e.g., Mitenyi Biotec MACS® GMP Cell Differentiation Bags, Origen Biomedical PermaLife® Cell Culture Bags). In some embodiments, the closed system is at least 500 cm². 2 It has an internal surface area of ​​at least 1000 cm². In some embodiments, the closure system has an internal surface area of ​​at least 1000 cm². 2 , 1200cm 2 , 1400cm 2 , 1500cm 2 , 1600cm 2 , 1800cm 2 , 2000cm 2 , 2200cm 2 , 2500cm 2 , or 3000cm 2 It has an internal surface area of ​​1500 cm². In some embodiments, the closure system has an internal surface area of ​​1500 cm². 2 , 1600cm 2 , 1800cm 2 , 2000cm 2 , 2200cm 2 , 2500cm 2 , or 3000cm 2 It has the following internal surface area.

[0164] In some embodiments, cell culture bags used in a closed system are coated with recombinant human fibronectin protein. The recombinant human fibronectin fragment may contain three functional domains: a central cell-binding domain, a heparin-binding domain II, and a CS1 sequence. Recombinant human fibronectin protein or its fragments can be used to increase the gene efficiency of viral transduction of immune cells by assisting the co-localization of target cells or vectors. In certain embodiments, the recombinant human fibronectin fragment is RetroNectin® (Takara Bio, Japan). In certain embodiments, cell culture bags may be coated with recombinant human fibronectin fragments at concentrations of approximately 0.1–60 μg / mL, preferably 0.5–40 μg / mL. In certain embodiments, cell culture bags may be coated with recombinant human fibronectin fragments at concentrations of approximately 0.5–20 μg / mL, 20–40 μg / mL, or 40–60 μg / mL. In certain embodiments, cell culture bags may be coated with recombinant human fibronectin fragments in concentrations of approximately 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, 10 μg / mL, 11 μg / mL, 12 μg / mL, 13 μg / mL, 14 μg / mL, 15 μg / mL, 16 μg / mL, 17 μg / mL, 18 μg / mL, 19 μg / mL, or 20 μg / mL. In certain embodiments, cell culture bags may be coated with recombinant human fibronectin fragments at concentrations of approximately 2–5 μg / mL, 2–10 μg / mL, 2–20 μg / mL, 2–25 μg / mL, 2–30 μg / mL, 2–35 μg / mL, 2–40 μg / mL, 2–50 μg / mL, or 2–60 μg / mL.In certain embodiments, the cell culture bag may be coated with recombinant human fibronectin fragments at a concentration of at least about 2 μg / mL, at least about 5 μg / mL, at least about 10 μg / mL, at least about 15 μg / mL, at least about 20 μg / mL, at least about 25 μg / mL, at least about 30 μg / mL, at least about 40 μg / mL, at least about 50 μg / mL, or at least about 60 μg / mL. In certain embodiments, the cell culture bag may be coated with recombinant human fibronectin fragments at a concentration of at least about 10 μg / mL.

[0165] In some embodiments, a transduction enhancer is introduced into a closed system. A non-limiting example of such a transduction enhancer is the Vectofusin® transduction mixture.

[0166] In certain embodiments, the cell culture bags used in a closed-bag culture system may be blocked with human albumin serum (HSA). In alternative embodiments, the cell culture bags are not blocked with HSA.

[0167] Once the closed system is coated with recombinant fibronectin, a solution containing the vector is added to the closed system so that the vector can be immobilized on the inner surface of the closed system by the recombinant fibronectin. Such immobilization can improve the transduction efficiency when cells are added.

[0168] In some embodiments, the vector may be a viral vector, such as a lentiviral vector or a retroviral vector. Several recombinant viruses are used as viral vectors to deliver genetic material to cells. Viral vectors that may be used according to the transduction process include, but are not limited to, recombinant retroviral vectors, recombinant lentiviral vectors, recombinant adenovirus vectors, and recombinant adeno-associated viral (AAV) vectors, and may be any ecotropic or amphotropic viral vector. In one embodiment, the viral vector is an MSGV1 gamma retroviral vector. In some embodiments, the vector is a nonviral vector.

[0169] In some embodiments, a solution containing a vector with a total volume of at least 10 mL is used. In some embodiments, a solution containing a vector with a total volume of at least 100 mL is used. In some embodiments, a solution containing a vector with a total volume of at least 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 110 mL, 120 mL, 130 mL, 140 mL, 150 mL, 160 mL, 170 mL, 180 mL, 190 mL, 200 mL, 210 mL, 220 mL, 230 mL, 240 mL, 250 mL, 260 mL, 270 mL, 280 mL, 290 mL, 300 mL, 350 mL, or 400 mL is used. In some embodiments, a solution containing a vector with a total volume of 150 mL, 160 mL, 170 mL, 180 mL, 190 mL, 200 mL, 210 mL, 220 mL, 230 mL, 240 mL, 250 mL, 260 mL, 270 mL, 280 mL, 290 mL, 300 mL, 350 mL, 400 mL, or 500 mL or less is used.

[0170] In some embodiments, the vector solution is 1 × 10⁶ per milliliter. 3 ~1 × 10 12 Contains a viral vector with transduction units (TU / ml).

[0171] Once the closed system is coated with recombinant fibronectin and the vector is immobilized, the vector solution can be removed. In some embodiments, the closed system does not contain recombinant fibronectin. In some embodiments, removal of the vector solution is performed by gravity or syringe drain, which helps to retain the immobilized vector on the inner surface while removing impurities.

[0172] Lymphocyte transduction can be performed in a closed system coated with an immobilized vector. In some embodiments, transduction is carried out using a sample containing lymphocytes. In some embodiments, the sample contains at least 2.5 × 10⁻⁶ cells. 7 It contains 10 lymphocytes (e.g., T cells). In some embodiments, the sample contains at least 3 × 10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 , 1 x 10 8 , 1.2 × 10 8 , 1.5×10 8 , 1.8×10 8 , 2×10 8 , 2.2 × 10 8 , 2.5×10 8 , 2.6×10 8 , 2.7×10 8 , 2.8×10 8 , 2.9×10 8 , 3 x 10 8 , 3.1×10 8 , 3.2×10 8 , 3.3×10 8 , 3.4×10 8 , 3.5×10 8 , 3.6×10 8 , 3.7×10 8 , 3.8×10 8 , 3.9×10 8 , 4×10 8 , 4.1×10 8 , 4.2×10 8, 4.3×10 8 , 4.4×10 8 , 4.5×10 8 , 4.6×10 8 , 4.7×10 8 , 4.8×10 8 , 4.9×10 8 , 5×10 8 , 5.1×10 8 , 5.2×10 8 , 5.3×10 8 , 5.4×10 8 , 5.5×10 8 , 5.6×10 8 , 5.7×10 8 , 5.8×10 8 , 5.9×10 8 , 6×10 8 , 6.1×10 8 , 6.2×10 8 , 6.3×10 8 , 6.4×10 8 , 6.5×10 8 , 6.6×10 8 , 6.7×10 8 , 6.8×10 8 , 6.9×10 8 , 7×10 8 , 7.5×10 8 , 8×10 8 , 9×10 8 , or 10×10 8 contains lymphocytes (e.g., T cells). In some embodiments, the sample is 3×10 8 , 3.1×10 8 , 3.2×10 8 , 3.3×10 8 , 3.4×10 8 , 3.5×10 8 , 3.6×10 8 , 3.7×10 8 , 3.8×10 8 , 3.9×10 8 , 4×10 8 , 4.1×10 8 , 4.2×10 8 , 4.3×10 8 , 4.4×10 8 , 4.5×10 8 , 4.6×10 8, 4.7×10 8 , 4.8×10 8 , 4.9×10 8 , 5×10 8 , 5.1×10 8 , 5.2×10 8 , 5.3×10 8 , 5.4×10 8 , 5.5×10 8 , 5.6×10 8 , 5.7×10 8 , 5.8×10 8 , 5.9×10 8 , 6×10 8 , 6.1×10 8 , 6.2×10 8 , 6.3×10 8 , 6.4×10 8 , 6.5×10 8 , 6.6×10 8 , 6.7×10 8 , 6.8×10 8 , 6.9×10 8 , 7×10 8 , 7.5×10 8 , 8×10 8 , 9×10 8 , or 10 x 10 8 It contains fewer than 100 lymphocytes (e.g., T cells).

[0173] Lymphocytes used in the methods disclosed herein are typically obtained from a donor subject, which may be a cancer patient to be treated with the cell population produced by the methods herein (i.e., an autologous donor) or an individual to provide a lymphocyte sample to be used to treat a different individual or cancer patient at the time of production of the cell population produced by the methods herein (i.e., an allogeneic donor). Lymphocytes may be obtained from a donor subject by any preferred method used in the art. For example, lymphocytes may be obtained by any preferred extracorporeal method, venipuncture, or other blood collection method that yields a sample of blood and / or lymphocytes. In one embodiment, lymphocytes are obtained by apheresis.

[0174] Optionally, in some embodiments, the methods described herein further include a step of enriching the lymphocyte population obtained from the donor subject prior to transduction. Lymphocyte enrichment can be achieved by any preferred separation method, including but not limited to the use of a separation medium (e.g., nonionic iodixanol-based media such as Ficoll-Paque®, RosetteSep® HLA whole lymphocyte enrichment cocktail, Lymphocyte Separation Medium (LSA) (MP Biomedical catalog no. 0850494X), OptiPrep®), cell size, shape, or density separation by filtration or elution, immunomagnetic separation (e.g., magnetic-activated cell sorting system, MACS), fluorescence separation (e.g., fluorescence-activated cell sorting system, FACS), or bead-based column separation.

[0175] In some embodiments, circulating lymphoma cells are controlled by the use of a selective reagent for CD4 + / CD8 + Cells are removed from the sample via positive enrichment. In some such embodiments, after incubation with a selection reagent, the incubated cells, which contain cells bound to the selection reagent, are transferred to a system for immunoaffinity-based separation of the cells. In some embodiments, the system for immunoaffinity-based separation is or includes a magnetic separation column.

[0176] In some such embodiments, the isolation method includes the separation of different cell types based on the expression or presence of one or more specific molecules in cells, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acids. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is based on affinity or immunoaffinity. For example, in some embodiments, the isolation includes the separation of cells and cell populations based on the expression or expression level of one or more markers, typically cell surface markers, e.g., incubation with an antibody or binding partner that specifically binds to such markers, usually followed by a washing step and separation of cells bound to the antibody or binding partner from cells that did not bind to the antibody or binding partner. Such a separation step may be based on positive selection, where cells bound to the reagent are reserved for further use, and / or negative selection, where cells that did not bind to the antibody or binding partner are reserved. In some examples, both fractions are reserved for further use.

[0177] In some such embodiments, negative selection may be particularly useful, where antibodies that specifically identify cell types within a heterogeneous population are unavailable, so that separation is best performed based on markers expressed by cells other than the desired population.

[0178] Isolation does not necessarily result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection or enrichment of a particular type of cell, e.g., cells expressing a marker, means increasing the number or percentage of such cells, but does not necessarily result in the complete absence of cells that do not express the marker. Similarly, negative selection, removal, or depletion of a particular type of cell, e.g., cells expressing a marker, means decreasing the number or percentage of such cells, but does not necessarily result in the complete removal of all such cells.

[0179] In some cases, a single step may lead to the execution of multiple rounds of separation steps, where positively or negatively selected fractions are subjected to further separation steps, such as subsequent positive or negative selection. In some cases, cells expressing multiple markers simultaneously may be removed by a single separation step, for example, by incubating cells with multiple antibodies or binding partners specific to markers targeted for negative selection, each of which is specific to a marker. Similarly, multiple cell types may be positively selected simultaneously by incubating cells with multiple antibodies or binding partners expressed in various cell types.

[0180] For example, in some embodiments, a specific subpopulation of T cells, e.g., cells that are positive for or express high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+ T cells, is isolated by positive or negative selection techniques. For example, CD3+, CD28+ T cells can be positively selected using anti-CD3 / anti-CD28 conjugate magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander). In some embodiments, a population of cells is enriched with T cells having a naive phenotype (CD45RA+CCR7+).

[0181] 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 achieved by incubating cells with one or more antibodies or other conjugates that specifically bind to one or more surface markers expressed on the cells to be positively or negatively selected, or expressed at relatively higher levels (marker high) (marker+).

[0182] In certain embodiments, a biological sample, such as a PBMC or other leukocyte sample, is subjected to the selection of CD4+ T cells, ensuring both negative and positive fractions are obtained. In certain embodiments, CD8+ T cells are obtained from the negative fraction. In some embodiments, a biological sample is subjected to the selection of CD8+ T cells, ensuring both negative and positive fractions are obtained. In certain embodiments, CD4+ T cells are obtained from the negative fraction.

