Accelerated administration of engineered lymphocytes

Abstract

Provided herein are methods for accelerated manufacture of engineered lymphocytes that are demonstrated to be associated with advantageous complete reaction rates and overall survival and reduce the risk of long-term thrombocytopenia. Accelerated manufacturing processes can produce transduced lymphocytes with improved efficacy or reduced adverse effects in the treatment of cancer. An example process includes obtaining lymphocytes from a patient by apheresis; incubating the lymphocytes with a polynucleotide vector to transduce the lymphocytes, thereby producing transduced lymphocytes; culturing the transduced lymphocytes; and infusing these transduced lymphocytes into the patient, thereby predicting the likelihood of complete response, overall survival, and risk of long term thrombocytopenia in a subject receiving immunotherapy.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 381,507 filed on October 28, 2022, U.S. Provisional Patent Application No. 63 / 386,831 filed on December 9, 2022, and U.S. Provisional Patent Application No. 63 / 506,288 filed on June 5, 2023, each of which is hereby incorporated in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which is electronically submitted in XML file format and is hereby incorporated by reference in its entirety. The XML copy was created on October 11, 2023, is named K-1143-WO-PCT_SL.xml, and is 28,791 bytes in size. Background Art

[0005] Chimeric antigen receptors (CARs) and engineered T-cell receptors (TCRs) include binding domains capable of interacting with specific tumor antigens. This binding ability allows immune cells to target and kill cancer cells. The highly complex and lengthy autologous cell engineering and production process presents significant challenges. During this process, lymphocytes collected from the patient must be transported to a processing center, and the resulting cells must be cryopreserved and then returned to the patient for transplantation. This highly complex process inevitably leads to high costs and limited clinical application. Therefore, there is a strong unmet need for developing approaches with shorter durations to improve patient outcomes. Summary of the Invention

[0006] Provided herein are methods for accelerating the manufacture and administration of engineered lymphocytes, which have been shown to be associated with favorable complete response rates and overall survival, and to reduce the risk of long-term thrombocytopenia. Accelerating the manufacturing process, particularly one with reduced veno-venous time, is associated with improved efficacy or reduced adverse effects in treating cancer. Also provided herein are methods for predicting the likelihood of a complete response, overall survival, and long-term thrombocytopenia risk in subjects receiving immunotherapy.

[0007] One embodiment of the present disclosure relates to a method for preparing lymphocytes with improved efficacy and / or reduced adverse effects in treating cancer, the method comprising obtaining lymphocytes from a patient by apheresis; incubating the lymphocytes with a polynucleotide vector to transduce the lymphocytes, thereby producing transduced lymphocytes; culturing the transduced lymphocytes to obtain a cultured lymphocyte sample; and infusing the sample into the patient, wherein the time taken from obtaining the lymphocytes to infusing the sample is no longer than 28 days.

[0008] In one embodiment of the present disclosure, the patient has a greater than 55% likelihood of having a complete response; a greater than 45% likelihood of having overall survival at 24 months; and / or a less than 30% likelihood of developing long-term thrombocytopenia.

[0009] In one embodiment of the present disclosure, the time taken from obtaining the lymphocytes to infusing the sample is no longer than 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.

[0010] In one embodiment of the present disclosure, the method further comprises administering lymphodepleting chemotherapy, wherein the lymphodepleting chemotherapy is administered within 5 days, 4 days, 3 days, 2 days, or 1 day of the infusing step.

[0011] One embodiment of the present disclosure is directed to a method for preventing and / or reducing the likelihood of long-term thrombocytopenia in a patient with r / r LBCL, the method comprising obtaining lymphocytes from the patient by apheresis; incubating the lymphocytes with a polynucleotide vector to transduce the lymphocytes, thereby producing transduced lymphocytes; culturing the transduced lymphocytes to obtain a cultured lymphocyte sample; and infusing the sample into the patient, wherein the time elapsed from obtaining the lymphocytes to infusing the sample is no longer than 28 days.

[0012] One embodiment of the present disclosure is directed to a method for predicting a patient's likelihood of a complete response to an immunotherapy, the method comprising: determining a time period from a leukapheresis step for the patient to administration of the immunotherapy to the patient; grouping the patient into one of a plurality of groups based on the determined time period, the plurality of groups comprising: a first group characterized in that the time period from the leukapheresis step to the administration of the immunotherapy to the patient is up to 28 days; a second group characterized in that the time period from the leukapheresis step to the administration of the immunotherapy to the patient is between 28 days and 40 days; and a third group characterized in that the time period from the leukapheresis step to the administration of the immunotherapy to the patient is at least 40 days; and determining the likelihood of a complete response in the patient based at least in part on which of the plurality of groups the patient is grouped into, wherein if the patient is grouped into the first group or the second group, the patient has a likelihood of a complete response of at least about 55%, and wherein if the patient is grouped into the third group, the patient has a likelihood of a complete response of at least about 42%.

[0013] In some embodiments of the present disclosure, the patient has about a 60% likelihood of a complete response if the patient is grouped into the first group or the second group.

[0014] One embodiment of the present disclosure relates to a method for predicting overall survival of a patient receiving immunotherapy, the method comprising: determining a time period from a leukocyte separation step to administration of the immunotherapy to the patient; grouping the patient into one of a plurality of groups based on the determined time period, the plurality of groups comprising: a first group characterized in that a time period from the leukocyte separation step to administration of the immunotherapy to the patient is up to 28 days; and a second group characterized in that a time period from the leukocyte separation step to administration of the immunotherapy to the patient is between 28 days and 40 days. and a third group characterized in that a time period from the leukapheresis step to the administration of the immunotherapy to the patient is at least 40 days; and determining the overall survival rate in the patient based at least in part on which of the plurality of groups the patient is grouped, wherein if the patient is grouped within the first group, the patient has an overall survival rate of at least about 49%, wherein if the patient is grouped within the second group, the patient has an overall survival rate of at least about 48%, and wherein if the patient is grouped within the third group, the patient has an overall survival rate of at least about 30%.

[0015] One embodiment of the present disclosure relates to a method for predicting a risk of thrombocytopenia in a patient receiving immunotherapy, the method comprising: determining a time period from a leukocyte separation step of the patient to administration of the immunotherapy to the patient; grouping the patient into one of a plurality of groups based on the determined time period, the plurality of groups comprising: a first group characterized in that a time period from the leukocyte separation step to the administration of the immunotherapy to the patient is up to 28 days; a second group characterized in that a time period from the leukocyte separation step to the administration of the immunotherapy to the patient is between 28 days and 40 days; and a third group characterized in that a time period from the leukapheresis step to the administration of the immunotherapy to the patient is at least 40 days; and the risk of thrombocytopenia in the patient is determined at least in part based on which of the multiple groups the patient is grouped into, wherein if the patient is grouped into the first group, the patient has an approximately 18% risk of thrombocytopenia, wherein if the patient is grouped into the second group, the patient has an approximately 25% risk of thrombocytopenia, and wherein if the patient is grouped into the third group, the patient has an approximately 34% risk of thrombocytopenia.

[0016] One embodiment of the present disclosure relates to a method for predicting life expectancy and quality-adjusted life years of a patient who has received immunotherapy, the method comprising: determining a time period from a leukapheresis step for the patient to administration of the immunotherapy to the patient, wherein the time period is a short time period or a long time period; assigning a probability of successful transfusion based on the time period; and inputting the patient information into a survival model to determine the patient's life expectancy and quality-adjusted life years.

[0017] In one embodiment of the present disclosure, the immunotherapy comprises one or more CARs that recognize one or more tumor antigens.

[0018] In one embodiment of the present disclosure, the immunotherapy is axicabtagene ciloleucel or brexucabtagene autoleucel.

[0019] In one embodiment of the present disclosure, the patient has relapsed or refractory (r / r) large B-cell lymphoma (LBCL). DETAILED DESCRIPTION

[0020] definition

[0021] In order to make the present disclosure more easily understood, certain terms are first defined below. Additional definitions of the following terms and other terms are set forth throughout the specification.

[0022] Unless otherwise stated or obvious from the context, as used herein, the term "or" is understood to be inclusive and encompasses both "or" and "and."

[0023] As used herein, the term "and / or" should be taken as a specific disclosure of each of the two specified features or components, with or without the other. Thus, 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 herein in phrases such as "A, B, and / or C," the term "and / or" is intended to encompass each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0024] Unless otherwise specified or apparent from the context, the term "about" refers to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, "about" or "consisting essentially of" can mean within one or more standard deviations. "About" or "consisting essentially of" can mean a range of up to 10% (i.e., ±10%). Thus, "about" can be understood to mean within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% greater or less than the stated value. For example, about 5 mg can include any amount between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, these terms can mean up to an order of magnitude or up to 5 times a value. When a specific value or composition is provided in this disclosure, unless otherwise stated, the meaning of "about" or "consisting essentially of" should be assumed to be within an acceptable error range for the specific value or composition.

[0025] "Administer" refers to the physical introduction of an agent into a subject using any of the various methods and delivery systems known to those skilled in the art, such as the modified T cells disclosed herein. Exemplary routes of administration for the preparations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration (e.g., by injection or infusion). The phrase "parenteral administration" means modes of administration (typically by injection) other than enteral and topical administration, and includes but is not limited to intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation. In some embodiments, the preparation is administered via a non-parenteral route (e.g., oral). Other non-parenteral routes include topical, epidermal or mucosal administration routes, such as intranasal, vaginal, rectal, sublingual or topical. Administration can also be performed, for example, once, multiple times, and / or over one or more extended periods of time.

[0026] The term "allogeneic" refers to any material derived from one individual and subsequently introduced into another individual of the same species.

[0027] The term "antibody" (Ab) includes, but is not limited to, immunoglobulins, glycoproteins that specifically bind to antigens. Generally speaking, an antibody comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigen-binding molecules 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 a single constant domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, termed complementarity-determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain the binding domains that interact with the antigen. The constant region of an Ab mediates the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Generally, human antibodies are tetrameric agents of approximately 150 kD, composed of two identical heavy (H) chain polypeptides (each approximately 50 kD) and two identical light (L) chain polypeptides (each approximately 25 kD), which associate with each other in a structure commonly referred to as a "Y-shaped" structure. The heavy and light chains are linked or connected to each other by a single disulfide bond; two additional disulfide bonds connect the heavy chain hinge regions, allowing dimers to connect and form tetramers. Naturally produced antibodies are also glycosylated, for example, at the CH2 domain.

[0028] "Antigen binding molecule," "antigen binding portion," "antigen binding fragment," and "antibody fragment" refer to any molecule that comprises an antigen-binding portion (e.g., CDR) of an antibody from which the molecule is derived. An antigen-binding molecule may comprise an antigen complementarity-determining region (CDR). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, dAbs, linear antibodies, scFv antibodies, and multispecific antibodies formed from antigen-binding molecules. Peptibodies (i.e., Fc fusion molecules comprising a peptide-binding domain) are another example of suitable antigen-binding molecules. In some embodiments, the antigen-binding molecule binds to an antigen on a tumor cell. In some embodiments, the antigen-binding molecule binds to an antigen on a cell involved in a hyperproliferative disease or 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).

[0029] The terms "variable region" or "variable domain" are used interchangeably. The variable region generally refers to a portion of an antibody, typically a portion of a light or heavy chain, typically the amino-terminal approximately 110 to 120 amino acids in a mature heavy chain and approximately 90 to 115 amino acids in a mature light chain. These regions vary widely in sequence between antibodies and are responsible for the binding and specificity of a particular antibody for its specific antigen. Sequence variability is concentrated in regions known as the complementarity determining regions (CDRs), while the more highly conserved regions of the variable domain are known as the framework regions (FRs). Without wishing to be bound by any particular mechanism or theory, it is believed that the light and heavy chain CDRs are primarily responsible for the antibody's interaction with the antigen and its specificity. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region includes rodent or murine CDRs and human framework regions (FRs). In specific embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region includes rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FRs).

[0030] The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody or antigen binding molecule thereof.

[0031] The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody or antigen binding molecule thereof.

[0032] 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 definitions used by Oxford Molecular's AbM antibody modeling software. The Contact definition is based on analysis of available complex crystal structures.

[0033] The term "autologous" refers to any material that originates from the same individual and is later reintroduced into that individual. For example, the engineered autologous cell therapy (eACT) described herein ™ ) method involves collecting lymphocytes from a patient, then engineering them to express, for example, a CAR construct, and subsequently administering them back to the same patient.

[0034] "Chimeric antigen receptor" or "CAR" refers to a molecule that is engineered to contain a binding motif and a means of activating an immune cell (e.g., a T cell, such as a naive T cell, a central memory T cell, an effector memory T cell, or a combination thereof) upon antigen binding. CAR is also known as an artificial T cell receptor, a chimeric T cell receptor, or a chimeric immunoreceptor. In some embodiments, a CAR comprises a binding motif, an extracellular domain, a transmembrane domain, one or more co-stimulatory domains, and an intracellular signaling domain. T cells that have been genetically engineered to express a chimeric antigen receptor can be referred to as CAR T cells. "Extracellular domain" (or "ECD") refers to a portion of a polypeptide that, when the polypeptide is present in a cell membrane, is understood to be located outside the cell membrane, in the extracellular space.

[0035] "T cell receptor" or "TCR" refers to the antigen recognition molecule 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, resulting in a highly diverse set of TCR proteins.

[0036] The term "heterologous" refers to any source other than a naturally occurring sequence. For example, a heterologous sequence included as part of a costimulatory protein is an amino acid sequence that does not occur naturally, i.e., is not identical to a wild-type human costimulatory protein. For example, a heterologous nucleotide sequence refers to a nucleotide sequence that is different from the sequence encoding a wild-type human costimulatory protein.

[0037] The term "identity" refers to the overall relatedness between polymeric molecules, such as nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules. Methods for calculating the percent identity between two provided polypeptide sequences are known. For example, the percent identity of two sequences can be calculated by aligning them for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second sequences for optimal alignment, and non-identical sequences can be ignored for comparison purposes). The nucleotides or amino acids at corresponding positions are then compared. When 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, the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, optionally taking into account the number and length of gaps, which may need to be introduced for optimal alignment of the two sequences. Sequence comparison or alignment and determination of percent identity between two sequences can be achieved using mathematical algorithms such as BLAST (Basic Local Alignment Search Tool). In some embodiments, polymer molecules are considered "homologous" to each other if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical (e.g., 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, or 95%-100% identical).

[0038] The immune cells for immunotherapy can be derived from any source known in the art. For example, immune cells can be differentiated from a hematopoietic stem cell population in vitro, or can be obtained from a subject. Immune cells can be obtained from, for example, peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In addition, immune cells can be derived from one or more immune cell lines available in the art. Various techniques known to those skilled in the art (such as FICOLL) can also be used. ™ Immune cells are obtained from a unit of blood collected from a subject by separation and / or apheresis. Additional methods for isolating immune cells for use in immune cell therapy are disclosed in U.S. Patent Publication No. 2013 / 0287748, which is incorporated herein by reference in its entirety.

[0039] A "patient" includes any person suffering from cancer (eg, lymphoma or leukemia). The terms "subject" and "patient" are used interchangeably herein.

[0040] The term "pharmaceutically acceptable" refers to a molecule or composition that is not harmful to the recipient when administered to the recipient, or any harmful effects are outweighed by the beneficial effects on the recipient. With respect to the carrier, diluent or excipient used to formulate the compositions disclosed herein, the pharmaceutically acceptable carrier, diluent or excipient must be compatible with the other ingredients of the composition and not harmful to the recipient, or any harmful effects must be outweighed by the beneficial effects on the recipient. The term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient or solvent encapsulating material, that is involved in carrying or transporting an agent from one part of the body to another (e.g., from one organ to another). Each carrier present in a pharmaceutical composition must be "acceptable" in the following sense: compatible with the other ingredients of the formulation and not harmful to the patient, or any harmful effects must be outweighed by the beneficial effects on the recipient. Some examples of materials that can serve 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; powdered gum tragacanth; 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 glycerol, 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; ethanol; pH buffered solutions; polyesters, polycarbonates, and / or polyanhydrides; and other nontoxic, compatible substances employed in pharmaceutical formulations.

[0041] The term "pharmaceutical composition" refers to a composition in which an active agent is formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dosage suitable for administration in a therapeutic regimen that, when administered to a relevant subject or population, shows a statistically significant probability of achieving a predetermined therapeutic effect. In some embodiments, the pharmaceutical composition can be formulated for administration in solid or liquid form, including but not limited to forms suitable for: oral administration, such as drenches (aqueous or non-aqueous solutions or suspensions), tablets (e.g., those targeted for buccal, sublingual, and systemic absorption), boluses, powders, granules, pastes for application to the tongue; parenteral administration, such as by subcutaneous, intramuscular, intravenous, or epidural injection, as, for example, sterile solutions or suspensions, or sustained-release formulations; topical application, such as as a cream, ointment, or controlled-release patch, or as a spray applied to the skin, lungs, or mouth; intravaginal or intrarectal administration, for example, as a vaginal suppository, cream, or foam; sublingual administration; ophthalmic administration; transdermal administration; or nasal, pulmonary, and other mucosal surfaces.

[0042] The terms "reduce" and "decrease" are used interchangeably herein and refer to any change that is less than the original value. "Reduce" and "decrease" are relative terms requiring a comparison between before and after measurement. "Reduce" and "decrease" include complete depletion.

[0043] The term "reference" describes a standard or control relative to which a comparison is made. For example, in some embodiments, an agent of interest, an animal, an individual, a colony, a sample, a sequence or a value is compared with a reference or control as an agent, an animal, an individual, a colony, a sample, a sequence or a value. In some embodiments, a reference or control is tested, measured and / or measured substantially simultaneously with an interested test, measurement or determination. In some embodiments, a reference or control is a historical reference or control, which is optionally embodied in a tangible medium. Generally speaking, a reference or control is measured or characterized under conditions or circumstances comparable to the conditions or circumstances of the assessment. When there are enough similarities to prove dependence and / or comparison to selected reference or control, the results are correct.

[0044] A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dose" of a therapeutic agent (e.g., 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 disease or promotes disease regression (as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic disease periods, or the prevention of impairment or disability resulting from the disease). The ability of a therapeutic agent to promote disease regression can be assessed using a variety of methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by measuring the activity of the agent in in vitro assays.

[0045] The terms "transduction" and "transduced" refer to the process of introducing exogenous nucleic acid into a cell 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, a DNA vector, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein-Barr virus vector, a papillomavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector, a lentiviral vector, or any combination thereof.

[0046] "Treatment" or "treatment" of a subject refers to any type of intervention or process performed on a subject, or the administration of an active agent to a subject, with the purpose of reversing, alleviating, improving, inhibiting, slowing down or preventing the onset, progression, development, severity or recurrence of symptoms, complications or conditions or biochemical indicators associated with a disease. In one embodiment, "treatment" includes partial remission. In another embodiment, "treatment" or "treatment" includes complete remission. In some embodiments, treatment can be treatment of subjects who do not show signs of the relevant disease, disorder and / or condition and / or only show early signs of the disease, disorder and / or condition. In some embodiments, such treatment can be treatment of subjects who show one or more established signs of the relevant disease, disorder and / or condition. In some embodiments, treatment can be treatment of subjects who have been diagnosed with the relevant disease, disorder and / or condition. In some embodiments, treatment can be treatment of subjects who are known to have one or more susceptibility factors that are statistically associated with an increased risk of the development of the relevant disease, disorder and / or condition.

[0047] The term "vector" refers to a receptor nucleic acid molecule modified to contain or 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 connected. Another type of vector is a viral vector, in which additional DNA segments can be connected to a viral genome. Certain vectors are capable of autonomous replication in the host cells they are introduced into (e.g., bacterial vectors with bacterial replication origins, and additional mammalian vectors). Other vectors (e.g., non-additional mammalian vectors) can be integrated into the genome of the host cell when introduced into the host cell, and thus replicated together with the host genome. In addition, certain vectors include sequences that guide the expression of inserted genes operably connected thereto. Such vectors may be referred to as "expression vectors" herein. Standard techniques may be used for vector engineering, for example, as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), which is incorporated herein by reference.

[0048] One embodiment of the present disclosure relates to a method for preparing lymphocytes with improved efficacy and / or reduced adverse effects in treating cancer, the method comprising obtaining lymphocytes from a patient by apheresis; incubating the lymphocytes with a polynucleotide vector to transduce the lymphocytes, thereby producing transduced lymphocytes; culturing the transduced lymphocytes to obtain a cultured lymphocyte sample; and infusing the sample into the patient. In this embodiment, the time from obtaining the lymphocytes to infusing the sample is no longer than 28 days.

[0049] One embodiment of the present disclosure relates to a method for preventing and / or reducing the likelihood of long-term thrombocytopenia in a patient with r / r LBCL, the method comprising: obtaining lymphocytes from the patient by apheresis; incubating the lymphocytes with a polynucleotide vector to transduce the lymphocytes, thereby producing transduced lymphocytes; culturing the transduced lymphocytes to obtain a cultured lymphocyte sample; and infusing the sample into the patient. In this embodiment, the time from obtaining the lymphocytes to infusing the sample is no longer than 28 days.

[0050] One embodiment of the present disclosure relates to any of the methods discussed above, wherein the patient has a greater than 55% likelihood of having a complete response; a greater than 45% likelihood of having overall survival at 24 months; and / or a less than 30% likelihood of developing long-term thrombocytopenia.

[0051] One embodiment of the present disclosure relates to any of the methods discussed above, wherein the time taken from obtaining the lymphocytes to infusing the sample is no longer than 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.

[0052] One embodiment of the present disclosure relates to any of the methods discussed above, further comprising administering lymphodepleting chemotherapy, and wherein the lymphodepleting chemotherapy is administered within 5 days, 4 days, 3 days, 2 days, or 1 day of the infusion step.

[0053] One embodiment of the present disclosure relates to any of the methods discussed above and does not include cryopreservation of cultured lymphocytes.

[0054] One embodiment of the present disclosure relates to any of the methods discussed above, wherein the transduced lymphocytes are cultured for less than 72 hours, 48 ​​hours, or 36 hours.

[0055] One embodiment of the present disclosure relates to any of the methods discussed above, wherein the incubation is performed in a closed system.

[0056] One embodiment of the present disclosure relates to any of the methods discussed above, wherein the closed system has a volume of at least 1500 cm 2 internal surface area.

[0057] 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 performed using a solution comprising about 1 μg / ml to 10 μg / ml of recombinant human fibronectin.

[0058] One embodiment of the present disclosure relates to any of the methods discussed above, wherein the inner surface is further contacted with a second solution comprising the polynucleotide carrier, and wherein the second solution has a volume of about 200 mL.

[0059] One embodiment of the present disclosure is directed to any of the methods discussed above, wherein the coating further comprises draining of the second solution.

[0060] One embodiment of the present disclosure relates to any of the methods discussed above, wherein the sample in the closed system has a 8 lymphocytes.

[0061] One embodiment of the present disclosure relates to any of the methods discussed above, wherein the sample has at least 4×10 8 lymphocytes.

[0062] 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.

[0063] 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.

[0064] 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 patient are administered to the patient.

[0065] 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 patient are administered to the patient.

[0066] One embodiment of the present disclosure relates to any of the methods discussed above, wherein at least 15% of the cultured lymphocytes are transduced with the vector.

[0067] One embodiment of the present disclosure relates to any of the methods discussed above, wherein the polynucleotide vector is a viral vector.

[0068] 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.

[0069] 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).

[0070] One embodiment of the present disclosure relates to any of the methods discussed above, wherein one or more CARs have an intracellular co-stimulatory domain.

[0071] One embodiment of the present disclosure relates to any of the methods discussed above, wherein the intracellular costimulatory domain is a signaling region of a protein selected from the group consisting of: DAP-10, CD28, OX-40, 4-1BB (CD137), CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell co-stimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD11a / CD18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), tumor necrosis factor superfamily member 14, TNFSF14, LIGHT), NKG2C, Igα (CD79a), Fcγ receptor, MHC Class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, CDS, GITR, BAFFR, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, 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), 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, ligands that specifically bind to CD83, and combinations thereof.

[0072] One embodiment of the present disclosure relates to any of the methods discussed above, wherein the intracellular costimulatory domain is the signaling region of CD28.

[0073] One embodiment of the present disclosure relates to any of the methods discussed above, wherein the one or more CARs recognize one or more tumor antigens.

[0074] One embodiment of the present disclosure relates to any of the methods discussed above, wherein the tumor antigen is CD19.

[0075] One embodiment of the present disclosure relates to any of the methods discussed above, wherein the lymphocytes comprising the CAR are akilenbine or brexucabtagene autoleucel.

[0076] One embodiment of the present disclosure relates to any of the methods discussed above, wherein the patient has relapsed or refractory (r / r) large B-cell lymphoma (LBCL).

[0077] One embodiment of the present disclosure relates to any of the methods discussed above, wherein the tumor antigens are CD19 and CD20.

[0078] One embodiment of the present disclosure relates to a method for predicting a patient's likelihood of a complete response to immunotherapy, the method comprising: determining a time period from a leukapheresis step for the patient to administration of the immunotherapy to the patient; grouping the patient into one of a plurality of groups based on the determined time period, the plurality of groups comprising: a first group characterized in that the time period from the leukapheresis step to administration of the immunotherapy to the patient is up to 28 days; a second group characterized in that the time period from the leukapheresis step to administration of the immunotherapy to the patient is between 28 days and 40 days; a third group characterized in that the time period from the leukapheresis step to administration of the immunotherapy to the patient is at least 40 days; and determining the likelihood of a complete response in the patient based at least in part on which of the plurality of groups the patient is grouped into. In this embodiment, if the patient is grouped into the first or second group, the patient has a likelihood of a complete response of at least about 55%, and if the patient is grouped into the third group, the patient has a likelihood of a complete response of at least about 42%.

[0079] One embodiment of the present disclosure relates to the method discussed above for predicting a patient's likelihood of a complete response to immunotherapy, wherein the patient has about a 60% likelihood of a complete response if the patient is grouped into the first group or the second group.

[0080] One embodiment of the present disclosure relates to the method discussed above for predicting a patient's likelihood of a complete response to an immunotherapy comprising one or more CARs that recognize one or more tumor antigens.

[0081] One embodiment of the present disclosure relates to the method discussed above for predicting a patient's likelihood of a complete response to immunotherapy, wherein the tumor antigen is CD19.

[0082] One embodiment of the present disclosure relates to the method discussed above for predicting a patient's likelihood of a complete response to immunotherapy, wherein the tumor antigens are CD19 and CD20.

[0083] One embodiment of the present disclosure relates to the method discussed above for predicting a patient's likelihood of a complete response to an immunotherapy, wherein the immunotherapy is akilenbine or autoleucel brexucabtagene.

[0084] One embodiment of the present disclosure relates to the method discussed above for predicting the likelihood of a complete response to immunotherapy in a patient, wherein the patient has relapsed or refractory (r / r) large B-cell lymphoma (LBCL).

[0085] One embodiment of the present disclosure relates to a method for predicting overall survival of a patient receiving immunotherapy, the method comprising: determining a time period from a leukapheresis step for the patient to administration of the immunotherapy to the patient; grouping the patient into one of a plurality of groups based on the determined time period, the plurality of groups comprising: a first group characterized in that the time period from the leukapheresis step to administration of the immunotherapy to the patient is up to 28 days; a second group characterized in that the time period from the leukapheresis step to administration of the immunotherapy to the patient is between 28 days and 40 days; a third group characterized in that the time period from the leukapheresis step to administration of the immunotherapy to the patient is at least 40 days; and determining the overall survival of the patient based at least in part on which of the plurality of groups the patient is grouped into. In this embodiment, if the patient is grouped into the first group, the patient has an overall survival rate of at least about 49%, if the patient is grouped into the second group, the patient has an overall survival rate of at least about 48%, and if the patient is grouped into the third group, the patient has an overall survival rate of at least about 30%.

[0086] One embodiment of the present disclosure relates to the method discussed above for predicting overall survival in a patient receiving immunotherapy, wherein the immunotherapy comprises one or more CARs that recognize one or more tumor antigens.

[0087] One embodiment of the present disclosure relates to the method discussed above for predicting overall survival in a patient receiving immunotherapy, wherein the tumor antigen is CD19.

[0088] One embodiment of the present disclosure relates to the method discussed above for predicting overall survival in a patient receiving immunotherapy, wherein the tumor antigens are CD19 and CD20.

[0089] One embodiment of the present disclosure relates to the method discussed above for predicting overall survival in a patient receiving immunotherapy, wherein the immunotherapy is akilenbine or brexucabtagene autoleucel.

[0090] One embodiment of the present disclosure relates to the method discussed above for predicting overall survival of a patient receiving immunotherapy, wherein the patient has relapsed or refractory (r / r) large B-cell lymphoma (LBCL).

[0091] One embodiment of the present disclosure relates to a method for predicting the risk of thrombocytopenia in a patient receiving immunotherapy, the method comprising: determining a time period from a leukapheresis step for the patient to the administration of the immunotherapy to the patient; grouping the patient into one of a plurality of groups based on the determined time period, the plurality of groups comprising: a first group characterized in that the time period from the leukapheresis step to the administration of the immunotherapy to the patient is up to 28 days; a second group characterized in that the time period from the leukapheresis step to the administration of the immunotherapy to the patient is between 28 days and 40 days; and a third group characterized in that the time period from the leukapheresis step to the administration of the immunotherapy to the patient is at least 40 days; and determining the risk of thrombocytopenia for the patient based at least in part on which of the plurality of groups the patient is grouped into. In this embodiment, if the patient is grouped into the first group, the patient has an approximately 18% risk of thrombocytopenia, if the patient is grouped into the second group, the patient has an approximately 25% risk of thrombocytopenia, and if the patient is grouped into the third group, the patient has an approximately 34% risk of thrombocytopenia.

[0092] One embodiment of the present disclosure relates to the method discussed above for predicting the risk of thrombocytopenia in a patient receiving immunotherapy, wherein the immunotherapy comprises one or more CARs that recognize one or more tumor antigens.

[0093] One embodiment of the present disclosure relates to the method discussed above for predicting the risk of thrombocytopenia in a patient receiving immunotherapy, wherein the tumor antigen is CD19.

[0094] One embodiment of the present disclosure relates to the method discussed above for predicting the risk of thrombocytopenia in a patient receiving immunotherapy, wherein the tumor antigens are CD19 and CD20.

[0095] One embodiment of the present disclosure relates to the method discussed above for predicting the risk of thrombocytopenia in a patient receiving immunotherapy, wherein the immunotherapy is akilencil or autoleucel brexucabtagene.

[0096] One embodiment of the present disclosure relates to the method discussed above for predicting the risk of thrombocytopenia in a patient receiving immunotherapy, wherein the patient has relapsed or refractory (r / r) large B-cell lymphoma (LBCL).

[0097] One embodiment of the present disclosure relates to a method for predicting life expectancy and quality-adjusted life years (QALYs) of a patient who has received immunotherapy, the method comprising: determining a time period from a leukapheresis step for a patient to administration of the immunotherapy to the patient, wherein the time period is a short time period or a long time period; assigning a probability of successful infusion based on the time period; and inputting the patient information into a survival model to determine the patient's life expectancy and quality-adjusted life years.

[0098] One embodiment of the present disclosure relates to the method discussed above for predicting life expectancy and quality-adjusted life years in a patient who has received immunotherapy, wherein the short time period is about 24 days or less.

[0099] One embodiment of the present disclosure relates to the method discussed above for predicting life expectancy and quality-adjusted life years in a patient who has received immunotherapy, wherein the long period of time is about 54 days or longer.

[0100] One embodiment of the present disclosure relates to the method discussed above for predicting life expectancy and quality-adjusted life years in a patient who has received immunotherapy, wherein the long period of time is about 37 days or longer.

[0101] One embodiment of the present disclosure relates to the method discussed above for predicting life expectancy and quality-adjusted life years for a patient who has received immunotherapy, wherein a short time period indicates an increase in life expectancy and an increase in quality-adjusted life years for the patient that is greater than 5 years, and wherein a long time period indicates an increase in life expectancy and an increase in quality-adjusted life years for the patient that is less than 5 years.

[0102] One embodiment of the present disclosure relates to the method discussed above for predicting life expectancy and quality-adjusted life years in a patient who has received immunotherapy, wherein the immunotherapy includes one or more CARs that recognize one or more tumor antigens.

[0103] One embodiment of the present disclosure relates to the method discussed above for predicting life expectancy and quality-adjusted life years in a patient who has received immunotherapy, wherein the tumor antigen is CD19.

[0104] One embodiment of the present disclosure relates to the method discussed above for predicting life expectancy and quality-adjusted life years in a patient who has received immunotherapy, wherein the tumor antigens are CD19 and CD20.

[0105] One embodiment of the present disclosure relates to the method discussed above for predicting life expectancy and quality-adjusted life years in a patient who has received immunotherapy, wherein the immunotherapy is akilenbine or autoleucel brexucabtagene.

[0106] One embodiment of the present disclosure relates to the method discussed above for predicting life expectancy and quality-adjusted life years in a patient who has received immunotherapy, wherein the patient has relapsed or refractory (r / r) large B-cell lymphoma (LBCL).

[0107] Accelerated manufacturing of engineered lymphocytes

[0108] The typical autologous CAR-T manufacturing process begins with leukapheresis. Apheresis material is collected from the patient at the treatment center. This process takes approximately 3 to 4 hours, during which time approximately 10 to 20 liters of blood are recirculated and approximately 100 to 500 mL of apheresis material is collected. The apheresis material collection bag is then shipped cold to a central manufacturing facility.

[0109] T cells are enriched from the apheresis material and then transduced with a retroviral vector containing the CAR gene. The transduced cells are expanded in culture until they reach the target dose, at which point the cells are washed and cryopreserved for shipment back to the treatment center. Each batch undergoes a series of tests to ensure it meets certain standards before release.

[0110] Once the treatment center receives the CAR-T cell product from the manufacturing facility, the patient undergoes lymphodepleting (LD) chemotherapy. Lymphodepleting chemotherapy for Akilencel consists of fludarabine (30 mg / m 2 ) and cyclophosphamide (500 mg / m 2 ) for 3 days (Day -5, Day -4, and Day -3). Currently, centers typically admit patients before some CAR-T cell infusion. CAR-T cells are administered 3 days after lymphodepletion is complete (Day 0), and for Akilencel, at a dose of, for example, 2 × 10 6 The target dose of CAR-T cells / kg was infused.

[0111] The entire process from apheresis to infusion (veno-venous) can take a median of 28 days for akilencel, 45 days for tisagenlecleucel, and 37 days for lisocabtagene maraleucel.