[0183] In some embodiments, T cells are isolated from PBMC samples by negative selection of markers expressed on non-T cells, e.g., B cells, monocytes, or other leukocytes, e.g., CD14. In some embodiments, a CD4+ or CD8+ selection step is used to isolate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations may be further sorted into subpopulations by positive or negative selection of markers expressed on or relatively more highly expressed on one or more naive T cell, memory T cell, and / or effector T cell subpopulations.

[0184] In one example, a monoclonal antibody cocktail for enriching CD4+ cells by negative selection typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or solid matrix, such as magnetic or paramagnetic beads, to enable the separation of cells by positive and / or negative selection. For example, in some embodiments, cells and cell populations are separated or isolated using immunomagnetic (or affinity magnetic) separation techniques. In some embodiments, a sample or composition of cells to be separated is incubated with a small magnetizable or magnetically responsive material, such as a paramagnetic bead (e.g., Dynabeads® or MACS beads), a magnetically responsive particle, or a microparticle. The magnetically responsive material, e.g., particles, is typically bound directly or indirectly to a binding partner, e.g., a molecule, e.g., an antibody that specifically binds to a surface marker present in the cells, a group of cells, or a population of cells that are to be separated, e.g., those to be negatively or positively selected.

[0185] In some embodiments, the magnetic particles or beads comprise a magnetically responsive material bound to a specifically binding member, such as an antibody or other binding partner. Numerous well-known magnetically responsive materials exist for use in magnetic separation methods. Incubation is typically performed under conditions where molecules such as an antibody or binding partner, or a secondary antibody or other reagent specifically binding to such an antibody or binding partner, which are bound to the magnetic particles or beads, specifically bind to cell surface molecules (if present on cells in the sample). In some embodiments, the sample is placed in a magnetic field, and cells to which magnetically responsive particles or magnetizable particles are bound are attracted to a magnet and separated from unlabeled cells. Positive selection ensures that cells attracted to the magnet are secured, while negative selection ensures that cells not attracted (unlabeled cells) are secured. In some embodiments, a combination of positive and negative selection is performed during the same selection step, where positive and negative fractions are secured and further processed or subjected to further separation steps. In some embodiments, the magnetically responsive particles are coated with a primary antibody or other binding partner, a secondary antibody, a lectin, an enzyme, or streptavidin. In certain embodiments, magnetic particles are bound to cells by coating with primary antibodies specific to one or more markers. In certain 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 (e.g., streptavidin) are added. In certain embodiments, streptavidin-coated magnetic particles are used together with a biotinylated primary or secondary antibody. In some embodiments, the magnetically responsive particles remain bound to cells to be subsequently incubated, cultured, and / or manipulated, and in some embodiments, the particles remain bound to cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, for example, the use of competitive unlabeled antibodies and magnetizable particles or antibodies conjugated with a cleavable linker.In some embodiments, the magnetizable particles are biodegradable.

[0186] In some embodiments, affinity-based selection is performed by magnetically activated cell sorting (MACS) (Miltenyi Biotec, Auburn, CA). The magnetically activated cell sorting (MACS) system allows for high-purity selection of cells bound to magnetized particles. In certain embodiments, MACS operates in a manner in which non-target and target species are sequentially eluted after the application of an external magnetic field. That is, cells bound to magnetized particles are retained in place, while unbound species are eluted. Subsequently, after this initial elution step is complete, species that were trapped in the magnetic field and whose elution was prevented are released in several ways so that they can be eluted and recovered. In certain embodiments, non-target cells are labeled and depleted from heterogeneous cell populations.

[0187] In some embodiments, isolation or separation is performed using a system, device, or apparatus that performs one or more of the isolation, cell preparation, separation, processing, incubation, culture, and / or formulation steps of the Method. In some embodiments, the system is used to perform each of these steps in a closed or sterile environment, for example, to minimize errors, user handling, and / or contamination. In one example, the system is described in International Patent Application Publication 2009 / 072003 or U.S. Patent Application Publication 20110003380(A1), which are each incorporated herein by reference. In some embodiments, the system or apparatus performs one or more of the isolation, processing, manipulation, and formulation steps, for example, all of them, in an integrated or self-contained system and / or in an automated or programmable manner. In some embodiments, the system or apparatus includes a computer and / or computer program communicating with the system or apparatus, which allows the user to program, control, evaluate the results of, and / or adjust various embodiments of processing, isolation, manipulation, and formulation steps. In some embodiments, separation and / or other steps are performed using a CliniMACS system (Miltenyi Biotec) for automated cell separation at a clinical scale within a closed, sterile system. Components may include an embedded microcomputer, a magnetic separation unit, a peristaltic pump, and various pinch valves. In some embodiments, the embedded microcomputer controls all components of the instrument and instructs the system to perform repetitive procedures in a standardized sequence. In some embodiments, the magnetic separation unit includes a movable permanent magnet and a holder for a selection column. The peristaltic pump controls the flow rate in the tube set and, together with the pinch valves, ensures a controlled flow of buffer and continuous suspension of cells through the system.

[0188] In some embodiments, the CliniMACS system uses magnetizable particles coupled to antibodies, supplied in a sterile, non-pyrogenic 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 tube set, which is then connected to a buffer bag and a cell collection bag. The tube set consists of pre-assembled sterile tubes containing a pre-column and a separation column, and is intended for single use only. After the separation program is started, the system automatically applies the cell sample onto the separation column. Labeled cells are retained in the column, while unlabeled cells are removed by a series of washing steps. In some embodiments, the cell population used in the method described herein is unlabeled and is not retained in the column. In some embodiments, the cell population used in the method described herein is labeled and is retained in the column. In some embodiments, the cell population for use in the method described herein is eluted from the column after the magnetic field is removed and collected in a cell collection bag.

[0189] In certain embodiments, separation and / or other steps are performed using a CliniMACS Prodigy system (Miltenyi Biotec). In some embodiments, the CliniMACS Prodigy system includes a cell processing unit that enables automated washing and fractionation of cells by centrifugation. The CliniMACS Prodigy system may also include an onboard camera and image recognition software that determines the optimal cell fractionation endpoint by identifying macroscopic layers of cell products from the source. For example, peripheral blood is automatically separated into red blood cells, white blood cells, and plasma layers. The CliniMACS Prodigy system may also include a built-in cell culture chamber that performs cell culture protocols such as cell differentiation and expansion, antigen loading, and long-term cell culture. An inlet may allow for sterile removal and replenishment of the culture medium, and cells may be monitored using a built-in microscope.

[0190] In some embodiments, the cell populations described herein are collected and enriched (or depleted) by flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluid flow. In some embodiments, the cell populations described herein are collected and enriched (or depleted) by sorting on a preparative scale (FACS). In certain embodiments, the cell populations described herein are collected and enriched (or depleted) by the use of a microelectromechanical system (MEMS) chip combined with a FACS-based detection system (see, for example, International Publication No. 2010 / 033140, Cho et al. (2010) Lab Chip 10, 1567-1573, and Godin et al. (2008) J Biophoton. l(5):355-376). In any case, the cells may be labeled with multiple markers, which allows for the isolation of a clearly defined T cell subset with high purity.

[0191] In some embodiments, the antibody or binding partner is 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 a fluorescently labeled antibody. In some examples, cell separation based on the binding of antibodies or other binding partners specific to one or more cell surface markers is performed in a fluid flow by, for example, a fluorescence-activated cell sorting (FACS) including a preparative scale (FACS) and / or a microelectromechanical system (MEMS) chip combined with, for example, a flow cytometry detection system. Such methods allow for simultaneous positive and negative selection based on multiple markers.

[0192] In some embodiments, at least 0.5 × 10 9 Individual lymphocytes are obtained from a donor and, optionally, enriched and / or subjected to stimulation. In some embodiments, at least 0.6 × 10⁶ lymphocytes are obtained. 9 , 0.7 × 10 9 , 0.8 × 10 9 , 0.9 × 10 9, 1 x 10 9 , 1.1 × 10 9 , 1.2 × 10 9 , 1.3 × 10 9 , 1.4×10 9 , 1.5×10 9 , 1.6×10 9 , 1.7×10 9 , 1.8×10 9 , 1.9 × 10 9 , 2×10 9 , 2.5×10 9 , or 3 × 10 9 Individual lymphocytes are obtained from a donor and, optionally, enriched and / or stimulated. In some embodiments, 1 × 10 9 , 1.1 × 10 9 , 1.2 × 10 9 , 1.3 × 10 9 , 1.4×10 9 , 1.5×10 9 , 1.6×10 9 , 1.7×10 9 , 1.8×10 9 , 1.9 × 10 9 , 2×10 9 , 2.5×10 9 , or 3 × 10 9 Lymphocytes of a certain number or less are obtained from a donor, and, at the discretion of the donor, are enriched and / or subjected to stimulation.

[0193] Optionally, the methods described herein further include the step of stimulating lymphocytes with one or more lymphocyte stimulants. In some embodiments, the stimulation is performed before the transduction step. In some embodiments, the stimulation is performed after the transduction step.

[0194] Any combination of one or more suitable lymphocyte stimulants may be used to stimulate (activate) lymphocytes. Non-limiting examples include antibodies or functional fragments thereof that target T cell stimulating or co-stimulating molecules (e.g., anti-CD2 antibodies, anti-CD3 antibodies, anti-CD28 antibodies, or their functional fragments), T cell cytokines (e.g., any isolated, wild-type, or recombinant cytokines such as interleukin 1 (IL-1), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 7 (IL-7), interleukin 15 (IL-15), tumor necrosis factor α (TNFα)), or any other suitable mitogens for T cell stimulating or co-stimulating molecules (e.g., tetradecanoyl phorbol acetate). Examples include acetate (TPA), phytohemagglutinin (PHA), concanavalin A (ConA), lipopolysaccharide (LPS), pokeweed mitogen (PWM), or natural ligands. In some embodiments, the stimulant is an anti-CD3 antibody and / or an anti-CD28 antibody.

[0195] In some embodiments, the lymphocyte stimulation steps described herein may involve stimulating lymphocytes with one or more stimulants at a predetermined temperature, for a predetermined duration, and / or in the presence of a predetermined level of CO2. In certain embodiments, the predetermined temperature for stimulation may be about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, or about 39°C. In certain embodiments, the predetermined temperature for stimulation may be about 34–39°C. In certain embodiments, the lymphocyte stimulation steps include stimulating lymphocytes with one or more stimulants for a predetermined duration. In certain embodiments, the predetermined duration for stimulation may be about 24–72 hours. In certain embodiments, the predetermined duration for stimulation may be about 24–36 hours. In certain embodiments, the lymphocyte stimulation steps may include stimulating lymphocytes with one or more stimulants in the presence of a predetermined level of CO2. In certain embodiments, the predetermined level of CO2 for stimulation may be about 1.0–10% CO2. In a particular embodiment, a predetermined level of CO2 for stimulation may be about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10.0% CO2.

[0196] In some embodiments, an anti-CD3 antibody (or a functional fragment thereof), an anti-CD28 antibody (or a functional fragment thereof), or a combination of an anti-CD3 antibody and an anti-CD28 antibody may be used in accordance with a step of stimulating a population of lymphocytes. Any soluble or immobilized anti-CD3 and / or anti-CD28 antibody or functional fragment thereof may be used (e.g., clone OKT3 (anti-CD3), clone 145-2C11 (anti-CD3), clone UCHT1 (anti-CD3), clone L293 (anti-CD28), clone 15E8 (anti-CD28)). In some embodiments, antibodies may be commercially purchased from distributors known in the art, including but not limited to Miltenyi Biotec, BD Biosciences (e.g., MACS GMP CD3 Pure 1 mg / mL, Part No. 170-076-116), and eBioscience, Inc. Furthermore, those skilled in the art will understand how to produce anti-CD3 antibodies and / or anti-CD28 antibodies by standard methods. Any antibodies used in the methods described herein must be produced under Good Manufacturing Practices (GMP) in accordance with the relevant institutional guidelines for biological products.

[0197] In certain embodiments, the T-cell stimulant may include an anti-CD3 or anti-CD28 antibody at concentrations ranging from about 20 ng / mL to 100 ng / mL. In certain embodiments, the concentration of the anti-CD3 or anti-CD28 antibody may be about 20 ng / mL, about 30 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, or about 100 ng / mL.

[0198] As demonstrated in the 7-day, 5-day, and 3-day processes described above, the prepared lymphocytes contain a higher proportion of immature lymphocytes (e.g., naive T cells). Accordingly, one embodiment of the present disclosure provides a population of lymphocytes prepared by the present method, comprising CD4+ T cells and CD8+ T cells.