[0112] Veno-venous time refers to the time from leukapheresis to infusion. Veno-venous time can be affected by various factors, including, but not limited to, the time from leukapheresis to delivery of the product to an authorized treatment center, the time from leukapheresis to product shipment, transportation time, the time from leukapheresis to release of the product from the manufacturing site, and the time it takes to manufacture the product.

[0113] As demonstrated in Example 1, shorter veno-veno time is associated with improved complete response (CR) rates and overall survival (OS), as well as a reduced risk of long-term thrombocytopenia. Therefore, such findings emphasize the importance of accelerating the development and deployment of CAR-T manufacturing processes.

[0114] Without limitation, in one embodiment, veno-venous time can be reduced by shortening the time required for transport. In some embodiments, the time to transport apheresis material to a manufacturing facility and to transport transduced cells back to a treatment center can be reduced by using faster shipping or using a manufacturing facility closer to the treatment center.

[0115] In another embodiment, veno-venous time can be reduced 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 needed.

[0116] In another embodiment, the ability to manufacture product that meets specification requirements (within specification) can reduce veno-veno time.

[0117] The veno-venous time can also depend on the quality and quantity of the starting apheresis material. When the starting apheresis material contains fewer or lower-quality lymphocytes, a longer veno-venous time may be required. Therefore, based on the improved performance associated with a shorter veno-venous time, the time used to expand lymphocytes can be reduced, even if this may result in a reduced number of transduced cells available for infusion.

[0118] Important parts of the manufacturing process include lymphocyte transduction and expansion. Through years of refinement, a transduction / expansion method has been developed that takes 7 days (after the enrichment step), as described below. Building on these successes, further refined methods have been developed that can be completed in 5 days or even 3 days (counting after the enrichment step) and are also described below. As described herein, the 5-day process includes transduction preparation and specific implementation steps, in which a larger number of lymphocytes are contacted with a vector immobilized on a recombinant fibronectin coated on the inner surface of a closed system. This improved transduction procedure allows for a significant shortening of the post-transduction cell expansion step. Transduced cells prepared from the 5-day process are biased towards younger cells, which leads to better in vivo anti-tumor efficacy.

[0119] In some embodiments, when post-transduction expansion is eliminated, the transduction and expansion (after the enrichment step) procedures can be completed in just 3 days. Compared to a 5-day process, this 3-day process produces a cell population with a higher percentage of young cells and a reduced percentage of more mature, differentiated, and activated cells. Thus, not only do cell products from a 3-day process exhibit optimal in vivo anti-tumor efficacy, this greatly improved efficacy can also be achieved at a much lower dose.

[0120] Example processes for 7-day, 5-day, and 3-day lymphocyte production processes are described below.

[0121] Seven-day lymphocyte production process

[0122] In some embodiments, a therapeutic cell product is produced after a 7-day process of preparing lymphocytes that have been transduced with a polynucleotide vector (e.g., a viral vector) encoding a therapeutic protein. The prepared lymphocytes can be used to treat various diseases such as cancer, particularly when the therapeutic protein is a chimeric antigen receptor (CAR) or T cell receptor (TCR) designed to target cancer cells.

[0123] As used throughout the text, the terms "7-day process" and "7-day lymphocyte manufacturing process" are used interchangeably and refer to a CAR cell manufacturing process that requires about 7 days after the initial enrichment and activation steps. From the initial enrichment and activation steps to the harvest step, the 7-day process is at least 8 days in length and can be 8 to 11 days in total when the enrichment and activation steps are included.

[0124] Apheresis can be performed using standard apheresis equipment (such as Cobe ® Spectra, Spectra Optia ® 、Fenwal ™ Amicus ® The leukocyte apheresis process typically produces approximately 200 mL to 400 mL of apheresis product from the patient. The apheresis product can be processed on-site or optionally shipped to a facility at a temperature of 1°C to 10°C for manufacturing at another location. Additional process steps can be performed in an ISO 7 cell culture process suite (or similar cleanroom-type environment).

[0125] Where appropriate, cell processing devices (such as Sepax ®The volume reduction step was performed using 2 laboratory instruments (Biosafe SA; Houston, TX) or equivalent instruments and standard sterile tubing kits. Given the variability in the number of cells and input source material volume from each subject (approximately 200 mL to 400 mL), the volume reduction step was designed to standardize the cell volume to approximately 120 mL. In cases where the apheresis volume is less than 120 mL, the volume reduction step is not required and the cells proceed directly to the lymphocyte enrichment step. The volume reduction step standardizes the volume of cells received from each subject, retains mononuclear cells, achieves consistent cell yield and high cell viability, and maintains a closed system to minimize contamination risk.

[0126] Lymphocyte enrichment. Following the volume reduction step, the cells can be isolated using a cell processing device (such as Sepax) using a separation protocol developed and recommended by the device manufacturer (NeatCell procedure) and standard sterile tubing kits. ® 2 or equivalent). 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 Ficoll), and prepares the cells in growth medium to prepare for cell activation, achieving consistent cell yields and high cell viability. The closed system minimizes environmental contamination.

[0127] The process can be performed in an ISO 7 zone at ambient temperature, with all connections made using a sterile tubing welder, or in an ISO 5 laminar flow hood.

[0128] Lymphocyte Activation. The lymphocyte activation step can be performed with freshly processed cells from lymphocyte enrichment or with previously cryopreserved cells. In the case of cryopreserved cells, the cells can be thawed using a developed protocol prior to use.

[0129] The lymphocyte activation step selectively activates lymphocytes, making them susceptible to retroviral vector transduction, reducing the viable cell population of all other cell types, achieving consistent cell yields and high lymphocyte viability, and maintaining a closed system to minimize contamination risk. Lymphocyte activation can be achieved using lymphocyte stimulators such as anti-CD3 antibodies and IL-2. In some embodiments, the lymphocyte activation step occurs in a 100 μm spherical microporous filter having a volume of at least 700 cm 2 In a closed system with an internal surface area of ​​​​

[0130] Wash 1. After the lymphocyte activation step, the cells can be washed using a cell processing device such as Sepax ®2 or equivalent), wash the cells using fresh medium from the standard sterile kit using the manufacturer's protocol. Optionally, concentrate the cells to a final volume of approximately 100 mL in preparation for retroviral vector transduction. The Wash 1 step reduces process-related carryover (such as anti-CD3 antibodies), spent growth medium, and cell debris; achieves consistent cell yields and high T cell viability, maintaining a closed system to minimize contamination risk; and concentrates and delivers sufficient numbers of viable T cells in a small volume suitable for initiating transduction.

[0131] Transduction. The activated cells from the wash 1 step in fresh cell growth medium can be transferred to a cell culture bag (Origen Biomedical PL240 or similar) that has been previously transduced by first incubating with recombinant fibronectin or a fragment thereof (such as RetroNectin). ® (Takara Bio, Japan) and then incubated with the retroviral vector according to a defined procedure before introduction into activated cells. RetroNectin can be performed at a temperature of 2°C to 8°C. ® Coating (10µg / mL) lasts for 20±4 hours and is then incubated with thawed retroviral vector for approximately 180 to 210 minutes at 37±1°C and 5±0.5% CO2. After adding cells to the bag, transduction can be performed at 37±1°C and 5±0.5% CO2 for up to 20±4 hours. The retroviral transduction step involves culturing activated T cells in the presence of retroviral vector under controlled conditions to allow for efficient transduction, consistent cell yield and high cell viability, and maintaining a closed system to minimize contamination risk.

[0132] Wash 2. Following the retroviral transduction step, the cells can be transfected using a cell handling device (such as Sepax) in a standard sterile kit using the manufacturer's developed protocol. ® Wash 2 or equivalent equipment) with fresh growth medium and concentrate the cells to a final volume of approximately 100 mL in preparation for the expansion step. This Wash 2 step reduces process-related carryover, such as retroviral vector particles, vector production process residues, spent growth medium, and cell debris, enabling consistent cell yields and high cell viability; maintains a closed system to minimize contamination risk; and replaces spent growth medium with the target number of cells in a specified volume suitable for starting the expansion step.

[0133] Lymphocyte Expansion. Cells from the Wash 2 step can be aseptically transferred to a culture bag (Origen Biomedical PL325 or equivalent) and diluted with fresh cell growth medium and cultured at 37 ± 1°C and 5 ± 0.5% CO2 for approximately 72 hours. Starting on day 5, cell density is measured daily. Because the doubling time of T cells can vary slightly from subject to subject, additional growth time exceeding 72 hours (i.e., 3 to 6 days) may be required if the total cell number is insufficient to deliver the target dose of CAR-positive T cells per kilogram of subject body weight. The lymphocyte expansion step is designed to culture cells under controlled conditions in order to produce sufficient numbers of transduced cells for delivery of an efficacious dose, maintain a closed system to minimize the risk of contamination, and achieve consistent cell yield and high cell viability. One such efficacious dose or target dose includes 2 × 10 6 FMC63-28Z CAR-positive or FMC63-CD828BBZ CAR-positive T cells / kg (±20%) of subject body weight, generated by transduction with the MSGV-FMC63-28Z retroviral vector or the MSGV-FMC63-CD828BBZ retroviral vector, respectively, both of which are described in detail in the following references: Kochenderfer et al., J Immunother. 2009 Sep;32(7):689–702.

[0134] Wash 3 and concentration. Following the lymphocyte expansion step, the lymphocytes can be harvested using cell processing equipment (such as Sepax spp.) in a standard sterile kit using the manufacturer's developed protocol. ® 2 or equivalent), wash the cells with 0.9% saline and concentrate them to a final volume of approximately 35 mL in preparation for formulation and cryopreservation. The Wash 3 step is designed to reduce process-related carryover (such as from retroviral production), spent growth medium, and cell debris; achieve consistent cell yields and high cell viability; and maintain a closed system to minimize contamination risk.

[0135] Once the cells are concentrated and washed into 0.9% saline, the appropriate cell dose can be formulated to prepare the final cryopreserved product.

[0136] The embodiments described herein efficiently produced engineered lymphocyte therapy within 7 days.

[0137] Five-day lymphocyte production process

[0138] In some embodiments, a therapeutic cell product is produced after a 5-day process of preparing lymphocytes that have been transduced with a polynucleotide vector (e.g., a viral vector) encoding a therapeutic protein. The prepared lymphocytes can be used to treat various diseases, such as cancer, particularly when the therapeutic protein is a chimeric antigen receptor (CAR) or T cell receptor (TCR) designed to target cancer cells. This 5-day process is based on the 7-day process described above.

[0139] As used throughout the text, the terms "5-day process" and "5-day lymphocyte manufacturing process" are used interchangeably and refer to a CAR cell manufacturing process that requires approximately 5 days after the initial enrichment and activation steps. From the initial enrichment and activation steps to the harvest step, the 5-day process is 6 days in length and can be 6 to 9 days in total when the enrichment and activation steps are included.

[0140] During the 7-day process, lymphocytes were enriched and activated on day 0; the transduction bag was coated with recombinant fibronectin on day 1; viral transduction was performed on day 2; the transduced lymphocytes were washed and then expanded on days 3 and 4; expansion continued on days 5 and 6 with daily medium changes; and the final cell product was harvested on day 7. 9 lymphocytes, about 2.4×10 8 Lymphocytes were incubated with the viral vectors used for transduction.

[0141] During the 5-day process, no changes were made to the procedure on Day 0. However, on Days 1 and 2, larger bags were used. Instead of using an Origen Biomedical PL240 bag for transduction, an Origen Biomedical PL325 bag, or more preferably a PL750 bag, was used. The larger bag allowed for the use of a larger volume of vector (200 mL instead of 100 mL) and a larger number of lymphocytes (at 3.2 × 10 8 and 6×10 8 between, rather than 2.4×10 8 ).

[0142] Interestingly, increasing the transduction volume and adding more vector and starting lymphocytes did not result in an unacceptable decrease in transduction efficiency (54% to 35.15%) or cell viability (92% to 92.4%). Thus, the cell expansion step, which typically takes 4 days over a 7-day process, could be reduced to 2 days, allowing the final cell product to be harvested on day 5.

[0143] However, the modest reduction in transduction rate was not associated with clinical efficacy or patient safety. Equally important, the cell product from the 5-day process was found to include an increased percentage of young cells in both the CD4+ T and CD8+ T cell populations, which is believed to correlate with improved therapeutic efficacy. It is important to note that these changes are within the historical range for donor runs collected from the 7-day process.

[0144] The shortened 5-day process meets regulatory requirements for transduction efficiency, therapeutic efficacy, and safety. At the same time, the 5-day product exhibits a more juvenile phenotype within the historical range.

[0145] Three-day lymphocyte production process

[0146] In some embodiments, a therapeutic cell product is produced after a 3-day process of preparing lymphocytes that have been transduced with a polynucleotide vector (e.g., a viral vector) encoding a therapeutic protein. The prepared lymphocytes can be used to treat various diseases, such as cancer, particularly when the therapeutic protein is a chimeric antigen receptor (CAR) or T cell receptor (TCR) designed to target cancer cells. The 3-day process is based on the 7-day and 5-day processes described above.

[0147] As used throughout the text, the terms "3-day process" and "3-day lymphocyte manufacturing process" are used interchangeably and refer to a CAR cell manufacturing process that takes about 3 days starting from the initial enrichment and activation steps. The length of the 3-day process is about 4 days from the initial enrichment and activation steps to the harvest step. The 3-day process does not include the cell expansion step, which includes one or more days after the transduction step and before the harvest step.

[0148] In the 3-day process, the procedure for Day 0 to Day 1 is similar to the 5-day process, including fibronectin coating of larger bags (e.g., Origen Biomedical PL325 or preferably PL750) for subsequent transduction on Day 2. However, in some embodiments, only approximately 4.8 × 10 8 Lymphocytes, the same amount of viral vector (200 mL) was used. Alternatively, in some embodiments, about 6×10 8 Lymphocytes can be used with 200 mL of viral vector for the transduction step. Alternatively, in some embodiments, only about 4.8 × 10 8 Each lymphocyte can be used with 100 mL of viral vector for the transduction step. Another important difference is that, unlike the 5-day and 7-day processes, a step-down step is performed for T cell expansion. Instead, transduced lymphocytes can be harvested on day 3, allowing the entire process to be completed within 3 days from the initial enrichment and activation steps.

[0149] Given the lack of a specific T cell expansion step in the 3-day process, the harvested cell product includes a slightly smaller percentage of T cells (CD3+). Importantly, however, the 3-day product can include an even greater percentage of young (naive) T cells compared to the 7-day process.

[0150] Although not tested with lymphocytes from the same donor, the data indicate that the percentage of naive T cells from the 3-day process was significantly higher than that from the 5-day process. Among CD4+ T cells, the 3-day process produced approximately 55.75% naive T cells, while the 5-day process produced approximately 40.65%; among CD8+ T cells, the 3-day process produced approximately 37.35% naive T cells, while the 5-day process produced approximately 3.93%.

[0151] In other words, a 3-day course can produce an approximately 1.4-fold increase in the percentage of CD4+ naive T cells compared to the percentage of such cells observed over a 5-day course, and an approximately 9.5-fold increase in the percentage of CD8+ naive T cells compared to the percentage of such cells observed over a 5-day course. Additionally, a 3-day course can produce an approximately 3.0-fold increase in the percentage of such cells compared to the historical average number of CD4+ naive T cells observed over a 7-day course, and an approximately 18.0-fold increase in the percentage of such cells compared to the historical average number of CD8+ naive T cells observed over a 7-day course.

[0152] In contrast, in CD4+ T cells, a 3-day process can only produce about 4.35% of effector memory T cells, while a 5-day process can produce about 8.55%; in CD8+ T cells, a 3-day process can only produce about 9.85% of effector memory T cells, while a 5-day process can produce about 15.85%.

[0153] In other words, the percentage of CD4+ effector memory T cells produced over a 3-day course can be reduced by about 2.0-fold compared to the percentage of CD8+ effector memory T cells observed over a 5-day course, and the percentage of such cells can be reduced by about 3.6-fold compared to the percentage of CD4+ effector memory T cells observed over a 7-day course. Additionally, the percentage of such cells produced over a 3-day course can be reduced by about 6.0-fold compared to the historical average number of CD4+ effector memory T cells observed over a 7-day course, and the percentage of such cells can be reduced by about 3.5-fold compared to the historical average number of CD8+ effector memory T cells observed over a 7-day course.

[0154] Cell viability measurements showed that cell viability remained high (>90%) throughout the 3-day process, with the wash step resulting in the greatest decrease in cell viability on day 2. In contrast, the 5-day process and the 7-day process included additional wash steps, each of which resulted in an additional decrease in cell viability.

[0155] Thus, according to one embodiment of the present disclosure, a method for preparing transduced lymphocytes with improved efficacy or reduced adverse effects in treating cancer is provided. In some embodiments, the method entails obtaining lymphocytes from a patient by apheresis; incubating the lymphocytes with a polynucleotide vector to transduce the lymphocytes, thereby producing transduced lymphocytes; culturing the transduced lymphocytes; and infusing the transduced lymphocytes into the patient. In some embodiments, the method entails shortening the manufacturing time of the therapeutic cell product (i.e., the transduced lymphocytes) by using any of the 7-day, 5-day, or 3-day manufacturing processes described above.

[0156] In some embodiments, the time it takes from harvesting lymphocytes to infusing the transduced lymphocytes (venovenous time) is reduced. The venovenous time can be determined by a variety of factors, such as the efficiency of cell transduction and expansion, and the quality and quantity of the starting apheresis material. In some embodiments, the venovenous time can be predicted taking into account such factors. According to one embodiment of the present technology, the predicted venovenous time is reduced by at least 1 day. In another embodiment, the predicted venovenous time is reduced by at least 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or 21 days.

[0157] In some embodiments, the veno-venous time is no longer than 28 days. In some embodiments, the time taken from obtaining lymphocytes to infusion of the transduced lymphocytes is no longer than 27 days. In some embodiments, the veno-venous time is no longer than 26 days. In some embodiments, the time taken from obtaining lymphocytes to infusion of the transduced lymphocytes is no longer than 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.

[0158] The venovenous time can also depend on the time from the start of LD chemotherapy to the infusion. Thus, in some embodiments, the venovenous time can be reduced by reducing the time from the start of LD chemotherapy to the infusion. In some embodiments, the time from the start of LD chemotherapy to the infusion is no more than 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day. In some embodiments, the time from the start of LD chemotherapy to the infusion is no more than 5 days.

[0159] Using this reduced veno-veno time, the method can produce transduced lymphocytes such that patients receiving the infusion have a greater than 55% likelihood of a complete response (CR). In some embodiments, the patient has a greater than 51%, or 52%, 53%, 54%, 56%, 57%, 58%, 59%, or 60% likelihood of having a complete response.

[0160] In some embodiments, the patient has a greater than 45% likelihood of having overall survival (OS) measured at 24 months (post-infusion). In some embodiments, the patient has a greater than 39%, or 40%, 41%, 42%, 43%, 44%, 46%, 47%, 48%, 49%, 50%, 51%, 52% or 53% likelihood of having overall survival (OS) at 24 months (post-infusion).

[0161] In some embodiments, the patient has a likelihood of developing long-term thrombocytopenia of less than 30%. In some embodiments, the patient has a likelihood of developing long-term thrombocytopenia of less than 32%, 31%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20% or 19%.

[0162] The term "complete response" (CR) refers to a treatment outcome in which a patient treated with evaluable but non-measurable disease has resolved their tumor and all evidence of disease. CR rates can be determined by methods known in the art (e.g., Cheson et al., J Clin Oncol, 2014). In some embodiments, CR rates can be assessed based on the response achieved after initial administration of the product and prior to any subsequent treatment (e.g., retreatment with the product, subsequent hematopoietic stem cell transplantation, and / or other anti-cancer therapies) that may be administered in response to relapse or disease progression.

[0163] The term "overall survival" means that at the time of measurement, the patient has not died from any cause.

[0164] "Thrombocytopenia" is a condition characterized by abnormally low levels of platelets (also called thrombocytes) in the blood. A normal human platelet count ranges from 150,000 to 450,000 platelets per microliter of blood. In patients with thrombocytopenia, the platelet count can drop below 50,000 per microliter. "Long-term thrombocytopenia" refers to a condition in which a patient has thrombocytopenia for at least 30 days after the initial transfusion.

[0165] According to one embodiment of the present disclosure, the accelerated manufacturing process includes an improved transduction / culture procedure. In some embodiments, the transduction / culture procedure requires incubating a lymphocyte sample with a polynucleotide vector to transduce the lymphocytes to produce transduced lymphocytes, and culturing the sample containing the transduced lymphocytes before harvesting the lymphocytes to produce a harvested sample.

[0166] In some embodiments, the culturing step is shortened compared to a conventional process that requires about 4 days. In some embodiments, the culturing step 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.

[0167] In some embodiments, the time for the culturing step is counted from the completion of the transduction step (eg, removal of cells from a system with immobilized vectors) to the harvesting of cells for storage, transport, or clinical use.

[0168] The culture of transduced lymphocytes can be carried out in culture media and under conditions known in the art. In some embodiments, the culture of transduced lymphocytes can be carried out at a certain temperature and / or in the presence of CO2. In certain embodiments, the temperature can 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 can be about 34°C to 39°C. In certain embodiments, the predetermined temperature can be about 35°C to 37°C. In certain embodiments, the preferred predetermined temperature can be about 36°C to 38°C. In certain embodiments, the preferred predetermined temperature can be about 36°C to 37°C, or more preferably about 37°C.

[0169] In some embodiments, the culture of transduced lymphocytes can be carried out in the presence of a predetermined level of CO . In certain embodiments, the predetermined level of CO can be 1.0% to 10% CO . In certain embodiments, the predetermined level of CO can 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% CO . In certain embodiments, the predetermined level of CO can be about 4.5% to 5.5% CO . In certain embodiments, the predetermined level of CO can be about 5% CO . In certain embodiments, the predetermined level of CO can be about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5% or about 6.5% CO . In some embodiments, the step of amplifying the transduced T cell population can be performed in any combination at a predetermined temperature and / or in the presence of a predetermined level of CO . For example, in one embodiment, the step of expanding the transduced T cell population can include a predetermined temperature of about 36° C. to 38° C. and in the presence of a predetermined level of CO 2 of about 4.5% to 5.5% CO 2 .

[0170] Any suitable medium T cell growth medium can be used to culture cells in suspension. For example, T cell growth medium can include, but is not limited to, a sterile low glucose solution comprising appropriate amounts of a buffer, magnesium pyruvate, calcium pyruvate, sodium pyruvate, and sodium bicarbonate. In one embodiment, the medium is OpTmizer ™ (Life Technologies), but those skilled in the art will understand how to generate similar culture media.

[0171] The incubation (and / or transduction) step can be performed without limitation in a closed system. In certain embodiments, the closed system is a cell culture bag using any suitable cell culture bag (e.g., Mitenyi Biotec MACS ® GMP cell differentiation bags, OrigenBiomedical PermaLife ™ In some embodiments, the closed system has a volume of at least 500 cm 2 In some embodiments, the closed system has an internal surface area of ​​at least 1000 cm 2 , 1200cm 2 , 1400cm 2 , 1500cm 2 , 1600cm 2 , 1800cm 2 , 2000cm 2 , 2200cm 2 , 2500cm 2or 3000cm 2 In some embodiments, the closed system has an internal surface area of ​​no more than 1500 cm 2 , 1600cm 2 , 1800cm 2 , 2000cm 2 , 2200cm 2 , 2500cm 2 or 3000cm 2 internal surface area.

[0172] In some embodiments, the cell culture bag used in the closed system is coated with recombinant human fibronectin. The recombinant human fibronectin fragment can include three functional domains: a central cell binding domain, a heparin binding domain II, and a CS1 sequence. Recombinant human fibronectin or its fragment can 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. ®(TakaraBio, Japan). In certain embodiments, the cell culture bag can be coated with a recombinant human fibronectin fragment at a concentration of about 0.1µg / mL to 60µg / mL, preferably 0.5µg / mL to 40µg / mL. In certain embodiments, the cell culture bag can be coated with a recombinant human fibronectin fragment at a concentration of about 0.5µg / mL to 20µg / mL, 20µg / mL to 40µg / mL, or 40µg / mL to 60µg / mL. In certain embodiments, the cell culture bags may be coated with about 0.5 µg / mL, 1 µg / mL, about 2 µg / mL, about 3 µg / mL, about 4 µg / mL, about 5 µg / mL, about 6 µg / mL, about 7 µg / mL, about 8 µg / mL, about 9 µg / mL, about 10 µg / mL, about 11 µg / mL, about 12 µg / mL, about 13 µg / mL, about 14 µg / mL, about 15 µg / mL, about 16 µg / mL, about 17 µg / mL, about 18 µg / mL, about 19 µg / mL, or about 20 µg / mL of a recombinant human fibronectin fragment. In certain embodiments, the cell culture bag can be coated with about 2µg / mL to 5µg / mL, about 2µg / mL to 10µg / mL, about 2µg / mL to 20µg / mL, about 2µg / mL to 25µg / mL, about 2µg / mL to 30µg / mL, about 2µg / mL to 35µg / mL, about 2µg / mL to 40µg / mL, about 2µg / mL to 50µg / mL, or about 2µg / mL to 60µg / mL of a recombinant human fibronectin fragment. In certain embodiments, the cell culture bag can be coated with at least about 2µg / mL, at least about 5µg / mL, at least about 10µg / mL, at least about 15µg / mL, at least about 20µg / mL, at least about 25µg / mL, at least about 30µg / mL, at least about 40µg / mL, at least about 50µg / mL, or at least about 60µg / mL of a recombinant human fibronectin fragment. In certain embodiments, the cell culture bag can be coated with at least about 10 μg / mL of a recombinant human fibronectin fragment.

[0173] In some embodiments, a transduction enhancer is introduced into the closed system. Non-limiting examples of such transduction enhancers include Vectofusin ™ Transduction mix.

[0174] In certain embodiments, the cell culture bags used in the closed bag culture system can be sealed with human albumin serum (HSA). In alternative embodiments, the cell culture bags are not sealed with HSA.

[0175] 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. This immobilization can improve transduction efficiency once cells are added.

[0176] In some embodiments, the vector can be a viral vector, such as a lentiviral vector and a retroviral vector. Several recombinant viruses have been used as viral vectors to deliver genetic material to cells. The viral vector that can be used in the transduction step can be any tropic or bitropic viral vector, including but not limited to recombinant retroviral vectors, recombinant lentiviral vectors, recombinant adenoviral vectors, and recombinant adeno-associated virus (AAV) vectors. In one embodiment, the viral vector is an MSGV1 gamma-retroviral vector. In some embodiments, the vector is a non-viral vector.

[0177] In some embodiments, a total volume of at least 100 mL of a solution containing the carrier is used. In some embodiments, a total volume of at least 100 mL of a solution containing the carrier is used. In some embodiments, a total volume of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, or 400 mL of a solution containing the carrier is used. In some embodiments, a total volume of no more than 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 of a solution containing the vector is used.

[0178] In some embodiments, the carrier solution comprises 1×10 3 Transducing units per milliliter (TU / ml) up to 1 × 10 12 Transducing units per milliliter (TU / ml) of viral vector.

[0179] Once the closed system is coated with recombinant fibronectin and the carrier is immobilized, the carrier solution can be removed. In some embodiments, the closed system does not include recombinant fibronectin. In some embodiments, removal of the carrier solution is performed by gravity or syringe discharge, which helps to retain the immobilized carrier on the inner surface while removing impurities.

[0180] Lymphocyte transduction can be performed in a coated closed system with a fixed carrier. In some embodiments, transduction is performed using a sample containing lymphocytes. In some embodiments, the sample includes at least 2.5×10 7 In some embodiments, the sample includes at least 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 , 1×10 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 , 3×10 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 , 5.7×108 , 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 In some embodiments, the sample includes no more than 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 , 6.1×108 , 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 lymphocytes (e.g., T cells).

[0181] The lymphocytes used in the presently disclosed methods are typically obtained from a donor subject, which can be a cancer patient to be treated with a cell population produced by the methods described herein (i.e., an autologous donor), or an individual who donates a lymphocyte sample (i.e., an allogeneic donor) that will be used to treat a different individual or cancer patient after generating a cell population produced by the methods described herein. Lymphocytes can be obtained from a donor subject by any suitable method used in the art. For example, lymphocytes can be obtained by any suitable in vitro method, venipuncture, or other blood collection method by which a blood sample and / or lymphocytes are obtained. In one embodiment, lymphocytes are obtained by apheresis.

[0182] Optionally, in some embodiments, the methods described herein further comprise the step of enriching the lymphocyte population obtained from the donor subject prior to transduction. Enrichment of lymphocytes can be accomplished by any suitable separation method, including but not limited to the use of a separation medium (e.g., Ficoll-Paque ™ 、RosetteSep ™ HLA Total Lymphocyte Enrichment Cocktail, Lymphocyte Separation Medium (LSA) (MP Biomedical Cat. No. 0850494X), etc.), non-ionic iodixanol-based media such as OptiPrep ™ etc.), cell size, shape, or density separation by filtration or panning, 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.

[0183] Optionally, in some embodiments, the CD4 + / CD8 +The cells are positively enriched to remove circulating lymphoma cells from the sample. In some such embodiments, after incubation with the selection agent, the incubated cells (including cells to which the selection agent has bound) are transferred to a system for immunoaffinity-based cell separation. In some embodiments, the system for immunoaffinity-based separation is or comprises a magnetic separation column.

[0184] In some such embodiments, the separation method includes separating different cell types based on the expression or presence of one or more specific molecules in the cell, such as surface markers, for example, surface proteins, intracellular markers or nucleic acids. In some embodiments, any known separation method based on such markers can be used. In some embodiments, separation is based on the separation of affinity or immunoaffinity. For example, separation in some embodiments includes separating cells and cell colonies based on the cellular expression or expression level of one or more markers (typically cell surface markers), such as by incubating with antibodies or binding partners that specifically bind to these markers, followed by a washing step and separating cells that have bound the antibody or binding partner from cells that have not bound the antibody or binding partner. Such separation steps can be based on positive selection and / or negative selection, retaining cells that have bound the reagent for further use in the positive selection and retaining cells that have not bound the antibody or binding partner in the negative selection. In some examples, two parts are retained for further use.

[0185] In some such embodiments, negative selection may be particularly useful when antibodies that specifically identify cell types within a heterogeneous population are not available, such that separation is best based on markers expressed by cells other than the desired population.

[0186] Separation need not result in 100% enrichment or depletion of a particular cell population or cells expressing a particular marker. For example, positive selection or enrichment of a particular type of cell (such as cells expressing a marker) refers to 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, positive selection, depletion, or depletion of a particular type of cell (such as cells expressing a marker) refers to decreasing the number or percentage of such cells, but does not necessarily result in the complete removal of all such cells.

[0187] In some examples, multiple rounds of separation steps are performed, wherein the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells that simultaneously express multiple markers, such as by incubating the cells with multiple antibodies or binding partners, each of which is specific for the marker targeted by the negative selection. Similarly, multiple cell types can be positively selected simultaneously by incubating the cells with multiple antibodies or binding partners expressed on multiple cell types.

[0188] For example, in some embodiments, specific subpopulations of T cells, such as cells that are positive 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, are isolated by positive or negative selection techniques. For example, anti-CD3 / anti-CD28 conjugated magnetic beads (e.g., DYNABEADS ® M-450 CD3 / CD28 T cell expander) to positively select CD3+, CD28+ T cells. In some embodiments, the cell population is enriched for T cells with a naive phenotype (CD45RA+ CCR7+).

[0189] 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 accomplished by incubating the cells with one or more antibodies or other binding agents that specifically bind to one or more surface markers expressed (marker+) or expressed at relatively high levels (markerhigh) on the positively or negatively selected cells, respectively.

[0190] In specific embodiments, a biological sample (e.g., a sample of PBMCs or other white blood cells) is subjected to selection for CD4+ T cells, wherein both the negative and positive fractions are retained. In certain embodiments, CD8+ T cells are selected from the negative fraction. In some embodiments, a biological sample is subjected to selection for CD8+ T cells, wherein both the negative and positive fractions are retained. In certain embodiments, CD4+ T cells are selected from the negative fraction.

[0191] In some embodiments, T cells are isolated from PBMC samples by negative selection for markers expressed on non-T cells such as B cells, monocytes or other white blood cells such as CD14. In some embodiments, CD4+ or CD8+ selection steps are used to separate CD4+ helper T cells and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into subpopulations by positive or negative selection for markers expressed or expressed at a relatively high level on one or more naive, memory and / or effector T cell subsets.

[0192] In one example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibodies or binding partners are bound to a solid support or matrix, such as magnetic or paramagnetic beads, to allow separation of cells for positive and / or negative selection. For example, in some embodiments, cells and cell populations are separated or separated 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 magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., such as Dynabeads ™ The magnetically responsive material (e.g., particle) is typically attached directly or indirectly to a binding partner (e.g., antibody) that specifically binds to a molecule (e.g., surface marker) present on one or more cells or cell populations for which isolation (e.g., negative or positive selection) is desired.

[0193] In some embodiments, the magnetic particles or beads comprise a magnetically responsive material bound to a specific binding member (such as an antibody or other binding partner). Many well-known magnetically responsive materials are used in magnetic separation methods. Incubation is typically performed under conditions in which the antibody or binding partner, or a molecule that specifically binds to such an antibody or binding partner attached to the magnetic particles or beads (such as a secondary antibody or other reagent), specifically binds to the cell surface molecule (if present on cells within the sample). In some embodiments, the sample is placed in a magnetic field, and those cells to which the magnetically responsive or magnetizable particles are attached are attracted to the magnet and separated from unlabeled cells. For positive selection, cells attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained. In some embodiments, a combination of positive and negative selection is performed during the same selection step, with the positive and negative fractions retained and further processed or subjected to further separation steps. In some embodiments, the magnetically responsive particles are coated in a primary antibody or other binding partner, a secondary antibody, a lectin, an enzyme, or streptavidin. In certain embodiments, the magnetic particles are attached to the cells via a coating of a primary antibody specific for one or more markers. In certain embodiments, cells (rather than beads) are labeled with a primary antibody or binding partner, and magnetic particles coated with a cell type-specific secondary antibody or other binding partner (e.g., streptavidin) are then added. In certain embodiments, streptavidin-coated magnetic particles are used in conjunction with a biotinylated primary or secondary antibody. In some embodiments, the magnetically responsive particles are attached to cells that are subsequently incubated, cultured, and / or engineered; in some embodiments, the particles remain attached to the 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 competing, unlabeled antibodies, and magnetizable particles or antibodies conjugated to cleavable linkers. In some embodiments, the magnetizable particles are biodegradable.