[0199] In some embodiments, at least 20% of CD4+ T cells are naive T cells. In some embodiments, at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% of CD4+ T cells are naive T cells.

[0200] In some embodiments, less than 25% of CD4+ T cells are effector memory T cells. In some embodiments, less than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, or 5% of CD4+ T cells are effector memory T cells.

[0201] In some embodiments, less than 44% of CD4+ T cells are central memory T cells. In some embodiments, less than 43%, 42%, 41%, or 40% of CD4+ T cells are central memory T cells.

[0202] In some embodiments, less than 1.5% of CD4+ T cells are effector T cells. In some embodiments, less than 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, or 0.5% of CD4+ T cells are effector T cells.

[0203] In some embodiments, at least 5% of CD8+ T cells are naive T cells. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, or 35% of CD8+ T cells are naive T cells.

[0204] In some embodiments, less than 30% of CD8+ T cells are effector memory T cells. In some embodiments, less than 28%, 27%, 25%, 22%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, or 10% of CD8+ T cells are effector memory T cells.

[0205] In some embodiments, less than 60% of CD8+ T cells are central memory T cells. In some embodiments, less than 58%, 56%, 55%, 54%, 52%, or 50% of CD8+ T cells are central memory T cells.

[0206] Each type of T cell can be characterized by cell surface markers well known in the field. For example, naive T cells can be characterized as CCR7+, CD45RO-, and CD95-. Additional markers for naive T cells include CD45RA+, CD62L+, CD27+, CD28+, CD127+, CD132+, CD25-, CD44-, and HLA-DR-.

[0207] Surface markers for stem memory T cells (Tscm) include, but are not limited to, CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, IL-7Ra+, CD95+, IL-2RP+, CXCR3+, and LFA-.

[0208] Surface markers for effector memory T cells (Tem) include, but are not limited to, CCR7-, CD45RO+, and CD95+. A further marker for effector memory T cells is IL-2Rβ+. For central memory T cells (Tcm), preferred markers include CD45RO+, CD95+, IL-2Rβ+, CCR7+, and CD62L+. For effector T cells (Teff), preferred markers include, but are not limited to, CD45RA+, CD95+, IL-2Rβ+, CCR7-, and CD62L-.

[0209] The collected lymphocytes preferably contain a good proportion of CD3+ T cells. In some embodiments, at least 25%, 35%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the collected lymphocytes are CD3+ T cells.

[0210] The collected lymphocytes preferably include a good proportion of transduced cells. In some embodiments, at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the collected lymphocytes are transduced with a vector. In some embodiments, each transduced lymphocyte contains at least one copy of the vector (or the coding sequence it contains) integrated into the host genome. In some embodiments, each transduced lymphocyte contains at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the vector integrated into the host genome.

[0211] In some embodiments, the vector includes a transgene encoding a polypeptide. The polypeptide may be, but is not limited to, a CAR or a TCR. In some embodiments, the CAR or TCR includes an antigen-binding molecule. In some embodiments, the antigen-binding molecule has binding specificity to an antigenic moiety. In some embodiments, the antigen-binding molecule has binding specificity to one or more antigenic moieties (e.g., 1, 2, 3, or 4 antigenic moieties). In some embodiments, the antigen-binding molecule has binding specificity to two different antigenic moieties.

[0212] In some embodiments, antigenic moieties are associated with cancer or cancer cells. Such antigenic moieties include 707-AP (707-alanine proline), AFP (alpha(a)-fetoprotein), ART-4 (adenocarcinoma antigen recognized by T4 cells), BAGE (B antigen, β-catenin / m, β-catenin / mutant), BCMA (B cell maturation antigen), Bcr-abl (easily cleaved region-Abelson), CAIX (carbonic anhydrase IX), CD19 (surface antigen classification 19), CD20 (surface antigen classification 20), CD22 (surface antigen classification 22), CD30 (surface antigen classification 30), CD33 (surface antigen classification 33), and CD 44v7 / 8 (Surface antigen classification 44, exon 7 / 8), CAMEL (CTL-recognizing antigen on melanoma), CAP-1 (Carcinoembryonic antigen peptide-1), CASP-8 (Caspase-8), CDC27m (Cell division cycle 27, variant), CDK4 / m (Cyrin-dependent kinase 4, variant), CEA (Carcinoembryonic antigen), CT (Cancer / testis (antigen)), Cyp-B (Cyclophyllin B), DAM (Differentiation antigen, melanoma), EGFR (Epidermal growth factor receptor), EGFRvIII (Epidermal growth factor receptor, variant III), EGP-2 (Epidermal glycoside) Glycoprotein 2), EGP-40 (epithelial glycoprotein 40), Erbb2, 3, 4 (erythroblastic leukemia virus oncogene homologs -2, -3, 4), ELF2M (elongation factor 2, variant), ETV6-AML1 (Ets variant gene 6 / acute myeloid leukemia 1 gene ETS), FBP (folate-binding protein), fAchR (fetal acetylcholine receptor), G250 (glycoprotein 250), GAGE ​​(G antigen), GD2 (dicialoganglioside 2), GD3 (dicialoganglioside 3), GnT-V (N-acetylglucosamine transferase) IV), Gp100 (glycoprotein 100kD), HAGE (helicose antigen), HER-2 / neu (human epithelial receptor-2 / neuronal, also known as EGFR2), HLA-A (human leukocyte antigen-A), HPV (human papillomavirus), HSP70-2M (heat shock protein 70-2 variant), HST-2 (human signet ring tumor-2), hTERT or hTRT (human telomerase reverse transcriptase), iCE (intestinal carboxylesterase), IL-13R-a2 (interleukin-13 receptor subunit alpha-2),KIAA0205, KDR (kinase insertion domain receptor), κ-light chain, LAGE (L antigen), LDLR / FUT (low-density lipid receptor / GDP-L-fucose:bD-galactosidase 2-αL-fucosyltransferase), LeY (Lewis-Y antibody), L1CAM (L1 cell adhesion molecule), MAGE (melanoma antigen), MAGE-A1 (melanoma-associated antigen 1), mesothelin, mouse CMV-infected cells, MART-1 / Melan-A (melanoma antigen-1 recognized by T cells / melanoma antigen A), MC1R (melanocortin 1 receptor), myosin / m (myosin variant), MUC1 (mucin 1), MUM-1, -2, -3 (melanoma ubiquitous, variants 1, 2, 3), NA88-A (NA of patient M88) cDNA clone), NKG2D (natural killer group 2, member D) ligand, NY-BR-1 (New York mammary gland differentiation antigen 1), NY-ESO-1 (New York esophageal squamous cell carcinoma-1), tumor fetal antigen (h5T4), P15 (protein 15), p190 minor bcr-abl (190KD bcr-abl protein), Pml / RARa (promyelocytic leukemia / retinoic acid receptor a), PRAME (preferential expression antigen for melanoma), PSA (prostate-specific antigen), PSCA (prostate stem cell antigen), PSMA (prostate-specific membrane antigen), RAGE (renal antigen), RU1 or RU2 (renal eccentric 1 or 2), SAGE (sarcoma antigen), SART-1 or This may include, but is not limited to, SART-3 (tumor rejection squamous cell antigen 1 or 3), SSX1, -2, -3, 4 (synovial sarcoma X1, -2, -3, -4), TAA (tumor-associated antigen), TAG-72 (tumor-associated glycoprotein 72), TEL / AML1 (translocation Ets-family leukemia / acute myeloid leukemia 1), TPI / m (triose phosphate isomerase variant), TRP-1 (tyrosinase-related protein 1 or gp75), TRP-2 (tyrosinase-related protein 2), TRP-2 / INT2 (TRP-2 / intron 2), VEGF-R2 (vascular endothelial growth factor receptor 2), or WT1 (Wilms oncogene), or any combination thereof.

[0213] Additional examples of cancer cell-associated antigens include 2B4 (CD244), 4-1BB, 5T4, A33 antigen, adenocarcinoma antigen, adrenoceptor beta 3 (ADRB3), A kinase anchor protein 4 (AKAP-4), alpha-fetoprotein (AFP), anaplastic lymphoma kinase (ALK), androgen receptor, B7H3 (CD276), β2-integrin, BAFF, B lymphoma cells, B cell maturation antigen (BCMA), and bcr-abl (breakpoint cluster region). Oncogene fusion protein consisting of region (BCR) and Abelson mouse leukemia virus oncogene homolog 1 (Abl), BhCG, bone marrow stromal cell antigen 2 (BST2), CCCTC binding factor (zinc finger protein)-like (sibling of BORIS or imprint site regulatory factor), BST2, C242 antigen, 9-O-acetyl-CA19-9 marker, CA-125, CAEX, calreticulin, carbonic anhydrase 9 (CAIX), C-MET, CCR4, CCR5, CCR8, CD2, CD3, CD4, CD5, CD8, CD7, CD10, CD16, CD19, CD20, CD22, CD23 (IgE receptor), CD24, CD25, CD27, CD28, CD30 (TNFRSF8), C D33, CD34, CD38, CD40, CD40L, CD41, CD44, CD44V6, CD49f, CD51, CD52, CD56, CD63, CD70, CD72, CD 74, CD79a, CD79b, CD80, CD84, CD96, CD97, CD100, CD123, CD125, CD133, CD137, CD138, CD150, CD1 52 (CTLA-4), CD160, CD171, CD179a, CD200, CD221, CD229, CD244, CD272 (BTLA), CD274 (PDL-1, B7H1), CD279 (PD-1), CD352, CD358, CD300 molecular-like family member f (CD300LF), carcinoembryonic antigensantigen (CEA), claudin 6 (CLDN6), C-type lectin-like molecule-1 (CLL-1 or CLECL1), C-type lectin domain family 12 member A (CLEC12A), cytomegalovirus (CMV) infected cell antigen, CNT0888, CRTAM (CD355), CS-1 (CD2 subset 1, also called CRACC, CD319, and 19A24), CTLA-4, cyclin B1, chromosome X open reading frame 61 (CXORF61), cytochrome P450 1B1 1, CYP1B1), DNAM-1 (CD226), desmoglein 4, DR3, DR5, E-cadherin neoepitope, epidermal growth factor receptor (EGFR), EGF1R, epidermal growth factor receptor variant III (EGFRvIII), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), elongation factor 2 mutated (ELF2M), endothialin, epithelial cell adhesion moleculemolecule (EPCAM), ephrin type A receptor 2 (EphA2), ephrin B2, receptor tyrosine protein kinase erb-B2, 3, 4 (erb-B2, 3, 4), ERBB, ERBB2 (Her2 / neu), ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), ETA, ETS translocation variant gene 6 (ETV6-AML) located on chromosome 12p, IgA receptor Fc fragment (FCAR or CD89), fibroblast activation protein alpha (FAP), FBP, Fc receptor-like 5 (FCRL5), fetal acetylcholine receptor (AChR), fibronectin extradomain B, Fms-like tyrosine kinase 3 (FLT3), folate-binding protein protein, FBP), folate receptor 1, folate receptor α, folate receptor β, Fos-related antigen 1, fucosyl, fucosyl GM1, GM2, ganglioside G2 (GD2), ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer), o-acetyl-GD2 ganglioside (OAcGD2), GITR( TNFRSF18), GM1, ganglioside GM3 (aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(ll)Cer), GP100, GloboH glycoceramide hexasaccharide portion (GloboH), glycoprotein 75, glypican-3 (GPC3), glycoprotein 100 (gp100), GPNMB, G protein-coupled receptor 20 (GPR20), G protein-coupled receptor class C group 5, member D (GPRC5D), Hepatitis A virus cellular receptor 1 (HAVCR1), human epidermal growth factor receptor 2 (HPV20)2. HER-2), HER2 / neu, HER3, HER4, HGF, high molecular weight-melanoma-associated antigen (HMWMAA), human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPV E7), Heat shock protein 70-2 mutant (mut hsp70-2), human scattering factor receptor kinase, human telomerase reverse transcriptase (hTERT), HVEM, ICOS, insulin-like growth factor receptor 1 (IGF-1 receptor), IGF-I, IgGl, immunoglobulin lambda-like polypeptide 1 (IGLL1), IL-6, interleukin 11 receptor alpha (IL-11Ra), IL-13, interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2), insulin-like growth factor I receptor IGF1-R receptor, integrin α5β1, integrin ανβ3, intestinal carboxylesterase, κ light chain, KCS1, kinase insert domain receptor (KDR), KIR, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL2, KIR-L, KG2D ligand, KIT (CD117), KLRGI, LAGE-LA, LAG3, lymphocyte-specific protein tyrosine kinase (LCK), leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), regmine, leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Lewis (Y) antigen, LeY, LG, LI cell adhesion moleculemolecule, LI-CAM), LIGHT, LMP2, lymphocyte antigen 6 complex, LTBR, ​​gene locus K9 (LY6K), Ly-6, lymphocyte antigen 75 (LY75), melanoma cancer testicular antigen-1 (MAD-CT-1), melanoma cancer testicular antigen-2 (MAD-CT-2), MAGE, melanoma-associated antigen 1 (MAGE-A1), MAGE-A3 melanoma antigen 1 recognized by T cells (MelanA or MARTI), MelanA / MARTI, mesothelin, MAGE A3, apoptosis-induced melanoma inhibitor (ML-IAP), melanoma-specific chondroitin sulfate proteoglycan proteoglycan (MCSCP), MORAb-009, MS4A1, Mucin1 (MUC1), MUC2, MUC3, MUC4, MUC5AC, MUC5b, MUC7, MUC16, mucin CanAg, Müllerian inhibitory substance (MIS) receptor type II, neuroblastoma-derived homolog of v-myc avian myelocytosis viral oncogene (MYCN), N-glycolylneuraminic acid, N-acetylglucosaminyltransferase V (NA17), neural cell adhesion molecule (NCAM), NKG2A, NKG2C, NKG2D, NKG2E ligand, NKR-P IA, NPC-1C, NTB-A, mammary gland differentiation antigen (NY-BR-1), NY-ESO-1, tumor fetal antigen (h5T4), olfactory receptor 51E2 51E2, OR51E2), OX40, plasma cell antigen, polySA, proacrosin-binding protein sp32 (OY-TES 1), p53, p53 variant, pannexin 3 (PANX3), prostatic acid phosphatase (PAP), paired box protein Pax-3 (PAX3), paired box protein Pax-5 (PAX5), prostate carcinoma tumor antigen-1 (PCTA-1 or galectin 8), PD-1H, platelet-derived growth factor receptor alpha (PAX3), prostate carcinoma tumor antigen-1 (PCTA-1 or galectin 8), PD-1H, platelet-derived growth factor receptor alpha (PAX3), prostate carcinoma tumor antigen-1 (PCTA-1 or galectin 8), platelet-derived growth factor receptor alpha (PAX3), prostate carcinoma tumor antigen-1 (PCTA-1 or galectin 8), prostate carcinoma tumor antigen-1 (PCTA-1 or galectin 8), prostate carcinoma tumor antigen-1 (PCTA-1 or galectin 8), prostate carcinoma tumor antigen-1 (PD-1H), platelet-derived growth factor receptor alpha (PAX3), prostate carcinoma tumor antigen-1 (PCTA-1 or galectin 8), prostate carcinoma tumor antigen-1 (PCTA-1 or galectin 8), prostate carcinoma tumor antigen-1 (PD3H), platelet-derived growth factor receptor alpha (PAX3), prostate carcinoma tumor antigen-1 (PCTA-1 or galectin 8Factor receptor alpha (PDGFR-alpha), PDGFR-beta, PDL192, PEN-5, phosphatidylserine, placenta-specific receptor 1 (PLAC1), polysialic acid, prostase, prostate cancer cells, prostain, protease serine 21 (testicin or PRSS21), proteinase 3 (PR1), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), proteasome (Macropain) subunit, beta type, Receptor for Advanced Glycation Endproducts (RAGE-1), RANKL, Ras variant, Ras homolog family member C (RhoC), RON, Receptor tyrosine kinase-like orphan receptor 1 (ROR1), renal ubiquitous receptor 1 1, RU1), Renal ubiquitous 2 (RU2), Sarcoma translocation breakpoint, Squamous cell carcinoma antigen recognized by T cells (SART3), SAS, SDC1, SLAMF7, Sialyl Lewis adhesion molecule (sLe), Siglec-3, Siglec-7, Siglec-9, Sonic hedgehog (SHH), Sperm protein 17 (SPA17), Stage-specific embryonic antigen-4 (SSEA-4), STEAP, sTn antigen, Synovial sarcoma, X breakpoint 2 (SSX2), Sulbibin, Tumor-associated glycoprotein 72 72, TAG72), TCR5y, TCRa, TCRB, TCR gamma alternative leading frame protein (TARP), telomerase, TIGITTNF-α precursor, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), tenascin C, TGF-beta 2, TGF-β, transglutaminase 5 (TGS5), angiopoietin-binding cell surface receptor 2 (Tie 2), TIM1, TIM2, TIM3, Tn Ag, TRAIL-R1, TRAIL-R2, tyrosinase-related protein 2 (TRP-2), thyroid stimulating hormone receptor (TSHR), tumor antigen CTAA16.88, tyrosinase, ROR1, TAG-72, uroplakin 2 (UPK2), VEGF-A, VEGFR-1, vascular endothelial growth factor receptor 2 Examples include 2. VEGFR2), and vimentin, Wilms oncoprotein (WT1), or the X antigen family, member 1A (XAGE1), or combinations thereof.

[0214] In other embodiments, the antigenic moiety is associated with a virus-infected cell (i.e., a viral antigenic moiety). Such antigenic moieties include Epstein-Barr virus (EBV) antigens (e.g., EBNA-1, EBNA-2, EBNA-3, LMP-1, LMP-2), hepatitis A virus antigens (e.g., VP1, VP2, VP3), hepatitis B virus antigens (e.g., HBsAg, HBcAg, HBeAg), hepatitis C virus antigens (e.g., envelope glycoproteins E1 and E2), herpes simplex virus types 1, 2, or 8 (HSV1, HSV2, or HSV8 virus antigens (e.g., glycoproteins gB, gC, gC, gE, gG, gH, gI, gJ, gK, gL, gM, UL20, UL32, US43, UL45, UL49A), cytomegalovirus (CMV) virus antigens (e.g., glycoproteins gB, gC, gC, gE, gG, gH, gI, gJ, gK, gL, gM, or other envelope proteins), human immunodeficiency virus (human) The antigens may include, but are not limited to, immunodeficiency virus (HIV) virus antigens (glycoproteins gp120, gp41, or p24), influenza virus antigens (e.g., hemagglutinin (HA) or neuraminidase (NA)), measles or mumps virus antigens, human papillomavirus (HPV) virus antigens (e.g., L1, L2), parainfluenza virus antigens, rubella virus antigens, respiratory syncytial virus (RSV) virus antigens, or varicella-zoster virus antigens, or combinations thereof. In such embodiments, the cell surface receptor may be any TCR or any CAR that recognizes any of the aforementioned viral antigens on a cell infected with the target virus.

[0215] In other embodiments, the antigenic moiety is associated with cells having immune or inflammatory dysfunction. Examples of such antigenic moieties include, but are not limited to, myelin basic protein (MBP), myelin proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG), carcinoembryonic antigen (CEA), proinsulin, glutamine decarboxylase (GAD65, GAD67), heat shock protein (HSP), or any other tissue-specific antigen, or combination thereof, that is involved in or associated with pathogenic autoimmune processes.

[0216] In some embodiments, the TCR has specificity for antigenic moieties on cancer cells. Non-limiting examples of TCRs include anti-707-AP TCR, anti-AFP TCR, anti-ART-4 TCR, anti-BAGE TCR, anti-Bcr-abl TCR, anti-CAMEL TCR, anti-CAP-1 TCR, anti-CASP-8 TCR, anti-CDC27m TCR, anti-CDK4 / m TCR, anti-CEA TCR, anti-CT TCR, anti-Cyp-B TCR, anti-DAM TCR, anti-TCR, anti-EGFRvIII TCR, anti-ELF2M TCR, anti-ETV6-AML1 TCR, anti-G250 TCR, GAGE ​​TCR, anti-GnT-V TCR, anti-Gp100 TCR, anti-HAGE TCR, anti-HER-2 / neu TCR, anti-HLA-A TCR, anti-HPV TCR, anti-HSP70-2M TCR, anti-HST-2 TCR, anti-hTERT TCR or anti-hTRT TCR, anti-iCE TCR, anti-KIAA0205, anti-LAGE (L antigen), anti-LDLR / FUT TCR, anti-MAGE TCR, anti-MART-1 / Melan-A TCR, anti-MC1R TCR, anti-Myosin / m TCR, anti-MUC1 TCR, anti-MUM-1, -2, -3 TCR, anti-NA88-A TCR, anti-NY-ESO-1 TCR, anti-P15 TCR, anti-p190 minor bcr-abl TCR, anti-Pml / RARa TCR, anti-PRAME TCR, anti-PSA TCR, anti-PSMA TCR, anti-RAGE TCR, anti-RU1 TCR or anti-RU2 TCR, anti-SAGE TCR, anti-SART-1 TCR or anti-SART-3 TCR, anti-SSX1, -2, -3, 4 TCR, anti-TEL / AML1 TCR, anti-TPI / m TCR, anti-TRP-1 TCR, anti-TRP-2 TCR, anti-TRP-2 / INT2 Examples include TCRs, anti-WT1 TCRs, or combinations thereof.

[0217] In some embodiments, the TCR can bind to a first antigenic moiety and a second antigenic moiety. In some embodiments, the first TCR binds to the first antigenic moiety and the second TCR binds to the second antigenic moiety.

[0218] This disclosure may include the use of a dual-target antigen-binding system. A dual-target antigen-binding system may include a bispecific CAR or TCR and / or a bisistronic CAR or TCR. A bispecific and bisistronic CAR may include two binding motifs (in a single CAR molecule or two CAR molecules, respectively). In some embodiments, the vector encodes a bisistronic and / or bispecific CAR (e.g., a bisistronic and / or bispecific CAR that binds to CD20 and CD19). Exemplary bispecific and bisistronic CARs are described in International Publication No. 2020 / 123691, which is incorporated herein by reference.

[0219] In some embodiments, the CAR includes a first scFv that binds to CD19 and a second scFv that binds to CD20. In some embodiments, the first CAR includes a first scFv that binds to CD19, and the second CAR includes a second scFv that binds to CD20. Examples of CD19-binding sequences or CD20-binding sequences are shown in Table 1. TIFF2026510004000001.tif242170TIFF2026510004000002.tif201170TIFF2026510004000003.tif164170

[0220] The CARs of this disclosure may include a hinge, a transmembrane domain, and / or an intracellular domain, in addition to the antigen-binding molecule. In some embodiments, the intracellular domain may include a co-stimulatory domain and an activating domain.

[0221] A hinge can be an extracellular domain of an antigen-binding system located between the binding motif and the transmembrane domain. The hinge may also be referred to as an extracellular domain or "spacer." The hinge may contribute to receptor expression, activity, and / or stability. The hinge may also provide flexibility for accessing the target antigen. In some embodiments, the hinge domain is located between the binding motif and the transmembrane domain.

[0222] In some embodiments, the hinge is an immunoglobulin-like hinge domain, derived from an immunoglobulin-like hinge domain, or originates from an immunoglobulin-like hinge domain (e.g., including all or fragments of an immunoglobulin-like hinge domain). In some embodiments, the hinge domain is derived from or from an immunoglobulin. In some embodiments, the hinge domain is selected from hinges or fragments thereof of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, or IgM.

[0223] In some embodiments, the hinges are CD2, CD3 Delta, CD3 Epsilon, CD3 Gamma, CD4, CD7, CD8 Alpha, CD8 Beta, CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (TNFRSF7), CD28, CD28T, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49 f(ITGA6), CD66a(CEACAM1), CD66b(CEACAM8), CD66c(CEACAM6), CD66d(CEACAM3), CD66e(CEACAM5), CD69(CLEC2), CD79A(B cell antigen receptor complex-associated alpha chain), CD79B(B cell antigen receptor complex-associated beta chain), CD84(SLAMF5), CD96(Tactile), CD100(SEMA4D), CD103(ITGAE), CD134(OX40), CD137(4-1BB), CD150(SLAMF1), CD158A(KI R2DL1), CD158B1(KIR2DL2), CD158B2(KIR2DL3), CD158C(KIR3DP1), CD158D(KIRDL4), CD158F1(KIR2DL5A), CD158F2(KIR2DL5B), CD158K(KIR3DL2), CD160(BY55), CD162(SELPLG), CD226(DNAM1), CD229(SLAMF3), CD244(SLAMF4), CD247(CD3-zeta), CD258(LIGHT), CD268(BAFFR), CD270(TNFSF14), C D272(BTLA), CD276(B7-H3), CD279(PD-1), CD314(NKG2D), CD319(SLAMF7), CD335(NK-p46), CD336(NK-p44), CD337(NK-p30), CD352(SLAMF6), CD353 (SLAMF8), CD355 (CRTAM), CD357 (TNFRSF18), inducible T cell costimulatory factor (ICOS), LFA-1 (CD11a / CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL-2R beta, IL-2R gamma,IL-7R alpha, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fc gamma receptor, MHC class 1 molecule, MHC class 2 molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, activated NK cell receptor, or Toll ligand receptor, or fragments or combinations thereof, derived from or from them (e.g., including all or fragments thereof).