[0194] In some embodiments, affinity-based selection is performed via magnetic-activated cell sorting (MACS) (Miltenyi Biotec, Auburn, CA). The magnetic-activated cell sorting (MACS) system enables high-purity selection of cells attached to magnetized particles. In certain embodiments, MACS operates in a mode in which non-target and target materials are eluted sequentially after application of an external magnetic field. That is, cells attached to the magnetized particles are held in place, while unattached materials are eluted. Then, after this first elution step is complete, materials trapped in the magnetic field and prevented from eluting are released in a manner that allows them to be eluted and recovered. In certain embodiments, non-target cells are labeled and removed from the heterogeneous cell population.

[0195] In some embodiments, use the system, device or equipment of one or more steps in the separation, cell preparation, separation, processing, incubation, cultivation and / or formulation step of carrying out these methods to separate or separate.In some embodiments, this system is for carrying out each step in these steps in a closed or sterile environment, for example, to minimize error, user processing and / or pollution.In one example, system is the system as described in International Patent Application Publication No. WO 2009 / 072003 or US20110003380 A1, and this International Patent Application is incorporated herein by reference separately.In some embodiments, system or equipment are with integrated or independent system and / or carry out one or more (for example, all) separation, processing, engineering approaches and formulation step in an automatic or programmable manner.In some embodiments, system or equipment comprise the computer and / or computer program communicated with this system or equipment, which allows the user to program, control the various embodiments of processing, separation, engineering approaches and formulation step, assess their result and / or adjust the various embodiments of processing, separation, engineering approaches and formulation step. In some embodiments, separation and / or other steps are performed using a CliniMACS system (Miltenyi Biotec), for example, which is used to automate cell separation at a clinical scale in a closed, sterile system. Components may include an integrated microcomputer, a magnetic separation unit, a peristaltic pump, and various pinch valves. In some embodiments, the integrated computer controls the instrument components and directs 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 selecting a column. The peristaltic pump controls the flow rate throughout the tubing set and, together with the pinch valves, ensures controlled flow of buffer through the system and continuous suspension of the cells.

[0196] In some embodiments, the CliniMACS system uses magnetizable particles coupled to antibodies provided in a sterile, pyrogen-free solution. In some embodiments, after labeling cells with magnetic particles, the cells are washed to remove excess particles. The cell preparation bag is then connected to a tubing set, which is in turn connected to a bag containing a buffer and a cell collection bag. The tubing set consists of pre-assembled sterile tubing, including a pre-column and a separation column, and is only for single use. After starting the separation procedure, the system automatically applies the cell sample to the separation column. The labeled cells remain in the column, while the unlabeled cells are removed by a series of washing steps. In some embodiments, the cell colony used for the method described herein is not labeled and is not retained in the column. In some embodiments, the cell colony used for the method described herein is labeled and retained in the column. In some embodiments, the cell colony used for the method described herein is eluted from the column after removing the magnetic field and is collected in a cell collection bag.

[0197] In certain embodiments, separation and / or other steps are performed using the CliniMACS Prodigy system (Miltenyi Biotec). In some embodiments, the CliniMACS Prodigy system is equipped with a cell processing unit that allows for automated washing and fractionation of cells by centrifugation. The CliniMACS Prodigy system can also include an onboard camera and image recognition software to determine the optimal cell fractionation endpoint by identifying macroscopic layers of source cell products. For example, peripheral blood is automatically separated into red blood cells, white blood cells, and plasma layers. The CliniMACS Prodigy system can also include an integrated cell culture chamber that completes cell culture protocols such as cell differentiation and expansion, antigen loading, and long-term cell culture. Input ports can allow for aseptic removal and replenishment of culture medium, and cells can be monitored using an integrated microscope.

[0198] In some embodiments, the cell populations described herein are collected and enriched (or depleted) by flow cytometry, wherein cells stained for multiple cell surface markers are carried in the fluid stream. In some embodiments, the cell populations described herein are collected and enriched (or depleted) by preparative scale (FACS) sorting. In certain embodiments, the cell populations described herein are collected and enriched (or depleted) by using a combination of a microelectromechanical system (MEMS) chip and a FACS-based detection system (see, e.g., WO 2010 / 033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. 1(5):355-376). In both cases, cells can be labeled with multiple markers, allowing the isolation of well-defined T cell subsets with high purity.

[0199] In some embodiments, the antibody or binding partner is labeled with one or more detectable markers to facilitate separation of positive and / or negative selection. For example, separation can be based on binding to a fluorescently labeled antibody. In some examples, cell separation based on binding of an antibody or other binding partner specific for one or more cell surface markers is performed in a fluid flow, such as by fluorescence activated cell sorting (FACS), including preparative (FACS) and / or microelectromechanical systems (MEMS) chips, for example, in combination with a flow cytometry detection system. Such methods allow for simultaneous positive and negative selection based on multiple markers.

[0200] In some embodiments, at least 0.5×10 9 In some embodiments, at least 0.6×10 lymphocytes are obtained from a donor and optionally enriched and / or stimulated. 9 , 0.7×109 , 0.8×10 9 , 0.9×10 9 , 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 In some embodiments, no more than 1×10 lymphocytes are obtained from a donor. 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, and optionally enrich and / or stimulate them.

[0201] Optionally, the methods described herein further comprise the step of stimulating lymphocytes with one or more lymphocyte stimulators. In some embodiments, stimulation is performed before the transduction step. In some embodiments, stimulation is performed after the transduction step.

[0202] Any combination of one or more suitable lymphocyte stimulators can be used to stimulate (activate) lymphocytes. Non-limiting examples include antibodies or functional fragments thereof that target T cell stimulatory or costimulatory molecules (e.g., anti-CD2 antibodies, anti-CD3 antibodies, anti-CD28 antibodies, or functional fragments thereof), 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 alpha (TNFα)), or any other suitable mitogen (e.g., tetradecanoylphorbol acetate (TPA), phytohemagglutinin (PHA), concanavalin A (conA), lipopolysaccharide (LPS), pokeweed mitogen (PWM)), or natural ligands of T cell stimulatory or costimulatory molecules. In some embodiments, the stimulator is an anti-CD3 antibody and / or an anti-CD28 antibody.

[0203] In some embodiments, the steps of stimulating lymphocytes described herein may require stimulating lymphocytes with one or more stimulating agents at a predetermined temperature, within a predetermined amount of time, 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°C to 39°C. In certain embodiments, the step of stimulating lymphocytes includes stimulating lymphocytes with one or more stimulating agents for a predetermined time. In certain embodiments, the predetermined time for stimulation may be about 24 hours to 72 hours. In certain embodiments, the predetermined time for stimulation may be about 24 hours to 36 hours. In certain embodiments, the step of stimulating lymphocytes may include stimulating lymphocytes with one or more stimulating agents in the presence of a predetermined level of CO2. In certain embodiments, the predetermined level of CO2 may be about 1.0% to about 10% CO2. In certain embodiments, the predetermined level of CO2 for stimulation can 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.

[0204] 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 anti-CD3 and anti-CD28 antibodies may be used in conjunction with the step of stimulating a lymphocyte population. 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 aspects, antibodies can be purchased commercially from suppliers known in the art, including, but not limited to, Technion Biotech, BD Biosciences (e.g., 1 mg / mL pure MACS GMP CD3, part number 170-076-116), and eBioscience. Furthermore, one skilled in the art will understand how to generate anti-CD3 and / or anti-CD28 antibodies using standard methods. Any antibodies used in the methods described herein should be produced under Good Manufacturing Practice (GMP) in compliance with relevant agency guidelines for biological products.

[0205] In certain embodiments, the T cell stimulator may include an anti-CD3 antibody or an anti-CD28 antibody at a concentration of about 20 ng / mL to 100 ng / mL. In certain embodiments, the concentration of the anti-CD3 antibody 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.

[0206] As demonstrated in the 7-day, 5-day, and 3-day processes described above, the prepared lymphocytes include a higher ratio of young cells (e.g., naive T cells). Thus, one embodiment of the present disclosure provides a lymphocyte population comprising CD4+ T and CD8+ T cells prepared by the present method.

[0207] In some embodiments, at least 20% of the CD4+ T cells are naive T cells. In some embodiments, at least 25%, 30%, 35%, 40%, 45%, 50%, 55% or 60% of the CD4+ T cells are naive T cells.

[0208] In some embodiments, no more than 25% of CD4+ T cells are effector memory T cells. In some embodiments, no more 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.

[0209] In some embodiments, no more than 44% of CD4+ T cells are central memory T cells. In some embodiments, no more than 43%, 42%, 41%, or 40% of CD4+ T cells are central memory T cells.

[0210] In some embodiments, no more than 1.5% of CD4+ T cells are effector T cells. In some embodiments, no more 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.

[0211] In some embodiments, at least 5% of the CD8+ T cells are naive T cells. In some embodiments, at least 10%, 15%, 20%, 25%, 30% or 35% of the CD8+ T cells are naive T cells.

[0212] In some embodiments, no more than 30% of the CD8+ T cells are effector memory T cells. In some embodiments, no more than 28%, 27%, 25%, 22%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, or 10% of the CD8+ T cells are effector memory T cells.

[0213] In some embodiments, no more than 60% of the CD8+ T cells are central memory T cells. In some embodiments, no more than 58%, 56%, 55%, 54%, 52% or 50% of the CD8+ T cells are central memory T cells.

[0214] As is well known in the art, each type of T cell can be characterized by cell surface markers. 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-.

[0215] Surface markers of stem cell 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-.

[0216] Surface markers for effector memory T cells (TEM) include, but are not limited to, CCR7-, CD45RO+, and CD95+. An additional marker for effector memory T cells is IL-2Rβ+. For central memory T cells (TCM), suitable markers include CD45RO+, CD95+, IL-2Rβ+, CCR7+, and CD62L+. For effector T cells (Teff), suitable markers include, but are not limited to, CD45RA+, CD95+, IL-2Rβ+, CCR7-, and CD62L-.

[0217] The harvested lymphocytes preferably include a substantial 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 harvested lymphocytes are CD3+ T cells.

[0218] The harvested lymphocytes preferably include a substantial proportion that have been transduced. In some embodiments, at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the harvested lymphocytes are transduced with the vector. In some embodiments, each transduced lymphocyte includes at least one copy of the vector (or the coding sequence included therein) integrated into the host genome. In some embodiments, each transduced lymphocyte includes at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the vector integrated into the host genome.

[0219] In some embodiments, the vector includes a transgene encoding a polypeptide. The polypeptide may be a CAR or a TCR, but is not limited thereto. In some embodiments, the CAR or TCR includes an antigen binding molecule. In some embodiments, the antigen binding molecule has binding specificity for an antigen portion. In some embodiments, the antigen binding molecule has binding specificity for one or more antigen portions (e.g., 1, 2, 3, or 4 antigen portions). In some embodiments, the antigen binding molecule has binding specificity for two different antigen portions.

[0220] In some aspects, the antigenic portion is associated with cancer or cancer cells. Such antigenic portions may include, but are not limited to, 707-AP (707 Alanine Proline), AFP (α (a)-fetoprotein), ART-4 (adenocarcinoma antigen recognized by T4 cells), BAGE (B antigen; b-catenin / m, b-catenin / mutated), BCMA (B cell maturation antigen), Bcr-abl (cluster of differentiation region-Abelson), CAIX (carbonic anhydrase IX), CD19 (cluster of differentiation 19), CD20 (cluster of differentiation 20), CD22 (cluster of differentiation 22), CD30 (cluster of differentiation 30), CD33 (cluster of differentiation 33), CD44v7 / 8 (cluster of differentiation 44, exon 7 / 8), CAMEL (antigen recognized by CTLs on melanoma), CAP-1 (carcinoembryonic antigen peptide 1), CASP-8 (caspase 8), CDC27m (mutated cyclin 27), CDK4 / m (mutated cyclin-dependent kinase 4), CEA (carcinoembryonic antigen), CT (carcinoma / testis (antigen)), Cyp-B (cyclophilin B ), DAM (differentiation antigen on melanoma), EGFR (epidermal growth factor receptor), EGFRvIII (epidermal growth factor receptor variant III), EGP-2 (epidermal glycoprotein 2), EGP-40 (epidermal glycoprotein 40), Erbb2, 3, 4 (erythroblastic leukemia viral oncogene homolog-2, -3, 4), ELF2M (mutated elongation factor 2), 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 (disialoganglioside 2), GD3 (disialoganglioside 3), GnT-V (N-acetylglucosamine transferase V), Gp100 (glycoprotein 100kD), HAGE (helicase antigen), HER-2 / neu (human epidermal receptor 2 / neural;Also known as EGFR2), HLA-A (human leukocyte antigen A), HPV (human papillomavirus), HSP70-2M (mutated heat shock protein 70-2), HST-2 (human cyclic tumor factor 2), hTERT or hTRT (human telomerase reverse transcriptase), iCE (intestinal carboxylesterase), IL-13R-a2 (interleukin-13 receptor subunit alpha-2), KIAA0205, KDR (kinase insert domain receptor), kappa light chain, LAGE (L antigen), LDLR / FUT (low-density lipoprotein receptor / GDP-L-fucose: bD-galactosidase 2-aL Fucosyltransferase), LeY (Lewis-Y antibody), L1CAM (L1 cell adhesion molecule), MAGE (melanoma antigen), MAGE-A1 (melanoma-associated antigen 1), mesothelin, murine CMV-infected cells, MART-1 / Melan-A (melanoma antigen 1 recognized by T cells / melanoma antigen A), MC1R (melanocortin 1 receptor), myosin / m (mutated myosin), MUC1 (mucin 1), MUM-1, -2, -3 (melanoma ubiquitously mutated proteins 1, 2, 3), NA88-A (NA from patient M88) cDNA clone), NKG2D (natural killer group 2, member D) ligand, NY-BR-1 (New York breast differentiation antigen 1), NY-ESO-1 (New York esophageal squamous cell carcinoma-1), carcinoembryonic antigen (h5T4), P15 (protein 15), p190 smaller bcr-abl (190KD protein bcr-abl), Pml / RARa (promyelocytic leukemia / retinoic acid receptor alpha), PRAME (preferentially expressed antigen in melanoma), PSA (prostate-specific antigen), PSCA (prostate stem cell antigen), PSMA (prostate-specific membrane antigen), RAGE (renal antigen), RU1 or RU2 (renal ubiquitin 1 or 2), SAGE (sarcoma antigen), SART-1 or SART-3 (squamous antigen for tumor rejection 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 (mutated triosephosphate isomerase), 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), WT1 (Wilm's tumor gene), or a combination thereof.

[0221] Additional examples of cancer cell-associated antigens include 2B4 (CD244), 4-1BB, 5T4, A33 antigen, adenocarcinoma antigen, adrenergic receptor beta 3 (ADRB3), A kinase anchoring protein 4 (AKAP-4), alpha-fetoprotein (AFP), anaplastic lymphoma kinase (ALK), androgen receptor, B7H3 (CD276), beta2-integrin, BAFF, B lymphoma cells, B cell maturation antigen (BCMA), bcr-abl (an oncogene fusion protein composed of the breakpoint cluster region (BCR) and Aberson murine leukemia virus oncogene homolog 1 (Abl)), BhCG, bone marrow stromal cell antigen 2 (BST2), CCCTC-binding factor (zinc finger protein)-like (BORIS or imprinting site regulator brother) brother), 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), CD33, CD34, CD38, CD40, CD40L, CD41, C D44, CD44V6, CD49f, CD51, CD52, CD56, CD63, CD70, CD72, CD74, CD79a, CD79b, CD80, CD84, CD96, CD97, CD100, CD123, CD125, CD1 33. CD137, CD138, CD150, CD152 (CTLA-4), CD160, CD171, CD179a, CD200, CD221, CD229, CD244, CD272 (BTLA), CD274 (PDL-1, B7H 1), CD279 (PD-1), CD352, CD358, CD300 molecule-like family member f (CD300LF), carcinoembryonic antigen (CEA), occludin 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 (also known as CD2 subclass 1, CRACC, CD319, and 19A24), CTLA-4, cyclin B l, chromosome X open reading frame 61 (CXORF61), cytochrome P450 1B1 (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), mucin-like hormone receptor-like 2 containing an EGF-like module (EMR2), elongation factor 2 mutant (ELF2M), endosialin, epithelial cell adhesion molecule (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 6 on chromosome 12p (ETV6-AML), Fc fragment of IgA receptor (FCAR or CD89), fibroblast activation protein alpha (FAP), FBP, Fc receptor-like 5 (FCRL5), fetal acetylcholine receptor (AChR), fibronectin extra domain-B, Fms-like tyrosine kinase 3 (FLT3), folate binding protein (FBP), folate receptor 1, folate receptor alpha, folate receptor beta, Fos-related antigen 1, fucosyl, fucosyl GM1; GM2, ganglioside G2 (GD2), ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(ll)Cer), o-acetyl-GD2 ganglioside (OAcGD2), GITR (TNFRSF 18), GM1, ganglioside GM3 (aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(ll)Cer), GP 100, the hexose portion of globoH glycoceramide (GloboH), glycoprotein 75, glypican 3 (GPC3), glycoprotein 100 (gp1OO), 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 (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 (muthsp70-2), human scatter factor receptor kinase, human telomerase reverse transcriptase (hTERT), HVEM, ICOS, insulin-like growth factor receptor 1 (IGF-1 receptor), IGF-I, IgG1, 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), integrin α5β1, integrin ανβ3, intestinal carboxylesterase, kappa 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), legumin, leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Lewis (Y) antigen, LeY, LG, LI cell adhesion molecule (LI-CAM), LIGHT, LMP2, lymphocyte antigen 6 complex, LTBR, ​​locus K 9 (LY6K), Ly-6, Lymphocyte antigen 75 (LY75), Melanoma cancer testis antigen-1 (MAD-CT-1); Melanoma cancer testis antigen-2 (MAD-CT-2), MAGE, Melanoma-associated antigen 1 (MAGE-A1), MAGE-A3 melanoma antigen recognized by T cells 1 (MelanA or MARTI), MelanA / MARTl, Mesothelin, MAGE A3, melanoma cell inhibitor of apoptosis (ML-IAP), melanoma-specific chondroitin sulfate proteoglycan (MCSCP), MORAb-009, MS4A1, mucin 1 (MUCl), MUC2, MUC3, MUC4, MUC5AC, MUC5b, MUC7, MUC16, mucin CanAg, Müllerian inhibitory substance (MIS) type II receptor, v-myc avian myelocytic tumor viral oncogene neuroblastoma-derived homolog (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 differentiation antigen (NY-BR-1), NY-ESO-1, carcinoembryonic antigen (h5T4), olfactory receptor 51E2 (OR51E2), OX40, plasma cell antigen, poly SA, acrosomal protease binding protein sp32 (OY-TESl), p53, p53 mutant, pan-linked protein 3 (PANX3), prostatic acid phosphatase (PAP), paired box protein Pax-3 (PAX3), paired box protein Pax-5 (PAX5), prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), PD-1H, platelet-derived growth factor receptor alpha (PDGFR-α), PDGFR-β, PDL192, PEN-5, phosphatidylserine, placenta-specific 1 (PLAC1), polysialic acid, prostate enzyme, prostate cancer cell, prostaglandin, protease serine 21 (testosterone or PRSS21), protease 3 (PR1), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), proteasome (pro, megalin factor) subunit beta type, advanced glycation end product receptor RAGE-1, RANKL, Ras mutant, Ras homolog family member C (RhoC), RON, receptor tyrosine kinase-like orphan receptor 1 (ROR1), renal ubiquitin 1 (RU1), renal ubiquitin 2 (RU2), sarcoma translocation breakpoints, squamous cell carcinoma antigen recognized by T cells 3 (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), survivin, tumor-associated glycoprotein 72 (TAG72), TCR5y, TCRa, TCRB, TCR gamma alternate reading frame protein (TARP), telomerase, TIGIT, TNF-α precursor, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), tenascin C, TGFβ2, 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, urokinase 2 (UPK2), VEGF-A, VEGFR-1, vascular endothelial growth factor receptor 2 (VEGFR2) and vimentin, Wilms' tumor protein (WT1), or X antigen family member 1A (XAGE1), or a combination thereof.

[0222] In other embodiments, the antigenic portion is associated with a virus-infected cell (i.e., a viral antigenic portion). Such antigenic portions can include, but are not limited to, 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 type 1, 2, or 8 (HSV1, HSV2, or HSV8) viral antigens (e.g., glycoproteins gB, gC, gC, gE, gG, gH, gI, gJ, gK, gL, gM, UL20, UL32, US4, 3, UL45, UL49A), cytomegalovirus (CMV) viral antigens (e.g., glycoprotein gB, gC, gC, gE, gG, gH, gI, gJ, gK, gL, gM or other envelope proteins), human immunodeficiency virus (HIV) viral antigens (glycoprotein gp120, gp41 or p24), influenza virus antigens (e.g., hemagglutinin (HA) or neuraminidase (NA)), measles or mumps virus antigens, human papillomavirus (HPV) viral antigens (e.g., L1, L2), viral antigens of parainfluenza virus, viral antigens of rubella virus, viral antigens of respiratory syncytial virus (RSV) virus, or viral antigens of varicella-zoster virus, or a combination thereof. In this embodiment, the cell surface receptor can be any TCR, or any CAR that recognizes any of the aforementioned viral antigens on a virus-infected target cell.

[0223] In other embodiments, the antigenic portion is associated with cells with immune or inflammatory dysfunction. Such antigenic portions may 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 involved in or associated with pathogenic autoimmune processes, or combinations thereof.

[0224] In some embodiments, the TCR is specific for a portion of an antigen on a cancer cell. 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-BTCR, 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-human papillomavirus 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-MC1RTCR, 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 TCR or anti-WT1 TCR or their combination.

[0225] In some embodiments, the TCR can bind to a first antigen portion and a second antigen portion. In some embodiments, the first TCR binds to the first antigen portion and the second TCR binds to the second antigen portion.

[0226] The present disclosure may involve the use of a dual-targeted antigen binding system. The dual-targeted antigen binding system may include a bispecific CAR or TCR and / or a bicistronic CAR or TCR. The bispecific and bicistronic CAR may include two binding motifs (in a single CAR molecule or two CAR molecules, respectively). In some embodiments, the vector encodes a bicistronic and / or bispecific CAR (e.g., a bicistronic and / or bispecific CAR that binds to CD20 and CD19). Exemplary bispecific and bicistronic CARs are described in WO2020 / 123691, which is incorporated herein by reference.

[0227] In some embodiments, the CAR comprises a first scFv that binds to CD19 and a second scFv that binds to CD20. In some embodiments, the first CAR comprises a first scFv that binds to CD19, and the second CAR comprises a second scFv that binds to CD20. Example CD19-binding sequences or CD20-binding sequences are provided in Table 1.

[0228] Table 1. Example antigen binding sequences

[0229]

[0230]

[0231]

[0232] In addition to the antigen binding molecules, the CAR of the present disclosure may include a hinge, a transmembrane domain and / or an intracellular domain. In some embodiments, the lymphocyte domain may include a costimulatory domain and an activation domain.

[0233] The hinge can be the extracellular domain of the antigen binding system located between the binding motif and the transmembrane domain. The hinge can also be referred to as the extracellular domain or "spacer". The hinge can contribute to receptor expression, activity and / or stability. The hinge can also provide flexibility in accessing the target antigen. In some embodiments, the hinge domain is located between the binding motif and the transmembrane domain.

[0234] In some embodiments, the hinge is, is derived from, or is derived from an immunoglobulin-like hinge domain (e.g., including all or fragments thereof). In some embodiments, the hinge domain is or is derived from an immunoglobulin. In some embodiments, the hinge domain is selected from the hinge of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, or IgM, or a fragment thereof.

[0235] In some embodiments, the hinge is, is from, or is derived from (e.g., comprises all or a fragment of) 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), CD49f (ITGA6), CD66a (CEACAM1), CD66 (CEACAM2), CD66 (CEACAM3), CD66 (CEACAM4), CD66 (CEACAM1), CD66 (CEACAM1), CD66 (CEACAM2), CD66 (CEACAM3), CD66 (CEACAM1), CD66 (CEACAM2), CD66 (CEACAM3), CD66 (CEACAM1), CD66 (CEACAM2), CD66 (CEACAM3), CD66 (CEACAM1), CD66 (CEACAM1), CD66 (CEACAM2), CD66 (CEACAM3), CD66 (CEACAM1), CD66 (CEACAM2 ... 6b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (B cell antigen receptor complex-associated α chain), CD79B (B cell antigen receptor complex-associated β chain), CD84 (SLAMF5), CD96 (Tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD158A (KIR2DL1), CD158B1 (K IR2DL2), CD158B2 (KIR2DL3), CD158C (KIR3DP1), CD158D (KIRDL4), CD158F1 (KIR2DL5A), CD158F2 (KIR2DL5B), CD158K (KIR3DL2), CD160 (BY 55), CD162 (SELPLG), CD226 (DNAM1), CD229 (SLAMF3), CD244 (SLAMF4), CD247 (CD3-ζ), CD258 (LIGHT), CD268 (BAFFR), CD270 (TNFSF14), CD27 2 (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 co-stimulator (ICOS), LFA-1 (CD11a / CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL-2Rβ, IL-2R γ, IL-7R α, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fc γ receptor, MHC class 1 molecule, MHC class 2 molecule, TNF receptor protein, immunoglobulin, cytokine receptor, integrin, activating NK cell receptor, or Toll ligand receptor or fragments or combinations thereof.

[0236] In some embodiments, the hinge is, is derived from, or is derived from the hinge of CD8 alpha (e.g., including all or fragments thereof). In some embodiments, the hinge is, is derived from, or is derived from the hinge of CD28. In some embodiments, the hinge is, is derived from, or is derived from a fragment of a CD8 alpha hinge or a fragment of a CD28 hinge, wherein the fragment is any fragment that is less than the entire hinge. In some embodiments, the fragment of a CD8 alpha hinge or a fragment of a CD28 hinge comprises 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 from the N-terminus or C-terminus or both of the CD8 alpha hinge or the CD28 hinge.

[0237] A "transmembrane domain" refers to a domain that, when present in a molecule on the cell surface or cell membrane (e.g., spanning part or all of the cell membrane), has the property of being present in the membrane. It is not necessary for every amino acid in a transmembrane domain to be present in the membrane. For example, in some embodiments, a transmembrane domain is characterized by a specified segment or portion of a protein being substantially localized in the membrane. Various algorithms can be used to analyze amino acid or nucleic acid sequences to predict protein subcellular localization (e.g., transmembrane localization). Examples of such programs include psort (PSORT.org) and Prosite (prosite.expasy.org).

[0238] The transmembrane domain can be derived from any membrane-bound or transmembrane protein, such as the α, β, or ζ chains of the T-cell receptor, CD28, CD3 ε, CD3 δ, CD3 γ, CD45, CD4, CD5, CD7, CD8, CD8 α, CD8 β, CD9, CD11a, CD11b, CD11c, CD11d, CD16, CD22, CD27, CD33, CD37, CD64, CD80, CD86, CD134, CD137, TNFSFR25, CD154, 4-1BB / CD137, activating NK cell receptor, immunoglobulin, 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 γ receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Ig α (CD79a), IL-2R β, IL-2R γ, IL-7R α, inducible T cell co-stimulator (ICOS), integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, ligand binding to CD83, LIGHT, LIGHT, LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1; CD1-1a / 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 activation molecule (SLAM protein), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Ly108), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1 or VLA-6, or fragments, truncated forms, or combinations thereof.

[0239] The intracellular domain (or cytoplasmic domain) comprises one or more signaling domains that, upon binding of the target antigen to the binding motif, induce and / or mediate intracellular signals, such as intracellular signals that activate 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 activation of at least one normal effector function of an immune cell. Effector functions of T cells can, for example, be 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 comprise a signaling domain that serves as an activation domain. The intracellular domain may comprise a signaling domain that is a co-stimulatory signaling domain.

[0240] Intracellular signaling domains are known to transduce signals upon antigen binding to immune cells. For example, cytoplasmic sequences of T cell receptors (TCRs) are known to initiate signal transduction upon TCR binding to antigens (see, e.g., Brownlie et al., Nature Rev. Immunol. 13:257-269 (2013)).

[0241] In certain embodiments, suitable signaling domains include, but are not limited to, 4-1BB / CD137, activating NK cell receptors, immunoglobulins, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 δ, CD3 ε, CD3 γ, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8α, CD8β, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CD5, CEACAM1, CRT AM, cytokine receptors, DAP-10, DNAM1 (CD226), Fc γ receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Ig α (CD79a), IL-2R β, IL-2Rγ, IL-7R α, inducible T cell co-stimulator (ICOS), integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, ligand binding to CD83, LIGHT, LIGHT, LTBR, ​​Ly9 (CD229), Ly108, lymphocyte function-associated antigen-1 (LFA-1; CD1-1a / 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 activation molecule (SLAM protein), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1 or VLA-6 or fragments, truncated forms or combinations thereof.

[0242] CARs may also include a co-stimulatory signaling domain, for example, to increase signaling efficacy. See U.S. Patent Nos. 7,741,465 and 6,319,494, as well as Krause et al. and Finney et al. (supra), 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 by the TCR alone may not be sufficient to fully activate T cells, and secondary or co-stimulatory signals can increase activation. Therefore, in some embodiments, the signaling domain further includes one or more additional signaling domains (e.g., co-stimulatory signaling domains) that activate one or more immune cell effector functions (e.g., the innate immune cell effector functions described herein). In some embodiments, a portion of such a costimulatory signaling domain can be used, as long as the portion transduces the effector function signal. In some embodiments, the cytoplasmic domain described herein comprises one or more cytoplasmic sequences of a T cell co-receptor (or fragment thereof). Non-limiting examples of such T cell co-receptors include 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 ligands that bind to CD83. Exemplary costimulatory proteins have the amino acid sequence of a costimulatory protein naturally found on T cells, the complete native amino acid sequence of which is described in NCBI Reference Sequence: NP 0.1. In some cases, the CAR includes a 4-1BB costimulatory domain. In some cases, the CAR includes a CD28 costimulatory domain. In some cases, the CAR includes a DAP-10 costimulatory domain.

[0243] In some embodiments, the co-stimulatory signaling domain is the signaling domain of CD28. As shown in the experimental examples, CAR molecules with CD28 co-stimulatory signaling domains can particularly benefit from the newly developed accelerated manufacturing process.

[0244] In some embodiments, CAR also includes ITAM. Examples of primary cytoplasmic signaling sequences containing ITAM that are particularly useful in the present disclosure include those derived from TCR ζ, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CD5, CD22, CD79a, CD79b, and CD66d. In some embodiments, ITAM includes CD3 ζ.

[0245] In some embodiments, the CAR molecule is any anti-CD19 CAR molecule. On the one hand, as described in WO 2015120096 or WO 2016191755, anti-CD19 CAR includes extracellular scFv domains, the intracellular part and / or transmembrane part of CD28 molecules, the optional extracellular part and intracellular cd3 ζ domains of CD28 molecules, each of which is incorporated herein in its entirety.

[0246] In certain embodiments, the anti-CD19 CAR may further include additional domains, such as the extracellular and / or transmembrane regions of CD8, an extracellular immunoglobulin Fc domain (e.g., IgG1, IgG2, IgG3, IgG4), or one or more additional signaling domains, such as 41BB, OX40, CD2 CD16, CD27, CD30 CD40, PD-1, ICOS, LFA-1, IL-2 receptor, Fcγ receptor, or any other costimulatory domain with an immunoreceptor tyrosine-based activation motif.

[0247] In certain embodiments, the cell surface receptor is an anti-CD19 CAR, such as the FMC63-28Z CAR or FMC63-CD828BBZ CAR described in Kochenderfer et al., J Immunother. 2009 Sep;32(7):689-702, “Construction and Preclinical Evaluation of an Anti-CD19 Chimeric Antigen Receptor,” the subject matter of which is hereby incorporated by reference for the purpose of providing methods for constructing vectors for generating T cells expressing the FMC63-28Z CAR or FMC63-CD828BBZ CAR.

[0248] In some embodiments, the T cell comprising the CAR molecule is Yescarta ® (Akilenbine). In some embodiments, the T cell comprising the CAR molecule is Tecartus ®(brexucabtagene autoleucel). In some embodiments, the T cells include one or more CAR molecules that can bind one or more antigen moieties.

[0249] In some embodiments, a pharmaceutical composition is provided, which comprises an engineered lymphocyte colony produced by the methods described herein. In certain embodiments, the pharmaceutical composition may also include a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier may be a pharmaceutically acceptable material, composition or vehicle that participates in carrying or transporting the cell of interest from a tissue, organ or part of a body to another tissue, organ or part of the body. For example, the carrier may be a liquid or solid filler, a diluent, an excipient, a solvent or an encapsulating material or a certain combination thereof. Each component of the carrier must be "pharmaceutically acceptable" because it must be compatible with the other ingredients of the formulation. It must also be suitable for contacting any body tissue, organ or part it may encounter, which means that it must not carry the risk of toxicity, irritation, allergic reaction, immunogenicity or any other complication that far exceeds its therapeutic benefit.

[0250] Treatment and use and optional storage

[0251] Lymphocytes prepared by the present method or lymphocyte populations as disclosed herein can be used to treat a variety of diseases and disorders.

[0252] In some embodiments, if the lymphocytes are not used immediately, they can be cryopreserved for later use. This method can include the steps of washing and concentrating the engineered lymphocyte population using a diluent solution. In some aspects, the diluent solution is normal saline, 0.9% saline, PlasmaLyte A (PL), 5% dextrose / 0.45% NaCl saline solution (D5), human serum albumin (HSA), or a combination thereof. In some aspects, HSA can be added to the washed and concentrated cells to improve cell viability and cell recovery after thawing. In another aspect, the wash solution is normal saline, and the washed and concentrated cells are supplemented with 5% HSA. The method can also include the step of creating a cryopreservation mixture, wherein the cryopreservation mixture comprises the diluted cell population in the diluent solution and a suitable cryopreservation solution. In some aspects, the cryopreservation solution can be any suitable cryopreservation solution, including but not limited to CryoStor10 (BioLifeSolution), mixed with the engineered lymphocyte diluent solution in a 1:1 or 2:1 ratio.