[0224] In some embodiments, the hinge is the hinge of CD8 alpha, is from the hinge of CD8 alpha, or originates from the hinge of CD8 alpha (e.g., including all or a fragment of the hinge of CD8 alpha). In some embodiments, the hinge is the hinge of CD28, is from the hinge of CD28, or originates from the hinge of CD28. In some embodiments, the hinge is a fragment of the hinge of CD8 alpha or a fragment of the hinge of CD28, or is from a fragment of the hinge of CD8 alpha or a fragment of the hinge of CD28, the fragment being smaller than the whole. In some embodiments, the CD8 alpha hinge fragment or the CD28 hinge fragment includes an amino acid sequence that excludes at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acids at the N-terminus or C-terminus of the CD8 alpha hinge or the CD28 hinge, or both.

[0225] A "transmembrane domain" refers to a domain that, when present on a cell surface or cell membrane molecule, has the attribute of being intramembrane (e.g., spanning part or all of the cell membrane). Not all amino acids in a transmembrane domain need to be intramembrane. For example, in some embodiments, a transmembrane domain is characterized by a designated sequence or portion of a protein being substantially intramembrane. Amino acid sequences or nucleic acid sequences can be analyzed using various algorithms to predict the intracellular localization (e.g., transmembrane localization) of a protein. The programs psort (PSORT.org) and Prosite (prosite.expasy.org) are examples of such programs.

[0226] The transmembrane domains include the alpha, beta, or zeta chains of T cell receptors, CD28, CD3 epsilon, CD3 delta, CD3 gamma, CD45, CD4, CD5, CD7, CD8, CD8 alpha, CD8 beta, CD9, CD11a, CD11b, CD11c, CD11d, CD16, CD22, CD27, CD33, CD37, CD64, CD80, CD86, CD134, CD137, TNFSFR25, CD154, and 4-1BB / CD1 37, activated NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD276(B7-H3), CD29, CD30, CD40, CD49a, CD49D, CD49f, CD69, CD84, CD96(Tactile), CD5, CEACAM1, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, CD83 binding Gand, LIGHT, LIGHT, LTBR, ​​Ly9 (CD229), Lymphocyte function-associated antigen-1 (LFA-1; CD1-1a / CD18), MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signal transduction lymphocyte activating molecule (SLAM protein), SLAM (SLAMF1, CD150, IPO-3), SLAMF4 (CD244, 2B4), SLAMF6 (NTB-A;Ly108) may be derived from any membrane-bound or transmembrane protein, such as SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or fragments, cleavages, or combinations thereof.

[0227] The intracellular domain (or cytoplasmic domain) includes one or more signaling domains that, upon binding of a target antigen to a binding motif, trigger and / or mediate an intracellular signal that activates, for example, one or more immune cell effector functions (e.g., innate immune cell effector functions). In some embodiments, the signaling domain of the intracellular domain mediates the activation of at least one of the normal effector functions of an immune cell. The effector function of a T cell may be, for example, cytolytic activity or helper activity including cytokine secretion. In some embodiments, the signaling domain of the intracellular domain mediates T cell activation, proliferation, survival, and / or other T cell functions. The intracellular domain may include a signaling domain that is an activating domain. The intracellular domain may include a signaling domain that is a co-stimulatory signaling domain.

[0228] Intracellular signaling domains are known to transmit signals during antigen binding to immune cells. For example, the cytoplasmic sequence of the T cell receptor (TCR) is known to initiate signal transduction after TCR binding to an antigen (e.g., Brownlie et al., Nature Rev. Immunol. 13:257-269 (2013)).

[0229] In certain embodiments, preferred signaling domains include 4-1BB / CD137, activated NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME(SLAMF8), BTLA, CD100(SEMA4D), CD103, CD160(BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276(B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8 alpha, CD8 beta, CD96(Tactile), CD11a, CD11b, CD11c, CD11d, CD5, CEACAM1, and CRT. AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, ligands that bind to CD83, LIGHT, LIGHT, LTBR, ​​Ly9 (CD229), Ly108), lymphocyte function-associated antigen-1 (LFA-1, CD1- This includes, but is not limited to, 1a / CD18), MHC class 1 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activating molecules (SLAM proteins), SLAM (SLAMF1, CD150, IPO-3), SLAMF4 (CD244, 2B4), SLAMF6 (NTB-A, SLAMF7, SLP-76), TNF receptor proteins, TNFR2, TNFSF14, Toll ligand receptors, TRANCE / RANKL, VLA1, or VLA-6, or fragments, cleavages, or combinations thereof.

[0230] CARs may also include co-stimulatory signaling domains, for example, to enhance signaling efficacy. See U.S. Patents 7,741,465 and 6,319,494, as well as Krause et al. and Finney et al. (above), Song et al., Blood 119:696-706 (2012), Kalos et al., Sci Transl. Med. 3:95 (2011), Porter et al., N.Engl. J. Med. 365:725-33 (2011), and Gross et al., Annu. Rev. Pharmacol. Toxicol. 56:59-83 (2016). Signals generated solely through TCRs may be insufficient for complete T cell activation, and secondary or co-stimulatory signals may increase activation. Therefore, in some embodiments, the signaling domain further comprises one or more additional signaling domains (e.g., co-stimulatory signaling domains) that activate one or more immune cell effector functions (e.g., innate immune cell effector functions described herein). In some embodiments, a portion of such co-stimulatory signaling domains may be used insofar as the portion transmits effector function signals. In some embodiments, the cytoplasmic domain described herein comprises one or more cytoplasmic sequences of T cell co-receptors (or fragments thereof). Non-limiting examples of such T cell co-receptors include ligands that bind to CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), MYD88, CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83. Exemplary co-stimulatory proteins have the amino acid sequence of a naturally occurring co-stimulatory protein on T cells, the complete natural amino acid sequence of that co-stimulatory protein is described in NCBI reference sequence: NP 0.1. In certain cases, the CAR contains a 4-1BB costimulatory domain. In certain cases, the CAR contains a CD28 costimulatory domain. In certain cases, the CAR contains a DAP-10 costimulatory domain.

[0231] In some embodiments, the co-stimulatory signaling domain is the signaling domain of CD28. As shown in the experimental examples, CAR molecules having the CD28 co-stimulatory signaling domain can particularly benefit from the newly developed rapid manufacturing process.

[0232] In some embodiments, the CAR further comprises an ITAM. Examples of ITAM-containing primary cytoplasmic signaling sequences that are particularly useful in the present disclosure include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In some embodiments, the ITAM comprises CD3 zeta.

[0233] In some embodiments, the CAR molecule is any anti-CD19 CAR molecule. In one aspect, the anti-CD19 CAR comprises an extracellular scFv domain, an intracellular and / or transmembrane portion of the CD28 molecule, an optional extracellular portion of the CD28 molecule, and an intracellular CD3 zeta domain as described in International Publication No. WO 2015 / 120096 or International Publication No. WO 2016 / 191755, each of which is incorporated herein by reference in its entirety.

[0234] In certain embodiments, the anti-CD19 CAR may also comprise additional domains such as the CD8 extracellular and / or transmembrane region, an extracellular immunoglobulin Fc domain (e.g., IgG1, IgG2, IgG3, IgG4), or one or more additional signaling domains such as 4-1BB, OX40, CD2, CD16, CD27, CD30, CD40, PD-1, ICOS, LFA-1, IL-2 receptor, Fc gamma receptor, or any other co-stimulatory domain having an immunoreceptor tyrosine-based activation motif.

[0235] In certain embodiments, the cell surface receptor is an anti-CD19 CAR, such as the FMC63-28Z CAR or the FMC63-CD828BBZ CAR described in Kochenderfer et al., J Immunother. 2009 September;32(7):689-702, "Construction and Preclinical Evaluation of an Anti-CD19 Chimeric Antigen Receptor," which is incorporated herein by reference for the purpose of providing methods for constructing vectors used to produce T cells expressing the FMC63-28Z CAR or the FMC63-CD828BBZ CAR.

[0236] In some embodiments, the T cells comprising the CAR molecule are Yescarta® (axicabtagene ciloleucel). In some embodiments, the T cells comprising the CAR molecule are Tecartus® (brexucabtagene autoleucel). In some embodiments, the T cells comprise one or more CAR molecules capable of binding to one or more antigenic moieties.

[0237] In some embodiments, pharmaceutical compositions are provided that comprise a population of engineered lymphocytes produced by the methods described herein. In certain embodiments, the pharmaceutical composition may also comprise a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier can be a pharmaceutically acceptable material, composition, or vehicle involved in the transport or delivery of the desired cells from one tissue, organ, or part of the body to another. For example, the carrier can be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or some combination thereof. Each component of the carrier needs to be "pharmaceutically acceptable" in that it must be compatible with the other components of the formulation. It also needs to be suitable for contact with any tissue, organ, or part of the body that it may encounter, meaning that it should not have a risk of toxicity, irritation, allergic reaction, immunogenicity, or any other complication that unduly outweighs its therapeutic benefit.

[0238] Treatment and use, and optional storage Lymphocytes prepared by this method, or lymphocyte populations disclosed herein, may be used to treat a variety of diseases and conditions.

[0239] In some embodiments, if lymphocytes are not to be used immediately, they may be cryopreserved for later use. Such a method may include the step of washing and concentrating a population of manipulated lymphocytes with a diluent. In some embodiments, the diluent is ordinary saline, 0.9% saline, PlasmaLyte A (PlasmaLyte, PL), 5% dextrose / 0.45% NaCl saline solution (D5), human serum albumin (HSA), or a combination thereof. In some embodiments, HSA may be added to the washed and concentrated cells to improve cell viability and cell recovery after thawing. In another embodiment, the washing solution is saline, and HSA (5%) is added to the washed and concentrated cells. This method may also include the step of producing a cryopreservation mixture, in which the cryopreservation mixture comprises a population of diluted cells in a diluent and a suitable cryopreservation solution. In some embodiments, the cryopreservation solution may include, but is not limited to, any suitable cryopreservation solution, CryoStor10 (BioLife Solution), and is mixed with the diluent of manipulated lymphocytes in a 1:1 or 2:1 ratio.

[0240] In certain embodiments, HSA may be added to provide a final concentration of about 1.0–10% HSA in the mixture to be cryopreserved. In certain embodiments, HSA may be added to provide a final concentration of about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10.0% HSA in the mixture to be cryopreserved. In certain embodiments, HSA may be added to provide a final concentration of about 1–3% HSA, about 1–4% HSA, about 1–5% HSA, about 1–7% HSA, about 2–4% HSA, about 2–5% HSA, about 2–6% HSA, or about 2–7% HSA in the cryopreserved mixture. In certain embodiments, HSA may be added to provide a final concentration of about 2.5% HSA in the cryopreserved mixture. For example, in a particular embodiment, cryopreservation of an engineered T cell population may include washing the cells with 0.9% saline, adding HSA to the washed cells at a final concentration of 5%, and diluting the cells 1:1 with CryoStor® CS10 (resulting in a final concentration of 2.5% HSA in the final cryopreservation mixture). In some embodiments, the method also includes the step of freezing the cryopreservation mixture. In one embodiment, the cryopreservation mixture is frozen in a controlled-rate freezer using a defined freezing cycle at a cell concentration of about 1e6 to about 1.5e7 cells per mL of the cryopreservation mixture. The method may also include the step of storing the cryopreservation mixture in gas-phase liquid nitrogen.

[0241] Methods and uses for treating diseases or pathological conditions in subjects having a disease or pathological condition are also provided. In some embodiments, the method involves administering a therapeutically effective amount or therapeutically effective dose of engineered lymphocytes to a subject. Pathogenic conditions that can be treated with engineered T cells produced by the methods described herein include, but are not limited to, cancer, viral infections, acute or chronic inflammation, autoimmune diseases, or any other immunodeficiency.