[0253] In certain embodiments, HSA can be added to provide a final concentration of about 1.0% to 10% HSA in a frozen mixture. In certain embodiments, HSA can 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 a frozen mixture. In certain embodiments, HSA can be added to provide a final concentration of about 1% to 3% HSA, about 1% to 4% HSA, about 1% to 5% HSA, about 1% to 7% HSA, about 2% to 4% HSA, about 2% to 5% HSA, about 2% to 6% HSA or about 2% to 7% HSA in a frozen mixture. In certain embodiments, HSA can be added to provide a final concentration of about 2.5% HSA in a frozen mixture. For example, in certain embodiments, cryopreservation of engineered T cell populations may comprise washing the cells with 0.9% saline, adding HSA to a final concentration of 5% to the washed cells, and cryopreserving the cells with a CryoStor ™ CS10 dilutes the cells 1:1 (for a final concentration of 2.5% HSA in the final cryopreservation mixture). In some embodiments, the method further comprises the step of freezing the cryopreservation mixture. In one aspect, the cryopreservation mixture is frozen in a controlled rate freezer using defined freezing cycles at a cell concentration of between about 1e6 and about 1.5e7 cells / mL of cryopreservation mixture. The method may further comprise the step of storing the cryopreservation mixture in vapor phase liquid nitrogen.

[0254] Also provided are methods and uses for treating a disease or pathological condition in a subject suffering from the disease or pathological condition. In some embodiments, the method requires administering to the subject a therapeutically effective amount or a therapeutically effective dose of engineered lymphocytes. Pathological conditions that can be treated with engineered T cells produced by the methods described herein include, but are not limited to, cancer, viral infection, acute or chronic inflammation, autoimmune disease, or any other immune dysfunction.

[0255] As referred to herein, "cancer" may be a cancer associated with a surface antigen or cancer marker, including but not limited to acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adenoid cystic carcinoma, adrenocortical carcinoma, cancer, AIDS-related cancer, anal cancer, appendix cancer, astrocytoma, atypical teratoid / rhabdoma, central nervous system cancer, B cell leukemia, lymphoma or other B cell malignancies, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, osteosarcoma and malignant fibrous histiocytoma, brain stem glioma, brain tumor, breast cancer, bronchial tumor, Burkitt's lymphoma, carcinoid tumor, central nervous system cancer, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML ), chronic myeloid proliferative disorder, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, embryonal tumor, central nervous system cancer, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, esophagus neuroblastoma, Ewing sarcoma family of tumors, extracranial germ cell tumor, extracranial germ cell tumor, extrahepatic bile duct cancer, eye cancer, bone fibrous histiocytoma, malignant tumor, osteosarcoma, gallbladder cancer, gastric (gastric / stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), soft tissue sarcoma, germ cell tumor, gestational trophoblastic tumor, glioma, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, Islet cell tumor (endocrine pancreas), Kaposi sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cancer, liver cancer (primary), lobular carcinoma in situ (LCIS), lung cancer, lymphoma, macroglobulinemia, male breast cancer, malignant fibrous histiocytoma and osteosarcoma of bone, medulloblastoma, medulloepithelioma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary midline tract cancer (involving the NUT gene), oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasms, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasms, chronic myeloid leukemia (CML), acute myeloid leukemia (AML) , multiple myeloma, myeloproliferative disorders, nasal cavity and sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oral cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, intermediate differentiation pinealoma, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, plasma cell neoplasms / multiple myeloma, pleuropulmonary blastoma, pregnancy and breast cancer, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, transitional cell cancer of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland cancer,Sarcoma, Sézary syndrome, small cell lung cancer, small intestinal cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer, gastric cancer, supratentorial primitive neuroectodermal tumor, T-cell lymphoma, skin cancer, testicular cancer, laryngeal cancer, thymoma and thymic cancer, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, trophoblastic tumor, ureter and renal pelvis cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, Wilms' tumor.

[0256] In some aspects, the cancer is a B-cell malignancy. Examples of B-cell malignancies include, but are not limited to, non-Hodgkin 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 (monocytoid B-cell lymphoma), splenic diffuse large cell lymphoma, B-cell chronic lymphocytic leukemia / lymphoma, Burkitt lymphoma, and lymphoblastic lymphoma.

[0257] As referred to herein, a "viral infection" can be 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 infection can cause or contribute to the development of cancer in a subject with the viral infection (e.g., HPV infection can cause or be associated with the development of several cancers, including cervical, vulvar, vaginal, penile, anal, oropharyngeal cancer, and HIV infection can cause the development of Kaposi's sarcoma).

[0258] Examples of chronic inflammatory diseases, autoimmune diseases, or any other immune dysfunction that can be treated using engineered T cells generated by the methods described herein include, but are not limited to, multiple sclerosis, lupus, and psoriasis.

[0259] The term "treating," "treating," or "treating" as used herein with respect to a disorder or disease may refer to preventing the disorder or disease, slowing the rate of onset or development of a disorder or disease, reducing the risk of developing a disorder or condition, preventing or delaying the development of symptoms associated with the disorder or disease, alleviating or ending symptoms associated with the disorder or disease, causing complete or partial regression of the disorder or disease, or some combination thereof.

[0260] A "therapeutically effective amount" or "therapeutically effective dose" is the amount of engineered lymphocytes that produces the desired therapeutic effect in a subject, such as preventing or treating a target condition by killing target cells or alleviating symptoms associated with the condition. The most effective result for a given subject will depend on various factors, including but not limited to the characteristics of the engineered lymphocytes (including lifespan, activity, pharmacokinetics, pharmacodynamics, and bioavailability), the subject's physiological condition (including age, sex, disease type and stage, general physical condition, responsiveness to a given dose, and type of drug), the nature of any one or more pharmaceutically acceptable carriers in any composition used, and the route of administration. The therapeutically effective dose of the engineered lymphocytes also depends on the cell surface receptors expressed by the lymphocytes (e.g., the affinity and density of the cell surface receptors expressed on the cells), the type of target cells, the nature of the disease or pathological condition being treated, or a combination of both.

[0261] As shown in the examples, the in vivo efficacy of engineered lymphocytes prepared by this process is greatly increased compared to conventional techniques, thereby requiring much lower doses.

[0262] Thus, in some aspects, the therapeutically effective dose of engineered lymphocytes is less than about 2 million engineered lymphocytes per kilogram of subject body weight to be treated (cells / kg). Thus, in some aspects, the therapeutically effective dose of engineered lymphocytes is about 10,000 to about 1,500,000 engineered lymphocytes / kg. In certain embodiments, the therapeutically effective dose is about 20 billion to about 1,200 billion engineered lymphocytes / kg. In certain embodiments, the therapeutically effective dose is about 20 billion to about 1,000 billion engineered lymphocytes / kg. In certain embodiments, the therapeutically effective dose is about 20 billion to about 500 billion engineered lymphocytes / kg. In certain embodiments, the therapeutically effective dose is about 20 billion to about 400 billion engineered lymphocytes / kg. In certain embodiments, the therapeutically effective dose is about 40 billion to about 400 billion engineered lymphocytes / kg. In certain embodiments, the therapeutically effective dose is about 50 billion to about 200 billion engineered lymphocytes per kilogram. In certain embodiments, the therapeutically effective dose is about 50 billion to about 100 billion engineered lymphocytes per kilogram.

[0263] In some embodiments, the administered T cells are Yescarta ® (Akirame). In some embodiments, the T cells administered are Tecartus ® (brexucabtagene autoleucel).

[0264] One embodiment of the present disclosure relates to a method for predicting the likelihood of a complete response to an immunotherapy in a patient, the method comprising: determining a time period from a leukocyte separation step for the patient to administration of the immunotherapy to the patient; grouping the patient into one of a plurality of groups based on the determined time period, the plurality of groups comprising: a first group characterized in that the time period from the leukocyte separation step to the administration of the immunotherapy to the patient is up to 28 days (e.g., 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); and a second group characterized in that the time period from the leukocyte separation step to the administration of the immunotherapy to the patient is 28 days. days to 40 days (e.g., 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, or 39 days); a third group characterized in that the time period from the leukapheresis step to the administration of the immunotherapy to the patient is at least 40 days (e.g., 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 ​​days, 49 days, or 50 days); and determining the likelihood of a complete response in the patient based at least in part on which of the multiple groups the patient is grouped into, wherein the patient has a likelihood of a complete response of at least about 55% if the patient is grouped into the first group or the second group, and wherein the patient has a likelihood of a complete response of at least about 42% if the patient is grouped into the third group.

[0265] As used herein, the term up to 28 days may mean less than (<) 28 days (eg, 27 days, 26 days, 20 days, 10 days, or 5 days).

[0266] As used herein, the term 28 days to 40 days may mean greater than or equal to (≧) 28 days to less than (<) 40 days.

[0267] As used herein, the term at least 40 days may mean greater than or equal to (≧) 40 days.

[0268] In some embodiments of the present disclosure, the patient has about a 60% likelihood of a complete response if the patient is grouped into the first group or the second group.

[0269] One embodiment of the present disclosure relates to a method for predicting overall survival of a patient receiving immunotherapy, the method comprising: determining a time period from a leukocyte separation step to administration of the immunotherapy to the patient; grouping the patient into one of a plurality of groups based on the determined time period, the plurality of groups comprising: a first group characterized in that a time period from the leukocyte separation step to administration of the immunotherapy to the patient is up to 28 days; and a second group characterized in that a time period from the leukocyte separation step to administration of the immunotherapy to the patient is between 28 days and less than 40 days. ; and a third group characterized in that a time period from the leukapheresis step to the administration of the immunotherapy to the patient is at least 40 days; and determining the overall survival rate in the patient based at least in part on which of the plurality of groups the patient is grouped, wherein if the patient is grouped within the first group, the patient has an overall survival rate of at least about 49%, wherein if the patient is grouped within the second group, the patient has an overall survival rate of at least about 48%, and wherein if the patient is grouped within the third group, the patient has an overall survival rate of at least about 30%.

[0270] One embodiment of the present disclosure relates to a method for predicting a risk of thrombocytopenia in a patient receiving immunotherapy, the method comprising: determining a time period from a leukocyte separation step of the patient to administration of the immunotherapy to the patient; grouping the patient into one of a plurality of groups based on the determined time period, the plurality of groups comprising: a first group characterized in that a time period from the leukocyte separation step to the administration of the immunotherapy to the patient is up to 28 days; a second group characterized in that a time period from the leukocyte separation step to the administration of the immunotherapy to the patient is between 28 days and 40 days; and a third group characterized in that a time period from the leukapheresis step to the administration of the immunotherapy to the patient is at least 40 days; and the risk of thrombocytopenia in the patient is determined at least in part based on which of the multiple groups the patient is grouped into, wherein if the patient is grouped into the first group, the patient has an approximately 18% risk of thrombocytopenia, wherein if the patient is grouped into the second group, the patient has an approximately 25% risk of thrombocytopenia, and wherein if the patient is grouped into the third group, the patient has an approximately 34% risk of thrombocytopenia.

[0271] One embodiment of the present disclosure relates to a method for predicting life expectancy and quality-adjusted life years of a patient who has received immunotherapy, the method comprising: determining a time period from a leukapheresis step for the patient to administration of the immunotherapy to the patient, wherein the time period is a short time period or a long time period; assigning a probability of successful transfusion based on the time period; and inputting the patient information into a survival model to determine the patient's life expectancy and quality-adjusted life years.

[0272] In some embodiments, the models described herein can be decision tree models with outcomes associated with long or short V2VT.

[0273] In some embodiments, model inputs may include: long V2VT, short V2VT, probability of transfusion, lifetime outcome (non-transfused patients), lifetime outcome (transfused patients), efficacy of long V2VT versus short V2VT for transfused patients, and / or QALYs.

[0274] In some embodiments, model outputs may include life-years (LYs) and / or quality-adjusted life-years (QALYs).

[0275] In some embodiments, the models described herein may include decision gates / nodes and probability nodes. Decision gates may represent the point at which a decision is made (e.g., long V2VT or short V2VT). Probability nodes may represent the probability of certain outcomes (e.g., infusion or no infusion). Graph survival predictions may be used to represent modeled lifetime outcomes based on data inputs.

[0276] As used herein, the term "life expectancy" refers to the number of years a subject or patient can be expected to live. In some embodiments, the increase in life expectancy can be measured from the time the patient is informed that they will receive immunotherapy. In some embodiments, the increase in life expectancy can be measured from the time the immunotherapy is infused into the patient.

[0277] As used herein, the term "quality-adjusted life year" or "QALY" refers to a measure of disease burden or health outcomes that includes both the quality and quantity of life. In some embodiments, the gain in QALYs can be measured starting from the time a patient is notified that they will be receiving immunotherapy. In some embodiments, the gain in QALYs can be measured starting from the time an immunotherapy is infused into the patient.

[0278] As used herein, the term "short time period" may refer to a time period (eg, veno-veno time) from a patient's leukapheresis procedure to administration of immunotherapy to the patient of 24 days or less.

[0279] As used herein, the term "long period of time" can refer to a period of time (e.g., veno-venous time) from the patient's leukapheresis procedure to the administration of immunotherapy to the patient of 37 days or longer, 54 days or longer, or between 37 and 54 days.

[0280] In some embodiments, a short time period may indicate an increase in the patient's life expectancy and / or quality-adjusted life years of greater 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).

[0281] In some embodiments, a long period of time may indicate an increase in the patient's life expectancy and / or an increase in quality-adjusted life years of less than about 5 years (e.g., about 4.5 years, 4 years, 3.5 years, 3 years, 2.5 years, 2 years, 1.5 years, or 1 year or less).

[0282] In one embodiment of the present disclosure, the immunotherapy comprises one or more CARs that recognize one or more tumor antigens.

[0283] In one embodiment of the present disclosure, the immunotherapy is axicabtagene ciloleucel or brexucabtagene autoleucel.

[0284] In one embodiment of the present disclosure, the patient has relapsed or refractory (r / r) large B-cell lymphoma (LBCL).

[0285] The following examples are intended to illustrate various embodiments of the present invention. Therefore, the specific embodiments discussed should not be interpreted as limiting the scope of the invention. For example, although the following examples are directed to T cells transduced with anti-CD19 chimeric antigen receptors (CARs), those skilled in the art will understand that the methods described herein are applicable 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 may be made without departing from the scope of the present invention, and it will be understood that such equivalent embodiments will be included herein. In addition, all references cited in this disclosure are hereby incorporated by reference in their entirety, as if fully set forth herein.

[0286] Example 1

[0287] Compared to other CAR T-cell products, axi-cel has a shorter median wait time from leukapheresis to infusion, known as venovenous-to-venous time (real-world: 28 days for axi-cel vs. 45 days for axi-cel; clinical trials: 36-37 days for axi-cel; Riedell et al. Transplant Cell Ther 2022; Abramson et al. Lancet 2020). Studies based on the JULIET trial have shown that reduced wait time is associated with increased efficacy (Chen et al. Value Health 2022). This example evaluates the real-world impact of venovenous-to-venous time on axi-cel outcomes in r / r LBCL.

[0288] method

[0289] A total of 1383 patients with r / r LBCL treated with commercial axi-cel from 78 US centers were identified from a noninterventional postauthorization safety study using the Center for International Blood and Marrow Transplant Research (CIBMTR) registry. Patients with the following information were excluded: primary central nervous system lymphoma or lymphoma other than LBCL, prior nontransplant cell therapy, missing comorbidity data (Sorror et al. Blood 2005), unknown or unrelated date of leukapheresis (≤2 days before lymphodepleting [LD] chemotherapy or ≥144 days before infusion), or no follow-up.

[0290] Efficacy outcomes were overall response rate (ORR) and complete response rate (CR), duration of response (DOR), and progression-free and overall survival (PFS and OS). Adverse events of interest included cytokine release syndrome (CRS) (Lee 2014 criteria), immune effector cell-associated neurotoxicity syndrome (ICANS) (ASTCT criteria), prolonged neutropenia, and thrombocytopenia. Logistic regression and Cox regression were used to estimate odds ratios (OR) and hazard ratios (HR) after adjustment for key prognostic factors such as age, comorbidities, Eastern Cooperative Oncology Group performance status, disease characteristics at diagnosis, and bridging therapy. Adjusted curves were generated based on directly adjusted survival functions (Makuch J Chronic Dis 1982).

[0291] result

[0292] Overall, the median venovenous-to-venovenous time (from leukapheresis to infusion) for axi-cel was 27 days (interquartile range [IQR], 26-32 days), including a median of 5 days (IQR, 5-5 days) from the start of LD chemotherapy to infusion. The venovenous-to-venovenous time was consistent regardless of the following baseline characteristics: disease histology, sex, race, ethnicity, Eastern Cooperative Oncology Group performance status before infusion, or chemosensitivity (Table 2).

[0293] Patients with a shorter venovenous time appeared to be younger and less likely to have comorbidities (Table 3). Patients with a venovenous time of ≥40 days were more heavily pretreated and more likely to receive bridging therapy.

[0294] At a median follow-up of 24.2 months, better outcomes were observed in patients with shorter IV-IV times. CR rates were 60%, 61%, and 50% for patients with IV-IV times <28 days, ≥28 to <40 days, and ≥40 days, respectively (ORR 77%, 77%, and 70%). OS at 24 months was 53% for patients with IV-IV times <28 days and ≥28 to <40 days, compared to 38% for patients with a wait time ≥40 days. After adjusting for other key prognostic factors, patients with a veno-veno time ≥40 days had significantly lower CR rates and OS compared with patients with a wait time <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 ≥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).

[0295] Adjusted analyses of progression-free survival (PFS), overall survival (OS), and duration of response (DOR) 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) were performed to balance differences in baseline characteristics. A sensitivity analysis comparing outcomes in patients with a veno-venous time <36 days versus ≥36 days was also performed to assess the validity of the veno-venous time categorization used in the primary analysis.