[0242] Where used herein, “cancer” means acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adenoid cystic carcinoma, adrenocortical carcinoma, AIDS-related cancer, anal cancer, appendiceal cancer, astrocytoma, atypical teratomatoid / rhabdomyosarcoma-like tumor, central nervous system cancer, B-cell leukemia, lymphoma or other B-cell malignancies, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, osteosarcoma and malignant fibrous histiocytoma, brainstem glioma, brain tumor, breast cancer, bronchial tumor, Burkitt lymphoma, carcinoid tumor, central nervous system cancer, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CLL) Leukemia (CML), chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, germ blastoma, central nervous system cancer, endometrial cancer, ependymoblastoma, esophageal cancer, sensory neuroblastoma, Ewing's sarcoma family tumors, extracranial germ cell tumors, extragonadal germ cell tumors, extrahepatic cholangiocarcinoma, ocular cancer, malignant fibrous histiocytoma of bone, and osteosarcoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors. tumor (GIST), soft tissue sarcoma, germ cell tumor, gestational trophoblastoma, glioma, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular carcinoma (liver), histiocytic hyperplasia, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor (pancreatic islet), Kaposi's sarcoma, renal cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, oral cavity cancer, liver cancer (primary), lobular carcinoma in situ (LCIS), lung cancer, lymphoma, macroglobulinemia, male breast cancer, malignant fibrous histiocytoma and osteosarcoma of bone, medulloblastoma, medullary epithelioma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell carcinoma with occult primary midline cancer associated with the NUT gene, oral cancer (mouth cancer), multiple endocrine neoplasia syndrome, multiple myeloma / plasmacytic neoplasm, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, myeloid leukemia, chronic (myelogenous leukemia,Chronic CML, myeloid leukemia, acute AML, myeloma, multiple myeloproliferative disorders, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer, pancreatic cancer, papilloma, paraganglioma, paranasal and nasal sinus cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, intermediate pineal parenchymal tumor, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, plasma cell tumor / multiple myeloma, pleuroblastoma, pregnancy and breast cancer, primary central nervous system cancer This includes, but is not limited to, any cancer associated with surface antigens or cancer markers, including (CNS) lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureter, transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sézary syndrome, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, cervical squamous cell carcinoma, gastric cancer, supratentorial primitive neuroectodermal tumor, T-cell lymphoma, skin cancer, testicular cancer, pharyngeal cancer, thymoma and thymic cancer, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, trophoblastic neoplasm, ureter and renal pelvis cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström type macroglobulinemia, and Wilms' tumor.

[0243] In some aspects, cancer is a B-cell malignancy. Examples of B-cell malignancies include, but are not limited to, non-Hodgkin's lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), small lymphocytic lymphoma (SLL / CLL), mantle cell lymphoma (MCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), extranodal (MALT lymphoma), nodal (monocytic B-cell lymphoma), splenic, diffuse large B-cell lymphoma, chronic B-cell lymphocytic leukemia / lymphoma, Burkitt lymphoma, and lymphoblastic lymphoma.

[0244] Where used herein, “viral infection” can mean an infection caused by any virus that causes a disease or pathological condition in a host. Examples of viral infections that can be treated with engineered T cells produced by the methods described herein include, but are not limited to, viral infections caused by Epstein-Barr virus (EBV); viral infections caused by hepatitis A virus, hepatitis B virus, or hepatitis C virus; viral infections caused by herpes simplex virus type 1, herpes simplex virus type 2, or herpes simplex virus type 8; viral infections caused by cytomegalovirus (CMV); viral infections caused by human immunodeficiency virus (HIV); viral infections caused by influenza virus; viral infections caused by measles or mumps virus; viral infections caused by human papillomavirus (HPV); viral infections caused by parainfluenza virus; viral infections caused by rubella virus; viral infections caused by respiratory syncytial virus (RSV); or viral infections caused by varicella-zoster virus. In some aspects, viral infections can lead to or cause cancer in individuals with viral infections (for example, HPV infection may cause or be associated with the development of several cancers, including cervical cancer, vulvar cancer, vaginal cancer, penile cancer, anal cancer, and oropharyngeal cancer; and HIV infection may cause the development of Kaposi's sarcoma).

[0245] Further examples of chronic inflammatory diseases, autoimmune diseases, or any other immunodeficiencies that can be treated with engineered T cells produced by the methods described herein include, but are not limited to, multiple sclerosis, lupus, and psoriasis.

[0246] As used herein with respect to a condition or disease, the terms “to treat,” “to treat,” or “to cure” may mean preventing a condition or disease, slowing the onset or progression of a condition or disease, reducing the risk of developing a condition or disease, preventing or delaying the onset of symptoms associated with a condition or disease, reducing or terminating symptoms associated with a condition or disease, causing complete or partial regression of a condition or disease, or any combination thereof.

[0247] The "therapeutic dose" is the amount of engineered lymphocytes that produce the desired therapeutic effect in a subject, such as preventing or treating a target condition or alleviating symptoms associated with the condition by killing target cells. The most effective outcome in terms of therapeutic efficacy in a given subject will vary depending on a variety of factors, including but not limited to the properties of the engineered lymphocytes (including lifespan, activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological state of the subject (including age, sex, type and stage of disease, general physical condition, responsiveness to a given dose, and type of drug), the properties of any pharmaceutically acceptable carrier(s) in any composition used, and the route of administration. The therapeutic dose of engineered lymphocytes may also depend on the cell surface receptors expressed by the lymphocytes (e.g., affinity and density of cell surface receptors expressed on the cells), the type of target cell, the nature of the disease or condition being treated, or a combination of both.

[0248] As shown in the examples, the manipulated lymphocytes prepared by this process have significantly increased in vivo efficacy compared to conventional techniques, and therefore require a much lower dose.

[0249] Therefore, in some embodiments, the therapeutically effective dose of manipulated lymphocytes is less than approximately 2,000,000 manipulated lymphocytes (cells / kg) per kilogram of body weight of the subject requiring treatment. Therefore, in some embodiments, the therapeutically effective dose of manipulated lymphocytes is approximately 10,000 to approximately 1,500,000 manipulated lymphocytes / kg. In certain embodiments, the therapeutically effective dose is approximately 20,000 to approximately 1,200,000 million manipulated lymphocytes / kg. In certain embodiments, the therapeutically effective dose is approximately 20,000 to approximately 1,000,000 million manipulated lymphocytes / kg. In certain embodiments, the therapeutically effective dose is approximately 20,000 to approximately 500,000 million manipulated lymphocytes / kg. In certain embodiments, the therapeutically effective dose is approximately 20,000 to approximately 400,000 million manipulated lymphocytes / kg. In certain embodiments, the therapeutically effective dose is approximately 40,000 to 400,000 million manipulated lymphocytes / kg. In certain embodiments, the therapeutically effective dose is approximately 50,000 to 200,000 million manipulated lymphocytes / kg. In certain embodiments, the therapeutically effective dose is approximately 50,000 to 100,000 million manipulated lymphocytes / kg.

[0250] In some embodiments, the administered T cells are Yescarta® (axicabutagen silol-ucells). In some embodiments, the administered T cells are Tecartus® (brexcabutagen autol-ucells).

[0251] One embodiment of the present disclosure is a method for predicting the likelihood of a complete response in a patient to immunotherapy, comprising determining the period from the leukocyte apheresis process in the patient to the administration of the immunotherapy to the patient, and, based on the determination of the period, dividing the patient into multiple groups, the period from the leukocyte apheresis process to the administration of the immunotherapy to the patient is up to 28 days (e.g., 2) A first group characterized by periods of 7, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 days, and a second group characterized by 28 to 40 days (for example, 28, 29, 30, 31, 32, 33, 34 days) from the leukocyte apheresis process to the administration of the immunotherapy to the patient. The method includes classifying a patient into one of several groups, including a second group characterized by a period of at least 40 days (e.g., 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 days) from the leukocyte apheresis process to the administration of the immunotherapy to the patient, and determining the likelihood of a complete response in the patient based at least partially on which of the several groups the patient is classified into, wherein if the patient is classified into the first or second group, the patient has at least about a 55% chance of a complete response, and if the patient is classified into the third group, the patient has at least about a 42% chance of a complete response.

[0252] As used herein, the term "up to 28 days" may mean less than 28 days (<28 days) (for example, 27 days, 26 days, 20 days, 10 days, or 5 days).

[0253] As used herein, the term 28 to 40 days may mean 28 days or more (≧28 days) to less than 40 days (<40 days).

[0254] As used herein, the term "at least 40 days" can mean 40 days or more (≥ 40 days).

[0255] In some embodiments of the present disclosure, if the patient is classified within the first group or the second group, the patient has a probability of about 60% complete response.

[0256] One embodiment of the present disclosure is a method for predicting the overall survival rate in a patient for immunotherapy, comprising determining the period from the leukapheresis step of the patient to the administration of the immunotherapy to the patient, and based on the determination of the period, classifying the patient into one of a plurality of groups, the plurality of groups including a first group characterized by a period of up to 28 days from the leukapheresis step to the administration of the immunotherapy to the patient, a second group characterized by a period of 28 days to less than 40 days from the leukapheresis step to the administration of the immunotherapy to the patient, and a third group characterized by a period of at least 40 days from the leukapheresis step to the administration of the immunotherapy to the patient, and determining the overall survival rate in the patient based at least in part on into which of the plurality of groups the patient is classified, wherein if the patient is classified within the first group, the patient has an overall survival rate of at least about 49%, if the patient is classified within the second group, the patient has an overall survival rate of at least about 48%, and if the patient is classified within the third group, the patient has an overall survival rate of at least about 30%.

[0257] One embodiment of the present disclosure is a method for predicting the risk of thrombocytopenia in a patient receiving immunotherapy, comprising determining the period from the leukocyte apheresis process to the administration of the immunotherapy to the patient, and, based on the determination of the period, classifying the patient into several groups, the first group characterized by a period of up to 28 days from the leukocyte apheresis process to the administration of the immunotherapy to the patient, the second group characterized by a period of 28 to 40 days from the leukocyte apheresis process to the administration of the immunotherapy to the patient, and from the leukocyte apheresis process to the patient The method comprises classifying a patient into one of several groups, including a third group characterized by a period of at least 40 days prior to the administration of the immunotherapy, and determining the risk of thrombocytopenia in the patient, at least in part, based on which of the several groups the patient is classified into, wherein if the patient is classified into the first group, the patient has a risk of approximately 18% of thrombocytopenia; if the patient is classified into the second group, the patient has a risk of approximately 25% of thrombocytopenia; and if the patient is classified into the third group, the patient has a risk of approximately 34% of thrombocytopenia.

[0258] One embodiment of the present disclosure is a method for predicting life expectancy and quality-adjusted life years in a patient who has received immunotherapy, the method comprising: determining a period of time from the leukocyte apheresis process in the patient to the administration of the immunotherapy to the patient, which is either short-term or long-term; assigning a probability of successful infusion based on that period; and inputting patient information into a survival model to determine the patient's life expectancy and quality-adjusted life years.

[0259] In some embodiments, the models described herein may be decision tree models having outcomes related to long V2VT or short V2VT.

[0260] In some embodiments, the model input may include long V2VT, short V2VT, probability of infusion, lifetime outcome (non-infusion patients), lifetime outcome (infusion patients), effectiveness of long V2VT versus short V2VT for infusion patients, and / or QALYs.

[0261] In some embodiments, the model output may include life years (LYs) and / or quality-adjusted life years (QALYs).

[0262] In some embodiments, the models described herein may comprise decision gates / nodes and stochastic nodes. Decision gates may represent points where decisions are made (e.g., long V2VT or short V2VT). Stochastic nodes may represent the probability of a particular outcome (e.g., injection or non-injection). Graph survival predictions may be used to represent lifetime outcomes modeled based on data inputs.

[0263] As used herein, the term “life expectancy” refers to the number of years a subject or patient is expected to survive. In some embodiments, the increase in life expectancy may be measured from the time the patient is informed of receiving immunotherapy. In some embodiments, the increase in life expectancy may be measured from the time the patient is infused with immunotherapy.

[0264] As used herein, the term “Quality-Adjusted Life Years” or “QALY” refers to a measure of disease burden or health outcome, encompassing both quality and quantity of life. In some embodiments, the increase in QALYs may be measured from the time the patient is informed of receiving immunotherapy. In some embodiments, the increase in QALYs may be measured from the time the patient is infused with immunotherapy.

[0265] As used herein, the term “short duration” may mean a period of 24 days or less between the patient’s leukocyte apheresis procedure and the administration of immunotherapy to the patient (e.g., vein-to-vein time).

[0266] As used herein, the term “long duration” may mean a period of 37 days or more, 54 days or more, or 37 to 54 days from the patient’s leukocyte apheresis process to the administration of immunotherapy to the patient (e.g., venous-to-venous time).

[0267] In some embodiments, a short term may refer to an increase in the patient's mean life expectancy and / or quality-adjusted life years of more than about 5 years (e.g., about 5.5 years, 6 years, 6.5 years, 7 years, 7.5 years, 8 years, 8.5 years, 9 years, 9.5 years, or 10 years or more).

[0268] In some embodiments, a long term may refer to an increase in the patient's mean life expectancy and / or an increase in quality-adjusted life years of less than approximately 5 years (e.g., approximately 4.5 years, 4 years, 3.5 years, 3 years, 2.5 years, 2 years, 1.5 years, or 1 year or less).

[0269] In one embodiment of the present disclosure, the immunotherapy comprises one or more CARs that recognize one or more tumor antigens.