[0296] Among patients who achieved a CR / partial response (PR) as their best response, the DOR at 12 months was 61% for patients with an IV-IV time of <28 days, 60% for patients with an IV-IV time of ≥28 to <40 days, and 61% for patients with an IV-IV time of ≥40 days. Sensitivity analyses of the DOR were consistent with the primary analysis.

[0297] Adjusted PFS and OS at 24 months appeared to be lower for patients with a venous-to-venous time ≥40 days compared with those with a venous-to-venous time <28 days or ≥28 days to <40 days. Sensitivity analyses of OS and PFS were consistent with the primary analysis, with patients with a venous-to-venous time ≥36 days having a significantly shorter OS compared with those with a venous-to-venous time <36 days (hazard ratio [HR], 1.25 [95% CI, 1.02-1.53]).

[0298] ICANS of any grade or grade ≥3 and prolonged neutropenia were consistent regardless of venous-venous time. Patients with an venous-venous time of <28 days had more ICANS of any grade compared with those with a wait time of ≥28 to <40 days (OR 1.34 [95% CI 1.06-1.71]), whereas ICANS of grade ≥3 did not differ significantly between the two groups (Table 2). Among patients alive at day 30, those with venous-venous times of ≥28 to <40 days and ≥40 days had higher rates of prolonged thrombocytopenia compared with those with a wait time of <28 days (OR 1.44 [95% CI 1.07-1.92] and 1.95 [95% CI 1.29-2.95], respectively).

[0299] The CRS grade is based on criteria from Lee, DW, et al. Blood, 2014; 124(2):188-195. The ICANS grade is based on criteria from Lee, DW, et al. Biol Blood Marrow Transplant. 2019; 25(4):625-638.

[0300] Regardless of venous-venous time, most CRS and ICANS resolved by 21 days after onset. The cumulative incidence of CRS resolution by 21 days after onset was 92%, 92%, and 94% for patients with venous-venous time <28 days, ≥28 days to <40 days, and ≥40 days, respectively. The cumulative incidence of ICANS resolution by 21 days after onset was 79%, 76%, and 64% for patients with venous-venous time <28 days, ≥28 days to <40 days, and ≥40 days, respectively.

[0301] Sensitivity analyses of safety results were consistent with the primary analysis.

[0302] The multivariate results of the sensitivity analysis comparing patients with a veno-venous time ≥36 days with patients with a veno-venous time <36 days are shown in Table 4 .

[0303] In this real-world analysis, most patients with r / r LBCL received axi-cel infusion within 5 weeks after apheresis. A shorter venovenous-to-venous time was associated with favorable CR rates, OS, and a reduced risk of long-term thrombocytopenia even after adjustment for key prognostic factors; however, ICANS of any grade may be higher. Taken together, these findings highlight the importance of shortening the venovenous-to-venous time in patients treated with axi-cel.

[0304] Table 2: Baseline Characteristics by Veno-Venous Time

[0305]

[0306] a Percentages are based on non-missing cases. b Previous transplants were not included. c Definition based on the hematopoietic cell transplantation-specific comorbidity index (Sorror, ML et al. Blood, 2005; 106(8): 2912-2919). ECOG PS, Eastern Cooperative Oncology Group performance status.

[0307] Table 3: Baseline characteristics, efficacy, and treatment outcomes of axi-cel by veno-venous time (time from leukapheresis to infusion). Sexual and safety outcomes

[0308]

[0309]

[0310] Table 4: Efficacy and Safety Results in Patients with IV-IV Time ≥36 Days vs. <36 Days

[0311]

[0312] Example 2

[0313] Chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment of hematologic cancers. However, production requires a complex, multistep process from leukocyte separation, manufacturing, transport, and storage prior to final infusion. This time, known as venovenous-to-venovenous time (V2VT), during which patients' disease may worsen, thus highlighting the potential importance of V2VT on patient outcomes. This modeling study was designed to compare the potential outcomes of "long" versus "short" V2VT in patients with relapsed / refractory large B-cell lymphoma (r / r LBCL) treated with CAR T-cell therapy in the 3L+ setting.

[0314] The aim of this study was to compare lifetime outcomes in hypothetical cohorts of patients who received CAR T therapy in the setting of r / r LBCL but with different V2VTs in the 3L+ setting.

[0315] method

[0316] A hypothetical cohort of patients entered the decision tree model at the time of leukapheresis and were then assigned a probability of successful transfusion based on V2VT.

[0317] Patients were then entered into a zonal survival model that estimated life-years (LY) and quality-adjusted life-years (QALYs) within a lifetime horizon based on real-world axi-cel OS data. Other CAR T efficacy data were not included to provide conservative estimates and avoid confounding. The model was informed by published literature, including studies examining how many patients ultimately underwent a transfusion versus the time elapsed from leukapheresis (based on the ZUMA-1, JULIET, and TRANSCEND-NHL-001 studies), the relationship between V2VT and survival (Locke et al.

[2022] ), and studies examining survival differences between transfused and non-transfused patients (Kuhl et al.

[2022] and Bachy et al.

[2022] ) (Table 5).

[0318] Epidemiological modeling was used to extrapolate the results to US patients eligible for CAR T-cell therapy. Based on V2VT allocation, the probability of successful infusion was determined and applied to a decision tree. Post-V2VT survival was estimated separately for patients who received a successful infusion versus those who did not. Finally, a protocol analysis was performed to assess the robustness of the results to key assumptions.

[0319] Based on the best available evidence of reported V2VT, three hypothetical V2VT cases were explored: 54 days (tisa-cel median V2VT; JULIET), 37 days (liso-cel median V2VT; TRANSCEND-NHL-001), and 24 days (axi-cel median V2VT; ZUMA-1).

[0320] Table 5: Data inputs used in the model

[0321]

[0322] To isolate the effect of V2VT on survival, we assumed that efficacy outcomes were equivalent across different CAR T therapies and, due to the limited availability of relevant published data to inform model inputs, we applied efficacy data for axi-cel to the model. To test the robustness of the results, we performed a series of sensitivity analyses.

[0323] Finally, an epidemiological model was used to scale the results for each patient to estimate the population outcome if all patients eligible for CAR T had reduced V2VT. Epidemiological estimates were taken from the NICE resource impact report (National Institute for Health and Care Excellence Resource impact report: Axicabtagene ciloleucel for treating diffuse large B-cell lymphoma and primary mediastinal large B-cell lymphoma after 2 or more systemic therapies, March 2023), but modified for the US population. In the US, an estimated 2700 patients were assumed to be eligible for CAR T.

[0324] result

[0325] 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).

[0326] 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.

[0327] Reducing V2VT from 54 days (tisa-cel median V2VT; JULIET) to 24 days (axi-cel median V2VT; ZUMA-1) resulted in a 3-year gain in life expectancy (4.2 vs. 7.7 LYs) and 2 additional QALYs per patient (2.9 vs. 5.3). If all ≈2710 eligible patients in the United States received “short” V2VT (24 days) rather than long (54 days), this would translate to 9328 additional LYs and 6385 additional QALYs per year. See Table 6. A smaller difference in V2VT use (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 a population-level gain of 7040 LYs and 4819 QALYs. The results were consistently positive in all sensitivity analyses.

[0328] Table 6: Base case and population-level outcomes per patient

[0329]

[0330] Extensive sensitivity analyses were performed, and all showed that shorter V2VT time resulted in improved outcomes (Table 7).

[0331] Sensitivity analyses indicated that the results were primarily driven by post-infusion outcomes as a function of V2VT and probability of infusion as a function of V2VT parameters.

[0332] Table 7: Sensitivity analysis results for LY increment

[0333]

[0334] Sensitivity analysis yielded QALY gains per patient ranging from 0.94 (post-infusion survival was unaffected by V2VT) to 3.71 (increasing the cutoff for granular V2VT classification [<28 days vs. ≥28 days to <40 days vs. ≥40 days, rather than <36 days vs. ≥36 days]).

[0335] This study is the first to quantify potential lifetime survival and QALY outcomes in patients with r / r LBCL treated with CAR T cells in the 3L+ setting associated with reduced V2VT using currently available evidence.

[0336] In real-world settings, multiple factors can influence V2VT in patients receiving CAR T, and delays during this multi-step process can impact patient outcomes. This study synthesized publicly available real-world data to demonstrate potential differences in survival outcomes based on V2VT. The modeling described herein demonstrated that outcomes were primarily determined by a higher probability of reaching infusion, and subsequently, improved outcomes were demonstrated for infused patients compared to non-infused patients. Results were further enhanced in sensitivity analyses across a range of tests.

[0337] As is common in modeling studies, a number of key assumptions were made, including the induction of HRs (hazard ratios); the lack of formal interrogation of outcomes with bridging therapy; and the assumption that there were no differences in the efficacy of the treatments. However, a series of sensitivity analyses were performed to test the impact of these assumptions.

[0338] V2VT can be an important predictor of outcome in R / R LBCL, and aiming for short-term manufacturing, product release, delivery, and infusion is key to further improve CAR T-cell outcomes. This study shows that modest differences in V2VT can lead to significant effects on life expectancy.

[0339] ***

[0340] Although a number of embodiments have been described, it will be apparent that the present disclosure and embodiments may provide other embodiments that utilize or are encompassed by the compositions and methods described herein. Therefore, it will be understood that the scope of the present disclosure will be defined by what can be understood from this disclosure and the appended claims, rather than by the embodiments that have been presented by way of example.

Claims

1. A method for preparing lymphocytes having improved efficacy and / or reduced adverse effects in treating cancer, the method comprising Obtaining lymphocytes from the patient by apheresis; incubating the lymphocytes with the polynucleotide vector to transduce the lymphocytes, thereby producing transduced lymphocytes; culturing the transduced lymphocytes to obtain a cultured lymphocyte sample; as well as infusing the sample into the patient, The time from obtaining the lymphocytes to infusing the sample is no longer than 28 days.

2. A method for preventing and / or reducing the likelihood of long-term thrombocytopenia in a patient with r / r LBCL, the method comprising obtaining lymphocytes from the patient by apheresis; incubating lymphocytes with the polynucleotide vector to transduce the lymphocytes, thereby producing transduced lymphocytes; culturing the transduced lymphocytes to obtain a cultured lymphocyte sample; as well as infusing the sample into the patient, The time from obtaining the lymphocytes to infusing the sample is no longer than 28 days.

3. The method according to claim 1 or 2, wherein the patient has greater than 55% probability of having a complete response; Greater than 45% probability of overall survival at 24 months; and / or Less than 30% chance of developing long-term thrombocytopenia.

4. The method of claim 1 or 2, wherein the time taken from obtaining the lymphocytes to infusing the sample is no longer than 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.

5. The method of claim 1 or 2, further comprising administering lymphodepleting chemotherapy, and wherein the lymphodepleting chemotherapy is administered within 5 days, 4 days, 3 days, 2 days, or 1 day of the infusion step.

6. A method according to any preceding claim, which does not comprise cryopreservation of the cultured lymphocytes.

7. The method of any preceding claim, wherein the transduced lymphocytes are cultured for less than 72 hours, 48 hours or 36 hours.

8. The method according to any preceding claim, wherein the incubation is performed in a closed system.

9. The method of claim 8, wherein the closed system has a volume of at least 1500 cm 2 The 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, wherein the coating is performed using a solution comprising about 1 µg / ml to 10 µg / ml of the recombinant human fibronectin.

11. The method of claim 10, wherein the inner surface is further contacted with a second solution comprising the polynucleotide carrier, wherein the second solution has a volume of about 200 mL of solution.

12. The method of claim 11, wherein the coating further comprises draining the second solution.

13. The method according to any one of claims 8 to 12, wherein the sample in the closed system comprises at least 1.5×10 8 Lymphocytes.

14. The method according to claim 12, wherein the sample comprises at least 4×10 8 Lymphocytes.

15. The method according to any preceding claim, wherein the lymphocytes are peripheral blood mononuclear cells (PBMC) or T cells.

16. The method according to any preceding claim, wherein the sample comprises CD4+ T cells and CD8+ T cells.

17. The method of any preceding claim, wherein a total of 10,000 to 1,000,000 cultured lymphocytes per kg of the patient are administered to the patient.

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

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

20. The method according to any preceding claim, wherein the polynucleotide vector is a viral vector.

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

22. The method of any preceding claim, wherein the vector encodes one or more chimeric antigen receptors (CARs) or one or more T cell receptors (TCRs).

23. The method of claim 22, wherein the one or more CARs comprise an intracellular co-stimulatory domain.

24. The method of claim 23, wherein the intracellular co-stimulatory domain is a signaling region of a protein selected from the group consisting of: DAP-10, CD28, OX-40, 4-1BB (CD137), CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T-cell co-stimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD11a / CD18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), tumor necrosis factor superfamily member 14, TNFSF14, LIGHT), NKG2C, Ig α (CD79a), Fc γ receptor, MHC Class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, CDS, GITR, BAFFR, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 α, CD8 β, IL2R β, IL2R γ, IL7R α, ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD (CD11d), ITGAE (CD103), ITGAL (CD11a), ITGAM (CD11b), IT GAX (CD11c), ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, TNFR2, TRANCE (RANKL), DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, C D96 (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, ligands that specifically bind to CD83, and combinations thereof.

25. The method of claim 24, wherein the intracellular co-stimulatory domain is the signaling region of CD28.

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

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

28. The method of claim 27, wherein the lymphocytes comprising the CAR are akilenbine or brexucabtagene autoleucel.

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

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

31. A method for predicting the likelihood of a complete response to immunotherapy in a patient, the method comprising: determining a time period from a leukapheresis step of said patient to administration of said immunotherapy to said patient; grouping the patients into one of a plurality of groups based on the determined time period, the plurality of groups comprising: a first group characterized in that the time period from said leukapheresis step to said administration of said immunotherapy to said patient is up to 28 days; A second group characterized in that the time period from said leukapheresis step to said administration of said immunotherapy to said patient is between 28 days and 40 days; and A third group, characterized in that the time period from said leukapheresis step to said administration of said immunotherapy to said patient is at least 40 days; and determining a likelihood of a complete response in the patient based at least in part on which of the plurality of groups the patient is grouped into, wherein the patient has a likelihood of at least about 55% of a complete response if the patient is grouped into the first group or the second group, and Wherein if the patient is grouped into the third group, the patient has a likelihood of at least about 42% of a complete response.

32. The method of claim 31 , wherein the patient has about a 60% likelihood of a complete response if the patient is grouped into the first group or the second group.

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

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

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

36. The method of claim 31, wherein the immunotherapy is akilenbine or brexucabtagene autoleucel.

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

38. A method for predicting overall survival of a patient receiving immunotherapy, the method comprising: determining a time period from a leukapheresis step of said patient to administration of said immunotherapy to said patient; grouping the patients into one of a plurality of groups based on the determined time period, the plurality of groups comprising: a first group characterized in that the time period from said leukapheresis step to said administration of said immunotherapy to said patient is up to 28 days; A second group characterized in that the time period from said leukapheresis step to said administration of said immunotherapy to said patient is between 28 days and 40 days; and A third group, characterized in that the time period from said leukapheresis step to said administration of said immunotherapy to said patient is at least 40 days; and determining the overall survival rate in the patient based at least in part on which of the plurality of groups the patient is grouped into, wherein if the patient is grouped into the first group, the patient has an overall survival rate of at least about 49%, wherein if the patient is grouped into the second group, the patient has an overall survival rate of at least about 48%, and Wherein if the patient is grouped into the third group, the patient has an overall survival rate of at least about 30%.

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

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

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

42. The method of claim 38, wherein the immunotherapy is akilenbine or brexucabtagene autoleucel.

43. The method of 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 a patient receiving immunotherapy, the method comprising: determining a time period from a leukapheresis step of said patient to administration of said immunotherapy to said patient; grouping the patients into one of a plurality of groups based on the determined time period, the plurality of groups comprising: a first group characterized in that the time period from said leukapheresis step to said administration of said immunotherapy to said patient is up to 28 days; A second group characterized in that the time period from said leukapheresis step to said administration of said immunotherapy to said patient is between 28 days and 40 days; and A third group, characterized in that the time period from said leukapheresis step to said administration of said immunotherapy to said patient is at least 40 days; and determining the risk of thrombocytopenia in the patient based at least in part on which of the plurality of groups the patient is grouped into, wherein if the patient is grouped into the first group, the patient has a risk of thrombocytopenia of about 18%, wherein if the patient is grouped into the second group, the patient has a risk of thrombocytopenia of about 25%, and Wherein if the patient is grouped into the third group, the patient has a risk of approximately 34% for thrombocytopenia.

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

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

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

48. The method of claim 44, wherein the immunotherapy is akilenbine or brexucabtagene autoleucel.

49. The method of 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 a patient who has received immunotherapy, the method comprising: determining a time period from a leukapheresis step of the patient to administration of the immunotherapy to the patient, wherein the time period is a short time period or a long time period; assigning a probability of successful infusion based on the time period; The patient information is input into a survival model to determine the life expectancy and the quality-adjusted life years of the patient.

51. The method of claim 50, wherein the short period of time is about 24 days or less.

52. The method of claim 50, wherein the prolonged period is about 54 days or longer.

53. The method of claim 50, wherein the prolonged period is about 37 days or longer.

54. The method of claim 50, wherein the short time period indicates that the increase in the life expectancy and the increase in the quality-adjusted life years of the patient are greater than 5 years, and wherein the long time period indicates that the increase in the life expectancy and the increase in the quality-adjusted life years of the patient are less than 5 years.

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

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

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

58. The method of claim 50, wherein the immunotherapy is akilenbine or brexucabtagene autoleucel.

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

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