[0270] In one embodiment of this disclosure, the immunotherapy is axicaptagen silolucel or brexcaptagen autolucel.

[0271] In one embodiment of the present disclosure, the patient has relapsed or refractory (r / r) large B-cell lymphoma (LBCL).

[0272] The following examples are intended to illustrate various embodiments of the present invention. Therefore, any particular embodiment considered should not be construed as a limitation on the scope of the invention. For example, the following examples involve T cells transduced with an anti-CD19 chimeric antigen receptor (CAR), but those skilled in the art will understand that the methods described herein may be applied to T cells transduced with any CAR or TCR. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of the invention, and such equivalent embodiments are included herein. Furthermore, all references cited herein are incorporated herein in their entirety by reference as they are fully described herein.

[0273] Example 1 Compared to other CAR T cell products, axi-cel had a shorter median latency time from leukocyte apheresis to infusion (called vein-to-vein time) (real-world: axi-cel, 28 days vs. tisagenlecleucel, 45 days; clinical trial: lysokabutagenmaralucel, 36-37 days; Riedell et al. Transplant Cell Ther 2022, Abramson et al. Lancet 2020). A study based on the JULIET trial suggested that reduced latency was associated with increased efficacy (Chenet al. Value Health 2022). This example evaluated the real-world impact of vein-to-vein time on outcomes for axi-cel in r / r LBCL.

[0274] method A non-interventional post-approval safety study using the Center for International Blood and Marrow Transplant Research (CIBMTR) registry identified a total of 1,383 patients from 78 US centers treated with commercially available axi-cel for r / r LBCL. Patients with the following conditions were excluded: primary central nervous system lymphoma or lymphoma other than LBCL, prior non-transplant cell therapy, missing data on comorbidities (Sorror et al. Blood 2005), unknown or out-of-range leukocytosis dates (≤2 days prior to lymphocyte depletion [LD] chemotherapy or ≥144 days prior to infusion), or no follow-up.

[0275] Efficacy outcomes included overall response rate and complete response rate (ORR and CR), duration of response (DOR), and progression-free survival and overall survival (PFS and OS). Adverse events included cytokine release syndrome (CRS) (Lee 2014 criteria), immune effector cell-associated neurotoxicity syndrome (ICANS) (ASTCT criteria), persistent neutropenia, and thrombocytopenia. Odds ratios (OR) and hazard ratios (HR) were estimated using logistic and Cox regression after adjusting for important prognostic factors such as age, comorbidities, ECOG performance status, disease characteristics at diagnosis, and bridging therapy. Adjusted curves were created based on the directly adjusted survival function (Makuch J Chronic Dis 1982).

[0276] result Overall, the median venous-to-venous time (from leukocyte apheresis to infusion) for axi-cel was 27 days (interquartile range [IQR], 26–32 days), which included a median of 5 days (IQR, 5–5 days) from the start of LD chemotherapy to infusion. Venous-to-venous time was consistent regardless of the following baseline characteristics: disease histology, sex, race, ethnicity, pre-infusion ECOG performance status, or chemotherapy sensitivity (Table 2).

[0277] Patients with shorter vein-to-vein transition times appeared to be younger and less likely to have comorbidities (Table 3). Patients with vein-to-vein transition times of ≥40 days were more heavily pre-treated and more likely to receive bridging therapy.

[0278] At a median follow-up of 24.2 months, better outcomes were observed in patients with shorter venous-to-venous times. CR rates were 60%, 61%, and 50% (ORR 77%, 77%, and 70%) for patients with venous-to-venous times <28 days, ≥28 days to <40 days, and ≥40 days, respectively. OS at 24 months was 53% for patients with both venous-to-venous times <28 days and ≥28 days to <40 days, compared to 38% for patients with ≥40 day waiting times. After adjusting for other important prognostic factors, patients with a venous-to-venous time of ≥40 days had significantly lower CR rates and OS compared to patients with a waiting time of <28 days (OR 0.61 [95% CI 0.42-0.90] for CR, HR 1.33 [95% CI 1.05-1.70] for OS) and patients with a waiting time of ≥28 to <40 days (OR 0.66 [95% CI 0.45-0.97] for CR, HR 1.36 [95% CI 1.06-1.74] for OS).

[0279] Adjusted progression-free survival (PFS), overall survival (OS), and duration of response (DOR) were analyzed based on stratified Cox models (Sorror, ML, et al. Blood. 2005; 106(8): 2912-2919, Chang IM, et al. J Chronic Dis. 1982; 35: 669-674) to balance differences in baseline characteristics. To evaluate the validity of the venous-to-venous time classification used in the primary analysis, a sensitivity analysis was also performed comparing outcomes between patients with venous-to-venous time < 36 days and those with ≥ 36 days.

[0280] Among patients who achieved the best response (CR / partial response, PR), the 12-month duration of response (DOR) was 61% for patients with a venous-to-venous time <28 days, 60% for patients with a venous-to-venous time ≥28 days to <40 days, and 61% for patients with a venous-to-venous time ≥40 days. The sensitivity analysis of DOR was consistent with the primary analysis.

[0281] Adjusted PFS and OS at 24 months appeared to be lower in patients with ≥40 days of venous-to-venous time compared to patients with <28 days or ≥28-<40 days of venous-to-venous time. Sensitivity analyses for OS and PFS were consistent with the primary analysis, with OS being significantly shorter in patients with ≥36 days of venous-to-venous time compared to patients with <36 days of venous-to-venous time (hazard ratio [HR], 1.25 [95% CI, 1.02–1.53]).

[0282] CRS of any grade or grade ≥3 and persistent neutropenia were consistent regardless of venous-to-venous time. Patients with venous-to-venous time <28 days had more ICANs of any grade compared to patients with waiting times ≥28 to <40 days (OR 1.34 [95% CI 1.06 to 1.71]), but there was no significant difference between the two groups in ICANs of grade ≥3 (Table 2). Among patients surviving at day 30, patients with venous-to-venous time ≥28 to <40 days and ≥40 days had a higher rate of persistent thrombocytopenia compared to patients with waiting times <28 days (OR 1.44 [95% CI 1.07 to 1.92] and 1.95 [95% CI 1.29 to 2.95], respectively).

[0283] The CRS grade was based on the criteria from Lee, DW, et al. Blood. 2014;124(2):188-195. The ICANS grade was based on the criteria from Lee, DW, et al. Biol Blood Marrow Transplant. 2019;25(4):625-638.

[0284] Most CRS and ICANs resolved by day 21 from symptom onset, regardless of the time from venous transfer to venous access. The cumulative incidence of CRS resolution by day 21 from symptom onset was 92%, 92%, and 94% for patients with venous transfer times of <28 days, ≥28 to <40 days, and ≥40 days, respectively. The cumulative incidence of ICAN resolution by day 21 from symptom onset was 79%, 76%, and 64% for patients with venous transfer times of <28 days, ≥28 to <40 days, and ≥40 days, respectively.

[0285] Sensitivity analysis of safety results was consistent with the primary analysis.

[0286] Table 4 shows the multivariate results of a sensitivity analysis comparing patients with a vein-to-vein time of ≥36 days with patients with a vein-to-vein time of <36 days.

[0287] In this real-world analysis, most patients with r / r LBCL received axi-cel infusion within 5 weeks post-apheresis. Shorter venous-to-venous times were associated with favorable CR rates, OS, and a reduced risk of persistent thrombocytopenia, even after adjusting for key prognostic factors, although higher ICANS of any grade may be possible. Overall, these findings highlight the importance of reducing venous-to-venous times in patients treated with axi-cel. TIFF2026510004000004.tif162161 a The percentages are based on cases with no missing data. b Does not include previous transplants. c Defined based on the hematopoietic stem cell transplant-specific comorbidity index (Sorror, ML, et al. Blood. 2005;106(8):2912-2919). ECOG PS, Performance Status of the Eastern Cooperative Oncology Group (US East Coast Cancer Clinical Trials Group). TIFF2026510004000005.tif218170TIFF2026510004000006.tif104170TIFF2026510004000007.tif66170

[0288] Example 2 Chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment of hematological malignancies. However, its production requires a complex multi-step process from leukocyte apheresis to manufacturing, transport, and storage before final infusion. This time is known as vein-to-vein time (V2VT), and the potential importance of V2VT to patient outcomes is highlighted because the patient's condition may deteriorate during this time. This modeling study was designed to compare the potential outcomes of "long" V2VT versus "short" V2VT in patients with relapsed / refractory (r / r LBCL) large B-cell lymphoma treated with CAR T-cell therapy in a 3L+ setting.

[0289] The objective of this trial was to compare lifetime outcomes in a hypothetical cohort of patients receiving CAR T therapy for r / r LBCL in a 3L+ setting, but with different V2VTs.

[0290] method The hypothetical cohort of patients enters a decision tree model at the time of leukocyte apheresis, and is then assigned a probability of infusion success based on V2VT.

[0291] Next, patients enter a segmented survival model to estimate lifetime survival years (LYs) and quality-adjusted survival years (QALYs) based on real-world axi-cel OS data. Other CAR T efficacy data provided conservative estimates and were not included to avoid confusion. This model was informed by published literature, including studies that investigated the number of patients who ultimately received infusions relative to the time elapsed since leukocyte apheresis (based on the ZUMA-1, JULIET, and TRANSCEND-NHL-001 studies), studies that investigated the relationship between V2VT and survival (Locke et al.

[2022] ), and studies that investigated the survival difference between infused and uninfused patients (Kuhl et al.,

[2022] and Bachy et al.,

[2022] ) (Table 5).

[0292] Using an epidemiological model, the results were extrapolated to US patients eligible for CAR T. Based on V2VT assignment, the probability of infusion success was determined and applied to a decision tree. Survival after V2VT was estimated separately for those who received successful infusions and those who did not. Finally, scenario analyses were performed to assess the robustness of the results to key assumptions.

[0293] Based on the best available V2VT evidence reported, we investigated three hypothetical V2VT cases: 54 days (tisa-cel V2VT median; JULIET), 37 days (liso-cel V2VT median; TRANSCEND-NHL-001), and 24 days (axi-cel V2VT median; ZUMA-1). TIFF2026510004000008.tif128170

[0294] To isolate the effect of V2VT on survival, efficacy results were assumed to be equivalent across various CAR T therapies, and due to the limited availability of relevant public data to inform model inputs, efficacy data for axi-cel were applied to the model. Various sensitivity analyses were performed to test the robustness of the results.

[0295] Finally, we used an epidemiological model to scale patient-specific outcomes and estimate population outcomes assuming all CAR T-eligible patients in the U.S. had reduced V2VT. Epidemiological assessments were taken from the NICE resource impact report (National Institute for Health and Care Excellence. Resource impact report: Axicaptagen silol-Ucel for the treatment of diffuse large B-cell lymphoma and primary mediastinal large B-cell lymphoma after two or more systemic therapies, March 2023), but modified for the U.S. population. An estimated 2,700 patients were assumed to be eligible for CAR T in the U.S.

[0296] result Survival predictions were modeled for three hypothetical patient cohorts with different V2VTs (Case 1: 24 days, Case 2: 54 days, and Case 3: 37 days).

[0297] The median overall survival for the three hypothetical patient cohorts was 19.5 months, 8.5 months, and 10.5 months for cases 1, 2, and 3, respectively.

[0298] Reducing V2VT from 54 days (tisa-cel median V2VT; JULIET) to 24 days (axi-cel median V2VT; ZUMA-1) resulted in an increase in average life expectancy per patient in three years (4.2 vs. 7.7 LYs) and an additional 2 QALYs (2.9 vs. 5.3). This translates to 9,328 additional LYs and 6,385 additional QALYs per year if all approximately 2,710 eligible patients in the U.S. received the "shorter" V2VT (24 days) instead of the longer (54 days) V2VT. See Table 6. Smaller differences in V2VT (24 days vs. 37 days [liso-cel median V2VT; TRANSCEND-NHL-001]) resulted in 2.6 and 1.8 additional LYs and QALYs, respectively, equivalent to an increase of 7,040 LYs and 4,819 QALYs at the population level. The results were consistently positive across all sensitivity analyses. TIFF2026510004000009.tif32162

[0299] Extensive sensitivity analyses were conducted, and all analyses showed that shorter V2VT times led to improved outcomes (Table 7).

[0300] Sensitivity analysis demonstrates that the outcome is primarily determined by the post-injection outcome as a function of V2VT and the probability of injection as a function of the V2VT parameters. TIFF2026510004000010.tif62162

[0301] Sensitivity analysis yielded an increase in QALYs per patient ranging from 0.94 (not affected by post-injection survival V2VT) to 3.71 (increasing the granularity of the cutoff for V2VT classification [<28 vs ≥28 to <40 vs ≥40, rather than <36 days vs ≥36 days]).

[0302] This study is the first to quantify potential lifetime survival and QALY outcomes in r / r LBCL patients treated with CAR T cells in a 3L+ setting associated with V2VT reduction, utilizing currently available evidence.

[0303] In real-world settings, several factors can influence V2VT in patients undergoing CAR T, and delays during this multi-stage process can impact patient outcomes. This study integrates publicly available real-world data to demonstrate potential differences in survival outcomes based on V2VT. The modeling described herein demonstrates that outcomes are primarily determined by a higher probability of reaching infusion, and that infused patients subsequently show improved outcomes compared to non-infusion patients. Results improved across the range of sensitivity analyses tested.

[0304] As is common in modeling studies, several important assumptions were made, including the generalization of hazard ratios (HRs), the lack of formal studies on bridging therapies for outcomes, and the assumption that there was no difference in effectiveness across therapies. However, various sensitivity analyses were performed to test the effects of these assumptions.

[0305] V2VT may be a key predictor of outcomes in R / R LBCL, and aiming for short-term production, product release, transport, and injection is key to further improving CAR T cell outcomes. This study demonstrates that moderate differences in V2VT can have a significant effect on life expectancy. * * *

[0306] While many embodiments have been described, it is clear that this disclosure and examples may provide other embodiments utilizing the compositions and methods described herein, or other embodiments encompassed by such compositions and methods. Therefore, it will be understood that the scope of the invention should be defined not by the embodiments presented as examples, but by what can be understood from this disclosure and the appended claims.

Claims

1. A method for preparing lymphocytes that have improved efficacy and / or reduced adverse effects in the treatment of cancer, Obtaining lymphocytes from patients via apheresis, The lymphocytes are incubated with a polynucleotide vector to transduce the lymphocytes and produce transduced lymphocytes. The transduced lymphocytes are cultured to obtain a sample of the cultured lymphocytes. This includes injecting the aforementioned sample into the aforementioned patient, A method wherein the time elapsed from obtaining the lymphocytes to injecting the sample is 28 days or less.

2. A method for preventing and / or reducing the likelihood of persistent thrombocytopenia in patients with r / r LBCL, Obtaining lymphocytes from the patient via apheresis, The process involves incubating lymphocytes with a polynucleotide vector to transduce the lymphocytes and produce transduced lymphocytes, The transduced lymphocytes are cultured to obtain a sample of the cultured lymphocytes. This includes injecting the aforementioned sample into the aforementioned patient, A method wherein the time elapsed from obtaining the lymphocytes to injecting the sample is 28 days or less.

3. The aforementioned patient, The probability of achieving a complete response exceeds 55%. The probability of overall survival at 24 months is greater than 45%, and / or The method according to claim 1 or 2, wherein the likelihood of developing persistent thrombocytopenia is less than 30%.

4. The method according to claim 1 or 2, wherein the time taken from obtaining the lymphocytes to injecting the sample is 27 days, 26 days, 25 days, 24 days, 23 days, 22 days, 21 days, 20 days, 19 days, 18 days, 17 days, 16 days, 15 days, 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, or 6 days or less.

5. The method according to claim 1 or 2, further comprising administering lymphocyte depletion chemotherapy, wherein the lymphocyte depletion chemotherapy is administered within 5, 4, 3, 2, or 1 day of the infusion step.

6. The method according to any one of claims 1 to 5, wherein the cultured lymphocytes are not cryopreserved.

7. The method according to any one of claims 1 to 6, wherein the transduced lymphocytes are cultured for 72 hours, 48 ​​hours, or less than 36 hours.

8. The method according to any one of claims 1 to 7, wherein the incubation is performed in a closed system.

9. The aforementioned closing system is at least 1500 cm ２ The method according to claim 8, having the following internal surface area.

10. The method according to claim 8 or 9, wherein the closed system has an inner surface coated with recombinant human fibronectin, and the coating is carried out with a solution containing about 1 to 10 μg / ml of recombinant human fibronectin.

11. The method according to claim 10, wherein the inner surface is further in contact with a second solution containing the polynucleotide vector, and the second solution has a volume of about 200 mL.

12. The method according to claim 11, wherein the coating further includes a drain for the second solution.

13. The sample in the closed system is at least 1.5 × 10 ８ The method according to any one of claims 8 to 12, comprising [number] lymphocytes.

14. The aforementioned sample is at least 4 × 10 ８ The method according to claim 12, comprising [number] lymphocytes.

15. The method according to any one of claims 1 to 14, wherein the lymphocytes are peripheral blood mononuclear cells (PBMCs) or T cells.

16. The method according to any one of claims 1 to 15, wherein the sample comprises CD4+ T cells and CD8+ T cells.

17. The method according to any one of claims 1 to 16, wherein a total of 10,000 to 1,000,000 cultured lymphocytes per kilogram of the patient are administered to the patient.

18. The method according to claim 17, wherein a total of 20,000 to 400,000 cultured lymphocytes per kilogram of the patient are administered to the patient.

19. The method according to claim 17 or 18, wherein at least 15% of the cultured lymphocytes are transduced with the vector.

20. The method according to any one of claims 1 to 19, wherein the polynucleotide vector is a viral vector.

21. The method according to claim 20, wherein the viral vector is a retroviral vector or a lentiviral vector.

22. The method according to any one of claims 1 to 21, wherein the vector encodes one or more chimeric antigen receptors (CARs) or one or more T cell receptors (TCRs).

23. The method according to claim 22, wherein one or more CARs include an intracellular costimulatory domain.

24. The intracellular costimulatory domains include DAP-10, CD28, OX-40, 4-1BB (CD137), CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulatory factor (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD11a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), tumor necrosis factor superfamily member 14, TNFSF14, LIGHT), NKG2C, Ig alpha (CD79a), Fc gamma receptor, MHC class I molecule, TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, signal transduction lymphocyte activating molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, CDS, GITR, BAFFR, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL 7R Alpha, ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD (CD11d), ITGAE (CD103), ITGAL (CD11a), ITGAM (CD11b), ITGAX (CD11c) ), ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, TNFR2, TRANCE (RANKL), DN AM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CR The method according to claim 23, wherein the signaling region of a protein is selected from the group consisting of ligands that specifically bind to TAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG (Cbp), CD19a, CD83, and combinations thereof.

25. The method according to claim 24, wherein the intracellular co-stimulatory domain is the signal transduction region of CD28.

26. The method according to any one of claims 22 to 25, wherein the one or more CARs recognize one or more tumor antigens.

27. The method according to claim 26, wherein the tumor antigen is CD19.

28. The method according to claim 27, wherein the lymphocytes containing the CAR are axicaptagen silolucel or brexcaptagen autolucel.

29. The method according to claim 1, wherein the patient has relapsed or refractory (r / r) large B-cell lymphoma (LBCL).

30. The method according to claim 26, wherein the tumor antigens are CD19 and CD20.

31. A method for predicting the likelihood of complete response in patients to immunotherapy, To determine the period from the leukocyte apheresis process of the patient to the administration of the immunotherapy to the patient, Based on the determination of the aforementioned period, the patients are divided into multiple groups, and the multiple groups are, A first group characterized by a period of up to 28 days from the leukocyte apheresis process to the administration of the immunotherapy to the patient, A second group characterized by a period of 28 to 40 days from the leukocyte apheresis process to the administration of the immunotherapy to the patient, Classifying into one of a plurality of groups, including a third group characterized by a period of at least 40 days from the leukocyte apheresis process to the administration of the immunotherapy to the patient, This includes determining the likelihood of a complete response in the patient, at least in part, based on which of the plurality of groups the patient is classified into, If the patient is classified into the first group or the second group, the patient has at least approximately 55% chance of complete response. A method wherein, if the patient is classified within the third group, the patient has at least about a 42% chance of complete response.

32. The method according to claim 31, wherein, if the patient is classified into the first group or the second group, the patient has a probability of approximately 60% complete response.

33. The method according to claim 31, wherein the immunotherapy comprises one or more CARs that recognize one or more tumor antigens.

34. The method according to claim 33, wherein the tumor antigen is CD19.

35. The method according to claim 33, wherein the tumor antigens are CD19 and CD20.

36. The method according to claim 31, wherein the immunotherapy is axicaptagen silolucel or brexcaptagen autolucel.

37. The method according to claim 31, wherein the patient has relapsed or refractory (r / r) large B-cell lymphoma (LBCL).

38. A method for predicting the overall survival rate in patients undergoing immunotherapy, To determine the period from the leukocyte apheresis process of the patient to the administration of the immunotherapy to the patient, Based on the determination of the aforementioned period, the patients are divided into multiple groups, and the multiple groups are, A first group characterized by a period of up to 28 days from the leukocyte apheresis process to the administration of the immunotherapy to the patient, A second group characterized by a period of 28 to 40 days from the leukocyte apheresis process to the administration of the immunotherapy to the patient, Classifying into one of a plurality of groups, including a third group characterized by a period of at least 40 days from the leukocyte apheresis process to the administration of the immunotherapy to the patient, This includes determining the overall survival rate in the patient, at least in part, based on which of the plurality of groups the patient is classified into, If the patient is classified into the first group, the patient has an overall survival rate of at least about 49%. If the patient is classified into the second group, the patient has an overall survival rate of at least about 48%. A method wherein, if the patient is classified into the third group, the patient has an overall survival rate of at least about 30%.

39. The method according to claim 38, wherein the immunotherapy comprises one or more CARs that recognize one or more tumor antigens.

40. The method according to claim 39, wherein the tumor antigen is CD19.

41. The method according to claim 39, wherein the tumor antigens are CD19 and CD20.

42. The method according to claim 38, wherein the immunotherapy is axicaptagen silolucel or brexcaptagen autolucel.

43. The method according to claim 38, wherein the patient has relapsed or refractory (r / r) large B-cell lymphoma (LBCL).

44. A method for predicting the risk of thrombocytopenia in patients receiving immunotherapy, To determine the period from the leukocyte apheresis process of the patient to the administration of the immunotherapy to the patient, Based on the determination of the aforementioned period, the patients are divided into multiple groups, and the multiple groups are, A first group characterized by a period of up to 28 days from the leukocyte apheresis process to the administration of the immunotherapy to the patient, A second group characterized by a period of 28 to 40 days from the leukocyte apheresis process to the administration of the immunotherapy to the patient, Classifying into one of a plurality of groups, including a third group characterized by a period of at least 40 days from the leukocyte apheresis process to the administration of the immunotherapy to the patient, This includes determining the risk of thrombocytopenia in the patient, at least in part, based on which of the plurality of groups the patient is classified into, If the patient is classified into the first group, the patient has approximately an 18% risk of thrombocytopenia. If the patient is classified into the second group, the patient has approximately a 25% risk of thrombocytopenia. A method wherein, if the patient is classified within the third group, the patient has a risk of approximately 34% of thrombocytopenia.

45. The method according to claim 44, wherein the immunotherapy comprises one or more CARs that recognize one or more tumor antigens.

46. The method according to claim 45, wherein the tumor antigen is CD19.

47. The method according to claim 45, wherein the tumor antigens are CD19 and CD20.

48. The method according to claim 44, wherein the immunotherapy is axicaptagen silolucel or brexcaptagen autolucel.

49. The method according to claim 44, wherein the patient has relapsed or refractory (r / r) large B-cell lymphoma (LBCL).

50. A method for predicting life expectancy and quality-adjusted life years in patients who have received immunotherapy, The period from the leukocyte apheresis process of the patient to the administration of the immunotherapy to the patient, and determining whether the period is short or long. Assigning the probability of successful injection based on the aforementioned period, A method comprising inputting patient information into a survival model to determine the patient's life expectancy and quality-adjusted life years.

51. The method according to claim 50, wherein the aforementioned short period is approximately 24 days or less.

52. The method according to claim 50, wherein the aforementioned long period is approximately 54 days or more.

53. The method according to claim 50, wherein the aforementioned long period is approximately 37 days or more.

54. The method according to claim 50, wherein the short period indicates that the increase in the patient's life expectancy and the increase in quality-adjusted life years exceeds 5 years, and the long period indicates that the increase in the patient's life expectancy and the increase in quality-adjusted life years is less than 5 years.

55. The method according to claim 50, wherein the immunotherapy comprises one or more CARs that recognize one or more tumor antigens.

56. The method according to claim 50, wherein the tumor antigen is CD19.

57. The method according to claim 50, wherein the tumor antigens are CD19 and CD20.

58. The method according to claim 50, wherein the immunotherapy is axicaptagen silolucel or brexcaptagen autolucel.

59. The method according to claim 50, wherein the patient has relapsed or refractory (r / r) large B-cell lymphoma (LBCL).

Images